TY - JOUR T1 - Blood progenitor redox homeostasis through olfaction-derived systemic GABA in hematopoietic growth control in Drosophila. JF - Development Y1 - 2022 A1 - Goyal, Manisha A1 - Tomar, Ajay A1 - Madhwal, Sukanya A1 - Mukherjee, Tina AB -

The role of reactive oxygen species (ROS) in myeloid development is well established. However, its aberrant generation alters hematopoiesis. Thus, a comprehensive understanding of events controlling ROS homeostasis forms the central focus of this study. We show that, in homeostasis, myeloid-like blood progenitor cells of the Drosophila larvae, which reside in a specialized hematopoietic organ termed the lymph gland, use TCA to generate ROS. However, excessive ROS production leads to lymph gland growth retardation. Therefore, to moderate blood progenitor ROS, Drosophila larvae rely on olfaction and its downstream systemic GABA. GABA internalization and its breakdown into succinate by progenitor cells activates pyruvate dehydrogenase kinase (PDK), which controls inhibitory phosphorylation of pyruvate dehydrogenase (PDH). PDH is the rate-limiting enzyme that connects pyruvate to the TCA cycle and to oxidative phosphorylation. Thus, GABA metabolism via PDK activation maintains TCA activity and blood progenitor ROS homeostasis, and supports normal lymph gland growth. Consequently, animals that fail to smell also fail to sustain TCA activity and ROS homeostasis, which leads to lymph gland growth retardation. Overall, this study describes the requirement of animal odor-sensing and GABA in myeloid ROS regulation and hematopoietic growth control.

VL - 149 IS - 8 ER - TY - JOUR T1 - Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic Channel. JF - J Phys Chem B Y1 - 2022 A1 - Mukherjee, Dipanjan A1 - Hasan, Md Nur A1 - Ghosh, Ria A1 - Ghosh, Gourab A1 - Bera, Arpan A1 - Prasad, Sujanthi Easwara A1 - Hiwale, Ankita A1 - Vemula, Praveen K A1 - Das, Ranjan A1 - Pal, Samir Kumar AB -

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.

VL - 126 IS - 3 ER - TY - JOUR T1 - Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2. JF - Front Genome Ed Y1 - 2022 A1 - George, Anila A1 - Ravi, Nithin Sam A1 - Prasad, Kirti A1 - Panigrahi, Lokesh A1 - Koikkara, Sanya A1 - Rajendiran, Vignesh A1 - Devaraju, Nivedhitha A1 - Paul, Joshua A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - Mohankumar, Kumarasamypet M AB -

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.

VL - 4 ER - TY - JOUR T1 - FMRP protects the lung from xenobiotic stress by facilitating the Integrated Stress Response. JF - J Cell Sci Y1 - 2022 A1 - Basu, Deblina Sain A1 - Bhavsar, Rital A1 - Gulami, Imtiyaz A1 - Chavda, Saraswati A1 - Lingamallu, Sai Manoz A1 - Muddashetty, Ravi A1 - Veeranna, Chandrakanth A1 - Chattarji, Sumantra A1 - Thimmulappa, Rajesh A1 - Bhattacharya, Aditi A1 - Guha, Arjun AB -

Stress response pathways protect the lung from the damaging effects of environmental toxicants. Here we investigate the role of the Fragile X Mental Retardation Protein (FMRP), a multifunctional protein implicated in stress responses, in the lung. We report that FMRP is expressed in murine and human lungs, in the airways and more broadly. Analysis of airway stress responses in mice and in a murine cell line ex vivo, using the well-established Naphthalene (Nap) injury model, reveals that FMRP-deficient cells exhibit increased expression of markers of oxidative and genotoxic stress and increased cell death. Further inquiry shows that FMRP-deficient cells fail to actuate the Integrated Stress Response Pathway (ISR) and upregulate the transcription factor ATF4. Knockdown of ATF4 expression phenocopies the loss of FMRP. We extend our analysis of the role of FMRP to human bronchial BEAS-2B cells, using a 9, 10-Phenanthrenequinone air pollutant model, to find FMRP-deficient BEAS-2B also fail to actuate the ISR and exhibit greater susceptibility. Taken together, our data suggest that FMRP has a conserved role in protecting the airways by facilitating the ISR.

ER - TY - JOUR T1 - Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. JF - Elife Y1 - 2022 A1 - Ravi, Nithin Sam A1 - Wienert, Beeke A1 - Wyman, Stacia K A1 - Bell, Henry William A1 - George, Anila A1 - Mahalingam, Gokulnath A1 - Vu, Jonathan T A1 - Prasad, Kirti A1 - Bandlamudi, Bhanu Prasad A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Syedbasha, Nazar A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Narayanasamy, Muthuraman A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - DeWitt, Mark A A1 - Crossley, Merlin A1 - Corn, Jacob E A1 - Mohankumar, Kumarasamypet M KW - Adenine KW - Anemia, Sickle Cell KW - beta-Globins KW - beta-Thalassemia KW - Cell Line KW - Clustered Regularly Interspaced Short Palindromic Repeats KW - CRISPR-Cas Systems KW - Cytosine KW - Fetal Hemoglobin KW - gamma-Globins KW - Gene Editing KW - Hematopoietic Stem Cells KW - Humans KW - Point Mutation KW - Promoter Regions, Genetic AB -

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.

VL - 11 ER - TY - JOUR T1 - Intermittent scavenging of storage lesion from stored red blood cells by electrospun nanofibrous sheets enhances their quality and shelf-life. JF - Nat Commun Y1 - 2022 A1 - Pandey, Subhashini A1 - Mahato, Manohar A1 - Srinath, Preethem A1 - Bhutani, Utkarsh A1 - Goap, Tanu Jain A1 - Ravipati, Priusha A1 - Vemula, Praveen Kumar KW - Acridines KW - Animals KW - Drug-Related Side Effects and Adverse Reactions KW - Erythrocytes KW - Humans KW - Mice KW - Nanofibers KW - Research Personnel AB -

Transfusion of healthy red blood cells (RBCs) is a lifesaving process. However, upon storing RBCs, a wide range of damage-associate molecular patterns (DAMPs), such as cell-free DNA, nucleosomes, free-hemoglobin, and poly-unsaturated-fatty-acids are generated. DAMPs can further damage RBCs; thus, the quality of stored RBCs declines during the storage and limits their shelf-life. Since these DAMPs consist of either positive or negative charged species, we developed taurine and acridine containing electrospun-nanofibrous-sheets (Tau-AcrNFS), featuring anionic, cationic charges and an DNA intercalating group on their surfaces. We show that Tau-AcrNFS are efficient in scavenging DAMPs from stored human and mice RBCs ex vivo. We find that intermittent scavenging of DAMPs by Tau-AcrNFS during the storage reduces the loss of RBC membrane integrity and reduces discocytes-to-spheroechinocytes transformation in stored-old-RBCs. We perform RBC-transfusion studies in mice to reveal that intermittent removal of DAMPs enhances the quality of stored-old-RBCs equivalent to freshly collected RBCs, and increases their shelf-life by ~22%. Such prophylactic technology may lead to the development of novel blood bags or medical device, and may therefore impact healthcare by reducing transfusion-related adverse effects.

VL - 13 IS - 1 ER - TY - JOUR T1 - Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours. JF - Nat Cell Biol Y1 - 2022 A1 - Jijumon, A S A1 - Bodakuntla, Satish A1 - Genova, Mariya A1 - Bangera, Mamata A1 - Sackett, Violet A1 - Besse, Laetitia A1 - Maksut, Fatlinda A1 - Henriot, Veronique A1 - Magiera, Maria M A1 - Sirajuddin, Minhajuddin A1 - Janke, Carsten AB -

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

VL - 24 IS - 2 ER - TY - JOUR T1 - Methionine uptake via the SLC43A2 transporter is essential for regulatory T-cell survival. JF - Life Sci Alliance Y1 - 2022 A1 - Saini, Neetu A1 - Naaz, Afsana A1 - Metur, Shree Padma A1 - Gahlot, Pinki A1 - Walvekar, Adhish A1 - Dutta, Anupam A1 - Davathamizhan, Umamaheswari A1 - Sarin, Apurva A1 - Laxman, Sunil KW - Interleukin-2 KW - Methionine KW - Racemethionine KW - Solute Carrier Proteins KW - T-Lymphocytes, Regulatory AB -

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.

VL - 5 IS - 12 ER - TY - JOUR T1 - Microbial metabolite restricts 5-fluorouracil-resistant colonic tumor progression by sensitizing drug transporters via regulation of FOXO3-FOXM1 axis. JF - Theranostics Y1 - 2022 A1 - Ghosh, Sweta A1 - Singh, Rajbir A1 - Vanwinkle, Zachary Matthew A1 - Guo, Haixun A1 - Vemula, Praveen Kumar A1 - Goel, Ajay A1 - Haribabu, Bodduluri A1 - Jala, Venkatakrishna Rao KW - Animals KW - Antimetabolites, Antineoplastic KW - ATP Binding Cassette Transporter, Subfamily G, Member 2 KW - Azoxymethane KW - Cell Line, Tumor KW - Colonic Neoplasms KW - Coumarins KW - Drug Resistance, Neoplasm KW - Fluorouracil KW - Forkhead Box Protein M1 KW - Forkhead Box Protein O3 KW - Gastrointestinal Microbiome KW - Humans KW - Mice KW - Neoplasm Proteins AB -

The survival rate of colorectal cancer patients is adversely affected by the selection of tumors resistant to conventional anti-cancer drugs such as 5-fluorouracil (5FU). Although there is mounting evidence that commensal gut microbiota is essential for effective colon cancer treatment, the detailed molecular mechanisms and the role of gut microbial metabolites remain elusive. The goal of this study is to decipher the impact and mechanisms of gut microbial metabolite, urolithin A (UroA) and its structural analogue, UAS03 on reversal of 5FU-resistant (5FUR) colon cancers. We have utilized the SW480 and HCT-116 parental (5FU-sensitive) and 5FUR colon cancer cells to examine the chemosensitization effects of UroA or UAS03 by using both and models. The effects of mono (UroA/UAS03/5FU) and combinatorial therapy (UroA/UAS03 + 5FU) on cell proliferation, apoptosis, cell migration and invasion, regulation of epithelial mesenchymal transition (EMT) mediators, expression and activities of drug transporters, and their regulatory transcription factors were examined using molecular, cellular, immunological and flowcytometric methods. Further, the anti-tumor effects of mono/combination therapy (UroA or UAS03 or 5FU or UroA/UAS03 + 5FU) were examined using pre-clinical models of 5FUR-tumor xenografts in NRGS mice and azoxymethane (AOM)-dextran sodium sulfate (DSS)-induced colon tumors. Our data showed that UroA or UAS03 in combination with 5FU significantly inhibited cell viability, proliferation, invasiveness as well as induced apoptosis of the 5FUR colon cancer cells compared to mono treatments. Mechanistically, UroA or UAS03 chemosensitized the 5FUR cancer cells by downregulating the expression and activities of drug transporters (MDR1, BCRP, MRP2 and MRP7) leading to a decrease in the efflux of 5FU. Further, our data suggested the UroA or UAS03 chemosensitized 5FUR cancer cells to 5FU treatment through regulating FOXO3-FOXM1 axis. Oral treatment with UroA or UAS03 in combination with low dose i.p. 5FU significantly reduced the growth of 5FUR-tumor xenografts in NRGS mice. Further, combination therapy significantly abrogated colonic tumors in AOM-DSS-induced colon tumors in mice. In summary, gut microbial metabolite UroA and its structural analogue UAS03 chemosensitized the 5FUR colon cancers for effective 5FU chemotherapy. This study provided the novel characteristics of gut microbial metabolites to have significant translational implications in drug-resistant cancer therapeutics.

VL - 12 IS - 12 ER - TY - JOUR T1 - Modulation of biliverdin dynamics and spectral properties by Sandercyanin. JF - RSC Adv Y1 - 2022 A1 - Ghosh, Swagatha A1 - Mondal, Sayan A1 - Yadav, Keerti A1 - Aggarwal, Shantanu A1 - Schaefer, Wayne F A1 - Narayana, Chandrabhas A1 - Subramanian, Ramaswamy AB -

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

VL - 12 IS - 31 ER - TY - JOUR T1 - Nanoarchitectonics for Free-Standing Polyelectrolyte Multilayers Films: Exploring the Flipped Surfaces JF - ChemNanoMat Y1 - 2022 A1 - Suman Pahal A1 - Rakshith Boranna A1 - Abinash Tripathy A1 - Venkanagouda S. Goudar A1 - Vyshnavi T. Veetil A1 - Rajendra Kurapati A1 - Gurusiddappa R. Prashanth A1 - Praveen K. Vemula UR - https://doi.org/10.1002%2Fcnma.202200462 ER - TY - JOUR T1 - Nanobody derived using a peptide epitope from the spike protein receptor-binding motif inhibits entry of SARS-CoV-2 variants. JF - J Biol Chem Y1 - 2022 A1 - Mendon, Nivya A1 - Ganie, Rayees A A1 - Kesarwani, Shubham A1 - Dileep, Drisya A1 - Sasi, Sarika A1 - Lama, Prakash A1 - Chandra, Anchal A1 - Sirajuddin, Minhajuddin AB -

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.

VL - 299 IS - 1 ER - TY - JOUR T1 - NMDAR mediated dynamic changes in mA inversely correlates with neuronal translation. JF - Sci Rep Y1 - 2022 A1 - Gowda, Naveen Kumar Chandappa A1 - Nawalpuri, Bharti A1 - Ramakrishna, Sarayu A1 - Jhaveri, Vishwaja A1 - Muddashetty, Ravi S KW - Adenosine KW - Neurons KW - Phosphorylation KW - Receptors, N-Methyl-D-Aspartate KW - RNA AB -

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (mA) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of mA modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of mA modification in response to NMDAR stimulation. We have consistently observed that 5 min NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the mA signal at 5 min time point, correlating with the global translation inhibition. The NMDAR induced increase in the mA signal is accompanied by the redistribution of the mA marked RNAs from translating to the non-translating pool of ribosomes. The increased mA levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in mA levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.

VL - 12 IS - 1 ER - TY - JOUR T1 - S. mediterranea ETS-1 regulates the function of cathepsin-positive cells and the epidermal lineage landscape via basement membrane remodeling. JF - J Cell Sci Y1 - 2022 A1 - Dubey, Vinay Kumar A1 - Sarkar, Souradeep R A1 - Lakshmanan, Vairavan A1 - Dalmeida, Rimple A1 - Gulyani, Akash A1 - Palakodeti, Dasaradhi KW - Animals KW - Basement Membrane KW - Cathepsins KW - Cell Differentiation KW - Epidermis KW - Humans KW - Matrix Metalloproteinases KW - Mediterranea KW - Planarians KW - Transcription Factors AB -

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.

VL - 135 IS - 20 ER - TY - JOUR T1 - Single step fabrication of hollow microneedles and an experimental package for controlled drug delivery. JF - Int J Pharm Y1 - 2022 A1 - Ghate, Vivek A1 - Renjith, Anu A1 - Badnikar, Kedar A1 - Pahal, Suman A1 - Jayadevi, Shreyas N A1 - Nayak, Manjunatha M A1 - Vemula, Praveen K A1 - Subramanyam, Dinesh N AB -

Hollow microneedle arrays (HMNs) are an excellent choice for managing chronic diseases requiring the administration of multiple drug doses over a prolonged duration. However, HMNs have gained partial success due to limitations in their manufacturing capabilities, and cumbersome processes. In the present study, polymeric HMNs were fabricated using a novel single-step drop-casting process without needing cleanroom facilities, and sophisticated instrumentation. When drop casted on the pyramidal tip stainless steel needles, the optimized polymer solution allowed the reproducible formation of desired height HMMs on a detachable acrylic base. To enable broader applications, the base with HMNs was integrated into an experimental package built to deliver a dose of ∼ 5 µL per 30° clockwise rotation of the actuator, allowing multiple metered drug dose administrations. The fabricated HMNs were optically imaged, and tested for mechanical integrity and stability. The working and functional utility of the HMNs package in delivering metered drug doses was demonstrated by delivering vitamin B12 (ex vivo) and insulin (in vivo), respectively. The optimized process can be used for the large-scale manufacturing of HMNs and the experimental package shows the potential to be further developed into a wearable device.

VL - 632 ER - TY - JOUR T1 - Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of matricellular protein Mindin. JF - Cell Rep Y1 - 2022 A1 - Badarinath, Krithika A1 - Dam, Binita A1 - Kataria, Sunny A1 - Zirmire, Ravindra K A1 - Dey, Rakesh A1 - Kansagara, Gaurav A1 - Ajnabi, Johan A1 - Hegde, Akshay A1 - Singh, Randhir A1 - Masudi, Tafheem A1 - Sambath, Janani A1 - Sachithanandan, Sasikala P A1 - Kumar, Prashant A1 - Gulyani, Akash A1 - He, You-Wen A1 - Krishna, Sudhir A1 - Jamora, Colin KW - Carcinoma, Squamous Cell KW - Cell Line, Tumor KW - Epithelial Cells KW - Extracellular Matrix Proteins KW - Humans KW - Integrins KW - Neoplasm Proteins KW - Neoplasm Recurrence, Local KW - Neoplastic Stem Cells KW - Skin Neoplasms KW - Snail Family Transcription Factors AB -

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.

VL - 40 IS - 12 ER - TY - JOUR T1 - The story of rRNA expansion segments: Finding functionality amidst diversity. JF - Wiley Interdiscip Rev RNA Y1 - 2022 A1 - Hariharan, Nivedita A1 - Ghosh, Sumana A1 - Palakodeti, Dasaradhi AB -

Expansion segments (ESs) are multinucleotide insertions present across phyla at specific conserved positions in eukaryotic rRNAs. ESs are generally absent in bacterial rRNAs with some exceptions, while the archaeal rRNAs have microexpansions at regions that coincide with those of eukaryotic ESs. Although there is an increasing prominence of ribosomes, especially the ribosomal proteins, in fine-tuning gene expression through translation regulation, the role of rRNA ESs is relatively underexplored. While rRNAs have been established as the major catalytic hub in ribosome function, the presence of ESs widens their scope as a species-specific regulatory hub of protein synthesis. In this comprehensive review, we have elaborately discussed the current understanding of the functional aspects of rRNA ESs of cytoplasmic eukaryotic ribosomes and discuss their past, present, and future. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems Translation > Ribosome Structure/Function Translation > Regulation.

ER - TY - JOUR T1 - Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy. JF - NPJ Genom Med Y1 - 2022 A1 - Lesurf, Robert A1 - Said, Abdelrahman A1 - Akinrinade, Oyediran A1 - Breckpot, Jeroen A1 - Delfosse, Kathleen A1 - Liu, Ting A1 - Yao, Roderick A1 - Persad, Gabrielle A1 - McKenna, Fintan A1 - Noche, Ramil R A1 - Oliveros, Winona A1 - Mattioli, Kaia A1 - Shah, Shreya A1 - Miron, Anastasia A1 - Yang, Qian A1 - Meng, Guoliang A1 - Yue, Michelle Chan Seng A1 - Sung, Wilson W L A1 - Thiruvahindrapuram, Bhooma A1 - Lougheed, Jane A1 - Oechslin, Erwin A1 - Mondal, Tapas A1 - Bergin, Lynn A1 - Smythe, John A1 - Jayappa, Shashank A1 - Rao, Vinay J A1 - Shenthar, Jayaprakash A1 - Dhandapany, Perundurai S A1 - Semsarian, Christopher A1 - Weintraub, Robert G A1 - Bagnall, Richard D A1 - Ingles, Jodie A1 - Melé, Marta A1 - Maass, Philipp G A1 - Ellis, James A1 - Scherer, Stephen W A1 - Mital, Seema AB -

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 × 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.

VL - 7 IS - 1 ER - TY - JOUR T1 - APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis. JF - J Neurosci Y1 - 2021 A1 - Ramakrishna, Sarayu A1 - Jhaveri, Vishwaja A1 - Konings, Sabine C A1 - Nawalpuri, Bharti A1 - Chakraborty, Sumita A1 - Holst, Bjørn A1 - Schmid, Benjamin A1 - Gouras, Gunnar K A1 - Freude, Kristine K A1 - Muddashetty, Ravi S AB -

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis. Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

VL - 41 IS - 42 ER - TY - JOUR T1 - Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3. JF - Stem Cell Reports Y1 - 2021 A1 - Chandrasekaran, Abinaya A1 - Dittlau, Katarina Stoklund A1 - Corsi, Giulia I A1 - Haukedal, Henriette A1 - Doncheva, Nadezhda T A1 - Ramakrishna, Sarayu A1 - Ambardar, Sheetal A1 - Salcedo, Claudia A1 - Schmidt, Sissel I A1 - Zhang, Yu A1 - Cirera, Susanna A1 - Pihl, Maria A1 - Schmid, Benjamin A1 - Nielsen, Troels Tolstrup A1 - Nielsen, Jørgen E A1 - Kolko, Miriam A1 - Kobolák, Julianna A1 - Dinnyés, András A1 - Hyttel, Poul A1 - Palakodeti, Dasaradhi A1 - Gorodkin, Jan A1 - Muddashetty, Ravi S A1 - Meyer, Morten A1 - Aldana, Blanca I A1 - Freude, Kristine K AB -

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.

VL - 16 IS - 11 ER - TY - JOUR T1 - Biophysical properties of the isolated spike protein binding helix of human ACE2. JF - Biophys J Y1 - 2021 A1 - Das, Anirban A1 - Vishvakarma, Vicky A1 - Dey, Arpan A1 - Dey, Simli A1 - Gupta, Ankur A1 - Das, Mitradip A1 - Vishwakarma, Krishna Kant A1 - Roy, Debsankar Saha A1 - Yadav, Swati A1 - Kesarwani, Shubham A1 - Venkatramani, Ravindra A1 - Maiti, Sudipta KW - Angiotensin-Converting Enzyme 2 KW - COVID-19 KW - Humans KW - Peptidyl-Dipeptidase A KW - Protein Binding KW - SARS-CoV-2 KW - Spike Glycoprotein, Coronavirus AB -

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.

VL - 120 IS - 14 ER - TY - JOUR T1 - Cycles, sources, and sinks: Conceptualizing how phosphate balance modulates carbon flux using yeast metabolic networks. JF - Elife Y1 - 2021 A1 - Gupta, Ritu A1 - Laxman, Sunil AB -

Phosphates are ubiquitous molecules that enable critical intracellular biochemical reactions. Therefore, cells have elaborate responses to phosphate limitation. Our understanding of long-term transcriptional responses to phosphate limitation is extensive. Contrastingly, a systems-level perspective presenting unifying biochemical concepts to interpret how phosphate balance is critically coupled to (and controls) metabolic information flow is missing. To conceptualize such processes, utilizing yeast metabolic networks we categorize phosphates utilized in metabolism into cycles, sources and sinks. Through this, we identify metabolic reactions leading to putative phosphate sources or sinks. With this conceptualization, we illustrate how mass action driven flux towards sources and sinks enable cells to manage phosphate availability during transient/immediate phosphate limitations. We thereby identify how intracellular phosphate availability will predictably alter specific nodes in carbon metabolism, and determine signature cellular metabolic states. Finally, we identify a need to understand intracellular phosphate pools, in order to address mechanisms of phosphate regulation and restoration.

VL - 10 ER - TY - JOUR T1 - Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye-brain-independent movement and arousal. JF - Proc Natl Acad Sci U S A Y1 - 2021 A1 - Shettigar, Nishan A1 - Chakravarthy, Anirudh A1 - Umashankar, Suchitta A1 - Lakshmanan, Vairavan A1 - Palakodeti, Dasaradhi A1 - Gulyani, Akash AB -

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

VL - 118 IS - 20 ER - TY - JOUR T1 - Duox generated reactive oxygen species activate ATR/Chk1 to induce G2 arrest in tracheoblasts. JF - Elife Y1 - 2021 A1 - Kizhedathu, Amrutha A1 - Chhajed, Piyush A1 - Yeramala, Lahari A1 - Sain Basu, Deblina A1 - Mukherjee, Tina A1 - Vinothkumar, Kutti R A1 - Guha, Arjun AB -

Progenitors of the thoracic tracheal system of adult (tracheoblasts) arrest in G2 during larval life and rekindle a mitotic program subsequently. G2 arrest is dependent on ATR-dependent phosphorylation of Chk1 that is actuated in the absence of detectable DNA damage. We are interested in the mechanisms that activate ATR/Chk1 (Kizhedathu et al., 2018, 2020). Here we report that levels of reactive oxygen species (ROS) are high in arrested tracheoblasts and decrease upon mitotic re-entry. High ROS is dependent on expression of Duox, an HO generating-Dual Oxidase. ROS quenching by overexpression of Superoxide Dismutase 1, or by knockdown of Duox, abolishes Chk1 phosphorylation and results in precocious proliferation. Tracheae deficient in Duox, or deficient in both Duox and regulators of DNA damage-dependent ATR/Chk1 activation (ATRIP/TOPBP1/ Claspin), can induce phosphorylation of Chk1 in response to micromolar concentrations of HO in minutes. The findings presented reveal that HO activates ATR/Chk1 in tracheoblasts by a non-canonical, potentially direct, mechanism.

VL - 10 ER - TY - JOUR T1 - Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay. JF - Life Sci Alliance Y1 - 2021 A1 - Chakravarthy, Anirudh A1 - Nandakumar, Anirudh A1 - George, Geen A1 - Ranganathan, Shyamsundar A1 - Umashankar, Suchitta A1 - Shettigar, Nishan A1 - Palakodeti, Dasaradhi A1 - Gulyani, Akash A1 - Ramesh, Arati KW - Biosensing Techniques KW - COVID-19 KW - Humans KW - Luminescence KW - Nucleic Acid Amplification Techniques KW - RNA KW - RNA, Viral KW - SARS-CoV-2 AB -

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

VL - 4 IS - 12 ER - TY - JOUR T1 - Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi, India. JF - Science Y1 - 2021 A1 - Dhar, Mahesh S A1 - Marwal, Robin A1 - Vs, Radhakrishnan A1 - Ponnusamy, Kalaiarasan A1 - Jolly, Bani A1 - Bhoyar, Rahul C A1 - Sardana, Viren A1 - Naushin, Salwa A1 - Rophina, Mercy A1 - Mellan, Thomas A A1 - Mishra, Swapnil A1 - Whittaker, Charles A1 - Fatihi, Saman A1 - Datta, Meena A1 - Singh, Priyanka A1 - Sharma, Uma A1 - Ujjainiya, Rajat A1 - Bhatheja, Nitin A1 - Divakar, Mohit Kumar A1 - Singh, Manoj K A1 - Imran, Mohamed A1 - Senthivel, Vigneshwar A1 - Maurya, Ranjeet A1 - Jha, Neha A1 - Mehta, Priyanka A1 - A, Vivekanand A1 - Sharma, Pooja A1 - Vr, Arvinden A1 - Chaudhary, Urmila A1 - Soni, Namita A1 - Thukral, Lipi A1 - Flaxman, Seth A1 - Bhatt, Samir A1 - Pandey, Rajesh A1 - Dash, Debasis A1 - Faruq, Mohammed A1 - Lall, Hemlata A1 - Gogia, Hema A1 - Madan, Preeti A1 - Kulkarni, Sanket A1 - Chauhan, Himanshu A1 - Sengupta, Shantanu A1 - Kabra, Sandhya A1 - Gupta, Ravindra K A1 - Singh, Sujeet K A1 - Agrawal, Anurag A1 - Rakshit, Partha A1 - Nandicoori, Vinay A1 - Tallapaka, Karthik Bharadwaj A1 - Sowpati, Divya Tej A1 - Thangaraj, K A1 - Bashyam, Murali Dharan A1 - Dalal, Ashwin A1 - Sivasubbu, Sridhar A1 - Scaria, Vinod A1 - Parida, Ajay A1 - Raghav, Sunil K A1 - Prasad, Punit A1 - Sarin, Apurva A1 - Mayor, Satyajit A1 - Ramakrishnan, Uma A1 - Palakodeti, Dasaradhi A1 - Seshasayee, Aswin Sai Narain A1 - Bhat, Manoj A1 - Shouche, Yogesh A1 - Pillai, Ajay A1 - Dikid, Tanzin A1 - Das, Saumitra A1 - Maitra, Arindam A1 - Chinnaswamy, Sreedhar A1 - Biswas, Nidhan Kumar A1 - Desai, Anita Sudhir A1 - Pattabiraman, Chitra A1 - Manjunatha, M V A1 - Mani, Reeta S A1 - Arunachal Udupi, Gautam A1 - Abraham, Priya A1 - Atul, Potdar Varsha A1 - Cherian, Sarah S AB -

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; ×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.

ER - TY - JOUR T1 - Histological and Immunohistochemical Examination of Stem Cell Proliferation and Reepithelialization in the Wounded Skin. JF - Bio Protoc Y1 - 2021 A1 - Gund, Rupali A1 - Zirmire, Ravindra A1 - J, Haarshaadri A1 - Kansagara, Gaurav A1 - Jamora, Colin AB -

The skin is the largest organ that protects our body from the external environment and it is constantly exposed to pathogenic insults and injury. Repair of damage to this organ is carried out by a complex process involving three overlapping phases of inflammation, proliferation and remodeling. Histological analysis of wounded skin is a convenient approach to examine broad alterations in tissue architecture and investigate cells in their indigenous microenvironment. In this article we present a protocol for immunohistochemical examination of wounded skin to study mechanisms involved in regulating stem cell activity, which is a vital component in the repair of the damaged tissue. Performing such histological analysis enables the understanding of the spatial relationship between cells that interact in the specialized wound microenvironment. The analytical tools described herein permit the quantitative measurement of the regenerative ability of stem cells adjacent to the wound and the extent of re-epithelialization during wound closure. These protocols can be adapted to investigate numerous cellular processes and cell types within the wounded skin.

VL - 11 IS - 2 ER - TY - JOUR T1 - iRGD conjugated nimbolide liposomes protect against endotoxin induced acute respiratory distress syndrome. JF - Nanomedicine Y1 - 2021 A1 - Pooladanda, Venkatesh A1 - Thatikonda, Sowjanya A1 - Sunnapu, Omprakash A1 - Tiwary, Shristy A1 - Vemula, Praveen Kumar A1 - Talluri, M V N Kumar A1 - Godugu, Chandraiah AB -

Acute respiratory distress syndrome (ARDS) is a deadly respiratory illness associated with refractory hypoxemia and pulmonary edema. The recent pandemic outbreak of COVID-19 is associated with severe pneumonia and inflammatory cytokine storm in the lungs. The anti-inflammatory phytomedicine nimbolide (NIM) may not be feasible for clinical translation due to poor pharmacokinetic properties and lack of suitable delivery systems. To overcome these barriers, we have developed nimbolide liposomes conjugated with iRGD peptide (iRGD-NIMLip) for targeting lung inflammation. It was observed that iRGD-NIMLip treatment significantly inhibited oxidative stress and cytokine storm compared to nimbolide free-drug (f-NIM), nimbolide liposomes (NIMLip), and exhibited superior activity compared to dexamethasone (DEX). iRGD-NIMLip abrogated the LPS induced p65 NF-κB, Akt, MAPK, Integrin β3 and β5, STAT3, and DNMT1 expression. Collectively, our results demonstrate that iRGD-NIMLip could be a promising novel drug delivery system to target severe pathological consequences observed in ARDS and COVID-19 associated cytokine storm.

VL - 33 ER - TY - JOUR T1 - Microneedles for Extended Transdermal Therapeutics: A Route to Advanced Healthcare. JF - Eur J Pharm Biopharm Y1 - 2021 A1 - Pahal, Suman A1 - Badnikar, Kedar A1 - Ghate, Vivek A1 - Bhutani, Utkarsh A1 - Nayak, Mangalore Manjunatha A1 - Subramanyam, Dinesh Narasimhaiah A1 - Vemula, Praveen Kumar AB -

Sustained release of drugs over a pre-determined period is required to maintain an effective therapeutic dose for variety of drug delivery applications. Transdermal devices such as polymeric microneedle patches and other microneedle-based devices have been utilized for sustained release of their payload. Swift clearing of drugs can be prevented either by designing a slow-degrading polymeric matrix or by providing physiochemical triggers to different microneedle-based devices for on-demand release. These long-acting transdermal devices prevent the burst release of drugs. This review highlights the recent advances of microneedle-based devices for sustained release of vaccines, hormones, and antiretrovirals with their prospective safe clinical translation.

VL - 159 ER - TY - JOUR T1 - SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. JF - Nature Y1 - 2021 A1 - Mlcochova, Petra A1 - Kemp, Steven A A1 - Dhar, Mahesh Shanker A1 - Papa, Guido A1 - Meng, Bo A1 - Ferreira, Isabella A T M A1 - Datir, Rawlings A1 - Collier, Dami A A1 - Albecka, Anna A1 - Singh, Sujeet A1 - Pandey, Rajesh A1 - Brown, Jonathan A1 - Zhou, Jie A1 - Goonawardane, Niluka A1 - Mishra, Swapnil A1 - Whittaker, Charles A1 - Mellan, Thomas A1 - Marwal, Robin A1 - Datta, Meena A1 - Sengupta, Shantanu A1 - Ponnusamy, Kalaiarasan A1 - Radhakrishnan, Venkatraman Srinivasan A1 - Abdullahi, Adam A1 - Charles, Oscar A1 - Chattopadhyay, Partha A1 - Devi, Priti A1 - Caputo, Daniela A1 - Peacock, Tom A1 - Wattal, Chand A1 - Goel, Neeraj A1 - Satwik, Ambrish A1 - Vaishya, Raju A1 - Agarwal, Meenakshi A1 - Mavousian, Antranik A1 - Lee, Joo Hyeon A1 - Bassi, Jessica A1 - Silacci-Fegni, Chiara A1 - Saliba, Christian A1 - Pinto, Dora A1 - Irie, Takashi A1 - Yoshida, Isao A1 - Hamilton, William L A1 - Sato, Kei A1 - Bhatt, Samir A1 - Flaxman, Seth A1 - James, Leo C A1 - Corti, Davide A1 - Piccoli, Luca A1 - Barclay, Wendy S A1 - Rakshit, Partha A1 - Agrawal, Anurag A1 - Gupta, Ravindra K KW - Antibodies, Neutralizing KW - Cell Fusion KW - Cell Line KW - COVID-19 Vaccines KW - Female KW - Health Personnel KW - Humans KW - Immune Evasion KW - India KW - Kinetics KW - Male KW - SARS-CoV-2 KW - Spike Glycoprotein, Coronavirus KW - Vaccination KW - Virus Replication AB -

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.

VL - 599 IS - 7883 ER - TY - JOUR T1 - Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors. JF - PLoS Pathog Y1 - 2021 A1 - Prabhakara, Chaitra A1 - Godbole, Rashmi A1 - Sil, Parijat A1 - Jahnavi, Sowmya A1 - Gulzar, Shah-E-Jahan A1 - van Zanten, Thomas S A1 - Sheth, Dhruv A1 - Subhash, Neeraja A1 - Chandra, Anchal A1 - Shivaraj, Akshatha A1 - Panikulam, Patricia A1 - U, Ibrahim A1 - Nuthakki, Vijay Kumar A1 - Puthiyapurayil, Theja Parassini A1 - Ahmed, Riyaz A1 - Najar, Ashaq Hussain A1 - Lingamallu, Sai Manoz A1 - Das, Snigdhadev A1 - Mahajan, Bhagyashri A1 - Vemula, Praveen A1 - Bharate, Sandip B A1 - Singh, Parvinder Pal A1 - Vishwakarma, Ram A1 - Guha, Arjun A1 - Sundaramurthy, Varadharajan A1 - Mayor, Satyajit KW - Ammonium Chloride KW - Angiotensin-Converting Enzyme 2 KW - Animals KW - Antiviral Agents KW - Cell Line KW - Chlorocebus aethiops KW - Chloroquine KW - Clathrin KW - COVID-19 KW - Drug Synergism KW - Endocytosis KW - Endosomes KW - Humans KW - Hydrogen-Ion Concentration KW - Hydroxychloroquine KW - Macrolides KW - Niclosamide KW - Protein Binding KW - Protein Domains KW - SARS-CoV-2 KW - Spike Glycoprotein, Coronavirus KW - Vero Cells KW - Virus Internalization AB -

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.

VL - 17 IS - 7 ER - TY - JOUR T1 - Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library. JF - Cell Chem Biol Y1 - 2021 A1 - Emery, Amy A1 - Hardwick, Bryn S A1 - Crooks, Alex T A1 - Milech, Nadia A1 - Watt, Paul M A1 - Mithra, Chandan A1 - Kumar, Vikrant A1 - Giridharan, Saranya A1 - Sadasivam, Gayathri A1 - Mathivanan, Subashini A1 - Sudhakar, Sneha A1 - Bairy, Sneha A1 - Bharatham, Kavitha A1 - Hurakadli, Manjunath A A1 - Prasad, Thazhe K A1 - Kamariah, Neelagandan A1 - Muellner, Markus A1 - Coelho, Miguel A1 - Torrance, Christopher J A1 - McKenzie, Grahame J A1 - Venkitaraman, Ashok R AB -

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

ER - TY - JOUR T1 - Adverse childhood experiences in families with multiple members diagnosed to have psychiatric illnesses. JF - Aust N Z J Psychiatry Y1 - 2020 A1 - Someshwar, Amala A1 - Holla, Bharath A1 - Pansari Agarwal, Preeti A1 - Thomas, Anza A1 - Jose, Anand A1 - Joseph, Boban A1 - Raju, Birudu A1 - Karle, Hariprasad A1 - Muthukumaran, M A1 - Kodancha, Prabhath G A1 - Kumar, Pramod A1 - Reddy, Preethi V A1 - Kumar Nadella, Ravi A1 - Naik, Sanjay T A1 - Mitra, Sayantanava A1 - Mallappagiri, Sreenivasulu A1 - Sreeraj, Vanteemar S A1 - Balachander, Srinivas A1 - Ganesh, Suhas A1 - Murthy, Pratima A1 - Benegal, Vivek A1 - Reddy, Janardhan Yc A1 - Jain, Sanjeev A1 - Mahadevan, Jayant A1 - Viswanath, Biju AB -

OBJECTIVE: Adverse childhood experiences are linked to the development of a number of psychiatric illnesses in adulthood. Our study examined the pattern of adverse childhood experiences and their relation to the age of onset of major psychiatric conditions in individuals from families that had ⩾2 first-degree relatives with major psychiatric conditions (multiplex families), identified as part of an ongoing longitudinal study.

METHODS: Our sample consisted of 509 individuals from 215 families. Of these, 268 were affected, i.e., diagnosed with bipolar disorder ( = 61), obsessive-compulsive disorder ( = 58), schizophrenia ( = 52), substance dependence ( = 59) or co-occurring diagnoses ( = 38), while 241 were at-risk first-degree relatives who were either unaffected ( = 210) or had other depressive or anxiety disorders ( = 31). All individuals were evaluated using the Adverse Childhood Experiences - International Questionnaire and total adverse childhood experiences exposure and severity scores were calculated.

RESULTS: It was seen that affected males, as a group, had the greatest adverse childhood experiences exposure and severity scores in our sample. A Cox mixed effects model fit by gender revealed that a higher total adverse childhood experiences severity score was associated with significantly increased risk for an earlier age of onset of psychiatric diagnoses in males. A similar model that evaluated the interaction of diagnosis revealed an earlier age of onset in obsessive-compulsive disorder and substance dependence, but not in schizophrenia and bipolar disorder.

CONCLUSION: Our study indicates that adverse childhood experiences were associated with an earlier onset of major psychiatric conditions in men and individuals diagnosed with obsessive-compulsive disorder and substance dependence. Ongoing longitudinal assessments in first-degree relatives from these families are expected to identify mechanisms underlying this relationship.

VL - 54 IS - 11 ER - TY - JOUR T1 - Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3 variant. JF - Biophys Rev Y1 - 2020 A1 - Arif, Mohammed A1 - Nabavizadeh, Pooneh A1 - Song, Taejeong A1 - Desai, Darshini A1 - Singh, Rohit A1 - Bazrafshan, Sholeh A1 - Kumar, Mohit A1 - Wang, Yigang A1 - Gilbert, Richard J A1 - Dhandapany, Perundurai S A1 - Becker, Richard C A1 - Kranias, Evangelia G A1 - Sadayappan, Sakthivel AB -

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.

VL - 12 IS - 4 ER - TY - JOUR T1 - Improved detection of RNA foci in amyotrophic lateral sclerosis post-mortem tissue using BaseScope™ shows a lack of association with cognitive dysfunction. JF - Brain Commun Y1 - 2020 A1 - Mehta, Arpan R A1 - Selvaraj, Bhuvaneish T A1 - Barton, Samantha K A1 - McDade, Karina A1 - Abrahams, Sharon A1 - Chandran, Siddharthan A1 - Smith, Colin A1 - Gregory, Jenna M AB -

The hexanucleotide repeat expansion is the commonest known genetic mutation in amyotrophic lateral sclerosis. A neuropathological hallmark is the intracellular accumulation of RNA foci. The role that RNA foci play in the pathogenesis of amyotrophic lateral sclerosis is widely debated. Historically, RNA foci have been identified using hybridization. Here, we have implemented BaseScope™, a high-resolution modified hybridization technique. We demonstrate that previous studies have underestimated the abundance of RNA foci in neurons and glia. This improved detection allowed us to investigate the abundance, regional distribution and cell type specificity of antisense RNA foci in post-mortem brain and spinal cord tissue of six deeply clinically phenotyped patients and six age- and sex-matched controls. We find a correlation between RNA foci and the accumulation of transactive response DNA-binding protein of 43 kDa in spinal motor neurons ( = 0.93; = 0.008), but not in glia or cortical motor neurons. We also demonstrate that there is no correlation between the presence of RNA foci and the accumulation of transactive response DNA binding protein of 43 kDa in extra-motor brain regions. Furthermore, there is no association between the presence of RNA foci and cognitive indices. These results highlight the utility of BaseScope™ in the clinicopathological assessment of the role of antisense RNA foci in .

VL - 2 IS - 1 ER - TY - JOUR T1 - Metabolic control of cellular immune-competency by odors in . JF - Elife Y1 - 2020 A1 - Madhwal, Sukanya A1 - Shin, Mingyu A1 - Kapoor, Ankita A1 - Goyal, Manisha A1 - Joshi, Manish K A1 - Ur Rehman, Pirzada Mujeeb A1 - Gor, Kavan A1 - Shim, Jiwon A1 - Mukherjee, Tina AB -

Studies in different animal model systems have revealed the impact of odors on immune cells; however, any understanding on why and how odors control cellular immunity remained unclear. We find that employ an olfactory-immune cross-talk to tune a specific cell type, the lamellocytes, from hematopoietic-progenitor cells. We show that neuronally released GABA derived upon olfactory stimulation is utilized by blood-progenitor cells as a metabolite and through its catabolism, these cells stabilize Sima/HIFα protein. Sima capacitates blood-progenitor cells with the ability to initiate lamellocyte differentiation. This systemic axis becomes relevant for larvae dwelling in wasp-infested environments where chances of infection are high. By co-opting the olfactory route, the preconditioned animals elevate their systemic GABA levels leading to the upregulation of blood-progenitor cell Sima expression. This elevates their immune-potential and primes them to respond rapidly when infected with parasitic wasps. The present work highlights the importance of the olfaction in immunity and shows how odor detection during animal development is utilized to establish a long-range axis in the control of blood-progenitor competency and immune-priming.

VL - 9 ER - TY - JOUR T1 - Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program. JF - J Biol Chem Y1 - 2020 A1 - Walvekar, Adhish S A1 - Kadamur, Ganesh A1 - Sreedharan, Sreesa A1 - Gupta, Ritu A1 - Srinivasan, Rajalakshmi A1 - Laxman, Sunil AB -

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (where the roles of Gcn4 are not well studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase PP2A; our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 towards a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs a conserved methyltransferase Ppm1, via its target phosphatase PP2A, to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master-regulator and drive anabolism.

ER - TY - JOUR T1 - Methylated PP2A stabilizes Gcn4 to enable a methionine-induced anabolic program. JF - J Biol Chem Y1 - 2020 A1 - Walvekar, Adhish S A1 - Kadamur, Ganesh A1 - Sreedharan, Sreesa A1 - Gupta, Ritu A1 - Srinivasan, Rajalakshmi A1 - Laxman, Sunil AB -

Methionine, through S-adenosylmethionine, activates a multifaceted growth program in which ribosome biogenesis, carbon metabolism, and amino acid and nucleotide biosynthesis are induced. This growth program requires the activity of the Gcn4 transcription factor (called ATF4 in mammals), which facilitates the supply of metabolic precursors that are essential for anabolism. However, how Gcn4 itself is regulated in the presence of methionine is unknown. Here, we discover that Gcn4 protein levels are increased by methionine, despite conditions of high cell growth and translation (in which the roles of Gcn4 are not well-studied). We demonstrate that this mechanism of Gcn4 induction is independent of transcription, as well as the conventional Gcn2/eIF2α-mediated increased translation of Gcn4. Instead, when methionine is abundant, Gcn4 phosphorylation is decreased, which reduces its ubiquitination and therefore degradation. Gcn4 is dephosphorylated by the protein phosphatase 2A (PP2A); our data show that when methionine is abundant, the conserved methyltransferase Ppm1 methylates and alters the activity of the catalytic subunit of PP2A, shifting the balance of Gcn4 toward a dephosphorylated, stable state. The absence of Ppm1 or the loss of the PP2A methylation destabilizes Gcn4 even when methionine is abundant, leading to collapse of the Gcn4-dependent anabolic program. These findings reveal a novel, methionine-dependent signaling and regulatory axis. Here methionine directs the conserved methyltransferase Ppm1 via its target phosphatase PP2A to selectively stabilize Gcn4. Through this, cells conditionally modify a major phosphatase to stabilize a metabolic master regulator and drive anabolism.

VL - 295 IS - 52 ER - TY - JOUR T1 - Multiple Wnts act synergistically to induce Chk1/Grapes expression and mediate G2 arrest in tracheoblasts. JF - Elife Y1 - 2020 A1 - Kizhedathu, Amrutha A1 - Kunnappallil, Rose Sebastian A1 - Bagul, Archit V A1 - Verma, Puja A1 - Guha, Arjun AB -

Larval tracheae of harbour progenitors of the adult tracheal system (tracheoblasts). Thoracic tracheoblasts are arrested in the G2 phase of the cell cycle in an ATR (mei-41)-Checkpoint Kinase1 (grapes, Chk1) dependent manner prior to mitotic re-entry. Here we investigate developmental regulation of Chk1 activation. We report that Wnt signaling is high in tracheoblasts and this is necessary for high levels of activated (phosphorylated) Chk1. We find that canonical Wnt signaling facilitates this by transcriptional upregulation of Chk1 expression in cells that have ATR kinase activity. Wnt signaling is dependent on four Wnts (Wg, Wnt5, 6,10) that are expressed at high levels in arrested tracheoblasts and are downregulated at mitotic re-entry. Interestingly, none of the Wnts are dispensable and act synergistically to induce Chk1. Finally, we show that downregulation of Wnt signaling and Chk1 expression leads to mitotic re-entry and the concomitant upregulation of Dpp signaling, driving tracheoblast proliferation.

VL - 9 ER - TY - JOUR T1 - The primary cilium dampens proliferative signaling and represses a G2/M transcriptional network in quiescent myoblasts. JF - BMC Mol Cell Biol Y1 - 2020 A1 - Venugopal, Nisha A1 - Ghosh, Ananga A1 - Gala, Hardik A1 - Aloysius, Ajoy A1 - Vyas, Neha A1 - Dhawan, Jyotsna AB -

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.

VL - 21 IS - 1 ER - TY - JOUR T1 - Rethinking Exosomes: From Cell-to-Cell Courier Services to Individualized Medicines JF - AAPS News magazine Y1 - 2020 A1 - Ghate, V A1 - Chaudhari, P A1 - Maxwell, A A1 - Lewis, S A1 - Pahal, S A1 - Vemula PK IS - June ER - TY - JOUR T1 - Temporal specificity and heterogeneity of Drosophila immune cells. JF - EMBO J Y1 - 2020 A1 - Cattenoz, Pierre B A1 - Sakr, Rosy A1 - Pavlidaki, Alexia A1 - Delaporte, Claude A1 - Riba, Andrea A1 - Molina, Nacho A1 - Hariharan, Nivedita A1 - Mukherjee, Tina A1 - Giangrande, Angela AB -

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.

ER - TY - JOUR T1 - Temporal specificity and heterogeneity of the fly immune cells’ transcriptional landscape JF - The EMBO Journal (in press) Y1 - 2020 A1 - Cattenoz, Pierre B. A1 - Sakr, Rosy A1 - Pavlidaki, Alexia A1 - Delaporte, Claude A1 - Riba, Andrea A1 - Molina, Nacho A1 - Hariharan, Nivedita A1 - Mukherjee, Tina A1 - Giangrande, Angela AB -

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.

ER - TY - JOUR T1 - VEGFA Promoter Polymorphisms rs699947 and rs35569394 Are Associated With the Risk of Anterior Cruciate Ligament Ruptures Among Indian Athletes: A Cross-sectional Study. JF - Orthop J Sports Med Y1 - 2020 A1 - Shukla, Manish A1 - Gupta, Rahul A1 - Pandey, Vivek A1 - Rochette, Jacques A1 - Dhandapany, Perundurai S A1 - Tiwari, Pramod Kumar A1 - Amrathlal, Rabbind Singh AB -

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' = 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.

VL - 8 IS - 12 ER - TY - JOUR T1 - Chemical fuel-driven living and transient supramolecular polymerization. JF - Nat Commun Y1 - 2019 A1 - Jain, Ankit A1 - Dhiman, Shikha A1 - Dhayani, Ashish A1 - Vemula, Praveen K A1 - George, Subi J AB -

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.

VL - 10 IS - 1 ER - TY - JOUR T1 - Dynamic expression of tRNA-derived small RNAs define cellular states. JF - EMBO Rep Y1 - 2019 A1 - Krishna, Srikar A1 - Yim, Daniel Gr A1 - Lakshmanan, Vairavan A1 - Tirumalai, Varsha A1 - Koh, Judice Ly A1 - Park, Jung Eun A1 - Cheong, Jit Kong A1 - Low, Joo Leng A1 - Lim, Michelle Js A1 - Sze, Siu Kwan A1 - Shivaprasad, Padubidri A1 - Gulyani, Akash A1 - Raghavan, Srikala A1 - Palakodeti, Dasaradhi A1 - DasGupta, Ramanuj AB -

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'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-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'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.

VL - 20 IS - 7 ER - TY - JOUR T1 - Efficient allelic-drive in Drosophila. JF - Nat Commun Y1 - 2019 A1 - Guichard, Annabel A1 - Haque, Tisha A1 - Bobik, Marketta A1 - Xu, Xiang-Ru S A1 - Klanseck, Carissa A1 - Kushwah, Raja Babu Singh A1 - Berni, Mateus A1 - Kaduskar, Bhagyashree A1 - Gantz, Valentino M A1 - Bier, Ethan KW - Agriculture KW - Alleles KW - Animals KW - Animals, Genetically Modified KW - CRISPR-Cas Systems KW - DNA End-Joining Repair KW - DNA Mutational Analysis KW - Drosophila KW - Female KW - Gene Drive Technology KW - Gene Editing KW - Inheritance Patterns KW - Male KW - Mosaicism KW - RNA, Guide AB -

Gene-drive systems developed in several organisms result in super-Mendelian inheritance of transgenic insertions. Here, we generalize this "active genetic" approach to preferentially transmit allelic variants (allelic-drive) resulting from only a single or a few nucleotide alterations. We test two configurations for allelic-drive: one, copy-cutting, in which a non-preferred allele is selectively targeted for Cas9/guide RNA (gRNA) cleavage, and a more general approach, copy-grafting, that permits selective inheritance of a desired allele located in close proximity to the gRNA cut site. We also characterize a phenomenon we refer to as lethal-mosaicism that dominantly eliminates NHEJ-induced mutations and favors inheritance of functional cleavage-resistant alleles. These two efficient allelic-drive methods, enhanced by lethal mosaicism and a trans-generational drive process we refer to as "shadow-drive", have broad practical applications in improving health and agriculture and greatly extend the active genetics toolbox.

VL - 10 IS - 1 ER - TY - JOUR T1 - Exome sequencing in families with severe mental illness identifies novel and rare variants in genes implicated in Mendelian neuropsychiatric syndromes. JF - Psychiatry Clin Neurosci Y1 - 2019 A1 - Ganesh, Suhas A1 - Ahmed P, Husayn A1 - Nadella, Ravi K A1 - More, Ravi P A1 - Seshadri, Manasa A1 - Viswanath, Biju A1 - Rao, Mahendra A1 - Jain, Sanjeev A1 - Mukherjee, Odity KW - Bipolar Disorder KW - Exome KW - Female KW - Genetic Predisposition to Disease KW - Genetic Variation KW - Genome-Wide Association Study KW - Humans KW - Male KW - Pedigree KW - Phenotype KW - Schizophrenia AB -

AIM: Severe mental illnesses (SMI), such as bipolar disorder and schizophrenia, are highly heritable, and have a complex pattern of inheritance. Genome-wide association studies detect a part of the heritability, which can be attributed to common genetic variation. Examination of rare variants with next-generation sequencing may add to the understanding of the genetic architecture of SMI.

METHODS: We analyzed 32 ill subjects from eight multiplex families and 33 healthy individuals using whole-exome sequencing. Prioritized variants were selected by a three-step filtering process, which included: deleteriousness by five in silico algorithms; sharing within families by affected individuals; rarity in South Asian sample estimated using the Exome Aggregation Consortium data; and complete absence of these variants in control individuals from the same gene pool.

RESULTS: We identified 42 rare, non-synonymous deleterious variants (~5 per pedigree) in this study. None of the variants were shared across families, indicating a 'private' mutational profile. Twenty (47.6%) of the variant harboring genes were previously reported to contribute to the risk of diverse neuropsychiatric syndromes, nine (21.4%) of which were of Mendelian inheritance. These included genes carrying novel deleterious variants, such as the GRM1 gene implicated in spinocerebellar ataxia 44 and the NIPBL gene implicated in Cornelia de Lange syndrome.

CONCLUSION: Next-generation sequencing approaches in family-based studies are useful to identify novel and rare variants in genes for complex disorders like SMI. The findings of the study suggest a potential phenotypic burden of rare variants in Mendelian disease genes, indicating pleiotropic effects in the etiology of SMI.

VL - 73 IS - 1 ER - TY - JOUR T1 - Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing. JF - Stem Cell Res Y1 - 2019 A1 - Giri, Subhajit A1 - Purushottam, Meera A1 - Viswanath, Biju A1 - Muddashetty, Ravi S AB -

Mutations in FMR1 gene is the cause of Fragile X Syndrome (FXS) leading inherited cause of intellectual disability and autism spectrum disorders. FMR1 gene encodes Fragile X Mental Retardation Protein (FMRP) which is a RNA binding protein and play important role in synaptic plasticity and translational regulation in neurons. We have generated a homozygous FMR1 knockout (FMR1-KO) hESC line using CRISPR/Cas9 based genome editing. It created a homozygous 280 nucleotide deletion at exon1, removing the start codon. This FMR1-KO cell line maintains stem cell like morphology, pluripotency, normal karyotype and ability to in-vitro differentiation.

VL - 39 ER - TY - JOUR T1 - Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line. JF - Stem Cell Res Y1 - 2019 A1 - Schmid, Benjamin A1 - Prehn, Kennie R A1 - Nimsanor, Natakarn A1 - Garcia, Blanca Irene Aldana A1 - Poulsen, Ulla A1 - Jørring, Ida A1 - Rasmussen, Mikkel A A1 - Clausen, Christian A1 - Mau-Holzmann, Ulrike A A1 - Ramakrishna, Sarayu A1 - Muddashetty, Ravi A1 - Steeg, Rachel A1 - Bruce, Kevin A1 - Mackintosh, Peter A1 - Ebneth, Andreas A1 - Holst, Bjørn A1 - Cabrera-Socorro, Alfredo AB -

Alzheimer's disease (AD) is the most frequent neurodegenerative disease amongst the elderly. The SNPs rs429358 and rs7412 in the APOE gene are the most common risk factor for sporadic AD, and there are three different alleles commonly referred to as APOE-ε2, APOE-ε3 and APOE-ε4. Induced pluripotent stem cells (iPSCs) hold great promise to model AD as such cells can be differentiated in vitro to the required cell type. Here we report the use of CRISPR/Cas9 technology employed on iPSCs from a healthy individual with an APOE-ε3/ε4 genotype to obtain isogenic APOE-ε2/ε2, APOE-ε3/ε3, APOE-ε4/ε4 lines as well as an APOE-knock-out line.

VL - 34 ER - TY - JOUR T1 - Graft-implanted, enzyme responsive, tacrolimus-eluting hydrogel enables long-term survival of orthotopic porcine limb vascularized composite allografts: A proof of concept study. JF - PLoS One Y1 - 2019 A1 - Fries, C Anton A1 - Lawson, Shari D A1 - Wang, Lin C A1 - Slaughter, Kai V A1 - Vemula, Praveen K A1 - Dhayani, Ashish A1 - Joshi, Nitin A1 - Karp, Jeffrey M A1 - Rickard, Rory F A1 - Gorantla, Vijay S A1 - Davis, Michael R AB -

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.

VL - 14 IS - 1 ER - TY - JOUR T1 - Highly Responsive Fluorescent Assemblies Allow for Unique, Multiparametric Sensing of the Phospholipid Membrane Environment. JF - Chemistry Y1 - 2019 A1 - Gulyani, Akash A1 - Dey, Nilanjan A1 - Bhattacharya, Santanu KW - Fluorescence Polarization KW - Fluorescent Dyes KW - Lipid Bilayers KW - Phospholipids KW - Pyrenes KW - Spectrometry, Fluorescence KW - Water AB -

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.

VL - 25 IS - 6 ER - TY - JOUR T1 - Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells. JF - Methods Mol Biol Y1 - 2019 A1 - Badarinath, Krithika A1 - Dutta, Abhik A1 - Hegde, Akshay A1 - Pincha, Neha A1 - Gund, Rupali A1 - Jamora, Colin AB -

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.

VL - 1879 ER - TY - JOUR T1 - KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation. JF - EMBO Rep Y1 - 2019 A1 - Rao, Radhika Arasala A1 - Ketkar, Alhad Ashok A1 - Kedia, Neelam A1 - Krishnamoorthy, Vignesh K A1 - Lakshmanan, Vairavan A1 - Kumar, Pankaj A1 - Mohanty, Abhishek A1 - Kumar, Shilpa Dilip A1 - Raja, Sufi O A1 - Gulyani, Akash A1 - Chaturvedi, Chandra Prakash A1 - Brand, Marjorie A1 - Palakodeti, Dasaradhi A1 - Rampalli, Shravanti AB -

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

ER - TY - JOUR T1 - Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway. JF - Nat Commun Y1 - 2019 A1 - Sathyanarayanan, Nitish A1 - Cannone, Giuseppe A1 - Gakhar, Lokesh A1 - Katagihallimath, Nainesh A1 - Sowdhamini, Ramanathan A1 - Ramaswamy, Subramanian A1 - Vinothkumar, Kutti R AB -

Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

VL - 10 IS - 1 ER - TY - JOUR T1 - Novel Series of Methyl 3-(Substituted Benzoyl)-7-Substituted-2-Phenylindolizine-1-Carboxylates as Promising Anti-Inflammatory Agents: Molecular Modeling Studies. JF - Biomolecules Y1 - 2019 A1 - Venugopala, Katharigatta N A1 - Al-Attraqchi, Omar H A A1 - Tratrat, Christophe A1 - Nayak, Susanta K A1 - Morsy, Mohamed A A1 - Aldhubiab, Bandar E A1 - Attimarad, Mahesh A1 - Nair, Anroop B A1 - Sreeharsha, Nagaraja A1 - Venugopala, Rashmi A1 - Haroun, Michelyne A1 - Girish, Meravanige B A1 - Chandrashekharappa, Sandeep A1 - Alwassil, Osama I A1 - Odhav, Bharti AB -

The cyclooxygenase-2 (COX-2) enzyme is considered to be an important target for developing novel anti-inflammatory agents. Selective COX-2 inhibitors offer the advantage of lower adverse effects that are commonly associated with non-selective COX inhibitors. In this work, a novel series of methyl 3-(substituted benzoyl)-7-substituted-2-phenylindolizine-1-carboxylates was synthesized and evaluated for COX-2 inhibitory activity. Compound was identified as the most active compound of the series with an IC of 6.71 M, which is comparable to the IC of indomethacin, a marketed non-steroidal anti-inflammatory drug (NSAID). Molecular modeling and crystallographic studies were conducted to further characterize the compounds and gain better understanding of the binding interactions between the compounds and the residues at the active site of the COX-2 enzyme. The pharmacokinetic properties and potential toxic effects were predicted for all the synthesized compounds, which indicated good drug-like properties. Thus, these synthesized compounds can be considered as potential lead compounds for developing effective anti-inflammatory therapeutic agents.

VL - 9 IS - 11 ER - TY - JOUR T1 - Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia. JF - Cell Death Dis Y1 - 2019 A1 - Ganesan, Saravanan A1 - Palani, Hamenth Kumar A1 - Lakshmanan, Vairavan A1 - Balasundaram, Nithya A1 - Alex, Ansu Abu A1 - David, Sachin A1 - Venkatraman, Arvind A1 - Korula, Anu A1 - George, Biju A1 - Balasubramanian, Poonkuzhali A1 - Palakodeti, Dasaradhi A1 - Vyas, Neha A1 - Mathews, Vikram AB -

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.

VL - 10 IS - 10 ER - TY - JOUR T1 - Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain. JF - ChemMedChem Y1 - 2019 A1 - Kurdekar, Vadiraj A1 - Giridharan, Saranya A1 - Subbarao, Jasti A1 - Nijaguna, Mamatha B A1 - Periasamy, Jayaprakash A1 - Boggaram, Sanjana A1 - Shivange, Amol V A1 - Sadasivam, Gayathri A1 - Padigaru, Muralidhara A1 - Potluri, Vijay A1 - Venkitaraman, Ashok R A1 - Bharatham, Kavitha AB -

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.

VL - 14 IS - 18 ER - TY - JOUR T1 - A tRNA modification balances carbon and nitrogen metabolism by regulating phosphate homeostasis. JF - Elife Y1 - 2019 A1 - Gupta, Ritu A1 - Walvekar, Adhish A1 - Liang, Shun A1 - Rashida, Zeenat A1 - Shah, Premal A1 - Laxman, Sunil AB -

Cells must appropriately sense and integrate multiple metabolic resources to commit to proliferation. Here, we report that cells regulate carbon and nitrogen metabolic homeostasis through tRNA U-thiolation. Despite amino acid sufficiency, tRNA-thiolation deficient cells appear amino acid starved. In these cells, carbon flux towards nucleotide synthesis decreases, and trehalose synthesis increases, resulting in a starvation-like metabolic signature. Thiolation mutants have only minor translation defects. However, in these cells phosphate homeostasis genes are strongly down-regulated, resulting in an effectively phosphate-limited state. Reduced phosphate enforces a metabolic switch, where glucose-6-phosphate is routed towards storage carbohydrates. Notably, trehalose synthesis, which releases phosphate and thereby restores phosphate availability, is central to this metabolic rewiring. Thus, cells use thiolated tRNAs to perceive amino acid sufficiency, balance carbon and amino acid metabolic flux and grow optimally, by controlling phosphate availability. These results further biochemically explain how phosphate availability determines a switch to a 'starvation-state'.

VL - 8 ER - TY - JOUR T1 - tRNA wobble-uridine modifications as amino acid sensors and regulators of cellular metabolic state. JF - Curr Genet Y1 - 2019 A1 - Gupta, Ritu A1 - Laxman, Sunil AB -

Cells must appropriately sense available nutrients and accordingly regulate their metabolic outputs, to survive. This mini-review considers the idea that conserved chemical modifications of wobble (U34) position tRNA uridines enable cells to sense nutrients and regulate their metabolic state. tRNA wobble uridines are chemically modified at the 2- and 5- positions, with a thiol (s2), and (commonly) a methoxycarbonylmethyl (mcm5) modification, respectively. These modifications reflect sulfur amino acid (methionine and cysteine) availability. The loss of these modifications has minor translation defects. However, they result in striking phenotypes consistent with an altered metabolic state. Using yeast, we recently discovered that the s2 modification regulates overall carbon and nitrogen metabolism, dependent on methionine availability. The loss of this modification results in rewired carbon (glucose) metabolism. Cells have reduced carbon flux towards the pentose phosphate pathway and instead increased flux towards storage carbohydrates-primarily trehalose, along with reduced nucleotide synthesis, and perceived amino acid starvation signatures. Remarkably, this metabolic rewiring in the s2U mutants is caused by mechanisms leading to intracellular phosphate limitation. Thus this U34 tRNA modification responds to methionine availability and integratively regulates carbon and nitrogen homeostasis, wiring cells to a 'growth' state. We interpret the importance of U34 modifications in the context of metabolic sensing and anabolism, emphasizing their intimate coupling to methionine metabolism.

ER - TY - JOUR T1 - Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer. JF - Mol Cancer Ther Y1 - 2019 A1 - Totiger, Tulasigeri M A1 - Srinivasan, Supriya A1 - Jala, Venkatakrishna R A1 - Lamichhane, Purushottam A1 - Dosch, Austin R A1 - Gaidarski, Alexander A A1 - Joshi, Chandrashekhar A1 - Rangappa, Shobith A1 - Castellanos, Jason A1 - Vemula, Praveen Kumar A1 - Chen, Xi A1 - Kwon, Deukwoo A1 - Kashikar, Nilesh A1 - VanSaun, Michael A1 - Merchant, Nipun B A1 - Nagathihalli, Nagaraj S AB -

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.

VL - 18 IS - 2 ER - TY - JOUR T1 - Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. JF - Microb Biotechnol Y1 - 2018 A1 - Bairy, Sneha A1 - Gopalan, Lakshmi Narayanan A1 - Setty, Thanuja Gangi A1 - Srinivasachari, Sathya A1 - Manjunath, Lavanyaa A1 - Kumar, Jay Prakash A1 - Guntupalli, Sai R A1 - Bose, Sucharita A1 - Nayak, Vinod A1 - Ghosh, Swagatha A1 - Sathyanarayanan, Nitish A1 - Caing-Carlsson, Rhawnie A1 - Wahlgren, Weixiao Yuan A1 - Friemann, Rosmarie A1 - Ramaswamy, S A1 - Neerathilingam, Muniasamy AB -

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

VL - 11 IS - 2 ER - TY - JOUR T1 - Chemically diverse small molecule fluorescent chemosensors for copper ion JF - Coordination Chemistry Reviews Y1 - 2018 A1 - Gandhi Sivaraman A1 - Murugan Iniya A1 - Thangaraj Anand A1 - Niranjan G. Kotla A1 - Omprakash Sunnapu A1 - Subramanian Singaravadivel A1 - Akash Gulyani A1 - Duraisamy Chellappa KW - Chemosensors KW - Fluorescence KW - Live cell imaging KW - Small molecules KW - Turn-off KW - Turn-on VL - 357 UR - http://www.sciencedirect.com/science/article/pii/S0010854517306537 ER - TY - JOUR T1 - Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas. JF - Tumour Biol Y1 - 2018 A1 - Mishra, Amrendra A1 - Sriram, Harshini A1 - Chandarana, Pinal A1 - Tanavde, Vivek A1 - Kumar, Rekha V A1 - Gopinath, Ashok A1 - Govindarajan, Raman A1 - Ramaswamy, S A1 - Sadasivam, Subhashini AB -

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44Lin and CD44Lin subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44Lin compared to CD44Lin cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

VL - 40 IS - 5 ER - TY - JOUR T1 - Discovery biology of neuropsychiatric syndromes (DBNS): a center for integrating clinical medicine and basic science. JF - BMC Psychiatry Y1 - 2018 A1 - Viswanath, Biju A1 - Rao, Naren P A1 - Narayanaswamy, Janardhanan C A1 - Sivakumar, Palanimuthu T A1 - Kandasamy, Arun A1 - Kesavan, Muralidharan A1 - Mehta, Urvakhsh Meherwan A1 - Venkatasubramanian, Ganesan A1 - John, John P A1 - Mukherjee, Odity A1 - Purushottam, Meera A1 - Kannan, Ramakrishnan A1 - Mehta, Bhupesh A1 - Kandavel, Thennarasu A1 - Binukumar, B A1 - Saini, Jitender A1 - Jayarajan, Deepak A1 - Shyamsundar, A A1 - Moirangthem, Sydney A1 - Vijay Kumar, K G A1 - Thirthalli, Jagadisha A1 - Chandra, Prabha S A1 - Gangadhar, Bangalore N A1 - Murthy, Pratima A1 - Panicker, Mitradas M A1 - Bhalla, Upinder S A1 - Chattarji, Sumantra A1 - Benegal, Vivek A1 - Varghese, Mathew A1 - Reddy, Janardhan Y C A1 - Raghu, Padinjat A1 - Rao, Mahendra A1 - Jain, Sanjeev AB -

BACKGROUND: There is emerging evidence that there are shared genetic, environmental and developmental risk factors in psychiatry, that cut across traditional diagnostic boundaries. With this background, the Discovery biology of neuropsychiatric syndromes (DBNS) proposes to recruit patients from five different syndromes (schizophrenia, bipolar disorder, obsessive compulsive disorder, Alzheimer's dementia and substance use disorders), identify those with multiple affected relatives, and invite these families to participate in this study. The families will be assessed: 1) To compare neuro-endophenotype measures between patients, first degree relatives (FDR) and healthy controls., 2) To identify cellular phenotypes which differentiate the groups., 3) To examine the longitudinal course of neuro-endophenotype measures., 4) To identify measures which correlate with outcome, and 5) To create a unified digital database and biorepository.

METHODS: The identification of the index participants will occur at well-established specialty clinics. The selected individuals will have a strong family history (with at least another affected FDR) of mental illness. We will also recruit healthy controls without family history of such illness. All recruited individuals (N = 4500) will undergo brief clinical assessments and a blood sample will be drawn for isolation of DNA and peripheral blood mononuclear cells (PBMCs). From among this set, a subset of 1500 individuals (300 families and 300 controls) will be assessed on several additional assessments [detailed clinical assessments, endophenotype measures (neuroimaging- structural and functional, neuropsychology, psychophysics-electroencephalography, functional near infrared spectroscopy, eye movement tracking)], with the intention of conducting repeated measurements every alternate year. PBMCs from this set will be used to generate lymphoblastoid cell lines, and a subset of these would be converted to induced pluripotent stem cell lines and also undergo whole exome sequencing.

DISCUSSION: We hope to identify unique and overlapping brain endophenotypes for major psychiatric syndromes. In a proportion of subjects, we expect these neuro-endophenotypes to progress over time and to predict treatment outcome. Similarly, cellular assays could differentiate cell lines derived from such groups. The repository of biomaterials as well as digital datasets of clinical parameters, will serve as a valuable resource for the broader scientific community who wish to address research questions in the area.

VL - 18 IS - 1 ER - TY - JOUR T1 - Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis. JF - Methods Mol Biol Y1 - 2018 A1 - Srivastava, Surabhi A1 - Gala, Hardik P A1 - Mishra, Rakesh K A1 - Dhawan, Jyotsna AB -

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.

VL - 1686 ER - TY - JOUR T1 - FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation. JF - iScience Y1 - 2018 A1 - D'Souza, Michelle Ninochka A1 - Gowda, Naveen Kumar Chandappa A1 - Tiwari, Vishal A1 - Babu, Rosana Ottakandathil A1 - Anand, Praveen A1 - Dastidar, Sudhriti Ghosh A1 - Singh, Randhir A1 - James, Owen G A1 - Selvaraj, Bhuvaneish A1 - Pal, Rakhi A1 - Ramesh, Arati A1 - Chattarji, Sumantra A1 - Chandran, Siddharthan A1 - Gulyani, Akash A1 - Palakodeti, Dasaradhi A1 - Muddashetty, Ravi S AB -

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'O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2'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.

VL - 9 ER - TY - JOUR T1 - Hippocampal and amygdalar cell-specific translation is similar soon after stress but diverge over time. JF - Hippocampus Y1 - 2018 A1 - Madan, Jesvin S A1 - Gupta, Kanika A1 - Chattarji, Sumantra A1 - Bhattacharya, Aditi AB -

Stress is known to cause contrasting patterns of morphological and physiological plasticity in the hippocampus and amygdala. An obligatory cellular process underlying such neural changes is de novo translation and alterations in protein expression. Yet the nature of the translational response to stress in neurons remains largely unexplored. Even less is known about how glia are affected. Using a click-chemistry-based method to label the de novo proteome in live brain slices, we monitored translation in neurons and astrocytes of the basolateral amygdala (BLA) and dorsal hippocampal area CA3 (dCA3) in rats at different time-points after a single 2-hr exposure to immobilization stress. We observed enhancements in neuronal translation in both brain regions 1 hour after stress. This initial increase persisted in the BLA up to 10 days afterwards. In contrast, dCA3 neuronal translation gradually decreased to below control levels 10 days later. Translation profiles of dCA3 astrocytes followed timelines similar to neurons, but in BLA astrocytes translation peaked 1 day later and remained elevated 10 days later. Together our results demonstrate that stress causes an immediate upregulation of protein synthesis in both amygdalar and hippocampal neurons and astrocytes. However, these two areas eventually exhibit opposite temporal profiles of protein expression well after the end of stress. These findings identify new metrics of stress-induced plasticity at the level of cell-type specific proteomic landscape that may provide important insights into the molecular basis of the divergent temporal effects of stress across brain regions and biological scales.

VL - 28 IS - 6 ER - TY - JOUR T1 - "Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes. JF - Biophys Rev Y1 - 2018 A1 - North, Rachel A A1 - Horne, Christopher R A1 - Davies, James S A1 - Remus, Daniela M A1 - Muscroft-Taylor, Andrew C A1 - Goyal, Parveen A1 - Wahlgren, Weixiao Yuan A1 - Ramaswamy, S A1 - Friemann, Rosmarie A1 - Dobson, Renwick C J AB -

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.

VL - 10 IS - 2 ER - TY - JOUR T1 - Methionine coordinates a hierarchically organized anabolic program enabling proliferation. JF - Mol Biol Cell Y1 - 2018 A1 - Walvekar, Adhish S A1 - Srinivasan, Rajalakshmi A1 - Gupta, Ritu A1 - Laxman, Sunil AB -

Methionine availability during overall amino acid limitation metabolically reprograms cells to support proliferation, the underlying basis for which remains unclear. Here, we construct the organization of this methionine mediated anabolic program, using yeast. Combining comparative transcriptome analysis, biochemical and metabolic flux based approaches, we discover that methionine rewires overall metabolic outputs by increasing the activity of a key regulatory node. This comprises of: the pentose phosphate pathway (PPP) coupled with reductive biosynthesis, the glutamate dehydrogenase (GDH) dependent synthesis of glutamate/glutamine, and pyridoxal-5-phosphate (PLP) dependent transamination capacity. This PPP-GDH-PLP node provides the required cofactors and/or substrates for subsequent rate-limiting reactions in the synthesis of amino acids, and therefore nucleotides. These rate-limiting steps in amino acid biosynthesis are also induced in a methionine-dependent manner. This thereby results in a biochemical cascade establishing a hierarchically organized anabolic program. For this methionine mediated anabolic program to be sustained, cells co-opt a "starvation stress response" regulator, Gcn4p. Collectively, our data suggest a hierarchical metabolic framework explaining how methionine mediates an anabolic switch.

ER - TY - JOUR T1 - Negative regulation of G2-M by ATR (mei-41)/Chk1(Grapes) facilitates tracheoblast growth and tracheal hypertrophy in Drosophila. JF - Elife Y1 - 2018 A1 - Kizhedathu, Amrutha A1 - Bagul, Archit V A1 - Guha, Arjun AB -

Imaginal progenitors in Drosophila are known to arrest in G2 during larval stages and proliferate thereafter. Here we investigate the mechanism and implications of G2 arrest in progenitors of the adult thoracic tracheal epithelium (tracheoblasts). We report that tracheoblasts pause in G2 for ~48-56 h and grow in size over this period. Surprisingly, tracheoblasts arrested in G2 express drivers of G2-M like Cdc25/String (Stg). We find that mechanisms that prevent G2-M are also in place in this interval. Tracheoblasts activate Checkpoint Kinase 1/Grapes (Chk1/Grp) in an ATR/mei-41-dependent manner. Loss of ATR/Chk1 led to precocious mitotic entry ~24-32 h earlier. These divisions were apparently normal as there was no evidence of increased DNA damage or cell death. However, induction of precocious mitoses impaired growth of tracheoblasts and the tracheae they comprise. We propose that ATR/Chk1 negatively regulate G2-M in developing tracheoblasts and that G2 arrest facilitates cellular and hypertrophic organ growth.

VL - 7 ER - TY - JOUR T1 - PAI1 mediates fibroblast-mast cell interactions in skin fibrosis. JF - J Clin Invest Y1 - 2018 A1 - Pincha, Neha A1 - Hajam, Edries Yousaf A1 - Badarinath, Krithika A1 - Batta, Surya Prakash Rao A1 - Masudi, Tafheem A1 - Dey, Rakesh A1 - Andreasen, Peter A1 - Kawakami, Toshiaki A1 - Samuel, Rekha A1 - George, Renu A1 - Danda, Debashish A1 - Jacob, Paul Mazhuvanchary A1 - Jamora, Colin AB -

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.

VL - 128 IS - 5 ER - TY - JOUR T1 - Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel. JF - J Biol Chem Y1 - 2018 A1 - Rostovtseva, Tatiana K A1 - Gurnev, Philip A A1 - Hoogerheide, David P A1 - Rovini, Amandine A1 - Sirajuddin, Minhajuddin A1 - Bezrukov, Sergey M AB -

The microtubule protein tubulin is a heterodimer comprising α/β subunits, in which each subunit features multiple isotypes in vertebrates. For example, seven α-tubulin and eight β-tubulin isotypes in the human tubulin gene family vary mostly in the length and primary sequence of the disordered anionic C-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. Here, we demonstrate that it may be a key feature of tubulin's role in regulation of the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC). Using recombinant yeast α/β-tubulin constructs with α-CTTs, β-CTTs, or both from various human tubulin isotypes, we probed their interactions with VDAC reconstituted into planar lipid bilayers. A comparative study of the blockage kinetics revealed that either α-CTTs or β-CTTs block VDAC pore and that the efficiency of blockage by individual CTTs spans two orders of magnitude, depending on the CTT isotype. β-Tubulin constructs, notably β3, blocked VDAC most effectively. We quantitatively describe these experimental results using a physical model that accounts only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. Based on these results, we speculate that the effectiveness of VDAC regulation by tubulin depends on the predominant tubulin isotype in a cell. Consequently, the fluxes of ATP/ADP through the channel could vary significantly depending on the isotype, thus suggesting an intriguing link between VDAC regulation and the diversity of tubulin isotypes present in vertebrates.

ER - TY - JOUR T1 - Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site. JF - Nat Commun Y1 - 2018 A1 - Wahlgren, Weixiao Y A1 - Dunevall, Elin A1 - North, Rachel A A1 - Paz, Aviv A1 - Scalise, Mariafrancesca A1 - Bisignano, Paola A1 - Bengtsson-Palme, Johan A1 - Goyal, Parveen A1 - Claesson, Elin A1 - Caing-Carlsson, Rhawnie A1 - Andersson, Rebecka A1 - Beis, Konstantinos A1 - Nilsson, Ulf J A1 - Farewell, Anne A1 - Pochini, Lorena A1 - Indiveri, Cesare A1 - Grabe, Michael A1 - Dobson, Renwick C J A1 - Abramson, Jeff A1 - Ramaswamy, S A1 - Friemann, Rosmarie AB -

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 Å 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.

VL - 9 IS - 1 ER - TY - JOUR T1 - Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling. JF - Cell Chem Biol Y1 - 2018 A1 - Periasamy, Jayaprakash A1 - Kurdekar, Vadiraj A1 - Jasti, Subbarao A1 - Nijaguna, Mamatha B A1 - Boggaram, Sanjana A1 - Hurakadli, Manjunath A A1 - Raina, Dhruv A1 - Kurup, Lokavya Meenakshi A1 - Chintha, Chetan A1 - Manjunath, Kavyashree A1 - Goyal, Aneesh A1 - Sadasivam, Gayathri A1 - Bharatham, Kavitha A1 - Padigaru, Muralidhara A1 - Potluri, Vijay A1 - Venkitaraman, Ashok R AB -

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.

VL - 25 IS - 6 ER - TY - JOUR T1 - Towards an arthritis flare-responsive drug delivery system. JF - Nat Commun Y1 - 2018 A1 - Joshi, Nitin A1 - Yan, Jing A1 - Levy, Seth A1 - Bhagchandani, Sachin A1 - Slaughter, Kai V A1 - Sherman, Nicholas E A1 - Amirault, Julian A1 - Wang, Yufeng A1 - Riegel, Logan A1 - He, Xueyin A1 - Rui, Tan Shi A1 - Valic, Michael A1 - Vemula, Praveen K A1 - Miranda, Oscar R A1 - Levy, Oren A1 - Gravallese, Ellen M A1 - Aliprantis, Antonios O A1 - Ermann, Joerg A1 - Karp, Jeffrey M AB -

Local delivery of therapeutics for the treatment of inflammatory arthritis (IA) is limited by short intra-articular half-lives. Since IA severity often fluctuates over time, a local drug delivery method that titrates drug release to arthritis activity would represent an attractive paradigm in IA therapy. Here we report the development of a hydrogel platform that exhibits disassembly and drug release controlled by the concentration of enzymes expressed during arthritis flares. In vitro, hydrogel loaded with triamcinolone acetonide (TA) releases drug on-demand upon exposure to enzymes or synovial fluid from patients with rheumatoid arthritis. In arthritic mice, hydrogel loaded with a fluorescent dye demonstrates flare-dependent disassembly measured as loss of fluorescence. Moreover, a single dose of TA-loaded hydrogel but not the equivalent dose of locally injected free TA reduces arthritis activity in the injected paw. Together, our data suggest flare-responsive hydrogel as a promising next-generation drug delivery approach for the treatment of IA.

VL - 9 IS - 1 ER - TY - JOUR T1 - A unique self-assembly-driven probe for sensing a lipid bilayer: ratiometric probing of vesicle to micelle transition JF - Chem. Commun. Y1 - 2018 A1 - Gulyani, Akash A1 - Dey, Nilanjan A1 - Bhattacharya, Santanu AB -

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 {'}rim{'} phases that co-exist in mixed assemblies (bicelles).

VL - 54 UR - http://dx.doi.org/10.1039/C8CC01635F ER - TY - JOUR T1 - Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. JF - Acta Crystallogr F Struct Biol Commun Y1 - 2017 A1 - Caing-Carlsson, Rhawnie A1 - Goyal, Parveen A1 - Sharma, Amit A1 - Ghosh, Swagatha A1 - Setty, Thanuja Gangi A1 - North, Rachel A A1 - Friemann, Rosmarie A1 - Ramaswamy, S KW - Adenosine Triphosphate KW - Amino Acid Sequence KW - Bacterial Proteins KW - Binding Sites KW - Cloning, Molecular KW - Crystallography, X-Ray KW - Escherichia coli KW - Fusobacterium nucleatum KW - Gene Expression KW - Genetic Vectors KW - Hexosamines KW - Models, Molecular KW - Phosphotransferases (Alcohol Group Acceptor) KW - Protein Binding KW - Protein Conformation, alpha-Helical KW - Protein Conformation, beta-Strand KW - Protein Interaction Domains and Motifs KW - Protein Multimerization KW - Recombinant Proteins KW - Sequence Alignment KW - Sequence Homology, Amino Acid KW - Substrate Specificity AB -

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

VL - 73 IS - Pt 6 ER - TY - JOUR T1 - Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian . JF - Development Y1 - 2017 A1 - Bansal, Dhiru A1 - Kulkarni, Jahnavi A1 - Nadahalli, Kavana A1 - Lakshmanan, Vairavan A1 - Krishna, Srikar A1 - Sasidharan, Vidyanand A1 - Geo, Jini A1 - Dilipkumar, Shilpa A1 - Pasricha, Renu A1 - Gulyani, Akash A1 - Raghavan, Srikala A1 - Palakodeti, Dasaradhi KW - Animals KW - Cell Lineage KW - Cell Proliferation KW - Cytoplasm KW - Epidermis KW - Epithelium KW - Extracellular Matrix KW - Gene Knockdown Techniques KW - Homeostasis KW - Models, Biological KW - Planarians KW - Poly(A)-Binding Protein I KW - Regeneration KW - RNA, Messenger KW - Wound Healing AB -

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.

VL - 144 IS - 17 ER - TY - JOUR T1 - Facile Synthesis of Highly Sensitive, Red‐Emitting, Fluorogenic Dye for Microviscosity and Mitochondrial Imaging in Embryonic Stem Cells JF - ChemistrySelect Y1 - 2017 A1 - Raja Sufi O. A1 - Sivaraman Gandhi A1 - Mukherjee Ananya A1 - Duraisamy Chellappa A1 - Gulyani Akash KW - Embryonic Stem Cells KW - Live cell imaging KW - Microviscosity KW - Mitochondrial fluorescent probe KW - Photoisomerization AB -

Abstract Bright, sensitive fluorescent probes that respond to changes in the cellular microenvironment are extremely valuable for imaging cellular dynamics. We report a simple, one‐step synthesis of a new hemicaynine (HC‐1) dye as a sensitive, red‐emitting (λmax‐610 nm) fluorogenic probe for micro‐viscosity and local order in diverse environments, including live cells. HC‐1 responds to increasing micro‐viscosity through changes in fluorescence intensity and lifetime, and is sensitive enough to report dynamic micellar self‐assembly. While HC‐1 shows properties of a molecular ‘rotor’, time‐dependent density functional theoretical analysis reveals that in HC‐1, an inhibition of photo‐isomerization in viscous environment is the likely cause of fluorescence enhancement. HC‐1 localizes to mitochondria in live cells and responds to mitochondrial ordering through a significant increase in fluorescence. Strikingly, we show that HC‐1 is also a sensitive probe for the spatial heterogeneity of mitochondrial organization in embryonic stem cells as well as dynamic remodeling of the mitochondria in early‐differentiated cells.

VL - 2 UR - https://onlinelibrary.wiley.com/doi/abs/10.1002/slct.201700463 ER - TY - JOUR T1 - Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm. JF - Sci Adv Y1 - 2017 A1 - Shettigar, Nishan A1 - Joshi, Asawari A1 - Dalmeida, Rimple A1 - Gopalkrishna, Rohini A1 - Chakravarthy, Anirudh A1 - Patnaik, Siddharth A1 - Mathew, Manoj A1 - Palakodeti, Dasaradhi A1 - Gulyani, Akash AB -

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.

VL - 3 IS - 7 ER - TY - JOUR T1 - Mimicking Muscle Stem Cell Quiescence in Culture: Methods for Synchronization in Reversible Arrest. JF - Methods Mol Biol Y1 - 2017 A1 - Arora, Reety A1 - Rumman, Mohammed A1 - Venugopal, Nisha A1 - Gala, Hardik A1 - Dhawan, Jyotsna KW - Actins KW - Adult Stem Cells KW - Animals KW - Biomarkers KW - Cell Culture Techniques KW - Cell Differentiation KW - Cell Line KW - Cell Proliferation KW - Fluorescent Antibody Technique KW - Humans KW - Mice KW - Microscopy, Fluorescence KW - Muscle, Skeletal KW - Myoblasts KW - Resting Phase, Cell Cycle KW - Satellite Cells, Skeletal Muscle KW - Stem Cells AB -

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.

VL - 1556 ER - TY - JOUR T1 - Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region. JF - Proc Natl Acad Sci U S A Y1 - 2017 A1 - Gangadharan, Binnu A1 - Sunitha, Margaret S A1 - Mukherjee, Souhrid A1 - Chowdhury, Ritu Roy A1 - Haque, Farah A1 - Sekar, Narendrakumar A1 - Sowdhamini, Ramanathan A1 - Spudich, James A A1 - Mercer, John A AB -

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80-180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

VL - 114 IS - 42 ER - TY - JOUR T1 - Scaling the effect of hydrophobic chain length on gene transfer properties of di-alkyl{,} di-hydroxy ethylammonium chloride based cationic amphiphiles JF - RSC Adv. Y1 - 2017 A1 - Hiwale, Ankita A. A1 - Voshavar, Chandrashekhar A1 - Dharmalingam, Priya A1 - Dhayani, Ashish A1 - Mukthavaram, Rajesh A1 - Nadella, Rasajna A1 - Sunnapu, Omprakash A1 - Gandhi, Sivaraman A1 - Naidu, V. G. M. A1 - Chaudhuri, Arabinda A1 - Marepally, Srujan A1 - Vemula, Praveen Kumar AB -

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.

VL - 7 UR - http://dx.doi.org/10.1039/C7RA02271A ER - TY - JOUR T1 - Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells. JF - Elife Y1 - 2017 A1 - Lee, Pedro A1 - Gund, Rupali A1 - Dutta, Abhik A1 - Pincha, Neha A1 - Rana, Isha A1 - Ghosh, Subhasri A1 - Witherden, Deborah A1 - Kandyba, Eve A1 - MacLeod, Amanda A1 - Kobielak, Krzysztof A1 - Havran, Wendy L A1 - Jamora, Colin AB -

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.

VL - 6 ER - TY - JOUR T1 - Blue protein with red fluorescence. JF - Proc Natl Acad Sci U S A Y1 - 2016 A1 - Ghosh, Swagatha A1 - Yu, Chi-Li A1 - Ferraro, Daniel J A1 - Sudha, Sai A1 - Pal, Samir Kumar A1 - Schaefer, Wayne F A1 - Gibson, David T A1 - Ramaswamy, S KW - Biliverdine KW - Crystallography, X-Ray KW - Fluorescence KW - Models, Molecular KW - Proteins KW - Recombinant Proteins AB -

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λ of 375 nm) and upon excitation at this wavelength emits in the red region (λ of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

VL - 113 IS - 41 ER - TY - JOUR T1 - Stochastic steps in secondary active sugar transport. JF - Proc Natl Acad Sci U S A Y1 - 2016 A1 - Adelman, Joshua L A1 - Ghezzi, Chiara A1 - Bisignano, Paola A1 - Loo, Donald D F A1 - Choe, Seungho A1 - Abramson, Jeff A1 - Rosenberg, John M A1 - Wright, Ernest M A1 - Grabe, Michael KW - Glucose KW - HEK293 Cells KW - Humans KW - Markov Chains KW - Molecular Dynamics Simulation KW - Monte Carlo Method KW - Patch-Clamp Techniques KW - Sodium KW - Sodium-Glucose Transporter 1 AB -

Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

VL - 113 IS - 27 ER - TY - JOUR T1 - Structure of a heterogeneous, glycosylated, lipid-bound, ıt in vivo-grown protein crystal at atomic resolution from the viviparous cockroach ıt Diploptera punctata JF - IUCrJ Y1 - 2016 A1 - Banerjee, Sanchari A1 - Coussens, Nathan P. A1 - Gallat, François-Xavier A1 - Sathyanarayanan, Nitish A1 - Srikanth, Jandhyam A1 - Yagi, Koichiro J. A1 - Gray, James S. S. A1 - Tobe, Stephen S. A1 - Stay, Barbara A1 - Chavas, Leonard M. G. A1 - Ramaswamy, Subramanian KW - glycosylation KW - protein heterogeneity KW - sulfur-SAD KW - viviparity in cockroach AB -

Macromolecular crystals for X-ray diffraction studies are typically grown ıt in vitro} from pure and homogeneous samples; however, there are examples of protein crystals that have been identified ıt in vivo}. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grown ıt in cellulo}. Here, an atomic resolution (1.2{\AA}) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. These ıt in vivo}-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach, ıt Diploptera punctata}. The milk proteins crystallized in space group ıt P}1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a single ıt in vivo}-grown crystal of a natural protein in its native environment at atomic resolution.

VL - 3 UR - http://dx.doi.org/10.1107/S2052252516008903 ER - TY - JOUR T1 - Mechanistic heterogeneity in contractile properties of α-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies. JF - J Biol Chem Y1 - 2015 A1 - Gupte, Tejas M A1 - Haque, Farah A1 - Gangadharan, Binnu A1 - Sunitha, Margaret S A1 - Mukherjee, Souhrid A1 - Anandhan, Swetha A1 - Rani, Deepa Selvi A1 - Mukundan, Namita A1 - Jambekar, Amruta A1 - Thangaraj, Kumarasamy A1 - Sowdhamini, Ramanathan A1 - Sommese, Ruth F A1 - Nag, Suman A1 - Spudich, James A A1 - Mercer, John A KW - Actins KW - Adenosine Triphosphatases KW - Calcium KW - Cardiomyopathies KW - Humans KW - Models, Molecular KW - Myosins KW - Point Mutation KW - Protein Stability KW - Tropomyosin AB -

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

VL - 290 IS - 11 ER -