Co-occurrence of acrocentric chromosome associations with dicentric chromosomes in irradiated human lymphocytes.

PURPOSE: The occurrence of acrocentric chromosome association (ACA) after radiation exposure is an interesting cytogenetic endpoint, known to show a dose-dependent increase in irradiated lymphocytes suggesting its potential use in radiation biodosimetry. Here, an attempt was made to study the complexity and correlation of the occurrence of ACA with dicentric chromosomes (DC) in lymphocytes exposed to gamma radiation. METHODS: Ninety metaphases each with DC and without DC were chosen randomly from lymphocytes irradiated with different doses (0, 1, 2, 3, 4 and 5 Gy) of gamma radiation. ACA along with chromosomal types of aberrations were scored and analyzed for complexity and co-occurrence, retrospectively. RESULTS: The number of associations between 2 and ≥ 3 acrocentric chromosomes showed an increase with each irradiation dose. Concomitantly, the total number of chromosomal type of aberrations showed an increase in number at each radiation dose studied. The number of DC showed an increase, however, metaphases containing 1DC decreased while ≥ 2DC increased as the radiation dose increased. The number of tricentric chromosomes increased at doses higher than 2 Gy. Importantly, the association of DC with an acrocentric chromosome was noticed at doses 2 Gy and above. A significant (p < 0.05) increase was noticed in ACA frequency in 1DC and ≥ 2DC metaphases at 1 and 2 Gy, in 1DC at 3 Gy, and in ≥ 2DC 4 and 5 Gy compared to the frequency in no DC metaphases. When average ACA frequency was plotted against DC frequency, a significant (p = 0.0009) correlation was observed, producing regression equation y = 0.9025x + 0.1283; R2 = 0.9522. CONCLUSION: The present analysis showed increasing ACA complexity with increasing radiation dose. Furthermore, a higher frequency of ACA in cells with 1DC or ≥ 2DC compared to the ACA in cells without DC from the same sample of irradiated lymphocytes demonstrated the co-occurrence of ACA and DC in the same cells.

Linear dose response of acrocentric chromosome associations to gamma irradiation in human lymphocytes.

PURPOSE: The frequency of acrocentric chromosome associations (ACA) was studied to determine the possible dose-response relationship of gamma irradiation in human lymphocytes. METHODS: Peripheral blood collected from three healthy donors was irradiated with 0, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy of gamma radiation. Chromosomal preparations were made after 48 h of culture as per standard guidelines. The experiment was repeated three times, with a different donor each time. RESULTS: The ACA frequency in irradiated lymphocytes increased with radiation dose. The D-G type of association was most prominent and showed a significant dose-dependent increase in frequency. The dose response of ACA frequency to radiation was found to be linear: ACA frequency = 0.2923 (±0.0276) + 0.1846 (±0.0307) × D (correlation coefficient r = 0.9442). As expected, dicentric chromosome (DC) frequencies followed the linear quadratic fit model, with DC frequency = 0.0015 (±0.0013) + 0.0220 (±0.0059) × D + 0.0215 (±0.0018) × D^2 (correlation coefficient r = 0.9982). A correlation curve was prepared for ACA frequency versus DC frequency, resulting in the regression equation y = 1.130x + 0.4051 (R2 = 0.7408; p = 0.0014). CONCLUSION: Our results showed an increase in ACA frequency in irradiated lymphocytes with an increase in radiation dose; thus, ACA may serve as a candidate cytogenetic biomarker for radiation biodosimetry.

The road-map for establishment of a prognostic molecular marker panel in glioma using liquid biopsy: current status and future directions

Gliomas are primary intracranial tumors with defined molecular markers available for precise diagnosis. The prognosis of glioma is bleak as there is an overlook of the dynamic crosstalk between tumor cells and components of the microenvironment. Herein, different phases of gliomagenesis are presented with reference to the role and involvement of secreted proteomic markers at various stages of tumor initiation and development. The secreted markers of inflammatory response, namely interleukin-6, tumor necrosis factor-α, interferon-ϒ, and kynurenine, proliferation markers human telomerase reverse transcriptase and microtubule-associated-protein-Tau, and stemness marker human-mobility-group-AThook-1 are involved in glial tumor initiation and growth. Further, hypoxia and angiogenic factors, heat-shock-protein-70, endothelial-growth-factor-receptor-1 and vascular endothelial growth factor play a major role in promoting vascularization and tumor volume expansion. Eventually, molecules such as matrix-metalloprotease-7 and intercellular adhesion molecule-1 contribute to the degradation and remodeling of the extracellular matrix, ultimately leading to glioma progression. Our study delineates the roadmap to develop and evaluate a non-invasive panel of secreted biomarkers using liquid biopsy for precisely evaluating disease progression, to accomplish a clinical translation. (AU)

The road-map for establishment of a prognostic molecular marker panel in glioma using liquid biopsy: current status and future directions.

Gliomas are primary intracranial tumors with defined molecular markers available for precise diagnosis. The prognosis of glioma is bleak as there is an overlook of the dynamic crosstalk between tumor cells and components of the microenvironment. Herein, different phases of gliomagenesis are presented with reference to the role and involvement of secreted proteomic markers at various stages of tumor initiation and development. The secreted markers of inflammatory response, namely interleukin-6, tumor necrosis factor-α, interferon-ϒ, and kynurenine, proliferation markers human telomerase reverse transcriptase and microtubule-associated-protein-Tau, and stemness marker human-mobility-group-AThook-1 are involved in glial tumor initiation and growth. Further, hypoxia and angiogenic factors, heat-shock-protein-70, endothelial-growth-factor-receptor-1 and vascular endothelial growth factor play a major role in promoting vascularization and tumor volume expansion. Eventually, molecules such as matrix-metalloprotease-7 and intercellular adhesion molecule-1 contribute to the degradation and remodeling of the extracellular matrix, ultimately leading to glioma progression. Our study delineates the roadmap to develop and evaluate a non-invasive panel of secreted biomarkers using liquid biopsy for precisely evaluating disease progression, to accomplish a clinical translation.

Can a signature molecular-profile define disparate survival in BRAF-positive Gliosarcoma and identify novel targets for therapeutic intervention?

Purpose: Gliosarcoma (GS) has a low incidence but is aggressively invasive, with poor-survival. Even though GS is recognized as a different subgroup from glioblastoma (GB), there is no molecular panel available to define its clinical outcome. The objective was to identify the molecular imprint of GS in terms of expression of human telomerase reverse transcriptase (hTERT), high mobility group A1 (HMGA-1), kinesin superfamily protein-14 (KIF-14), epidermal growth factor receptor (EGFR) markers with reference to disparate prognosis and identify plausible targets for intervention. Materials and Methods: We retrieved 9-GS samples from a cohort of 57-GB patients during a 36 months study period and compared them with 10 molecularly typed GB-samples and 15 controls. Conventional-immunohistochemistry (IHC) was used for histopathology of GS and immunofluorescence-IHC was performed for quantification of identified marker-panel. Statistical tools for non-parametric data were used for inferring results. Results: GS was confirmed by reticulin-staining and positivity for glial fibrillary acidic protein, Vimentin, smooth muscle actin. Immune-reactivity for BRAF-V600Ewas present in both glial and sarcomatous cells and negative expression of isocitrate dehydrogenase, ATRX, TP53.Comparison between GS, GB, and control tissues showed that the expression of markers reached significance (P < 0.0001), without the influence of confounders. Significant correlation of EGFR was found with hTERT (r = 0.77), HMGA-1 (r = 0.72), KIF-14 (r = 0.82) suggesting that their combined analysis can define prognosis. To establish the diagnostic accuracy (threshold ≥80% specificity), AUC for EGFR was 0.78 (>3.95), KIF-14 0.97 (>7.45), hTERT 0.63 (>23.86), and HMGA-1 0.53 (>15.45). Conclusion: This is the first evidence-based investigation presenting differential expression of proliferation and stemness markers hTERT, HMGA-1, KIF-14 in-correlation with EGFR, indicating a plausible-association between survival and disease-progression in individual GS-cases. It can serve as a model for further studies in this glioma-subgroup and the designing of a target panel for personalized treatment.

Novel molecular panel for evaluating systemic inflammation and survival in therapy naïve glioma patients.

BACKGROUND: Inflammation is crucial to tumor progression. A traumatic event at a specific site in the brain activates the signaling molecules, which triggers inflammation as the initial response within the tumor and its surroundings. The educated immune cells and secreted proteins then initiate the inflammatory cascade leading to persistent chronic inflammation. Therefore, estimation of the circulating inflammatory indicators kynurenine (KYN), interleukin-6 (IL-6), tissue-inhibitor of matrix-metalloproteinase-1 and human telomerase reverse transcriptase (hTERT) along with neutrophil-lymphocyte ratio (NLR) has prognostic value. AIM: To assess the utility of chosen inflammatory marker panel in estimating systemic inflammation. METHODS: The chosen markers were quantitatively evaluated in 90 naive, molecularly sub-typed plasma samples of glioma. A correlation between the markers and confounders was assessed to establish their prognostication power. Follow-up on the levels of the indicators was done 3-mo post-surgery. To establish the validity of circulating KYN, it was also screened qualitatively by dot-immune-assay and by immunofluorescence-immunohistochemistry in tumor tissues. RESULTS: Median values of circulating KYN, IL-6, hTERT, tissue-inhibitor of matrix-metalloproteinase-1 and NLR in isocitrate-dehydrogenase-mutant/wildtype and within the astrocytic sub-groups were estimated, which differed from controls, reaching statistical significance (P < 0.0001). All markers negatively correlated with mortality (P < 0.0001). Applying combination-statistics, the panel of KYN, IL-6, hTERT and NLR achieved higher sensitivity and specificity (> 90%) than stand-alone markers, to define survival. The inflammatory panel could discriminate between WHO grades, and isocitrate-dehydrogenase-mutant/wildtype and define differential survival between astrocytic isocitrate-dehydrogenase-mutant/wildtype. Therefore, its assessment for precise disease prognosis is indicated. Association of KYN with NLR, IL-6 and hTERT was significant. Cox-regression described KYN, IL-6, NLR, and hTERT as good prognostic markers, independent of confounders. Multivariate linear-regression analysis confirmed the association of KYN and hTERT with inflammation marker IL-6.There was a concomitant significant decrease in their levels in a 3-mo follow-up. CONCLUSION: The first evidence-based study of circulating-KYN in molecularly defined gliomas, wherein the tissue expression was found to be concomitant with plasma levels. A non-invasive model for assessing indicators of chronic systemic inflammation is proposed.

Ensemble based machine learning approach for prediction of glioma and multi-grade classification.

Glioma is the most pernicious cancer of the nervous system, with histological grade influencing the survival of patients. Despite many studies on the multimodal treatment approach, survival time remains brief. In this study, a novel two-stage ensemble of an ensemble-type machine learning-based predictive framework for glioma detection and its histograde classification is proposed. In the proposed framework, five characteristics belonging to 135 subjects were considered: human telomerase reverse transcriptase (hTERT), chitinase-like protein (YKL-40), interleukin 6 (IL-6), tissue inhibitor of metalloproteinase-1 (TIMP-1) and neutrophil/lymphocyte ratio (NLR). These characteristics were examined using distinctive ensemble-based machine learning classifiers and combination strategies to develop a computer-aided diagnostic system for the non-invasive prediction of glioma cases and their grade. In the first stage, the analysis was conducted to classify glioma cases and control subjects. Machine learning approaches were applied in the second stage to classify the recognised glioma cases into three grades, from grade II, which has a good prognosis, to grade IV, which is also known as glioblastoma. All experiments were evaluated with a five-fold cross-validation method, and the classification results were analysed using different statistical parameters. The proposed approach obtained a high value of accuracy and other statistical parameters compared with other state-of-the-art machine learning classifiers. Therefore, the proposed framework can be utilised for designing other intervention strategies for the prediction of glioma cases and their grades.

Curcumin in Combination with Other Adjunct Therapies for Brain Tumor Treatment: Existing Knowledge and Blueprint for Future Research.

Malignant brain tumors proliferate aggressively and have a debilitating outcome. Surgery followed by chemo-radiotherapy has been the standard procedure of care since 2005 but issues of therapeutic toxicity and relapse still remain unaddressed. Repurposing of drugs to develop novel combinations that can augment existing treatment regimens for brain tumors is the need of the hour. Herein, we discuss studies documenting the use of curcumin as an adjuvant to conventional and alternative therapies for brain tumors. Comprehensive analysis of data suggests that curcumin together with available therapies can generate a synergistic action achieved through multiple molecular targeting, which results in simultaneous inhibition of tumor growth, and reduced treatment-induced toxicity as well as resistance. The review also highlights approaches to increase bioavailability and bioaccumulation of drugs when co-delivered with curcumin using nano-cargos. Despite substantial preclinical work on radio-chemo sensitizing effects of curcumin, to date, there is only a single clinical report on brain tumors. Based on available lab evidence, it is proposed that antibody-conjugated nano-curcumin in combination with sub-toxic doses of conventional or repurposed therapeutics should be designed and tested in clinical studies. This will increase tumor targeting, the bioavailability of the drug combination, reduce therapy resistance, and tumor recurrence through modulation of aberrant signaling cascades; thus improving clinical outcomes in brain malignancies.

MicroRNA-30e-5p has an Integrated Role in the Regulation of the Innate Immune Response during Virus Infection and Systemic Lupus Erythematosus.

Precise regulation of innate immunity is crucial for development of appropriate host immunity against microbial infections and maintenance of immune homeostasis. MicroRNAs are small non-coding RNAs, post-transcriptional regulator of multiple genes, and act as a rheostat for protein expression. Here, we identified microRNA-30e-5p induced by hepatitis B virus and other viruses that act as a master regulator for innate immunity. Moreover, pegylated interferons treatment of patients with HBV for viral reduction also reduces miRNA. Additionally, we have also shown the immuno-pathological effects of miR-30e in patients with systemic lupus erythematosus (SLE) and mouse model. Mechanistically, miR-30e targets multiple negative regulators of innate immune signaling and enhances immune responses. Furthermore, sequestering of miR-30e in patients with SLE and mouse model significantly reduces type-I interferon and pro-inflammatory cytokines. Collectively, our study demonstrates the novel role of miR-30e in innate immunity and its prognostic and therapeutic potential in infectious and autoimmune diseases.

Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review.

The emergence of antibiotic resistant bacteria in the healthcare is a serious concern. In the Healthcare premises precisely intensive care unit are major sources of microbial diversity. Recent findings have demonstrated not only microbial diversity but also drug resistant microbes largely habitat in ICU. Pseudomonas aeruginosa found as a part of normal intestinal flora and a significant pathogen responsible for wide range of ICU acquired infection in critically ill patients. Nosocomial infection associated with this organism including gastrointestinal infection, urinary tract infections and blood stream infection. Infection caused by this organism are difficult to treat because of the presence of its innate resistance to many antibiotics (ß-lactam and penem group of antibiotics), and its ability to acquire further resistance mechanism to multiple class of antibiotics, including Beta-lactams, aminoglycosides and fluoroquinolones. In the molecular evolution microbes adopted several mechanism to maintain genomic plasticity. The tool microbe use for its survival is mainly biofilm formation, quorum sensing, and horizontal gene transfer and enzyme promiscuity. Such genomic plasticity provide an ideal habitat to grow and survive in hearse environment mainly antibiotics pressure. This review focus on infection caused by Pseudomonas aeruginosa, its mechanisms of resistance and available treatment options. The present study provides a systemic review on major source of Pseudomonas aeruginosa in ICU. Further, study also emphasizes virulence gene/s associated with Pseudomonas aeruginosa genome for extended drug resistance. Study gives detailed overview of antibiotic drug resistance mechanism.

Circulatory YKL-40 & NLR: Underestimated Prognostic Indicators in Diffuse Glioma.

In addition to histopathological parameters, evaluation of associated hematological factors is essential for devising a sensitive prognostic scale in glioma. Increased neutrophil-lymphocyte ratio (NLR), a marker of systemic inflammatory response, has recently been associated with worse outcome in various cancers. Given that glioma progression is characterized by inflammation, aggressive angiogenesis, and invasion, increased levels of systemic human-chitinase-3-like-one protein (YKL-40) have also been linked to poor prognosis. The aim of the present study was to assess the plausible association of YKL-40, NLR, and platelet count with increasing tumor grade, and evaluate their status as independent prognostic factors in terms of overall survival (OS) in treatment naive patients with diffuse glioma. Plasma levels of both biochemical markers in 72 diffuse gliomas, median age 42 years, were compared with 36 controls. Comparison of YKL-40, NLR, and PC with respect to tumor grade was found to be significant for each of the markers (P <0.0001) while an inverse significant correlation was seen for YKL-40 and NLR with OS (r = -0.4619, P <0.0001, and r = -0.5561, P < 0.0001, respectively). NLR was the best performing marker with AUC 0.9417 at 97% specificity. In addition, YKL-40 had a positive correlation with NLR (r = 0.4902, P <0.0001), indicating that expression of both markers was linked to inflammation and tumor progression as they were significantly correlated with tumor grade. Expression of YKL-40 and NLR was independently associated with worse survival (HR 1.0062, P = 0.039, and HR 1.1787, P = 0.0003, respectively), thus establishing their clinical utility as prognosticators for diffuse gliomas.

A Unique Immunofluorescence Protocol to Detect Protein Expression in Vascular Tissues: Tacking a Long Standing Pathological Hitch.

OBJECTIVE: Autofluorescence induced interference is one of the major drawbacks in immunofluorescence analysis of formalin-fixed paraffin-embedded tissues, as it decreases the signal-to-noise ratio of specific labeling. Apart from aldehyde-fixation induced artifacts; collagen and elastin, red blood cells and endogenous fluorescent pigment lipofuscin are prime sources of autofluorescence in vascular and aging tissues. We describe herein, an optimized indirect-immunofluorescence method for archival formalin-fixed paraffin-embedded tissues tissues and cryo sections, using a combination of 3-reagents in a specific order, to achieve optimal fluorescence signals and imaging. MATERIAL AND METHOD: Human telomerase reverse transcriptase, a protein implicated as a proliferation marker, was chosen relevant to its expression in solid tumors along with 3 other intracellular proteins exhibiting nuclear and/or cytoplasmic expression. Staining was performed on 10 glioma tissue sections along with 5 of their cryo sections, 5 sections each of hepatocellular, lung, papillary-thyroid and renal cell carcinoma, with 10 non-malignant brain tissue samples serving as control. Specimens were imaged using epifluorescence microscopy, followed by software-based quantification of fluorescence signals for statistical analysis and validation. RESULTS: We observed that the combined application of sodium-borohydride followed by crystal violet before antigen retrieval and a Sudan black B treatment after secondary antibody application proved to be most efficacious for masking autofluorescence/non-specific background in vascular tissues. CONCLUSION: This unique trio-methodology provides quantifiable observations with maximized fluorescence signal intensity of the target protein for longer retention time of the signal even after prolonged storage. The results can be extrapolated to other human tissues for different protein targets.

Evaluating the potential of circulating hTERT levels in glioma: can plasma levels serve as an independent prognostic marker?

Glioma is an aggressive primary Neuro-epithelial tumor with dismal prognosis, since there is a lack of molecular work-up during routine radiological monitoring of the disease. Currently, a number of potential molecular prognostic and predictive biomarkers are being characterized in line with structured diagnosis defined in World Health Organization guidelines 2016. Human-telomerase reverse-transcriptase (hTERT), a marker of proliferation and maintenance of genomic integrity has thus been investigated for its clinical relevance as an independent prognosticator in glioma. Expression of the protein in tumor tissue and in plasma of 72 diffuse glioma patients (astrocytoma) grade II-IV was determined and compared with relevant controls using immunofluorescence-immunohistochemistry and enzyme-linked immuno-sorbent assay. Appropriate statistical tests were applied to establish a correlation between on-site tumor and circulating levels of the marker and its independence of covariates. Expression of the marker in glioma tissues was significantly different from controls (p < 0.0001) and could discriminate within grades with ≥80% sensitivity. The tissue and plasma levels were positively associated with grades (r = 0.8845, p < 0.0001) and (r = 0.2834, p = 0.0158) respectively, while an inverse correlation with overall survival (r = -0.6558, p < 0.0001) and (r = -0.3941, p = 0.0006) respectively, was recorded. Plasma hTERT was significantly correlated with corresponding intra-tumor expression of hTERT (r = 0.2794, p = 0.0175). Multivariate Cox-regression identified plasma hTERT (p < 0.0005) as a prognostic factor; not associated with age, site or extent of resection (p > 0.05). This is the first experimental evidence for association of higher plasma levels of hTERT with overall survival in both low and high grade glioma.

Immunophenotypic signature of primary glioblastoma multiforme: A case of extended progression free survival.

Glioblastoma-multiforme (GBM), the most aggressive glial tumor, has a worldwide age-adjusted incidence ranging from 0.59-3.69/100000 persons. Despite current multimodal-treatment approach, median-survival time and progression-free survival (PFS) remains short. Glioblastomas display a variety of molecular alterations, which necessitates determining which of these have a prognostic significance. This is a case of a 45-year-old patient who presented with progressive slurring of speech and features of raised intracranial pressure. Computed tomography (CT) scan revealed a large heterogeneously enhancing lesion in the left front-temporal-perisylvian region with solid, cystic areas, suggestive of malignant glioma. Partial tumor-excision was followed by concurrent chemo-radiotherapy. Histopathologically, the tumor was astrocytoma grade-IV. Patient had an extended PFS of 12 mo, with an overall survival of 26 mo. Primary-GBM was confirmed using molecular markers and the immunophenotypic signature was defined by evaluating systemic expression of human telomerase reverse transcriptase, interleukin-6, neutrophil-lymphocyte ratio, tissue inhibitor of metalloproteinases-1, human chitinase-3-like-protein-1 (YKL-40) and high mobility group-A1. Current findings suggest that this signature can identify worst outcomes, independent of clinical criteria.

Unique case of oligoastrocytoma with recurrence and grade progression: Exhibiting differential expression of high mobility group-A1 and human telomerase reverse transcriptase.

Mixed gliomas, primarily oligoastrocytomas, account for about 5%-10% of all gliomas. Distinguishing oligoastrocytoma based on histological features alone has limitations in predicting the exact biological behavior, necessitating ancillary markers for greater specificity. In this case report, human telomerase reverse transcriptase (hTERT) and high mobility group-A1 (HMGA1); markers of proliferation and stemness, have been quantitatively analyzed in formalin-fixed paraffin-embedded tissue samples of a 34 years old patient with oligoastrocytoma. Customized florescence-based immunohistochemistry protocol with enhanced sensitivity and specificity is used in the study. The patient presented with a history of generalized seizures and his magnetic resonance imaging scans revealed infiltrative ill-defined mass lesion with calcified foci within the left frontal white matter, suggestive of glioma. He was surgically treated at our center for four consecutive clinical events. Histopathologically, the tumor was identified as oligoastrocytoma-grade II followed by two recurrence events and final progression to grade III. Overall survival of the patient without adjuvant therapy was more than 9 years. Glial fibrillary acidic protein, p53, Ki-67, nuclear atypia index, pre-operative neutrophil-lymphocyte ratio, are the other parameters assessed. Findings suggest that hTERT and HMGA1 are linked to tumor recurrence and progression. Established markers can assist in defining precise histopathological grade in conjuction with conventional markers in clinical setup.

A retrospective review of cytogenetic studies on methyl isocyanate with special reference to the Bhopal gas tragedy: is the next generation also at risk?

The world's worst industrial disaster, at Union Carbide, Bhopal, India, took place on 2-3 December 1984, leading to the leakage of poisonous methyl-isocyanate into the environment, causing thousands of deaths, pregnancy loss and for some, incapacitation for life. More than a quarter of a century later, the Indian Council of Medical Research undertook to redefine the abysmal consequences of the toxic gas exposure on the exposed population. This invigorated the interest of scientific community in the evaluation of the long-term effects, with reference to cytogenetic parameters. The thrust area was identified in terms of genetic disorders, low birth weight, developmental/growth disorders and congenital malformations. Also the impact on epigenetic factors, which may have contributed to variations in the functional expression of genes, was not negated, stimulating intense scientific research on in utero exposure and the progeny of the exposed population. To accomplish this mammoth task, molecular cytogenetic investigations must be undertaken in conjunction with conventional cytogenetics, using techniques such as FISH, Immuno-FISH, SKY and SNP analysis, to build up a cytogenetic database of the surviving population.

Mosaicism for trisomy 21 and ring (21) in a male born to normal parents: a case report.

We present a case of a ring (21) in a mentally challenged patient with mosaicism for trisomy 21 showing karyotype 47, XY,+21/47,XY,+21(r)/46,XY, born to normal parents. The parents and female sibling were phenotypically normal. This is a unique case report from Central India, on occurrence of trisomy 21 and r (21) in the same individual born to normal parents. Also being documented for the first time is the immuno-FISH analysis revealing differential expression of hTERT and a linked over expression of TRF2 in proband, probably corresponding to a high percentage of acrocentric associations.

Novel approach for quantification of hepatitis C virus in liver cirrhosis using real-time reverse transcriptase PCR.

BACKGROUND: Hepatitis C virus (HCV) infects nearly 3% of the population worldwide and is a major cause of acute and chronic infections leading to fibrosis, cirrhosis, and hepatocellular carcinoma. Current laboratory diagnosis of HCV is based on specific antibody detection (anti-hepatitis C virus (anti-HCV)) in serum. As HCV replicates in the liver cells, detection and localization of HCV RNA in liver tissue are vital for diagnosis. METHODS: Ten biopsy samples diagnosed for cryptogenic liver cirrhosis, negative for the presence of anti-HCV and serum HCV RNA, were studied for analyzing presence of viral nucleic acid in liver tissues. Qualitative screening for HCV was done through ELISA while the nucleic acid analysis was performed through COBAS Amplicor. Detection of HCV RNA in liver tissue biopsies was performed following standard protocol of HCV detection kit (Shenzhen PG Biotech) with modifications using Light Cycler 2.0 (minimum detection limit 10 copies/ml). RESULT: Quantitative detection in liver biopsies following the modified method showed the presence of HCV RNA in three samples out of the ten studied. CONCLUSION: The results indicate that using Light Cycler 2.0, following the modified technique described, constitutes a reliable method of quantitative detection and localization of HCV in tissue in "serosilent" HCV infection.