TY - JOUR T1 - Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System. JF - Methods Mol Biol Y1 - 2022 A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Ravi, Nithin Sam A1 - Mohankumar, Kumarasamypet M KW - Animals KW - CRISPR-Cas Systems KW - Gene Editing KW - Hematopoietic Stem Cell Transplantation KW - Hematopoietic Stem Cells KW - Mice KW - Transplantation, Autologous AB -

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

VL - 2429 ER - TY - JOUR T1 - Gene Editing in Human Induced Pluripotent Stem Cells Using Doxycycline-Inducible CRISPR-Cas9 System. JF - Methods Mol Biol Y1 - 2021 A1 - Thamodaran, Vasanth A1 - Rani, Sonam A1 - Velayudhan, Shaji R AB -

Induced pluripotent stem cells (iPSCs) generated from patients are a valuable tool for disease modelling, drug screening, and studying the functions of cell/tissue-specific genes. However, for this research, isogenic iPSC lines are important for comparison of phenotypes in the wild type and mutant differentiated cells generated from the iPSCs. The advent of gene editing technologies to correct or generate mutations helps in the generation of isogenic iPSC lines with the same genetic background. Due to the ease of programming, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-based gene editing tools have gained pace in gene manipulation studies, including investigating complex diseases like cancer. An iPSC line with drug inducible Cas9 expression from the Adeno-Associated Virus Integration Site 1 (AAVS1) safe harbor locus offers a controllable expression of Cas9 with robust gene editing. Here, we describe a stepwise protocol for the generation and characterization of such an iPSC line (AAVS1-PDi-Cas9 iPSC) with a doxycycline (dox)-inducible Cas9 expression cassette from the AAVS1 safe harbor site and efficient editing of target genes with lentiviral vectors expressing gRNAs. This approach with a tunable Cas9 expression that allows investigating gene functions in iPSCs or in the differentiated cells can serve as a versatile tool in disease modelling studies.

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 - Generic Molding Platform for Simple, Low‐Cost Fabrication of Polymeric Microneedles JF - Macromolecular Materials and Engineering Y1 - 2020 A1 - 7. Badnikar, K. A1 - Jayadevi, S. N. A1 - Pahal, S. A1 - Sripada, S. A1 - Nayak, M. M. A1 - Vemula, P. K. A1 - Subrahmanyam, D. N. AB -

Micromolding technology is widely used for the fabrication of polymer microneedles for transdermal and intradermal drug delivery applications. Geometric features of microneedles in molding are solely determined by geometry of the master mold template. Fabrication of master mold template usually involves costly and cumbersome technologies due to small feature sizes typical of microneedles. In this research, a novel molding platform is designed that is fabricated using low‐cost and simple techniques with flexibility of producing large number of microneedle geometries. The proposed molding platform eliminates need for developing multiple mold templates for fabrication of various geometries of polymer microneedles. Utility of this molding platform is demonstrated in polylactic acid‐based solid thermoplastic microneedles and polyacrylic acid‐based dissolvable microneedles with various aspect ratio settings. Various microneedles fabricated at heights differing with resolution of as low as 100 µm are successfully achieved using specified settings in the molding platform. The suitability of fabricated microneedles for drug delivery applications is evaluated by in vitro and in vivo testing.

VL - 305 IS - 5 ER - TY - JOUR T1 - Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3 variant. JF - Biophys Rev Y1 - 2020 A1 - Arif, Mohammed A1 - Nabavizadeh, Pooneh A1 - Song, Taejeong A1 - Desai, Darshini A1 - Singh, Rohit A1 - Bazrafshan, Sholeh A1 - Kumar, Mohit A1 - Wang, Yigang A1 - Gilbert, Richard J A1 - Dhandapany, Perundurai S A1 - Becker, Richard C A1 - Kranias, Evangelia G A1 - Sadayappan, Sakthivel AB -

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

VL - 12 IS - 4 ER - TY - JOUR T1 - Genetic drift and bottleneck do not influence diversity in Toll-like receptor genes at a small spatial scale in a Himalayan passerine. JF - Ecol Evol Y1 - 2020 A1 - Nandakumar, Mridula A1 - Ishtiaq, Farah AB -

Genetic diversity is important for long-term viability of a population. Low genetic diversity reduces persistence and survival of populations and increases susceptibility to diseases. Comparisons of the neutral markers with functional loci such as immune genes [Toll-like receptors; TLR] can provide useful insights into evolutionary potential of a species and how the diversity of pathogens and selection pressures on their hosts are directly linked to their environment. In this study, we compare genetic diversity in neutral (eleven microsatellite loci) and adaptive (seven TLR loci) loci to determine genetic variation in a nonmigratory western Himalayan passerine, the black-throated tit (), distributed across an elevation gradient with varying degree of pathogen-mediated selection pressure. We further compare the diversity in TLR loci with a high-elevation sister species, the white-throated tit (). Our results indicate a lack of population genetic structure in the black-throated tit and signatures of a past bottleneck. In contrast, we found high diversity in TLR loci and locus-specific (TLR7) signatures of pathogen-mediated selection, which was comparable to diversity in the white-throated tit. Levels of diversity at TLR5 locus corresponded very closely with neutral microsatellite variation. We found evidence of positive selection in TLR1LA, TLR5, and TLR7 loci highlighting the importance in pathogen recognition. Our finding demonstrates that reduction in neutral variation does not necessarily lead to reduction in functional genetic diversity and probably helps in revival of population in a widespread species.

VL - 10 IS - 21 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 - Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing. JF - Stem Cell Res Y1 - 2019 A1 - Giri, Subhajit A1 - Purushottam, Meera A1 - Viswanath, Biju A1 - Muddashetty, Ravi S AB -

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

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

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

VL - 34 ER - TY - JOUR T1 - Generation of two iPSC lines with either a heterozygous V717I or a heterozygous KM670/671NL mutation in the APP gene. JF - Stem Cell Res Y1 - 2019 A1 - Frederiksen, Henriette R A1 - Holst, Bjørn A1 - Ramakrishna, Sarayu A1 - Muddashetty, Ravi A1 - Schmid, Benjamin A1 - Freude, Kristine AB -

Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people worldwide. Mutations in the genes PSEN1, PSEN2 or APP are known to cause familial forms of AD with an early age of onset. In this study, specific pathogenic mutations in the APP gene were introduced into an iPSC line from a healthy individual by the use of CRISPR-Cas9. The study resulted in the generation of two new cell lines, one carrying the V717I APP mutation and one with the KM670/671NL APP mutation.

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

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

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

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

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

VL - 14 IS - 1 ER - TY - JOUR T1 - 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 - Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases. JF - PLoS One Y1 - 2016 A1 - Saha, Piu A1 - Yeoh, Beng San A1 - Singh, Rajbir A1 - Chandrasekar, Bhargavi A1 - Vemula, Praveen Kumar A1 - Haribabu, Bodduluri A1 - Vijay-Kumar, Matam A1 - Jala, Venkatakrishna R AB -

Numerous studies signify that diets rich in phytochemicals offer many beneficial functions specifically during pathologic conditions, yet their effects are often not uniform due to inter-individual variation. The host indigenous gut microbiota and their modifications of dietary phytochemicals have emerged as factors that greatly influence the efficacy of phytoceutical-based intervention. Here, we investigated the biological activities of one such active microbial metabolite, Urolithin A (UA or 3,8-dihydroxybenzo[c]chromen-6-one), which is derived from the ellagic acid (EA). Our study demonstrates that UA potently inhibits heme peroxidases i.e. myeloperoxidase (MPO) and lactoperoxidase (LPO) when compared to the parent compound EA. In addition, chrome azurol S (CAS) assay suggests that EA, but not UA, is capable of binding to Fe3+, due to its catechol-like structure, although its modest heme peroxidase inhibitory activity is abrogated upon Fe3+-binding. Interestingly, UA-mediated MPO and LPO inhibition can be prevented by innate immune protein human NGAL or its murine ortholog lipocalin 2 (Lcn2), implying the complex nature of host innate immunity-microbiota interactions. Spectral analysis indicates that UA inhibits heme peroxidase-catalyzed reaction by reverting the peroxidase back to its inactive native state. In support of these in vitro results, UA significantly reduced phorbol myristate acetate (PMA)-induced superoxide generation in neutrophils, however, EA failed to block the superoxide generation. Treatment with UA significantly reduced PMA-induced mouse ear edema and MPO activity compared to EA treated mice. Collectively, our results demonstrate that microbiota-mediated conversion of EA to UA is advantageous to both host and microbiota i.e. UA-mediated inhibition of pro-oxidant enzymes reduce tissue inflammation, mitigate non-specific killing of gut bacteria, and abrogate iron-binding property of EA, thus providing a competitive edge to the microbiota in acquiring limiting nutrient iron and thrive in the gut.

VL - 11 IS - 6 ER -