Dysregulation of noncoding RNA in chordoma; implications in identifying potential targets for novel therapeutic approaches.

Chordoma is a rare form of bone cancer develops in the spinal cord and skull. Instead of conventional (radio/chemotherapies) and targeted therapies, the disease is associated with high rate of recurrence and poor patient survival. Thus, for better disease management, the molecular pathogenesis of chordoma should be studied in detail to identify dysregulated biomolecules that can be targeted by novel therapeutics. Recent research showed frequent dysregulation of long noncoding RNA (lncRNA), microRNA (miRNA), and circular RNA (circRNA) in association with aggressive tumor phenotypes like cell proliferation, migration, invasion, and metastasis in a variety of cancers, including chordoma. Apart from diagnostic and prognostic importance, noncoding RNAs may serve as promising targets for novel therapeutics in cancer. In this review, we summarized a list of miRNAs, lncRNAs, and circRNA found to be dysregulated in chordoma from available data published in relevant databases (PubMed), as such an approach seems to be rare to date. The dysregulated noncoding RNAs were also associated with adverse tumor phenotypes to assess the impact on disease pathogenesis and, associated downstream molecular pathways were focused. Synthetic compounds and natural products that were reported to target the noncoding RNAs in other malignancies were also listed from published literature and proposed as potential therapeutic agents in chordoma. This review will provide information for further research on chordoma focusing on detailed characterization of dysregulated lncRNAs, miRNAs, and circRNA to understand the disease pathogenesis and, exploration of suitable natural and synthetic products targeting dysregulated non-coding RNAs to develop effective therapeutic measures.

Differential operation of MLH1/MSH2 and FANCD2 crosstalk in chemotolerant bladder carcinoma: a clinical and therapeutic intervening study.

We aimed to understand the crosstalk between mismatch repair (MMR) and FA-BRCA pathway in primary bladder carcinoma (BlCa) samples as well as in chemotolerant cell line. We analysed the genetic alterations of MLH1 and MSH2 (MMR-related genes) and after that we correlated it with the nuclear translocation of FANCD2 protein. Next, we evaluated this crosstalk in T24 BlCa cell line in response to doxorubicin treatment. In primary BlCa tumors, infrequent genetic deletion (17-20%) but frequent promoter methylation (28-55%) of MLH1 and MSH2 was observed, where MLH1 was significantly (p < 0.05) more methylated among the early staged samples (NMIBC). However, MSH2 was significantly more altered among the NMIBC samples, signifying the importance of MMR pathway during the early pathogenesis of the disease. Furthermore, BlCa samples with underexpressed MLH1/MSH2 protein possessed cytoplasmic FANCD2 protein; encouraging that inefficiency of MMR proteins might restrict FANCD2 nuclear translocation. Next, we analysed publicly available data in GEO2R tool where we observed that in response to chemotherapeutic drugs, expression of MLH1, MSH2 and FANCD2 were diminishing. Validating this result in doxorubicin tolerant T24 cells, we found that expression of MLH1 and MSH2 was gradually decreased with increasing dose of doxorubicin. Interestingly, FANCD2 mono-ubiquitination (L-form) was also reduced in chemotolerant T24 cells. The crosstalk between MMR and FA-BRCA pathway was substantiated in the primary BlCa tumors. Further, in response to doxorubicin, this crosstalk was found to be hampered due to under-expression of MLH1 and MSH2 gene, thereby rendering chemotolerance.

Co-Occurrence of Bromine and Iodine Species in US Drinking Water Sources That Can Impact Disinfection Byproduct Formation.

Bromine and iodine species are precursors for forming disinfection byproducts in finished drinking waters. Our study incorporates spatial and temporal data to quantify concentrations of inorganic (bromide (Br-), iodide (I-), and iodate (IO3-)), organic, and total bromine (BrT) and iodine (IT) species from 286 drinking water sources and 7 wastewater effluents across the United States. Br- ranged from <5-7800 µg/L (median of 62 µg/L in surface water (SW) and 95 µg/L in groundwater (GW)). I- was detected in 41% of SW (1-72 µg/L, median = <1 µg/L) and 62% of GW (<1-250 µg/L, median = 3 µg/L) samples. The median Br-/I- ratio in SW and GW was 22 µg/µg and 16 µg/µg, respectively, in paired samples with detect Br- and I-. BrT existed primarily as Br-, while IT was present as I-, IO3-, and/or total organic iodine (TOI). Inorganic iodine species (I- and IO3-) were predominant in GW samples, accounting for 60-100% of IT; however, they contributed to only 20-50% of IT in SW samples. The unknown fraction of IT was attributed to TOI. In lakes, seasonal cycling of I-species was observed and was presumably due to algal productivity. Finally, Spearman Rank Correlation tests revealed a strong correlation between Br- and IT in SW (RBr-,IT = 0.83) following the log10 (Br-, µg/L) = 0.65 × log10 (IT, µg/L) - 0.17 relationship. Br- and I- in treated wastewater effluents (median Br- = 234 µg/L, median I- = 5 µg/L) were higher than drinking water sources.

PRNCR1: a long non-coding RNA with a pivotal oncogenic role in cancer.

Long non-coding RNAs (lncRNAs) have been gaining importance in the field of cancer research in recent years. PRNCR1 (prostate cancer-associated non-coding RNA1) is a 12.7 kb, intron-less lncRNA found to play an oncogenic role in malignancy of diverse organs including prostate, breast, lung, oral cavity, colon and rectum. Single-nucleotide polymorphisms (SNPs) of PRNCR1 locus have been found to be associated with cancer susceptibility in different populations. In this review, an attempt has been made for the first time to summarize all sorts of available data on PRNCR1 to date from relevant databases (GeneCard, LncExpDB, Ensembl genome browser, and PubMed). As functional roles of PRNCR1, miRNA (microRNA) sponging was mostly highlighted in the pathogenesis of different cancer; in addition, an association of the lncRNA with chromatin-modifying complex to enhance androgen receptor-mediated gene transcription was reported in prostate cancer. Diagnostic and prognostic importance of PRNCR1 was found in some malignancies suggesting potency of the lncRNA to serve as a clinical biomarker. For PRNCR1 SNPs, although cancer susceptibility of the risk alleles/genotypes was reported in different populations, majorities of the findings were not replicated and underlying molecular mechanisms remained unexplored. Therapeutic implication of PRNCR1 was not studied well and future research may come up in this direction for intervening novel strategies to fight against cancer.

Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats.

BACKGROUND: On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose transporters (GLUT1/GLUT3/GLUT4), if any, in such putative association. METHODS AND RESULTS: Oral carcinogenesis was induced by 4-nitroquinoline N-oxide in 10 non-diabetic and 10 diabetic rats; 8 non-diabetic and 7 diabetic rats served as controls. Expressions of selected GLUTs at mRNA and protein levels were analyzed in oral tissue (normal/lesion) by quantitative real-time PCR and immunohistochemistry respectively. Premalignant lesions (hyperplasia/dysplasia/carcinoma-in-situ) appeared on tongues of carcinogen-treated animals. Significant increase of GLUT1mRNA level was seen from normal to lesion tongues, along increasing lesion grades (from hyperplasia/mild dysplasia to moderate/severe dysplasia) and in lesions induced under hyperglycemic condition than that induced under normoglycemic one; a similar trend was found in transcript variant-1 of GLUT1, but not in variant-2. GLUT3 and GLUT4 mRNA levels were comparable among lesions irrespective of grades and glycemic status. Concordant to mRNA level, overall expression of GLUT1 protein was higher in tongue lesions in presence of hyperglycemia than in absence of such condition; non-lesion portions of tongues exposed to carcinogen showed a similar trend. Moreover in carcinogen-treated groups, non-lesion and lesion portions of tongues under hyperglycemic condition showed predominantly membranous expression for GLUT1 which was again significantly higher than equivalent portions of tongue under normoglycemic condition. CONCLUSION: Hyperglycemia seemed to favor GLUT1 over-expression and membrane localization of the protein during oral carcinogenesis. GLUT1 transcript variant-1 appeared to be more important than variant-2 in disease pathogenesis.

Arsenic level in bladder tumor of patients from an exposed population: association with progression and prognosis.

Aims: To elucidate the impact of arsenic on progression and prognosis of bladder cancer. Patients & methods: Total arsenic in 145 tumors (80 non-muscle-invasive [NMIBC] and 65 muscle-invasive bladder cancer [MIBC]) was measured and associated with Ki67 expression, tumor-clinicopathological parameters and patient outcome. Results: Tumor arsenic concentration was higher in exposed than unexposed patients (256 µg/kg vs 77 µg/kg; p < 0.0001) and positively correlated (r = 0.65; p < 0.0001) with arsenic content of patient's drinking water. Arsenic concentration showed significant association with Ki67-overexpression (p = 0.001) and advanced tumor stages (NMIBC vs MIBC; p = 0.0009). In NMIBC, high tumor arsenic (>100 µg/kg) and Ki67 overexpression was established as predictors for recurrence (hazard ratio [HR]: 4.68; p = 0.005 and HR: 3.91; p = 0.018) and progression (HR: 6.04; p = 0.023 and HR: 6.87; p = 0.013). In MIBC, association of high arsenic remained significant with increased risk of recurrence (HR: 4.58; p = 0.04). Conclusion: In NMIBC, high arsenic and Ki67 overexpression and in MIBC, only high arsenic showed prognostic importance in predicting poor patient outcome.

Lay abstract Research work suggests arsenic as risk factor for bladder cancer. In developing countries such as India, arsenic contamination of underground drinking water is a major health problem. The present study aimed to evaluate impact of arsenic on parameters of bladder cancer aggressiveness and clinical outcome of patients from West Bengal, India. Our data showed accumulation of arsenic in bladder tumor of patients exposed mainly through contaminated drinking water. Arsenic content in tumor favored aggressive phenotypes in bladder cancer (higher cell proliferation and advanced tumor stages) and was found to be a potential predictor for cases of death and disease recurrence in patients after receiving primary treatment measures. Therefore, arsenic content in bladder tumor may be used to improve existing protocols for better prediction of patient outcomes in populations with a similar type of exposure.

Integrative genomics and pathway analysis identified prevalent FA-BRCA pathway alterations in arsenic-associated urinary bladder carcinoma: Chronic arsenic accumulation in cancer tissues hampers the FA-BRCA pathway.

Arsenic in drinking water is one of the major etiological factors in urinary bladder carcinoma (BlCa). Here, high-resolution CGH-SNP microarray analysis in arsenic accumulated BlCa tissues showed significant (p < 0.05) association of chromosomal alterations with high arsenic (≥112 ng/g) accumulation, further corroborated by high γH2AX nuclear expression. Cytobands 5q11-35, 9p24.3-21.5, 18q11.1-25, etc. showed deletion, whereas 12q was amplified in high arsenic samples (AsH). Consecutively, IPA® found FA-BRCA pathway to be exclusively altered in AsH group. Validation of several key regulatory genes (RAD50, BRIP1, UIMC1, FANCD2, BRCA2 and BRCA1) of the pathway, were performed in independent BlCa cases (n = 81). UIMC1, RAD50 and BRIP1 were differentially deleted and associated with poor survival of AsH samples. Moreover, reduced nuclear expression with diffused cytoplasmic expression of FANCD2 was higher in AsH samples. Collectively, frequent deregulation of RAD50, UIMC1 and BRIP1 may result in reduced nuclear translocation of FANCD2, which may cause more chromosomal aberrations among AsH samples.

Association of HLA-DQ and IL13 gene variants with challenge-proven shrimp allergy in West Bengal, India.

Little is known about genetic factors and mechanisms underlying shrimp allergy. Genome-wide association studies identified HLA class-II and IL13 genes as highly plausible candidates for shrimp allergy. The present study was designed to investigate potential associations of HLA-DQ rs9275596, IL13 rs20541, and IL13 rs1800925 polymorphisms with challenge-proven shrimp allergy using the data from 532 people of West Bengal, India; selected on basis of positive skin prick test, elevated specific IgE and medical history. Risk genotypes, i.e., HLA-DQ rs9275596 CC, IL13 rs20541 AA, and IL13 rs1800925 TT, were found to be significantly associated with challenge positive shrimp allergy (P = 0.04, 0.01, and 0.03, respectively). Distribution of genotypes for HLA-DQ and IL13 polymorphisms in allergic and control subjects showed significant difference between younger (20-40 years) and older (> 40 years) age group (P = 0.006). Risk genotypes significantly associated with elevated shrimp-specific IgE. IL13 TA haplotype significantly associated with shrimp allergy and elevated specific IgE (P = 0.02). Synergistic effect of IL13 TA haplotype-HLA-DQ rs9275596 CC genotype interaction significantly elevated specific IgE (P = 0.03). The present study suggests that HLA-DQ and IL13 polymorphisms pose major risk for shrimp allergic patients in West Bengal, India and thus could be helpful for early target-specific therapeutic intervention in near future.

Association of the STAT6 rs3024974 (C/T) Polymorphism with IgE-Mediated Food Sensitization among West Bengal Population in India.

INTRODUCTION: Genetic polymorphisms associated with IgE-mediated food sensitization have been a robust area of research for decades. A genome-wide search for susceptible loci regulating the IgE response (atopy) identified the candidate gene STAT6, which is important in the context of food allergic manifestations. OBJECTIVE: The present study was designed to investigate the sensitization of West Bengal population against some common allergenic food items and to study the role of the STAT6 gene polymorphism in elevating food-specific IgE levels among sensitized individuals. METHODS: Skin prick test was performed for 6 food items among 501 patients (126 children, 85 adolescents, and 290 adults)from West Bengal, India. Among them, 165 patients were selected for measurement of total IgE and food-specific IgE levels along with 165 controls. Finally, the STAT6 (rs3024974 (C/T) polymorphism was genotyped in 139 cases and control subjects. RESULTS: Shrimp was identified as a dominant food allergen in adolescents and adults, whereas milk sensitization was highest in children. Food-sensitized patients with onset during childhood had significantly higher total IgE levels compared to patients with onset during adulthood (p < 0.00001). The frequency of the rs3024974 CC genotype in both cases and control subjects (55.40 and 46.76%, respectively) was higher than that of CT or TT. Patients with childhood onset bearing the CC genotype had significantly higher specific IgE levels in comparison to those with adult onset (p = 0.001). CONCLUSION: Food sensitization has a genetic background and the rs3024974 polymorphism is associated with susceptibility and reaction severity in food-sensitized patients in West Bengal population in India.

Divergent molecular profile of PIK3CA gene in arsenic-associated bladder carcinoma.

The activation of PIK3CA in bladder carcinoma (BlCa) with its recurrent mutations in exon 9 and 20 were well reported. But the association of arsenic on the activation of the pathway is not well elucidated. Therefore, we aimed to analyse the effect of arsenic on the genetic (copy number variation/mutation) and expression profiles of PIK3CA in primary BlCa samples. Infrequent amplification (16%) of the PIK3CA locus was observed, with higher frequency among the arsenic-high (AsH) than arsenic-low (AsL) samples. Frequent (54%) tumour-specific mutations in exon 9 and 20 of PIK3CA were observed in the BlCa samples with prevalent (47%) C>T transition mutations. Exon 9 and 20 harboured 48% and 73% of the total mutations, respectively, with 37% in E542K/E545K and 25% of the mutation in H1047Y/R. Though mutation frequency in AsH and AsL was found to be comparable, we observed some arsenic-specific mutation at c.1633G>A, c.1634A>C (E545K) and c.2985C>T and c.3130G>T mutations, as well as prevalent transverse mutations of A>C and G>T in AsH group. Furthermore, 73% of the BlCa samples showed overexpression (mRNA/protein) of PIK3CA with genetic alterations (amplification/mutation), significantly (P = 0.01) higher in AsH group. However, 36% of the samples showed overexpressed PIK3CA, independent of mutation or amplification, signifying a transcriptional upregulation of PIK3CA gene. Therefore, the expression status of NFκB, a transcription factor of PIK3CA, was assessed and found to be significantly correlated with the overexpression of PIK3CA (mRNA/protein) in AsH group. Similarly, the expression pattern of pAKT1 (Thr 308) was also found to be significantly correlated with PIK3CA overexpression. Finally, AsH patients with the overexpression of PIK3CA or NFκB had the worst overall survival, signifying a strong impact of arsenic on this pathway and outcome of the patients. Thus, our study showed that the arsenic-associated differential molecular profile of PIK3CA/AKT1/NFkB in BlCa has an important role in the molecular pathogenesis of the disease.

Atlas of the Immune Cell Repertoire in Mouse Atherosclerosis Defined by Single-Cell RNA-Sequencing and Mass Cytometry.

RATIONALE: Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is poorly understood. OBJECTIVE: We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single-cell RNA-sequencing and mass cytometry (cytometry by time of flight) to define an atlas of the immune cell landscape in atherosclerosis. METHODS AND RESULTS: Using single-cell RNA-sequencing of aortic leukocytes from chow diet- and Western diet-fed Apoe-/- and Ldlr-/- mice, we detected 11 principal leukocyte clusters with distinct phenotypic and spatial characteristics while the cellular repertoire in healthy aortas was less diverse. Gene set enrichment analysis on the single-cell level established that multiple pathways, such as for lipid metabolism, proliferation, and cytokine secretion, were confined to particular leukocyte clusters. Leukocyte populations were differentially regulated in atherosclerotic Apoe-/- and Ldlr-/- mice. We confirmed the phenotypic diversity of these clusters with a novel mass cytometry 35-marker panel with metal-labeled antibodies and conventional flow cytometry. Cell populations retrieved by these protein-based approaches were highly correlated to transcriptionally defined clusters. In an integrated screening strategy of single-cell RNA-sequencing, mass cytometry, and fluorescence-activated cell sorting, we detected 3 principal B-cell subsets with alterations in surface markers, functional pathways, and in vitro cytokine secretion. Leukocyte cluster gene signatures revealed leukocyte frequencies in 126 human plaques by a genetic deconvolution strategy. This approach revealed that human carotid plaques and microdissected mouse plaques were mostly populated by macrophages, T-cells, and monocytes. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. CONCLUSIONS: The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunologic mechanisms and cell-type-specific pathways, and establishes a functional relevance for lesional leukocytes in human atherosclerosis.

IL-15 stimulates NKG2D while promoting IgM expression of B-1a cells.

Interleukin (IL)-15, a key manipulator of T-cell function also modulates B-1a cell activity by augmenting activation markers, turning them towards type 1 polarization and immunoglobulin (Ig) expression which is significant in the context of gut immunity. Here we show, for the first time, IL-15 mediated up-regulation of the activation receptor NKG2D and its adaptor DAP10 in B-1a cells indicating their essential coupling with IL-15 receptor signaling pathway. Our results demonstrate IL-15 treatment increases phosphorylation of STAT5 and p38 leading to translocation of NF-κB onto the nucleus, an attribute that delineates activation of B-1a cells and its role in inflammation. In parallel, increase of anti-apoptotic Bcl-xL suggests its role in long term survival of B-1a cells in culture by IL-15. The cytokine induced overexpression of the plasma cell differentiation transcription factor BLIMP-1 while reducing PAX-5a that could be responsible for the spontaneous Ig secretion by B-1a cells. Up-regulation of IgM transcripts in presence of IL-15 validates mucosal response of the cells through natural Abs to counter pathogens.

Using a relative health indicator (RHI) metric to estimate health risk reductions in drinking water.

When a new drinking water regulation is being developed, the USEPA conducts a health risk reduction and cost analysis to, in part, estimate quantifiable and non-quantifiable cost and benefits of the various regulatory alternatives. Numerous methodologies are available for cumulative risk assessment ranging from primarily qualitative to primarily quantitative. This research developed a summary metric of relative cumulative health impacts resulting from drinking water, the relative health indicator (RHI). An intermediate level of quantification and modeling was chosen, one which retains the concept of an aggregated metric of public health impact and hence allows for comparisons to be made across "cups of water," but avoids the need for development and use of complex models that are beyond the existing state of the science. Using the USEPA Six-Year Review data and available national occurrence surveys of drinking water contaminants, the metric is used to test risk reduction as it pertains to the implementation of the arsenic and uranium maximum contaminant levels and quantify "meaningful" risk reduction. Uranium represented the threshold risk reduction against which national non-compliance risk reduction was compared for arsenic, nitrate, and radium. Arsenic non-compliance is most significant and efforts focused on bringing those non-compliant utilities into compliance with the 10 µg/L maximum contaminant level would meet the threshold for meaningful risk reduction.

SNP rs1049430 in the 3'-UTR of SH3GL2 regulates its expression: Clinical and prognostic implications in head and neck squamous cell carcinoma.

Single nucleotide polymorphisms (SNPs) in the 3'-UTR region are emerging cis-regulatory factors associated with the occurrences of several human diseases. SH3GL2, which is located at chromosome 9p21-22, is associated with hyperplastic/mildly dysplastic lesions of the head and neck and has a long 3'-UTR with multiple SNPs. The aim of the present study was to determine the susceptible allele(s) in the 3'-UTR SNPs of SH3GL2 in head and neck squamous cell carcinoma (HNSCC). First, we screened the genotypes of all SNPs located in the 3'-UTR of SH3GL2 in 110 controls and 147 cases in Indian populations by sequencing. A SNP (rs1049430:>G/T) that showed only heterozygosity was further confirmed by genotyping with an Illumina GoldenGate platform in 530 controls and 764 cases. Genotype-specific survival analysis of the HNSCC patients was performed. In addition, genotype-specific mRNA stability, isoform expression and protein expression were analyzed. SNP rs1049430 was not associated with disease occurrence, but it was associated with poor patient outcome. The G allele was associated with decreased SH3GL2 mRNA stability, differential splicing and low protein expression. Thus, our data demonstrate that the presence of the susceptible G allele in SNP rs1049430 is associated with the inactivation of SH3GL2 and could be used as a prognostic marker of HNSCC.

Porin differentiates TLR mediated proinflammatory response of follicular zone B cell from TLR-unresponsive IL-10 expressing marginal zone B cell.

TLR-ligands are frequently chosen as candidates for vaccine or adjuvant development because they can primarily bridge innate signaling with adaptive immune responses. Since the adjuvant action of porin, the major outer membrane protein commonly present on Gram-negative bacteria, has been tested on several antigen-presenting cells, we investigated its role in driving systemic immunity which is considered a benchmark for a successful adjuvant. Here, we show porin differentially regulated splenic marginal zone (MZ) and follicular zone (FO) B cell responses in contrast to other classical TLR2-ligands FSL-1 and Pam3CSK4. The protein up-regulated TLR2 and TLR6 and stimulated the activation and costimulatory molecules on FO B cells skewing the cells toward TLR-dependent type-1 cytokine response. However, porin could not up-regulate the TLRs and activate MZ B cells. These cells responded to porin by expressing toll-interacting protein (TOLLIP), the TLR2 and -4 signaling inhibitor along with stimulation of the intracellular pathogen recognition receptor NLR caspase recruitment domain containing protein 5 (NLRC5). The CD1d(hi) MZ B cells released IL-10 unequivocally demonstrating regulatory B cell feature. Immunization with porin also resulted in transient IL-10 expression by the CD19(+)CD21(hi) B cells prior to plasma cell formation. Moreover, the plasma cells developed from the B-2 cell subsets show marked variation in generation of immunoglobulin subclasses. The work delineates multi-faceted role of B cell subsets induced by porin for robust immunity without compromising with the checks and controls.

LPS stimulates and Hsp70 down-regulates TLR4 to orchestrate differential cytokine response of culture-differentiated innate memory CD8(+) T cells.

Nonconventional innate memory CD8(+) T cells characteristically expressing CD44, CD122, eomesodermin (Eomes) and promyelocytic leukemia zinc finger (PLZF) were derived in culture from CD4(+)CD8(+) double positive (DP) thymocytes of normal BALB/c and C57BL/6 mice. These culture-differentiated cells constitutively express toll-like receptor (TLR)4 and release interferon (IFN)-γ and interleukin (IL)-10. We show the TLR4-ligand lipopolysaccharide (LPS) stimulate the TLR and up-regulate IFN-γ skewing the cells towards type 1 polarization. In presence of LPS these cells also express suppressor of cytokine signaling (SOCS)1 and thus suppress IL-10 expression. In contrast, heat shock protein (Hsp)70 down-regulated TLR4 augmenting the anti-inflammatory cytokine IL-10. In association with IL-10 release IFN-γ was abrogated. The programmed cell death (PD)-1 mostly present in regulatory T cells was stimulated in these IL-10 producing cells by Hsp70 and not LPS indicating the cells can be driven to two contrast outcomes by the two TLR4 ligands. Our work provides a scope for in vitro monitoring of CD8(+) T cells to decipher important immune therapeutic option during infection or sepsis.

Culture-differentiated CD8(+) T cells acquire innate memory-like traits and respond to a pathogen-associated molecule.

Selection of conventional CD4(+) or CD8(+) T cells is usually driven by the interaction of double-positive CD4(+)CD8(+) thymocytes with epithelial cells. Here, we demonstrate preferential selection of CD8(+) thymocytes from in vitro differentiation of CD4(+)CD8(+) double-positive thymocytes exhibiting the characteristics of nonconventional innate memory CD8(+) cells. In contrast to conventional CD8(+) thymocytes, these culture-differentiated CD8(+) cells are eomesodermin positive and robustly express CXCR3, CD44, CD122 and TLR2. Interestingly, the pathogen-associated molecule porin promotes preferential differentiation of the CD8(+) single-positive subset in association with promyelocytic leukemia zinc-finger upregulation and interleukin (IL)-4 production. On priming with anti-CD3 antibody, porin augmented TLR2 and IFN-γ indicating a role of the TLR ligand in acquisition of innate memory response of CD8(+) thymocytes. In addition, porin has a cooperative role with IL-15 on the expansion of memory-phenotype CD8(+) T cells along with its effector function. Thus, the study opens an avenue to unfold the cues for development of these cells and the strategies adopted for bolstering immunity during primary infection.

Association of common variants of TCF7L2 and PCSK2 with gestational diabetes mellitus in West Bengal, India.

The genetic etiology of gestational diabetes mellitus (GDM) was suggested to overlap with type-2 diabetes(T2D). Transcription factor 7-like 2 (TCF7L2) and Proprotein Convertase Subtilisin/Kexin type 2 (PCSK2) are T2D susceptibility genes of the insulin synthesis/processing pathway. We analyzed associations of TCF7L2 and PCSK2 variants with GDM risk and evaluated their potential impact on impaired insulin processing in an eastern Indian population. The study included 114 GDM (case) and 228 non-GDM pregnant women (control). rs7903146, rs4132670, rs12255372 of TCF7L2, and rs2269023 of PCSK2 were genotyped by PCR-RFLP, and genotype distributions were compared between case and control. Fasting serum proinsulin and C-peptide levels were measured by ELISA and the Proinsulin/C-peptide ratio was considered an indicator of proinsulin conversion. Significantly higher frequency of risk allele (T) of rs12255372 (p = 0.02, OR = 2.0, 95%CI = 1.11-3.64) and rs4132670 (p = 0.002, OR = 2.26, 95%CI = 1.32-3.87) of TCF7L2 was found in GDM cases than non-GDM controls; TT genotype was associated with significantly increased disease risk. In rs7903146 (TCF7L2) and rs2269023 (PCSK2), although the frequency of risk allele (T) was not significantly higher in cases than controls, an association of TT for both variants remained significant with higher GDM risk in the recessive model. Increased serum pro-insulin and proinsulin:c-peptide ratio was found in GDM than non-GDM women and the phenomenon showed significant association with careers of risk alleles for TCF7L2 variants. In conclusion, TCF7L2 and PCSK2 variants are related to GDM risk in the studied population and hence may serve as potential biomarkers for assessing the disease risk. TCF7L2 variants contribute to impaired insulin processing.

Dysregulation of DNA epigenetic modulators during prostate carcinogenesis in an eastern Indian patient population: Prognostic implications.

The role of epigenetic alteration in prostate cancer pathogenesis was reported. We aimed to analyze dysregulation of DNA methylase (DNA methyl transferase/DNMT) and demethylase (ten eleven translocase/TET) and the associated interplay between them during prostate tumorigenesis. Promoter methylation and RNA/protein expression of selected DNMT and TETs were analysed in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer (PCa). Genomic 5-hydroxymethylcytosine (5hmC) level was detected and correlated with DNMT and TET proteins. Clinicopathological association of molecular data was done. Our data revealed a very low frequency of promoter methylation for DNMT1 (5-3% and high frequency for TET1 (22-38%), TET2 (68-90 %), and TET3 (43-32 %) in BPH and PCa. The promoter methylation of DNMT1 (p = 0.019) showed a significantly decreasing trend, while that of TET1 (p = 0.0005) and TET2 (p < 0.0001) showed an increasing trend from normal prostate to BPH to PCa, indicating their epigenetic dysregulation during prostate tumorigenesis. RNA/protein overexpression of DNMT1 and reduced expression of TET1 and TET2 in PCa compared to BPH were associated with the promoter methylation status of genes. The 5hmC level was significantly lower in PCa than in BPH and correlated negatively with DNMT1 but positively with TET1 and TET2 proteins, suggesting dysregulation of DNA methylase and de-methylase activities during prostate tumorigenesis. Lastly, tumors having methylated TET1 and TET2 promoters showed advanced clinicopathological features (a higher PSA level/Gleason score) and increased risk of bone metastasis. In conclusion, DNMT1 upregulation and epigenetic silencing of TET1 and TET2 was seen during PCa development. TET1 and TET2 promoter methylation has prognostic importance.