@article {147302, title = {Computational Analysis of the Accumulation of Mutations in Therapeutically Important RNA Viral Proteins During Pandemics with Special Emphasis on SARS-CoV-2.}, journal = {J Mol Biol}, volume = {436}, year = {2024}, month = {2024 Jul 22}, pages = {168716}, abstract = {

Single stranded RNA viruses are primary causative agents for pandemics, causing extensive morbidity and mortality worldwide. A pivotal question in pandemic preparedness and therapeutic intervention is what are the specific mutations which are more likely to emerge during such global health crises? This study aims to identify markers for mutations with the highest probability of emergence in these pandemics, focusing on the SARS-CoV-2 spike protein, an essential and therapeutically significant viral protein, starting from sequence information from the onset of the pandemic until July 2022. Quite consistently, we observed that emerged mutations tended to demonstrate a high genetic score, which reflects high similarity of the type of codon required for translation between an amino acid and to the mutated one. Further, this pattern is also observed in therapeutically significant proteins of other ssRNA pandemic viruses, including influenza (HA, NA), spike proteins of Ebola, envelope of Dengue and Chikungunya. We propose that the genetic score serves as an initial indicator, preceding the actual impact of the mutation on viral fitness. Finally, we developed a comprehensive computational pipeline to further explore and predict the subsequent effects of mutations on viral fitness. We believe that our pipeline can narrow down and predict future mutations in therapeutically important viral proteins during a pandemic.

}, issn = {1089-8638}, doi = {10.1016/j.jmb.2024.168716}, author = {Sharma, Abhishek and Chandrashekar, C R and Krishna, Sudhir and Sowdhamini, Ramanathan} } @article {124446, title = {Bioinformatics analysis of mutations sheds light on the evolution of Dengue NS1 protein with implications in the identification of potential functional and druggable sites.}, journal = {Mol Biol Evol}, year = {2023}, month = {2023 Feb 16}, abstract = {

Non-structural protein (NS1) is a 350 amino acid long conserved protein in the dengue virus. Conservation of NS1 is expected due to its importance in dengue pathogenesis. The protein is known to exist in dimeric and hexameric states. The dimeric state is involved in its interaction with host proteins and viral replication, whereas the hexameric state is involved in viral invasion. In this work, we performed extensive structure and sequence analysis of NS1 protein, and uncovered the role of NS1 quaternary states in its evolution. A three-dimensional modeling of unresolved loop regions in NS1 structure is performed. {\textquoteright}Conserved{\textquoteright} and {\textquoteright}Variable{\textquoteright} regions within NS1 protein were identified from sequences obtained from patient samples and the role of compensatory mutations in selecting destabilizing mutations was identified. Molecular dynamics (MD) simulations were performed to extensively study the effect of a few mutations on NS1 structure stability and study compensatory mutations. Virtual saturation mutagenesis, predicting the effect of every individual amino acid substitution on NS1 stability sequentially, revealed virtual-conserved and variable sites. The increase in number of observed and virtual conserved regions across NS1 quaternary states suggests the role of higher order structure formation in its evolutionary conservation. Our sequence and structure analysis could enable in identifying possible protein-protein interfaces and druggable sites. Virtual screening of nearly 10,000 small molecules, including FDA-approved drugs, permitted us to recognize six drug-like molecules targeting the dimeric sites. These molecules could be promising due to their stable interactions with NS1 throughout the simulation.

}, issn = {1537-1719}, doi = {10.1093/molbev/msad033}, author = {Sharma, Abhishek and Krishna, Sudhir and Sowdhamini, Ramanathan} } @article {135854, title = {Building Indian Biomedical Leadership to Bridge the Gap Between Science, Primary Health Care and Public Health}, journal = {JOURNAL OF THE INDIAN INSTITUTE OF SCIENCE }, year = {2023}, month = {09/2023}, abstract = {

The Indian biomedical landscape has been characterized by the existence of somewhat polarized institutional structures and professional growth. While some scientific and public health challenges have been met with existing structures, there is still a large unmet scientific and public health need. Broadly, the physical separation of science, engineering, medical campuses and industry has led to silos of excellence and accomplishment with huge gaps in innovation and implementation. The lack of inter-disciplinary educational options has further reinforced the cultural underpinning of "guilds" that have found it difficult to collaborate. Strikingly, with almost a comparable number of institutions that train doctors in the allopathic or traditional disciplines such as Ayurveda, Unani etc., an "integrative medicine" framework has not emerged, apart from an over reliance on specialization at the expense of primary care. This paper is written by two physician-scientists, the first is located in a basic life science research center. The second, a practicing family physician, from the institutional anchor of a life sciences research institution. In this, we trace our experiences, primarily from a principal investigator{\textquoteright}s perspective, describing the scientific projects and try to explore the lessons learnt along the way. We will first describe the research in the lab{\textquoteright}s core area of human cervical cancer progression and our more recent effort with Dengue genomics and vaccine design. We then describe the lab{\textquoteright}s engagement with medical campuses and other agencies as well as review our various meetings and interactions so far with our colleagues from Africa to grasp what might be the "generalizable lessons" for the future. The Indian council of medical research initiated a program with Africa in health sciences. Building upon those interactions, we have taken some incremental steps in that direction and described our efforts.

}, doi = {10.1007/s41745-022-00320-1}, author = {Krishna, Sudhir and Prasad, Ramakrishna} } @article {126683, title = {Evolutionary dynamics of dengue virus in India.}, journal = {PLoS Pathog}, volume = {19}, year = {2023}, month = {2023 Apr 03}, pages = {e1010862}, abstract = {

More than a hundred thousand dengue cases are diagnosed in India annually, and about half of the country{\textquoteright}s population carries dengue virus-specific antibodies. Dengue propagates and adapts to the selection pressures imposed by a multitude of factors that can lead to the emergence of new variants. Yet, there has been no systematic analysis of the evolution of the dengue virus in the country. Here, we present a comprehensive analysis of all DENV gene sequences collected between 1956 and 2018 from India. We examine the spatio-temporal dynamics of India-specific genotypes, their evolutionary relationship with global and local dengue virus strains, interserotype dynamics and their divergence from the vaccine strains. Our analysis highlights the co-circulation of all DENV serotypes in India with cyclical outbreaks every 3-4 years. Since 2000, genotype III of DENV-1, cosmopolitan genotype of DENV-2, genotype III of DENV-3 and genotype I of DENV-4 have been dominating across the country. Substitution rates are comparable across the serotypes, suggesting a lack of serotype-specific evolutionary divergence. Yet, the envelope (E) protein displays strong signatures of evolution under immune selection. Apart from drifting away from its ancestors and other contemporary serotypes in general, we find evidence for recurring interserotype drift towards each other, suggesting selection via cross-reactive antibody-dependent enhancement. We identify the emergence of the highly divergent DENV-4-Id lineage in South India, which has acquired half of all E gene mutations in the antigenic sites. Moreover, the DENV-4-Id is drifting towards DENV-1 and DENV-3 clades, suggesting the role of cross-reactive antibodies in its evolution. Due to the regional restriction of the Indian genotypes and immunity-driven virus evolution in the country, ~50\% of all E gene differences with the current vaccines are focused on the antigenic sites. Our study shows how the dengue virus evolution in India is being shaped in complex ways.

}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1010862}, author = {Jagtap, Suraj and Pattabiraman, Chitra and Sankaradoss, Arun and Krishna, Sudhir and Roy, Rahul} } @article {116720, title = {Building Indian Biomedical Leadership to Bridge the Gap Between Science, Primary Health Care and Public Health.}, journal = {J Indian Inst Sci}, year = {2022}, month = {2022 Sep 07}, pages = {1-7}, abstract = {

The Indian biomedical landscape has been characterized by the existence of somewhat polarized institutional structures and professional growth. While some scientific and public health challenges have been met with existing structures, there is still a large unmet scientific and public health need. Broadly, the physical separation of science, engineering, medical campuses and industry has led to silos of excellence and accomplishment with huge gaps in innovation and implementation. The lack of inter-disciplinary educational options has further reinforced the cultural underpinning of "guilds" that have found it difficult to collaborate. Strikingly, with almost a comparable number of institutions that train doctors in the allopathic or traditional disciplines such as Ayurveda, Unani etc., an "integrative medicine" framework has not emerged, apart from an over reliance on specialization at the expense of primary care. This paper is written by two physician-scientists, the first is located in a basic life science research center. The second, a practicing family physician, from the institutional anchor of a life sciences research institution. In this, we trace our experiences, primarily from a principal investigator{\textquoteright}s perspective, describing the scientific projects and try to explore the lessons learnt along the way. We will first describe the research in the lab{\textquoteright}s core area of human cervical cancer progression and our more recent effort with Dengue genomics and vaccine design. We then describe the lab{\textquoteright}s engagement with medical campuses and other agencies as well as review our various meetings and interactions so far with our colleagues from Africa to grasp what might be the "generalizable lessons" for the future. The Indian council of medical research initiated a program with Africa in health sciences. Building upon those interactions, we have taken some incremental steps in that direction and described our efforts.

}, issn = {0970-4140}, doi = {10.1007/s41745-022-00320-1}, author = {Krishna, Sudhir and Prasad, Ramakrishna} } @article {104897, title = {HT-SELEX-based identification of binding pre-miRNA hairpin-motif for small molecules.}, journal = {Mol Ther Nucleic Acids}, volume = {27}, year = {2022}, month = {2022 Mar 08}, pages = {165-174}, abstract = {

Selective targeting of biologically relevant RNAs with small molecules is a long-standing challenge due to the lack of clear understanding of the binding RNA motifs for small molecules. The standard SELEX procedure allows the identification of specific RNA binders (aptamers) for the target of interest. However, more effort is needed to identify and characterize the sequence-structure motifs in the aptamers important for binding to the target. Herein, we described a strategy integrating high-throughput (HT) sequencing with conventional SELEX followed by bioinformatic analysis to identify aptamers with high binding affinity and target specificity to unravel the sequence-structure motifs of pre-miRNA, which is essential for binding to the recently developed new water-soluble small-molecule CMBL3aL. To confirm the fidelity of this approach, we investigated the binding of CMBL3aL to the identified motifs by surface plasmon resonance (SPR) spectroscopy and its potential regulatory activity on dicer-mediated cleavage of the obtained aptamers and endogenous pre-miRNAs comprising the identified motif in its hairpin loop. This new approach would significantly accelerate the identification process of binding sequence-structure motifs of pre-miRNA for the compound of interest and would contribute to increase the spectrum of biomedical application.

}, issn = {2162-2531}, doi = {10.1016/j.omtn.2021.11.021}, author = {Mukherjee, Sanjukta and Murata, Asako and Ishida, Ryoga and Sugai, Ayako and Dohno, Chikara and Hamada, Michiaki and Krishna, Sudhir and Nakatani, Kazuhiko} } @article {105164, title = {Immune profile and responses of a novel Dengue DNA vaccine encoding EDIII-NS1 consensus design based on Indo-African sequences.}, journal = {Mol Ther}, year = {2022}, month = {2022 Jan 06}, abstract = {

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a DENV-2 NS1 protein coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying of intracellular IFN-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios and immune gene profiling suggest a strong Th1-dominant immune response. Finally, passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design; offering a novel vaccine candidate with a possible broad spectrum protection and successful clinical translation either as a stand-alone or in a mix and match strategy.

}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2022.01.013}, author = {Sankaradoss, Arun and Jagtap, Suraj and Nazir, Junaid and Moula, Shefta-E and Modak, Ayan and Fialho, Joshuah and Iyer, Meenakshi and Shastri, Jayanthi S and Dias, Mary and Gadepalli, Ravisekhar and Aggarwal, Alisha and Vedpathak, Manoj and Agrawal, Sachee and Pandit, Awadhesh and Nisheetha, Amul and Kumar, Anuj and Bordoloi, Mahasweta and Shafi, Mohamed and Shelar, Bhagyashree and Balachandra, Swathi S and Damodar, Tina and Masika, Moses Muia and Mwaura, Patrick and Anzala, Omu and Muthumani, Kar and Sowdhamini, Ramanathan and Medigeshi, Guruprasad R and Roy, Rahul and Pattabiraman, Chitra and Krishna, Sudhir and Sreekumar, Easwaran} } @article {117964, title = {Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of matricellular protein Mindin.}, journal = {Cell Rep}, volume = {40}, year = {2022}, month = {2022 Sep 20}, pages = {111390}, abstract = {

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

}, keywords = {Carcinoma, Squamous Cell, Cell Line, Tumor, Epithelial Cells, Extracellular Matrix Proteins, Humans, Integrins, Neoplasm Proteins, Neoplasm Recurrence, Local, Neoplastic Stem Cells, Skin Neoplasms, Snail Family Transcription Factors}, issn = {2211-1247}, doi = {10.1016/j.celrep.2022.111390}, author = {Badarinath, Krithika and Dam, Binita and Kataria, Sunny and Zirmire, Ravindra K and Dey, Rakesh and Kansagara, Gaurav and Ajnabi, Johan and Hegde, Akshay and Singh, Randhir and Masudi, Tafheem and Sambath, Janani and Sachithanandan, Sasikala P and Kumar, Prashant and Gulyani, Akash and He, You-Wen and Krishna, Sudhir and Jamora, Colin} } @article {106150, title = {Versatile role of miR-24/24-1*/24-2*expression in cancer and other human diseases}, journal = {AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH }, volume = {14}, year = {2022}, month = {01/2022}, pages = {20-54}, abstract = {

MiRNAs (miRs) have been proven to be well-validated therapeutic targets. Emerging evidence has demonstrated that intricate, intrinsic and paradoxical functions of miRs are context-dependent because of their multiple upstream regulators, broad spectrum of downstream molecular targets and distinct expression in various tissues, organs and disease states. Targeted therapy has become an emerging field of research. One key for the development of successful miR-based/targeted therapy is to acquire integrated knowledge of its regulatory network and its association with disease phenotypes to identify critical nodes of the underlying pathogenesis. Herein, we systematically summarized the comprehensive role of miR-24-3p (miR-24), along with its passenger strands miR24-1-5p* (miR-24-1) and miR-24-2-5p* (miR-24-2), emphasizing their microenvironment, intracellular targets, and associated gene networks and regulatory phenotypes in 18 different cancer types and 13 types of other disorders. MiR-24 targets and regulates numerous genes in various cancer types and enhances the expression of several oncogenes (e.g., cMyc, BCL2 and HIF1), which are challenging in terms of druggability. In contrast, several tumor suppressor proteins (p21 and p53) have been reported to be downregulated by miR-24. MiR-24 also regulates the cell cycle and is associated with numerous cancer hallmarks such as apoptosis, proliferation, metastasis, invasion, angiogenesis, autophagy, drug resistance and other diseases pathogenesis. Overall, miR-24 plays an emerging role in the diagnosis, prognosis and pathobiology of various diseases. MiR-24 is a potential target for targeted therapy in the era of precision medicine, which expands the landscape of targetable macromolecules, including undruggable proteins.

}, url = {https://e-century.us/files/ajtr/14/1/ajtr0135181.pdf}, author = {Mukherjee, Sanjukta and Shelar, Bhagyashree and Krishna, Sudhir} } @article {96657, title = {Antimicrobial Resistance Profiling and Phylogenetic Analysis of Neisseria gonorrhoeae Clinical Isolates From Kenya in a Resource-Limited Setting}, journal = {Front. Microbiol}, year = {2021}, month = {07/2021}, abstract = {

Background: Africa has one of the highest incidences of gonorrhea. Neisseria gonorrhoeae is gaining resistance to most of the available antibiotics, compromising treatment across the world. Whole-genome sequencing (WGS) is an efficient way of predicting AMR determinants and their spread in the population. Recent advances in next-generation sequencing technologies like Oxford Nanopore Technology (ONT) have helped in the generation of longer reads of DNA in a shorter duration with lower cost. Increasing accuracy of base-calling algorithms, high throughput, error-correction strategies, and ease of using the mobile sequencer MinION in remote areas lead to its adoption for routine microbial genome sequencing. To investigate whether MinION-only sequencing is sufficient for WGS and downstream analysis in resource-limited settings, we sequenced the genomes of 14 suspected N. gonorrhoeae isolates from Nairobi, Kenya.

}, doi = {10.3389/fmicb.2021.647565}, author = {Juma, Meshack and Sankaradoss, Arun and Ndombi, Redcliff and Mwaura, Patrick and Damoda, Tina and Nazir, Junaid and Pandit, Awadhesh and Khurana, Rupsy and Masika, Moses and Chirchir, Ruth and Gachie, John and Krishna, Sudhir and Sowdhamini, Ramanathan and Anzala, Omu and Meenakshi, Iyer S.} } @article {92432, title = {CD24 gene inhibition and TIMP-4 gene upregulation by Imperata cylindrica{\textquoteright}s root extract prevents metastasis of CaSki cells via inhibiting PI3K/Akt/snail signaling pathway and blocking EMT}, journal = {J Ethnopharmacol}, year = {2021}, month = {04/2021}, abstract = {

Ethnopharmacological relevance: Imperata cylindrica (L.) Raeusch (Gramineae) is a medicinal spice traditionally used in the treatment of hypertension and cancer.

Aim of the study: To assess the anti-metastatic potential of the methanol extract of I. cylindrica roots and determined its mechanisms of action.

Material and methods: The growth inhibition activity of I. cylindrica root extract in vitro and in vivo in human cervical cancer. The scratch assay and Boyden Chamber assay were used to determine the anti-migrative and anti-invasion actions of the plant extract. The whole-genome gene expression profiling using RNA-Seq was performed to determine the differentially expressed genes in CaSki cells after exposure to I. cylindrica to identify its targeted genes related to metastasis. Using protein analysis (western blotting) and gene expression analysis (RTqPCR), the targeted pathways of the key genes that were initially identified with RNA-Seq, were evaluated.

Results: I. cylindrica extract showed dose-dependent cytotoxicity in vitro and in vivo in mice bearing tumors. Furthermore, I. cylindrica root extract significantly inhibited cell migration and cell invasion. After the genome-wide transcriptome analysis, we found that important genes involved in cancer progression and metastasis of cervical cancer, that is, CD24 and TIMP-4 were significantly downregulated and upregulated, respectively. Moreover, I. cylindrica root extract significantly inhibited the PI3/AKT/Snail signaling pathway and blocked the EMT of CaSki cells.

Conclusion: These findings provide an anti-metastatic mechanism of action of I. cylindrica root extract toward the human cervical cancer suggesting that this plant maybe developed into selective chemotherapy.

}, doi = {10.1016/j.jep.2021.114111}, author = {Nayim, P and Mbaveng, AT and Sanjukta, M and Rikesh, J and Kuete, V and Krishna, Sudhir} } @article {74444, title = {DETECTing Merkel Cell Polyomavirus in Merkel Tumors.}, journal = {Front Mol Biosci}, volume = {7}, year = {2020}, month = {2020}, pages = {10}, abstract = {

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post-integration (~80\% cases), or by UV-mediated DNA damage. Interestingly, overall survival of MCV-positive Merkel cell carcinoma patients is better, making this differential information of significant diagnostic and prognostic value. Also, MCV provides a direct target for therapy in MCC patients. Currently, the methods used for diagnosis of MCV in tumors are often discordant and unreliable. Here we used a guided molecular scissors based-DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an molecular diagnostic tool for MCV-positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme-gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that MCV DETECTR system can detect MCV integrated in Merkel tumor rapidly, specifically and with femto-molar sensitivity. Our study is a preliminary, proof-of-principle analysis showing the use of CRISPR for MCV diagnosis. Further validation in human tumor samples is needed for its clinical use in the near future. This new system is promising and we hope it can be coupled with immunohistochemical studies to diagnose the viral status of MCC in clinics soon.

}, issn = {2296-889X}, doi = {10.3389/fmolb.2020.00010}, author = {Arora, Reety and Gupta, Komal and Vijaykumar, Anjali and Krishna, Sudhir} } @article {43846, title = {Complete assembly of a dengue virus type 3 genome from a recent genotype III clade by metagenomic sequencing of serum.}, journal = {Wellcome Open Res}, volume = {3}, year = {2019}, month = {01/2019}, pages = {44}, abstract = {

Mosquito-borne flaviviruses, such as dengue and Japanese encephalitis virus (JEV), cause life-threatening diseases, particularly in the tropics. Here we performed unbiased metagenomic sequencing of RNA extracted from the serum of four patients and the plasma of one patient, all hospitalized at a tertiary care centre in South India with severe or prolonged febrile illness, together with the serum from one healthy control, in 2014. We identified and assembled a complete dengue virus type 3 sequence from a case of severe dengue fever. We also identified a small number of JEV sequences in the serum of two adults with febrile illness, including one with severe dengue. Phylogenetic analysis revealed that the dengue sequence belonged to genotype III. It has an estimated divergence time of 13.86 years from the most highly related Indian strains. In total, 11 amino acid substitutions were predicted for this strain in the antigenic envelope protein, when compared to the parent strain used for development of the first commercial dengue vaccine.\  We demonstrate that both genome assembly and detection of a low number of viral sequences are possible through the unbiased sequencing of clinical material. These methods may help ascertain causal agents for febrile illnesses with no known cause.

}, issn = {2398-502X}, doi = {10.12688/wellcomeopenres.14438.1}, author = {Dias, Mary and Pattabiraman, Chitra and Siddappa, Shilpa and Gowda, Malali and Shet, Anita and Smith, Derek and Muehlemann, Barbara and Tamma, Krishnapriya and Solomon, Tom and Jones, Terry and Krishna, Sudhir} } @inbook {79737, title = {Immunomodulatory and Therapeutic Potential of Marine Flora Products in the Treatment of Cancer}, booktitle = {Bioactive Natural Products for the Management of Cancer: from Bench to Bedside }, year = {2019}, pages = {139-166}, publisher = {Springer}, organization = {Springer}, chapter = {7}, abstract = {

Marine floras, constituting over 90\% of the oceanic biomass, are rich sources of potent chemicals predominantly belonging to polyphenols and sulphated polysaccharides. They act as potential sources of drugs with various pharmacological properties (such as antioxidant, anti-inflammatory, antibiotic, immunostimulatory, and anticancer). Although human history depicts the use of marine floras as medicine and nutrient supplement, the search for novel pharmaceutical compounds has mostly been limited to terrestrial floras. Marine floras are taxonomically diverse, largely productive, biologically active, and chemically unique, thereby offering great scope for discovery of new anticancer drugs. The mode of action of marine flora products is through activation of macrophages, induction of apoptosis, and prevention of oxidative damage of DNA, thereby controlling carcinogenesis. This chapter focuses on the immunomodulatory and therapeutic properties of marine flora-derived products in the context of increasing cancer incidences and demand for cheaper, safer, and potent anticancer drugs. It highlights the excellent therapeutic potential of the known marine flora-derived anticancer compounds, thereby encouraging researchers to actively participate in the marine natural product drug discovery for treatment and prevention of cancer.

}, isbn = {978-981-13-7606-1}, doi = {10.1007/978-981-13-7607-8_7}, author = {Singh, A and Krishna, Sudhir} } @article {48889, title = {Isolation and molecular characterization of Dengue virus clinical isolates from pediatric patients in New Delhi.}, journal = {Int J Infect Dis}, volume = {84}, year = {2019}, month = {07/2019}, pages = {S25-S33}, abstract = {

OBJECTIVES: To characterize the in vitro replication fitness, viral diversity and phylogeny of dengue viruses (DENV) isolated from Indian patients.

METHODS: DENV was isolated from whole blood collected from patients by passaging in cell culture. Passage 3 viruses were used for growth kinetics in C6/36 mosquito cells. Parallel efforts also focused on isolation of DENV RNA from plasma samples of the same patients and processed for next generation sequencing.

RESULTS: We were able to isolate 64 clinical isolates, mostly DENV-2, of which 25 were further used for growth curve analysis in vitro which showed a wide range of replication kinetics. Highest viral titers were of isolates from dengue with warning signs and severe dengue cases. We obtained full genome sequences of 21 DENV isolates. Genome analysis mapped the circulating DENV-2 strains to the Cosmopolitan genotype.

CONCLUSIONS: The replication kinetics of isolates from patients with mild or severe infection was not significantly different but the viral titers between the isolates varied by two orders of magnitude suggesting differences in replication fitness among the circulating DENV-2 isolates.

}, issn = {1878-3511}, doi = {10.1016/j.ijid.2018.12.003}, author = {Kar, Meenakshi and Nisheetha, Amul and Kumar, Anuj and Jagtap, Suraj and Shinde, Jitendra and Singla, Mohit and M, Saranya and Pandit, Awadhesh and Chandele, Anmol and Kabra, Sushil K and Krishna, Sudhir and Roy, Rahul and Lodha, Rakesh and Pattabiraman, Chitra and Medigeshi, Guruprasad R} } @article {66090, title = {Merkel cell polyomavirus is implicated in a subset of Merkel cell carcinomas, in the Indian subcontinent.}, journal = {Microb Pathog}, year = {2019}, month = {2019 Oct 07}, pages = {103778}, abstract = {

Merkel cell carcinoma is a rare, lethal cancer histopathologically composed of cells showing similarity with mechanoreceptor Merkel cells. Merkel cell tumors manifest in two distinct forms. While a virus called Merkel cell polyomavirus is involved in the pathogenesis of one form of Merkel tumor, the other is driven by ultraviolet (UV)-linked mutations. In this study we investigated 18 cases, from the Indian population, of Merkel cell carcinoma for immunohistochemical (IHC) expression of Merkel cell polyomavirus (MCV) T antigen, including 12 cases tested by PCR, to identify viral etiopathology. We tested the tumors with two sensitive antibodies (CM2B4 and Ab3), targeting the viral large T antigen protein and with PCR primers targeting the N terminus of T antigen. Overall, we observed 38.8\% (7/18) tumors displaying positive IHC expression of Merkel cell polyomavirus T antigen and 25\% (3/12) tumors showing positive results, by both, immunohistochemistry and PCR. This constitutes the first report from India showing implication of MCV in Merkel cell carcinomas. Moreover, this is one of the larger series of Merkel cell carcinomas, tested for MCV, by both immunohistochemistry and PCR, in this part of the world. These results further indicate that a slightly more number of such cases in India are likely to be caused by UV-linked damage, as opposed to Merkel cell polyomavirus mediated tumorigenesis, which is definitely implicated in a subset of cases.

}, issn = {1096-1208}, doi = {10.1016/j.micpath.2019.103778}, author = {Arora, Reety and Rekhi, Bharat and Chandrani, Pratik and Krishna, Sudhir and Dutt, Amit} } @article {66851, title = {Notch signalling in cervical cancer.}, journal = {Exp Cell Res}, year = {2019}, month = {2019 Oct 18}, pages = {111682}, abstract = {

The initial discovery of key developmental signalling pathways, largely using classical genetic approaches in model organisms, was followed by an intense burst of characterisation of the molecular components. Studies also began demonstrating a role for these pathways in oncogenesis. Patterns of mutations in Notch pathway components, such as those reported in sub-sets of hematological malignancies, have been easier to study, and the cumulative information is leading to potentially new therapies. However, it has been more challenging to clearly define the role of the Notch pathway in human solid tumours, given the absence of widespread specific activating or repressive mutations in key components of the pathway. In this review, we trace more than two decades of work looking at the role of Notch signalling in human cervical cancer progression. We document the contrasting reports on a tumour suppressive role and pro-oncogenic role in cervical cancers. However, an analysis of recent genomic data strikingly shows both widespread features of Notch expression and genetic changes that largely amplify positive regulators and delete negative controllers of the Notch pathway. This analysis reinforces a largely pro-oncogenic role for Notch signalling and lays the foundation for a nuanced exploration of synergistic and targeted therapies. Lastly, we further trace some of the complex challenges in advanced cervical cancer progression, including issues of cancer stem cells and metastasis.

}, issn = {1090-2422}, doi = {10.1016/j.yexcr.2019.111682}, author = {Rodrigues, Calvin and Joy, Leanna R and Sachithanandan, Sasikala P and Krishna, Sudhir} } @article {64329, title = {RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer.}, journal = {J Exp Clin Cancer Res}, volume = {38}, year = {2019}, month = {2019 Sep 05}, pages = {392}, abstract = {

BACKGROUND: Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response.

METHODS: Biochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies.

RESULTS: Enhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation.

CONCLUSIONS: Our data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment.

}, issn = {1756-9966}, doi = {10.1186/s13046-019-1385-7}, author = {Pranatharthi, Annapurna and Thomas, Pavana and Udayashankar, Avinash H and Bhavani, Chandra and Suresh, Srinag Bangalore and Krishna, Sudhir and Thatte, Jayashree and Srikantia, Nirmala and Ross, Cecil R and Srivastava, Sweta} } @article {53190, title = {A SUV39H1-low chromatin state characterises and promotes migratory properties of cervical cancer cells.}, journal = {Exp Cell Res}, year = {2019}, month = {2019 Feb 14}, abstract = {

Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1 cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.

}, issn = {1090-2422}, doi = {10.1016/j.yexcr.2019.02.010}, author = {Rodrigues, Calvin and Pattabiraman, Chitra and Vijaykumar, Anjali and Arora, Reety and Narayana, Suma Mysore and Kumar, Rekha V and Notani, Dimple and Varga-Weisz, Patrick and Krishna, Sudhir} } @article {36950, title = {Recovery of Five Complete Influenza A(H1N1)pdm09 Genome Sequences from the 2015 Influenza Outbreak in India by Metagenomic Sequencing.}, journal = {Genome Announc}, volume = {6}, year = {2018}, month = {2018 Jun 28}, abstract = {

Five complete (H1N1)pdm09 viral sequences were recovered from hospitalized individuals during the 2015 influenza outbreak by metagenomic sequencing. Four of the genomes are from oropharyngeal swabs, and one is from an isolate. All five sequences belong to an emerging 6B clade. Studying them further is critical for outbreak preparedness.

}, issn = {2169-8287}, doi = {10.1128/genomeA.00511-18}, author = {Dash, Paban Kumar and Pattabiraman, Chitra and Tandel, Kundan and Sharma, Shashi and Kumar, Jyoti S and Siddappa, Shilpa and Gowda, Malali and Krishna, Sudhir and Parida, Manmohan} } @article {12201, title = {MiRNA182 regulates percentage of myeloid and erythroid cells in chronic myeloid leukemia.}, journal = {Cell Death Dis}, volume = {8}, year = {2017}, month = {2017 Jan 12}, pages = {e2547}, abstract = {

The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.

}, issn = {2041-4889}, doi = {10.1038/cddis.2016.471}, author = {Arya, Deepak and Sachithanandan, Sasikala P and Ross, Cecil and Palakodeti, Dasaradhi and Li, Shang and Krishna, Sudhir} } @article {28247, title = {Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer.}, journal = {Sci Rep}, volume = {7}, year = {2017}, month = {2017 Dec 11}, pages = {17289}, abstract = {

Oestrogen controls Foxp3 expression in regulatory T cells (Treg cells) via a mechanism thought to involve oestrogen receptor alpha (ERα), but the molecular basis and functional impact of ERα signalling in Treg cells remain unclear. We report that ERα ligand oestradiol (E2) is significantly increased in human cervical cancer (CxCa) tissues and tumour-infiltrating Treg cells (CD4+CD25hiCD127low), whereas blocking ERα with the antagonist ICI 182,780 abolishes FOXP3 expression and impairs the function of CxCa infiltrating Treg cells. Using a novel approach of co-immunoprecipitation with antibodies to E2 for capture, we identified binding of E2:ERα complexes to FOXP3 protein in CxCa-derived Treg cells. Chromatin immunoprecipitation analyses of male blood Treg cells revealed ERα occupancy at the FOXP3 promoter and conserved non-coding DNA elements 2 and 3. Accordingly, computational analyses of the enriched regions uncovered eight putative oestrogen response elements predicted to form a loop that can activate the FOXP3 promoter. Together, these data suggest that E2-mediated ERα signalling is critical for the sustenance of FOXP3 expression and Treg cell function in human CxCa via direct interaction of ERα with FOXP3 promoter. Overall, our work gives a molecular insight into ERα signalling and highlights a fundamental role of E2 in controlling human Treg cell physiology.

}, issn = {2045-2322}, doi = {10.1038/s41598-017-17102-w}, author = {Adurthi, Sreenivas and Kumar, Mahesh M and Vinodkumar, H S and Mukherjee, Geetashree and Krishnamurthy, H and Acharya, K Kshitish and Bafna, U D and Uma, Devi K and Abhishekh, B and Krishna, Sudhir and Parchure, A and Alka, Murali and Jayshree, R S} } @article {3562, title = {CD66 and CD49f expressing cells are associated with distinct neoplastic phenotypes and progression in human cervical cancer.}, journal = {Eur J Cancer}, volume = {60}, year = {2016}, month = {2016 Apr 28}, pages = {166-178}, abstract = {

BACKGROUND: In this study, building on our recent work identifying a subset of CD66+ve cells with distinctive tumourigenic properties in human cervical cancers, we examine patterns of expression and function of these cells; to generate insights into the process of metastasis.

METHODS: Our broad approach in this study has been to compare the expression and function of two subsets marked by CD66 and CD49f. We use a combination of histopathology, immunostaining and flow cytometry, functional analysis of an established cervical cancer cell line and a retrospective analysis of a cohort of cervical cancer.

RESULTS: We noted CD66 expression associated with clusters of cells which are spindle shaped, SMA+ve, podoplanin+ve, phalloidin high, fibronectin high, plakoglobin low, ki67-ve and CK10+ve at the migratory phase along with features of partial EMT. Further, TGFβ1 a well known regulator of EMT, positively correlated with CD66 expression. The additional CD49f+ve subset at the leading invading front of migration was SMA-ve, phalloidin low, fibronectin low, plakoglobin high, Ki67+ve and CK14+ve. These data are consistent with a role for CD66 cells in metastatic invasion with a collective cell migration process co-opting the CD49f subset. Our retrospective analysis of a cohort is consistent with a role for CD66 in metastasis. However, the broad analysis of CD66, CD49f and TGFβ1 expression with patterns of overall survival points to a possible protective effect particularly for local recurrences. Hence, future studies focussing on potential heterogeneity within the CD66 subset\ along with the possible role of isoforms and intra-cellular roles would be essential.

}, issn = {1879-0852}, doi = {10.1016/j.ejca.2016.03.072}, author = {Ammothumkandy, Aswathy and Maliekal, Tessy Thomas and Bose, Mayil Vahanan and Rajkumar, Thangarajan and Shirley, Sundersingh and Thejaswini, B and Giri, Venkat G and Krishna, Sudhir} }