More than a hundred thousand dengue cases are diagnosed in India annually, and about half of the country'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.
Imperata cylindrica is traditionally used to cure several diseases including cancer, wounds, and hypertension. The present study was designed to investigate the anticancer activity of the methanolic ...root extract of I. cylindrica (IC-MeOH). The water-soluble tetrazolium-1 and colony formation assays were used to check the proliferation ability of the cells. Cell apoptosis and cell cycle were measured by flow cytometry-based fluorescence-activated cell sorting. The ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis was used for the metabolites profiling of IC-MeOH. Based on high-mass accuracy, spectral data, and previous reports, tentative compound identifications were assigned. Our findings revealed that IC-MeOH inhibited the proliferation of HeLa and CaSki cells. The plant extract was also found to induce a concentration- and time-dependent apoptosis and cell cycle arrest in the G0/G1 phase (IC50 value) in CaSki cell line. Analysis of IC-MeOH permitted the identification of 10 compounds already reported for their anticancer activity, epicatechin, curcumin, (-)-yatein, caffeic acid, myricetin, jatrorrhizine, harmaline, cinnamaldehyde, dobutamine, and syringin. In conclusion, IC-MeOH is a rich source of cytotoxic metabolites that inhibits human cervical cancer proliferation via apoptosis and cell cycle arrest.
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 tumors, 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.
•We investigated 18 Merkel cell carcinoma cases, from the Indian population to identify viral etiopathology.•Overall, 38.8% (7/18) tumors displayed positive IHC expression for MCV T antigen using antibodies CM2B4 and Ab3.•Three of the twelve tumors, (25%) showed positive MCV presence, by both, immunohistochemistry and PCR.•Our study is the first report from India showing implication of Merkel cell polyomavirus (MCV) in Merkel cell carcinomas.
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.
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.
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.
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.
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.
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.
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Using HT-SELEX, a binding sequence-structure motif in pre-miRNA for the small molecule CMBL3aL was identified. The fidelity of this approach was validated by SPR-based binding assays and in vitro dicer cleavage reactions of CMBL3aL with enriched RNA aptamer sequences. We further evaluated the potential applicability using endogenous pre-miRNAs comprising the characteristic binding sequence-structure motif for CMBL3aL.
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.
Methods
Using WGS, the isolates were confirmed to be cases of
N. gonorrhoeae
(
n
= 9), and there were three co-occurrences of
N. gonorrhoeae
with
Moraxella osloensis
and
N. meningitidis
(
n
= 2).
N. meningitidis
has been implicated in sexually transmitted infections in recent years. The near-complete
N. gonorrhoeae
genomes (
n
= 10) were analyzed further for mutations/factors causing AMR using an in-house database of mutations curated from the literature.
Results
We observe that ciprofloxacin resistance is associated with multiple mutations in both gyrA and parC. Mutations conferring tetracycline (
rpsJ
) and sulfonamide (
folP
) resistance and plasmids encoding beta-lactamase were seen in all the strains, and
tet(M)
-containing plasmids were identified in nine strains. Phylogenetic analysis clustered the 10 isolates into clades containing previously sequenced genomes from Kenya and countries across the world. Based on homology modeling of AMR targets, we see that the mutations in GyrA and ParC disrupt the hydrogen bonding with quinolone drugs and mutations in FolP may affect interaction with the antibiotic.
Conclusion
Here, we demonstrate the utility of mobile DNA sequencing technology in producing a consensus genome for sequence typing and detection of genetic determinants of AMR. The workflow followed in the study, including AMR mutation dataset creation and the genome identification, assembly, and analysis, can be used for any clinical isolate. Further studies are required to determine the utility of real-time sequencing in outbreak investigations, diagnosis, and management of infections, especially in resource-limited settings.
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