Structural elucidation of HIV-1 G-quadruplexes in a cellular environment and their ligand binding using responsive 19F-labeled nucleoside probes.

Understanding the structure and recognition of highly conserved regulatory segments of the integrated viral DNA genome that forms unique topologies can greatly aid in devising novel therapeutic strategies to counter chronic infections. In this study, we configured a probe system using highly environment-sensitive nucleoside analogs, 5-fluoro-2'-deoxyuridine (FdU) and 5-fluorobenzofuran-2'-deoxyuridine (FBFdU), to investigate the structural polymorphism of HIV-1 long terminal repeat (LTR) G-quadruplexes (GQs) by fluorescence and 19F NMR. FdU and FBFdU, serving as hairpin and GQ sensors, produced distinct spectral signatures for different GQ topologies adopted by LTR G-rich oligonucleotides. Importantly, systematic 19F NMR analysis in Xenopus laevis oocytes gave unprecedented information on the structure adopted by the LTR G-rich region in the cellular environment. The results indicate that it forms a unique GQ-hairpin hybrid architecture, a potent hotspot for selective targeting. Furthermore, structural models generated using MD simulations provided insights on how the probe system senses different GQs. Using the responsiveness of the probes and Taq DNA polymerase stop assay, we monitored GQ- and hairpin-specific ligand interactions and their synergistic inhibitory effect on the replication process. Our findings suggest that targeting GQ and hairpin motifs simultaneously using bimodal ligands could be a new strategy to selectively block the viral replication.

The Impact of Diabetes Mellitus on Sensorineural Hearing Loss: A Cross-Sectional Study in Eastern India.

Objective Hearing loss as a comorbidity of type 2 diabetes mellitus (type 2 DM) is frequently overlooked by patients and healthcare professionals because of a lack of awareness. This cross-sectional study aims to investigate the impact of DM on sensorineural hearing loss (SNHL) in the population of Eastern India. The primary objectives are to assess the prevalence and severity of SNHL among individuals with DM, explore demographic and clinical factors associated with hearing impairment, and contribute valuable insights to the understanding of this relationship in a specific regional context. Methods An institutional-based cross-sectional study was conducted on 198 patients with type 2 DM. Of these, 46 patients were excluded based on exclusion criteria. All patients underwent detailed demographic and clinical assessments, including glycemic control, DM duration, and associated complications. Pure tone audiometry was used to evaluate hearing thresholds. Otoacoustic emission testing was performed to assess cochlear dysfunction. Results A high prevalence of SNHL (70.4%) was observed among the 152 participants meeting the inclusion criteria. Females exhibited a higher prevalence than males, and most participants experienced mild SNHL. Rural residence, lower socioeconomic status, and poor glycemic control were associated with increased SNHL. Significant associations were found between hearing loss severity and DM duration, glycosylated hemoglobin (HbA1c) levels, and complications. Among complications, a strong association was noted with diabetic neuropathy. No significant association was observed with the presence or absence of otoacoustic-emission. Conclusion This study reveals a substantial impact of DM on SNHL in Eastern India, emphasizing the importance of routine hearing assessments in diabetic populations. The findings contribute to regional understanding and have implications for targeted healthcare interventions and preventive strategies.

A novel rspA gene regulates biofilm formation and virulence of Salmonella Typhimurium.

Salmonella spp. are facultative anaerobic, Gram-negative, rod-shaped bacteria and belongs to the Enterobacteriaceae family. Although much has been known about Salmonella pathogenesis, the functional characterizations of certain genes are yet to be explored. The rspA (STM14_1818) is one such gene with putative dehydratase function, and its role in pathogenesis is unknown. The background information showed that rspA gene is upregulated in Salmonella when it resides inside macrophages, which led us to investigate its role in Salmonella pathogenesis. We generated the rspA knockout strain and complement strain in S. Typhimurium 14028. Ex-vivo and in-vivo infectivity was looked at macrophage and epithelial cell lines and Caenorhabditis elegans (C. elegans). The mutant strain differentially formed the biofilm at different temperatures by altering the expression of genes involved in the synthesis of cellulose and curli. Besides, the mutant strain is hyperproliferative intracellularly and showed increased bacterial burden in C. elegans. The mutant strain became more infectious and lethal, causing faster death of the worms than the wild type, and also modulates the worm's innate immunity. Thus, we found that the rspA deletion mutant was more pathogenic. In this study, we concluded that the rspA gene differentially regulates the biofilm formation in a temperature dependent manner by modulating the genes involved in the synthesis of cellulose and curli and negatively regulates the Salmonella virulence for longer persistence inside the host.

Probing juxtaposed G-quadruplex and hairpin motifs using a responsive nucleoside probe: a unique scaffold for chemotherapy.

Paucity of efficient probes and small molecule ligands that can distinguish different G-quadruplex (GQ) topologies poses challenges not only in understanding their basic structure but also in targeting an individual GQ form from others. Alternatively, G-rich sequences that harbour unique chimeric structural motifs (e.g., GQ-duplex or GQ-hairpin junctions) are perceived as new therapeutic hotspots. In this context, the epidermal growth factor receptor (EGFR) gene, implicated in many cancers, contains a 30 nucleotide G-rich segment in the promoter region, which adopts in vitro two unique architectures each composed of a GQ topology (parallel and hybrid-type) juxtaposed with a hairpin domain. Here, we report the use of a novel dual-app probe, C5-trifluoromethyl benzofuran-modified 2'-deoxyuridine (TFBF-dU), in the systematic analysis of EGFR GQs and their interaction with small molecules by fluorescence and 19F NMR techniques. Notably, distinct fluorescence and 19F NMR signals exhibited by the probe enabled the quantification of the relative population of random, parallel and hybrid-type GQ structures under different conditions, which could not be obtained by conventional CD and 1H NMR techniques. Using the fluorescence component, we quantified ligand binding properties of GQs, whereas the 19F label enabled the assessment of ligand-induced changes in GQ dynamics. Studies also revealed that mutations in the hairpin domain affected GQ formation and stability, which was further functionally verified in polymerase stop assay. We anticipate that these findings and useful properties of the nucleoside probe could be utilized in designing and evaluating binders that jointly target both GQ and hairpin domains for enhanced selectivity and druggability.

Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies.

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Laryngocele: Experience at a Tertiary Care Hospital of Eastern India.

OBJECTIVES: To study the details of clinical profile and management of laryngocele at a tertiary care teaching hospital of eastern India. STUDY DESIGN: A prospective study. Case series of six patients of laryngocele. METHODS: Six patients of laryngocele were examined at the Outpatient Department of ENT of Institute of Medical Sciences & SUM Hospital, Bhubaneswar, Odisha, India, between August 2010 and January 2014. The details of the patients, such as age, gender, occupations, clinical presentations, imaging modalities and treatment options, are discussed. RESULTS: The common clinical presentations of laryngocele are hoarseness of voice and swelling in the neck. Sometimes, laryngocele patients are asymptomatic. The patients were in the range of 45-70 years old, among them five were males and one was female. The diagnosis was made clinically and radiologically. Among the six patients, five were treated by surgical approach. CONCLUSIONS: Laryngocele is an abnormal dilatation of the laryngeal saccule. It is a very rare clinical condition. Hoarseness of voice and swelling in the neck are common clinical presentations in laryngocele. Imaging studies are essential for making diagnosis, determining the type, localization, extent of laryngocele and for treatment. Surgery is the treatment of choice in laryngocele.