A multicentre study to evaluate the diagnostic performance of a novel CAD software, DecXpert, for radiological diagnosis of tuberculosis in the northern Indian population.

Tuberculosis (TB) is the leading cause of mortality among infectious diseases globally. Effectively managing TB requires early identification of individuals with TB disease. Resource-constrained settings often lack skilled professionals for interpreting chest X-rays (CXRs) used in TB diagnosis. To address this challenge, we developed "DecXpert" a novel Computer-Aided Detection (CAD) software solution based on deep neural networks for early TB diagnosis from CXRs, aiming to detect subtle abnormalities that may be overlooked by human interpretation alone. This study was conducted on the largest cohort size to date, where the performance of a CAD software (DecXpert version 1.4) was validated against the gold standard molecular diagnostic technique, GeneXpert MTB/RIF, analyzing data from 4363 individuals across 12 primary health care centers and one tertiary hospital in North India. DecXpert demonstrated 88% sensitivity (95% CI 0.85-0.93) and 85% specificity (95% CI 0.82-0.91) for active TB detection. Incorporating demographics, DecXpert achieved an area under the curve of 0.91 (95% CI 0.88-0.94), indicating robust diagnostic performance. Our findings establish DecXpert's potential as an accurate, efficient AI solution for early identification of active TB cases. Deployed as a screening tool in resource-limited settings, DecXpert could enable early identification of individuals with TB disease and facilitate effective TB management where skilled radiological interpretation is limited.

Estrogen receptor alpha deficiency in cardiomyocytes reprograms the heart-derived extracellular vesicle proteome and induces obesity in female mice.

Dysregulation of estrogen receptor alpha (ERα) has been linked with increased metabolic and cardiovascular disease risk. Here, we generate and characterize cardiomyocyte-specific ERα knockout (ERαHKO) mice to assess the role of ERα in the heart. The most striking phenotype was obesity in female ERαHKO but not male ERαHKO mice. Female ERαHKO mice showed cardiac dysfunction, mild glucose and insulin intolerance and reduced ERα gene expression in skeletal muscle and white adipose tissue. Transcriptomic, proteomic, lipidomic and metabolomic analyses revealed evidence of contractile and/or metabolic dysregulation in heart, skeletal muscle and white adipose tissue. We show that heart-derived extracellular vesicles from female ERαHKO mice contain a distinct proteome associated with lipid and metabolic regulation, and have the capacity to metabolically reprogram the target skeletal myocyte proteome with functional impacts on glycolytic capacity and reserve. This multi-omics study uncovers a cardiac-initiated and sex-specific cardiometabolic phenotype regulated by ERα and provides insights into extracellular vesicle-mediated interorgan communication.

PICH acts as a force-dependent nucleosome remodeler.

In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. To prevent genome instability, chromatin bridges must be resolved prior to cytokinesis. The SNF2 protein PICH has been proposed to play a direct role in this process through the remodeling of nucleosomes. However, direct evidence of nucleosome remodeling by PICH has remained elusive. Here, we present an in vitro single-molecule assay that mimics chromatin under tension, as is found in anaphase chromatin bridges. Applying a combination of dual-trap optical tweezers and fluorescence imaging of PICH and histones bound to a nucleosome-array construct, we show that PICH is a tension- and ATP-dependent nucleosome remodeler that facilitates nucleosome unwrapping and then subsequently slides remaining histones along the DNA. This work elucidates the role of PICH in chromatin-bridge dissolution, and might provide molecular insights into the mechanisms of related SNF2 proteins.

From Processivity to Genome Maintenance: The Many Roles of Sliding Clamps.

Sliding clamps play a pivotal role in the process of replication by increasing the processivity of the replicative polymerase. They also serve as an interacting platform for a plethora of other proteins, which have an important role in other DNA metabolic processes, including DNA repair. In other words, clamps have evolved, as has been correctly referred to, into a mobile "tool-belt" on the DNA, and provide a platform for several proteins that are involved in maintaining genome integrity. Because of the central role played by the sliding clamp in various processes, its study becomes essential and relevant in understanding these processes and exploring the protein as an important drug target. In this review, we provide an updated report on the functioning, interactions, and moonlighting roles of the sliding clamps in various organisms and its utilization as a drug target.

RAD51 protects human cells from transcription-replication conflicts.

Oncogene activation during tumorigenesis promotes DNA replication stress (RS), which subsequently drives the formation of cancer-associated chromosomal rearrangements. Many episodes of physiological RS likely arise due to conflicts between the DNA replication and transcription machineries operating simultaneously at the same loci. One role of the RAD51 recombinase in human cells is to protect replication forks undergoing RS. Here, we have identified a key role for RAD51 in preventing transcription-replication conflicts (TRCs) from triggering replication fork breakage. The genomic regions most affected by RAD51 deficiency are characterized by being replicated and transcribed in early S-phase and show significant overlap with loci prone to cancer-associated amplification. Consistent with a role for RAD51 in protecting against transcription-replication conflicts, many of the adverse effects of RAD51 depletion are ameliorated by inhibiting early S-phase transcription. We propose a model whereby RAD51 suppresses fork breakage and subsequent inadvertent amplification of genomic loci prone to experiencing TRCs.

PICH and TOP3A cooperate to induce positive DNA supercoiling.

All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.

Identification of a Novel Splice Variant of Neural Cell Adhesion Molecule in Glioblastoma Through Proteogenomics Analysis.

Splice variants are known to be important in the pathophysiology of tumors, including the brain cancers. We applied a proteogenomics pipeline to identify splice variants in glioblastoma (GBM, grade IV glioma), a highly malignant brain tumor, using in-house generated mass spectrometric proteomic data and public domain RNASeq dataset. Our analysis led to the identification of a novel exon that maps to the long isoform of Neural cell adhesion molecule 1 (NCAM1), expressed on the surface of glial cells and neurons, important for cell adhesion and cell signaling. The presence of the novel exon is supported with the identification of five peptides spanning it. Additional peptides were also detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel separated proteins from GBM patient tissue, underscoring the presence of the novel peptides in the intact brain protein. The novel exon was detected in the RNASeq dataset in 18 of 25 GBM samples and separately validated in additional 10 GBM tumor tissues using quantitative real-time-polymerase chain reaction (qRT-PCR). Both transcriptomic and proteomic data indicate downregulation of NCAM1, including the novel variant, in GBM. Domain analysis of the novel NCAM1 sequence indicates that the insertion of the novel exon contributes extra low-complexity region in the protein that may be important for protein-protein interactions and hence for cell signaling associated with tumor development. Taken together, the novel NCAM1 variant reported in this study exemplifies the importance of future multiomics research and systems biology applications in GBM.

Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp.

Protein folding process involves formation of transiently occurring intermediates that are difficult to isolate and characterize. It is both necessary and interesting to characterize the structural conformations adopted by these intermediates, also called molten globules (MG), to understand protein folding. Here, we investigated the equilibrium (un)folding intermediate state of T4 phage gene product 45 (gp45, also known as DNA polymerase processivity factor or sliding clamp) obtained during chemical denaturation. We show that gp45 undergoes substantial conformational rearrangement during unfolding and forms an expanded dry-MG. By monitoring the fluorescence of tryptophans that were strategically introduced at various sites, we demonstrate that the urea-treated molecule has its surface residues flip inside the core, and closely placed residues move farther. We were also able to isolate and purify the MG form of gp45 in native condition (i.e., nondenaturing buffer, at physiological pH and temperature); characteristics of this purified molecule substantially match with urea-treated wild-type gp45. To the best of our knowledge, this is one of the few reports that demonstrate the isolation and purification of a protein folding intermediate in native condition. We believe that our work not only allows us to dissect the process of protein folding, but will also help in the designing of folding inhibitors against sliding clamps to treat a wide variety of diseases from bacterial infection to cancer, due to the vast presence of clamps in all the domains of life.

Bioinformatics Methods to Deduce Biological Interpretation from Proteomics Data.

High-throughput proteomics studies generate large amounts of data. Biological interpretation of these large scale datasets is often challenging. Over the years, several computational tools have been developed to facilitate meaningful interpretation of large-scale proteomics data. In this chapter, we describe various analyses that can be performed and bioinformatics tools and resources that enable users to do the analyses. Many Web-based and stand-alone tools are relatively user-friendly and can be used by most biologists without significant assistance.

On the domains of T4 phage sliding clamp gp45: An intermolecular crosstalk governs structural stability and biological activity.

BACKGROUND: DNA polymerase processivity factors are ubiquitously present in all living organisms. Notwithstanding their high significance, the molecular details of clamps pertaining to the factors contributing to their stability are presently lacking. The bacteriophage T4 sliding clamp gp45 forms a homotrimer that besides being involved in DNA replication, moonlights as a transcription factor. Here we have carried out a detailed characterization of gp45 to understand the role of monomer-monomer interface interactions in stability and functioning of the protein. METHODS: We generated several gp45 mutants harboring either Ala or Pro substitutions at the interface residues and performed a detailed investigation using biochemical and biophysical methods including circular dichroism, fluorescence anisotropy and quenching, differential scanning calorimetry, blue-native PAGE, cross-linking, size exclusion chromatography, and dynamic light scattering. We also carried out both transcription and DNA replication to understand the properties of the wild-type and the mutant proteins. RESULTS: One specific mutation S88P leads not only to monomerization, but also results in an unstable molecule. Most interestingly, mutating either Q125 or K164 in the gp45 C-terminal domain negatively affects the stability of the N-terminal domain. We also report that these residues upon mutation to alanine make gp45 inactive for late promoter transcription, whereas strand-displacement DNA replication ability remains unaltered. CONCLUSIONS AND GENERAL SIGNIFICANCE: The results suggest that the two domains of gp45 demonstrate an "inter-monomer" crosstalk that stabilizes the trimer. We also conclude that the residue-specific interactions at the interface allow the protein to function distinctly as replication and transcription factors.

Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.

We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.

Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics.

Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function.

Serum vitamin c and iron levels in oral submucous fibrosis.

BACKGROUND: In this study Serum Vitamin C and Iron levels in Oral submucous fibrosis (OSMF) were estimated. The objective was to evaluate the correlation between Serum Vitamin C and Iron levels in OSMF individuals. Serum Iron content can be a predictor for the progression of this condition. OSMF is basically a disorder of collagen metabolism where Vitamin C gets utilized in conversion of proline into hydroxyproline, this hydroxylation reaction requires ferrous Iron and Vitamin C. Many studies regarding micronutrients and other antioxidants levels have been emphasized, but very few studies are done on the Serum levels of Vitamin C and its correlation with Iron in OSMF patients. METHODS: Thirty five OSMF patients and 50 deleterious habit free healthy individuals (controls) were selected. Two ml of venous blood was collected from each individual. Vitamin C level in serum was estimated by 2-4 dinitrophenylhydrazine method and Iron estimated by Tripyridyl method. RESULTS: The level of Serum Vitamin-C and Iron was significantly decreased in OSMF patients when compared to controls which were statistically significant. CONCLUSION: On the basis of these observations, it seems possible that the chemical, thermal and/or mechanical factors associated with the use of areca nut may act in conjunction with the Vitamin C and Iron deficiency leading to the development of OSMF. Therapeutic substitution of vitamin C and Iron may be recommended in the management of OSMF.

HDAC5, a key component in temporal regulation of p53-mediated transactivation in response to genotoxic stress.

Despite being one of the most well-studied transcription factors, the temporal regulation of p53-mediated transcription is not very well understood. Recent data suggest that target specificity of p53-mediated transactivation is achieved by posttranslational modifications of p53. K120 acetylation is a modification critical for recruitment of p53 to proapoptotic targets. Our data reveal that histone deacetylase 5 (HDAC5) binds to p53 and abrogates K120 acetylation, resulting in preferential recruitment of p53 to proarrest and antioxidant targets at early phases of stress. However, upon prolonged genotoxic stress, HDAC5 undergoes nuclear export. Concomitantly, p53 is acetylated at the K120 residue and selectively transactivates proapoptotic target genes, leading to onset of apoptosis. Furthermore, upon genotoxic stress in mice where HDAC5 expression is downregulated, the onset of apoptosis is accelerated in the highly vulnerable tissues. These findings suggest that HDAC5 is a key determinant of p53-mediated cell fate decisions in response to genotoxic stress.

Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.

We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag.

Quantitative evaluation and correlation of serum glycoconjugates: Protein bound hexoses, sialic acid and fucose in leukoplakia, oral sub mucous fibrosis and oral cancer.

BACKGROUND: Cancer is one of the most common causes of mortality and morbidity today, with more than 10 million new cases and more than 6 million deaths each year worldwide. Globally Oral Cancer is the sixth most common cause of cancer related death. India accounts for 86% of the world's oral cancer cases. Often it proceeds by pre cancerous conditions and lesions. In search for biological markers with diagnostic value, we investigated serum glycoconjugates like protein bound hexoses, fucose and sialic acid in these diseases. MATERIALS AND METHODS: For this Study 27 newly diagnosed Oral leukoplakia, 27 OSMF and 26 Oral Cancer patients, 40 healthy controls who are non tobacco users and 40 healthy controls who are tobacco users were selected. In all these groups we estimated serum glycoconjugates. RESULTS: We observed no difference in serum glycoconjugates levels between tobacco and non tobacco controls (P > 0.05), but very high levels in oral cancer, Leukoplakia and oral sub mucous fibrosis (OSMF) patients (P < 0.001) when compared to control groups. Fucose levels were significant (P < 0.05) of all the glycoconjugates between OSMF and Leukoplakia. CONCLUSION: The serum glycoconjugates whose levels were very high in OSMF, Leukoplakia and Oral Cancer, do have a significant diagnostic and prognostic value in these diseases.

Plasma zinc antioxidant vitamins, glutathione levels and total antioxidant activity in oral leukoplakia.

BACKGROUND: Leukoplakia is a common, potentially premalignant lesion with malignant transformation rate from 1 to 17% with highest transformation rate for the lesions on the floor of the mouth, soft palate and tongue. One of the main etiological factors is consuming areca nut and its commercial preparations which generate high levels of reactive oxygen species during their metabolism. So the aim of this present study is to evaluate the plasma levels of antioxidant vitamins, antioxidant mineral zinc, glutathione and total antioxidant status (TAS) in leukoplakia patients. MATERIALS AND METHODS: For this cross-sectional study, we selected 23 newly diagnosed oral leukoplakia patients of both sexes within the age group 28-40 years and the same number of age and sex matched healthy individuals without having history of any systemic illness were selected as control group. In both the groups, we measured plasma antioxidant vitamins A, C, E, antioxidant mineral zinc, GSH and TAS. Student's t test was applied and the P value <0.001 was considered as statistically significant. RESULTS: We observed very low levels of antioxidant vitamins A, C, E, antioxidant mineral zinc and antioxidant metabolite GSH (P<0.001) and at the same time we also observed very poor (TAS) (P<0.001) in leukoplakia patients when compared to patients in control group. CONCLUSION: The consumption of tobacco or areca quid which contains high copper levels creates an oxidative stress like environment during their metabolism, might play a major role in causation and propagation of oral leukoplakia.

A case of ruptured uterus at 27 weeks of gestation complicated by placenta accreta.

INTRODUCTION: Placenta accreta is a rare complication, which can lead to maternal morbidity and mortality. MATERIALS AND METHODS: This is a interesting case report where uterine rupture in the fundal region complicated with placenta accreta. CONCLUSION: Undiagnosed placenta accreta can lead to catastrophic consequences.

Puerperal group A Streptococcus infection.

Group A Streptococci infection during antenatal period as well as postnally can be very serious and would require intense management-both surgical and medical. Many authors believe the incidence is rising. High index of clinical suspicion is necessary in order to early intervention.