Molecular Chaperonin HSP60: Current Understanding and Future Prospects.

Molecular chaperones are highly conserved across evolution and play a crucial role in preserving protein homeostasis. The 60 kDa heat shock protein (HSP60), also referred to as chaperonin 60 (Cpn60), resides within mitochondria and is involved in maintaining the organelle's proteome integrity and homeostasis. The HSP60 family, encompassing Cpn60, plays diverse roles in cellular processes, including protein folding, cell signaling, and managing high-temperature stress. In prokaryotes, HSP60 is well understood as a GroEL/GroES complex, which forms a double-ring cavity and aids in protein folding. In eukaryotes, HSP60 is implicated in numerous biological functions, like facilitating the folding of native proteins and influencing disease and development processes. Notably, research highlights its critical involvement in sustaining oxidative stress and preserving mitochondrial integrity. HSP60 perturbation results in the loss of the mitochondria integrity and activates apoptosis. Currently, numerous clinical investigations are in progress to explore targeting HSP60 both in vivo and in vitro across various disease models. These studies aim to enhance our comprehension of disease mechanisms and potentially harness HSP60 as a therapeutic target for various conditions, including cancer, inflammatory disorders, and neurodegenerative diseases. This review delves into the diverse functions of HSP60 in regulating proteo-homeostasis, oxidative stress, ROS, apoptosis, and its implications in diseases like cancer and neurodegeneration.

The small heat shock protein Hsp27: Present understanding and future prospects.

Heat shock proteins are important for maintaining protein homeostasis and cell survival. Among different classes of highly conserved Hsps, low molecular weight Hsps (sHsps) have significant place, particularly Hsp27, whose role has been demonstrated in wide range of biological processes, including development, immunity, diseases and therapy. In this review, the structure and functions of Hsp27 and related genes, their role in different cellular processes as well as in stress tolerance, is highlighted.

Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer.

BACKGROUND: Poor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer. METHODS: Proteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA. RESULTS: The quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells. CONCLUSIONS: Our findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.

Identification of prosaposin and transgelin as potential biomarkers for gallbladder cancer using quantitative proteomics.

Gallbladder cancer is an uncommon but lethal malignancy with particularly high incidence in Chile, India, Japan and China. There is a paucity of unbiased large-scale studies investigating molecular basis of gallbladder cancer. To systematically identify differentially regulated proteins in gallbladder cancer, iTRAQ-based quantitative proteomics of gallbladder cancer was carried out using Fourier transform high resolution mass spectrometry. Of the 2575 proteins identified, proteins upregulated in gallbladder cancer included several lysosomal proteins such as prosaposin, cathepsin Z and cathepsin H. Downregulated proteins included serine protease HTRA1 and transgelin, which have been reported to be downregulated in several other cancers. Novel biomarker candidates including prosaposin and transgelin were validated to be upregulated and downregulated, respectively, in gallbladder cancer using tissue microarrays. Our study provides the first large scale proteomic characterization of gallbladder cancer which will serve as a resource for future discovery of biomarkers for gallbladder cancer.

Epigenetic Biomarkers in Gallbladder Cancer.

Gallbladder cancer (GBC) is associated with various nongenetic and genetic factors. Lack of specific and sensitive diagnostic markers has significantly impacted the mortality of this disease. Here we discuss the recent discovery of epigenetic changes that show great promise as diagnostic biomarkers as well as potential therapeutic targets for GBC.

Exploring the Role of C-C Motif Chemokine Ligand-2 Single Nucleotide Polymorphism in Pulmonary Tuberculosis: A Genetic Association Study from North India.

The C-C motif chemokine ligand-2 (CCL2) was evidenced to be associated with tuberculosis susceptibility in some ethnic groups. In the present study, effort was made to find out the association of CCL2-2518 A>G and -362 G>C variants with susceptibility to TB in a population from North India. The genotyping was carried out in 373 participants with pulmonary TB (PTB) and 248 healthy controls (HCs) for CCL2-2518 A>G and -362 G>C polymorphisms by PCR-RFLP and by melting curve analysis using fluorescence-labeled hybridization fluorescent resonance energy transfer (FRET) probes, respectively, followed by DNA sequencing in a few representative samples. Genotype and allele frequencies were compared by the chi-squared test and crude and Mantel-Haenszel (M-H) odds ratio (OR). OR was calculated using STATA/MP16.1 software. Further, CCL2, IL-12p70, IFN-γ, TNF-α, and TGF-ß levels were measured in serum samples of these participants using commercially available kits. Our analysis indicated that the homozygous mutant in both -2518 GG (OR = 2.07, p = 0.02) and -362 CC (OR = 1.92, p = 0.03) genotypes was associated with susceptibility to pulmonary TB. Further, heterozygous genotypes -2518AG (OR = 0.60, p = 0.003) and -362GC (OR = 0.64, p = 0.013) provide resistance from PTB disease. Haplotype analysis revealed AC haplotype (p = 0.006) to be a risk factor associated with PTB susceptibility. The serum CCL2 level was significantly elevated among participants with -2518 AA genotype compared to -2518 GG genotype. CCL2 level was observed to be positively correlated with IL12p70, IFN-γ and TNF-α, thus suggesting the immunological regulatory role of CCL2 against pulmonary tuberculosis. CCL2-2518 GG and -362 CC genotypes were found to be associated with susceptibility to pulmonary tuberculosis and CCL2-2518AG and CCL2-362GC with resistance from PTB. AC haplotype was found to be a risk factor for PTB in the present study. It may be hypothesized from the findings that -2518G allele could be responsible for lower production of CCL2 which leads to defective Th1 response and makes a host susceptible for pulmonary tuberculosis.

Spoligotyping, phenotypic and genotypic characterization of katG, rpoB gene of M. tuberculosis isolates from Sahariya tribe of Madhya Pradesh India.

BACKGROUND: Sahariya, a primitive tribal group, residing in Gwalior and Sheopur districts of Madhya Pradesh, India, show high incidence and prevalence of pulmonary tuberculosis (PTB). The study was designed to understand the genetic diversity and phenotype - genotype association of drug resistant M. tuberculosis strains, infecting Sahariya tribe. MATERIALS AND METHODS: A total of 103 pulmonary tuberculosis patients from Sahariya tribe were recruited from Gwalior and Sheopur districts. Sputum samples were collected and cultured on LJ slants and drug sensitivity tests were carried out. Genomic DNA was extracted, followed by spoligotyping and genotyping of drug target genes, katG and rpoB, using MAS-PCR, PCR-RFLP and DNA sequencing. RESULT: Seventeen different spoligotypes were identified, in which, EAI3_IND/ST11 M. tuberculosis strain appeared predominant, followed by CAS1_Delhi/ST26. Results of our phenotypic drug susceptibility test identified high incidence (12.6%) of isoniazid-resistant tuberculosis, while 4.85% isolates were multi drug resistant (MDR). Further genotyping of drug target genes identified 8.7% of isoniazid-R isolates to have a mutation at katG codon 463, while 3.8% isolates showed mutations at two sites, katG codons 315 and 463. In case of MDR-TB isolates, all from CAS lineage, 3.85% had mutations on katG and rpoB genes, at codon 463 and codon 526, respectively, while 0.97% isolates were harbouring mutations at codons 315, 463 and 531. CONCLUSION: Our findings have revealed that EAI3_IND strain is the predominant strain infecting Sahariya. The incidence of isoniazid-R M. tuberculosis strain infection is high, with an increased propensity to evolve into MDR-TB. Therefore, the TB centres should also consider isoniazid-R status of the isolates along with CBNAAT before deciding the drug regimen for the patients.

HLA-A*02 repertoires in three defined population groups from North and Central India: Punjabi Khatries, Kashmiri Brahmins and Sahariya Tribe.

The allelic family of HLA-A*02 with a repertoire of approximately 1022 alleles represents the predominant and most heterogeneous group at the HLA-A locus. This remarkable diversity signifies its evolutionary relevance. Its population-specific diversity is attributed to environmental factors and pathogen pressure and can be harnessed in biology and medicine, particularly in disease association and for HLA-based vaccination approaches. We therefore investigated the HLA-A*02 repertoire in two North Indian caste populations, viz Punjabi Khatries (PK, N = 250), Kashmiri Brahmins (KB, N = 160) and a Central Indian tribe Sahariya (ST, N = 100) using Luminex-based high-resolution rSSO method. When required, results were confirmed with high-resolution PCR-SSP and/or next-generation sequencing (NGS). In the three populations evaluated, HLA-A*02 was observed with an overall high phenotypic/allelic frequency, however, A*02 repertoire differed among them. A total of six alleles were observed (A*02:01, *02:03, *02:05, *02:06, *02:07 and *02:11) in the caste groups, compared with four (except *02:05 and *02:07) in the tribals. Our striking observation was the high occurrence of A*02:11 at the repertoire level (80.6% in ST, 39% in PK, 31.8% in KB). Globally, this allele is rare, observed with low frequencies in limited ethnic groups. The primordial A*02:01 allele, representative A*02 allele in most ethnicities was observed as the second predominant allele (PK = 27.3%, KB = 31.8% and ST = 11.9%). Extremely high occurrence of A*02:11 in ST may be representation of ancient Austro-Asiatic genetic pool. In caste populations, the observed A*02 repertoire may be a consequence of natural selection and/or admixture from different populations.

PIM1 kinase promotes gallbladder cancer cell proliferation via inhibition of proline-rich Akt substrate of 40 kDa (PRAS40).

Gallbladder cancer (GBC) is a rare malignancy, associated with poor disease prognosis with a 5-year survival of only 20%. This has been attributed to late presentation of the disease, lack of early diagnostic markers and limited efficacy of therapeutic interventions. Elucidation of molecular events in GBC can contribute to better management of the disease by aiding in the identification of therapeutic targets. To identify aberrantly activated signaling events in GBC, tandem mass tag-based quantitative phosphoproteomic analysis of five GBC cell lines was carried out. Proline-rich Akt substrate 40 kDa (PRAS40) was one of the proteins found to be hyperphosphorylated in all the invasive GBC cell lines. Tissue microarray-based immunohistochemical labeling of phospho-PRAS40 (T246) revealed moderate to strong staining in 77% of the primary gallbladder adenocarcinoma cases. Regulation of PRAS40 activity by inhibiting its upstream kinase PIM1 resulted in a significant decrease in cell proliferation, colony forming and invasive ability of GBC cells. Our results support the role of PRAS40 phosphorylation in GBC cell survival and aggressiveness. This study also elucidates phospho-PRAS40 as a clinical marker in GBC and the role of PIM1 as a therapeutic target in GBC.

Restriction fragment length polymorphism of mitochondrial DNA and phylogenetic relationships among five species of Indian freshwater turtles.

DNA-based identification of species for phylogenetic analysis as well as forensic identification is widely being carried out with the help of polymerase chain reaction (PCR). In this study, a successful effort has been made to identify 5 species of Indian freshwater turtles, including 3 hard-shell turtles (Geoemydidae), i.e. Kachuga dhongoka, K. kachuga and Geoclemys hamiltoni, and 2 species of soft-shell turtles (Trionychidae), i.e. Aspideretes gangeticus and Lissemys punctata punctata, by using a well-optimized PCR-RFLP method. The analysis of nucleotide sequence variations in the PCR-amplified mitochondrial cyt-b genes (encoding cytochrome b) from the 5 species revealed its usefulness in the taxonomic differentiation of these species. On the basis of cyt-b sequence data and the PCR-RFLP pattern, a phylogeny was developed to resolve the genetic relationships between these species, living in the same habitat type. In comparison, the PCR-RFLP of mitochondrial 16S rDNA genes appeared less decisive in analysing phylogenetic relationships or even in species differentiation. Further, the molecular method (PCR-RFLP) developed here is simple, rapid, reliable and reproducible; hence it can be routinely applied for species identification, essential for conservation and management of endangered chelonian species.

Molecular modelling and docking analysis of katG and rpoB gene in MDR-TB isolates from North Central Indian population.

Tuberculosis caused by Mycobacterium tuberculosis, requires multi drug therapy approach. Drug resistance in M. tuberculosis is caused by mutations in specific regions in drug target genes. The study aimed to identify mutations in katG and rpoB genes and investigate the drug-drug target interactions. A total of 27 MDR-TB isolates were sequenced for katG and rpoB genes and docking and MIC analysis were performed. Three types of mutations for katG gene (Arg463Leu in all isolates of Sahariya and non-tribes; Asp529Thr and Asp529His, each in two isolates only, in Sahariya) were observed. In rpoB gene, the Ser531Leu change was observed in 17/21 isolates in Sahariya and 3/6 isolates in non-tribes. The docking analysis revealed that the drugs isoniazid and rifampicin bind to different residues in mutant forms than their proposed active sites, making active binding sites rigid and causing resistance. The MIC for isoniazid was found to range from 0.2 to 5µg/ml in Sahariya tribe, whereas, in non-tribes, it is 0.2µg/ml and 1µg/ml. The MIC for rifampicin was observed at 64µg/ml in both the population groups. The study explored the possible functional variation in isoniazid and rifampicin resistance with respect to the identified mutations. The present results indicate that these mutations affect the drug binding affinity and are causing resistance.

Cloning & sequence identification of Hsp27 gene and expression analysis of the protein on thermal stress in Lucilia cuprina.

Hsp27, a highly conserved small molecular weight heat shock protein, is widely known to be developmentally regulated and heat inducible. Its role in thermotolerance is also implicated. This study is a sequel of our earlier studies to understand the molecular organization of heat shock genes/proteins and their role in development and thermal adaptation in a sheep pest, Lucilia cuprina (blowfly), which exhibits unusually high adaptability to a variety of environmental stresses, including heat and chemicals. In this report our aim was to understand the evolutionary relationship of Lucilia hsp27 gene/protein with those of other species and its role in thermal adaptation. We sequence characterized the Lchsp27 gene (coding region) and analyzed its expression in various larval and adult tissues under normal as well as heat shock conditions. The nucleotide sequence analysis of 678 bps long-coding region of Lchsp27 exhibited closest evolutionary proximity with Drosophila (90.09%), which belongs to the same order, Diptera. Heat shock caused significant enhancement in the expression of Lchsp27 gene in all the larval and adult tissues examined, however, in a tissue specific manner. Significantly, in Malpighian tubules, while the heat-induced level of hsp27 transcript (mRNA) appeared increased as compared to control, the protein level remained unaltered and nuclear localized. We infer that Lchsp27 may have significant role in the maintenance of cellular homeostasis, particularly, during summer months, when the fly remains exposed to high heat in its natural habitat.

Status of multidrug resistant tuberculosis (MDR-TB) among the Sahariya tribe of North Central India.

BACKGROUND: The incidence/prevalence of tuberculosis (TB) is reported to be high in the Sahariya tribe of North Central India. The outbreaks of different drug-resistant isolates of Mycobacterium tuberculosis emphasized the need for continuous monitoring of resistance to anti-tuberculosis drugs. This study aimed to assess the profile of multidrug resistant TB among the Sahariya tribe and their non-tribal neighbors for first line drugs through field-based investigations. METHODOLOGY: A total of 274 sputum positive pulmonary TB individuals were enrolled and studied for their drug susceptibility profile by the proportion method. RESULTS: A total of 21 cases from Sahariya and 6 from non-tribes were identified with MDR-TB. Thus Sahariya tribe showed a 1.95-fold increased risk of developing drug resistance than non-tribes. Significant differences were observed for developing drug sensitivity between Sahariya males and females when analyzed for resistance developed to any drug and overall drug resistance vs. sensitive isolates, respectively. A 4.46-fold risk was found for MDR-TB among the smokers of Sahariya tribe, whereas, the non-tribes did not show any significant association. CONCLUSION: The drug susceptibility profile developed in the present study indicates that drug-resistant tuberculosis is emerging as a serious public health concern in Sahariya tribe. Urgent and effective control measures and better management policies are needed for the prevention of MDR-TB in the tribe.

Fine needle aspiration cytology of lesions of liver and gallbladder: An analysis of 400 consecutive aspirations.

BACKGROUND: Patients presenting with mass lesions of liver and gallbladder are a common occurrence in a cancer hospital in north central part of India. Fine-needle aspiration cytology (FNAC) serves as first line of pathological investigations, but there are pros and cons involved. AIM: The main objective of the present study was to establish adequacy of the procedure and to find out diagnostic pitfalls. An attempt was made to analyze inconclusive and inadequate aspirations. MATERIALS AND METHODS: A total of 400 consecutive fine-needle aspirates of liver, belonging to 328 cases over a period of 2 years, were analyzed. Hematoxylin and eosin and May-Grόnwald-Giemsa stains were used. Chi-square test was carried out to compare significant degree of difference in different kind of diagnosis. RESULTS: Out of 400 aspirations, 289 (72.2%) were adequate, 75 (18.7%), inconclusive and 36 (9%), inadequate. Among positive aspirations the most common was metastatic adenocarcinoma, 128 (44.2%). The positive diagnosis and adequate aspirations were significantly high (P < 0.0001). Major differential diagnostic problems were: Distinguishing the poorly differentiated hepatocellular carcinoma from the metastatic adenocarcinoma; and leukemia/lymphoma from other malignant round cell tumors. Common diagnostic pitfalls were repeated aspirations from the necrotic area and aspiration of atypical, disorganized and reactive hepatocytes, adjacent to a metastasis. No complications were observed. CONCLUSION: FNAC can be used successfully for the diagnosis of liver and gallbladder lesions, thus avoiding open biopsy. Study indicates the potential of using FNAC in clinical intervention where the incidence of gall-bladder and liver cancer is very high and open biopsy and surgery are not an option.