TY - JOUR T1 - Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways JF - Frontiers in Bioengineering and Biotechnology Y1 - 2023 A1 - Brijesh Lohchania A1 - Abisha Crystal Christopher A1 - Porkizhi Arjunan A1 - Gokulnath Mahalingam A1 - Durga Kathirvelu A1 - Aishwarya Prasannan A1 - Vigneshwaran Venkatesan A1 - Pankaj Taneja A1 - Mohan Kumar KM A1 - Saravanabhavan Thangavel A1 - Srujan Marepally VL - 10 UR - https://doi.org/10.3389%2Ffbioe.2022.1031049 ER - TY - JOUR T1 - Age-stratified adeno-associated virus serotype 3 neutralizing and total antibody prevalence in hemophilia A patients from India. JF - J Med Virol Y1 - 2022 A1 - Daniel, Hubert D-J A1 - Kumar, Sanjay A1 - Kannangai, Rajesh A1 - J, Farzana A1 - Joel, Joseph N A1 - Abraham, Aby A1 - Lakshmi, Kavitha M A1 - Agbandje-McKenna, Mavis A1 - Coleman, Kirsten E A1 - Srivastava, Arun A1 - Srivastava, Alok A1 - Abraham, Asha M KW - Adult KW - Animals KW - Antibodies, Neutralizing KW - Antibodies, Viral KW - Child KW - Dependovirus KW - Genetic Vectors KW - Hemophilia A KW - Humans KW - Prevalence KW - Serogroup AB -

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

VL - 94 IS - 9 ER - TY - JOUR T1 - DDX24 is required for muscle fiber organization and the suppression of wound-induced Wnt activity necessary for pole re-establishment during planarian regeneration. JF - Dev Biol Y1 - 2022 A1 - Sarkar, Souradeep R A1 - Dubey, Vinay Kumar A1 - Jahagirdar, Anusha A1 - Lakshmanan, Vairavan A1 - Haroon, Mohamed Mohamed A1 - Sowndarya, Sai A1 - Sowdhamini, Ramanathan A1 - Palakodeti, Dasaradhi AB -

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

VL - 488 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 - 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 - 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 - 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 - 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 - Adiponectin receptor 1 variants contribute to hypertrophic cardiomyopathy that can be reversed by rapamycin. JF - Sci Adv Y1 - 2021 A1 - Dhandapany, Perundurai S A1 - Kang, Soojeong A1 - Kashyap, Deepak K A1 - Rajagopal, Raksha A1 - Sundaresan, Nagalingam R A1 - Singh, Rajvir A1 - Thangaraj, Kumarasamy A1 - Jayaprakash, Shilpa A1 - Manjunath, Cholenahally N A1 - Shenthar, Jayaprakash A1 - Lebeche, Djamel AB -

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

VL - 7 IS - 2 ER - TY - JOUR T1 - The bacterial social network and beyond. JF - Nat Rev Mol Cell Biol Y1 - 2021 A1 - Laxman, Sunil VL - 22 IS - 7 ER - TY - JOUR T1 - Bend or break: how biochemically versatile molecules enable metabolic division of labor in clonal microbial communities. JF - Genetics Y1 - 2021 A1 - Varahan, Sriram A1 - Laxman, Sunil AB -

In fluctuating nutrient environments, isogenic microbial cells transition into "multicellular" communities composed of phenotypically heterogeneous cells, showing functional specialization. In fungi (such as budding yeast), phenotypic heterogeneity is often described in the context of cells switching between different morphotypes (e.g., yeast to hyphae/pseudohyphae or white/opaque transitions in Candida albicans). However, more fundamental forms of metabolic heterogeneity are seen in clonal Saccharomyces cerevisiae communities growing in nutrient-limited conditions. Cells within such communities exhibit contrasting, specialized metabolic states, and are arranged in distinct, spatially organized groups. In this study, we explain how such an organization can stem from self-organizing biochemical reactions that depend on special metabolites. These metabolites exhibit plasticity in function, wherein the same metabolites are metabolized and utilized for distinct purposes by different cells. This in turn allows cell groups to function as specialized, interdependent cross-feeding systems which support distinct metabolic processes. Exemplifying a system where cells exhibit either gluconeogenic or glycolytic states, we highlight how available metabolites can drive favored biochemical pathways to produce new, limiting resources. These new resources can themselves be consumed or utilized distinctly by cells in different metabolic states. This thereby enables cell groups to sustain contrasting, even apparently impossible metabolic states with stable transcriptional and metabolic signatures for a given environment, and divide labor in order to increase community fitness or survival. We speculate on possible evolutionary implications of such metabolic specialization and division of labor in isogenic microbial communities.

VL - 219 IS - 2 ER - TY - JOUR T1 - Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs). JF - RNA Y1 - 2021 A1 - Lakshmanan, Vairavan A1 - T N, Sujith A1 - Bansal, Dhiru A1 - Padubidri, Shivaprasad V A1 - Palakodeti, Dasaradhi A1 - Krishna, Srikar AB -

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological process such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterise planarian tRFs, we first annotated 457 tRNAs in S.mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians - the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of Ago1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalogue of post-transcriptional regulatory systems in planarian, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.

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 - 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 - Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea. JF - Glycobiology Y1 - 2021 A1 - Subramanian, Sabarinath Peruvemba A1 - Lakshmanan, Vairavan A1 - Palakodeti, Dasaradhi A1 - Subramanian, Ramaswamy AB -

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix, and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and di-substituted O-GalNAc structures. Extensions made of hexoses and 3-O methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, sixty genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. While a majority of the genes involved in mucin-type O-glycan biosynthesis was highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

ER - TY - JOUR T1 - Hair Follicle Grafting Therapy Promotes Re-Emergence of Critical Skin Components in Chronic Nonhealing Wounds JF - JID Innovations Y1 - 2021 A1 - Saha, D ED - Thannimangalath, S AB -

An exploding public health crisis is the exponential growth in the incidence of chronic nonhealing ulcers associated with diseases such as diabetes. Various modalities have been developed to stimulate wound closure that is otherwise recalcitrant to standard clinical treatments. However, these approaches primarily focus on the process of re-epithelialization and are often deficient in regenerating the full spectrum of structures necessary for normal skin function. Autologous hair follicle grafting is a recent therapy to stimulate the closure of such nonhealing wounds, and we observed effects beyond the epidermis to other important components of the dermis. We found that hair follicle grafting facilitated the reappearance of various undifferentiated and differentiated layers of the epidermis with the restoration of epidermal junctions. In addition, other important structures that are critical for cutaneous health and function such as the blood and lymph vasculature, nerve fibers, and sweat gland structures were restored in postgrafted wounds. Interestingly, both immune cells and inflammatory signals were substantially decreased, indicating a reduction in the chronic inflammation that is a hallmark of nonhealing wounds. Our observation that punch wounds created on the postgrafted area likewise healed suggests that this is a self-sustaining long-term therapy for patients with chronic wounds.

VL - 1 IS - 3 ER - TY - JOUR T1 - Kog1/Raptor mediates metabolic rewiring during nutrient limitation by controlling SNF1/AMPK activity. JF - Sci Adv Y1 - 2021 A1 - Rashida, Zeenat A1 - Srinivasan, Rajalakshmi A1 - Cyanam, Meghana A1 - Laxman, Sunil AB -

In changing environments, cells modulate resource budgeting through distinct metabolic routes to control growth. Accordingly, the TORC1 and SNF1/AMPK pathways operate contrastingly in nutrient replete or limited environments to maintain homeostasis. The functions of TORC1 under glucose and amino acid limitation are relatively unknown. We identified a modified form of the yeast TORC1 component Kog1/Raptor, which exhibits delayed growth exclusively during glucose and amino acid limitations. Using this, we found a necessary function for Kog1 in these conditions where TORC1 kinase activity is undetectable. Metabolic flux and transcriptome analysis revealed that Kog1 controls SNF1-dependent carbon flux apportioning between glutamate/amino acid biosynthesis and gluconeogenesis. Kog1 regulates SNF1/AMPK activity and outputs and mediates a rapamycin-independent activation of the SNF1 targets Mig1 and Cat8. This enables effective glucose derepression, gluconeogenesis activation, and carbon allocation through different pathways. Therefore, Kog1 centrally regulates metabolic homeostasis and carbon utilization during nutrient limitation by managing SNF1 activity.

VL - 7 IS - 16 ER - TY - JOUR T1 - Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells. JF - Dev Cell Y1 - 2021 A1 - Biswas, Ritusree A1 - Banerjee, Avinanda A1 - Lembo, Sergio A1 - Zhao, Zhihai A1 - Lakshmanan, Vairavan A1 - Lim, Ryan A1 - Le, Shimin A1 - Nakasaki, Manando A1 - Kutyavin, Vassily A1 - Wright, Graham A1 - Palakodeti, Dasaradhi A1 - Ross, Robert S A1 - Jamora, Colin A1 - Vasioukhin, Valeri A1 - Jie, Yan A1 - Raghavan, Srikala AB -

Vinculin, a mechanotransducer associated with both adherens junctions (AJs) and focal adhesions (FAs), plays a central role in force transmission through cell-cell and cell-substratum contacts. We generated the conditional knockout (cKO) of vinculin in murine skin that results in the loss of bulge stem cell (BuSC) quiescence and promotes continual cycling of the hair follicles. Surprisingly, we find that the AJs in vinculin cKO cells are mechanically weak and impaired in force generation despite increased junctional expression of E-cadherin and α-catenin. Mechanistically, we demonstrate that vinculin functions by keeping α-catenin in a stretched/open conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, at the AJs. Altogether, our data provide mechanistic insights into the hitherto-unexplored regulatory link between the mechanical stability of cell junctions and contact-inhibition-mediated maintenance of BuSC quiescence.

VL - 56 IS - 6 ER - TY - JOUR T1 - Mitochondrial state determines functionally divergent stem cell population in planaria. JF - Stem Cell Reports Y1 - 2021 A1 - Mohamed Haroon, Mohamed A1 - Lakshmanan, Vairavan A1 - Sarkar, Souradeep R A1 - Lei, Kai A1 - Vemula, Praveen Kumar A1 - Palakodeti, Dasaradhi AB -

Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.

VL - 16 IS - 5 ER - TY - JOUR T1 - Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis. JF - Cell Metab Y1 - 2021 A1 - Wiley, Christopher D A1 - Sharma, Rishi A1 - Davis, Sonnet S A1 - Lopez-Dominguez, Jose Alberto A1 - Mitchell, Kylie P A1 - Wiley, Samantha A1 - Alimirah, Fatouma A1 - Kim, Dong Eun A1 - Payne, Therese A1 - Rosko, Andrew A1 - Aimontche, Eliezer A1 - Deshpande, Sharvari M A1 - Neri, Francesco A1 - Kuehnemann, Chisaka A1 - Demaria, Marco A1 - Ramanathan, Arvind A1 - Campisi, Judith AB -

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

ER - TY - JOUR T1 - Proteome plasticity in response to persistent environmental change. JF - Mol Cell Y1 - 2021 A1 - Domnauer, Matthew A1 - Zheng, Fan A1 - Li, Liying A1 - Zhang, Yanxiao A1 - Chang, Catherine E A1 - Unruh, Jay R A1 - Conkright-Fincham, Juliana A1 - McCroskey, Scott A1 - Florens, Laurence A1 - Zhang, Ying A1 - Seidel, Christopher A1 - Fong, Benjamin A1 - Schilling, Birgit A1 - Sharma, Rishi A1 - Ramanathan, Arvind A1 - Si, Kausik A1 - Zhou, Chuankai KW - Acclimatization KW - Adaptation, Physiological KW - Animals KW - Environmental Exposure KW - Gene Expression Regulation, Fungal KW - Hot Temperature KW - Proteome KW - Saccharomycetales KW - Stress, Physiological KW - Transcriptome AB -

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

VL - 81 IS - 16 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 - Scanning electron microscopy of murine skin ultrathin sections and cultured keratinocytes. JF - STAR Protoc Y1 - 2021 A1 - Banerjee, Avinanda A1 - Biswas, Ritusree A1 - Lim, Ryan A1 - Pasolli, Hilda Amalia A1 - Raghavan, Srikala AB -

Generating high-quality electron microscopy images of the skin and keratinocytes can be challenging. Here we describe a simple protocol for scanning electron microscopy (SEM) of murine skin. The protocol enables characterization of the ultrastructure of the epidermis, dermis, hair follicles, basement membrane, and cell-cell junctions. We detail the specific steps for sample preparation and highlight the critical need for proper orientation of the sample for ultrathin sectioning. We also describe the isolation and preparation of primary keratinocyte monolayers for SEM. For complete details on the use and execution of this protocol, please refer to Biswas et al. (2021).

VL - 2 IS - 3 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 - Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism. JF - J Biol Chem Y1 - 2020 A1 - Bhatia, Muskan A1 - Thakur, Jyotika A1 - Suyal, Shradha A1 - Oniel, Ruchika A1 - Chakraborty, Rahul A1 - Pradhan, Shalini A1 - Sharma, Monika A1 - Sengupta, Shantanu A1 - Laxman, Sunil A1 - Masakapalli, Shyam Kumar A1 - Bachhawat, Anand Kumar KW - Adenosine Triphosphate KW - Allosteric Regulation KW - Humans KW - Methylation KW - Methylenetetrahydrofolate Reductase (NADPH2) KW - S-Adenosylmethionine KW - Saccharomyces cerevisiae KW - Saccharomyces cerevisiae Proteins AB -

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CHTHF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.

VL - 295 IS - 47 ER - TY - JOUR T1 - Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria. JF - Mol Cell Biol Y1 - 2020 A1 - Shaw, Eisha A1 - Talwadekar, Manasi A1 - Rashida, Zeenat A1 - Mohan, Nitya A1 - Acharya, Aishwarya A1 - Khatri, Subhash A1 - Laxman, Sunil A1 - Kolthur-Seetharam, Ullas AB -

Anabolic and catabolic signaling mediated via mTOR and AMPK (AMP-activated kinase) have to be intrinsically coupled to mitochondrial functions for maintaining homeostasis and mitigate cellular/organismal stress. Although glutamine is known to activate mTOR, whether and how differential mitochondrial utilization of glutamine impinges on mTOR signaling has been less explored. Mitochondrial SIRT4, which unlike other sirtuins is induced in a fed state, is known to inhibit catabolic signaling/pathways through the AMPK-PGC1α/SIRT1-peroxisome proliferator-activated receptor α (PPARα) axis and negatively regulate glutamine metabolism via the tricarboxylic acid cycle. However, physiological significance of SIRT4 functions during a fed state is still unknown. Here, we establish SIRT4 as key anabolic factor that activates TORC1 signaling and regulates lipogenesis, autophagy, and cell proliferation. Mechanistically, we demonstrate that the ability of SIRT4 to inhibit anaplerotic conversion of glutamine to α-ketoglutarate potentiates TORC1. Interestingly, we also show that mitochondrial glutamine sparing or utilization is critical for differentially regulating TORC1 under fed and fasted conditions. Moreover, we conclusively show that differential expression of SIRT4 during fed and fasted states is vital for coupling mitochondrial energetics and glutamine utilization with anabolic pathways. These significant findings also illustrate that SIRT4 integrates nutrient inputs with mitochondrial retrograde signals to maintain a balance between anabolic and catabolic pathways.

VL - 40 IS - 2 ER - TY - JOUR T1 - Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns. JF - Mol Autism Y1 - 2020 A1 - Das Sharma, Shreya A1 - Pal, Rakhi A1 - Reddy, Bharath Kumar A1 - Selvaraj, Bhuvaneish T A1 - Raj, Nisha A1 - Samaga, Krishna Kumar A1 - Srinivasan, Durga J A1 - Ornelas, Loren A1 - Sareen, Dhruv A1 - Livesey, Matthew R A1 - Bassell, Gary J A1 - Svendsen, Clive N A1 - Kind, Peter C A1 - Chandran, Siddharthan A1 - Chattarji, Sumantra A1 - Wyllie, David J A AB -

BACKGROUND: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP).

METHODS: Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced.

RESULTS: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca-activated K currents and the persistent Na current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines.

CONCLUSIONS: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

VL - 11 IS - 1 ER - TY - JOUR T1 - Genetically encoded live-cell sensor for tyrosinated microtubules. JF - J Cell Biol Y1 - 2020 A1 - Kesarwani, Shubham A1 - Lama, Prakash A1 - Chandra, Anchal A1 - Reddy, P Purushotam A1 - Jijumon, A S A1 - Bodakuntla, Satish A1 - Rao, Balaji M A1 - Janke, Carsten A1 - Das, Ranabir A1 - Sirajuddin, Minhajuddin AB -

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

VL - 219 IS - 10 ER - TY - JOUR T1 - Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program. JF - PLoS Genet Y1 - 2020 A1 - Srinivasan, Rajalakshmi A1 - Walvekar, Adhish S A1 - Rashida, Zeenat A1 - Seshasayee, Aswin A1 - Laxman, Sunil KW - Basic-Leucine Zipper Transcription Factors KW - Cell Proliferation KW - Gene Expression Regulation, Fungal KW - Gene Regulatory Networks KW - Genome, Fungal KW - Ribosomes KW - Saccharomyces cerevisiae KW - Saccharomyces cerevisiae Proteins KW - Transcriptional Activation AB -

Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells use coordinated gene expression programs and manage biomolecule supply in order to match the demands of metabolism and translation. Global growth programs complement increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis. How these resources are collectively managed is a fundamental question. The role of the Gcn4/ATF4 transcription factor has been best studied in contexts where cells encounter amino acid starvation. However, high Gcn4 activity has been observed in contexts of rapid cell proliferation, and the roles of Gcn4 in such growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum that maintains metabolic supply in order to sustain translation outputs. By integrating matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lysine and arginine enriched transcripts, which include genes encoding the translation machinery. The Gcn4 dependent lysine and arginine supply thereby maintains the synthesis of the translation machinery. This is required to maintain translation capacity. Gcn4 consequently enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controlled using the same Gcn4 transcriptional outputs that function in distinct contexts.

VL - 16 IS - 12 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 - Modulation of β-catenin levels regulates cranial neural crest patterning and dispersal into first pharyngeal arch. JF - Dev Dyn Y1 - 2020 A1 - Javali, Alok A1 - Lakshmanan, Vairavan A1 - Palakodeti, Dasaradhi A1 - Sambasivan, Ramkumar AB -

BACKGROUND: Vertebrate cranial neural crest cells (CNCCs) are multipotent, proximal to the source CNCC form the cranial ganglia. Distally, in the pharyngeal arches, they give rise to the craniofacial skeleton and connective tissues. Fate choices are made as CNCC pattern into distinct destination compartments. In spite of this importance, the mechanism patterning CNCC is poorly defined.

RESULTS: Here, we report that a novel β-catenin-dependent regulation of N-Cadherin levels may drive CNCC patterning. In mouse embryos, at the first pharyngeal arch axial level, membrane β-catenin levels correlate with the extent of N-cadherin-mediated adhesion and thus suggest the presence of collective and dispersed states of CNCC. Using in vitro human neural crest model and chemical modulators of β-catenin levels, we show a requirement for down-modulating β-catenin for regulating N-cadherin levels and cell-cell adhesion. Similarly, in β-catenin gain-of-function mutant mouse embryos, CNCC fail to lower N-cadherin levels. This indicates a failure to reduce cell-cell adhesion, which may underlie the failure of mutant CNCC to populate first pharyngeal arch.

CONCLUSION: We suggest that β-catenin-mediated regulation of CNCC adhesion, a previously underappreciated mechanism, underlies the patterning of CNCC into fate-specific compartments.

ER - TY - JOUR T1 - A novel polyubiquitin chain linkage formed by viral Ubiquitin is resistant to host deubiquitinating enzymes. JF - Biochem J Y1 - 2020 A1 - Negi, Hitendra A1 - Reddy, Pothula Purushotham A1 - Vengayil, Vineeth A1 - Patole, Chhaya A1 - Laxman, Sunil A1 - Das, Ranabir AB -

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

VL - 477 IS - 12 ER - TY - JOUR T1 - Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. JF - J Proteome Res Y1 - 2020 A1 - Bowling, Heather L A1 - Kasper, Amanda A1 - Patole, Chhaya A1 - Venkatasubramani, Janani Priya A1 - Leventer, Sarah Parker A1 - Carmody, Erin A1 - Sharp, Kevin A1 - Berry-Kravis, Elizabeth A1 - Kirshenbaum, Kent A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

VL - 19 IS - 9 ER - TY - JOUR T1 - The Rad53-Spt21 and Tel1 axes couple glucose tolerance to histone dosage and subtelomeric silencing. JF - Nat Commun Y1 - 2020 A1 - Bruhn, Christopher A1 - Ajazi, Arta A1 - Ferrari, Elisa A1 - Lanz, Michael Charles A1 - Batrin, Renaud A1 - Choudhary, Ramveer A1 - Walvekar, Adhish A1 - Laxman, Sunil A1 - Longhese, Maria Pia A1 - Fabre, Emmanuelle A1 - Smolka, Marcus Bustamente A1 - Foiani, Marco KW - Acetylation KW - Ataxia Telangiectasia Mutated Proteins KW - Cell Cycle Proteins KW - Checkpoint Kinase 2 KW - DNA Damage KW - DNA Repair KW - Gene Silencing KW - Glucose KW - Histone Deacetylases KW - Histones KW - Intracellular Signaling Peptides and Proteins KW - Mutation KW - Phosphorylation KW - Protein-Serine-Threonine Kinases KW - Saccharomyces cerevisiae KW - Saccharomyces cerevisiae Proteins KW - Serine KW - Telomere KW - Transcription Factors AB -

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21 on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1 and Rpd3 activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.

VL - 11 IS - 1 ER - TY - JOUR T1 - Resource plasticity-driven carbon-nitrogen budgeting enables specialization and division of labor in a clonal community. JF - Elife Y1 - 2020 A1 - Varahan, Sriram A1 - Sinha, Vaibhhav A1 - Walvekar, Adhish A1 - Krishna, Sandeep A1 - Laxman, Sunil AB -

Previously, we found that in glucose-limited colonies, metabolic constraints drive cells into groups exhibiting gluconeogenic or glycolytic states. In that study, threshold amounts of trehalose - a limiting, produced carbon-resource, controls the emergence and self-organization of cells exhibiting the glycolytic state, serving as a carbon source that fuels glycolysis (Varahan et al., 2019). We now discover that the plasticity of use of a non-limiting resource, aspartate, controls both resource production and the emergence of heterogeneous cell states, based on differential metabolic budgeting. In gluconeogenic cells, aspartate is a carbon source for trehalose production, while in glycolytic cells using trehalose for carbon, aspartate is predominantly a nitrogen source for nucleotide synthesis. This metabolic plasticity of aspartate enables carbon-nitrogen budgeting, thereby driving the biochemical self-organization of distinct cell states. Through this organization, cells in each state exhibit true division of labor, providing growth/survival advantages for the whole community.

VL - 9 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 - Altered steady state and activity-dependent de novo protein expression in fragile X syndrome. JF - Nat Commun Y1 - 2019 A1 - Bowling, Heather A1 - Bhattacharya, Aditi A1 - Zhang, Guoan A1 - Alam, Danyal A1 - Lebowitz, Joseph Z A1 - Bohm-Levine, Nathaniel A1 - Lin, Derek A1 - Singha, Priyangvada A1 - Mamcarz, Maggie A1 - Puckett, Rosemary A1 - Zhou, Lili A1 - Aryal, Sameer A1 - Sharp, Kevin A1 - Kirshenbaum, Kent A1 - Berry-Kravis, Elizabeth A1 - Neubert, Thomas A A1 - Klann, Eric AB -

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase 1 and Ras, also are expressed in the blood of FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.

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 - The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown upon glucose availability. JF - J Biol Chem Y1 - 2019 A1 - Vengayil, Vineeth A1 - Rashida, Zeenat A1 - Laxman, Sunil AB -

Cells use multiple mechanisms to regulate their metabolic states in response to changes in their nutrient environment. One example is the response of cells to glucose. In S. cerevisiae growing in glucose-depleted medium, the re-availability of glucose leads to the downregulation of gluconeogenesis, and the activation of glycolysis, leading to 'glucose repression'. However, our knowledge of the mechanisms mediating the glucose dependent downregulation of the gluconeogenic transcription factors is limited. Using a major gluconeogenic transcription factor Rds2 as a candidate, here we identify a novel role for the E3 ubiquitin ligase Pib1 in regulating the stability and degradation of Rds2. Glucose addition to cells growing in glucose limitation results in rapid ubiquitination of Rds2, followed by its proteasomal degradation. Through in vivo and in vitro experiments, we establish Pib1 as the ubiquitin E3 ligase that regulates Rds2 ubiquitination and stability. Notably, this Pib1 mediated Rds2 ubiquitination, followed by proteasomal degradation, is specific to the presence of glucose. This Pib1 mediated ubiquitination of Rds2 depends on the phosphorylation state of Rds2, suggesting a cross-talk between ubiquitination and phosphorylation to achieve a metabolic state change. Using stable-isotope based metabolic flux experiments we find that the loss of Pib1 results in an imbalanced gluconeogenic state, regardless of glucose availability. Pib1 is required for complete glucose repression, and enables cells to optimally grow in competitive environments when glucose becomes re-available. Our results reveal the existence of a Pib1 mediated regulatory program that mediates glucose-repression when glucose availability is restored.

ER - TY - JOUR T1 - Effect of early maternal separation stress on attention, spatial learning and social interaction behaviour. JF - Exp Brain Res Y1 - 2019 A1 - Kambali, Maltesh Y A1 - Anshu, Kumari A1 - Kutty, Bindu M A1 - Muddashetty, Ravi S A1 - Laxmi, T Rao AB -

Early life stress is known to influence affective and cognitive functions in later life but comprehensive explanation for the impact of early life stress on attentional functions, behavioural control and social behaviour is inadequate. The early life stress was induced by exposing rat pups to 6 h of maternal separation and isolation (MS) stress from postnatal days 4-14 i.e. during SHRP period. The long-term impact of MS in these rats was evaluated by assessing anxiety, sociability, social preference, spatial learning and memory along with a detailed evaluation of attentional functions during young adulthood period. Adult male MS rats showed increased anxiety-like behaviour, impaired flexibility in social interactions, and increased reward-seeking behaviour. MS rats also showed faster spatial learning in the partially baited radial arm maze and exhibited moderately enhanced sustained attention in the 5-choice serial reaction time task (5CSRTT). These results suggest that early MS has both positive and negative consequences in adulthood. Increased cognitive ability in MS rats, as evidenced by the improved sustained attention and spatial learning and memory, is usually advantageous and indicates positive influences of early stressors that might lead to the development of resilience and enhanced compensatory mechanisms later in adulthood. MS stress has compromised flexibility in social behaviour that promotes solitary lifestyle and social isolation. Heightened reward-seeking behaviour, as shown by the MS rats, could be a predisposing factor for substance abuse and addiction. Thus, our study highlights the crucial and differential impact of early life challenges on behaviour during adulthood and suggests that the positive aspects could be an asset that may be utilized to suppress the negative effects of early life stress in adulthood.

VL - 237 IS - 8 ER - TY - JOUR T1 - Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway. JF - Nat Commun Y1 - 2019 A1 - Singh, Rajbir A1 - Chandrashekharappa, Sandeep A1 - Bodduluri, Sobha R A1 - Baby, Becca V A1 - Hegde, Bindu A1 - Kotla, Niranjan G A1 - Hiwale, Ankita A A1 - Saiyed, Taslimarif A1 - Patel, Paresh A1 - Vijay-Kumar, Matam A1 - Langille, Morgan G I A1 - Douglas, Gavin M A1 - Cheng, Xi A1 - Rouchka, Eric C A1 - Waigel, Sabine J A1 - Dryden, Gerald W A1 - Alatassi, Houda A1 - Zhang, Huang-Ge A1 - Haribabu, Bodduluri A1 - Vemula, Praveen K A1 - Jala, Venkatakrishna R KW - Animals KW - Basic Helix-Loop-Helix Transcription Factors KW - Caco-2 Cells KW - Coumarins KW - Epithelial Cells KW - Gene Expression Regulation KW - HT29 Cells KW - Humans KW - Intestinal Mucosa KW - Macrophages KW - Mice KW - Mice, Inbred C57BL KW - Mice, Knockout KW - NF-E2-Related Factor 2 KW - Receptors, Aryl Hydrocarbon KW - Specific Pathogen-Free Organisms KW - Tight Junction Proteins AB -

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

VL - 10 IS - 1 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 - 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 - Metabolic constraints drive self-organization of specialized cell groups. JF - Elife Y1 - 2019 A1 - Varahan, Sriram A1 - Walvekar, Adhish A1 - Sinha, Vaibhhav A1 - Krishna, Sandeep A1 - Laxman, Sunil AB -

How phenotypically distinct states in isogenic cell populations appear and stably co-exist remains unresolved. We find that within a mature, clonal yeast colony developing in low glucose, cells arrange into metabolically disparate cell groups. Using this system, we model and experimentally identify metabolic constraints sufficient to drive such self-assembly. Beginning in a uniformly gluconeogenic state, cells exhibiting a contrary, high pentose phosphate pathway activity state, spontaneously appear and proliferate, in a spatially constrained manner. Gluconeogenic cells in the colony produce and provide a resource, which we identify as trehalose. Above threshold concentrations of external trehalose, cells switch to the new metabolic state and proliferate. A self-organized system establishes, where cells in this new state are sustained by trehalose consumption, which thereby restrains other cells in the trehalose producing, gluconeogenic state. Our work suggests simple physico-chemical principles that determine how isogenic cells spontaneously self-organize into structured assemblies in complimentary, specialized states.

VL - 8 ER - TY - JOUR T1 - Methionine at the Heart of Anabolism and Signaling: Perspectives From Budding Yeast. JF - Front Microbiol Y1 - 2019 A1 - Walvekar, Adhish S A1 - Laxman, Sunil AB -

Studies using a fungal model, , have been instrumental in advancing our understanding of sulfur metabolism in eukaryotes. Sulfur metabolites, particularly methionine and its derivatives, induce anabolic programs in yeast, and drive various processes integral to metabolism (one-carbon metabolism, nucleotide synthesis, and redox balance). Thereby, methionine also connects these processes with autophagy and epigenetic regulation. The direct involvement of methionine-derived metabolites in diverse chemistries such as transsulfuration and methylation reactions comes from the elegant positioning and safe handling of sulfur through these molecules. In this mini-review, we highlight studies from yeast that reveal how this amino acid holds a unique position in both metabolism and cell signaling, and illustrate cell fate decisions that methionine governs. We further discuss the interconnections between sulfur and NADPH metabolism, and highlight critical nodes around methionine metabolism that are promising for antifungal drug development.

VL - 10 ER - TY - JOUR T1 - Secretion of leukotrienes by senescent lung fibroblasts promotes pulmonary fibrosis. JF - JCI Insight Y1 - 2019 A1 - Wiley, Christopher D A1 - Brumwell, Alexis N A1 - Davis, Sonnet S A1 - Jackson, Julia R A1 - Valdovinos, Alexis A1 - Calhoun, Cheresa A1 - Alimirah, Fatouma A1 - Castellanos, Carlos A A1 - Ruan, Richard A1 - Wei, Ying A1 - Chapman, Harold A A1 - Ramanathan, Arvind A1 - Campisi, Judith A1 - Jourdan Le Saux, Claude AB -

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

VL - 4 IS - 24 ER - TY - JOUR T1 - Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea. JF - FEBS Lett Y1 - 2019 A1 - Sarkar, Arunabha A1 - Mukundan, Namita A1 - Sowndarya, Sai A1 - Dubey, Vinay Kumar A1 - Babu, Rosana A1 - Lakshmanan, Vairavan A1 - Rangiah, Kannan A1 - Panicker, Mitradas M A1 - Palakodeti, Dasaradhi A1 - Subramanian, Sabarinath Peruvemba A1 - Ramaswamy, Subramanian AB -

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

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 - Sustained correction of associative learning deficits after brief, early treatment in a rat model of Fragile X Syndrome. JF - Sci Transl Med Y1 - 2019 A1 - Asiminas, Antonis A1 - Jackson, Adam D A1 - Louros, Susana R A1 - Till, Sally M A1 - Spano, Teresa A1 - Dando, Owen A1 - Bear, Mark F A1 - Chattarji, Sumantra A1 - Hardingham, Giles E A1 - Osterweil, Emily K A1 - Wyllie, David J A A1 - Wood, Emma R A1 - Kind, Peter C AB -

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

VL - 11 IS - 494 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 - Exploring membrane permeability of Tomatidine to enhance lipid mediated nucleic acid transfections JF - Biochimica et Biophysica Acta (BBA) - Biomembranes Y1 - 2018 A1 - Vignesh K. Rangasami A1 - Brijesh Lohchania A1 - Chandrashekhar Voshavar A1 - Harikrishna R. Rachamalla A1 - Rajkumar Banerjee A1 - Ashish Dhayani A1 - Saravanabhavan Thangavel A1 - Praveen K. Vemula A1 - Srujan Marepally KW - Endocytosis AB -

Abstract Intracellular delivery of nucleic acids is one of the critical steps in the transfections. Prior findings demonstrated various strategies including membrane fusion, endosomal escape for the efficient cytoplasmic delivery. In our continuing efforts to improve the nucleic acids transfections, we harnessed cell permeable properties of Tomatidine (T), a steroidal alkaloid abundantly found in green tomatoes for maximizing intracellular delivery of lipoplexes. We doped Tomatidine into liposomes of cationic lipid with amide linker (A) from our lipid library. Six liposomal formulations (AT) of Lipid A (1 mM) with varying concentrations of Tomatidine (0–1 mM) were prepared and evaluated for their transfection efficacies. Owing to its signature characteristic of cell membrane permeability, Tomatidine modulated endocytosis process, enhanced the intracellular delivery of the lipoplexes, and in turn increased the transfection efficacy of cationic liposomes. Our findings provide ‘proof of concept’ for enhancing transfections in gene delivery applications with Tomatidine in cationic liposomal formulations. These findings can be further applied in lipid mediated gene therapy and drug delivery applications.

UR - https://www.sciencedirect.com/science/article/pii/S0005273618301780 ER - TY - JOUR T1 - Local injections of tacrolimus-loaded hydrogel reduce systemic immunosuppression-related toxicity in vascularized composite allotransplantation. JF - Transplantation Y1 - 2018 A1 - Dzhonova, Dzhuliya V A1 - Olariu, Radu A1 - Leckenby, Jonathan A1 - Banz, Yara A1 - Prost, Jean-Christophe A1 - Dhayani, Ashish A1 - Vemula, Praveen K A1 - Voegelin, Esther A1 - Taddeo, Adriano A1 - Rieben, Robert AB -

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

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

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

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

ER - TY - JOUR T1 - Local release of tacrolimus from hydrogel-based drug delivery system is controlled by inflammatory enzymes in vivo and can be monitored non-invasively using in vivo imaging. JF - PLoS One Y1 - 2018 A1 - Dzhonova, Dzhuliya A1 - Olariu, Radu A1 - Leckenby, Jonathan A1 - Dhayani, Ashish A1 - Vemula, Praveen Kumar A1 - Prost, Jean-Christophe A1 - Banz, Yara A1 - Taddeo, Adriano A1 - Rieben, Robert KW - Animals KW - Drug Delivery Systems KW - Humans KW - Hydrogels KW - Immunosuppressive Agents KW - Inflammation KW - Male KW - Rats KW - Rats, Inbred BN KW - Rats, Inbred Lew KW - Tacrolimus AB -

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

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

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

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

VL - 13 IS - 8 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 - A minimal "push-pull" bistability model explains oscillations between quiescent and proliferative cell states. JF - Mol Biol Cell Y1 - 2018 A1 - Krishna, Sandeep A1 - Laxman, Sunil AB -

A minimal model for oscillating between quiescent and growth/proliferation states, dependent on the availability of a central metabolic resource, is presented. From the yeast metabolic cycles, metabolic oscillations in oxygen consumption are represented as transitions between quiescent and growth states. We consider metabolic resource availability, growth rates, and switching rates (between states) to model a relaxation oscillator explaining transitions between these states. This frustrated bistability model reveals a required communication between the metabolic resource that determines oscillations and the quiescent and growth state cells. Cells in each state reflect memory, or hysteresis of their current state, and "push-pull" cells from the other state. Finally, a parsimonious argument is made for a specific central metabolite as the controller of switching between quiescence and growth states. We discuss how an oscillator built around the availability of such a metabolic resource is sufficient to generally regulate oscillations between growth and quiescence through committed transitions.

VL - 29 IS - 19 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 versatile LC-MS/MS approach for comprehensive, quantitative analysis of central metabolic pathways. JF - Wellcome Open Res Y1 - 2018 A1 - Walvekar, Adhish A1 - Rashida, Zeenat A1 - Maddali, Hemanth A1 - Laxman, Sunil AB -

Liquid chromatography-mass spectrometry (LC-MS/MS) based approaches are widely used for the identification and quantitation of specific metabolites, and are a preferred approach towards analyzing cellular metabolism. Most methods developed come with specific requirements such as unique columns, ion-pairing reagents and pH conditions, and typically allow measurements in a specific pathway alone. Here, we present a single column-based set of methods for simultaneous coverage of multiple pathways, primarily focusing on central carbon, amino acid, and nucleotide metabolism. We further demonstrate the use of this method for quantitative, stable isotope-based metabolic flux experiments, expanding its use beyond steady-state level measurements of metabolites. The expected kinetics of label accumulation pertinent to the pathway under study are presented with some examples. The methods discussed here are broadly applicable, minimize the need for multiple chromatographic resolution methods, and highlight how simple labeling experiments can be valuable in facilitating a comprehensive understanding of the metabolic state of cells.

VL - 3 ER - TY - JOUR T1 - Co-expression of Tbx6 and Sox2 identifies a novel transient neuromesoderm progenitor cell state. JF - Development Y1 - 2017 A1 - Javali, Alok A1 - Misra, Aritra A1 - Leonavicius, Karolis A1 - Acharyya, Debalina A1 - Vyas, Bhakti A1 - Sambasivan, Ramkumar KW - Animals KW - Body Patterning KW - Cell Differentiation KW - Cell Lineage KW - Embryonic Stem Cells KW - Gene Expression Regulation, Developmental KW - Mesoderm KW - Mice KW - Mice, Transgenic KW - Neural Tube KW - SOXB1 Transcription Factors KW - Spinal Cord KW - Transcription Factors AB -

Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6 cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the NMP cell state. Furthermore, we report a novel aspect of the documented mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate 'on' and progenitor state 'off', and thus has implications for the molecular mechanism driving NMP fate choice.

VL - 144 IS - 24 ER - TY - JOUR T1 - Conceptualizing Eukaryotic Metabolic Sensing and Signaling. JF - J Indian Inst Sci Y1 - 2017 A1 - Laxman, Sunil AB -

For almost all cells, nutrient availability, from glucose to amino acids, dictates their growth or developmental programs. This nutrient availability is closely coupled to the overall intracellular metabolic state of the cell. Therefore, cells have evolved diverse, robust and versatile modules to sense intracellular metabolic states, activate signaling outputs and regulate outcomes to these states. Yet, signaling and metabolism have been viewed as important but separate. This short review attempts to position aspects of intracellular signaling from a metabolic perspective, highlighting how conserved, core principles of metabolic sensing and signaling can emerge from an understanding of metabolic regulation. I briefly explain the nature of metabolic sensors, using the example of the AMP activated protein kinase (AMPK) as an "energy sensing" hub. Subsequently, I explore how specific central metabolites, particularly acetyl-CoA, but also -adenosyl methionine and SAICAR, can act as signaling molecules. I extensively illustrate the nature of a metabolic signaling hub using the specific example of the Target of Rapamycin Complex 1 (TORC1), and amino acid sensing. A highlight is the emergence of the lysosome/vacuole as a metabolic and signaling hub. Finally, the need to expand our understanding of the intracellular dynamics (in concentration and localization) of several metabolites, and their signaling hubs is emphasized.

VL - 97 IS - 1 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 - The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea JF - Development Y1 - 2017 A1 - Sasidharan, Vidyanand A1 - Marepally, Srujan A1 - Elliott, Sarah A. A1 - Baid, Srishti A1 - Lakshmanan, Vairavan A1 - Nayyar, Nishtha A1 - Bansal, Dhiru A1 - Sánchez Alvarado, Alejandro A1 - Vemula, Praveen Kumar A1 - Palakodeti, Dasaradhi AB -

Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system.

VL - 144 UR - http://dev.biologists.org/content/144/18/3211 ER - TY - JOUR T1 - Morphology transition in helical tubules of a supramolecular gel driven by metal ions. JF - Chem Commun (Camb) Y1 - 2017 A1 - Lalitha, Krishnamoorthy A1 - Sridharan, Vellaisamy A1 - Maheswari, C Uma A1 - Vemula, Praveen Kumar A1 - Nagarajan, Subbiah AB -

Our aim to access a particular chemical functionality on helical tubules has been achieved by the rational molecular design and synthesis of glucono-appended cardanol derivatives. For the first time, we report a chiral molecular packing with α-helical tubules, and chiral symmetry-breaking upon exposure to Cu that generated the final ordered structure via an in situ morphological transition without undergoing any phase change.

VL - 53 IS - 9 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 - Thiol trapping and metabolic redistribution of sulfur metabolites enable cells to overcome cysteine overload. JF - Microb Cell Y1 - 2017 A1 - Deshpande, Anup Arunrao A1 - Bhatia, Muskan A1 - Laxman, Sunil A1 - Bachhawat, Anand Kumar AB -

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

VL - 4 IS - 4 ER - TY - JOUR T1 - Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea. JF - G3 (Bethesda) Y1 - 2016 A1 - Lakshmanan, Vairavan A1 - Bansal, Dhiru A1 - Kulkarni, Jahnavi A1 - Poduval, Deepak A1 - Krishna, Srikar A1 - Sasidharan, Vidyanand A1 - Anand, Praveen A1 - Seshasayee, Aswin A1 - Palakodeti, Dasaradhi KW - 3' Untranslated Regions KW - Animals KW - Computational Biology KW - Genome, Helminth KW - Genome-Wide Association Study KW - High-Throughput Nucleotide Sequencing KW - MicroRNAs KW - Molecular Sequence Annotation KW - Platyhelminths KW - Poly A KW - Polyadenylation KW - Reproducibility of Results KW - RNA Interference KW - RNA Processing, Post-Transcriptional KW - RNA, Messenger AB -

In eukaryotes, 3' untranslated regions (UTRs) play important roles in regulating posttranscriptional gene expression. The 3'UTR is defined by regulated cleavage/polyadenylation of the pre-mRNA. The advent of next-generation sequencing technology has now enabled us to identify these events on a genome-wide scale. In this study, we used poly(A)-position profiling by sequencing (3P-Seq) to capture all poly(A) sites across the genome of the freshwater planarian, Schmidtea mediterranea, an ideal model system for exploring the process of regeneration and stem cell function. We identified the 3'UTRs for ∼14,000 transcripts and thus improved the existing gene annotations. We found 97 transcripts, which are polyadenylated within an internal exon, resulting in the shrinking of the ORF and loss of a predicted protein domain. Around 40% of the transcripts in planaria were alternatively polyadenylated (ApA), resulting either in an altered 3'UTR or a change in coding sequence. We identified specific ApA transcript isoforms that were subjected to miRNA mediated gene regulation using degradome sequencing. In this study, we also confirmed a tissue-specific expression pattern for alternate polyadenylated transcripts. The insights from this study highlight the potential role of ApA in regulating the gene expression essential for planarian regeneration.

VL - 6 IS - 10 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 - Decoding the stem cell quiescence cycle--lessons from yeast for regenerative biology. JF - J Cell Sci Y1 - 2015 A1 - Dhawan, Jyotsna A1 - Laxman, Sunil AB -

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

VL - 128 IS - 24 ER - TY - JOUR T1 - A quantitative metabolomics peek into planarian regeneration. JF - Analyst Y1 - 2015 A1 - Natarajan, Nivedita A1 - Ramakrishnan, Padma A1 - Lakshmanan, Vairavan A1 - Palakodeti, Dasaradhi A1 - Rangiah, Kannan KW - Animals KW - Calibration KW - Chromatography, High Pressure Liquid KW - Limit of Detection KW - Metabolomics KW - Planarians KW - Reference Standards KW - Regeneration KW - Reproduction, Asexual KW - Species Specificity KW - Tandem Mass Spectrometry AB -

The fresh water planarian species Schmidtea mediterranea is an emerging stem cell model because of its capability to regenerate a whole animal from a small piece of tissue. It is one of the best model systems to address the basic mechanisms essential for regeneration. Here, we are interested in studying the roles of various amines, thiols and nucleotides in planarian regeneration, stem cell function and growth. We developed mass spectrometry based quantitative methods and validated the differential enrichment of 35 amines, 7 thiol metabolites and 4 nucleotides from both intact and regenerating planarians. Among the amines, alanine in sexual and asparagine in asexual are the highest (>1000 ng/mg) in the intact planarians. The levels of thiols such as cysteine and GSH are 651 and 1107 ng mg(-1) in planarians. Among the nucleotides, the level of cGMP is the lowest (0.03 ng mg(-1)) and the level of AMP is the highest (187 ng mg(-1)) in both of the planarian strains. We also noticed increasing levels of amines in both anterior and posterior regenerating planarians. The blastema from day 3 regenerating planarians also showed higher amounts of many amines. Interestingly, the thiol (cysteine and GSH) levels are well maintained during planarian regeneration. This suggests an inherent and effective mechanism to control induced oxidative stress because of the robust regeneration and stem cell proliferation. Like in intact planarians, the level of cGMP is also very low in regenerating planarians. Surprisingly, the levels of amines and thiols in head regenerating blastemas are ∼3 times higher compared to those for tail regenerating blastemas. Thus our results strongly indicate the potential roles of amines, thiols and nucleotides in planarian regeneration.

VL - 140 IS - 10 ER -