Oxidative versus Reductive Stress in Breast Cancer Development and Cellular Mechanism of Alleviation: A Current Perspective with Anti-breast Cancer Drug Resistance.

Redox homeostasis is essential for keeping our bodies healthy, but it also helps breast cancer cells grow, stay alive, and resist treatment. Changes in the redox balance and problems with redox signaling can make breast cancer cells grow and spread and make them resistant to chemotherapy and radiation therapy. Reactive oxygen species/reactive nitrogen species (ROS/RNS) generation and the oxidant defense system are out of equilibrium, which causes oxidative stress. Many studies have shown that oxidative stress can affect the start and spread of cancer by interfering with redox (reduction-oxidation) signaling and damaging molecules. The oxidation of invariant cysteine residues in FNIP1 is reversed by reductive stress, which is brought on by protracted antioxidant signaling or mitochondrial inactivity. This permits CUL2FEM1B to recognize its intended target. After the proteasome breaks down FNIP1, mitochondrial function is restored to keep redox balance and cell integrity. Reductive stress is caused by unchecked amplification of antioxidant signaling, and changes in metabolic pathways are a big part of breast tumors' growth. Also, redox reactions make pathways like PI3K, PKC, and protein kinases of the MAPK cascade work better. Kinases and phosphatases control the phosphorylation status of transcription factors like APE1/Ref-1, HIF-1, AP-1, Nrf2, NF-B, p53, FOXO, STAT, and - catenin. Also, how well anti-breast cancer drugs, especially those that cause cytotoxicity by making ROS, treat patients depends on how well the elements that support a cell's redox environment work together. Even though chemotherapy aims to kill cancer cells, which it does by making ROS, this can lead to drug resistance in the long run. The development of novel therapeutic approaches for treating breast cancer will be facilitated by a better understanding of the reductive stress and metabolic pathways in tumor microenvironments.

Comparative Analysis of Published Database Predicting MicroRNA Binding in 3'UTR of mRNA in Diverse Species.

BACKGROUND: Micro-RNAs are endogenous non-coding RNA moieties of 22-27 nucleotides that play a crucial role in the regulation of various biological processes and make them useful prognostic and diagnostic biomarkers. Discovery and experimental validation of miRNA is a laborious and time-consuming process. For early prediction, multiple bioinformatics databases are available for miRNA target prediction; however, their utility can confuse amateur researchers in selecting the most appropriate tools for their study. OBJECTIVE: This descriptive review aimed to analyse the usability of the existing database based on the following criteria: accessibility, efficiency, interpretability, updatability, and flexibility for miRNA target prediction of 3'UTR of mRNA in diverse species so that the researchers can utilize the database most appropriate to their research. METHODS: A systematic literature search was performed in PubMed, Google Scholar and Scopus databases up to November 2022. ≥10,000 articles found online, including ⁓130 miRNA tools, which contain various information on miRNA. Out of them, 31 databases that provide information on validated 3'UTR miRNAs target databases were included and analysed in this review. RESULTS: These miRNA database tools are being used in varied areas of biological research to select the most suitable miRNA for their experimental validation. These databases, updated until the year 2021, consist of miRNA-related data from humans, animals, mice, plants, viruses etc. They contain 525-29806351 data entries, and information from most databases is freely available on the online platform. CONCLUSION: Reviewed databases provide significant information, but not all information is accurate or up-to-date. Therefore, Diana-TarBase and miRWalk are the most comprehensive and up-to-date databases.

Overexpression of XRCC1 is Associated with Poor Survival in Patients with Head and Neck Squamous Carcinoma and Has Potential to Be Used as Targeted Therapy by Synthetic Lethality.

Background: Head neck squamous cell carcinoma (HNSC) is globally prevalent cancer attributed to tobacco habit. Despite the significant advances in early diagnosis and treatment of HNSC chemo-radio resistance are routinely observed in patients. Aberrant DNA repair mechanisms mainly microhomology mediated DNA end joining (MMEJ) pathway causing deleterious mutations and is implicated in treatment resistance. X-ray cross complimenting group 1 (XRCC1) has recently been shown to play an essential role in MMEJ making XRCC1 a potential therapeutic target to render tumors chemo-radiosensitive. This study analyzes the correlation between the expression level of XRCC1 gene with survival, regulation by miRNA and synthetic lethality partners in HNSCC. Materials and Methods: XRCC1 gene expression was evaluated in 520 HNSC patients and 44 of normal tissues using the UALCAN (TCGA) database and its correlation with survival outcome of HNSC patients was analyzed by Kaplan-Meier plot. Infiltration of immune cells in tumors was analyzed by "Tumor-Infiltrating Immune Estimation Resource (TIMER) and promoter methylation status of XRCC1 in samples was analysed by UALCAN. STRING was used to find gene interacting partners of XRCC1.  Results: XRCC1 was significantly overexpressed in primary tumor of HNSCC and significantly increased with tumor stages and grade and associated with poor survival rate. High XRCC1 expression in HNSC was positively correlated with infiltration level of B cells naïsve, CD4+ and macrophages. Conclusion: These results indicate that XRCC1 is a prognostic marker for predicting survival in HNSC patients. Understanding how XRCC1 leads to treatment resistance and modulate immune response can lead to development of targeted therapy.

Aetiologies of Acute Complications in Autoimmune Rheumatologic Diseases: A Hospital-Based Cross-Sectional Study.

BACKGROUND: Autoimmune rheumatic diseases (ARD) present unique challenges in clinical practice. Many of them present in medical emergencies in an unstable state and need immediate evaluation for further plans of action. The clinical conundrum is to distinguish between sepsis, disease flare, or Addisonian crisis (AC) (secondary to steroid withdrawal). This may be further complicated by overlapping clinical features like shock/fever and the coexistence of a combination of the above pathophysiologic mechanisms (e.g. AC with sepsis or AC with disease flare). The known biomarkers may not perform optimally to distinguish them and additional supportive investigations like imaging, cultures, autoimmune serological markers, etc. are needed. Ultimately the boundaries between "the art of medicine" and "the science of medicine" may get blurred, as the established literature evidence falls short and the expert opinion is needed in a time-sensitive manner. In this pragmatic study, researchers have attempted to explore the presentation of rheumatologic emergencies on the above three differentials (sepsis, disease flare, and AC). MATERIALS AND METHODS:  In this hospital-based cross-sectional study, adult patients (age >18 years) with ARD who had unplanned hospital admission due to acute worsening were enrolled. This study was conducted over one year, after getting the Institutional Human Ethics Committee's approval. All relevant hematological, immunological, and hormonal parameters (specifically morning cortisol) were collected and analyzed. The aim was to find the individual and combined prevalence of sepsis, disease flare, or AC in this study group. RESULTS: Forty-one patients were analyzed, with females in the majority (95%) and the dominant age group being 26-49 years (56.1%). A majority had a diagnosis of rheumatoid arthritis (RA) (56.1%) or systemic lupus erythematosus (SLE) (31.7%); the rest were other connective tissue diseases (12.2%). High-risk Quick Sequential Organ Failure Assessment score (qSOFA) score 2-3 was present in 29.3% while the rest had low-risk scores (qSOFA score 0-1). Thirty-two percent had severe disease activity, 46% had mild to moderate disease activity, and 22% of patients had no disease activity. While 78% of patients had low procalcitonin (PCT) values <0.5 microgm/L (low risk of sepsis), 15% had <20 microgm/L, and 7% percentage of patients had serum levels >20 microgm/L (high risk of sepsis). A total of 73.2% of patients had no evidence of infection while 26.8% had either microbiological/radiological evidence of infection. Only 7% of all patients had the presence of an AC. qSOFA scores didn't statistically correlate with a diagnosis of infection or AC but positively correlated with PCT and C-reactive protein (CRP) values. Serum PCT didn't correlate with the presence of infection with statistically significance (p-value 0.217). CONCLUSION: Infections and sepsis are the most important considerations in the emergency presentations of ARDs. Disease flare and AC are also important differentials. Current inflammatory biomarkers like serum CRP and PCT may be less valuable for discriminating between infectious and non-infectious sepsis, especially in chronic inflammatory diseases like ARDs. qSOFA scores may have a prognostic role with less discriminant value. Management of ARD emergencies needs better biomarkers and more research is warranted.

Blood-based biomarkers for diagnosis, prognosis, and severity prediction of COVID-19: Opportunities and challenges.

The reasons for high morbidity and mortality with Corona virus disease (COVID-19) disease remain unanswered with extremes of manifestation and uncertainty of modes of transmission for which biomarkers are urgently needed for early prediction of severity and prompt treatment. We have reviewed publications from PubMed (years 2019-2021) analysing the biochemical, immune-inflammatory, nucleic acid, and cellular biomarkers that predict infection, disease progression in COVID-19 with emphasis on organ-specific damage. Our analysis of 65 biomarkers assessing the impact of SCoV-2 infection on five organs (lung, liver, cardiac, kidney, and neural) reported that increased levels of CRP, TNF-α, ferritin, IL-6, D-dimer, Procalcitonin, Fibrinogen to Albumin Ratio (FAR), and decrease platelet count (PC), lymphocyte count, leukocyte count, and CD4+/CD8 + ratio shows promising association in the early diagnosis, prediction of prognosis and severity disease and also correlates with cytokine storm a cardinal feature of COVID-19 progression. In the above scenario, this review has put forth the most promising biomarkers for COVID diagnosis and prognosis based on the reported literature. In recent year's chemically synthesized antibody-like biomolecules, aptamers were also used in the diagnosis of COVID-19 which could be preferably used for diagnosis over antibodies. Biomarkers including increase in free DNA and Fibrinogen-to-Albumin Ratio, CRP, PCT, and Ferritin along with a consequential decrease of CD3+ T, CD4+ T, CD8+ T, NK cells with corresponding increase in CD4+/CD8+ ratio following SARS CoV-2 infection has been consistently correlated with disease severity. Despite the two waves of COVID-19 pandemic, currently there is no standard clinical practice guideline for evaluating the severity of the devastating pandemic of COVID-19, hence these biomarkers will have immense relevance for the third and subsequent wave of COVID-19 and related pandemic.

Current Scenario of Clinical Diagnosis to Identify Inborn Errors of Metabolism with Precision Profiling for Expanded Screening in Infancy in a Resource-limited Setting.

Inborn errors of metabolism (IEM) are a diverse collection of abnormalities that cause a variety of morbidities and mortality in children and are classified as uncommon genetic diseases. Early and accurate detection of the condition can save a patient's life. By aiding families as they navigate the experience of having a child with an IEM, healthcare practitioners have the chance to reduce the burden of negative emotional consequences. New therapeutic techniques, such as enzyme replacement and small chemical therapies, organ transplantation, and cellular and gene-based therapies using whole-genome sequencing, have become available in addition to traditional medical intake and cofactor treatments. In the realm of metabolic medicine and metabolomics, the twentyfirst century is an exciting time to be alive. The availability of metabolomics and genomic analysis has led to the identification of a slew of novel diseases. Due to the rarity of individual illnesses, obtaining high-quality data for these treatments in clinical trials and real-world settings has proven difficult. Guidelines produced using standardized techniques have helped enhance treatment delivery and clinical outcomes over time. This article gives a comprehensive description of IEM and how to diagnose it in patients who have developed clinical signs early or late. The appropriate use of standard laboratory outcomes in the preliminary patient assessment is also emphasized that can aid in the ordering of specific laboratory tests to confirm a suspected diagnosis, in addition, to begin treatment as soon as possible in a resource limiting setting where genomic analysis or newborn screening facility is not available.

Elucidating the Role of Cardiac Biomarkers in COVID-19: A Narrative Evaluation with Clinical Standpoints and a Pragmatic Approach for Therapeutics.

With the incidence of the unabated spreading of the COVID-19 (coronavirus disease 2019) pandemic with an increase in heart-related complications in COVID-19 patients, laboratory investigations on general health and diseases of heart have greater importance. The production of a higher level of clots in the blood in COVID-19 individuals carries a high risk of severe lethal pneumonia, pulmonary embolism, or widespread thromboembolism. The COVID-19 pandemic has raised awareness regarding the severe consequences for the cardiac system that might cause due to severe acute respiratory distress syndrome (SARS-CoV-2). COVID-19 causes acute respiratory distress syndrome (ARDS), acute myocardial infarction, venous thromboembolism, and acute heart failure in people with preexisting cardiac illness. However, as COVID-19 is primarily a respiratory infectious disease, there is still a lot of debate on whether and how cardiac biomarkers should be used in COVID-19 patients. Considering the most practical elucidation of cardiac biomarkers in COVID-19, it is important to note that recent findings on the prognostic role of cardiac biomarkers in COVID-19 patients are similar to those found in pneumonia and ARDS studies. The use of natriuretic peptides and cardiac troponin concentrations as quantitative variables should help with COVID-19/pneumonia risk classification and ensure that these biomarkers sustain their high diagnostic precision for acute myocardial infarction and heart failure. Serial assessment of D-dimers will possibly aid clinicians in the assortment of patients for venous thromboembolism imaging in addition to the increase of anticoagulation from preventive to marginally higher or even therapeutic dosages because of the central involvement of endothelitis and thromboembolism in COVID-19. Therefore, cardiac biomarkers are produced in this phase because of some pathological processes; this review will focus on major cardiac biomarkers and their significant role in COVID-19.

Assessment of zinc inadequacy among tribal adolescent population of central India - A cross-sectional study.

Background & objectives: Zinc is a crucial micronutrient in adolescence, required for promoting growth and sexual maturation. Adolescents of some tribes may be at high risk of zinc deficiency due to dietary inadequacy and poor bioavailability of zinc from plant-based diets. This study aimed to evaluate the risk of zinc deficiency by estimating prevalence of inadequate zinc intake, prevalence of low serum zinc and stunting among tribal adolescents. Methods: A cross-sectional community-based survey was conducted among adolescents (10-19 yr) in three purposively selected districts where Bhil, Korku and Gond tribes were in majority. Structured data collection instrument comprising information about sociodemographic characteristics and dietary recall data was used. Anthropometric assessment was conducted by standardized weighing scales and anthropometry tapes, and blood sample was collected from antecubital vein into trace element-free vacutainers. Serum zinc was estimated using an atomic absorption spectrophotometer. Results: A total of 2310 households were approached for participation in the study, of which 2224 households having 5151 adolescents participated. Out of these enlisted adolescents, 4673 responded to dietary recall (90.7% response rate). Anthropometry of 2437 participants was carried out, and serum zinc was analyzed in 844 adolescents. The overall prevalence of dietary zinc inadequacy was 42.6 per cent [95% confidence interval (CI) 41.2 to 44.1] with reference to the estimated average requirement suggested by International Zinc Nutrition Consultative Group (IZiNCG) and 64.8 per cent (95% CI 63.4 to 66.2) with Indian Council of Medical Research-recommended requirements. Stunting was observed in 29 per cent (95% CI 27.2 to 30.8) participants. According to IZiNCG cut-offs, low serum zinc was detected in 57.5 per cent (95% CI 54.1 to 60.8) of adolescents, whereas it was 34.4 per cent (95% CI: 31.2-37.5) according to the national level cut-off. Interpretation & conclusions: Risk of dietary zinc inadequacy and low serum zinc concentration amongst adolescents of the Gond, Bhil and Korku tribes is a public health concern.

Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch vaccines, designed by reverse epitomics approach, show potential to cover large ethnically distributed human population worldwide.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is responsible for the COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. A novel reverse epitomics approach, 'overlapping-epitope-clusters-to-patches' method is utilized to identify the antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are named as 'Ag-Patch or Ag-Patches', for Antigenic Patch or Patches. The identification of Ag-Patches is based on the clusters of overlapping epitopes rising from SARS-CoV-2 proteins. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel method for the vaccine design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. We identified 73 CTL (Cytotoxic T-Lymphocyte) and 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 overlapping epitopes targeting 55 different HLA alleles leading to 99.98% of world human population coverage. The MPVs and Toll-Like Receptor ectodomain complex shows stable complex formation tendency. Further, the cDNA analysis favors high expression of the MPVs constructs in a human cell line. We identified highly immunogenic novel Ag-Patches from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach and utilized them to design MPVs. We conclude that the novel MPVs could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. Communicated by Ramaswamy H. Sarma.

Determinants of behavioural and biological risk factors for cardiovascular diseases from state level STEPS survey (2017-19) in Madhya Pradesh.

BACKGROUND: National and statewide assessment of cardiovascular risk factors needs to be conducted periodically in order to inform public health policy and prioritise allocation of funds, especially in LMICs. Although there have been studies from India which have explored the determinants of cardiovascular risk factors, they have mostly been from high epidemiological transition states. The present study assessed the determinants of cardiovascular (CVD) risk factors in a low epidemiological transition state (Madhya Pradesh) using the WHO STEPwise approach to surveillance (STEPS). METHODS: A total of 5,680 persons aged 18-69 years were selected from the state of Madhya Pradesh through multi-stage cluster random sampling. Key CVD risk factors we sought to evaluate were from behavioural (tobacco, alcohol, physical activity, diet) and biological domains (overweight or obese, Hypertension, Diabetes, and Raised serum cholesterol). Key socio-demographic factors of interest were the caste and tribe groups, and rural vs urban location, in addition to known influencers of CVD risk such as age, gender and education level. RESULTS: Those belonging to the scheduled tribes were more at risk of consuming tobacco (OR 2.13 (95% CI [1.52-2.98]), and a diet with less than five servings of fruits and vegetables (OR 2.78 (95% CI [1.06-7.24]), but had had the least risk of physical inactivity (OR 0.31 (95% CI [0.02-0.54]). Residence in a rural area also reduced the odds of physical inactivity (OR 0.65 (95% CI [0.46-0.92])). Lack of formal education was a risk factor for both tobacco consumption and alcohol intake (OR 1.40 (95% CI [1.08-1.82]) for tobacco use; 1.68 (95% CI [1.14-2.49]) for alcohol intake). Those belonging to schedules tribes had much lower risk of being obese (OR 0.25; 95% CI [0.17-0.37]), but were at similar risk of all other clinical CVD risk factors as compared to other caste groups. CONCLUSION: In the current study we explored socio-demographic determinants of behavioural and biological CVD risks, and found that in Madhya Pradesh, belonging to a scheduled tribe or living in a rural location, protects against being physically inactive or being overweight or obese. Increasing age confers a greater CVD-risk in all domains. Being a male, and lack of formal education confers a greater risk for behavioural domains, but not for most clinical risk domains. Future efforts at curbing CVDs should be therefore two pronged -a population-based strategy targeting biological risk factors, and a more focussed approach directed at those displaying risky behaviour.

Descriptive profile of risk factors for cardiovascular diseases using WHO STEP wise approach in Madhya Pradesh.

BACKGROUND: Periodic information on risk factor distribution is critical for public health response for reduction in non-communicable disease (NCDs). For this purpose, the WHO has developed STEPs wise approach. State representative population-based STEPS survey was last conducted in 2007-08 in seven states of In India. Since then no such work has been reported from low ETL states. This survey was carried out to assess the prevalence of risk factors associated with NCDs and the prevalence of NCDs in the low ETL state of Madhya Pradesh using the WHO STEPs approach. METHODS: A total of 5680 persons aged 18-69 years were selected from the state of Madhya Pradesh using multi-stage cluster random sampling. Using the WHO STEPs approach, details were collected on demographics, STEP 1 variables (tobacco consumption, alcohol consumption, physical activity, diet), STEP 2 variables (weight, height, waist circumference, blood pressure) and STEP 3 variables (fasting blood glucose, blood cholesterol). RESULTS: We found that 9.4% individuals smoked tobacco, 15.3% were overweight/obese, 22.3% had hypertension, and 6.8% have diabetes mellitus. As compared to women, men were less likely to be overweight or obese, but more likely to smoke tobacco, and have diabetes mellitus. Hypertension was also more common in men. Overall, about a fourth of all adults had three or more risk factors for cardiovascular disease. CONCLUSION: The survey shows that a large section of the population from Madhya Pradesh is either suffering from NCDs or have risk factors which predispose them to acquire NCDs. This state representative survey provides benchmarking information for behavioural and biological risk factor distribution for recently scaled up National Programme for the Prevention and Control of Cancer, Diabetes, Cardiovascular Diseases, and Stroke (NPCDCS).

Cytokines: the yin and yang of vitiligo pathogenesis.

INTRODUCTION: Dysregulation of melanocyte function is associated with vitiligo, an idiopathic autoimmune hypopigmentary skin disorder, caused by the selective destruction of melanocytes. Cytokines, the key mediators of immune response, which are pivotal in maintaining immune homeostasis, are crucial in vitiligo pathogenesis. Several studies indicate that there is an imbalance between pro- and anti-inflammatory cytokines in the skin and serum of vitiligo patients. Areas covered: In this comprehensive review, we have summarized the correlation of cytokine imbalance and vitiligo pathogenesis, its role in melanocyte biology, and its impact on vitiligo treatment. We have integrated various published reports on the levels of major cytokines from skin and serum samples of vitiligo patients. We have also discussed the role of endoplasmic reticulum and oxidative stress on cytokine imbalance and vice versa leading to destruction of melanocytes. Expert commentary: The review reflects that dysregulation of cytokines is multifactorial, ranging from genetic predisposition to altered protein expression relevant to vitiligo pathogenesis. We emphasize that cytokine imbalance in systemic and skin microenvironment plays a crucial role in vitiligo pathogenesis and has promising potential as therapeutic targets for vitiligo.

Genetic susceptibility to multiple primary neoplasms in the upper aero-digestive tract: genotype score and phenotype correlation.

Early detection and treatment of head and neck cancer has led to increased patient survival. However such patients are at a high risk for multiple primary neoplasm(s) (MPN). In order to study the genetic susceptibility to MPN, 22 candidate SNPs were genotyped based on which a distinctive Genotype Score was created using Additive, Dominant and Recessive models. Using lymphoblastoid cell lines (LCLs) generated from these individuals, the Genotype Score was correlated with carcinogen sensitivity in vitro. LCLs from MPN patients exhibited significantly higher Genotype Score and showed resistance to genotoxic agents compared to matched controls. This report demonstrates quantitative assessment of cumulative effect of gene polymorphisms and its correlation with carcinogen sensitivity for predicting susceptibility to MPN.

Establishment & characterization of lymphoblastoid cell lines from patients with multiple primary neoplasms in the upper aero-digestive tract & healthy individuals.

BACKGROUND & OBJECTIVES: A major drawback for genetic studies as well as long-term genotype-phenotype correlation studies in cancer is lack of representative human cell lines providing a continuous source of basic biomolecules and a system to carry out various experimental investigations. This can be overcome to some extent by establishing lymphoblastoid cell lines (LCLs) by infecting peripheral blood lymphocytes with Epstein Barr virus (EBV) which is known to immortalize human resting B cells in vitro giving rise to actively proliferating B-lymphoblastoid cell lines. The present study involves preparation and characterization of LCLs generated from patients with multiple primary neoplasms (MPN) of upper aero-digestive tract (UADT). METHODS: Thirty seven LCLs were established from UADT MPN patients and healthy age, sex and habit matched controls using EBV crude stock. Characterization was done with respect to expression of CD-19 (Pan B-cell marker), CD3 (T cell specific marker), CD56 (NK-cell specific marker), cell morphology, ploidy analysis, genotype and gene expression comparison with the parent lymphocytes. RESULTS: LCLs showed rosette morphology with doubling time of approximately 24 h. Ploidy analysis showed diploid DNA content which was maintained for at least 30 population doublings. When compared with parent lymphocytes there appeared no change at genetic and gene expression level. INTERPRETATION & CONCLUSIONS: Our results show that lymphoblastoid cell lines are a good surrogate of isolated lymphocytes bearing their close resemblance at genetic and phenotypic level to parent lymphocytes and are a valuable resource for understanding genotype-phenotype interactions.

Cernunnos influences human immunoglobulin class switch recombination and may be associated with B cell lymphomagenesis.

Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells, with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.

Multiple pathway-based genetic variations associated with tobacco related multiple primary neoplasms.

BACKGROUND: In order to elucidate a combination of genetic alterations that drive tobacco carcinogenesis we have explored a unique model system and analytical method for an unbiased qualitative and quantitative assessment of gene-gene and gene-environment interactions. The objective of this case control study was to assess genetic predisposition in a biologically enriched clinical model system of tobacco related cancers (TRC), occurring as Multiple Primary Neoplasms (MPN). METHODS: Genotyping of 21 candidate Single Nucleotide Polymorphisms (SNP) from major metabolic pathways was performed in a cohort of 151 MPN cases and 210 cancer-free controls. Statistical analysis using logistic regression and Multifactor Dimensionality Reduction (MDR) analysis was performed for studying higher order interactions among various SNPs and tobacco habit. RESULTS: Increased risk association was observed for patients with at least one TRC in the upper aero digestive tract (UADT) for variations in SULT1A1 Arg²¹³His, mEH Tyr¹¹³His, hOGG1 Ser³²6Cys, XRCC1 Arg²8°His and BRCA2 Asn³7²His. Gene-environment interactions were assessed using MDR analysis. The overall best model by MDR was tobacco habit/p53(Arg/Arg)/XRCC1(Arg³99His)/mEH(Tyr¹¹³His) that had highest Cross Validation Consistency (8.3) and test accuracy (0.69). This model also showed significant association using logistic regression analysis. CONCLUSION: This is the first Indian study on a multipathway based approach to study genetic susceptibility to cancer in tobacco associated MPN. This approach could assist in planning additional studies for comprehensive understanding of tobacco carcinogenesis.

Non-homologous end joining in class switch recombination: the beginning of the end.

Immunoglobulin class switch recombination (CSR) is initiated by a B-cell-specific factor, activation-induced deaminase, probably through deamination of deoxycytidine residues within the switch (S) regions. The initial lesions in the S regions are subsequently processed, resulting in the production of DNA double-strand breaks (DSBs). These breaks will then be recognized, edited and repaired, finally leading to the recombination of the two S regions. Two major repair pathways have been implicated in CSR, the predominant non-homologous end joining (NHEJ) and the alternative end-joining (A-EJ) pathways. The former requires not only components of the 'classical' NHEJ machinery, i.e. Ku70/Ku80, DNA-dependent protein kinase catalytic subunit, DNA ligase IV and XRCC4, but also a number of DNA-damage sensors or adaptors, such as ataxia-telangiectasia mutated, gammaH2AX, 53BP1, MDC1, the Mre11-Rad50-NBS1 complex and the ataxia telangiectasia and Rad3-related protein (ATR). The latter pathway is not well characterized yet and probably requires microhomologies. In this review, we will focus on the current knowledge of the predominant NHEJ pathway in CSR and will also give a perspective on the A-EJ pathway.

Involvement of Artemis in nonhomologous end-joining during immunoglobulin class switch recombination.

DNA double-strand breaks (DSBs) introduced in the switch (S) regions are intermediates during immunoglobulin class switch recombination (CSR). These breaks are subsequently recognized, processed, and joined, leading to recombination of the two S regions. Nonhomologous end-joining (NHEJ) is believed to be the principle mechanism involved in DSB repair during CSR. One important component in NHEJ, Artemis, has however been considered to be dispensable for efficient CSR. In this study, we have characterized the S recombinational junctions from Artemis-deficient human B cells. Smu-Salpha junctions could be amplified from all patients tested and were characterized by a complete lack of "direct" end-joining and a remarkable shift in the use of an alternative, microhomology-based end-joining pathway. Smu-Sgamma junctions could only be amplified from one patient who carries "hypomorphic" mutations. Although these Smu-Sgamma junctions appear to be normal, a significant increase of an unusual type of sequential switching from immunoglobulin (Ig)M, through one IgG subclass, to a different IgG subclass was observed, and the Sgamma-Sgamma junctions showed long microhomologies. Thus, when the function of Artemis is impaired, varying modes of CSR junction resolution may be used for different S regions. Our findings strongly link Artemis to the predominant NHEJ pathway during CSR.

A regulatory role for NBS1 in strand-specific mutagenesis during somatic hypermutation.

Activation-induced cytidine deaminase (AID) is believed to initiate somatic hypermutation (SHM) by deamination of deoxycytidines to deoxyuridines within the immunoglobulin variable regions genes. The deaminated bases can subsequently be replicated over, processed by base excision repair or mismatch repair, leading to introduction of different types of point mutations (G/C transitions, G/C transversions and A/T mutations). It is evident that the base excision repair pathway is largely dependent on uracil-DNA glycosylase (UNG) through its uracil excision activity. It is not known, however, which endonuclease acts in the step immediately downstream of UNG, i.e. that cleaves at the abasic sites generated by the latter. Two candidates have been proposed, an apurinic/apyrimidinic endonuclease (APE) and the Mre11-Rad50-NBS1 complex. The latter is intriguing as this might explain how the mutagenic pathway is primed during SHM. We have investigated the latter possibility by studying the in vivo SHM pattern in B cells from ataxia-telangiectasia-like disorder (Mre11 deficient) and Nijmegen breakage syndrome (NBS1 deficient) patients. Our results show that, although the pattern of mutations in the variable heavy chain (V(H)) genes was altered in NBS1 deficient patients, with a significantly increased number of G (but not C) transversions occurring in the SHM and/or AID targeting hotspots, the general pattern of mutations in the V(H) genes in Mre11 deficient patients was only slightly altered, with an increased frequency of A to C transversions. The Mre11-Rad50-NBS1 complex is thus unlikely to be the major nuclease involved in cleavage of the abasic sites during SHM, whereas NBS1 might have a specific role in regulating the strand-biased repair during phase Ib mutagenesis.