Publications by authors named "Arunachalam Ramaiah"

22 Publications

  • Page 1 of 1

ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling.

bioRxiv 2021 May 4. Epub 2021 May 4.

ORAI1 and STIM1 are the critical mediators of store-operated Ca entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using and knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, knockout cells showed reduced homeostatic cytoplasmic Ca concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca -dependent transcription factors of the activator protein 1 (AP-1) family and . Our results identify a novel role of ORAI1-mediated Ca signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.
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http://dx.doi.org/10.1101/2021.05.04.442548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109209PMC
May 2021

Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication.

Cell Rep 2021 04 18;35(1):108940. Epub 2021 Mar 18.

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA 90095, USA; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA 90095, USA; California NanoSystems Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA. Electronic address:

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.
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http://dx.doi.org/10.1016/j.celrep.2021.108940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969873PMC
April 2021

SARS-CoV-2 infection rewires host cell metabolism and is potentially susceptible to mTORC1 inhibition.

Nat Commun 2021 03 25;12(1):1876. Epub 2021 Mar 25.

Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles (UCLA), Los Angeles, CA, USA.

Viruses hijack host cell metabolism to acquire the building blocks required for replication. Understanding how SARS-CoV-2 alters host cell metabolism may lead to potential treatments for COVID-19. Here we profile metabolic changes conferred by SARS-CoV-2 infection in kidney epithelial cells and lung air-liquid interface (ALI) cultures, and show that SARS-CoV-2 infection increases glucose carbon entry into the TCA cycle via increased pyruvate carboxylase expression. SARS-CoV-2 also reduces oxidative glutamine metabolism while maintaining reductive carboxylation. Consistent with these changes, SARS-CoV-2 infection increases the activity of mTORC1 in cell lines and lung ALI cultures. Lastly, we show evidence of mTORC1 activation in COVID-19 patient lung tissue, and that mTORC1 inhibitors reduce viral replication in kidney epithelial cells and lung ALI cultures. Our results suggest that targeting mTORC1 may be a feasible treatment strategy for COVID-19 patients, although further studies are required to determine the mechanism of inhibition and potential efficacy in patients.
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http://dx.doi.org/10.1038/s41467-021-22166-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994801PMC
March 2021

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly.

BMC Biol 2021 02 10;19(1):28. Epub 2021 Feb 10.

Department of Ecology and Evolutionary Biology, University of California, Irvine, CA, 92697, USA.

Background: The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector's genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution.

Results: To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role.

Conclusion: Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.
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http://dx.doi.org/10.1186/s12915-021-00963-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876825PMC
February 2021

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.

Nat Commun 2020 11 3;11(1):5553. Epub 2020 Nov 3.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, 92697-3900, USA.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
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http://dx.doi.org/10.1038/s41467-020-19426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609566PMC
November 2020

Hippo Signaling Pathway Has a Critical Role in Zika Virus Replication and in the Pathogenesis of Neuroinflammation.

Am J Pathol 2020 04 5;190(4):844-861. Epub 2020 Feb 5.

Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California; Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, California; Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California. Electronic address:

Zika virus (ZIKV) is a reemerging human pathogen that causes congenital abnormalities, including microcephaly and eye disease. The cellular/molecular basis of ZIKV and host interactions inducing ocular and neuronal pathogenesis are unclear. Herein, we noted that the Hippo/Salvador-Warts-Hippo signaling pathway, which controls organ size through progenitor cell proliferation and differentiation, is dysregulated after ZIKV infection. In human fetal retinal pigment epithelial cells, there is an early induction of transcriptional coactivator, Yes-associated protein (YAP), which is later degraded with a corresponding activation of the TANK binding kinase 1/interferon regulatory factor 3 type I interferon pathway. YAP/transcriptional co-activator with a PDZ-binding domain (TAZ) silencing results in reduced ZIKV replication, indicating a direct role of Hippo pathway in regulating ZIKV infection. Using an in vivo Ifnar1 knockout mouse model, ZIKV infection was found to reduce YAP/TAZ protein levels while increasing phosphorylated YAP Ser127 in the retina and brain. Hippo pathway is activated in major cellular components of the blood-brain barrier, including endothelial cells and astrocytes. In addition, this result suggests AMP-activated protein kinase signaling pathway's role in regulating YAP/TAZ in ZIKV-infected cells. These data demonstrate that ZIKV infection might initiate a cross talk among AMP-activated protein kinase-Hippo-TBK1 pathways, which could regulate antiviral and energy stress responses during oculoneuronal inflammation.
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http://dx.doi.org/10.1016/j.ajpath.2019.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163250PMC
April 2020

Evolution and functional classification of mammalian copper amine oxidases.

Mol Phylogenet Evol 2019 10 24;139:106571. Epub 2019 Jul 24.

Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Turku, Finland. Electronic address:

Mammalian copper-containing amine oxidases (CAOs), encoded by four genes (AOC1-4) and catalyzing the oxidation of primary amines to aldehydes, regulate many biological processes and are linked to various diseases including inflammatory conditions and histamine intolerance. Despite the known differences in their substrate preferences, CAOs are currently classified based on their preference for either primary monoamines (EC 1.4.3.21) or diamines (EC 1.4.3.22). Here, we present the first extensive phylogenetic study of CAOs that, combined with structural analyses of the CAO active sites, provides in-depth knowledge of their relationships and guidelines for classification of mammalian CAOs into AOC1-4 sub-families. The phylogenetic results show that CAOs can be classified based on two residues, X1 and X2, from the active site motif: T/S-X1-X2-N-Y-D. Residue X2 discriminates among the AOC1 (Tyr), AOC2 (Gly), and AOC3/AOC4 (Leu) proteins, while residue X1 further classifies the AOC3 (Leu) and AOC4 (Met) proteins that so far have been poorly identified and annotated. Residues X1 and X2 conserved within each sub-family and located in the catalytic site seem to be the key determinants for the unique substrate preference of each CAO sub-family. Furthermore, one residue located at 10 Å distance from the catalytic site is different between the sub-families but highly conserved within each sub-family (Asp in AOC1, His in AOC2, Thr in AOC3 and Asn in AOC4) and likely contributes to substrate selectivity. Altogether, our results will benefit the design of new sub-family specific inhibitors and the design of in vitro tests to detect individual CAO levels for diagnostic purposes.
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http://dx.doi.org/10.1016/j.ympev.2019.106571DOI Listing
October 2019

Evidence for Highly Variable, Region-Specific Patterns of T-Cell Epitope Mutations Accumulating in Strains.

Front Immunol 2019 13;10:195. Epub 2019 Feb 13.

Centre for Infectious Disease Research, Indian Institute of Science, Bangalore, India.

Vaccines that confer protection through induction of adaptive T-cell immunity rely on understanding T-cell epitope (TCE) evolution induced by immune escape. This is poorly understood in tuberculosis (TB), an ancient, chronic disease, where CD4 T-cell immunity is of recognized importance. We probed 905 functionally validated, curated human CD4 T cell epitopes in 79 (Mtb) whole genomes from India. This screen resulted in identifying 64 mutated epitopes in these strains initially using a computational pipeline and subsequently verified by single nucleotide polymorphism (SNP) analysis. SNP based phylogeny revealed the 79 Mtb strains to cluster to East African Indian (EAI), Central Asian Strain (CAS), and Beijing (BEI) lineages. Eighty-nine percent of the mutated T-cell epitopes (mTCEs) identified in the 79 Mtb strains from India has not previously been reported. These mTCEs were encoded by genes with high nucleotide diversity scores including seven mTCEs encoded by six antigens in the top 10% of rapidly divergent Mtb genes encoded by these strains. Using a T cell functional assay readout, we demonstrate 62% of mTCEs tested to significantly alter CD4 T-cell IFNγ and/or IL2 secretion with associated changes in predicted HLA-DR binding affinity: the gain of function mutations displayed higher predicted HLA-DR binding affinity and conversely mutations resulting in loss of function displayed lower predicted HLA-DR binding affinity. Most mutated antigens belonged to the cell wall/cell processes, and, intermediary metabolism and respiration families though all known Mtb proteins encoded mutations. Analysis of the mTCEs in an SNP database of 5,310 global Mtb strains identified 82% mTCEs to be significantly more prevalent in Mtb strains isolated from India, including 36 mTCEs identified exclusively in strains from India. These epitopes had a significantly higher predicted binding affinity to HLA-DR alleles that were highly prevalent in India compared to HLA-DR alleles rare in India, highlighting HLA-DR maybe an important driver of these mutations. This first evidence of region-specific TCE mutations potentially employed by Mtb to escape host immunity has important implications for TB vaccine design.
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http://dx.doi.org/10.3389/fimmu.2019.00195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381025PMC
January 2020

Detection and distribution of Sca autotransporter protein antigens in diverse isolates of Orientia tsutsugamushi.

PLoS Negl Trop Dis 2018 09 20;12(9):e0006784. Epub 2018 Sep 20.

Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA, United States of America.

Orientia tsutsugamushi (Ots) frequently causes severe scrub typhus infections in the Asia-Pacific region. Korean investigators have demonstrated that Ots encodes five different autotransporter domain (ATD) proteins (ScaA-ScaE). ScaA functions as an adhesin and confers protective immunity in a lethal mouse model of Ots infection. Specific antibodies are detected against ScaA and ScaC in Korean scrub typhus patients. However, there is limited data on the distribution of the Sca protein genes in diverse isolates of Ots. By BLAST analysis with the conserved beta barrel autotransporter domain (ATD) regions of the sca proteins, we discovered a sixth gene scaF among 3 of 10 new partial Ots genome sequences available at NCBI GenBank (Sido, Karp, AFSC7). We designed two to seven specific TaqMan assays to detect the ATD for each of the six sca genes. The TaqMan assays among those for each sca gene which gave the greatest sensitivity and linearity with DNA log dilutions were then used for screening DNAs from Ots isolates grown in L929 mouse cells for sca genes. The sca prevalence survey was performed for all six sca genes with 178 DNAs from isolates from 12 countries. The survey results were confirmed by conventional PCR with primers from conserved regions of the passenger domains (PD) and ATD of the sca proteins. The ATD was highly conserved between the DNAs of different genotypes compared to the sca PD but each TaqMan assay was sca specific. The percentage positivity for 56 kDa and scaA genes in the 178 DNAs using Ha primers was 59.6% and 62.4%, respectively. Our scaA conventional ATD PCR assay was positive in 98.3% but scaA was present in all 178 DNAs (100%) by ATD TaqMan. scaB, scaC, scaD, scaE and scaF were detected in 33.7%, 97.8%, 93.8%, 97.2% and 43.3% isolates by ATD TaqMan, respectively. The ATDs of Ots sca genes are thus sufficiently conserved between different genotypes for molecular assay design. Four sca genes are widely distributed among diverse Ots isolates from diverse geographical areas. scaB and scaF were detected in fewer Ots isolates and absent from some available genome sequences. Whether the utility of the ScaA, ScaC, ScaD, and ScaE antigenic passenger protein domains exceeds that of the mixed 56 kDa type surface antigens of Ots now used in combination diagnostic assays needs to be determined before they can be considered as suitable alternative serological antigens for diagnosis of scrub typhus.
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http://dx.doi.org/10.1371/journal.pntd.0006784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168176PMC
September 2018

Biosynthesis of zinc oxide nanoparticles usingMangifera indica leaves and evaluation of their antioxidant and cytotoxic properties in lung cancer (A549) cells.

Enzyme Microb Technol 2018 Oct 25;117:91-95. Epub 2018 Jun 25.

Chemistry of Heterocycles & Natural Product Research Laboratory, Department of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore, TN 632014, India. Electronic address:

Green synthesis is an eco-friendly approach to nanoparticle production, which eliminates the use of toxic chemicals, high temperatures, and costly equipment needed for traditional physical and chemical synthesis methods. This eco-friendly approach was used in the present study to biosynthesize zinc oxide nanoparticles (ZnO NPs) from Mangifera indica (mango) leaves which were then evaluated for their antioxidant activity and cytotoxic effects on lung cancer A549 cells. Synthesized ZnO nanoparticles were characterized using UV-vis spectroscopy, XRD, SEM, and EDX analyses. The XRD and SEM analyses showed 45-60 nm as the size of synthesized nanoparticles, the pure crystal form of ZnO NPs, and the shape of the NPs as nearly spherical and hexagonal quartzite. The antioxidant potential of nanoparticles was estimated using a DPPH free radical scavenging assay. The percent of viable cells was inversely proportional to the concentration of ZnO nanoparticles at 25 μg/mL concentration. The MTT assay used for cytotoxicity evaluation depicted the significant cytotoxic effect of ZnO NPs against the A549 lung cancer cell line. The drop in the proportion of viable A549 cells after exposure to ZnO NPs was comparable to the effects of the standard drug used i.e. cyclophosphamide. Antioxidant activity of NPs was increased by increasing the concentration of NPs. The present biosynthesis approach is rapid, inexpensive and eco-friendly and it yielded highly stable ZnO NPs with significant antioxidant and anticancer potential. This is the first report of M. indicia -mediated synthesis of ZnO NPs as antioxidant and, anticancer agents for the treatment of lung cancer and subsequent therapeutic applications.
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http://dx.doi.org/10.1016/j.enzmictec.2018.06.009DOI Listing
October 2018

Complexity of type-specific 56 kDa antigen CD4 T-cell epitopes of Orientia tsutsugamushi strains causing scrub typhus in India.

PLoS One 2018 26;13(4):e0196240. Epub 2018 Apr 26.

Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, GA, United States.

Orientia tsutsugamushi (Ots) is an obligate, intracellular, mite-transmitted human pathogen which causes scrub typhus. Understanding the diversity of Ots antigens is essential for designing specific diagnostic assays and efficient vaccines. The protective immunodominant type-specific 56 kDa antigen (TSA) of Ots varies locally and across its geographic distribution. TSA contains four hypervariable domains. We bioinformatically analyzed 345 partial sequences of TSA available from India, most of which contain only the three variable domains (VDI-III) and three spacer conserved domains (SVDI, SVDII/III, SVDIII). The total number (152) of antigenic types (amino acid variants) varied from 14-36 in the six domains of TSA that we studied. Notably, 55% (787/1435) of the predicted CD4 T-cell epitopes (TCEs) from all the six domains had high binding affinities (HBA) to at least one of the prevalent Indian human leukocyte antigen (HLA) alleles. A surprisingly high proportion (61%) of such TCEs were from spacer domains; indeed 100% of the CD4 TCEs in the SVDI were HBA. TSA sequences from India had more antigenic types (AT) than TSA from Korea. Overall, >90% of predicted CD4 TCEs from spacer domains were predicted to have HBA against one or more prevalent HLA types from Indian, Korean, Asia-Pacific region or global population data sets, while only <50% of CD4 TCEs in variable domains exhibited such HBA. The phylogenetically and immunologically important amino acids in the conserved spacer domains were identified. Our results suggest that the conserved spacer domains are predicted to be functionally more important than previously appreciated in immune responses to Ots infections. Changes occurring at the TCE level of TSA may contribute to the wide range of pathogenicity of Ots in humans and mouse models. CD4 T-cell functional experiments are needed to assess the immunological significance of these HBA spacer domains and their role in clearance of Ots from Indian patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196240PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919512PMC
July 2018

Genome Sequence of Coxiella-Like Endosymbiont Strain CLE-RmD, a Bacterial Agent in the Cattle Tick (Rhipicephalus microplus) Deutsch Strain.

Genome Announc 2018 Mar 29;6(13). Epub 2018 Mar 29.

Rickettsial Zoonoses Branch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA

We report a partial genome sequence for the -like endosymbiont strain CLE-RmD, assembled from metagenomics data obtained from the southern cattle tick () Deutsch strain.
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http://dx.doi.org/10.1128/genomeA.00003-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876479PMC
March 2018

Comparative analysis of protein evolution in the genome of pre-epidemic and epidemic Zika virus.

Infect Genet Evol 2017 07 14;51:74-85. Epub 2017 Mar 14.

Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, United States; Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, United States; Department of Surgery, David Geffen School of Medicine, University of California at Los Angeles, CA 90095, United States. Electronic address:

Zika virus (ZIKV) causes microcephaly in congenital infection, neurological disorders, and poor pregnancy outcome and no vaccine is available for use in humans or approved. Although ZIKV was first discovered in 1947, the exact mechanism of virus replication and pathogenesis remains unknown. Recent outbreaks of Zika virus in the Americas clearly suggest a human-mosquito cycle or urban cycle of transmission. Understanding the conserved and adaptive features in the evolution of ZIKV genome will provide a hint on the mechanism of ZIKV adaptation to a new cycle of transmission. Here, we show comprehensive analysis of protein evolution of ZIKV strains including the current 2015-16 outbreak. To identify the constraints on ZIKV evolution, selection pressure at individual codons, immune epitopes and co-evolving sites were analyzed. Phylogenetic trees show that the ZIKV strains of the Asian genotype form distinct cluster and share a common ancestor with African genotype. The TMRCA (Time to the Most Recent Common Ancestor) for the Asian lineage and the subsequently evolved Asian human strains was calculated at 88 and 34years ago, respectively. The proteome of current 2015/16 epidemic ZIKV strains of Asian genotype was found to be genetically conserved due to genome-wide negative selection, with limited positive selection. We identified a total of 16 amino acid substitutions in the epidemic and pre-epidemic strains from human, mosquito, and monkey hosts. Negatively selected amino acid sites of Envelope protein (E-protein) (positions 69, 166, and 174) and NS5 (292, 345, and 587) were located in central dimerization domains and C-terminal RNA-directed RNA polymerase regions, respectively. The predicted 137 (92 CD4 TCEs; 45 CD8 TCEs) immunogenic peptide chains comprising negatively selected amino acid sites can be considered as suitable target for sub-unit vaccine development, as these sites are less likely to generate immune-escape variants due to strong functional constrains operating on them. The targeted changes at the amino acid level may contribute to better adaptation of ZIKV strains to human-mosquito cycle or urban cycle of transmission.
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http://dx.doi.org/10.1016/j.meegid.2017.03.012DOI Listing
July 2017

Ebolavirus evolves in human to minimize the detection by immune cells by accumulating adaptive mutations.

Virusdisease 2016 Jun 22;27(2):136-44. Epub 2016 Feb 22.

Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048 USA ; Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048 USA ; Department of Surgery, University of California at Los Angeles, Los Angeles, CA 90095 USA.

The current outbreak of Zaire ebolavirus (EBOV) lasted longer than the previous outbreaks and there is as yet no proven treatment or vaccine available. Understanding host immune pressure and associated EBOV immune evasion that drive the evolution of EBOV is vital for diagnosis as well as designing a highly effective vaccine. The aim of this study was to deduce adaptive selection pressure acting on each amino acid sites of EBOV responsible for the recent 2014 outbreak. Multiple statistical methods employed in the study include SLAC, FEL, REL, IFEL, FUBAR and MEME. Results show that a total of 11 amino acid sites from sGP and ssGP, and 14 sites from NP, VP40, VP24 and L proteins were inferred as positively and negatively selected, respectively. Overall, the function of 11 out of 25 amino acid sites under selection pressure exactly found to be involved in T cell and B-cell epitopes. We identified that the EBOV had evolved through purifying selection pressure, which is a predictor that is known to aid the virus to adapt better to the human host and subsequently reduce the efficiency of existing immunity. Furthermore, computational RNA structure prediction showed that the three synonymous nucleotide mutations in NP gene altered the RNA secondary structure and optimal base-pairing energy, implicating a possible effect on genome replication. Here, we have provided evidence that the EBOV strains involved in the recent 2014 outbreak have evolved to minimize the detection by T and B cells by accumulating adaptive mutations to increase the survival fitness.
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http://dx.doi.org/10.1007/s13337-016-0305-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908996PMC
June 2016

Profile of Inflammation-associated genes during Hepatic Differentiation of Human Pluripotent Stem Cells.

Data Brief 2015 Dec 9;5:871-8. Epub 2015 Nov 9.

Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA ; Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA ; Department of Surgery, University of California at Los Angeles, Los Angeles, CA 90095, USA.

Expression of genes associated with inflammation was analyzed during differentiation of human pluripotent stem cells (PSCs) to hepatic cells. Messenger RNA transcript profiles of differentiated endoderm (day 5), hepatoblast (day 15) and hepatocyte-like cells (day 21) were obtained by RNA sequencing analysis. When compared to endoderm cells an immature cell type, the hepatic cells (days 15 and 21) had significantly higher expression of acute phase protein genes including complement factors, coagulation factors, serum amyloid A and serpins. Furthermore, hepatic phase of cells expressed proinflammatory cytokines IL18 and IL32 as well as cytokine receptors IL18R1, IL1R1, IL1RAP, IL2RG, IL6R, IL6ST and IL10RB. These cells also produced CCL14, CCL15, and CXCL- 1, 2, 3, 16 and 17 chemokines. Endoderm cells had higher levels of chemokine receptors, CXCR4 and CXCR7, than that of hepatic cells. Sirtuin family of genes involved in aging, inflammation and metabolism were differentially regulated in endoderm and hepatic phase cells. Ligands and receptors of the tumor necrosis factor (TNF) family as well as downstream signaling factors TRAF2, TRAF4, FADD, NFKB1 and NFKBIB were differentially expressed during hepatic differentiation.
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http://dx.doi.org/10.1016/j.dib.2015.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669432PMC
December 2015

Characterization of type I interferon pathway during hepatic differentiation of human pluripotent stem cells and hepatitis C virus infection.

Stem Cell Res 2015 Sep 15;15(2):354-364. Epub 2015 Aug 15.

Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048, USA.

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-α and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-α treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs--LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival.
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http://dx.doi.org/10.1016/j.scr.2015.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4600668PMC
September 2015

Adaptive evolution of a novel avian-origin influenza A/H7N9 virus.

Genomics 2014 Dec 1;104(6 Pt B):545-53. Epub 2014 Nov 1.

Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627 412, Tamil Nadu, India; Centre for Infectious Disease Research, Indian Institute of Science, Bangalore 560 012, India(1). Electronic address:

In China, the recent outbreak of novel influenza A/H7N9 virus has been assumed to be severe, and it may possibly turn brutal in the near future. In order to develop highly protective vaccines and drugs for the A/H7N9 virus, it is critical to find out the selection pressure of each amino acid site. In the present study, six different statistical methods consisting of four independent codon-based maximum likelihood (CML) methods, one hierarchical Bayesian (HB) method and one branch-site (BS) method, were employed to determine if each amino acid site of A/H7N9 virus is under natural selection pressure. Functions for both positively and negatively selected sites were inferred by annotating these sites with experimentally verified amino acid sites. Comprehensively, the single amino acid site 627 of PB2 protein was inferred as positively selected and it function was identified as a T-cell epitope (TCE). Among the 26 negatively selected amino acid sites of PB2, PB1, PA, HA, NP, NA, M1 and NS2 proteins, only 16 amino acid sites were identified to be involved in TCEs. In addition, 7 amino acid sites including, 608 and 609 of PA, 480 of NP, and 24, 25, 109 and 205 of M1, were identified to be involved in both B-cell epitopes (BCEs) and TCEs. Conversely, the function of positions 62 of PA, and, 43 and 113 of HA was unknown. In conclusion, the seven amino acid sites engaged in both BCEs and TCEs were identified as highly suitable targets, as these sites will be predicted to play a principal role in inducing strong humoral and cellular immune responses against A/H7N9 virus.
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http://dx.doi.org/10.1016/j.ygeno.2014.10.012DOI Listing
December 2014

Detection of site-specific positive Darwinian selection on pandemic influenza A/H1N1 virus genome: integrative approaches.

Genetica 2013 Jun 26;141(4-6):143-55. Epub 2013 Mar 26.

Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi 627412, Tamil Nadu, India.

In the twenty-first century, the first pandemic novel human influenza A/H1N1virus (NIV) outbreak was reported at Mexico and USA on March and early April, 2009 respectively. The outbreak occurred among human populations due to the presence of meager or no immune response against newly emerged viruses. The success of vaccines and drugs depends on their low susceptibility to the formation of escape mutants in virus. Identification of excess, non-synonymous substitutions over synonymous ones is a main indicator of positive Darwinian selection in protein-coding genes of NIVs. The positive Darwinian selection operating on each site of proteins were inferred by computing ω, the ratio of the non-synonymous/synonymous substitutions [dN/dS (or) Ka/Ks], which was calculated by three different methods in terms of codon-based maximum likelihood, branch-site and empirical Bayesian methods under various models. Totally, nine sites from PB2, PB1, HA, M2 and NS1 are inferred as positively selected. The function for amino acid sites of NIVs proteins under positive selection are inferred by comparing the sites with experimentally determined functionally known amino acid sites. Completely 4 positively selected sites of PB1, HA and M2 are found to be involved in B-cell epitopes (BCEs). Interestingly, most of these sites are also involving in T-cell epitopes (TCEs). However, more sites under positive selection forces are involved in TCEs than those of BCEs. Amino acid sites engaged in both BCEs and TCEs should be measured as highly suitable targets, because these sites could induce the strong humoral and cellular immune responses against targets.
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http://dx.doi.org/10.1007/s10709-013-9713-xDOI Listing
June 2013

Genetic ancestor of external antigens of pandemic influenza A/H1N1 virus.

Interdiscip Sci 2012 Dec 26;4(4):282-90. Epub 2013 Jan 26.

Environmental Nanotechnology Division, Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, Tamilnadu, India.

The aim of the present investigation was to discover the genetic relationships of 2009 pandemic novel influenza A/H1N1 virus (NIV) external antigens Hemagglutinin (HA) and Neuraminidase (NA) with other influenza viruses by performing phylogenetic, comparative and statistical analyses. Phylogenetic trees of these two antigens show that the sequences of the NIV viruses are relatively homogeneous and these were derived from several viruses circulating in swine. The phylogenetic tree of HA shows that NIV had the closest relationship with North-American pig lineages whereas NA had with European pig lineages. In both segments, NIVs had the closest genetic relationship with swine influenza virus lineages. It strongly suggests that pigs are the most possible animal reservoir. Comparative analysis shows that among clade A, NIVs had very low genetic divergence as well as high similarity and also suffered strong purifying selection whereas neighbor clade B shows moderate values when compared to those of clades C-F. It indicates that classical swine influenza viruses present in clade B might be an ancestor of NIVs external antigens. The process of re-assortment occurred in classical swine influenza viruses. The mutation sites exclusively fixed in the NIV of swine and human along with vaccine strain provide an important suggestion for disease diagnosis and vaccine research.
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http://dx.doi.org/10.1007/s12539-012-0136-7DOI Listing
December 2012

Structural comparison of the active site channels in rodent and primate vascular adhesion protein-1.

J Neural Transm (Vienna) 2013 Jun 18;120(6):947-50. Epub 2013 Jan 18.

Structural Bioinformatics Laboratory, Department of Biosciences, Åbo Akademi University, Tykistökatu 6A, 20520, Turku, Finland.

In this study, we have made homology models of mouse, rat, and monkey vascular adhesion protein-1 (VAP-1) to reveal basis for the species-specific ligand recognition of VAP-1. Based on the structural comparisons, rodent VAP-1s have a narrower active site channel than primate VAP-1s. The variable residues in mouse and rat VAP-1, Phe447 from arm I and the polar residues from the first α-helix of the D3 domain together with C-terminal residues are likely to affect ligand recognition and binding.
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http://dx.doi.org/10.1007/s00702-013-0974-4DOI Listing
June 2013

Characterization and phylogenetic analysis of cellulase producing Streptomyces noboritoensis SPKC1.

Interdiscip Sci 2010 Jun 1;2(2):205-12. Epub 2010 May 1.

Division of Microbiology, Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, Kerala, India.

A cellulase producing strain of Actinomycetes was isolated from soil samples which were collected from Southwest ghats, Kerala, India at a depth of 6-12 inches and Actinomycetes was characterized by morphological, cultural, physiological, chemotaxonomical and phylogenetic analysis. The 16S rRNA region of this strain was amplified and sequenced. The Neighbor-joining and Maximum Parsimony algorithm with topology tree of 16S rRNA was constructed. Based on results of observation and phylogenetic analysis, the strain SPKC1 was proved to belong to the species Streptomyces noboritoensis with cellulase activity. The Carboxy Methyl Cellulose (CMCase) activities of the strain SPKC1 on eighth day an amount of 910 microg/ml of glucose, 210 microg/ml of protein and 850 mg/100 ml of growth (biomass) on ninth day were recorded. In exocellulase activity strain SPKC1 on first day an amount of 500 microg/ml glucose was produced.
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http://dx.doi.org/10.1007/s12539-010-0069-yDOI Listing
June 2010

Effect of ompR gene mutation in expression of ompC and ompF of Salmonella typhi.

Interdiscip Sci 2010 Jun 1;2(2):157-62. Epub 2010 May 1.

Centre for Biotechnology, Muthayammal College of Arts and Science, Rasipuram, TN, India.

In the present investigation, a total of 50 stool samples were collected from the food handlers to screen the typhoid asymptomatic carriers, positive result was yielded for 2 out of the 50 samples. Salmonella typhi was isolated and identified based on the cultural characteristics on BSA, Macconkey agar, XLD and phylogenetic analysis. The ompR region of these two strains was amplified, sequenced and a Neighbor-Joining algorithm tree of ompR was constructed. The isolates were designated as (Salmonella Strain) SS-3 and SS-5 respectively. The isolates were subjected to mutation using sodium chloride at various osmolarity concentrations in LB broth. Both the wild and mutant Salmonella typhi were used for the isolation of outer membrane protein. The outer membrane protein was isolated and compared with both the wild and mutated Salmonella typhi. The expression of outer membrane protein was showing the variation in the expression which were noticed by using SDS-PAGE. On the basis of the results, it was concluded that the ompR-envZ two component regulatory systems play an important role on the regulation of Vi polysaccharide synthesis in S. typhi, and that one of the environmental signals for this regulation may be osmolarity.
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http://dx.doi.org/10.1007/s12539-010-0067-0DOI Listing
June 2010
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