@article {6585, title = {Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.}, journal = {Mol Ther}, volume = {32}, year = {2024}, month = {2024 Mar 06}, pages = {663-677}, abstract = {

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in\ vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in\ vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

}, keywords = {Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Repressor Proteins, Zinc Fingers}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2024.01.023}, author = {Rajendiran, Vignesh and Devaraju, Nivedhitha and Haddad, Mahdi and Ravi, Nithin Sam and Panigrahi, Lokesh and Paul, Joshua and Gopalakrishnan, Chandrasekar and Wyman, Stacia and Ariudainambi, Keerthiga and Mahalingam, Gokulnath and Periyasami, Yogapriya and Prasad, Kirti and George, Anila and Sukumaran, Dhiyaneshwaran and Gopinathan, Sandhiya and Pai, Aswin Anand and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Ramalingam, Rajasekaran and Thangavel, Saravanabhavan and Velayudhan, Shaji R and Corn, Jacon E and Mackay, Joel P and Marepally, Srujan and Srivastava, Alok and Crossley, Merlin and Mohankumar, Kumarasamypet M} } @article {6592, title = {Cryo-EM research in India.}, journal = {Structure}, volume = {32}, year = {2024}, month = {2024 Feb 01}, pages = {113-119}, abstract = {

To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.

}, keywords = {Cryoelectron Microscopy, India}, issn = {1878-4186}, doi = {10.1016/j.str.2024.01.002}, author = {Shukla, Arun K and Banerjee, Manidipa and Singh, Appu K and Penmatsa, Aravind and Dutta, Somnath and Anand, Ruchi and Sirajuddin, Minhajuddin and Srivastava, Shanti Swaroop} } @article {6583, title = {Photostimulated Extended Nitric Oxide (NO) Release from Water-Soluble Block Copolymer to Enhance Antibacterial Activity.}, journal = {Biomacromolecules}, volume = {25}, year = {2024}, month = {2024 Jan 08}, pages = {77-88}, abstract = {

-Nitrosamines are well established motifs to release nitric oxide (NO) under photoirradiation. Herein, a series of amphiphilic -nitrosamine-based block copolymers () are developed to attain controlled NO release under photoirradiation (365 nm, 3.71 mW/cm). The water-soluble forms micellar architecture in aqueous medium and exhibits a sustained NO release of 92-160 μM within 11.5 h, which is 36.8-64.0\% of the calculated value. To understand the NO release mechanism, a small molecular NO donor () resembling the NO releasing functional motif of is synthesized, which displays a burst NO release in DMSO within 2.5 h. The radical nature of the released NO is confirmed by electron paramagnetic resonance (EPR) spectroscopy. The gradual NO release from micellar enhances antibacterial activity over and exhibits a superior bactericidal effect on Gram-positive . In relation to biomedical applications, this work offers a comprehensive insight into tuning light-triggered NO release to improve antibacterial activity.

}, keywords = {Anti-Bacterial Agents, Micelles, Nitric Oxide, Polymers, Staphylococcus aureus}, issn = {1526-4602}, doi = {10.1021/acs.biomac.3c00822}, author = {Paul, Soumya and Ashrit, Priya and Kumar, Manish and Mete, Sourav and Ghosh, Shilpendu and Vemula, Praveen Kumar and Mukherjee, Arindam and De, Priyadarsi} } @article {6587, title = {Sleep fragmentation induces heart failure in a hypertrophic cardiomyopathy mouse model by altering redox metabolism.}, journal = {iScience}, volume = {27}, year = {2024}, month = {2024 Mar 15}, pages = {109075}, abstract = {

Sleep fragmentation (SF) disrupts normal biological rhythms and has major impacts on cardiovascular health; however, it has never been shown to be a risk factor involved in the transition from cardiac hypertrophy to heart failure (HF). We now demonstrate devastating effects of SF on hypertrophic cardiomyopathy (HCM). We generated a transgenic mouse model harboring a patient-specific myosin binding protein C3 (MYBPC3) variant displaying HCM, and measured the progression of pathophysiology in the presence and absence of SF. SF induces mitochondrial damage, sarcomere disarray, and apoptosis in HCM mice; these changes result in a transition of hypertrophy to an HF phenotype by chiefly targeting redox metabolic pathways. Our findings for the first time show that SF is a risk factor for HF transition and have important implications in clinical settings where HCM patients with sleep disorders have worse prognosis, and strategic intervention with regularized sleep patterns might help such patients.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2024.109075}, author = {Bose, Karthikeyan and Agrawal, Radhika and Sairam, Thiagarajan and Mil, Jessenya and Butler, Matthew P and Dhandapany, Perundurai S} } @article {2881, title = {Age-stratified adeno-associated virus serotype 3 neutralizing and total antibody prevalence in hemophilia A patients from India.}, journal = {J Med Virol}, volume = {94}, year = {2022}, month = {2022 Sep}, pages = {4542-4547}, abstract = {

Gene therapy using an adeno-associated virus (AAV) vector offers a new treatment option for individuals with monogenetic disorders. The major bottleneck is the presence of pre-existing anti-AAV antibodies, which impacts its use. Even very low titers of neutralizing antibodies (NAb) to capsids from natural AAV infections have been reported to inhibit the transduction of intravenously administered AAV in animal models and are associated with limited efficacy in human trials. Assessing the level of pre-existing NAb is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Techniques used to screen AAV-antibodies include AAV capsid enzyme-linked immunosorbent assay\ (ELISA) and transduction inhibition assay (TIA) for detecting total capsid-binding (TAb) and Nab, respectively. In this study, we screened 521 individuals with hemophilia A from India for TAb and NAb using ELISA and TIA, respectively. The prevalence of TAb and NAb in hemophilia A patients from India were 96\% and 77.5\%, respectively. There was a significant increase in anti-AAV3 NAb prevalence with age in the hemophilia A patient group from India. There was a trend in anti-AAV3 TAb positivity between the pediatric age group (94.4\%) and the adult age group (97.4\%).

}, keywords = {Adult, Animals, Antibodies, Neutralizing, Antibodies, Viral, Child, Dependovirus, Genetic Vectors, Hemophilia A, Humans, Prevalence, Serogroup}, issn = {1096-9071}, doi = {10.1002/jmv.27859}, author = {Daniel, Hubert D-J and Kumar, Sanjay and Kannangai, Rajesh and J, Farzana and Joel, Joseph N and Abraham, Aby and Lakshmi, Kavitha M and Agbandje-McKenna, Mavis and Coleman, Kirsten E and Srivastava, Arun and Srivastava, Alok and Abraham, Asha M} } @article {2885, title = {An Amphiphilic Double-Brush Polymer Hydrogel for Sustained Release of Small Molecules and Biologics: Insulin Delivering-Hydrogel to Control Hyperglycemia}, year = {2022}, doi = { https://doi.org/10.1002/cnma.202200184}, author = {Dhayani, Ashish and Bej, Sujoy and Mudnakudu-Nagaraju, Kiran K and Chakraborty, Saheli and Srinath, Preetham and Kumar, Ashok H and PS, Ann Maria and Kristi, Anand and Ramakrishnan, S and Vemula PK} } @article {2466, title = {The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy}, journal = {Front. Immunol}, year = {2022}, abstract = {

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90\% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

}, doi = {https://doi.org/10.3389/fimmu.2022.792684}, author = {Karthik V. Karuppusamy and John Paul Demosthenes and Vigneshwaran Venkatesan and Abisha Crystal Christopher and Prathibha Babu and Manojkumar K. Azhagiri and Annlin Jacob and Veena Vadhini Ramalingam and Sumathi Rangaraj and Mohankumar Kumarasamypet Murugesan and Srujan Marepally and George Varghese and Alok Srivastava and Rajesh Kannangai and Saravanabhavan Thangavel} } @article {2542, title = {DDX24 is required for muscle fiber organization and the suppression of wound-induced Wnt activity necessary for pole re-establishment during planarian regeneration.}, journal = {Dev Biol}, volume = {488}, year = {2022}, month = {2022 May 04}, pages = {11-29}, abstract = {

Planarians have a remarkable ability to undergo whole-body regeneration. Successful regeneration outcome is determined by processes like polarity establishment at the wound site, which is followed by pole (organizer) specification. Interestingly, these determinants are almost exclusively expressed by muscles in these animals. However, the molecular toolkit that enables the functional versatility of planarian muscles remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase, is necessary for planarian survival and regeneration. We found that DDX24 is enriched in muscles and its knockdown disrupts muscle fiber organization. This leads to defective pole specification, which in turn results in misregulation of many positional control genes specifically during regeneration. ddx24 RNAi also upregulates wound-induced Wnt signalling. Suppressing this ectopic Wnt activity rescues the knockdown phenotype by enabling better anterior pole regeneration. To summarize, our work highlights the role of an RNA helicase in muscle fiber organization, and modulating amputation-induced wnt levels, both of which seem critical for pole re-organization, thereby regulating whole-body regeneration.

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2022.04.011}, author = {Sarkar, Souradeep R and Dubey, Vinay Kumar and Jahagirdar, Anusha and Lakshmanan, Vairavan and Haroon, Mohamed Mohamed and Sowndarya, Sai and Sowdhamini, Ramanathan and Palakodeti, Dasaradhi} } @article {2425, title = {Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic Channel.}, journal = {J Phys Chem B}, volume = {126}, year = {2022}, month = {2022 Jan 27}, pages = {588-600}, abstract = {

Complexes of cationic liposomes with DNA have emerged as promising nonviral vectors for delivering genetic information into cells for gene therapy. Kinetics of the liposome/DNA complex (lipoplex) formation on a millisecond time scale are studied by monitoring time evolution of fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) and ethidium bromide (EtBr) in a continuous flow microfluidic channel coupled to a fluorescence microscope. The formation of lipoplexes between calf thymus DNA and liposomes based on two novel cationic lipids (Lip1810 and Lip1814) are found to follow a two-step process with kinetic constants for the Lip1814/DNA complex ( = 1120-1383 s, = 0.227-1.45 s) being significantly different from those ( = 68.53-98.5 s, = 32.3-60.19 s) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is whereas the formation of Lip1810/DNA complex occurs by a . The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.1c07263}, author = {Mukherjee, Dipanjan and Hasan, Md Nur and Ghosh, Ria and Ghosh, Gourab and Bera, Arpan and Prasad, Sujanthi Easwara and Hiwale, Ankita and Vemula, Praveen K and Das, Ranjan and Pal, Samir Kumar} } @article {3715, title = {Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.}, journal = {Front Genome Ed}, volume = {4}, year = {2022}, month = {2022}, pages = {1085111}, abstract = {

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG \> GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A \> T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72\%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

}, issn = {2673-3439}, doi = {10.3389/fgeed.2022.1085111}, author = {George, Anila and Ravi, Nithin Sam and Prasad, Kirti and Panigrahi, Lokesh and Koikkara, Sanya and Rajendiran, Vignesh and Devaraju, Nivedhitha and Paul, Joshua and Pai, Aswin Anand and Nakamura, Yukio and Kurita, Ryo and Balasubramanian, Poonkuzhali and Thangavel, Saravanabhavan and Marepally, Srujan and Velayudhan, Shaji R and Srivastava, Alok and Mohankumar, Kumarasamypet M} } @article {3341, title = {Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications.}, journal = {Sci Rep}, volume = {12}, year = {2022}, month = {2022 08 18}, pages = {14033}, abstract = {

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel\ lineage-specific RNA interference\ (RNAi) lentiviral\ vectors, H23B-Ery-Lin-shRNA\ and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells\ without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by\ transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for\ any gene, and they also provide superior knockdown of the target gene expression with a\ single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A,\ two\ well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34 cells with these vectors resulted in\ \>80\% reduction in the target\ protein levels and up to 40\% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34 cells transduced with H23B-Ery-Lin-shBCL11A\ LV in immunocompromised mice showed ~ 60\% reduction in BCL11A protein expression with ~\ 40\% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34 cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a\ high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.

}, keywords = {Animals, Cell Line, Tumor, Cell Lineage, DNA-Binding Proteins, Genetic Vectors, Humans, Lentivirus, Mice, RNA Interference, RNA, Small Interfering, Transcription Factors, Transduction, Genetic}, issn = {2045-2322}, doi = {10.1038/s41598-022-13783-0}, author = {Bagchi, Abhirup and Devaraju, Nivedhitha and Chambayil, Karthik and Rajendiran, Vignesh and Venkatesan, Vigneshwaran and Sayed, Nilofer and Pai, Aswin Anand and Nath, Aneesha and David, Ernest and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Srivastava, Alok and Thangavel, Saravanabhavan and Mohankumar, Kumarasamypet M and Velayudhan, Shaji R} } @article {3346, title = {Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis.}, journal = {Mol Neurobiol}, volume = {59}, year = {2022}, month = {2022 Dec}, pages = {7370-7392}, abstract = {

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP{\textquoteright}s domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP{\textquoteright}s domains in translation regulation.

}, keywords = {Fragile X Mental Retardation Protein, Fragile X Syndrome, Humans, Microtubules, Neurons, Protein Biosynthesis, Ribosomes, RNA, Messenger}, issn = {1559-1182}, doi = {10.1007/s12035-022-03049-1}, author = {D{\textquoteright}Souza, Michelle Ninochka and Ramakrishna, Sarayu and Radhakrishna, Bindushree K and Jhaveri, Vishwaja and Ravindran, Sreenath and Yeramala, Lahari and Nair, Deepak and Palakodeti, Dasaradhi and Muddashetty, Ravi S} } @article {2545, title = {Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System.}, journal = {Methods Mol Biol}, volume = {2429}, year = {2022}, month = {2022}, pages = {307-331}, abstract = {

Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.

}, keywords = {Animals, CRISPR-Cas Systems, Gene Editing, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Mice, Transplantation, Autologous}, issn = {1940-6029}, doi = {10.1007/978-1-0716-1979-7_20}, author = {Devaraju, Nivedhitha and Rajendiran, Vignesh and Ravi, Nithin Sam and Mohankumar, Kumarasamypet M} } @article {2465, title = {Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.}, journal = {Elife}, volume = {11}, year = {2022}, month = {2022 02 11}, abstract = {

Naturally occurring point mutations in the promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T \> C and -124T \> C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

}, keywords = {Adenine, Anemia, Sickle Cell, beta-Globins, beta-Thalassemia, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Cytosine, Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Point Mutation, Promoter Regions, Genetic}, issn = {2050-084X}, doi = {10.7554/eLife.65421}, author = {Ravi, Nithin Sam and Wienert, Beeke and Wyman, Stacia K and Bell, Henry William and George, Anila and Mahalingam, Gokulnath and Vu, Jonathan T and Prasad, Kirti and Bandlamudi, Bhanu Prasad and Devaraju, Nivedhitha and Rajendiran, Vignesh and Syedbasha, Nazar and Pai, Aswin Anand and Nakamura, Yukio and Kurita, Ryo and Narayanasamy, Muthuraman and Balasubramanian, Poonkuzhali and Thangavel, Saravanabhavan and Marepally, Srujan and Velayudhan, Shaji R and Srivastava, Alok and DeWitt, Mark A and Crossley, Merlin and Corn, Jacob E and Mohankumar, Kumarasamypet M} } @article {3343, title = {Initiation of wound healing is regulated by the convergence of mechanical and epigenetic cues.}, journal = {PLoS Biol}, volume = {20}, year = {2022}, month = {2022 09}, pages = {e3001777}, abstract = {

Wound healing in the skin is a complex physiological process that is a product of a cell state transition from homeostasis to repair. Mechanical cues are increasingly being recognized as important regulators of cellular reprogramming, but the mechanism by which it is translated to changes in gene expression and ultimately cellular behavior remains largely a mystery. To probe the molecular underpinnings of this phenomenon further, we used the down-regulation of caspase-8 as a biomarker of a cell entering the wound healing program. We found that the wound-induced release of tension within the epidermis leads to the alteration of gene expression via the nuclear translocation of the DNA methyltransferase 3A (DNMT3a). This enzyme then methylates promoters of genes that are known to be down-regulated in response to wound stimuli as well as potentially novel players in the repair program. Overall, these findings illuminate the convergence of mechanical and epigenetic signaling modules that are important regulators of the transcriptome landscape required to initiate the tissue repair process in the differentiated layers of the epidermis.

}, keywords = {Biomarkers, Caspase 8, Cues, Epigenesis, Genetic, Wound Healing}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3001777}, author = {Bhatt, Tanay and Dey, Rakesh and Hegde, Akshay and Ketkar, Alhad Ashok and Pulianmackal, Ajai J and Deb, Ashim P and Rampalli, Shravanti and Jamora, Colin} } @article {3342, title = {Methionine uptake via the SLC43A2 transporter is essential for regulatory T-cell survival.}, journal = {Life Sci Alliance}, volume = {5}, year = {2022}, month = {2022 Sep 09}, abstract = {

Cell death, survival, or growth decisions in T-cell subsets depend on interplay between cytokine-dependent and metabolic processes. The metabolic requirements of T-regulatory cells (Tregs) for their survival and how these are satisfied remain unclear. Herein, we identified a necessary requirement of methionine uptake and usage for Tregs survival upon IL-2 deprivation. Activated Tregs have high methionine uptake and usage to S-adenosyl methionine, and this uptake is essential for Tregs survival in conditions of IL-2 deprivation. We identify a solute carrier protein SLC43A2 transporter, regulated in a Notch1-dependent manner that is necessary for this methionine uptake and Tregs viability. Collectively, we uncover a specifically regulated mechanism of methionine import in Tregs that is required for cells to adapt to cytokine withdrawal. We highlight the need for methionine availability and metabolism in contextually regulating cell death in this immunosuppressive population of T cells.

}, keywords = {Interleukin-2, Methionine, Racemethionine, Solute Carrier Proteins, T-Lymphocytes, Regulatory}, issn = {2575-1077}, doi = {10.26508/lsa.202201663}, author = {Saini, Neetu and Naaz, Afsana and Metur, Shree Padma and Gahlot, Pinki and Walvekar, Adhish and Dutta, Anupam and Davathamizhan, Umamaheswari and Sarin, Apurva and Laxman, Sunil} } @article {3640, title = {Nanobody derived using a peptide epitope from the spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants.}, journal = {J Biol Chem}, volume = {299}, year = {2022}, month = {2022 Nov 22}, pages = {102732}, abstract = {

The emergence of new escape mutants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has escalated its penetration among the human population and has reinstated its status as a global pandemic. Therefore, developing effective antiviral therapy against emerging SARS-CoV variants and other viruses in a short period becomes essential. Blocking SARS-CoV-2 entry into human host cells by disrupting the spike glycoprotein-angiotensin-converting enzyme 2 interaction has already been exploited for vaccine development and monoclonal antibody therapy. Unlike the previous reports, our study used a nine-amino acid peptide from the receptor-binding motif of the spike protein as an epitope. We report the identification of an efficacious nanobody N1.2 that blocks the entry of pseudovirus-containing SARS-CoV-2 spike as the surface glycoprotein. Moreover, using mCherry fluorescence-based reporter assay, we observe a more potent neutralizing effect against both the hCoV19 (Wuhan/WIV04/2019) and the Omicron (BA.1) pseudotyped spike virus with a bivalent version of the N1.2 nanobody. In summary, our study presents a rapid and efficient methodology to use peptide sequences from a protein-receptor interaction interface as epitopes for screening nanobodies against potential pathogenic targets. We propose that this approach can also be widely extended to target other viruses and pathogens in the future.

}, issn = {1083-351X}, doi = {10.1016/j.jbc.2022.102732}, author = {Mendon, Nivya and Ganie, Rayees A and Kesarwani, Shubham and Dileep, Drisya and Sasi, Sarika and Lama, Prakash and Chandra, Anchal and Sirajuddin, Minhajuddin} } @article {2422, title = {Novel Mutations in β- Gene in Indian Patients With Dilated Cardiomyopathy.}, journal = {CJC Open}, volume = {4}, year = {2022}, month = {2022 Jan}, pages = {1-11}, abstract = {

Background: Heart failure is a hallmark of severe hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Several mutations in the gene lead to hypertrophic cardiomyopathy. Recently, causative mutations in the gene have also been detected in DCM from different populations.

Methods: Here, we sequenced the gene in 137 Indian DCM patients and 167 ethnically matched healthy controls to detect the frequency of mutations and their association.

Results: Our study revealed 27 variations, of which 7 mutations (8.0\%) were detected exclusively in Indian DCM patients for the first time. These included 4 missense mutations-Arg723His, Phe510Leu, His358Leu, and Ser384Tyr (2.9\%); a frameshift mutation-Asn676_T-del (1.5\%); and 2 splice-site mutations (IVS17+2T) T\>G and (IVS19-1G) G\>A (3.6\%). Remarkably, all 4 missense mutations altered evolutionarily conserved amino acids. All 4 missense mutations were predicted to be pathogenic by 2 bioinformatics tools-polymorphism phenotyping v2 (PolyPhen-2) and sorting intolerant from tolerant (SIFT). In addition, the 4 homology models of β-MYH7-p.Leu358, p.Tyr384, p.Leu510, and p.His723-displayed root-mean-square deviations of \~{}2.55 {\r A}, \~{}1.24 {\r A}, \~{}3.36 {\r A}, and \~{}3.86 {\r A}, respectively.

Conclusions: In the present study, we detected numerous novel, unique, and rare mutations in the gene exclusively in Indian DCM patients (8.0\%). Here, we demonstrated how each mutant (missense) uniquely disrupts a critical network of non-bonding interactions at the mutation site (molecular level) and may contribute to development of dilated cardiomyopathy (DCM). Therefore, our findings may provide insight into the understanding of the molecular bases of disease and into diagnosis along with promoting novel therapeutic strategies (through personalized medicine).

}, issn = {2589-790X}, doi = {10.1016/j.cjco.2021.07.020}, author = {Rani, Deepa Selvi and Vijaya Kumar, Archana and Nallari, Pratibha and Sampathkumar, Katakam and Dhandapany, Perundurai S and Narasimhan, Calambur and Rathinavel, Andiappan and Thangaraj, Kumarasamy} } @article {3716, title = {Omicron infection increases IgG binding to spike protein of predecessor variants.}, journal = {J Med Virol}, year = {2022}, month = {2022 Dec 22}, abstract = {

BACKGROUND: SARS-CoV-2 transmission in India in 2020-2022 was driven predominantly by Wild (Wuhan-Hu-1and D614G), Delta, and Omicron variants. The aim of this study was to examine the effect of infections on the humoral immune response and cross-reactivity to spike proteins of Wuhan-Hu-1, Delta, C.1.2., and Omicron.

OBJECTIVES: Residual archival sera (N=81) received between January 2020 and March 2022 were included. Infection status was inferred by a positive SARS-CoV-2 RT-PCR and/or serology (anti-N and anti-S antibodies) and sequencing of contemporaneous samples (N=18) to infer lineage. We estimated the levels and cross-reactivity of infection-induced sera including Wild, Delta, Omicron as well as vaccine breakthrough infections (Delta and Omicron).

RESULTS: We found ~2-fold increase in spike-specific IgG antibody binding in post-Omicron infection compared to the pre-Omicron period, whilst the change in pre- and post-Delta infections were similar. Further investigation of Omicron-specific humoral responses revealed primary Omicron infection as an inducer of cross-reactive antibodies against predecessor variants, in spite of weaker degree of humoral response compared to Wuhan-Hu-1 and Delta infection. Intriguingly, Omicron vaccine-breakthrough infections when compared with primary infections, exhibited increased humoral responses against RBD (7.7-fold) and Trimeric S (Trimeric form of spike protein) (34.6-fold) in addition to increased binding of IgGs towards previously circulating variants (4.2 - 6.5-fold). Despite Delta breakthrough infections showing a higher level of humoral response against RBD (2.9-fold) and Trimeric S (5.7-fold) compared to primary Delta sera, a demonstrably reduced binding (36-49\%) was observed to Omicron spike protein.

CONCLUSIONS: Omicron vaccine breakthrough infection results in increased intensity of humoral response and wider breadth of IgG binding to spike proteins of antigenically-distinct, predecessor variants. This article is protected by copyright. All rights reserved.

}, issn = {1096-9071}, doi = {10.1002/jmv.28419}, author = {Mahalingam, Gokulnath and Periyasami, Yogapriya and Arjunan, Porkizhi and Subaschandrabose, Rajesh Kumar and Mathivanan, Tamil Venthan and Mathew, Roshlin Susan and Ramya Devi, Kt and Premkumar, Prasanna Samuel and Muliyil, Jayaprakash and Srivastava, Alok and Moorthy, Mahesh and Marepally, Srujan} } @article {3347, title = {S. mediterranea ETS-1 regulates the function of cathepsin-positive cells and the epidermal lineage landscape via basement membrane remodeling.}, journal = {J Cell Sci}, volume = {135}, year = {2022}, month = {2022 10 15}, abstract = {

Extracellular matrix (ECM) is an important component of stem cell niche. Remodeling of ECM mediated by ECM regulators, such as matrix metalloproteinases (MMPs) plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF) ETS-1, which is expressed in the cathepsin-positive cell population, in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outwards from this region and progressively differentiate to maintain the terminal epidermis. Our data shows that thickening of the basement membrane in the absence of ets-1 results in defective migration of stem cell progeny. Furthermore, the absence of ets-1 leads to a defective epidermal progenitor landscape, despite its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodeling in regulating tissue homeostasis and regeneration in the planarian Schmidtea mediterranea. This article has an associated First Person interview with one of the co-first authors of the paper.

}, keywords = {Animals, Basement Membrane, Cathepsins, Cell Differentiation, Epidermis, Humans, Matrix Metalloproteinases, Mediterranea, Planarians, Transcription Factors}, issn = {1477-9137}, doi = {10.1242/jcs.259900}, author = {Dubey, Vinay Kumar and Sarkar, Souradeep R and Lakshmanan, Vairavan and Dalmeida, Rimple and Gulyani, Akash and Palakodeti, Dasaradhi} } @article {3345, title = {Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of matricellular protein Mindin.}, journal = {Cell Rep}, volume = {40}, year = {2022}, month = {2022 09 20}, pages = {111390}, abstract = {

Preservation of a small population of cancer stem cells (CSCs) within a heterogeneous carcinoma serves as a paradigm to understand how select cells in a tissue maintain their undifferentiated status. In both embryogenesis and cancer, Snail has been correlated with stemness, but the molecular underpinning of this phenomenon remains largely ill-defined. In models of cutaneous squamous cell carcinoma (cSCC), we discovered a non-epithelial-mesenchymal transition function for the transcription factor Snail in maintaining the stemness of epidermal keratinocytes. Snail-expressing cells secrete the matricellular protein Mindin, which functions in an autocrine fashion to activate a Src-STAT3 pathway to reinforce their stem/progenitor phenotype. This pathway is activated by the engagement of Mindin with the leukocyte-specific integrin, CD11b (ITGAM), which is also unexpectedly expressed by epidermal keratinocytes. Interestingly, disruption of this signaling module in human cSCC attenuates tumorigenesis, suggesting that targeting Mindin would be a promising therapeutic approach to hinder cancer recurrence.

}, keywords = {Carcinoma, Squamous Cell, Cell Line, Tumor, Epithelial Cells, Extracellular Matrix Proteins, Humans, Integrins, Neoplasm Proteins, Neoplasm Recurrence, Local, Neoplastic Stem Cells, Skin Neoplasms, Snail Family Transcription Factors}, issn = {2211-1247}, doi = {10.1016/j.celrep.2022.111390}, author = {Badarinath, Krithika and Dam, Binita and Kataria, Sunny and Zirmire, Ravindra K and Dey, Rakesh and Kansagara, Gaurav and Ajnabi, Johan and Hegde, Akshay and Singh, Randhir and Masudi, Tafheem and Sambath, Janani and Sachithanandan, Sasikala P and Kumar, Prashant and Gulyani, Akash and He, You-Wen and Krishna, Sudhir and Jamora, Colin} } @article {2463, title = {Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy.}, journal = {NPJ Genom Med}, volume = {7}, year = {2022}, month = {2022 Mar 14}, pages = {18}, abstract = {

Cardiomyopathy (CMP) is a heritable disorder. Over 50\% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5\% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 {\texttimes} 10 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7-58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

}, issn = {2056-7944}, doi = {10.1038/s41525-022-00288-y}, author = {Lesurf, Robert and Said, Abdelrahman and Akinrinade, Oyediran and Breckpot, Jeroen and Delfosse, Kathleen and Liu, Ting and Yao, Roderick and Persad, Gabrielle and McKenna, Fintan and Noche, Ramil R and Oliveros, Winona and Mattioli, Kaia and Shah, Shreya and Miron, Anastasia and Yang, Qian and Meng, Guoliang and Yue, Michelle Chan Seng and Sung, Wilson W L and Thiruvahindrapuram, Bhooma and Lougheed, Jane and Oechslin, Erwin and Mondal, Tapas and Bergin, Lynn and Smythe, John and Jayappa, Shashank and Rao, Vinay J and Shenthar, Jayaprakash and Dhandapany, Perundurai S and Semsarian, Christopher and Weintraub, Robert G and Bagnall, Richard D and Ingles, Jodie and Mel{\'e}, Marta and Maass, Philipp G and Ellis, James and Scherer, Stephen W and Mital, Seema} } @article {2211, title = {Adiponectin receptor 1 variants contribute to hypertrophic cardiomyopathy that can be reversed by rapamycin.}, journal = {Sci Adv}, volume = {7}, year = {2021}, month = {2021 Jan}, abstract = {

Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart muscle disease characterized by hypertrophy with preserved or increased ejection fraction in the absence of secondary causes. However, recent studies have demonstrated that a substantial proportion of individuals with HCM also have comorbid diabetes mellitus (~10\%). Whether genetic variants may contribute a combined phenotype of HCM and diabetes mellitus is not known. Here, using next-generation sequencing methods, we identified novel and ultrarare variants in adiponectin receptor 1 () as risk factors for HCM. Biochemical studies showed that variants dysregulate glucose and lipid metabolism and cause cardiac hypertrophy through the p38/mammalian target of rapamycin and/or extracellular signal-regulated kinase pathways. A transgenic mouse model expressing an variant displayed cardiomyopathy that recapitulated the cellular findings, and these features were rescued by rapamycin. Our results provide the first evidence that variants can cause HCM and provide new insights into regulation.

}, issn = {2375-2548}, doi = {10.1126/sciadv.abb3991}, author = {Dhandapany, Perundurai S and Kang, Soojeong and Kashyap, Deepak K and Rajagopal, Raksha and Sundaresan, Nagalingam R and Singh, Rajvir and Thangaraj, Kumarasamy and Jayaprakash, Shilpa and Manjunath, Cholenahally N and Shenthar, Jayaprakash and Lebeche, Djamel} } @article {2362, title = {Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.}, journal = {Stem Cell Reports}, volume = {16}, year = {2021}, month = {2021 Nov 09}, pages = {2736-2751}, abstract = {

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2021.09.013}, author = {Chandrasekaran, Abinaya and Dittlau, Katarina Stoklund and Corsi, Giulia I and Haukedal, Henriette and Doncheva, Nadezhda T and Ramakrishna, Sarayu and Ambardar, Sheetal and Salcedo, Claudia and Schmidt, Sissel I and Zhang, Yu and Cirera, Susanna and Pihl, Maria and Schmid, Benjamin and Nielsen, Troels Tolstrup and Nielsen, J{\o}rgen E and Kolko, Miriam and Kobol{\'a}k, Julianna and Dinny{\'e}s, Andr{\'a}s and Hyttel, Poul and Palakodeti, Dasaradhi and Gorodkin, Jan and Muddashetty, Ravi S and Meyer, Morten and Aldana, Blanca I and Freude, Kristine K} } @article {2375, title = {Biophysical properties of the isolated spike protein binding helix of human ACE2.}, journal = {Biophys J}, volume = {120}, year = {2021}, month = {2021 07 20}, pages = {2785-2792}, abstract = {

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility \> 0.8\ mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel β-sheet content and no helicity. The helicity is substantial (\>20\%) only upon adding high concentrations (>=20\% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations \>50\ nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

}, keywords = {Angiotensin-Converting Enzyme 2, COVID-19, Humans, Peptidyl-Dipeptidase A, Protein Binding, SARS-CoV-2, Spike Glycoprotein, Coronavirus}, issn = {1542-0086}, doi = {10.1016/j.bpj.2021.06.017}, author = {Das, Anirban and Vishvakarma, Vicky and Dey, Arpan and Dey, Simli and Gupta, Ankur and Das, Mitradip and Vishwakarma, Krishna Kant and Roy, Debsankar Saha and Yadav, Swati and Kesarwani, Shubham and Venkatramani, Ravindra and Maiti, Sudipta} } @article {2272, title = {Chromatin remodelling complexes in cerebral cortex development and neurodevelopmental disorders.}, journal = {Neurochem Int}, volume = {147}, year = {2021}, month = {2021 Jul}, pages = {105055}, abstract = {

The diverse number of neurons in the cerebral cortex are generated during development by neural stem cells lining the ventricle, and they continue maturing postnatally. Dynamic chromatin regulation in these neural stem cells is a fundamental determinant of the emerging property of the functional neural network, and the chromatin remodellers are critical determinants of this process. Chromatin remodellers participate in several steps of this process from proliferation, differentiation, migration leading to complex network formation which forms the basis of higher-order functions of cognition and behaviour. Here we review the role of these ATP-dependent chromatin remodellers in cortical development in health and disease and highlight several key mouse mutants of the subunits of the complexes which have revealed how the remodelling mechanisms control the cortical stem cell chromatin landscape for expression of stage-specific transcripts. Consistent with their role in cortical development, several putative risk variants in the subunits of the remodelling complexes have been identified as the underlying causes of several neurodevelopmental disorders. A basic understanding of the detailed molecular mechanism of their action is key to understating how mutations in the same networks lead to disease pathologies and perhaps pave the way for therapeutic development for these complex multifactorial disorders.

}, issn = {1872-9754}, doi = {10.1016/j.neuint.2021.105055}, author = {D{\textquoteright}Souza, Leora and Channakkar, Asha S and Muralidharan, Bhavana} } @article {2326, title = {Correction of amygdalar dysfunction in a rat model of fragile X syndrome.}, journal = {Cell Rep}, volume = {37}, year = {2021}, month = {2021 Oct 12}, pages = {109805}, abstract = {

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in\ vitro and in\ vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.109805}, author = {Fernandes, Giselle and Mishra, Pradeep K and Nawaz, Mohammad Sarfaraz and Donlin-Asp, Paul G and Rahman, Mohammed Mostafizur and Hazra, Anupam and Kedia, Sonal and Kayenaat, Aiman and Songara, Dheeraj and Wyllie, David J A and Schuman, Erin M and Kind, Peter C and Chattarji, Sumantra} } @article {2215, title = {Decreased dendritic spine density in posterodorsal medial amygdala neurons of proactive coping rats.}, journal = {Behav Brain Res}, volume = {397}, year = {2021}, month = {2021 Jan 15}, pages = {112940}, abstract = {

There are large individual differences in the way animals, including humans, behaviorally and physiologically cope with environmental challenges and opportunities. Rodents with either a proactive or reactive coping style not only differ in their capacity to adapt successfully to environmental conditions, but also have a differential susceptibility to develop stress-related (psycho)pathologies when coping fails. In this study, we explored if there are structural neuronal differences in spine density in brain regions important for the regulation of stress coping styles. For this, the individual coping styles of wild-type Groningen (WTG) rats were determined using their level of offensive aggressiveness assessed in the resident-intruder paradigm. Subsequently, brains from proactive (high-aggressive) and reactive (low-aggressive) rats were Golgi-cox stained for spine quantification. The results reveal that dendritic spine densities in the dorsal hippocampal CA1 region and basolateral amygdala are similar in rats with proactive and reactive coping styles. Interestingly, however, dendritic spine density in the medial amygdala (MeA) is strikingly reduced in the proactive coping rats. This brain region is reported to be strongly involved in rivalry aggression which is the criterion by which the coping styles in our study are dissociated. The possibility that structural differences in spine density in the MeA are involved in other behavioral traits of distinct coping styles needs further investigation.

}, issn = {1872-7549}, doi = {10.1016/j.bbr.2020.112940}, author = {Anilkumar, Shobha and Patel, Deepika and de Boer, Sietse F and Chattarji, Sumantra and Buwalda, Bauke} } @article {2323, title = {Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi, India.}, journal = {Science}, year = {2021}, month = {2021 Oct 14}, pages = {eabj9932}, abstract = {

Delhi, the national capital of India, has experienced multiple SARS-CoV-2 outbreaks in 2020 and reached population seropositivity of over 50\% by 2021. During April 2021, the city became overwhelmed by COVID-19 cases and fatalities, as a new variant B.1.617.2 (Delta) replaced B.1.1.7 (Alpha). A Bayesian model explains the growth advantage of Delta through a combination of increased transmissibility and reduced sensitivity to immune responses generated against earlier variants (median estimates; {\texttimes}1.5-fold, 20\% reduction). Seropositivity of an employee and family cohort increased from 42\% to 87.5\% between March and July 2021, with 27\% reinfections, as judged by increased antibody concentration after a previous decline. The likely high transmissibility and partial evasion of immunity by the Delta variant contributed to an overwhelming surge in Delhi.

}, issn = {1095-9203}, doi = {10.1126/science.abj9932}, author = {Dhar, Mahesh S and Marwal, Robin and Vs, Radhakrishnan and Ponnusamy, Kalaiarasan and Jolly, Bani and Bhoyar, Rahul C and Sardana, Viren and Naushin, Salwa and Rophina, Mercy and Mellan, Thomas A and Mishra, Swapnil and Whittaker, Charles and Fatihi, Saman and Datta, Meena and Singh, Priyanka and Sharma, Uma and Ujjainiya, Rajat and Bhatheja, Nitin and Divakar, Mohit Kumar and Singh, Manoj K and Imran, Mohamed and Senthivel, Vigneshwar and Maurya, Ranjeet and Jha, Neha and Mehta, Priyanka and A, Vivekanand and Sharma, Pooja and Vr, Arvinden and Chaudhary, Urmila and Soni, Namita and Thukral, Lipi and Flaxman, Seth and Bhatt, Samir and Pandey, Rajesh and Dash, Debasis and Faruq, Mohammed and Lall, Hemlata and Gogia, Hema and Madan, Preeti and Kulkarni, Sanket and Chauhan, Himanshu and Sengupta, Shantanu and Kabra, Sandhya and Gupta, Ravindra K and Singh, Sujeet K and Agrawal, Anurag and Rakshit, Partha and Nandicoori, Vinay and Tallapaka, Karthik Bharadwaj and Sowpati, Divya Tej and Thangaraj, K and Bashyam, Murali Dharan and Dalal, Ashwin and Sivasubbu, Sridhar and Scaria, Vinod and Parida, Ajay and Raghav, Sunil K and Prasad, Punit and Sarin, Apurva and Mayor, Satyajit and Ramakrishnan, Uma and Palakodeti, Dasaradhi and Seshasayee, Aswin Sai Narain and Bhat, Manoj and Shouche, Yogesh and Pillai, Ajay and Dikid, Tanzin and Das, Saumitra and Maitra, Arindam and Chinnaswamy, Sreedhar and Biswas, Nidhan Kumar and Desai, Anita Sudhir and Pattabiraman, Chitra and Manjunatha, M V and Mani, Reeta S and Arunachal Udupi, Gautam and Abraham, Priya and Atul, Potdar Varsha and Cherian, Sarah S} } @article {2244, title = {Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis.}, journal = {Cell Metab}, year = {2021}, month = {2021 Mar 31}, abstract = {

Cellular senescence is a stress or damage response that causes a permanent proliferative arrest and secretion of numerous factors with potent biological activities. This senescence-associated secretory phenotype (SASP) has been characterized largely for secreted proteins that participate in embryogenesis, wound healing, inflammation, and many age-related pathologies. By contrast, lipid components of the SASP are understudied. We show that senescent cells activate the biosynthesis of several oxylipins that promote segments of the SASP and reinforce the proliferative arrest. Notably, senescent cells synthesize and accumulate an unstudied intracellular prostaglandin, 1a,1b-dihomo-15-deoxy-delta-12,14-prostaglandin J2. Released 15-deoxy-delta-12,14-prostaglandin J2 is a biomarker of senolysis in culture and in\ vivo. This and other prostaglandin D2-related lipids promote the senescence arrest and SASP by activating RAS signaling. These data identify an important aspect of cellular senescence and a method to detect senolysis.

}, issn = {1932-7420}, doi = {10.1016/j.cmet.2021.03.008}, author = {Wiley, Christopher D and Sharma, Rishi and Davis, Sonnet S and Lopez-Dominguez, Jose Alberto and Mitchell, Kylie P and Wiley, Samantha and Alimirah, Fatouma and Kim, Dong Eun and Payne, Therese and Rosko, Andrew and Aimontche, Eliezer and Deshpande, Sharvari M and Neri, Francesco and Kuehnemann, Chisaka and Demaria, Marco and Ramanathan, Arvind and Campisi, Judith} } @article {2376, title = {Proteome plasticity in response to persistent environmental change.}, journal = {Mol Cell}, volume = {81}, year = {2021}, month = {2021 08 19}, pages = {3294-3309.e12}, abstract = {

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

}, keywords = {Acclimatization, Adaptation, Physiological, Animals, Environmental Exposure, Gene Expression Regulation, Fungal, Hot Temperature, Proteome, Saccharomycetales, Stress, Physiological, Transcriptome}, issn = {1097-4164}, doi = {10.1016/j.molcel.2021.06.028}, author = {Domnauer, Matthew and Zheng, Fan and Li, Liying and Zhang, Yanxiao and Chang, Catherine E and Unruh, Jay R and Conkright-Fincham, Juliana and McCroskey, Scott and Florens, Laurence and Zhang, Ying and Seidel, Christopher and Fong, Benjamin and Schilling, Birgit and Sharma, Rishi and Ramanathan, Arvind and Si, Kausik and Zhou, Chuankai} } @article {2270, title = {Repeated victorious and defeat experiences induce similar apical dendritic spine remodeling in CA1 hippocampus of rats.}, journal = {Behav Brain Res}, volume = {406}, year = {2021}, month = {2021 May 21}, pages = {113243}, abstract = {

In this study, apical dendritic spine density of neurons in hippocampal, amygdalar and prefrontal cortical areas was compared in rats that were repeatedly winning or losing social conflicts. Territorial male wild-type Groningen (WTG) rats were allowed multiple daily attacks (\>20 times) on intruder males in the resident-intruder paradigm. Frequent winning experiences are known to facilitate uncontrolled aggressive behavior reflected in aggressive attacks on anesthetized males which was also observed in the winners in this study. Both winners and losers were socially housed during the experiments; winners with females to stimulate territorial behavior, and losers with two other losing male rats. Twenty-four hours after the last social encounter, brains from experienced residential winners and repeatedly defeated intruder rats were collected and neuronal morphology in selected brain regions was studied via Golgi-Cox staining. Results indicate that spine density in the apical dendrites of the hippocampal CA1 reduced similarly in both winners and losers. In addition, winners showed increased spine densities at the proximal segments (20-30 μm) of the basolateral amygdala neurons and losers tended to show a decreased spine density at the more proximal segments of the infralimbic region of prefrontal cortex neurons. No effect of winning and losing was observed in the medial amygdala. The atrophic effect of repeated defeats in hippocampal and prefrontal regions was anticipated despite the fact that social housing of the repeatedly losing intruder males may have played a protective role. The reduction of hippocampal spine density in the winners seems surprising but supports previous findings in hierarchical dominant males in rat colonies. The dominants showed even greater shrinkage of the apical dendritic arbors of hippocampal CA3 pyramidal neurons compared to the stressed subordinates.

}, issn = {1872-7549}, doi = {10.1016/j.bbr.2021.113243}, author = {Patel, Deepika and Anilkumar, Shobha and Chattarji, Sumantra and de Boer, Sietse F and Buwalda, Bauke} } @article {2372, title = {Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.}, journal = {J Med Genet}, year = {2021}, month = {2021 Dec 16}, abstract = {

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM.

METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 () gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect.

CONCLUSIONS: Our study demonstrates for the first time that the variants in the gene are associated with HCM, and early detection of the variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of gene variants.

}, issn = {1468-6244}, doi = {10.1136/jmedgenet-2021-107866}, author = {Jain, Pratul Kumar and Jayappa, Shashank and Sairam, Thiagarajan and Mittal, Anupam and Paul, Sayan and Rao, Vinay J and Chittora, Harshil and Kashyap, Deepak K and Palakodeti, Dasaradhi and Thangaraj, Kumarasamy and Shenthar, Jayaprakash and Koranchery, Rakesh and Rajendran, Ranjith and Alireza, Haghighi and Mohanan, Kurukkanparampil Sreedharan and Rathinavel, Andiappan and Dhandapany, Perundurai S} } @article {2363, title = {SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion.}, journal = {Nature}, volume = {599}, year = {2021}, month = {2021 11}, pages = {114-119}, abstract = {

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

}, keywords = {Antibodies, Neutralizing, Cell Fusion, Cell Line, COVID-19 Vaccines, Female, Health Personnel, Humans, Immune Evasion, India, Kinetics, Male, SARS-CoV-2, Spike Glycoprotein, Coronavirus, Vaccination, Virus Replication}, issn = {1476-4687}, doi = {10.1038/s41586-021-03944-y}, author = {Mlcochova, Petra and Kemp, Steven A and Dhar, Mahesh Shanker and Papa, Guido and Meng, Bo and Ferreira, Isabella A T M and Datir, Rawlings and Collier, Dami A and Albecka, Anna and Singh, Sujeet and Pandey, Rajesh and Brown, Jonathan and Zhou, Jie and Goonawardane, Niluka and Mishra, Swapnil and Whittaker, Charles and Mellan, Thomas and Marwal, Robin and Datta, Meena and Sengupta, Shantanu and Ponnusamy, Kalaiarasan and Radhakrishnan, Venkatraman Srinivasan and Abdullahi, Adam and Charles, Oscar and Chattopadhyay, Partha and Devi, Priti and Caputo, Daniela and Peacock, Tom and Wattal, Chand and Goel, Neeraj and Satwik, Ambrish and Vaishya, Raju and Agarwal, Meenakshi and Mavousian, Antranik and Lee, Joo Hyeon and Bassi, Jessica and Silacci-Fegni, Chiara and Saliba, Christian and Pinto, Dora and Irie, Takashi and Yoshida, Isao and Hamilton, William L and Sato, Kei and Bhatt, Samir and Flaxman, Seth and James, Leo C and Corti, Davide and Piccoli, Luca and Barclay, Wendy S and Rakshit, Partha and Agrawal, Anurag and Gupta, Ravindra K} } @article {2290, title = {Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.}, journal = {PLoS Pathog}, volume = {17}, year = {2021}, month = {2021 07}, pages = {e1009706}, abstract = {

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

}, keywords = {Ammonium Chloride, Angiotensin-Converting Enzyme 2, Animals, Antiviral Agents, Cell Line, Chlorocebus aethiops, Chloroquine, Clathrin, COVID-19, Drug Synergism, Endocytosis, Endosomes, Humans, Hydrogen-Ion Concentration, Hydroxychloroquine, Macrolides, Niclosamide, Protein Binding, Protein Domains, SARS-CoV-2, Spike Glycoprotein, Coronavirus, Vero Cells, Virus Internalization}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1009706}, author = {Prabhakara, Chaitra and Godbole, Rashmi and Sil, Parijat and Jahnavi, Sowmya and Gulzar, Shah-E-Jahan and van Zanten, Thomas S and Sheth, Dhruv and Subhash, Neeraja and Chandra, Anchal and Shivaraj, Akshatha and Panikulam, Patricia and U, Ibrahim and Nuthakki, Vijay Kumar and Puthiyapurayil, Theja Parassini and Ahmed, Riyaz and Najar, Ashaq Hussain and Lingamallu, Sai Manoz and Das, Snigdhadev and Mahajan, Bhagyashri and Vemula, Praveen and Bharate, Sandip B and Singh, Parvinder Pal and Vishwakarma, Ram and Guha, Arjun and Sundaramurthy, Varadharajan and Mayor, Satyajit} } @article {2200, title = {tRNA-derived fragments (tRFs): establishing their turf in post-transcriptional gene regulation.}, journal = {Cell Mol Life Sci}, year = {2021}, month = {2021 Jan 02}, abstract = {

Transfer RNA (tRNA)-derived fragments (tRFs) are an emerging class of conserved small non-coding RNAs that play important roles in post-transcriptional gene regulation. High-throughput sequencing of multiple biological samples have identified heterogeneous species of tRFs with distinct functionalities. These small RNAs have garnered a lot of scientific attention due to their ubiquitous expression and versatility in regulating various biological processes. In this review, we highlight our current understanding of tRF biogenesis and their regulatory functions. We summarize the diverse modes of biogenesis through which tRFs are generated and discuss the mechanism through which different tRF species regulate gene expression and the biological implications. Finally, we conceptualize research areas that require focus to strengthen our understanding of the biogenesis and function of tRFs.

}, issn = {1420-9071}, doi = {10.1007/s00018-020-03720-7}, author = {Krishna, Srikar and Raghavan, Srikala and DasGupta, Ramanuj and Palakodeti, Dasaradhi} } @article {2065, title = {Convolvulus pluricaulis extract can modulate synaptic plasticity in rat brain hippocampus.}, journal = {Neuroreport}, volume = {31}, year = {2020}, month = {2020 May 22}, pages = {597-604}, abstract = {

The memory-boosting property of Indian traditional herb, Convolvulus pluricaulis, has been documented in literature; however, its effect on synaptic plasticity has not yet been reported. Two important forms of synaptic plasticity known to be involved in the processes of memory formation are long-term potentiation (LTP) and long-term depression (LTD). In the present study, the effect of C. pluricaulis plant extract on LTP and LTD were evaluated. The adult male Wistar rats were fed orally with 250, 500 and 1000 mg/kg of this extract for 4 weeks and the effect was determined on LTP and LTD in the Schaffer collaterals of the hippocampal cornu ammonis region CA1. We found that the 500 mg/kg dose of the extract could significantly enhance LTP compared to the vehicle treated ones. Moreover, the same dose could also reduce LTD while used in a separate set of animals. Also, a fresh group of animals treated with the effective dose (500 mg/kg) of plant extract were examined for memory retention in two behavioral platforms namely, contextual fear conditioning (CFC) and novel object recognition test (NORT). Increased fear response to the conditioned stimulus and enhanced recognition of objects were observed in CFC and NORT, respectively, both indicating strengthening of memory. Following up, ex-vivo electrophysiology experiments were performed with the active single molecule scopoletin, present in C. pluricaulis extract and similar patterns in synaptic plasticity changes were obtained. These findings suggest that prolonged treatment of C. pluricaulis extract, at a specific dose in healthy animals, can augment memory functions by modulating hippocampal plasticity.

}, issn = {1473-558X}, doi = {10.1097/WNR.0000000000001446}, author = {Das, Rishi and Sengupta, Tathagata and Roy, Shubhrajit and Chattarji, Sumantra and Ray, Jharna} } @article {2112, title = {Corticosterone after acute stress prevents the delayed effects on the amygdala.}, journal = {Neuropsychopharmacology}, year = {2020}, month = {2020 Jul 06}, abstract = {

Even a single 2-hour episode of immobilization stress is known to trigger anxiety-like behavior and increase spine-density in the basolateral amygdala (BLA) of rats 10 days later. This delayed build-up of morphological and behavioral effects offers a stress-free time window of intervention after acute stress, which we used to test a protective role for glucocorticoids against stress. We observed that post-stress corticosterone, given 1 day after acute stress in drinking water, reversed enhanced anxiety-like behavior 10 days later. Quantification of spine-density on Golgi-stained BLA principal neurons showed that the same intervention also prevented the increase in spine numbers in the amygdala, at the same delayed time-point. Further, stress elevated serum corticosterone levels in rats that received vehicle in the drinking water. However, when stress was followed 24 h later by corticosterone in the drinking water, the surge in corticosterone was prevented. Together, these observations suggest that corticosterone, delivered through drinking water even 24 h after acute stress, is capable of reversing the delayed enhancing effects on BLA synaptic connectivity and anxiety-like behavior. Strikingly, although the immobilization-induced surge in corticosterone by itself has delayed detrimental effects on amygdalar structure and function, there exists a window of opportunity even after stress to mitigate its impact with a second surge of exogenously administered corticosterone. This provides a framework in the amygdala for analyzing how the initial physiological and endocrine processes triggered by traumatic stress eventually give rise to debilitating emotional symptoms, as well as the protective effects of glucocorticoids against their development.

}, issn = {1740-634X}, doi = {10.1038/s41386-020-0758-0}, author = {Chakraborty, Prabahan and Datta, Siddhartha and McEwen, Bruce S and Chattarji, Sumantra} } @article {2146, title = {Differential flexibility leading to crucial microelastic properties of asymmetric lipid vesicles for cellular transfection: A combined spectroscopic and atomic force microscopy studies.}, journal = {Colloids Surf B Biointerfaces}, volume = {196}, year = {2020}, month = {2020 Sep 21}, pages = {111363}, abstract = {

The role of microscopic elasticity of nano-carriers in cellular uptake is an important aspect in biomedical research. Herein we have used AFM nano-indentation force spectroscopy and F{\"o}rster resonance energy transfer (FRET) measurements to probe microelastic properties of three novel cationic liposomes based on di-alkyl dihydroxy ethyl ammonium chloride based lipids having asymmetry in their hydrophobic chains (Lip1818, Lip1814 and Lip1810). AFM data reveals that symmetry in hydrophobic chains of a cationic lipid (Lip1818) imparts higher rigidity to the resulting liposomes than those based on asymmetric lipids (Lip1814 and Lip1810). The stiffness of the cationic liposomes is found to decrease with increasing asymmetry in the hydrophobic lipid chains in the order of Lip1818 \> Lip1814 \> lip1810. FRET measurements between Coumarin 500 (Donor) and Merocyanine 540 (Acceptor) have revealed that full width at half-maxima (hw) of the probability distribution (P(r)) of donor-acceptor distance (r), increases in an order Lip1818 \< Lip1814 \< Lip1810 with increasing asymmetry of the hydrophobic lipid chains. This increase in width (hw) of the donor-acceptor distance distributions is reflective of increasing flexibility of the liposomes with increasing asymmetry of their constituent lipids. Thus, the results from AFM and FRET studies are complementary to each other and indicates that an increase in asymmetry of the hydrophobic lipid chains increases elasticity and or flexibility of the corresponding liposomes. Cell biology experiments confirm that liposomal flexibility or rigidity directly influences their cellular transfection efficiency, where Lip1814 is found to be superior than the other two liposomes manifesting that a critical balance between flexibility and rigidity of the cationic liposomes is key to efficient cellular uptake. Taken together, our studies reveal how asymmetry in the molecular architecture of the hydrophobic lipid chains influences the microelastic properties of the liposomes, and hence, their cellular uptake efficiency.

}, issn = {1873-4367}, doi = {10.1016/j.colsurfb.2020.111363}, author = {Mukherjee, Dipanjan and Rakshit, Tatini and Singh, Priya and Mondal, Suman and Paul, Debashish and Ahir, Manisha and Adhikari, Arghya and Puthiyapurayil, Theja P and Vemula, Praveen Kumar and Senapati, Dulal and Das, Ranjan and Pal, Samir Kumar} } @article {2067, title = {Distinct regulation of bioenergetics and translation by group I mGluR and NMDAR.}, journal = {EMBO Rep}, year = {2020}, month = {2020 Apr 29}, pages = {e48037}, abstract = {

Neuronal activity is responsible for the high energy consumption in the brain. However, the cellular mechanisms draining ATP upon the arrival of a stimulus are yet to be explored systematically at the post-synapse. Here, we provide evidence that a significant fraction of ATP is consumed upon glutamate stimulation to energize mGluR-induced protein synthesis. We find that both mGluR and NMDAR alter protein synthesis and ATP consumption with distinct kinetics at the synaptic-dendritic compartments. While mGluR activation leads to a rapid and sustained reduction in neuronal ATP levels, NMDAR activation has no immediate impact on the same. ATP consumption correlates inversely with the kinetics of protein synthesis for both receptors. We observe a persistent elevation in protein synthesis within 5\ minutes of mGluR activation and a robust inhibition of the same within 2\ minutes of NMDAR activation, assessed by the phosphorylation status of eEF2 and metabolic labeling. However, a delayed protein synthesis-dependent ATP expenditure ensues after 15\ minutes of NMDAR stimulation. We identify a central role for AMPK in the correlation between protein synthesis and ATP consumption. AMPK is dephosphorylated and inhibited upon mGluR activation, while it is phosphorylated upon NMDAR activation. Perturbing AMPK activity disrupts receptor-specific modulations of eEF2 phosphorylation and protein synthesis. Our observations, therefore, demonstrate that the regulation of the AMPK-eEF2 signaling axis by glutamate receptors alters neuronal protein synthesis and bioenergetics.

}, issn = {1469-3178}, doi = {10.15252/embr.201948037}, author = {Ghosh Dastidar, Sudhriti and Das Sharma, Shreya and Chakraborty, Sumita and Chattarji, Sumantra and Bhattacharya, Aditi and Muddashetty, Ravi S} } @article {2115, title = {A field-based quantitative analysis of sublethal effects of air pollution on pollinators.}, journal = {Proc Natl Acad Sci U S A}, volume = {117}, year = {2020}, month = {2020 Aug 25}, pages = {20653-20661}, abstract = {

While the impact of air pollution on human health is well studied, mechanistic impacts of air pollution on wild systems, including those providing essential ecosystem services, are largely unknown, but directly impact our health and well-being. India is the world{\textquoteright}s largest fruit producer, second most populous country, and contains 9 of the world{\textquoteright}s 10 most polluted cities. Here, we sampled Giant Asian honey bees, , at locations with varying air pollution levels in Bangalore, India. We observed significant correlations between increased respirable suspended particulate matter (RSPM) deposition and changes in bee survival, flower visitation, heart rate, hemocyte levels, and expression of genes related to lipid metabolism, stress, and immunity. Lab-reared exposed to these same sites also exhibited similar molecular and physiological differences. Our study offers a quantitative analysis on the current impacts of air pollution on insects, and indicates the urgency for more nonhuman studies to accurately assess the effects of pollution on our natural world.

}, issn = {1091-6490}, doi = {10.1073/pnas.2009074117}, author = {Thimmegowda, Geetha G and Mullen, Susan and Sottilare, Katie and Sharma, Ankit and Mohanta, Saptashi Soham and Brockmann, Axel and Dhandapany, Perundurai S and Olsson, Shannon B} } @article {2155, title = {Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3 variant.}, journal = {Biophys Rev}, volume = {12}, year = {2020}, month = {2020 Aug}, pages = {1065-1084}, abstract = {

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40\% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25-base pair deletion in intron 32 of MYBPC3 (MYBPC3) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3 carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3 variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3 pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.

}, issn = {1867-2450}, doi = {10.1007/s12551-020-00725-1}, author = {Arif, Mohammed and Nabavizadeh, Pooneh and Song, Taejeong and Desai, Darshini and Singh, Rohit and Bazrafshan, Sholeh and Kumar, Mohit and Wang, Yigang and Gilbert, Richard J and Dhandapany, Perundurai S and Becker, Richard C and Kranias, Evangelia G and Sadayappan, Sakthivel} } @article {2148, title = {Genetically encoded live-cell sensor for tyrosinated microtubules.}, journal = {J Cell Biol}, volume = {219}, year = {2020}, month = {2020 Oct 05}, abstract = {

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

}, issn = {1540-8140}, doi = {10.1083/jcb.201912107}, author = {Kesarwani, Shubham and Lama, Prakash and Chandra, Anchal and Reddy, P Purushotam and Jijumon, A S and Bodakuntla, Satish and Rao, Balaji M and Janke, Carsten and Das, Ranabir and Sirajuddin, Minhajuddin} } @article {2108, title = {A novel polyubiquitin chain linkage formed by viral Ubiquitin is resistant to host deubiquitinating enzymes.}, journal = {Biochem J}, volume = {477}, year = {2020}, month = {2020 Jun 26}, pages = {2193-2219}, abstract = {

The Baculoviridae family of viruses encode a viral Ubiquitin (vUb) gene. Though the vUb is homologous to the host eukaryotic Ubiquitin (Ub), its preservation in the viral genome indicates unique functions that are not compensated by the host Ub. We report the structural, biophysical, and biochemical properties of the vUb from Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV). The packing of central helix α1 to the beta-sheet β1-β5 is different between vUb and Ub. Consequently, its stability is lower compared with Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin-binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54), and the deubiquitinating enzymes are ineffective against the K54-linked polyubiquitin chains. We propose that the modification of host/viral proteins with the K54-linked chains is an effective way selected by the virus to protect the vUb signal from host DeUbiquitinases.

}, issn = {1470-8728}, doi = {10.1042/BCJ20200289}, author = {Negi, Hitendra and Reddy, Pothula Purushotham and Vengayil, Vineeth and Patole, Chhaya and Laxman, Sunil and Das, Ranabir} } @article {2066, title = {The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts.}, journal = {BMC Mol Cell Biol}, volume = {21}, year = {2020}, month = {2020 Apr 15}, pages = {25}, abstract = {

BACKGROUND: Reversible cell cycle arrest (quiescence/G0) is characteristic of adult stem cells and is actively controlled at multiple levels. Quiescent cells also extend a primary cilium, which functions as a signaling hub. Primary cilia have been shown to be important in multiple developmental processes, and are implicated in numerous developmental disorders. Although the association of the cilium with G0 is established, the role of the cilium in the control of the quiescence program is still poorly understood.

RESULTS: Primary cilia are dynamically regulated across different states of cell cycle exit in skeletal muscle myoblasts: quiescent myoblasts elaborate a primary cilium in vivo and in vitro, but terminally differentiated myofibers do not. Myoblasts where ciliogenesis is ablated using RNAi against a key ciliary assembly protein (IFT88) can exit the cell cycle but display an altered quiescence program and impaired self-renewal. Specifically, the G0 transcriptome in IFT88 knockdown cells is aberrantly enriched for G2/M regulators, suggesting a focused repression of this network by the cilium. Cilium-ablated cells also exhibit features of activation including enhanced activity of Wnt and mitogen signaling and elevated protein synthesis via inactivation of the translational repressor 4E-BP1.

CONCLUSIONS: Taken together, our results show that the primary cilium integrates and dampens proliferative signaling, represses translation and G2/M genes, and is integral to the establishment of the quiescence program.

}, issn = {2661-8850}, doi = {10.1186/s12860-020-00266-1}, author = {Venugopal, Nisha and Ghosh, Ananga and Gala, Hardik and Aloysius, Ajoy and Vyas, Neha and Dhawan, Jyotsna} } @article {2209, title = {Reply to Negri et al.: Air pollution and health impacts on bees: Signs of causation.}, journal = {Proc Natl Acad Sci U S A}, volume = {117}, year = {2020}, month = {2020 10 27}, pages = {26578-26579}, keywords = {Air Pollution, Animals, Bees, Health}, issn = {1091-6490}, doi = {10.1073/pnas.2017972117}, author = {Thimmegowda, Geetha G and Brockmann, Axel and Dhandapany, Perundurai S and Olsson, Shannon B} } @article {2064, title = {Temporal specificity and heterogeneity of Drosophila immune cells.}, journal = {EMBO J}, year = {2020}, month = {2020 Mar 12}, pages = {e104486}, abstract = {

Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

}, issn = {1460-2075}, doi = {10.15252/embj.2020104486}, author = {Cattenoz, Pierre B and Sakr, Rosy and Pavlidaki, Alexia and Delaporte, Claude and Riba, Andrea and Molina, Nacho and Hariharan, Nivedita and Mukherjee, Tina and Giangrande, Angela} } @article {2039, title = {Temporal specificity and heterogeneity of the fly immune cells{\textquoteright} transcriptional landscape}, journal = {The EMBO Journal (in press)}, year = {2020}, abstract = {

Immune cells provide defense against the non-self, however recent data suggest roles well beyond innate immunity, in processes as diverse as development, metabolism and tumor progression. Nevertheless, the heterogeneity of these cells remains an open question. Using bulk RNA sequencing we find that the Drosophila immune cells (hemocytes) display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single cell RNA sequencing we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct cellular processes e.g. proliferation, phagocytosis, metabolic homeostasis and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation that triggers the differentiation of a novel cell type, the lamellocyte. This first molecular atlas provides precious insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

}, author = {Cattenoz, Pierre B. and Sakr, Rosy and Pavlidaki, Alexia and Delaporte, Claude and Riba, Andrea and Molina, Nacho and Hariharan, Nivedita and Mukherjee, Tina and Giangrande, Angela} } @article {2210, title = {VEGFA Promoter Polymorphisms rs699947 and rs35569394 Are Associated With the Risk of Anterior Cruciate Ligament Ruptures Among Indian Athletes: A Cross-sectional Study.}, journal = {Orthop J Sports Med}, volume = {8}, year = {2020}, month = {2020 Dec}, pages = {2325967120964472}, abstract = {

Background: Associations of genetic variants within certain fibril-forming genes have previously been observed with anterior cruciate ligament (ACL) injuries. Evidence suggests a significant role of angiogenesis-associated cytokines in remodeling the ligament fibril matrix after mechanical loading and maintaining structural and functional integrity of the ligament. Functional polymorphisms within the vascular endothelial growth factor A (VEGFA) gene have emerged as plausible candidates owing to their role in the regulation of angiogenic responses.

Hypothesis: VEGFA promoter polymorphisms rs699947 and rs35569394 are associated with ACL injury risk among athletes.

Study Design: Cross-sectional study; Level of evidence, 3.

Methods: A total of 90 Indian athletes with radiologically confirmed or surgically proven isolated ACL tears and 76 matched-control athletes were selected for the present cross-sectional genetic association study. Oral mouthwash samples were collected from all the case and control athletes and genotyped for VEGFA rs699947 and rs35569394 using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Results: The A allele (rs699947) was significantly overrepresented in the ACL group (C vs A allele: odds ratio [OR], 1.68 [95\% CI, 1.08-2.60]; = .021) (CC vs CA + AA: OR, 2.69 [95\% CI, 1.37-5.26]; = .004). There was a greater frequency of the AA genotype in the ACL group in comparison with the control group (OR, 3.38 [95\% CI, 1.23-9.28]; = .016) when only male athletes were compared. Likewise, there was a greater frequency of the I allele (rs35569394) in the ACL group (D vs I allele: OR, 1.64 [95\% CI, 1.06-2.55]; = .025) (DD vs ID + II: OR, 2.61 [95\% CI, 1.31-5.21]; = .006). The A-I haplotype was overrepresented in the ACL group compared with the control group (OR, 1.68 [95\% CI, 1.08-2.60]; χ = 5.320; = .021), and both the polymorphisms were found to be in complete linkage disequilibrium ( = 0.929; logarithm of the odds score = 63.74; D{\textquoteright} = 1.0). Female athletes did not show any difference in genotype or allele frequency.

Conclusion: This is the first study to investigate the association of VEGFA promoter polymorphisms in ACL tears among Indian athletes. Increased frequencies of the A allele (rs699947) and I allele (rs35569394) were observed in the ACL group. These results suggest that sequence variants in the VEGF gene are associated with ACL injury risk among athletes. Further research with long-term follow-ups measuring VEGF expression levels during recovery is warranted to establish its role in ACL injuries and healing.

}, issn = {2325-9671}, doi = {10.1177/2325967120964472}, author = {Shukla, Manish and Gupta, Rahul and Pandey, Vivek and Rochette, Jacques and Dhandapany, Perundurai S and Tiwari, Pramod Kumar and Amrathlal, Rabbind Singh} } @article {1935, title = {Biomaterials for topical and transdermal drug delivery in reconstructive transplantation.}, journal = {Nanomedicine (Lond)}, year = {2019}, month = {2019 Oct 23}, abstract = {

Lifelong systemic immunosuppression remains the biggest challenge in vascularized composite allotransplantation (VCA) due to the adverse effects it causes. Since VCA is a life-enhancing procedure as compared with solid organ transplant which is life-saving;\ one needs to weigh the benefits and risks carefully. Thus, there is a huge unmet clinical need to design biomaterial-based vehicles that can deliver drugs more efficiently, topically and locally to eliminate adverse effects of systemic immune suppression. This review discusses several biomaterial-based systems that have been carefully designed, conceived and attempted to make VCA a more patient compliant approach. Variety of promising preclinical studies has shown the feasibility of the approaches, and clinical trials are required to bridge the gap. Several challenges for the future and new approaches have been discussed.

}, issn = {1748-6963}, doi = {10.2217/nnm-2019-0137}, author = {Dhayani, Ashish and Kalita, Sanjeeb and Mahato, Manohar and Srinath, Preethem and Vemula, Praveen K} } @article {1605, title = {Chemical fuel-driven living and transient supramolecular polymerization.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 25}, pages = {450}, abstract = {

Temporal control over self-assembly process is a desirable trait in the quest towards adaptable and controllable materials. The ability to devise synthetic ways to control the growth, as well as decay of materials has long been a property which only the biological systems could perform seamlessly. A common synthetic strategy which works on the biological principles such as chemical fuel-driven control over temporal self-assembly profile has not been completely realized synthetically. Here we show, we filled this dearth by showing that a chemical fuel driven self-assembling system can not only be grown in a controlled manner, but it can also result in precise control over the assembly and disassembly kinetics. Herein, we elaborate strategies which clearly show that once a chemical fuel driven self-assembly is established it can be made receptive to multiple molecular cues such that the inherent growth and decay characteristics are programmed into the ensemble.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08308-9}, author = {Jain, Ankit and Dhiman, Shikha and Dhayani, Ashish and Vemula, Praveen K and George, Subi J} } @article {1737, title = {Cytoplasmic sequestration of the RhoA effector mDiaphanous1 by Prohibitin2 promotes muscle differentiation.}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Jun 05}, pages = {8302}, abstract = {

Muscle differentiation is controlled by adhesion and growth factor-dependent signalling through common effectors that regulate muscle-specific transcriptional programs. Here we report that mDiaphanous1, an effector of adhesion-dependent RhoA-signalling, negatively regulates myogenesis at the level of Myogenin expression. In myotubes, over-expression of mDia1ΔN3, a RhoA-independent mutant, suppresses Myogenin promoter activity and expression. We investigated mDia1-interacting proteins that may counteract mDia1 to permit Myogenin expression and timely differentiation. Using yeast two-hybrid and mass-spectrometric analysis, we report that mDia1 has a stage-specific interactome, including Prohibitin2, MyoD, Akt2, and β-Catenin, along with a number of proteosomal and mitochondrial components. Of these interacting partners, Prohibitin2 colocalises with mDia1 in cytoplasmic punctae in myotubes. We mapped the interacting domains of mDia1 and Phb2, and used interacting (mDia1ΔN3/Phb2 FL or mDia1ΔN3/Phb2-Carboxy) and non-interacting pairs (mDia1H + P/Phb2 FL or mDia1ΔN3/Phb2-Amino) to dissect the functional consequences of this partnership on Myogenin promoter activity. Co-expression of full-length as well as mDia1-interacting domains of Prohibitin2 reverse the anti-myogenic effects of mDia1ΔN3, while non-interacting regions do not. Our results suggest that Prohibitin2 sequesters mDia1, dampens its anti-myogenic activity and fine-tunes RhoA-mDia1 signalling to promote differentiation. Overall, we report that mDia1 is multi-functional signalling effector whose anti-myogenic activity is modulated by a differentiation-dependent interactome.\ The data have been deposited to the ProteomeXchange with identifier PXD012257.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-44749-4}, author = {Saleh, Amena and Subramaniam, Gunasekaran and Raychaudhuri, Swasti and Dhawan, Jyotsna} } @article {1839, title = {Differential effects of unipolar versus bipolar depression on episodic memory updating.}, journal = {Neurobiol Learn Mem}, volume = {161}, year = {2019}, month = {2019 05}, pages = {158-168}, abstract = {

Episodic memories, when reactivated, can be modified or updated by new learning. Since such dynamic memory processes remain largely unexplored in psychiatric disorders, we examined the impact of depression on episodic memory updating. Unipolar and bipolar depression patients, and age/education matched controls, first learned a set of objects (List-1). Two days later, participants in all three groups were either reminded of the first learning session or not followed by the learning of a new set of objects (List-2). Forty-eight hours later, List-1 recall was impaired in unipolar and bipolar patients compared to control participants. Further, as expected, control participants who received a reminder spontaneously recalled items from List-2 during recall of List-1, indicative of an updated List-1 memory. Such spontaneous intrusions were also seen in the unipolar and bipolar patients that received the reminder, suggesting that memory updating was unaffected in these two patient groups despite impaired recall of List 1. Unexpectedly, we observed a trend towards higher intrusions, albeit statistically insignificant, not only in the reminder but also in the no-reminder subgroups of bipolar patients. We probed this further in a second cohort by testing recall of List-2, which was also impaired in both depression groups. Again bipolar patients showed intrusions, but this time in the reverse order from List-1 into List-2, independent of a reminder. Taken together, despite impaired recall, updating of episodic memories was intact and unidirectional in unipolar depression. In contrast, indiscriminate updating, as evidenced by bidirectional interference between episodic memories, was seen in bipolar depression. These findings reveal a novel distinction between unipolar versus bipolar depression using a reactivation-dependent memory updating paradigm.

}, issn = {1095-9564}, doi = {10.1016/j.nlm.2019.04.008}, author = {Dongaonkar, Bhaktee and Hupbach, Almut and Nadel, Lynn and Chattarji, Sumantra} } @article {1740, title = {Dynamic expression of tRNA-derived small RNAs define cellular states.}, journal = {EMBO Rep}, volume = {20}, year = {2019}, month = {2019 Jul}, pages = {e47789}, abstract = {

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5{\textquoteright}-tsRNAs. We report the identification of a set of 5{\textquoteright}-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5{\textquoteright}-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5{\textquoteright}-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5{\textquoteright}-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5{\textquoteright}-tsRNAs in defining distinct cell states.

}, issn = {1469-3178}, doi = {10.15252/embr.201947789}, author = {Krishna, Srikar and Yim, Daniel Gr and Lakshmanan, Vairavan and Tirumalai, Varsha and Koh, Judice Ly and Park, Jung Eun and Cheong, Jit Kong and Low, Joo Leng and Lim, Michelle Js and Sze, Siu Kwan and Shivaprasad, Padubidri and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi and DasGupta, Ramanuj} } @article {1603, title = {Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 09}, pages = {89}, abstract = {

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.

}, keywords = {Animals, Basic Helix-Loop-Helix Transcription Factors, Caco-2 Cells, Coumarins, Epithelial Cells, Gene Expression Regulation, HT29 Cells, Humans, Intestinal Mucosa, Macrophages, Mice, Mice, Inbred C57BL, Mice, Knockout, NF-E2-Related Factor 2, Receptors, Aryl Hydrocarbon, Specific Pathogen-Free Organisms, Tight Junction Proteins}, issn = {2041-1723}, doi = {10.1038/s41467-018-07859-7}, author = {Singh, Rajbir and Chandrashekharappa, Sandeep and Bodduluri, Sobha R and Baby, Becca V and Hegde, Bindu and Kotla, Niranjan G and Hiwale, Ankita A and Saiyed, Taslimarif and Patel, Paresh and Vijay-Kumar, Matam and Langille, Morgan G I and Douglas, Gavin M and Cheng, Xi and Rouchka, Eric C and Waigel, Sabine J and Dryden, Gerald W and Alatassi, Houda and Zhang, Huang-Ge and Haribabu, Bodduluri and Vemula, Praveen K and Jala, Venkatakrishna R} } @article {1608, title = {Graft-implanted, enzyme responsive, tacrolimus-eluting hydrogel enables long-term survival of orthotopic porcine limb vascularized composite allografts: A proof of concept study.}, journal = {PLoS One}, volume = {14}, year = {2019}, month = {2019}, pages = {e0210914}, abstract = {

BACKGROUND: Currently, patients receiving vascularized composite allotransplantation (VCA) grafts must take long-term systemic immunosuppressive therapy to prevent immunologic rejection. The morbidity and mortality associated with these medications is the single greatest barrier to more patients being able to receive these life-enhancing transplants. In contrast to solid organs, VCA, exemplified by hand or face transplants, allow visual diagnosis of clinical acute rejection (AR), directed biopsy and targeted graft therapies. Local immunosuppression in VCA could reduce systemic drug exposure and limit adverse effects. This proof of concept study evaluated, in a large animal forelimb VCA model, the efficacy and tolerability of a novel graft-implanted enzyme-responsive, tacrolimus (TAC)-eluting hydrogel platform, in achieving long-term graft survival.

METHODS: Orthotopic forelimb VCA were performed in single haplotype mismatched mini-swine. Controls (n = 2) received no treatment. Two groups received TAC hydrogel: high dose (n = 4, 91 mg TAC) and low dose (n = 4, 49 mg TAC). The goal was to find a dose that was tolerable and resulted in long-term graft survival. Limbs were evaluated for clinical and histopathological signs of AR. TAC levels were measured in serial blood and skin tissue samples. Tolerability of the dose was evaluated by monitoring animal feeding behavior and weight.

RESULTS: Control limbs underwent Banff Grade IV AR by post-operative day six. Low dose TAC hydrogel treatment resulted in long-term graft survival time to onset of Grade IV AR ranging from 56 days to 93 days. High dose TAC hydrogel also resulted in long-term graft survival (24 to 42 days), but was not well tolerated.

CONCLUSION: Graft-implanted TAC-loaded hydrogel delays the onset of Grade IV AR of mismatched porcine forelimb VCA grafts, resulting in long term graft survival and demonstrates dose-dependent tolerability.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0210914}, author = {Fries, C Anton and Lawson, Shari D and Wang, Lin C and Slaughter, Kai V and Vemula, Praveen K and Dhayani, Ashish and Joshi, Nitin and Karp, Jeffrey M and Rickard, Rory F and Gorantla, Vijay S and Davis, Michael R} } @article {1606, title = {Highly Responsive Fluorescent Assemblies Allow for Unique, Multiparametric Sensing of the Phospholipid Membrane Environment.}, journal = {Chemistry}, volume = {25}, year = {2019}, month = {2019 Jan 28}, pages = {1507-1514}, abstract = {

Despite decades-long extensive research, probes that provide a comprehensive description of the lipid membrane microenvironment are still lacking. Here, a "smart" pyrene-terpyridine probe for multiparametric sensing of lipid membranes is reported. The complexity of the associated local microenvironment can be described by the distinct features of the probe fluorescence. The self-assembly of the probe molecules in phospholipid bilayers was sensitive to membrane order and phase state. The self-assembled probes showed a unique emission, influenced by dye-dye interactions and excited-state charge transfer. Moreover, this emission was sensitive to interfacial hydration, with very specific changes in emission wavelengths and fluorescence lifetimes upon variation of lipid compositions and properties. In parallel, changes in the lipid order and hydration affected the ground-state interactions in the dye aggregates and, thus, could be measured through ratiometric changes in the excitation and emission readouts. In addition, fluorescence anisotropy measurements provided another way to study the nature of dye aggregates in lipid bilayers. Overall, this report demonstrates how multiple aspects of the membrane microenvironment can be sensed through the unique fluorescence signatures of this "smart" probe in lipid membranes, and it establishes a new paradigm in lipid-membrane sensing.

}, keywords = {Fluorescence Polarization, Fluorescent Dyes, Lipid Bilayers, Phospholipids, Pyrenes, Spectrometry, Fluorescence, Water}, issn = {1521-3765}, doi = {10.1002/chem.201803627}, author = {Gulyani, Akash and Dey, Nilanjan and Bhattacharya, Santanu} } @article {1578, title = {Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells.}, journal = {Methods Mol Biol}, volume = {1879}, year = {2019}, month = {2019}, pages = {285-297}, abstract = {

The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells.

}, issn = {1940-6029}, doi = {10.1007/7651_2018_155}, author = {Badarinath, Krithika and Dutta, Abhik and Hegde, Akshay and Pincha, Neha and Gund, Rupali and Jamora, Colin} } @article {1644, title = {Myocardin ablation in a cardiac-renal rat model.}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Apr 10}, pages = {5872}, abstract = {

Cardiorenal syndrome is defined by primary heart failure conditions influencing or leading to renal injury or dysfunction. Dilated cardiomyopathy (DCM) is a major co-existing form of heart failure (HF) with renal diseases. Myocardin (MYOCD), a cardiac-specific co-activator of serum response factor (SRF), is increased in DCM porcine and patient cardiac tissues and plays a crucial role in the pathophysiology of DCM. Inhibiting the increased MYOCD has shown to be partially rescuing the DCM phenotype in porcine model. However, expression levels of MYOCD in the cardiac tissues of the cardiorenal syndromic patients and the effect of inhibiting MYOCD in a cardiorenal syndrome model remains to be explored. Here, we analyzed the expression levels of MYOCD in the DCM patients with and without renal diseases. We also explored, whether cardiac specific silencing of MYOCD expression could ameliorate the cardiac remodeling and improve cardiac function in a renal artery ligated rat model (RAL). We observed an increase in MYOCD levels in the endomyocardial biopsies of DCM patients associated with renal failure compared to DCM alone. Silencing of MYOCD in RAL rats by a cardiac homing peptide conjugated MYOCD siRNA resulted in attenuation of cardiac hypertrophy, fibrosis and restoration of the left ventricular functions. Our data suggest hyper-activation of MYOCD in the pathogenesis of the cardiorenal failure cases. Also, MYOCD silencing showed beneficial effects by rescuing cardiac hypertrophy, fibrosis, size and function in a cardiorenal rat model.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-42009-z}, author = {Mittal, Anupam and Rana, Santanu and Sharma, Rajni and Kumar, Akhilesh and Prasad, Rishikesh and Raut, Satish K and Sarkar, Sagartirtha and Saikia, Uma Nahar and Bahl, Ajay and Dhandapany, Perundurai S and Khullar, Madhu} } @article {1989, title = {Secretion of leukotrienes by senescent lung fibroblasts promotes pulmonary fibrosis.}, journal = {JCI Insight}, volume = {4}, year = {2019}, month = {2019 Dec 19}, abstract = {

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+ cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.

}, issn = {2379-3708}, doi = {10.1172/jci.insight.130056}, author = {Wiley, Christopher D and Brumwell, Alexis N and Davis, Sonnet S and Jackson, Julia R and Valdovinos, Alexis and Calhoun, Cheresa and Alimirah, Fatouma and Castellanos, Carlos A and Ruan, Richard and Wei, Ying and Chapman, Harold A and Ramanathan, Arvind and Campisi, Judith and Jourdan Le Saux, Claude} } @article {1844, title = {Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea.}, journal = {FEBS Lett}, year = {2019}, month = {2019 Sep 17}, abstract = {

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a non-canonical enzyme, phenylalanine hydroxylase (PAH). Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescue the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

}, issn = {1873-3468}, doi = {10.1002/1873-3468.13607}, author = {Sarkar, Arunabha and Mukundan, Namita and Sowndarya, Sai and Dubey, Vinay Kumar and Babu, Rosana and Lakshmanan, Vairavan and Rangiah, Kannan and Panicker, Mitradas M and Palakodeti, Dasaradhi and Subramanian, Sabarinath Peruvemba and Ramaswamy, Subramanian} } @article {1981, title = {Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia.}, journal = {Cell Death Dis}, volume = {10}, year = {2019}, month = {2019 Sep 30}, pages = {736}, abstract = {

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

}, issn = {2041-4889}, doi = {10.1038/s41419-019-1964-8}, author = {Ganesan, Saravanan and Palani, Hamenth Kumar and Lakshmanan, Vairavan and Balasundaram, Nithya and Alex, Ansu Abu and David, Sachin and Venkatraman, Arvind and Korula, Anu and George, Biju and Balasubramanian, Poonkuzhali and Palakodeti, Dasaradhi and Vyas, Neha and Mathews, Vikram} } @article {1738, title = {Sustained correction of associative learning deficits after brief, early treatment in a rat model of Fragile X Syndrome.}, journal = {Sci Transl Med}, volume = {11}, year = {2019}, month = {2019 May 29}, abstract = {

Fragile X Syndrome (FXS) is one of the most common monogenic forms of autism and intellectual disability. Preclinical studies in animal models have highlighted the potential of pharmaceutical intervention strategies for alleviating the symptoms of FXS. However, whether treatment strategies can be tailored to developmental time windows that define the emergence of particular phenotypes is unknown. Similarly, whether a brief, early intervention can have long-lasting beneficial effects, even after treatment cessation, is also unknown. To address these questions, we first examined the developmental profile for the acquisition of associative learning in a rat model of FXS. Associative memory was tested using a range of behavioral paradigms that rely on an animal{\textquoteright}s innate tendency to explore novelty. knockout (KO) rats showed a developmental delay in their acquisition of object-place recognition and did not demonstrate object-place-context recognition paradigm at any age tested (up to 23 weeks of age). Treatment of KO rats with lovastatin between 5 and 9 weeks of age, during the normal developmental period that this associative memory capability is established, prevents the emergence of deficits but has no effect in wild-type animals. Moreover, we observe no regression of cognitive performance in the FXS rats over several months after treatment. This restoration of the normal developmental trajectory of cognitive function is associated with the sustained rescue of both synaptic plasticity and altered protein synthesis. The findings provide proof of concept that the impaired emergence of the cognitive repertoire in neurodevelopmental disorders may be prevented by brief, early pharmacological intervention.

}, issn = {1946-6242}, doi = {10.1126/scitranslmed.aao0498}, author = {Asiminas, Antonis and Jackson, Adam D and Louros, Susana R and Till, Sally M and Spano, Teresa and Dando, Owen and Bear, Mark F and Chattarji, Sumantra and Hardingham, Giles E and Osterweil, Emily K and Wyllie, David J A and Wood, Emma R and Kind, Peter C} } @article {1582, title = {Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer.}, journal = {Mol Cancer Ther}, volume = {18}, year = {2019}, month = {2019 Feb}, pages = {301-311}, abstract = {

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against , a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1a;LSL-Kras;Tgfbr2 (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A-treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.

}, issn = {1538-8514}, doi = {10.1158/1535-7163.MCT-18-0464}, author = {Totiger, Tulasigeri M and Srinivasan, Supriya and Jala, Venkatakrishna R and Lamichhane, Purushottam and Dosch, Austin R and Gaidarski, Alexander A and Joshi, Chandrashekhar and Rangappa, Shobith and Castellanos, Jason and Vemula, Praveen Kumar and Chen, Xi and Kwon, Deukwoo and Kashikar, Nilesh and VanSaun, Michael and Merchant, Nipun B and Nagathihalli, Nagaraj S} } @article {1209, title = {Cell density overrides the effect of substrate stiffness on human mesenchymal stem cells{\textquoteright} morphology and proliferation.}, journal = {Biomater Sci}, volume = {6}, year = {2018}, month = {2018 May 01}, pages = {1109-1119}, abstract = {

The effect of substrate stiffness on the cellular morphology, proliferation, and differentiation of human mesenchymal stem cells (hMSCs) has been extensively researched and well established. However, the majority of these studies are done with a low seeding density where cell to cell interactions do not play a significant role. While these conditions permit an analysis of cell-substratum interactions at the single cell level, such a model system fails to capture a critical aspect of the cellular micro-environment in vivo, i.e. the cell-cell interaction via matrix deformation (i.e., strain). To address this question, we seeded hMSCs on soft poly-acrylamide (PAA) gels, at a seeding density that permits cells to be mechanically interacting via the underlying substrate. We found that as the intercellular distance decreases with the increasing seeding density, cellular sensitivity towards the substrate rigidity becomes significantly diminished. With the increasing seeding density, the cell spread area increased on a soft substrate (500 Pa) but reduced on an even slightly stiffer substrate (2 kPa) as well as on glass making them indistinguishable at a high seeding density. Not only in terms of cell spread area but also at a high seeding density, cells formed mature focal adhesions and prominent stress fibres on a soft substrate similar to that of the cells being cultured on a stiff substrate. The decreased intercellular distance also influenced the proliferation rate of the cells: higher seeding density on the soft substrate showed cell cycle progression similar to that of the cells on glass substrates. In summary, this paper demonstrates how the effect of substrate rigidity on the cell morphology and fate is a function of inter-cellular distance when seeded on a soft substrate. Our AFM data suggest that such changes happen due to local strain stiffening of the soft PAA gel, an effect that has been rarely reported in the literature so far.

}, issn = {2047-4849}, doi = {10.1039/c7bm00853h}, author = {Venugopal, Balu and Mogha, Pankaj and Dhawan, Jyotsna and Majumder, Abhijit} } @article {1207, title = {Cycling to Meet Fate: Connecting Pluripotency to the Cell Cycle}, journal = {Frontiers in Cell and Developmental Biology}, volume = {6}, year = {2018}, pages = {57}, abstract = {

Pluripotent stem cells are characterised by their high proliferative rates, their ability to self-renew and their potential to differentiate to all the three germ layers. This rapid proliferation is brought about by a highly modified cell cycle that allows the cells to quickly shuttle from DNA synthesis to cell division, by reducing the time spent in the intervening gap phases. Many key regulators that define the somatic cell cycle are either absent or exhibit altered behaviour, allowing the pluripotent cell to bypass cell cycle checkpoints typical of somatic cells. Experimental analysis of this modified stem cell cycle has been challenging due to the strong link between rapid proliferation and pluripotency, since perturbations to the cell cycle or pluripotency factors result in differentiation. Despite these hurdles, our understanding of this unique cell cycle has greatly improved over the past decade, in part because of the availability of new technologies that permit the analysis of single cells in heterogeneous populations. This review aims to highlight some of the recent discoveries in this area with a special emphasis on different states of pluripotency. We also discuss the highly interlinked network that connects pluripotency factors and key cell cycle genes and review evidence for how this interdependency may promote the rapid cell cycle. This issue gains translational importance since disruptions in stem cell proliferation and differentiation can impact disorders at opposite ends of a spectrum, from cancer to degenerative disease.

}, issn = {2296-634X}, doi = {10.3389/fcell.2018.00057}, url = {https://www.frontiersin.org/article/10.3389/fcell.2018.00057}, author = {Zaveri, Lamuk and Dhawan, Jyotsna} } @article {1212, title = {Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis.}, journal = {Methods Mol Biol}, volume = {1686}, year = {2018}, month = {2018}, pages = {215-239}, abstract = {

Regenerative potential in adult stem cells is closely associated with the establishment of-and exit from-a temporary state of quiescence. Emerging evidence not only provides a rationale for the link between lineage determination programs and cell cycle regulation but also highlights the understanding of quiescence as an actively maintained cellular program, encompassing networks and mechanisms beyond mitotic inactivity or metabolic restriction. Interrogating the quiescent genome and transcriptome using deep-sequencing technologies offers an unprecedented view of the global mechanisms governing this reversibly arrested cellular state and its importance for cell identity. While many efforts have identified and isolated pure target stem cell populations from a variety of adult tissues, there is a growing appreciation that their isolation from the stem cell niche in vivo leads to activation and loss of hallmarks of quiescence. Thus, in vitro models that recapitulate the dynamic reversibly arrested stem cell state in culture and lend themselves to comparison with the activated or differentiated state are useful templates for genome-wide analysis of the quiescence network.In this chapter, we describe the methods that can be adopted for whole genome epigenomic and transcriptomic analysis of cells derived from one such established culture model where mouse myoblasts are triggered to enter or exit quiescence as homogeneous populations. The ability to synchronize myoblasts in G permits insights into the genome in "deep quiescence." The culture methods for generating large populations of quiescent myoblasts in either 2D or 3D culture formats are described in detail in a previous chapter in this series (Arora et al. Methods Mol Biol 1556:283-302, 2017). Among the attractive features of this model are that genes isolated from quiescent myoblasts in culture mark satellite cells in vivo (Sachidanandan et al., J Cell Sci 115:2701-2712, 2002) providing a validation of its approximation of the molecular state of true stem cells. Here, we provide our working protocols for ChIP-seq and RNA-seq analysis, focusing on those experimental elements that require standardization for optimal analysis of chromatin and RNA from quiescent myoblasts, and permitting useful and revealing comparisons with proliferating myoblasts or differentiated myotubes.

}, issn = {1940-6029}, doi = {10.1007/978-1-4939-7371-2_16}, author = {Srivastava, Surabhi and Gala, Hardik P and Mishra, Rakesh K and Dhawan, Jyotsna} } @article {1188, title = {Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections}, journal = {Biochimica et Biophysica Acta (BBA) - Biomembranes}, year = {2018}, pages = {-}, abstract = {

Abstract Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0{\textendash}1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide {\textquoteleft}proof of concept{\textquoteright} for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.

}, keywords = {Endocytosis}, issn = {0005-2736}, doi = {https://doi.org/10.1016/j.bbamem.2018.06.006}, url = {https://www.sciencedirect.com/science/article/pii/S0005273618301780}, author = {Vignesh K. Rangasami and Brijesh Lohchania and Chandrashekhar Voshavar and Harikrishna R. Rachamalla and Rajkumar Banerjee and Ashish Dhayani and Saravanabhavan Thangavel and Praveen K. Vemula and Srujan Marepally} } @article {1598, title = {FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.}, journal = {iScience}, volume = {9}, year = {2018}, month = {2018 Nov 30}, pages = {399-411}, abstract = {

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2{\textquoteright}O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2{\textquoteright}O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2018.11.007}, author = {D{\textquoteright}Souza, Michelle Ninochka and Gowda, Naveen Kumar Chandappa and Tiwari, Vishal and Babu, Rosana Ottakandathil and Anand, Praveen and Dastidar, Sudhriti Ghosh and Singh, Randhir and James, Owen G and Selvaraj, Bhuvaneish and Pal, Rakhi and Ramesh, Arati and Chattarji, Sumantra and Chandran, Siddharthan and Gulyani, Akash and Palakodeti, Dasaradhi and Muddashetty, Ravi S} } @article {1145, title = {Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics.}, journal = {Stem Cell Res}, volume = {30}, year = {2018}, month = {2018 May 17}, pages = {69-80}, abstract = {

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2018.05.010}, author = {Rumman, Mohammad and Majumder, Abhijit and Harkness, Linda and Venugopal, Balu and Vinay, M B and Pillai, Malini S and Kassem, Moustapha and Dhawan, Jyotsna} } @article {1155, title = {"Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes.}, journal = {Biophys Rev}, volume = {10}, year = {2018}, month = {2018 Apr}, pages = {219-227}, abstract = {

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

}, issn = {1867-2450}, doi = {10.1007/s12551-017-0343-x}, author = {North, Rachel A and Horne, Christopher R and Davies, James S and Remus, Daniela M and Muscroft-Taylor, Andrew C and Goyal, Parveen and Wahlgren, Weixiao Yuan and Ramaswamy, S and Friemann, Rosmarie and Dobson, Renwick C J} } @article {1189, title = {Local injections of tacrolimus-loaded hydrogel reduce systemic immunosuppression-related toxicity in vascularized composite allotransplantation.}, journal = {Transplantation}, year = {2018}, month = {2018 May 23}, abstract = {

BACKGROUND: Routine application of vascularized composite allotransplantation (VCA) is hampered by immunosuppression-related health comorbidities. To mitigate these we developed an inflammation-responsive hydrogel for local immunosuppression. Here we report on its long-term effect on graft survival, immunological and toxicological impact.

METHODS: Brown Norway-to-Lewis rat hind limb transplantations were treated either systemically with daily injections of 1 mg/kg tacrolimus or with subcutaneous intragraft injections of hydrogel containing 7 mg tacrolimus, every 70 days. Animals were monitored for rejection or other pathology for 280 days. Systemic and graft tacrolimus levels, regulatory T cells, and donor cell chimerism were measured periodically. At endpoint, markers for kidney, liver and metabolic state were compared to na{\"\i}ve age-matched rats.

RESULTS: Both daily systemic tacrolimus and subcutaneous intragraft tacrolimus hydrogel at 70 day intervals were able to sustain graft survival for \>280 days in 5 out of 6 recipients. In the hydrogel group, 1 graft progressed to grade 3 rejection at postoperative day (POD) 149. In systemic tacrolimus group, 1 animal was euthanized due to lymphoma on POD 275. Hydrogel treatment provided stable graft- and reduced systemic tacrolimus levels, and a 4 times smaller total tacrolimus dose compared with systemic immunosuppression. Hydrogel-treated animals showed preserved kidney function, absence of malignancies or opportunistic infections and increased hematopoietic chimerism compared to systemic immunosuppression.

CONCLUSIONS: Our findings demonstrate that localized immunosuppression with tacrolimus hydrogel is a long-term safe and reliable treatment. It may reduce the burden of systemic immunosuppression in VCA, potentially boosting the clinical application of this surgical intervention.

}, issn = {1534-6080}, doi = {10.1097/TP.0000000000002283}, author = {Dzhonova, Dzhuliya V and Olariu, Radu and Leckenby, Jonathan and Banz, Yara and Prost, Jean-Christophe and Dhayani, Ashish and Vemula, Praveen K and Voegelin, Esther and Taddeo, Adriano and Rieben, Robert} } @article {1592, title = {Local release of tacrolimus from hydrogel-based drug delivery system is controlled by inflammatory enzymes in vivo and can be monitored non-invasively using in vivo imaging.}, journal = {PLoS One}, volume = {13}, year = {2018}, month = {2018}, pages = {e0203409}, abstract = {

BACKGROUND: Local drug delivery systems that adjust the release of immunosuppressive drug in response to the nature and intensity of inflammation represent a promising approach to reduce systemic immunosuppression and its side effects in allotransplantation. Here we aimed to demonstrate that release of tacrolimus from triglycerol monostearate hydrogel is inflammation-dependent in vivo. We further report that by loading the hydrogel with a near-infrared dye, it is possible to monitor drug release non-invasively in an in vivo model of vascularized composite allotransplantation.

MATERIALS AND METHODS: Inflammation was induced by local challenge with lipopolysaccharides in na{\"\i}ve rats 7 days after injection of tacrolimus-loaded hydrogel in the hind limb. Tacrolimus levels in blood and tissues were measured at selected time points. A near-infrared dye was encapsulated in the hydrogel together with tacrolimus in order to monitor hydrogel deposits and drug release in vitro and in vivo in a model of vascularized composite allotransplantation.

RESULTS: Injection of lipopolysaccharides led to increased blood and skin tacrolimus levels (p = 0.0076, day 7 vs. day 12 in blood, and p = 0.0007 in treated limbs, 48 h after injection compared to controls). Moreover, lipopolysaccharides-injected animals had higher tacrolimus levels in treated limbs compared to contralateral limbs (p = 0.0003 for skin and p = 0.0053 for muscle). Imaging of hydrogel deposits and tacrolimus release was achieved by encapsulating near-infrared dye in the hydrogel for 160 days. The correlation of tacrolimus and near-infrared dye release from hydrogel was R2 = 0.6297 and R2 = 0.5619 in blood and grafts of transplanted animals respectively and R2 = 0.6066 in vitro.

CONCLUSIONS: Here we demonstrate the inflammation-responsiveness of a tacrolimus-loaded hydrogel in vivo. Moreover, we show that encapsulating a near-infrared dye in the hydrogel provides a reliable correlation of tacrolimus and dye release from the hydrogel, and an accessible non-invasive method for monitoring drug release from hydrogel deposits.

}, keywords = {Animals, Drug Delivery Systems, Humans, Hydrogels, Immunosuppressive Agents, Inflammation, Male, Rats, Rats, Inbred BN, Rats, Inbred Lew, Tacrolimus}, issn = {1932-6203}, doi = {10.1371/journal.pone.0203409}, author = {Dzhonova, Dzhuliya and Olariu, Radu and Leckenby, Jonathan and Dhayani, Ashish and Vemula, Praveen Kumar and Prost, Jean-Christophe and Banz, Yara and Taddeo, Adriano and Rieben, Robert} } @article {1152, title = {PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.}, journal = {J Clin Invest}, volume = {128}, year = {2018}, month = {2018 May 01}, pages = {1807-1819}, abstract = {

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

}, issn = {1558-8238}, doi = {10.1172/JCI99088}, author = {Pincha, Neha and Hajam, Edries Yousaf and Badarinath, Krithika and Batta, Surya Prakash Rao and Masudi, Tafheem and Dey, Rakesh and Andreasen, Peter and Kawakami, Toshiaki and Samuel, Rekha and George, Renu and Danda, Debashish and Jacob, Paul Mazhuvanchary and Jamora, Colin} } @article {1588, title = {The Sodium Sialic Acid Symporter From Has Altered Substrate Specificity.}, journal = {Front Chem}, volume = {6}, year = {2018}, month = {2018}, pages = {233}, abstract = {

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from (SiaT). We demonstrate that SiaT rescues an strain lacking its endogenous sialic acid transporter when grown on the sialic acids -acetylneuraminic acid (Neu5Ac) or -glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SiaT that may explain the altered specificity. SiaT is shown to be electrogenic, and transport is dependent upon more than one Na ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SiaT, and developing the and assay systems, our work underpins the design of inhibitors to this transporter.

}, issn = {2296-2646}, doi = {10.3389/fchem.2018.00233}, author = {North, Rachel A and Wahlgren, Weixiao Y and Remus, Daniela M and Scalise, Mariafrancesca and Kessans, Sarah A and Dunevall, Elin and Claesson, Elin and Soares da Costa, Tatiana P and Perugini, Matthew A and Ramaswamy, S and Allison, Jane R and Indiveri, Cesare and Friemann, Rosmarie and Dobson, Renwick C J} } @article {1599, title = {A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation.}, journal = {Microb Cell Fact}, volume = {17}, year = {2018}, month = {2018 Dec 03}, pages = {192}, abstract = {

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E.\ coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure\ to predict product chirality.

RESULTS: Six enzymes originating from Sulfolobus\ sulfotaricus, Zygosaccharomyces\ rouxii, Hansenula\ polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC\ 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E.\ coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z.\ rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565\ (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95\% (S)-specific alcohol with a chemical yield of 23-79\% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99\%.

CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces\ rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

}, keywords = {Biotransformation, Catalysis, Ethanol, Ketones}, issn = {1475-2859}, doi = {10.1186/s12934-018-1036-2}, author = {Haq, Saiful F and Shanbhag, Anirudh P and Karthikeyan, Subbulakshmi and Hassan, Imran and Thanukrishnan, Kannan and Ashok, Abhishek and Sukumaran, Sunilkumar and Ramaswamy, S and Bharatham, Nagakumar and Datta, Santanu and Samant, Shalaka and Katagihallimath, Nainesh} } @article {1147, title = {Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site.}, journal = {Nat Commun}, volume = {9}, year = {2018}, month = {2018 May 01}, pages = {1753}, abstract = {

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 {\r A} resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na ions. One Na binds to the conserved Na2 site, while the second Na binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na sites regulate N-acetylneuraminic acid transport.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-04045-7}, author = {Wahlgren, Weixiao Y and Dunevall, Elin and North, Rachel A and Paz, Aviv and Scalise, Mariafrancesca and Bisignano, Paola and Bengtsson-Palme, Johan and Goyal, Parveen and Claesson, Elin and Caing-Carlsson, Rhawnie and Andersson, Rebecka and Beis, Konstantinos and Nilsson, Ulf J and Farewell, Anne and Pochini, Lorena and Indiveri, Cesare and Grabe, Michael and Dobson, Renwick C J and Abramson, Jeff and Ramaswamy, S and Friemann, Rosmarie} } @article {1591, title = {The transcription factor Lef1 switches partners from β-catenin to Smad3 during muscle stem cell quiescence.}, journal = {Sci Signal}, volume = {11}, year = {2018}, month = {2018 Jul 24}, abstract = {

Skeletal muscle stem cells (MuSCs), also known as satellite cells, persist in adult mammals by entering a state of quiescence (G) during the early postnatal period. Quiescence is reversed during damage-induced regeneration and re-established after regeneration. Entry of cultured myoblasts into G is associated with a specific, reversible induction of Wnt target genes, thus implicating members of the Tcf and Lef1 (Tcf/Lef) transcription factor family, which mediate transcriptional responses to Wnt signaling, in the initiation of quiescence. We found that the canonical Wnt effector β-catenin, which cooperates with Tcf/Lef, was dispensable for myoblasts to enter quiescence. Using pharmacological and genetic approaches in cultured C2C12 myoblasts and in MuSCs, we demonstrated that Tcf/Lef activity during quiescence depended not on β-catenin but on the transforming growth factor-β (TGF-β) effector and transcriptional coactivator Smad3, which colocalized with Lef1 at canonical Wnt-responsive elements and directly interacted with Lef1 specifically in G Depletion of Smad3, but not β-catenin, reduced Lef1 occupancy at target promoters, Tcf/Lef target gene expression, and self-renewal of myoblasts. In vivo, MuSCs underwent a switch from β-catenin-Lef1 to Smad3-Lef1 interactions during the postnatal switch from proliferation to quiescence, with β-catenin-Lef1 interactions recurring during damage-induced reactivation. Our findings suggest that the interplay of Wnt-Tcf/Lef and TGF-β-Smad3 signaling activates canonical Wnt target promoters in a manner that depends on β-catenin during myoblast proliferation but is independent of β-catenin during MuSC quiescence.

}, issn = {1937-9145}, doi = {10.1126/scisignal.aan3000}, author = {Aloysius, Ajoy and DasGupta, Ramanuj and Dhawan, Jyotsna} } @article {1190, title = {A unique self-assembly-driven probe for sensing a lipid bilayer: ratiometric probing of vesicle to micelle transition}, journal = {Chem. Commun.}, volume = {54}, year = {2018}, pages = {5122-5125}, abstract = {

An amphiphilic pyrene-terpyridine (Pytpy) probe forms novel{,} fluorescent nanoaggregates in phospholipid membranes. This unique membrane-driven self-assembly of Pytpy shows large Stokes shifts and long-lived fluorescent states and efficiently reports on vesicle-micelle transition through ratiometric changes. Strikingly{,} Pytpy can even distinguish between bilayer-like domains and more-dynamic micelle-like {{\textquoteright}}rim{{\textquoteright}} phases that co-exist in mixed assemblies (bicelles).

}, doi = {10.1039/C8CC01635F}, url = {http://dx.doi.org/10.1039/C8CC01635F}, author = {Gulyani, Akash and Dey, Nilanjan and Bhattacharya, Santanu} } @article {1210, title = {C-State: an interactive web app for simultaneous multi-gene visualization and comparative epigenetic pattern search.}, journal = {BMC Bioinformatics}, volume = {18}, year = {2017}, month = {2017 Sep 13}, pages = {392}, abstract = {

BACKGROUND: Comparative epigenomic analysis across multiple genes presents a bottleneck for bench biologists working with NGS data. Despite the development of standardized peak analysis algorithms, the identification of novel epigenetic patterns and their visualization across gene subsets remains a challenge.

RESULTS: We developed a fast and interactive web app, C-State (Chromatin-State), to query and plot chromatin landscapes across multiple loci and cell types. C-State has an interactive, JavaScript-based graphical user interface and runs locally in modern web browsers that are pre-installed on all computers, thus eliminating the need for cumbersome data transfer, pre-processing and prior programming knowledge.

CONCLUSIONS: C-State is unique in its ability to extract and analyze multi-gene epigenetic information. It allows for powerful GUI-based pattern searching and visualization. We include a case study to demonstrate its potential for identifying user-defined epigenetic trends in context of gene expression profiles.

}, keywords = {Algorithms, Embryonic Stem Cells, Epigenomics, Genes, Genomics, HeLa Cells, Humans, Internet, K562 Cells, Promoter Regions, Genetic, Software, Transcription, Genetic, Web Browser}, issn = {1471-2105}, doi = {10.1186/s12859-017-1786-6}, author = {Sowpati, Divya Tej and Srivastava, Surabhi and Dhawan, Jyotsna and Mishra, Rakesh K} } @article {1160, title = {Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian .}, journal = {Development}, volume = {144}, year = {2017}, month = {2017 09 01}, pages = {3066-3079}, abstract = {

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including , are translationally regulated by SMED-PABPC2 Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.

}, keywords = {Animals, Cell Lineage, Cell Proliferation, Cytoplasm, Epidermis, Epithelium, Extracellular Matrix, Gene Knockdown Techniques, Homeostasis, Models, Biological, Planarians, Poly(A)-Binding Protein I, Regeneration, RNA, Messenger, Wound Healing}, issn = {1477-9129}, doi = {10.1242/dev.152942}, author = {Bansal, Dhiru and Kulkarni, Jahnavi and Nadahalli, Kavana and Lakshmanan, Vairavan and Krishna, Srikar and Sasidharan, Vidyanand and Geo, Jini and Dilipkumar, Shilpa and Pasricha, Renu and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi} } @article {1214, title = {Exosomes: mobile platforms for targeted and synergistic signaling across cell boundaries.}, journal = {Cell Mol Life Sci}, volume = {74}, year = {2017}, month = {2017 05}, pages = {1567-1576}, abstract = {

Intercellular communications play a vital role during tissue patterning, tissue repair, and immune reactions, in homeostasis as well as in disease. Exosomes are cell-derived secreted vesicles, extensively studied for their role in intercellular communication. Exosomes have the intrinsic ability to package multiple classes of proteins and nucleic acids within their lumens and on their membranes. Here, we explore the hypothesis that exosomal targeting may represent a cellular strategy that has evolved to deliver specific combinations of signals to specific target cells and influence normal or pathological processes. This review aims to evaluate the available evidence for this hypothesis and to identify open questions whose answers will illuminate our understanding and applications of exosome biology.

}, keywords = {Animals, Cell Communication, Cell Membrane, Exosomes, Humans, Models, Biological, RNA, Signal Transduction}, issn = {1420-9071}, doi = {10.1007/s00018-016-2413-9}, author = {Vyas, Neha and Dhawan, Jyotsna} } @article {1163, title = {Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm.}, journal = {Sci Adv}, volume = {3}, year = {2017}, month = {2017 Jul}, pages = {e1603025}, abstract = {

Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms. Unsurprisingly, mechanistic insights into how differential light processing can cause distinct behavioral outputs are limited. We show how an organism can achieve complex light processing with a simple "eye" while also having independent but mutually interacting light sensing networks. Although planarian flatworms lack wavelength-specific eye photoreceptors, a 25 nm change in light wavelength is sufficient to completely switch their phototactic behavior. Quantitative photoassays, eye-brain confocal imaging, and RNA interference/knockdown studies reveal that flatworms are able to compare small differences in the amounts of light absorbed at the eyes through a single eye opsin and convert them into binary behavioral outputs. Because planarians can fully regenerate, eye-brain injury-regeneration studies showed that this acute light intensity sensing and processing are layered on simple light detection. Unlike intact worms, partially regenerated animals with eyes can sense light but cannot sense finer gradients. Planarians also show a "reflex-like," eye-independent (extraocular/whole-body) response to low ultraviolet A light, apart from the "processive" eye-brain-mediated (ocular) response. Competition experiments between ocular and extraocular sensory systems reveal dynamic interchanging hierarchies. In intact worms, cerebral ocular response can override the reflex-like extraocular response. However, injury-regeneration again offers a time window wherein both responses coexist, but the dominance of the ocular response is reversed. Overall, we demonstrate acute light intensity-based behavioral switching and two evolutionarily distinct but interacting light sensing networks in a regenerating organism.

}, issn = {2375-2548}, doi = {10.1126/sciadv.1603025}, author = {Shettigar, Nishan and Joshi, Asawari and Dalmeida, Rimple and Gopalkrishna, Rohini and Chakravarthy, Anirudh and Patnaik, Siddharth and Mathew, Manoj and Palakodeti, Dasaradhi and Gulyani, Akash} } @article {1211, title = {Mimicking Muscle Stem Cell Quiescence in Culture: Methods for Synchronization in Reversible Arrest.}, journal = {Methods Mol Biol}, volume = {1556}, year = {2017}, month = {2017}, pages = {283-302}, abstract = {

Growing evidence supports the view that in adult stem cells, the defining stem cell features of potency and self-renewal are associated with the quiescent state. Thus, uncovering the molecular logic of this reversibly arrested state underlies not only a fundamental understanding of adult tissue dynamics but also hopes for therapeutic regeneration and rejuvenation of damaged or aging tissue. A key question concerns how adult stem cells use quiescence to establish or reinforce the property of self-renewal. Since self-renewal is largely studied by assays that measure proliferation, the concept of self-renewal programs imposed during non-proliferating conditions is counterintuitive. However, there is increasing evidence generated by deconstructing the quiescent state that highlights how programs characteristic of this particular cell cycle exit may enhance stem cell capabilities, through both cell-intrinsic and extrinsic programs.Toward this end, culture models that recapitulate key aspects of stem cell quiescence are useful for molecular analysis to explore attributes and regulation of the quiescent state. In this chapter, we review the different methods used to generate homogeneous populations of quiescent muscle cells, largely by manipulating culture conditions that feed into core signaling programs that regulate the cell cycle. We also provide detailed protocols developed or refined in our lab over the past two decades.

}, keywords = {Actins, Adult Stem Cells, Animals, Biomarkers, Cell Culture Techniques, Cell Differentiation, Cell Line, Cell Proliferation, Fluorescent Antibody Technique, Humans, Mice, Microscopy, Fluorescence, Muscle, Skeletal, Myoblasts, Resting Phase, Cell Cycle, Satellite Cells, Skeletal Muscle, Stem Cells}, issn = {1940-6029}, doi = {10.1007/978-1-4939-6771-1_15}, author = {Arora, Reety and Rumman, Mohammed and Venugopal, Nisha and Gala, Hardik and Dhawan, Jyotsna} } @article {1201, title = {Scaling the effect of hydrophobic chain length on gene transfer properties of di-alkyl{,} di-hydroxy ethylammonium chloride based cationic amphiphiles}, journal = {RSC Adv.}, volume = {7}, year = {2017}, pages = {25398-25405}, abstract = {

The success of gene therapy critically depends on the availability of efficient transfection vectors. Cationic lipids are the most widely studied non-viral vectors. The molecular architecture of the cationic lipid determines its transfection efficiency. Variations in alkyl chain lengths of lipids influence self-assembly and liposomal fusion with the cell membrane. These factors determine the transfection ability of the lipid. Thus{,} to probe the effect of asymmetry in hydrophobic chains on transfection efficiency{,} we designed and synthesized a series of cationic lipids by systematically varying one of the two alkyl chains linked to the quaternary nitrogen centre from C18 to C10 and keeping the other alkyl C18 chain constant (Lip1818-Lip1810). Transfection studies in multiple cultured mammalian cells (CHO{,} B16F10 and HeLa) revealed that the lipids with C18:C14 and C18:C12 alkyl chains (Lip1814 \& Lip1812) showed 20-30\% higher transfection efficacies than their counterparts at 2 : 1 and 4 : 1 lipid to pDNA charge ratios. Cryo-transmission electron images showed unilamellar vesicle structures for the liposomes of lipids. Mechanistic studies involving Small Angle X-ray Scattering (SAXS) revealed that asymmetry in the hydrophobic region has a significant impact on liposomal fusion with the plasma membrane model. Collectively{,} these findings demonstrate that chain length asymmetry in the hydrophobic region of cationic lipids has an important role in their liposome-DNA interactions at optimal 2 : 1 and 4 : 1 lipid to pDNA charge ratios{,} which in turn modulates their gene transfer properties.

}, doi = {10.1039/C7RA02271A}, url = {http://dx.doi.org/10.1039/C7RA02271A}, author = {Hiwale, Ankita A. and Voshavar, Chandrashekhar and Dharmalingam, Priya and Dhayani, Ashish and Mukthavaram, Rajesh and Nadella, Rasajna and Sunnapu, Omprakash and Gandhi, Sivaraman and Naidu, V. G. M. and Chaudhuri, Arabinda and Marepally, Srujan and Vemula, Praveen Kumar} } @article {1156, title = {Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells.}, journal = {Elife}, volume = {6}, year = {2017}, month = {2017 Dec 04}, abstract = {

The cutaneous wound-healing program is a product of a complex interplay among diverse cell types within the skin. One fundamental process that is mediated by these reciprocal interactions is the mobilization of local stem cell pools to promote tissue regeneration and repair. Using the ablation of epidermal caspase-8 as a model of wound healing in , we analyzed the signaling components responsible for epithelial stem cell proliferation. We found that IL-1α and IL-7 secreted from keratinocytes work in tandem to expand the activated population of resident epidermal γδT-cells. A downstream effect of activated γδT-cells is the preferential proliferation of hair follicle stem cells. By contrast, IL-1α-dependent stimulation of dermal fibroblasts optimally stimulates epidermal stem cell proliferation. These findings provide new mechanistic insights into the regulation and function of epidermal cell-immune cell interactions and into how components that are classically associated with inflammation can differentially influence distinct stem cell niches within a tissue.

}, issn = {2050-084X}, doi = {10.7554/eLife.28875}, author = {Lee, Pedro and Gund, Rupali and Dutta, Abhik and Pincha, Neha and Rana, Isha and Ghosh, Subhasri and Witherden, Deborah and Kandyba, Eve and MacLeod, Amanda and Kobielak, Krzysztof and Havran, Wendy L and Jamora, Colin} } @article {1164, title = {Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload.}, journal = {Microb Cell}, volume = {4}, year = {2017}, month = {2017 Mar 27}, pages = {112-126}, abstract = {

Cysteine is an essential requirement in living organisms. However, due to its reactive thiol side chain, elevated levels of intracellular cysteine can be toxic and therefore need to be rapidly eliminated from the cellular milieu. In mammals and many other organisms, excess cysteine is believed to be primarily eliminated by the cysteine dioxygenase dependent oxidative degradation of cysteine, followed by the removal of the oxidative products. However, other mechanisms of tackling excess cysteine are also likely to exist, but have not thus far been explored. In this study, we use , which naturally lacks a cysteine dioxygenase, to investigate mechanisms for tackling cysteine overload. Overexpressing the high affinity cysteine transporter, , enabled yeast cells to rapidly accumulate high levels of intracellular cysteine. Using targeted metabolite analysis, we observe that cysteine is initially rapidly interconverted to non-reactive cystine . A time course revealed that cells systematically convert excess cysteine to inert thiol forms; initially to cystine, and subsequently to cystathionine, S-Adenosyl-L-homocysteine (SAH) and S-Adenosyl L-methionine (SAM), in addition to eventually accumulating glutathione (GSH) and polyamines. Microarray based gene expression studies revealed the upregulation of arginine/ornithine biosynthesis a few hours after the cysteine overload, and suggest that the non-toxic, non-reactive thiol based metabolic products are eventually utilized for amino acid and polyamine biogenesis, thereby enabling cell growth. Thus, cells can handle potentially toxic amounts of cysteine by a combination of thiol trapping, metabolic redistribution to non-reactive thiols and subsequent consumption for anabolism.

}, issn = {2311-2638}, doi = {10.15698/mic2017.04.567}, author = {Deshpande, Anup Arunrao and Bhatia, Muskan and Laxman, Sunil and Bachhawat, Anand Kumar} } @article {1169, title = {In Situ Synthesis of Metal Nanoparticle Embedded Hybrid Soft Nanomaterials.}, journal = {Acc Chem Res}, volume = {49}, year = {2016}, month = {2016 Sep 20}, pages = {1671-80}, abstract = {

The allure of integrating the tunable properties of soft nanomaterials with the unique optical and electronic properties of metal nanoparticles has led to the development of organic-inorganic hybrid nanomaterials. A promising method for the synthesis of such organic-inorganic hybrid nanomaterials is afforded by the in situ generation of metal nanoparticles within a host organic template. Due to their tunable surface morphology and porosity, soft organic materials such as gels, liquid crystals, and polymers that are derived from various synthetic or natural compounds can act as templates for the synthesis of metal nanoparticles of different shapes and sizes. This method provides stabilization to the metal nanoparticles by the organic soft material and advantageously precludes the use of external reducing or capping agents in many instances. In this Account, we exemplify the green chemistry approach for synthesizing these materials, both in the choice of gelators as soft material frameworks and in the reduction mechanisms that generate the metal nanoparticles. Established herein is the core design principle centered on conceiving multifaceted amphiphilic soft materials that possess the ability to self-assemble and reduce metal ions into nanoparticles. Furthermore, these soft materials stabilize the in situ generated metal nanoparticles and retain their self-assembly ability to generate metal nanoparticle embedded homogeneous organic-inorganic hybrid materials. We discuss a remarkable example of vegetable-based drying oils as host templates for metal ions, resulting in the synthesis of novel hybrid nanomaterials. The synthesis of metal nanoparticles via polymers and self-assembled materials fabricated via cardanol (a bioorganic monomer derived from cashew nut shell liquid) are also explored in this Account. The organic-inorganic hybrid structures were characterized by several techniques such as UV-visible spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Utilization of silver nanoparticle-based hybrid nanomaterials as an antimicrobial material is another illustration of the advantage of hybrid nanomaterials. We envision that the results summarized in this Account will help the scientific community to design and develop diverse organic-inorganic hybrid materials using environmentally benign methods and that these materials will yield advanced properties that have multifaceted applications in various research fields.

}, issn = {1520-4898}, doi = {10.1021/acs.accounts.6b00201}, author = {Divya, Kizhmuri P and Miroshnikov, Mikhail and Dutta, Debjit and Vemula, Praveen Kumar and Ajayan, Pulickel M and John, George} } @article {1172, title = {Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest.}, journal = {PLoS One}, volume = {11}, year = {2016}, month = {2016}, pages = {e0158860}, abstract = {

OBJECTIVES: Most cell culture studies have been performed at atmospheric oxygen tension of 21\%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21\% O2 and in 1\% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts.

MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21\% O2 and 1\% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot.

RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1\% O2) compared to 21\% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1\% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21\% O2, while the canonical Wnt signaling may be induced in 1\% O2 during activation and proliferation of myoblasts.

}, keywords = {Adolescent, Cell Cycle Checkpoints, Cell Hypoxia, Cell Proliferation, Cell Separation, Cells, Cultured, Down-Regulation, Female, Gene Expression Regulation, Humans, Ki-67 Antigen, Male, Muscle Development, Muscle Proteins, Myoblasts, Oxygen, Receptors, Notch, Resting Phase, Cell Cycle, Transforming Growth Factor beta, Wnt Signaling Pathway, Young Adult}, issn = {1932-6203}, doi = {10.1371/journal.pone.0158860}, author = {Sellathurai, Jeeva and Nielsen, Joachim and Hejb{\o}l, Eva Kildall and J{\o}rgensen, Louise Helskov and Dhawan, Jyotsna and Nielsen, Michael Friberg Bruun and Schr{\o}der, Henrik Daa} } @article {1171, title = {Melatonin and Human Cardiovascular Disease.}, journal = {J Cardiovasc Pharmacol Ther}, year = {2016}, month = {2016 Jul 21}, abstract = {

The possible therapeutic role of melatonin in the pathophysiology of coronary artery disorder (CAD) is increasingly being recognized. In humans, exogenous melatonin has been shown to decrease nocturnal hypertension, improve systolic and diastolic blood pressure, reduce the pulsatility index in the internal carotid artery, decrease platelet aggregation, and reduce serum catecholamine levels. Low circulating levels of melatonin are reported in individuals with CAD, arterial hypertension, and congestive heart failure. This review assesses current literature on the cardiovascular effects of melatonin in humans. It can be concluded that melatonin deserves to be considered in clinical trials evaluating novel therapeutic interventions for cardiovascular disorders.

}, issn = {1940-4034}, doi = {10.1177/1074248416660622}, author = {Pandi-Perumal, Seithikurippu R and BaHammam, Ahmed S and Ojike, Nwakile I and Akinseye, Oluwaseun A and Kendzerska, Tetyana and Buttoo, Kenneth and Dhandapany, Perundurai S and Brown, Gregory M and Cardinali, Daniel P} } @article {618, title = {Decoding the stem cell quiescence cycle--lessons from yeast for regenerative biology.}, journal = {J Cell Sci}, volume = {128}, year = {2015}, month = {2015 Dec 15}, pages = {4467-74}, abstract = {

In the past decade, major advances have occurred in the understanding of mammalian stem cell biology, but roadblocks (including gaps in our fundamental understanding) remain in translating this knowledge to regenerative medicine. Interestingly, a close analysis of the Saccharomyces cerevisiae literature leads to an appreciation of how much yeast biology has contributed to the conceptual framework underpinning our understanding of stem cell behavior, to the point where such insights have been internalized into the realm of the known. This Opinion article focuses on one such example, the quiescent adult mammalian stem cell, and examines concepts underlying our understanding of quiescence that can be attributed to studies in yeast. We discuss the metabolic, signaling and gene regulatory events that control entry and exit into quiescence in yeast. These processes and events retain remarkable conservation and conceptual parallels in mammalian systems, and collectively suggest a regulated program beyond the cessation of cell division. We argue that studies in yeast will continue to not only reveal fundamental concepts in quiescence, but also leaven progress in regenerative medicine.

}, issn = {1477-9137}, doi = {10.1242/jcs.177758}, author = {Dhawan, Jyotsna and Laxman, Sunil} }