Evaluating Riboglow-FLIM probes for RNA sensing.

We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. In vitro measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.

G-quadruplex motifs in Neisseria gonorrhoeae as anti-gonococcal targets.

Neisseria gonorrhoeae is an obligate human pathogen that causes gonorrhea and has shown a vast emergence of multidrug resistance in recent times. It is necessary to develop novel therapeutic strategies to combat this multidrug-resistant pathogen. The non-canonical stable secondary structures of nucleic acids, G-quadruplexes (GQs), are reported to regulate gene expressions in viruses, prokaryotes, and eukaryotes. Herein, we explored the whole genome of N. gonorrhoeae to mine evolutionary conserved GQ motifs. The Ng-GQs were highly enriched in the genes involved in various important biological and molecular processes of N. gonorrhoeae. Five of these GQ motifs were characterized using biophysical and biomolecular techniques. The GQ-specific ligand, BRACO-19, showed a high affinity towards these GQ motifs and stabilized them in both in vitro and in vivo conditions. The ligand showed potent anti-gonococcal activity and modulated the gene expression of the GQ-harboring genes. Strikingly, BRACO-19 also altered the biofilm formation in N. gonorrhoeae and its adhesion and invasion of the human cervical epithelial cells. In summary, the present study showed a significant role of GQ motifs in N. gonorrhoeae biology and put forward a step closer towards the search for therapeutic measures in combating the emerging antimicrobial resistance in the pathogen. KEY POINTS: •Neisseria gonorrhoeae genome is enriched in non-canonical nucleic acid structures-G-quadruplexes. •These G-quadruplexes might regulate bacterial growth, virulence, and pathogenesis. •G-quadruplex ligands inhibit biofilm formation, adhesion, and invasion of the gonococcus bacterium.

Multifunctional Ultrasmall Theranostic Nanohybrids Developed by Ultrasonic Atomizer for Drug Delivery and Magnetic Resonance Imaging.

Theranostic nanoparticulate systems (TNPs) have shown potential in addressing problems related to spatial localization and temporally controlled release of drugs with the capabilities of real-time imaging to evaluate the progress of therapy. The current study reports the ultrasonic atomization-led synthesis of in vitro and in vivo evaluations of ultrasmall chitosan-based theranostic nanohybrid formulations with encapsulated doxorubicin (DOX) and iron-oxide magnetic nanoparticles. The nanohybrid particles are characterized using transmission electron microscopy, powder X-ray diffraction, FTIR, DOX encapsulation efficiency, in vitro release, cellular uptake, and toxicity. These formulations were also tested for the capability of invivo tumor reduction and simultaneous magnetic resonance imaging using Swiss albino mice. Ultrasonic atomizer-led synthesis resulted in chitosan-magnetic nanohybrids (CMNPs) having sizes of 15 ± 3 nm which comprise MNP of 10 ± 3 nm. The encapsulation of DOX in CMNP was around 25%, resulting in an 80% sustained release over 10 days at pH 5 and 7. CMNP was also found to be an efficient DOX delivery vehicle tested on cancer cells (HeLa). The CMNPs were able to reduce the tumor volume by 60% in 15 days. The inherent magnetic property and nanoscale size of CMNPs also provided for enhanced contrast efficiency in magnetic resonance imaging of tumors. Thus, such multifunctional theranostic nanoparticles can be an efficient tool for targeted diagnostic and therapeutic success.

Morpho-physiological and demographic responses of three threatened Ilex species to changing climate aligned with species distribution models in future climate scenarios.

The success of a species in future climate change scenarios depends on its morphological, physiological, and demographic adaptive responses to changing climate. The existence of threatened species against climate adversaries is constrained due to their small population size, narrow genetic base, and narrow niche breadth. We examined if ecological niche model (ENM)-based distribution predictions of species align with their morpho-physiological and demographic responses to future climate change scenarios. We studied three threatened Ilex species, viz., Ilex khasiana Purkay., I. venulosa Hook. f., and I. embelioides Hook. F, with restricted distribution in Indo-Burma biodiversity hotspot. Demographic analysis of the natural populations of each species in Meghalaya, India revealed an upright pyramid suggesting a stable population under the present climate scenario. I. khasiana was confined to higher elevations only while I. venulosa and I. embelioides had wider altitudinal distribution ranges. The bio-climatic niche of I. khasiana was narrow, while the other two species had relatively broader niches. The ENM-predicted potential distribution areas under the current (2022) and future (2050) climatic scenarios (General Circulation Models (GCMs): IPSL-CM5A-LR and NIMR-HADGEM2-AO) revealed that the distribution of highly suitable areas for the most climate-sensitive I. khasiana got drastically reduced. In I. venulosa and I. embelioides, there was an increase in highly suitable areas under the future scenarios. The eco-physiological studies showed marked variation among the species, sites, and treatments (p < 0.05), indicating the differential responses of the three species to varied climate scenarios, but followed a similar trend in species performance aligning with the model predictions.

DNA Aptamer Targets Mycobacterium tuberculosis DevR/DosR Response Regulator Function by Inhibiting Its Dimerization and DNA Binding Activity.

Tuberculosis is recognized as one of the major public health threats worldwide. The DevR-DevS (DosR/DosS) two-component system is considered a novel drug target in Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, owing to its central role in bacterial adaptation and long-term persistence. An increase in DevR levels and the decreased permeability of the mycobacterial cell wall during hypoxia-associated dormancy pose formidable challenges to the development of anti-DevR compounds. Using an in vitro evolution approach of Systematic Evolution of Ligands by EXponential enrichment (SELEX), we developed a panel of single-stranded DNA aptamers that interacted with Mtb DevR protein in solid-phase binding assays. The best-performing aptamer, APT-6, forms a G-quadruplex structure and inhibits DevR-dependent transcription in Mycobacterium smegmatis. Mechanistic studies indicate that APT-6 functions by inhibiting the dimerization and DNA binding activity of DevR protein. In silico studies reveal that APT-6 interacts majorly with C-terminal domain residues that participate in DNA binding and formation of active dimer species of DevR. To the best of our knowledge, this is the first report of a DNA aptamer that inhibits the function of a cytosolic bacterial response regulator. By inhibiting the dimerization of DevR, APT-6 targets an essential step in the DevR activation mechanism, and therefore, it has the potential to universally block the expression of DevR-regulated genes for intercepting dormancy pathways in mycobacteria. These findings also pave the way for exploring aptamer-based approaches to design and develop potent inhibitors against intracellular proteins of various bacterial pathogens of global concern.

Ni+2 permease system of Helicobacter pylori contains highly conserved G-quadruplex motifs.

The genome of a micro-organism contains all the information required for its survival inside its host cells. The guanine rich regions of the genome can form stable G-quadruplex structures that act as the regulators of gene expression. Herein, the completely sequenced genomes of Helicobacter pylori were explored for the identification and characterization of the conserved G-quadruplex motifs in this gastrointestinal pathogen. Initial in silico analysis revealed the presence of ~8241 GQ motifs in the H. pylori genome. Metal binding proteins of H. pylori are significantly enriched in the GQ motifs. Our study emphasizes the identification and characterization of four highly conserved G-quadruplex forming motifs (HPGQs) in the nickel transporter genes (nixA, niuB1, niuB2, and niuD) of the H. pylori. Nickel is a virulence determinant in H. pylori and is required as a co-factor for the urease and [NiFe] hydrogenase enzymes that are crucial for its survival in the stomach lining of humans. The presence of GQ motifs in these nickel transporter genes can affect their expression and may alter the functioning of Urease and [NiFe] hydrogenase. Similar to human and virus G-quadruplexes, targeting these conserved PGQs with bioactive molecules may represent a novel therapeutic avenue for combating infection of H. pylori. The identified HPGQs were characterized in-vitro by using CD spectroscopy, electrophoresis technique, and NMR spectroscopy at both acidic (4.5) and neutral pH (7.0). ITC revealed the specific interaction of these HPGQs with high affinity to the known G-quadruplex binding ligand, TMPyP4. The mTFP based reporter assay showed decrease in the gene expression of mTFP in the TMPyP4 treated cells as compared to the untreated and further affirmed the formation of stable G-quadruplex structures in the HPGQ motifs in vivo. This is the first report for characterizing G-quadruplex motifs in nickel transport-associated genes in the H. pylori bacterium.

Conserved G-Quadruplex Motifs Regulate Gene Expression in Neisseria meningitidis.

The noncanonical structures, G-quadruplexes (GQs), formed in the guanine-rich region of nucleic acids regulate various biological and molecular functions in prokaryotes and eukaryotes. Neisseria meningitidis is a commensal residing in a human's upper respiratory tract but occasionally becomes virulent, causing life-threatening septicemia and meningitis. The factors causing these changes in phenotypes are not fully understood. At the molecular level, regulatory components help in a clearer understanding of the pathogen's virulence and pathogenesis. Herein, genome analysis followed by biophysical assays and cell-based experiments revealed the presence of conserved GQ motifs in N. meningitidis. These GQs are linked to the essential genes involved in cell adhesion, pathogenesis, virulence, transport, DNA repair, and recombination. Primer extension stop assay, reporter assays, and quantitative real-time polymerase chain reaction (qRT-PCR) further affirmed the formation of stable GQs in vitro and in vivo. These results support the existence of evolutionarily conserved GQ motifs in N. meningitidis and uphold the usage of GQ-specific ligands as novel antimeningococcal therapeutics.

Access Leads to Success: Management of Gunshot Injury to Maxillofacial Region with Access Osteotomy for Bullet Retrieval.

Head, face and neck are three highly separate frame area that behave in a different way in phrases of gunshot injuries. Interpersonal violence, assaults, accidents and suicide attempts being the most common reason in most developed and developing countries. Morbidity and mortality to this area depends on the type of weapon used,entry and exit path and the distance from where it is fired. The complexity of facial skeleton and its close association with important vital structure makes the management of these gunshot wounds challenging in terms of accessibility, visibility and wound management. Here we present a case of access osteotomy in the form of maxillary Lefort I osteotomy for bullet retrieval lodged in nasopharyngeal area following gunshot injury due to interpersonal violence.

Mining of Ebola virus genome for the construction of multi-epitope vaccine to combat its infection.

Ebola virus is the primary causative agent of viral hemorrhagic fever that is an epidemic disease and responsible for the massive premature deaths in humans. Despite knowing the molecular mechanism of its pathogenesis, to date, no commercial or FDA approved multiepitope vaccine is available against Ebola infection. The current study focuses on designing a multi-epitope subunit vaccine for Ebola using a novel immunoinformatic approach. The best predicted antigenic epitopes of Cytotoxic-T cell (CTL), Helper-T cells (HTL), and B-cell epitopes (BCL) joined by various linkers were selected for the multi-epitope vaccine designing. For the enhanced immune response, two adjuvants were also added to the construct. Further analysis showed the vaccine to be immunogenic and non-allergenic, forming a stable and energetically favorable structure. The stability of the unbound vaccine construct and vaccine/TLR4 was elucidated via atomistic molecular dynamics simulations. The binding free energy analysis (ΔGBind = -194.2 ± 0.5 kcal/mol) via the molecular mechanics Poisson-Boltzmann docking scheme revealed a strong association and thus can initiate the maximal immune response. Next, for the optimal expression of the vaccine construct, its gene construct was cloned in the pET28a + vector system. In summary, the Ebola viral proteome was screened to identify the most potential HTLs, CTLs, and BCL epitopes. Along with various linkers and adjuvants, a multi-epitope vaccine is constructed that showed a high binding affinity with the immune receptor, TLR4. Thus, the current study provides a highly immunogenic multi-epitope subunit vaccine construct that may induce humoral and cellular immune responses against the Ebola infection.Communicated by Ramaswamy H. Sarma.

Design and Evaluation of Sustained Release of Ornidazole by Dental Inserts.

Aim & Background: Ornidazole is an antimicrobial drug used to treat certain types of vaginal, urinary tract, and interstitial infections. The study aims to formulate and evaluate the dental inserts by using a drug candidate to sustained drug release to improve patient compliance, reduce dosing frequency, reduce the risk of dose dumping, and avoid the first-pass metabolism. They have better therapeutic efficacy and fewer side effects. METHODS: The dental inserts were prepared using various polymers alone and in combination with the different ratios of polymers. The evaluation parameters like thickness, drug content, content uniformity, moisture reuptake, weight variation, swelling studies, and erosion studies of the optimized inserts were studied. The in-vivo studies were conducted to determine the reduction of pocket depth in human volunteers. RESULTS: The system containing ethylcellulose and hydroxyl methyl propyl cellulose K100M (4:1) formulation F6 was optimized because drug release was sustained up to 120 hrs concerning other formulations. Optimized formulation followed first-order kinetics and Peppas release kinetics via fickian diffusion. There was no swelling, itching, irritation, and no reduction in the pocket depth in in-vivo studies. CONCLUSION: The study concluded that dental inserts could extend the release of Ornidazole for many hours and also enhance bioavailability. Furthermore, they also help in avoiding the first-pass effect. In vivo studies' observations showed no itching, irritation, swelling, and pocket-depth reduction.

Inhibition of Zika virus replication by G-quadruplex-binding ligands.

Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control.

Scrutinizing the SARS-CoV-2 protein information for designing an effective vaccine encompassing both the T-cell and B-cell epitopes.

Novel SARS coronavirus (SARS-CoV-2) has caused a pandemic condition worldwide. It has been declared as a public health emergency of international concern by WHO in a very short span of time. The community transmission of this highly infectious virus has severely affected various parts of China, Italy, Spain, India, and USA, among others. The prophylactic solution against SARS-CoV-2 infection is challenging due to the high mutation rate of its RNA genome. Herein, we exploited a next-generation vaccinology approach to construct a multi-epitope vaccine candidate against SARS-CoV-2 that is predicted to have high antigenicity, safety, and efficacy to combat this deadly infectious agent. The whole proteome was scrutinized for the screening of highly conserved, antigenic, non-allergen, and non-toxic epitopes having high population coverage that can elicit both humoral and cellular mediated immune response against COVID-19 infection. These epitopes along with four different adjuvants, were utilized to construct a multi-epitope-vaccine candidate that can generate strong immunological memory response having high efficacy in humans. Various physiochemical analyses revealed the formation of a stable vaccine product having a high propensity to form a protective solution against the detrimental SARS-CoV-2 strain with high efficacy. The vaccine candidate interacted with immunological receptor TLR3 with a high affinity depicting the generation of innate immunity. Further, the codon optimization and in silico expression show the plausibility of the high expression and easy purification of the vaccine product. Thus, this present study provides an initial platform for the rapid generation of an efficacious protective vaccine for combating COVID-19.

Exploring Computational and Biophysical Tools to Study the Presence of G-Quadruplex Structures: A Promising Therapeutic Solution for Drug-Resistant Vibrio cholerae.

Vibrio cholerae, a gram-negative bacterium that causes cholera, has already caused seven major pandemics across the world and infects roughly 1.3-4 million people every year. Cholera treatment primarily involves oral rehydration therapy supplemented with antibiotics. But recently, multidrug-resistant strains of V. cholerae have emerged. High genomic plasticity further enhances the pathogenesis of this human pathogen. Guanines in DNA or RNA assemble to form G-quadruplex (GQ) structures which have begun to be seen as potential drug targeting sites for different pathogenic bacteria and viruses. In this perspective, we carried out a genome-wide hunt in V. cholerae using a bio-informatics approach and observed ∼85 G-quadruplex forming motifs (VC-PGQs) in chromosome I and ∼45 putative G-quadruplexs (PGQs) in chromosome II. Ten putative G-quadruplex forming motifs (VC-PGQs) were selected on the basis of conservation throughout the genus and functional analysis displayed their location in the essential genes encoding bacterial proteins, for example, methyl-accepting chemotaxis protein, orotate phosphoribosyl transferase protein, amidase proteins, etc. The predicted VC-PGQs were validated using different bio-physical techniques, including Nuclear Magnetic Resonance spectroscopy, Circular Dichroism spectroscopy, and electrophoretic mobility shift assay, which demonstrated the formation of highly stable GQ structures in the bacteria. The interaction of these VC-PGQs with the known specific GQ ligand, TMPyP4, was analyzed using ITC and molecular dynamics studies that displayed the stabilization of the VC-PGQs by the GQ ligands and thus represents a potential therapeutic strategy against this enteric pathogen by inhibiting the PGQ harboring gene expression, thereby inhibiting the bacterial growth and virulence. In summary, this study reveals the presence of conserved GQ forming motifs in the V. cholerae genome that has the potential to be used to treat the multi-drug resistance problem of the notorious enteric pathogen.

An Aptamer Linked Immobilized Sorbent Assay (ALISA) to Detect Circulatory IFN-α, an Inflammatory Protein among Tuberculosis Patients.

Dysregulation of IFN-α is the basis for pathogenesis of autoimmune as well as infectious diseases. Identifying inflammatory signatures in peripheral blood of patients is an approach for monitoring active infection. Hence, estimation of type I IFNs as an inflammatory biomarker to scrutinize disease status after treatment is useful. Accordingly, an Aptamer Linked Immobilized Sorbent Assay (ALISA) for the detection of IFN-α in serum samples was developed. Sixteen aptamers were screened for their ability to bind IFN-α. Aptamer IFNα-3 exhibited specificity for IFN-α with no cross-reactivity with interferons ß and γ and human serum albumin. The disassociation constant (Kd) was determined to be 3.96 ± 0.36 nM, and the limit of detection was ∼2 ng. The characterized IFNα-3 aptamer was used in ALISA to screen tuberculosis (TB) patients' sera. An elevated IFN-α level in sera derived from untreated TB patients (median = 0.31), compared to nontuberculous household contacts (median = 0.13) and healthy volunteers (median = 0.12), and further a decline in IFN-α level among treated patients (median = 0.13) were seen. The ALISA assay facilitates direct estimation of inflammatory protein(s) in circulation unlike mRNA estimation by real time PCR. Designing of aptamers similar to the IFNα-3 aptamer provides a novel approach to assess other inflammatory protein(s) in patients before, during, and after completion of treatment and would denote clinical improvement in successfully treated patients.

G-quadruplex stabilization in the ions and maltose transporters gene inhibit Salmonella enterica growth and virulence.

The G-quadruplex structure is a highly conserved drug target for preventing infection of several human pathogens. We tried to explore G-quadruplex forming motifs as promising drug targets in the genome of Salmonella enterica that causes enteric fever in humans. Herein, we report three highly conserved G-quadruplex motifs (SE-PGQ-1, 2, and 3) in the genome of Salmonella enterica. Bioinformatics analysis inferred the presence of SE-PGQ-1 in the regulatory region of mgtA, SE-PGQ-2 in ORF of entA, and SE-PGQ-3 in the promoter region of malE and malK genes. The G-quadruplex forming sequences were confirmed by biophysical and biomolecular techniques. Cellular studies affirm the inhibitory effect of G-quadruplex specific ligands on Salmonella enterica growth. Further, PCR inhibition, reporter based assay, and RT-qPCR assays emphasize the biological relevance of G-quadruplexes in these genes. Thus, this study confirmed the presence of G-quadruplex motifs in Salmonella enterica and characterized them as a promising drug target.

Conserved G-Quadruplex Motifs in Gene Promoter Region Reveals a Novel Therapeutic Approach to Target Multi-Drug Resistance Klebsiella pneumoniae.

An opportunistic pathogen, Klebsiella pneumoniae is known to cause life-threating nosocomial infection with a high rate of morbidity and mortality. Evolutions of multi-drug-resistant and hyper-virulent strains of K. pneumoniae make the situation worse. Currently, there is no incisive drug molecule available for drug-resistant hyper-virulent K. pneumoniae infection that emphasizes the need for identification of novel and more promising drug targets in K. pneumoniae. Recently, various non-canonical structures of nucleic acids especially G-quadruplex (G4) motifs have been identified as potential therapeutic targets against several human pathogenic bacteria and viruses including Mycobacterium tuberculosis, Streptococcus pneumoniae, human immunodeficiency virus (HIV), Ebola, and Nipah. Therefore, in present study we screened the K. pneumoniae genomes for identification of evolutionary conserved G4 structure-forming motifs as promising anti-bacterial drug targets. Bioinformatics analysis revealed the presence of six highly conserved G4 motifs in the promoter region of five essential genes that play a critical role in nutrient transport and metabolism. Biophysical studies showed the formation of G4 structure by these conserved motifs. Circular Dichroism melting analysis showed the stabilization of these G4 motifs by a well-known G4-stabilizing agent, BRACO-19. The stabilization of these motifs by BRACO-19 was also able to stop the primer extension process, which is an essential phenomenon for expression of the G4-harboring gene. The addition of G4-specific ligand at low micromolar range was observed to be lethal for the growth of this bacteria and negatively controlled the expression of the G4-harboring genes via G4 structure stabilization. These observations strengthen the formation of G4 structures by the predicted G4 motif in vivo, which can be stabilized by G4 ligands like BRACO-19. This stabilization of G4 structures can attenuate the expression of G4-harboring essential genes and thus play a critical role in the regulation of gene expression. Thus, taking all given result in consideration, for the first time, this study showed the new therapeutic avenue for combating K. pneumoniae infection by characterizing the conserved G4 motifs as promising therapeutic targets.

Discovery of New Hydroxyethylamine Analogs against 3CLpro Protein Target of SARS-CoV-2: Molecular Docking, Molecular Dynamics Simulation, and Structure-Activity Relationship Studies.

The novel coronavirus, SARS-CoV-2, has caused a recent pandemic called COVID-19 and a severe health threat around the world. In the current situation, the virus is rapidly spreading worldwide, and the discovery of a vaccine and potential therapeutics are critically essential. The crystal structure for the main protease (Mpro) of SARS-CoV-2, 3-chymotrypsin-like cysteine protease (3CLpro), was recently made available and is considerably similar to the previously reported SARS-CoV. Due to its essentiality in viral replication, it represents a potential drug target. Herein, a computer-aided drug design (CADD) approach was implemented for the initial screening of 13 approved antiviral drugs. Molecular docking of 13 antivirals against the 3-chymotrypsin-like cysteine protease (3CLpro) enzyme was accomplished, and indinavir was described as a lead drug with a docking score of -8.824 and a XP Gscore of -9.466 kcal/mol. Indinavir possesses an important pharmacophore, hydroxyethylamine (HEA), and thus, a new library of HEA compounds (>2500) was subjected to virtual screening that led to 25 hits with a docking score more than indinavir. Exclusively, compound 16 with a docking score of -8.955 adhered to drug-like parameters, and the structure-activity relationship (SAR) analysis was demonstrated to highlight the importance of chemical scaffolds therein. Molecular dynamics (MD) simulation analysis performed at 100 ns supported the stability of 16 within the binding pocket. Largely, our results supported that this novel compound 16 binds with domains I and II, and the domain II-III linker of the 3CLpro protein, suggesting its suitability as a strong candidate for therapeutic discovery against COVID-19.

Genome-wide analysis reveals a regulatory role for G-quadruplexes during Adenovirus multiplication.

The G-quadruplex (GQ) motifs have recently been gaining prominence because of their role as gene cis-regulatory elements in a variety of organisms and as potential druggable targets for anti-cancer therapy and ageing. Several studies have demonstrated the existence of GQs in the genomes of emerging and re-emerging human pathogens, such as hepatitis virus, herpesviruses, Ebola virus, Zika virus and Nipah virus. Human Adenovirus (HAdV) exhibits a large number of clinical manifestations especially infecting the children and the immunocompromised patients. Moreover, the HAdV-based vectors have been widely used to deliver foreign DNAs to cells in gene therapy. However, the DNA secondary structural elements in AdV-based vectors could significantly determine the gene delivery efficacy of the vectors. In this study, using a combination of whole genome sequence analysis, biochemical, biophysical and interaction assays, we revealed fifteen putative GQs that are conserved across the different species of HAdV. We further showed that the GQs are embedded in the sequences of essential viral genes, namely E1B, E2B, and L3 genes (among others), which are involved in the early and late stages of the viral life cycle. Notably, Braco-19 (a well-known GQ binding ligand) interacted specifically with the HAdV GQs and increased their stability and further blocked the HAdV multiplication in human cells. Taken together, our data strongly supported the existence of G-quadruplex structures in the HAdV genome that affect the virus multiplication and posit that such structures may influence the efficacy of the gene-delivery vectors or even the HAdV virus life-cycle.

Slow slip source characterized by lithological and geometric heterogeneity.

Slow slip events (SSEs) accommodate a significant proportion of tectonic plate motion at subduction zones, yet little is known about the faults that actually host them. The shallow depth (<2 km) of well-documented SSEs at the Hikurangi subduction zone offshore New Zealand offers a unique opportunity to link geophysical imaging of the subduction zone with direct access to incoming material that represents the megathrust fault rocks hosting slow slip. Two recent International Ocean Discovery Program Expeditions sampled this incoming material before it is entrained immediately down-dip along the shallow plate interface. Drilling results, tied to regional seismic reflection images, reveal heterogeneous lithologies with highly variable physical properties entering the SSE source region. These observations suggest that SSEs and associated slow earthquake phenomena are promoted by lithological, mechanical, and frictional heterogeneity within the fault zone, enhanced by geometric complexity associated with subduction of rough crust.

Identification and characterization of two conserved G-quadruplex forming motifs in the Nipah virus genome and their interaction with G-quadruplex specific ligands.

The G-quadruplex (GQ) motifs are considered as potential drug-target sites for several human pathogenic viruses such as Zika, Hepatitis, Ebola, and Human Herpesviruses. The recent outbreaks of Nipah virus (NiV) in India, the highly fatal emerging zoonotic virus is a potential threat to global health security as no anti-viral drug or vaccine in currently available. Therefore, here in the present study, we sought to assess the ability of the putative G-quadruplex forming sequences in the NiV genome to form G-quadruplex structures and act as targets for anti-viral compounds. Bioinformatics analysis underpinned by various biophysical and biochemical techniques (such as NMR, CD, EMSA, DMS footprinting assay) confirmed the presence of two highly conserved G-quadruplex forming sequences (HGQs) in the G and L genes of NiV. These genes encode the cell attachment glycoprotein and RNA-dependent RNA polymerase, respectively and are essential for the virus entry and replication within the host cell. It remains possible that stabilization of these HGQs by the known G-quadruplex binding ligands like TMPyP4 and Braco-19 represents a promising strategy to inhibit the expression of the HGQ harboring genes and thereby stop the viral entry and replication inside the host cell. Accordingly, we report for the first time, that HGQs in Nipah virus genome are targets for G-quadruplex specific ligands; therefore, could serve as potential targets for anti-viral therapy.