Furanocoumarins promote proteasomal degradation of viral HBx protein and down-regulate cccDNA transcription and replication of hepatitis B virus.

Nucleot(s)ide analogues, the current antiviral treatments against chronic hepatitis B (CHB) infection, are non-curative due to their inability to eliminate covalently closed circular DNA (cccDNA) from the infected hepatocytes. Preclinical studies have shown that coumarin derivatives can effectively reduce the HBV DNA replication. We evaluated the antiviral efficacy of thirty new coumarin derivatives in cell culture models for studying HBV. Furanocoumarins Fc-20 and Fc-31 suppressed the levels of pre-genomic RNA as well as cccDNA, and reduced the secretion of virions, HBsAg and HBeAg. The antiviral efficacies of Fc-20 and Fc31 improved further when used in combination with the hepatitis B antiviral drug Entecavir. There was a marked reduction in the intracellular HBx level in the presence of these furanocoumarins due to proteasomal degradation resulting in the down-regulation of HBx-dependent viral genes. Importantly, both Fc-20 and Fc-31 were non-cytotoxic to cells even at high concentrations. Further, our molecular docking studies confirmed a moderate to high affinity interaction between furanocoumarins and viral HBx via residues Ala3, Arg26 and Lys140. These data suggest that furanocoumarins could be developed as a new therapeutic for CHB infection.

RNA G-quadruplexes inhibit translation of the PE/PPE transcripts in Mycobacterium tuberculosis.

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.

Distinct epigenetic signatures of classical and hypervirulent Klebsiella pneumoniae.

Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical K. pneumoniae (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.

Genome profiling of uropathogenic E. coli from strictly defined community-acquired UTI in paediatric patients: a multicentric study.

BACKGROUND: Urinary tract infection (UTI) in children is a common bacterial infection. The emergence of extended-spectrum beta-lactamases (ESBLs) poses a major challenge against the treatment of uropathogens. We aimed to characterize the E. coli isolates recovered from children with UTI for their resistance profile and circulating sequence types (ST). METHODS: Children (> 1.5-18 years of age) from different community health centres of India with symptoms of UTI were enrolled. Isolates causing significant bacteriuria were identified by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and tested for antimicrobial susceptibility by the automated system, VITEK-2 (Biomeriux, Durhum, US). Nineteen E. coli isolates (15 ESBL positive and 4 ESBL negative) were sequenced in Oxford Nanopore platform followed by core-genome phylogeny, accessory genome cluster analysis, identification of sequence types, mobile genetic elements, genetic antimicrobial resistance markers. The correlation between detection of antimicrobial resistance genes with phenotypic resistance profiles was also investigated. RESULTS: Eleven percent of children had significant bacteriuria [male:female-1:1, > 50% were 11-18 years of age group]. E. coli was predominant (86%) followed by K. pneumoniae (11%). Susceptibility of E. coli was highest against fosfomycin (100%) followed by carbapenems (90.7%) and nitrofurantoin (88.8%). ST131 (15.8%) and ST167 (10.5%) found as high-risk clones with the presence of plasmid [IncFIB (63.1%), IncFIA (52.6%)], and composite transposon [Tn2680 (46.6%)] in many isolates. Few isolates coharboured multiple beta-lactamases including blaNDM-5 (33.3%), blaOXA-1 (53.3%), blaCTX-M-15 (60%) and blaTEM-4 (60%). CONCLUSIONS: This study highlights horizontal transmission of resistance genes and plasmids in paediatric patients at community centers across the nation harbouring multidrug-resistant genes such as blaNDM-5 and blaCTX-M-15 associated with high-risk clones ST131 and ST167. The data is alarming and emphasizes the need for rapid identification of resistance markers to reduce the spread in community. To our knowledge, this is the first multicentric study targeting paediatric UTI patients from the community setting of India.

Inhibition of Hepatitis B Virus (HBV) by Tachyplesin, a Marine Antimicrobial Cell-Penetrating Peptide.

We investigate the role of Tachyplesin (Tpl), a marine antimicrobial cell-penetrating peptide, as an anti-HBV agent. Our findings, using confocal microscopy and flow cytometry, demonstrate the internalization of FITC-Tpl in both Huh7 and HepG2 cell lines. Further, our results show that Tpl inhibits the expression of HBV proteins, including hepatitis B surface antigen (HBsAg) and hepatitis B 'e' antigen (HBeAg) in cell supernatants of human liver cell lines transfected with 1.3× pHBV. Interestingly Tpl also reduces levels of HBV pre-core RNA and HBV pregenomic RNA, suggesting that Tpl-mediated inhibition occurs at the early stages of HBV replication, including viral transcription. In addition, Tpl led to a significant reduction in levels of hepatitis B virion secretion. In sum, here we demonstrate the potent anti-HBV activity of Tpl at non-cytotoxic concentrations indicating the potential of Tpl to emerge as an effective therapeutic peptide against HBV.

Quantitative Assessment of Microbial Pathogens and Indicators of Wastewater Treatment Performance for Safe and Sustainable Water Reuse in India.

Currently, there is no data on the molecular quantification of microbial indicators of recycled water quality in India. In this study, multiple microbial pathogens and indicators of water quality were evaluated at three wastewater treatment plants located in two Indian cities (New Delhi and Jaipur) to determine the treatment performance and suitability of recycled water for safe and sustainable reuse applications. Real-time polymerase chain reaction (PCR) was used for the rapid evaluation of six human pathogens and six microbial indicators of fecal contamination. Among the microbial indicators, pepper mild mottle virus (PMMoV), F+RNA-GII bacteriophage, Bacteroides thetaiotamicron, and four human pathogens (Norovirus genogroups I & II, Giardia, and Campylobacter coli) were detected in all of the influent samples analyzed. This work suggests that the raw influents contain lower levels of noroviruses and adenoviruses and higher levels of Giardia compared to those reported from other geographic regions. Overall, the efficacy of the removal of microbial targets was over 93% in the final effluent samples, which is consistent with reports from across the world. PMMoV and Giardia were identified as the best microbial targets, from the microbial indicators spanning across bacteria, bacteriophages, DNA/RNA viruses, and protozoan parasites, by which to evaluate treatment performance and recycled water quality in Indian settings, as they were consistently present at high concentrations in untreated wastewater both within and across the sites. Also, they showed a strong correlation with other microbial agents in both the raw influent and in the final effluent. These findings provide valuable insights into the use of culture-independent molecular indicators that can be used to assess the microbial quality of recycled water in Indian settings. IMPORTANCE Wastewater treatment plants (WWTPs) have rapidly increased in India during the last decade. Nonetheless, there are only a few labs in India that can perform culture-based screening for microbial quality. In the last 2 years of the pandemic, India has witnessed a sharp increase in molecular biology labs. Therefore, it is evident that culture-independent real-time PCR will be increasingly used for the assessment of microbial indicators/pathogens in wastewater, especially in resource-limited settings. There is no data available on the molecular quantitation of microbial indicators from India. There is an urgent need to understand and evaluate the performance of widely used microbial indicators via molecular quantitation in Indian WWTPs. Our findings lay the groundwork for the molecular quantitation of microbial indicators in WWTPs in India. We have screened for 12 microbial targets (indicators and human pathogens) and have identified pepper mild mottle virus (PMMoV) and Giardia as the best molecular microbiological indicators in Indian settings.

Hepatitis B Virus-Encoded HBsAg Contributes to Hepatocarcinogenesis by Inducing the Oncogenic Long Noncoding RNA LINC00665 through the NF-κB Pathway.

Clinical and in vivo studies have demonstrated a role for hepatitis B virus (HBV)-encoded HBsAg (hepatitis B surface antigen) in HBV-related hepatocellular carcinoma (HCC); however, the underlying mechanisms remain largely unknown. Here, we investigated the role of HBsAg in regulating long noncoding RNAs (lncRNAs) involved in HCC progression. Our analysis of microarray data sets identified LINC00665 as an HBsAg-regulated lncRNA. Furthermore, LINC00665 is upregulated in liver samples from HBV-infected patients as well as in HCC, specifically in HBV-related HCC liver samples. These findings were supported by our in vitro data demonstrating that HBsAg, as well as HBV, positively regulates LINC00665 in multiple HBV cell culture models. Next, we evaluated the oncogenic potential of LINC00665 by its overexpression and CRISPR interference (CRISPRi)-based knockdown in various cell-based assays. LINC00665 promoted cell proliferation, migration, and colony formation but inhibited cell apoptosis in vitro. We then identified the underlying mechanism of HBsAg-mediated regulation of LINC00665. We used immunofluorescence assays to show that HBsAg enhanced the nuclear translocation of NF-κB factors in HepG2 cells, confirming that HBsAg activates NF-κB. Inhibition of NF-κB signaling nullified HBsAg-mediated LINC00665 upregulation, suggesting that HBsAg acts through NF-κB to regulate LINC00665. Furthermore, the LINC00665 promoter contains NF-κB binding sites, and their disruption abrogated HBsAg-induced LINC00665 upregulation. Finally, HBsAg facilitated the enrichment of the NF-κB factors NF-κB1, RelA, and c-Rel in the LINC00665 promoter. Taken together, this work shows that HBsAg can drive hepatocarcinogenesis by upregulating oncogenic LINC000665 through the NF-κB pathway, thereby identifying a novel mechanism in HBV-related HCC. IMPORTANCE Hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma (HCC). Numerous reports indicate an oncogenic role for HBV-encoded HBsAg; however, the underlying mechanisms are not well understood. Here, we studied the role of HBsAg in regulating lncRNAs involved in hepatocarcinogenesis. We demonstrate that HBsAg, as well as HBV, positively regulates oncogenic lncRNA LINC00665. The clinical significance of this lncRNA is highlighted by our observation that LINC00665 is upregulated in liver samples during HBV infection and HBV-related HCC. Furthermore, we show LINC00665 can drive hepatocarcinogenesis by promoting cell proliferation, colony formation, and cell migration and inhibiting apoptosis. Taken together, this work identified LINC00665 as a novel gene through which HBsAg can drive hepatocarcinogenesis. Finally, we show that HBsAg enhances LINC00665 levels in hepatocytes by activating the NF-κB pathway, thereby identifying a novel mechanism by which HBV may contribute to HCC.

A small molecule targeting hepatitis B surface antigen inhibits clinically relevant drug-resistant hepatitis B virus.

BACKGROUND: Currently approved oral antivirals for chronic HBV infection target the reverse transcriptase (RT) domain of the HBV polymerase. Emergence of drug resistance has been reported in a small proportion of chronic HBV patients on prolonged treatment with antivirals. We recently reported ZINC20451377, a small molecule targeting hepatitis B surface antigen (HBsAg) that effectively inhibits both WT HBV and tenofovir-resistant HBV. Due to the partial overlap between the RT domain and HBsAg, drug-resistant mutants are associated with corresponding mutations in HBsAg. OBJECTIVES: To evaluate the efficacy of ZINC20451377 against nine clinically relevant drug-resistant HBV mutants that lead to simultaneous mutations in the overlapping HBsAg gene. METHODS: Huh7 cells were transfected with 1.2× HBV replicons corresponding to WT HBV or drug-resistant HBV mutants and treated with different concentrations of ZINC20451377. We assessed the IC50 values of ZINC20451377 for HBsAg levels in the culture supernatants using ELISAs. HBV secretion was measured by immunocapture of secreted virions followed by real-time PCR quantitation of virion-associated DNA. RESULTS: ZINC20451377 led to a dose-dependent inhibition of secreted HBsAg encoded by WT HBV and all nine drug-resistant mutants tested and the IC50 values were in the low micromolar range. ZINC20451377 inhibited HBV secretion from drug-resistant mutants except for mutants harbouring the rtL180M + rtM204V (MV) mutation. CONCLUSIONS: The small molecule ZINC20451377 inhibits HBsAg and virion secretion in some of the clinically relevant drug-resistant HBV mutants. ZINC20451377 has a modest overall effect, and it was not effective against the MV mutants (lamivudine- and entecavir-resistant mutants).

Contrasting roles for G-quadruplexes in regulating human Bcl-2 and virus homologues KSHV KS-Bcl-2 and EBV BHRF1.

Herpesviruses are known to acquire several genes from their hosts during evolution. We found that a significant proportion of virus homologues encoded by HSV-1, HSV-2, EBV and KSHV and their human counterparts contain G-quadruplex motifs in their promoters. We sought to understand the role of G-quadruplexes in the regulatory regions of viral Bcl-2 homologues encoded by KSHV (KS-Bcl-2) and EBV (BHRF1). We demonstrate that the KSHV KS-Bcl-2 and the EBV BHRF1 promoter G-quadruplex motifs (KSHV-GQ and EBV-GQ) form stable intramolecular G-quadruplexes. Ligand-mediated stabilization of KS-Bcl-2 and BHRF1 promoter G-quadruplexes significantly increased the promoter activity resulting in enhanced transcription of these viral Bcl-2 homologues. Mutations disrupting KSHV-GQ and EBV-GQ inhibit promoter activity and render the KS-Bcl-2 and the BHRF1 promoters non-responsive to G-quadruplex ligand. In contrast, promoter G-quadruplexes of human bcl-2 gene inhibit promoter activity. Further, KS-Bcl-2 and BHRF1 promoter G-quadruplexes augment RTA (a virus-encoded transcription factor)-mediated increase in viral bcl-2 promoter activity. In sum, this work highlights how human herpesviruses have evolved to exploit promoter G-quadruplexes to regulate virus homologues to counter their cellular counterparts.

The Slowing Rate of CpG Depletion in SARS-CoV-2 Genomes Is Consistent with Adaptations to the Human Host.

Depletion of CpG dinucleotides in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes has been linked to virus evolution, host-switching, virus replication, and innate immune responses. Temporal variations, if any, in the rate of CpG depletion during virus evolution in the host remain poorly understood. Here, we analyzed the CpG content of over 1.4 million full-length SARS-CoV-2 genomes representing over 170 million documented infections during the first 17 months of the pandemic. Our findings suggest that the extent of CpG depletion in SARS-CoV-2 genomes is modest. Interestingly, the rate of CpG depletion is highest during early evolution in humans and it gradually tapers off, almost reaching an equilibrium; this is consistent with adaptations to the human host. Furthermore, within the coding regions, CpG depletion occurs predominantly at codon positions 2-3 and 3-1. Loss of ZAP (Zinc-finger antiviral protein)-binding motifs in SARS-CoV-2 genomes is primarily driven by the loss of the terminal CpG within the motifs. Nonetheless, majority of the CpG depletion in SARS-CoV-2 genomes occurs outside ZAP-binding motifs. SARS-CoV-2 genomes selectively lose CpGs-motifs from a U-rich context; this may help avoid immune recognition by TLR7. SARS-CoV-2 alpha-, beta-, and delta-variants of concern have reduced CpG content compared to sequences from the beginning of the pandemic. In sum, we provide evidence that the rate of CpG depletion in virus genomes is not uniform and it greatly varies over time and during adaptations to the host. This work highlights how temporal variations in selection pressures during virus adaption may impact the rate and the extent of CpG depletion in virus genomes.

Selective Depletion of ZAP-Binding CpG Motifs in HCV Evolution.

Hepatitis C virus (HCV) is a bloodborne pathogen that can cause chronic liver disease and hepatocellular carcinoma. The loss of CpGs from virus genomes allows escape from restriction by the host zinc-finger antiviral protein (ZAP). The evolution of HCV in the human host has not been explored in the context of CpG depletion. We analysed 2616 full-length HCV genomes from 1977 to 2021. During the four decades of evolution in humans, we found that HCV genomes have become significantly depleted in (a) CpG numbers, (b) CpG O/E ratios (i.e., relative abundance of CpGs), and (c) the number of ZAP-binding motifs. Interestingly, our data suggests that the loss of CpGs in HCV genomes over time is primarily driven by the loss of ZAP-binding motifs; thus suggesting a yet unknown role for ZAP-mediated selection pressures in HCV evolution. The HCV core gene is significantly enriched for the number of CpGs and ZAP-binding motifs. In contrast to the rest of the HCV genome, the loss of CpGs from the core gene does not appear to be driven by ZAP-mediated selection. This work highlights CpG depletion in HCV genomes during their evolution in humans and the role of ZAP-mediated selection in HCV evolution.

Biogenesis, characterization, and functions of mirtrons.

MicroRNAs (miRNAs) are major post-transcriptional regulators of gene expression. They base pair with the complementary target mRNA at the 3'UTR and modulate cellular processes by repressing the mRNA translation or degrading the mRNA. There are well-documented mechanisms of biogenesis of miRNA; however, a sizeable number of miRNAs are also produced by non-canonical pathways. Mirtrons represent a predominant class of non-canonical miRNAs. Mirtrons originate from intronic regions and are produced in a splicing-dependent and Drosha-independent manner. Mirtrons constitute about 15% of all miRNAs produced in a human body and have caught attention of researchers worldwide due to their unconventional origin, sequence characteristics, evolutionary dynamics, ability to regulate variety of cellular processes and their immense potential in disease therapeutics. In this comprehensive review we collate the research done in the past decade including biogenesis, sequence characteristics, regulation, and emerging therapeutic roles of mirtrons. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.

Vitamin D signaling inhibits HBV activity by directly targeting the HBV core promoter.

Clinical and epidemiological studies support a role for vitamin D in suppressing hepatitis B virus (HBV). This antiviral role of vitamin D is widely attributed to vitamin D receptor (VDR)/retinoid X receptor-mediated regulation of host immunomodulatory genes through vitamin D response elements (VDREs) in their promoters. Here, we investigated the ability of calcitriol (1α,25-dihydroxyvitamin D3, metabolically activated vitamin D) to directly regulate HBV activity through this signaling pathway. We observed that calcitriol selectively inhibited only the HBV core promoter without affecting the HBV-PreS1, HBV-PreS2/S, or HBx promoters. We then identified a VDRE cluster in the HBV core promoter that is highly conserved across most HBV genotypes. Disruption of this VDRE cluster abrogated calcitriol-mediated suppression of the HBV core promoter. Furthermore, we showed that VDR interacts directly with the VDRE cluster in the HBV core promoter independent of retinoid X receptor. This demonstrates that calcitriol inhibits HBV core promoter activity through a noncanonical calcitriol-activated VDR pathway. Finally, we observed that calcitriol suppressed expression of the canonical HBV core promoter transcripts, pregenomic RNA, and precore RNA in multiple HBV cell culture models. In addition, calcitriol inhibited the secretion of hepatitis B "e" antigen and hepatitis B surface antigen (HBV-encoded proteins linked to poor disease prognosis), without affecting virion secretion. Our findings identify VDR as a novel regulator of HBV core promoter activity and also explain at least in part the correlation of vitamin D levels to HBV activity observed in clinical studies. Furthermore, this study has implications on the potential use of vitamin D along with anti-HBV therapies, and lays the groundwork for studies on vitamin D-mediated regulation of viruses through VDREs in virus promoters.

A novel piperazine derivative that targets hepatitis B surface antigen effectively inhibits tenofovir resistant hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is a global problem. The loss of hepatitis B surface antigen (HBsAg) in serum is a therapeutic end point. Prolonged therapy with nucleoside/nucleotide analogues targeting the HBV-polymerase may lead to resistance and rarely results in the loss of HBsAg. Therefore, inhibitors targeting HBsAg may have potential therapeutic applications. Here, we used computational virtual screening, docking, and molecular dynamics simulations to identify potential small molecule inhibitors against HBsAg. After screening a million molecules from ZINC database, we identified small molecules with potential anti-HBV activity. Subsequently, cytotoxicity profiles and anti-HBV activities of these small molecules were tested using a widely used cell culture model for HBV. We identified a small molecule (ZINC20451377) which binds to HBsAg with high affinity, with a KD of 65.3 nM, as determined by Surface Plasmon Resonance spectroscopy. Notably, the small molecule inhibited HBsAg production and hepatitis B virion secretion (10 µM) at low micromolar concentrations and was also efficacious against a HBV quadruple mutant (CYEI mutant) resistant to tenofovir. We conclude that this small molecule exhibits strong anti-HBV properties and merits further testing.

KLa(0.95-x)GdxF4:Eu3+ hexagonal phase nanoparticles as luminescent probes for in vitro Huh-7 cancer cell imaging.

A facile chemical route is reported for synthesizing red-emitting photoluminescent/MRI multi-functional KLa(0.95-x)GdxF4:Eu3+ (x = 0 to 0.4) bio-compatible nanomaterials for targeted in vitro tumor imaging. Hexagonal phase pure nanoparticles show a significant and systematic change in morphology with enhanced photoluminescence due to the substitution of La3+ with Gd3+ ions. Single phase ß-KLa(0.95-x)GdxF4:Eu3+ exhibits multifunctional properties, both intense red emission and strong paramagnetism for high-contrast bioimaging applications. These silica capped magnetic/luminescent nanoparticles show long-term colloidal stability, optical transparency in water, strong red emission, and low cytotoxicity. The cellular uptake of coated nanoparticles was investigated in liver cancer cell line Huh-7. Our findings suggest that these nanoparticles can serve as highly luminescent imaging probes for in vitro applications with potential for in vivo and live cell imaging applications.

Insights on the biological role of ultrafine particles of size PM<0.25: A prospective study from New Delhi.

When the total ambient PM2.5 levels are several-fold higher than the recommended limit, it may be important to study the distributions of different sizes of particulate matter (PM). Here, we assess the distributions of various sizes of total PM2.5 for 12 months (on a monthly basis) in New Delhi, India. Importantly, we found that ultrafine particles (i.e., particles <0.5 µm) contribute significantly to total PM2.5. PM<0.25 were the most cytotoxic particles to human lung epithelial cells in all the 12 months. In addition, PM<0.25 were associated with significantly higher cytotoxicity per unit mass compared to other size fractions constituting PM2.5. For any given size of PM, the amount of reactive oxygen species (ROS) generated per unit mass is higher for the month of March as compared to that for the rest of the months in the year. The higher ROS generations for all sizes of PM collected in the month of March was not explained by differences in their metal content values. Our data suggests the lack of correlation between total PM2.5 levels and the highly cytotoxic PM<0.25. In summary, this work establishes the need for policy changes to routinely monitor PM<0.25 and the necessity to establish exposure limits for PM<0.25, especially when the total PM2.5 levels are breached.

Reactive oxygen species production and inflammatory effects of ambient PM2.5 -associated metals on human lung epithelial A549 cells "one year-long study": The Delhi chapter.

The fine particulate matter (PM2.5) was collected at academic campus of Indian Institute of Technology, Delhi, India from January-December 2017. The PM2.5 samples were analysed for carcinogenic (Cd, Cr, As, Ni, and Pb) and non-carcinogenic (V, Cu, Zn, Fe) trace metals and their elicited effects on carcinoma epithelial cell line A549. Toxicological testing was done with ELISA kit. Same analyses were repeated for standard reference material (NIST-1648a) represents urban particulate matter. The student-t test and spearman correlation were used for data analysis. The seasonality in PM2.5 mass concentration and chemical composition showed effect on biological outcomes. The PM2.5 in post-monsoon and winter had higher amount of trace metals compared to mass collected in pre-monsoon and monsoon. Following the trend in PM mass concentration significantly (p < 0.5) lower cell viability was observed in post-monsoon and winter compared to other two seasons. NIST UPM 1648(a) samples always had higher cytotoxicity compared to ambient PM2.5 Delhi sample. Strong association of Chromium, Nickel, Cadmium, and Zinc was observed with cell viability and reactive oxygen species (ROS) production. In winter IL-6, IL-8 production were 2.8 and 3 times higher than values observed in post-monsoon and 53 and 9 times higher than control. In winter season trace metals As, Cu, Fe, in pre-monsoon Cr, Ni, As, Pb, V, and Fe, in post-monsoon Cd and V strongly correlated with ROS generation. ROS production in winter and pre-monsoon seasons found to be 2.6 and 1.3 times higher than extremely polluted post-monsoon season which had 2 to 3 times higher PM2.5 concentration compared to winter and pre-monsoon. The result clearly indicated that the presence of Fe in winter and pre-monsoon seasons catalysed the ROS production, probably OH˙ radical caused high cytokines production which influenced the cell viability reduction, while in post-monsoon PM majorly composed of Pb, As, Fe and Cu and affected by photochemical smog formation showed significant association between ROS production with cell viability. Overall, in Delhi most toxic seasons for respiratory system are winter and post-monsoon and safest season is monsoon.

Analysis of G-quadruplexes upstream of herpesvirus miRNAs: evidence of G-quadruplex mediated regulation of KSHV miR-K12-1-9,11 cluster and HCMV miR-US33.

BACKGROUND: G-quadruplexes regulate gene expression, recombination, packaging and latency in herpesviruses. Herpesvirus-encoded miRNAs have been linked to important biological functions. The presence and the biological role of G-quadruplexes have not been studied in the regulatory regions of virus miRNA. We hypothesized that herpesvirus-encoded miRNAs are regulated by G-quadruplexes in their promoters. RESULTS: We analyzed the 1 kb regulatory regions of all herpesvirus-encoded miRNAs for the presence of putative quadruplex-forming sequences (PQS). Over two-third (67%) of the regulatory regions of herpesvirus miRNAs had atleast 1 PQS. The 200 bp region of the promoter proximal to herpesvirus miRNA is particularly enriched for PQS. We chose to study the G-quadruplex motifs in the promoters of miR-K12 cluster in Kaposi's sarcoma-associated Herpesvirus (KSHV miR-K12-1-9,11) and the miR-US33 encoded by Human Cytomegalovirus (HCMV miR-US33). Biophysical characterization indicates that the G-quadruplex motifs in the promoters of the KSHV miR-K12 cluster and the HCMV miR-US33 form stable intramolecular G-quadruplexes in vitro. Mutations disrupting the G-quadruplex motif in the promoter of the KSHV miR-K12 cluster significantly inhibits promoter activity, while those disrupting the motif in the promoter of HCMV miR-US33 significantly enhance the promoter activity as compared to that of the respective wild-type promoter. Similarly, the addition of G-quadruplex binding ligands resulted in the modulation of promoter activity of the wild-type promoters (with intact G-quadruplex) but not the mutant promoters (containing quadruplex-disrupting mutations). CONCLUSION: Our findings highlight previously unknown mechanisms of regulation of virus-encoded miRNA and also shed light on new roles for G-quadruplexes in herpesvirus biology.

Prion protein transcription is auto-regulated through dynamic interactions with G-quadruplex motifs in its own promoter.

Cellular prion protein (PrP) misfolds into an aberrant and infectious scrapie form (PrPSc) that lead to fatal transmissible spongiform encephalopathies (TSEs). Association of prions with G-quadruplex (GQ) forming nucleic acid motifs has been reported, but implications of these interactions remain elusive. Herein, we show that the promoter region of the human prion gene (PRNP) contains two putative GQ motifs (Q1 and Q2) that assume stable, hybrid, intra-molecular quadruplex structures and bind with high affinity to PrP. Here, we investigate the ability of PrP to bind to the quadruplexes in its own promoter. We used a battery of techniques including SPR, NMR, CD, MD simulations and cell culture-based reporter assays. Our results show that PrP auto-regulates its expression by binding and resolving the GQs present in its own promoter. Furthermore, we map this resolvase-like activity to the N-terminal region (residues 23-89) of PrP. Our findings highlight a positive transcriptional-translational feedback regulation of the PRNP gene by PrP through dynamic unwinding of GQs in its promoter. Taken together, our results shed light on a yet unknown mechanism of regulation of the PRNP gene. This work provides the necessary framework for a plethora of studies on understanding the regulation of PrP levels and its implications in prion pathogenesis.

A Prelude to Biogermylene Chemistry*.

The biological applications of germylenes remain unrealised owing to their unstable nature. We report the isolation of air-, water-, and culture-medium-stable germylene DPMGeOH (3; DPM=dipyrromethene ligand) and its potential biological application. Compound 3 exhibits antiproliferative effects comparable to that of cisplatin in human cancer cells. The cytotoxicity of compound 3 on normal epithelial cells is minimal and is similar to that of the currently used anticancer drugs. These findings provide a framework for a plethora of biological studies using germylenes and have important implications for low-valent main-group chemistry.