Characterization of G-quadruplex structures in genes involved in survival and pathogenesis of Acinetobacter baumannii as a potential drug target.

Acinetobacter baumannii is a notorious pathogen that commonly thrives in hospital environments and is responsible for numerous nosocomial infections in humans. The burgeoning multi-drug resistance leaves relatively minimal options for treating the bacterial infection, posing a significant problem and prompting the identification of new approaches for tackling the same. This motivated us to focus on non-canonical nucleic acid structures, mainly G-quadruplexes, as drug targets. G-quadruplexes have recently been gaining attention due to their involvement in multiple bacterial and viral pathogenesis. Herein, we sought to explore conserved putative G-quadruplex motifs in A. baumannii. In silico analysis revealed the presence of eight conserved motifs in genes involved in bacterial survival and pathogenesis. The biophysical and biomolecular analysis confirmed stable G-quadruplex formation by the motifs and showed a high binding affinity with the well-reported G-quadruplex binding ligand, BRACO-19. BRACO-19 exposure also decreased the growth of bacteria and downregulated the expression of G-quadruplex-harboring genes. The biofilm-forming ability of the bacteria was also affected by BRACO-19 addition. Taking all these observations into account, we have shown here for the first time the potential of G-quadruplex structures as a promising drug target in Acinetobacter baumannii, for addressing the challenges posed by this infamous pathogen.

Osteonecrosis in patients with inflammatory bowel disease: a systematic review and meta-analysis.

BACKGROUND: The relationship of inflammatory bowel disease (IBD) with osteonecrosis or avascular necrosis (AVN) is uncertain. METHODS: Systematic review to estimate the frequency of osteonecrosis in IBD was performed. Electronic databases were searched on 12 December 2022 to identify relevant studies. We planned to estimate the pooled prevalence of AVN in IBD, the risk in IBD when compared to the healthy population (without any chronic disease), and the impact of steroid use on osteonecrosis (IBD with and without steroid use). The risk of Bias was assessed with the Joanna Briggs Institute appraisal tool. RESULTS: Fifteen studies including 105 154 individuals were included. The pooled rate AVN was 10.39 per 1000 patients (95% confidence interval, 4.44-24.11, I 2  = 97%). Subgroup analysis suggested that the prevalence was lower in larger studies (>1000 participants) at 3.10, 1.07; 8.98, I 2  = 98% versus 21.03, 8.69; 50.01, I 2  = 83%. The use of steroids did not seem to increase the risk of osteonecrosis in the included studies (pooled odds ratio: 1.88, 0.55-6.41, I 2  = 39%). The systematic review was limited by the absence of comparison with the control population free of chronic disease. CONCLUSION: IBD may be associated with a risk of osteonecrosis. Future studies should assess the risk in comparison to the healthy population and the impact of disease activity and IBD therapies on the risk.

A DNA aptamer-based assay for the detection of soluble ST2, a prognostic biomarker for monitoring heart failure.

Heart failure (HF) is emerging as a leading cause of death worldwide. Estimation of BNP levels is a routine diagnosis in these patients. However, in patients having high body-mass index (BMI), renal disease or in geriatric patients, BNP level is reported to be noisy and leads to incongruous conclusion. Thus, for better risk stratification among heart failure patients, it is imperative to look for a superior biomarker. In recent times, sST2 has shown promise as a biomarker. Identifying such biomarkers in peripheral blood of HF patients, need an affine and selective molecular recognition element. Thus, in the current study an aptamer (sS9_P) against sST2 was identified from an aptamer library. Systematic Evolution of Ligands through Exponential enrichment (SELEX) derived aptamer evinced role of its primer binding domains in maintaining its selectivity. This aptamer candidate demonstrated dissociation constant (Kd) in low nanomolar range, and the Limit of Detection (LOD) was ~4 ng. Circular dichroism confirms the formation of complex stem-loop like structure. The well characterized sS9_P aptamer was used in an Aptamer Linked Immobilized Sorbent Assay (ALISA) to detect sST2 level in patients' serum (n = 99). Aptamer sS9_P has shown significant discrimination to differentiate HF patients and healthy volunteers with a reasonable specificity (~83 %) with a modest sensitivity of ~64 %. While sST-2 antibody has shown poor specificity of ~44% but good sensitivity (~87%). The insight obtained from this study indicates that a combination of aptamer and antibody-based assay can be used to design a point-of-care assay for the rapid detection of HF patients in emergency settings.

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.

Diagnosis of COVID-19 using chest X-ray images based on modified DarkCovidNet model.

Coronavirus disease, also known as COVID-19, is an infectious disease caused by SARS-CoV-2. It has a direct impact on the upper and lower respiratory tract and threatened the health of many people around the world. The latest statistics show that the number of people diagnosed with COVID-19 is growing exponentially. Diagnosing positive cases of COVID-19 is important for preventing further spread of the disease. Currently, Coronavirus is a serious threat to scientists, medical experts and researchers around the world from its detection to its treatment. It is currently detected using reverse transcription polymerase chain reaction (RT-PCR) analysis at the most test centers around the world. Yet, knowing the reliability of a deep learning based medical diagnosis is important for doctors to build confidence in the technology and improve treatment. The goal of this study is to develop a model that automatically identifies COVID-19 by using chest X-ray images. To achieve this, we modified the DarkCovidNet model which is based on a convolutional neural network (CNN) and plotted the experimental results for two scenarios: binary classification (COVID-19 versus No-findings) and multi-class classification (COVID-19 versus pneumonia versus No-findings). The model is trained on more than 10 thousand X-ray images and achieved an average accuracy of 99.53% and 94.18% for binary and multi-class classification, respectively. Therefore, the proposed method demonstrates the effectiveness of COVID-19 detection using X-ray images. Our model can be used to test the patient via cloud and also be used in situations where RT-PCR tests and other options aren't available.

Structural and Functional Characterization of Rv0792c from Mycobacterium tuberculosis: Identifying Small Molecule Inhibitor against HutC Protein.

In order to adapt in host tissues, microbial pathogens regulate their gene expression through a variety of transcription factors. Here, we have functionally characterized Rv0792c, a HutC homolog from Mycobacterium tuberculosis. In comparison to the parental strain, a strain of M. tuberculosis with a Rv0792c mutant was compromised for survival upon exposure to oxidative stress and infection in guinea pigs. RNA sequencing analysis revealed that Rv0792c regulates the expression of genes involved in stress adaptation and virulence of M. tuberculosis. Solution small-angle X-ray scattering (SAXS) data-steered model building confirmed that the C-terminal region plays a pivotal role in dimer formation. Systematic evolution of ligands by exponential enrichment (SELEX) resulted in the identification of single-strand DNA (ssDNA) aptamers that can be used as a tool to identify small-molecule inhibitors targeting Rv0792c. Using SELEX and SAXS data-based modeling, we identified residues essential for Rv0792c's aptamer binding activity. In this study, we also identified I-OMe-Tyrphostin as an inhibitor of Rv0792c's aptamer and DNA binding activity. The identified small molecule reduced the growth of intracellular M. tuberculosis in macrophages. The present study thus provides a detailed shape-function characterization of a HutC family of transcription factor from M. tuberculosis. IMPORTANCE Prokaryotes encode a large number of GntR family transcription factors that are involved in various fundamental biological processes, including stress adaptation and pathogenesis. Here, we investigated the structural and functional role of Rv0792c, a HutC homolog from M. tuberculosis. We demonstrated that Rv0792c is essential for M. tuberculosis to adapt to oxidative stress and establish disease in guinea pigs. Using a systematic evolution of ligands by exponential enrichment (SELEX) approach, we identified ssDNA aptamers from a random ssDNA library that bound to Rv0792c protein. These aptamers were thoroughly characterized using biochemical and biophysical assays. Using SAXS, we determined the structural model of Rv0792c in both the presence and absence of the aptamers. Further, using a combination of SELEX and SAXS methodologies, we identified I-OMe-Tyrphostin as a potential inhibitor of Rv0792c. Here we provide a detailed functional characterization of a transcription factor belonging to the HutC family from M. tuberculosis.

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.

Current Advancements and Future Road Map to Develop ASSURED Microfluidic Biosensors for Infectious and Non-Infectious Diseases.

Better diagnostics are always essential for the treatment and prevention of a disease. Existing technologies for detecting infectious and non-infectious diseases are mostly tedious, expensive, and do not meet the World Health Organization's (WHO) ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end user) criteria. Hence, more accurate, sensitive, and faster diagnostic technologies that meet the ASSURED criteria are highly required for timely and evidenced-based treatment. Presently, the diagnostics industry is finding interest in microfluidics-based biosensors, as this integration comprises all qualities, such as reduction in the size of the equipment, rapid turnaround time, possibility of parallel multiple analysis or multiplexing, etc. Microfluidics deal with the manipulation/analysis of fluid within micrometer-sized channels. Biosensors comprise biomolecules immobilized on a physicochemical transducer for the detection of a specific analyte. In this review article, we provide an outline of the history of microfluidics, current practices in the selection of materials in microfluidics, and how and where microfluidics-based biosensors have been used for the diagnosis of infectious and non-infectious diseases. Our inclination in this review article is toward the employment of microfluidics-based biosensors for the improvement of already existing/traditional methods in order to reduce efforts without compromising the accuracy of the diagnostic test. This article also suggests the possible improvements required in microfluidic chip-based biosensors in order to meet the ASSURED criteria.

Double-Edged Nanobiotic Platform with Protean Functionality: Leveraging the Synergistic Antibacterial Activity of a Food-Grade Peptide to Mitigate Multidrug-Resistant Bacterial Pathogens.

While persistent efforts are being made to develop a novel arsenal against bacterial pathogens, the development of such materials remains a formidable challenge. One such strategy is to develop a multimodel antibacterial agent which will synergistically combat bacterial pathogens, including multidrug-resistant bacteria. Herein, we used pediocin, a class IIa bacteriocin, to decorate Ag° and developed a double-edged nanoplatform (Pd-SNPs) that inherits intrinsic properties of both antibacterial moieties, which engenders strikingly high antibacterial potency against a broad spectrum of bacterial pathogens including the ESKAPE category without displaying adverse cytotoxicity. The enhanced antimicrobial activity of Pd-SNPs is due to their higher affinity with the bacterial cell wall, which allows Pd-SNPs to penetrate the outer membrane, inducing membrane depolarization and the disruption of membrane integrity. Bioreporter assays revealed the upregulation of cpxP, degP, and sosX genes, triggering the burst of reactive oxygen species which eventually cause bacterial cell death. Pd-SNPs prevented biofilm formation, eradicated established biofilms, and inhibited persister cells. Pd-SNPs display unprecedented advantages because they are heat-resistant, retain antibacterial activity in human serum, and alleviate vancomycin intermediate Staphylococcus aureus (VISA) infection in the mouse model. In addition, Pd-SNPs wrapped in biodegradable nanofibers mitigated Listeria monocytogenes in cheese samples. Collectively, Pd-SNPs exhibited excellent biocompatibility and in vivo therapeutic potency without allowing foreseeable resistance acquisition by pathogens. These findings underscore new avenues for using a potent biocompatible nanobiotic platform to combat a wide range of bacterial pathogens.

Assessment of DNA aptamers targeting GlcB and HspX antigens for application in the diagnosis of abdominal tuberculosis.

The diagnosis of abdominal tuberculosis (aTB) is challenging and there is an urgent need for an accurate diagnostic test. We have developed a high affinity DNA aptamer against GlcB antigen of Mycobacterium tuberculosis (Mtb). We further compared the diagnostic utility of in-house-generated high affinity DNA aptamers and polyclonal antibodies against two Mtb antigens, namely GlcB and HspX, in ascitic fluid samples. These diagnostic reagents were assessed in patients (n = 94) who were categorized as 'Definite TB', 'Probable TB', 'Possible TB' (taken together as aTB) and 'Non-TB' disease. Receiver operating characteristic curves were used to derive cut-off values to provide ≥93% specificity. Aptamer Linked Immobilized Sorbent Assay (ALISA) for HspX and GlcB exhibited a sensitivity of ∼84% and 50%, respectively (p-value <0.01). In contrast, antibody-based ELISA exhibited a lower sensitivity of ∼18% and ∼28% for HspX and GlcB, respectively (p-value <0.0001 and p = 0.05 for HspX and GlcB ELISA vs. ALISA, respectively). HspX ALISA detected 32/38 aTB cases, while Xpert detected only 9 samples. In conclusion, HspX aptamer-based test was found to be superior to the other tests for diagnosing aTB and it nearly fulfils the sensitivity criteria of WHO's 'Target Product Profile' for extrapulmonary tuberculosis (sensitivity ≥80%, specificity 98%).

Development of paper-based DNA sensor for detection of O. tsutsugamushi using sustainable GQDs@AuNPs nanocomposite.

The graphene quantum dots (GQDs) was synthesized using potato starch and water by hydrothermal method and further used for reduction of tetracholoroauric acid to form graphene quantum dots-gold (GQDs@AuNPs) nanocomposite. The GQDs/GQDs@AuNPs were analyzed using FTIR, UV-Vis, Flourometry and HR-TEM. The synthesized GQDs@AuNPs were further used for fabrication of cost-effective screen-printed paper electrode (SPPE) based DNA sensor for the detection of O. tsutsugamushi using htrA gene specific 5'NH2 linked DNA probe. Modification of SPPE using GQDs@AuNPs nanocomposite and ssDNA probe was monitored using EIS, FTIR, FE-SEM and AFM. The sensor detection limit (LOD) was assessed as 0.002 ng/µl from the standard calibration curve with the correlation coefficient, R2 = 0.993. The sensitivity of the DNA sensor was calculated as 7700 µA/cm2/ng for ssGDNA of O. tsutsugamushi using cyclic voltammetry. The sensor validation was done using scrub typhus patient's blood DNA samples. The sensor showed good storage stability at 4 °C for six months with just a loss of 12% of the initial current values. The SPPE/DNA sensor developed is very specific, sensitive, stable and detects O. tsutsugamushi in less time.

A paper microfluidic device based colorimetric sensor for the detection and discrimination of elapid versus viper envenomation.

Snake bites are a neglected tropical disease, causing mortality and severe damage to various vital organs like the nervous system, kidneys and heart. There is increasing interest in designing new antivenom treatments that are more specific to particular groups (either taxonomic or regional) of species, given the increasing evidence that current polyvalent Indian antivenom is ineffective in many situations. Under these circumstances, being able to detect the species, or a group of species, responsible for the envenomation becomes important. Unfortunately, no such diagnostic tool is available in the Indian market. Such a tool will need to be rapid, sensitive and affordable. To address this need, we have combined the power of nanotechnology and paper microfluidics and herein report a device that has the ability to detect and differentiate viper venom from elapid and scorpion venom. In principle, this assay is based on the release of the dye from the stimuli-responsive glutaraldehyde cross-linked methylene blue-loaded gelatin (GMG) nanoparticles in the presence of snake venom metalloproteases and serine proteases. The developed equipment-free assay can detect and discriminate viper venom from that of elapids and scorpions. The low-end detection limit of the sensor is ∼3.0 ng for the saw-scaled viper Echis carinatus, while the same for Russell's viper Daboia russelii is ∼6.0 ng. The performance of the sensor remains unaltered for different batches of GMG nanoparticles. Altogether, this finding establishes the role of nanotechnology and paper microfluidics in the rapid and accurate detection of viper venom.

Identification and Engineering of Aptamers for Theranostic Application in Human Health and Disorders.

An aptamer is a short sequence of synthetic oligonucleotides which bind to their cognate target, specifically while maintaining similar or higher sensitivity compared to an antibody. The in-vitro selection of an aptamer, applying a conjoining approach of chemistry and molecular biology, is referred as Systematic Evolution of Ligands by Exponential enrichment (SELEX). These initial products of SELEX are further modified chemically in an attempt to make them stable in biofluid, avoiding nuclease digestion and renal clearance. While the modification is incorporated, enough care should be taken to maintain its sensitivity and specificity. These modifications and several improvisations have widened the window frame of aptamer applications that are currently not only restricted to in-vitro systems, but have also been used in molecular imaging for disease pathology and treatment. In the food industry, it has been used as sensor for detection of different diseases and fungal infections. In this review, we have discussed a brief history of its journey, along with applications where its role as a therapeutic plus diagnostic (theranostic) tool has been demonstrated. We have also highlighted the potential aptamer-mediated strategies for molecular targeting of COVID-19. Finally, the review focused on its future prospective in immunotherapy, as well as in identification of novel biomarkers in stem cells and also in single cell proteomics (scProteomics) to study intra or inter-tumor heterogeneity at the protein level. Small size, chemical synthesis, low batch variation, cost effectiveness, long shelf life and low immunogenicity provide advantages to the aptamer over the antibody. These physical and chemical properties of aptamers render them as a strong biomedical tool for theranostic purposes over the existing ones. The significance of aptamers in human health was the key finding of this review.

Complex target SELEX-based identification of DNA aptamers against Bungarus caeruleus venom for the detection of envenomation using a paper-based device.

Complex target SELEX always have been an intriguing approach to the scientific community, as it offers the potential discovery of novel biomarkers. We herein successfully performed SELEX on Bungarus caeruleus venom to develop a panel of highly affine aptamers that specifically recognizes the B. caeruleus (common krait) venom and was able to discriminate the B. caeruleus venom from Cobra, Russell's, and Saw-scaled viper's venom. The aptamers generated against the crude venom also lead to the identification of the specific component of the venom, which is ß-Bungarotoxin, a toxin uniquely present in the B. caeruleus venom. The best performing aptamer candidates were used as a molecular recognition element in a paper-based device and were able to detect as low as 2 ng krait venom in human serum background. The developed aptamer-based paper device can be used for potential point-of-care venom detection applications due to its simplicity and affordability.

First stellar photons for an integrated optics discrete beam combiner at the William Herschel Telescope.

We present the first on-sky results of a four-telescope integrated optics discrete beam combiner (DBC) tested at the 4.2 m William Herschel Telescope. The device consists of a four-input pupil remapper followed by a DBC and a 23-output reformatter. The whole device was written monolithically in a single alumino-borosilicate substrate using ultrafast laser inscription. The device was operated at astronomical H-band (1.6 µm), and a deformable mirror along with a microlens array was used to inject stellar photons into the device. We report the measured visibility amplitudes and closure phases obtained on Vega and Altair that are retrieved using the calibrated transfer matrix of the device. While the coherence function can be reconstructed, the on-sky results show significant dispersion from the expected values. Based on the analysis of comparable simulations, we find that such dispersion is largely caused by the limited signal-to-noise ratio of our observations. This constitutes a first step toward an improved validation of the DBC as a possible beam combination scheme for long-baseline interferometry.

Graphene Oxide Nanoparticles Modified Paper Electrode as a Biosensing Platform for Detection of the htrA Gene of O. tsutsugamushi.

The unique structural and electrochemical properties of graphene oxide (GO) make it an ideal material for the fabrication of biosensing devices. Therefore, in the present study, graphene oxide nanoparticles modified paper electrodes were used as a low-cost matrix for the development of an amperometric DNA sensor. The graphene oxide was synthesized using the modified hummers method and drop cast on a screen-printed paper electrode (SPPE) to enhance its electrochemical properties. Further, the GO/SPPE electrode was modified with a 5'NH2 labeled ssDNA probe specific to the htrA gene of Orientia tsutsugamushi using carbodiimide cross-linking chemistry. The synthesized GO was characterized using UV-Vis, FTIR, and XRD. The layer-by-layer modification of the paper electrode was monitored via FE-SEM, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). The sensor response after hybridization with single-stranded genomic DNA (ssGDNA) of O. tsutsugamushi was recorded using differential pulse voltammetry (DPV). Methylene blue (1 mM in PBS buffer, pH 7.2) was used as a hybridization indicator and [Fe(CN)6]-3/-4 (2.5 mM in PBS buffer, pH 7.2) as a redox probe during electrochemical measurements. The developed DNA sensor shows excellent sensitivity (1228.4 µA/cm2/ng) and LOD (20 pg/µL) for detection of O. tsutsugamushi GDNA using differential pulse voltammetry (DPV).

A novel G-quadruplex aptamer-based spike trimeric antigen test for the detection of SARS-CoV-2.

The recent SARS-CoV-2 outbreak has been declared a global health emergency. It will take years to vaccinate the whole population to protect them from this deadly virus, hence the management of SARS-CoV-2 largely depends on the widespread availability of an accurate diagnostic test. Toward addressing the unmet need of a reliable diagnostic test in the current work by utilizing the power of Systematic Evolution of Ligands by EXponential enrichment, a 44-mer G-quadruplex-forming DNA aptamer against spike trimer antigen of SARS-CoV-2 was identified. The lead aptamer candidate (S14) was characterized thoroughly for its binding, selectivity, affinity, structure, and batch-to-batch variability by utilizing various biochemical, biophysical, and in silico techniques. S14 has demonstrated a low nanomolar KD, confirming its tight binding to a spike antigen of SARS-CoV-2. S14 can detect as low as 2 nM of antigen. The clinical evaluation of S14 aptamer on nasopharyngeal swab specimens (n = 232) has displayed a highly discriminatory response between SARS-CoV-2 infected individuals from the non-infected one with a sensitivity and specificity of ∼91% and 98%, respectively. Importantly, S14 aptamer-based test has evinced a comparable performance with that of RT-PCR-based assay. Altogether, this study established the utility of aptamer technology for the detection of SARS-CoV-2.

Wuhan to World: The COVID-19 Pandemic.

COVID-19 is a Severe Acute Respiratory Syndrome (SARS), caused by SARS-CoV-2, a novel virus which belongs to the family Coronaviridae. It was first reported in December 2019 in the Wuhan city of China and soon after, the virus and hence the disease got spread to the entire world. As of February 26, 2021, SARS-CoV-2 has infected ~112.20 million people and caused ~2.49 million deaths across the globe. Although the case fatality rate among SARS-CoV-2 patient is lower (~2.15%) than its earlier relatives, SARS-CoV (~9.5%) and MERS-CoV (~34.4%), the SARS-CoV-2 has been observed to be more infectious and caused higher morbidity and mortality worldwide. As of now, only the knowledge regarding potential transmission routes and the rapidly developed diagnostics has been guiding the world for managing the disease indicating an immediate need for a detailed understanding of the pathogen and the disease-biology. Over a very short period of time, researchers have generated a lot of information in unprecedented ways in the key areas, including viral entry into the host, dominant mutation, potential transmission routes, diagnostic targets and their detection assays, potential therapeutic targets and drug molecules for inhibiting viral entry and/or its replication in the host including cross-neutralizing antibodies and vaccine candidates that could help us to combat the ongoing COVID-19 pandemic. In the current review, we have summarized the available knowledge about the pathogen and the disease, COVID-19. We believe that this readily available knowledge base would serve as a valuable resource to the scientific and clinical community and may help in faster development of the solution to combat the disease.

G-Quadruplex Structures in Bacteria: Biological Relevance and Potential as an Antimicrobial Target.

DNA strands consisting of multiple runs of guanines can adopt a noncanonical, four-stranded DNA secondary structure known as G-quadruplex or G4 DNA. G4 DNA is thought to play an important role in transcriptional and translational regulation of genes, DNA replication, genome stability, and oncogene expression in eukaryotic genomes. In other organisms, including several bacterial pathogens and some plant species, the biological roles of G4 DNA and G4 RNA are starting to be explored. Recent investigations showed that G4 DNA and G4 RNA are generally conserved across plant species. In silico analyses of several bacterial genomes identified putative guanine-rich, G4 DNA-forming sequences in promoter regions. The sequences were particularly abundant in certain gene classes, suggesting that these highly diverse structures can be employed to regulate the expression of genes involved in secondary metabolite synthesis and signal transduction. Furthermore, in the pathogen Mycobacterium tuberculosis, the distribution of G4 motifs and their potential role in the regulation of gene transcription advocate for the use of G4 ligands to develop novel antitubercular therapies. In this review, we discuss the various roles of G4 structures in bacterial DNA and the application of G4 DNA as inhibitors or therapeutic agents to address bacterial pathogens.

Development of DNA Aptamers to Visualize Release of Mycobacterial Membrane-Derived Extracellular Vesicles in Infected Macrophages.

Extracellular vesicles (EVs) have emerged into a novel vaccine platform, a biomarker and a nano-carrier for approved drugs. Their accurate detection and visualization are central to their utility in varied biomedical fields. Owing to the limitations of fluorescent dyes and antibodies, here, we describe DNA aptamer as a promising tool for visualizing mycobacterial EVs in vitro. Employing SELEX from a large DNA aptamer library, we identified a best-performing aptamer that is highly specific and binds at nanomolar affinity to EVs derived from three diverse mycobacterial strains (pathogenic, attenuated and avirulent). Confocal microscopy revealed that this aptamer was not only bound to in vitro-enriched mycobacterial EVs but also detected EVs that were internalized by THP-1 macrophages and released by infecting mycobacteria. To the best of our knowledge, this is the first study that detects EVs released by mycobacteria during infection in host macrophages. Within 4 h, most released mycobacterial EVs spread to other parts of the host cell. We predict that this tool will soon hold huge potential in not only delineating mycobacterial EVs-driven pathogenic functions but also in harboring immense propensity to act as a non-invasive diagnostic tool against tuberculosis in general, and extra-pulmonary tuberculosis in particular.