Obstructive sleep apnea induced bilateral tonic- clonic seizure of unknown origin: A case series of a novel association.

Background: Obstructive sleep apnea is a highly prevalent disorder, characterized by recurrent events of upper airway obstruction during sleep and associated with recurrent cycles of desaturation and re-oxygenation, sympathetic hyperactivity, and intra-thoracic pressure fluctuations, resulting in fragmentation of sleep and subsequent daytime fatigue with excessive sleepiness. Obstructive sleep apnea-induced bilateral tonic-clonic seizures are unheard of. We aimed to report 3 patients with previously undiagnosed obstructive sleep apnea who presented to the emergency department with new onset bilateral tonic-clonic seizure without any evidential neurological or metabolic cause. Methods: Patient data were obtained from medical records from the Department of Internal Medicine, IPGMER and SSKM Hospital, Kolkata, and Belle Vue Clinic, Kolkata, India. Results: Three male patients (67, 58, and 44 years old) presented with bilateral tonic-clonic seizure disorder without any underlying cause of seizures after rigorous investigations except for moderate to severe obstructive sleep apnea on polysomnography. All 2 patients were seizure-free after being treated with levetiracetam, chronic continuous positive airway pressure therapy in 2, and only continuous positive airway pressure in the other. The patients remained seizure-free on continuous positive airway pressure, even when levetiracetam was withdrawn, suggesting obstructive sleep apnea's causality in their new-onset acute seizures. Conclusion: Although further investigation is required to clarify this association, underlying obstructive sleep apnea should be ruled out in patients with a first-ever bilateral tonic-clonic seizure. Whether or not continuous positive airway pressure alone could effectively treat hypoxia and deranged cortical excitability, which may lead to seizures in cases with longstanding obstructive sleep apnea, is yet to be explored.

Introducción: La apnea obstructiva del sueño es una enfermedad con una alta prevalencia que se caracteriza por episodios recurrentes de obstrucción de las vías respiratorias altas durante el sueño, lo que conlleva ciclos repetidos de hipoxia y reoxigenación, hiperactividad simpática y fluctuaciones en la presión intratorácica. Todos estos procesos dan lugar a una fragmentación del sueño, lo que provoca fatiga diurna y somnolencia excesiva. Las crisis tónico-clónicas bilaterales inducidas por apnea obstructiva del sueño son poco conocidas. Presentamos los casos de tres pacientes con apnea obstructiva del sueño sin diagnosticar previamente que acudieron a urgencias por crisis tónico-clónicas de nueva aparición sin causa neurológica o metabólica aparente. Métodos: Los datos de nuestros pacientes se recogieron de los historiales médicos del servicio de Medicina Interna del Institute of Post-Graduate Medical Education and Research and Seth Sukhlal Karnani Memorial Hospital y de la Belle Vue Clinic, ambos en Kolkata (India). Resultados: Tres pacientes varones de 67, 58 y 44 años de edad presentaron convulsiones tónico-clónicas bilaterales sin causa identificada tras examen riguroso, exceptuando una apnea obstructiva del sueño de gravedad moderada a grave observada en la polisomnografía. Los tres pacientes recibieron tratamiento con levetiracetam durante el ingreso; al alta, se pautó tratamiento crónico con presión positiva continua de las vías respiratorias más levetiracetam en dos pacientes, y en el tercero solo presión positiva continua de las vías respiratorias. Ninguno presentó nuevas crisis tras la retirada de levetiracetam, lo que sugiere que la causa de las convulsiones era la apnea obstructiva del sueño. Conclusión: Aunque es necesario realizar más estudios para aclarar esta asociación, debemos descartar la apnea obstructiva del sueño en pacientes con crisis tónico-clónicas bilaterales de nueva aparición. Queda aún por determinar si la presión positiva continua podría tratar de forma efectiva la hipoxia y las alteraciones en la excitabilidad cortical, que podrían provocar crisis en casos de apnea obstructiva del sueño de larga evolución.

Genome-wide DNA methylation profiling of stomach cancer in the ethnic population of Mizoram, North East India.

Stomach cancer is the fifth most common cancer in terms of prevalence and incidence and the fourth leading cause of mortality in men and women worldwide. It is well-established that aberrant DNA methylation in cells can lead to carcinogenesis. The primary objective of our study was to investigate the aberrant DNA methylation status of genes associated with stomach cancer with a particular reference to the ethnic population of Mizoram, North East India. The site-level analysis identified 2883 CpG sites differentially methylated, representing ∼922 genes. Out of which 476 Differentially Methylated Positions (DMPs) were promoter-associated, 452 DMPs were hypermethylated, and 24 were hypomethylated. The region-level analysis identified 462 Differentially Methylated Regions (DMRs) corresponding to ∼320 genes, of which ∼281 genes were hypermethylated and âˆ¼40 genes were hypomethylated. TCGA analysis showed that some of the genes had been previously implicated in other cancers including stomach cancer. Five hypermethylated genes were selected as candidate genes for further investigations and they have shown to be novel and could serve as candidate hypermethylation biomarkers for stomach cancer in this particular ethnic group.

Surfaceome profiling enables isolation of cancer-specific exosomal cargo in liquid biopsies from pancreatic cancer patients.

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling. Patients and methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis. Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation. Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA.

High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients.

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). Patients and methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

A predictive model of inflammatory markers and patient-reported symptoms for cachexia in newly diagnosed pancreatic cancer patients.

BACKGROUND: Cachexia is a frequent manifestation of pancreatic cancer, can limit a patient's ability to take chemotherapy, and is associated with shortened survival. We developed a model to predict the early onset of cachexia in advanced pancreatic cancer patients. METHODS: Patients with newly diagnosed, untreated metastatic or locally advanced pancreatic cancer were included. Serum cytokines were drawn prior to therapy. Patient symptoms were recorded using the M.D. Anderson Symptom Inventory (MDASI). Our primary endpoint was either 10% weight loss or death within 60 days of the start of therapy. RESULTS: Twenty-seven of 89 patients met the primary endpoint (either having lost 10% of body weight or having died within 60 days of the start of treatment). In a univariate analysis, smoking, history symptoms of pain and difficulty swallowing, high levels of MK, CXCL-16, IL-6, TNF-a, and low IL-1b all correlated with this endpoint. We used recursive partition to fit a regression tree model, selecting four of 26 variables (CXCL-16, IL-1b, pain, swallowing difficulty) as important in predicting cachexia. From these, a model of two cytokines (CXCL-16 > 5.135 ng/ml and IL-1b < 0.08 ng/ml) demonstrated a better sensitivity and specificity for this outcome (0.70 and 0.86, respectively) than any individual cytokine or tumor marker. CONCLUSIONS: Cachexia is frequent in pancreatic cancer; one in three patients met our endpoint of 10% weight loss or death within 60 days. Inflammatory cytokines are better than conventional tumor markers at predicting this outcome. Recursive partitioning analysis suggests that a model of CXCL-16 and IL-1B may offer a better ability than individual cytokines to predict this outcome.

Chronic inflammation initiates multiple forms of K-Ras-independent mouse pancreatic cancer in the absence of TP53.

Chronic inflammation (CI) is a risk factor for pancreatic cancer (PC) including the most common type, ductal adenocarcinoma (PDAC), but its role and the mechanisms involved are unclear. To investigate the role of CI in PC, we generated genetic mouse models with pancreatic specific CI in the presence or absence of TP53. Mice were engineered to express either cyclooxygenase-2 (COX-2) or IκB kinase-2 (IKK2), and TP53+/+ or TP53f/f specifically in adult pancreatic acinar cells by using a full-length pancreatic elastase promoter-driven Cre. Animals were followed for >80 weeks and pancreatic lesions were evaluated histologically and immunohistochemically. The presence of K-ras mutations was assessed by direct sequencing, locked nuclei acid (LNA)-based PCR, and immunohistochemistry. We observed that sustained COX-2/IKK2 expression caused histological abnormalities of pancreas, including increased immune cell infiltration, proliferation rate and DNA damage. A minority of animals with CI developed pre-neoplastic lesions, but cancer was not observed in any TP53+/+ animals within 84 weeks. In contrast, all animals with CI-lacking TP53 developed various subtypes of PC, including acinar cell carcinoma, ductal adenocarcinoma, sarcomatoid carcinoma and neuroendocrine tumors, and all died within 65 weeks. No evidence of K-ras mutations was observed. Variations in the activity of the Hippo, pERK and c-Myc pathways were found in the diverse cancer subtypes. In summary, chronic inflammation is extremely inefficient at inducing PC in the presence of TP53. However, in the absence of TP53, CI leads to the development of several rare K-ras-independent forms of PC, with infrequent PDAC. This may help explain the rarity of PDAC in persons with chronic inflammatory conditions.

Minimally invasive genomic and transcriptomic profiling of visceral cancers by next-generation sequencing of circulating exosomes.

BACKGROUND: The ability to perform comprehensive profiling of cancers at high resolution is essential for precision medicine. Liquid biopsies using shed exosomes provide high-quality nucleic acids to obtain molecular characterization, which may be especially useful for visceral cancers that are not amenable to routine biopsies. PATIENTS AND METHODS: We isolated shed exosomes in biofluids from three patients with pancreaticobiliary cancers (two pancreatic, one ampullary). We performed comprehensive profiling of exoDNA and exoRNA by whole genome, exome and transcriptome sequencing using the Illumina HiSeq 2500 sequencer. We assessed the feasibility of calling copy number events, detecting mutational signatures and identifying potentially actionable mutations in exoDNA sequencing data, as well as expressed point mutations and gene fusions in exoRNA sequencing data. RESULTS: Whole-exome sequencing resulted in 95%-99% of the target regions covered at a mean depth of 133-490×. Genome-wide copy number profiles, and high estimates of tumor fractions (ranging from 56% to 82%), suggest robust representation of the tumor DNA within the shed exosomal compartment. Multiple actionable mutations, including alterations in NOTCH1 and BRCA2, were found in patient exoDNA samples. Further, RNA sequencing of shed exosomes identified the presence of expressed fusion genes, representing an avenue for elucidation of tumor neoantigens. CONCLUSIONS: We have demonstrated high-resolution profiling of the genomic and transcriptomic landscapes of visceral cancers. A wide range of cancer-derived biomarkers could be detected within the nucleic acid cargo of shed exosomes, including copy number profiles, point mutations, insertions, deletions, gene fusions and mutational signatures. Liquid biopsies using shed exosomes has the potential to be used as a clinical tool for cancer diagnosis, therapeutic stratification and treatment monitoring, precluding the need for direct tumor sampling.

p53 mutations cooperate with oncogenic Kras to promote adenocarcinoma from pancreatic ductal cells.

Pancreatic cancer is one of the most lethal malignancies, with virtually all patients eventually succumbing to their disease. Mutations in p53 have been documented in >50% of pancreatic cancers. Owing to the high incidence of p53 mutations in PanIN 3 lesions and pancreatic tumors, we interrogated the comparative ability of adult pancreatic acinar and ductal cells to respond to oncogenic Kras and mutant Tp53(R172H) using Hnf1b:CreER(T2) and Mist1:CreER(T2) mice. These studies involved co-activation of a membrane-tethered GFP lineage label, allowing for direct visualization and isolation of cells undergoing Kras and mutant p53 activation. Kras activation in Mist1(+) adult acinar cells resulted in brisk PanIN formation, whereas no evidence of pancreatic neoplasia was observed for up to 6 months following Kras activation in Hnf1beta(+) adult ductal cells. In contrast to the lack of response to oncogenic Kras alone, simultaneous activation of Kras and mutant p53 in adult ductal epithelium generated invasive PDAC in 75% of mice as early as 2.5 months after tamoxifen administration. These data demonstrate that pancreatic ductal cells, whereas exhibiting relative resistance to oncogenic Kras alone, can serve as an effective cell of origin for pancreatic ductal adenocarcinoma in the setting of gain-of-function mutations in p53.

Validation of histomolecular classification utilizing histological subtype, MUC1, and CDX2 for prognostication of resected ampullary adenocarcinoma.

BACKGROUND: Outcomes for ampullary adenocarcinomas are heterogeneous, and numerous methods of categorisation exist. A histomolecular phenotype based on histology, caudal-type homeodomain transcription factor 2 (CDX2) staining and Mucin 1 (MUC1) staining has recently been tested and validated in two cohorts. We attempt to validate this classification in a large patient population. METHODS: Tissue samples from 163 patients with resected ampullary adenocarcinoma were classified based on histology and immunohistochemical expression of CDX2 and MUC1. A pancreaticobiliary histomolecular classification (PB) was defined as a sample with pancreaticobiliary histology, positive MUC1 and negative CDX2 expression. RESULTS: There were 82 deaths; median follow-up of 32.4 months; and median overall survival of 87.7 (95% CI 42.9-109.5) months. PB comprised 28.2% of the cases. Factors associated with overall survival were histological subtype (P=0.0340); T1/2 vs T3/4 (P=0.001); perineural (P<0.0001) and lymphovascular (P=0.0203) invasion; and histomolecular intestinal histomolecular phenotype (INT) vs PB phenotype (106.4 vs 21.2 months, P<0.0001). Neither MUC1 nor CDX2 was statistically significant, although MUC1 positivity defined as ⩾10% staining was significant (P=0.0023). In multivariate analysis, age (HR 1.03), PB phenotype (HR 2.26) and perineural invasion (PNI; HR 2.26) were associated with poor survival. CONCLUSIONS: The prognostic ability of histomolecular phenotype has been validated in an independent cohort of ampullary adenocarcinoma patients.

Active viscoelastic matter: from bacterial drag reduction to turbulent solids.

A paradigm for internally driven matter is the active nematic liquid crystal, whereby the equations of a conventional nematic are supplemented by a minimal active stress that violates time-reversal symmetry. In practice, active fluids may have not only liquid-crystalline but also viscoelastic polymer degrees of freedom. Here we explore the resulting interplay by coupling an active nematic to a minimal model of polymer rheology. We find that adding a polymer can greatly increase the complexity of spontaneous flow, but can also have calming effects, thereby increasing the net throughput of spontaneous flow along a pipe (a "drag-reduction" effect). Remarkably, active turbulence can also arise after switching on activity in a sufficiently soft elastomeric solid.

The emerging role of endoscopic ultrasound-guided core biopsy for the evaluation of solid pancreatic masses.

Pancreatic ductal adenocarcinoma is a lethal cancer with a 5-year survival rate of less than 5%. Surgical resection is the only curative treatment but only 20% are eligible for resection at the time of diagnosis. Early detection of cancer is of paramount importance in the management. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the preferred modality for obtaining tissue diagnosis of pancreatic masses. However, the diagnostic accuracy of EUS-FNA may be limited by several factors like availability of onsite cytopathology, adequacy of tissue core for histology, location of the mass, presence of underlying chronic pancreatitis, and experience of the endoscopist. Modern oncology is focusing on personalizing treatment based on tissue analysis of genetic aberrations and molecular biomarkers which are now available. Core tissue also aids in the diagnosis of disease entities like lymphoma, metastatic tumors, neuroendocrine tumors and autoimmune pancreatitis whose diagnosis rely on preserved tissue architecture and immunohistochemistry. Making accurate diagnosis of solid pancreatic masses is critical to avoid unnecessary resections in patients with benign lesions like focal lesions of chronic pancreatitis and autoimmune pancreatitis which mimic cancer. To overcome the limitations of FNA and to obtain adequate core tissue, a Tru-Cut biopsy needle was developed which met with variable success due to stiffness, cumbersome operation and technical failure using it in the duodenum/pancreatic head. More recently fine needle biopsy needles, with reverse bevel technology have become available in different sizes (19, 22, 25-gauge). The aim of this article was to review the emerging role of core biopsy needles in acquiring tissue in solid pancreatic masses and discuss its potential role in personalized medicine.

Differential in vivo tumorigenicity of diverse KRAS mutations in vertebrate pancreas: A comprehensive survey.

Somatic activation of the KRAS proto-oncogene is evident in almost all pancreatic cancers, and appears to represent an initiating event. These mutations occur primarily at codon 12 and less frequently at codons 13 and 61. Although some studies have suggested that different KRAS mutations may have variable oncogenic properties, to date there has been no comprehensive functional comparison of multiple KRAS mutations in an in vivo vertebrate tumorigenesis system. We generated a Gal4/UAS-based zebrafish model of pancreatic tumorigenesis in which the pancreatic expression of UAS-regulated oncogenes is driven by a ptf1a:Gal4-VP16 driver line. This system allowed us to rapidly compare the ability of 12 different KRAS mutations (G12A, G12C, G12D, G12F, G12R, G12S, G12V, G13C, G13D, Q61L, Q61R and A146T) to drive pancreatic tumorigenesis in vivo. Among fish injected with one of five KRAS mutations reported in other tumor types but not in human pancreatic cancer, 2/79 (2.5%) developed pancreatic tumors, with both tumors arising in fish injected with A146T. In contrast, among fish injected with one of seven KRAS mutations known to occur in human pancreatic cancer, 22/106 (20.8%) developed pancreatic cancer. All eight tumorigenic KRAS mutations were associated with downstream MAPK/ERK pathway activation in preneoplastic pancreatic epithelium, whereas nontumorigenic mutations were not. These results suggest that the spectrum of KRAS mutations observed in human pancreatic cancer reflects selection based on variable tumorigenic capacities, including the ability to activate MAPK/ERK signaling.

Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits.

KiSS1 is considered to be a key mediator of molecular mechanism of reproduction (puberty and prolificacy) in mammals. Kisspeptins are a family of structurally related peptides, encoded by KiSS1 gene, with ability to regulate gonadotropin-releasing hormone and hence hypothalamic-pituitary-gonadal axis. The present study investigated the polymorphism of caprine KiSS1 gene in 9 Indian goat breeds differing in sexual precocity and prolificacy. Comparison of KiSS1 amplified sequences of indigenous goats resulted in identification of nine SNPs (intron (1) G296C, T455G, T505A, T693C, T950C and intron (2) T1125C, A2510G, C2540T, A2803G) of which four are novel. These loci were not segregating together (r(2)<0.33). Mutations existed in both, sexually precocious and late-maturing goat breeds as well as low and high prolificacy goat breeds. Three loci reported to be associated with goat litter size (G296C, G2510A and C2540T) were identified in Indian goats as well. Association between loci of KiSS1 gene and age of puberty as well as litter size was explored in Black Bengal (N=158), a sexually precocious and prolific goat breed of India by designing PCR-RFLP. None of the mutations were found to be associated with reproductive traits however, difference in litter size as well age of sexual maturity for different genotypes indicates that the study on additional data based on more number of breeds and animals would be interesting to perform. Considering the importance of the reproductive trait in small ruminants, the results extend the limited information on genetic variation of the caprine KiSS1, which might contribute toward molecular breeding to enhance productivity of goat.

Adhesion forces and composition of planktonic and adhering oral microbiomes.

The oral microbiome consists of a planktonic microbiome residing in saliva and an adhering microbiome (the biofilm adhering to oral hard and soft tissues). Here we hypothesized that possible differences in microbial composition of the planktonic and adhering oral microbiome on teeth can be related to the forces by which different bacterial species are attracted to the tooth surface. The relative presence of 7 oral bacterial species in saliva and biofilm collected from 10 healthy human volunteers was determined twice in each volunteer by denaturing-gradient-gel electrophoresis. Analysis of both microbiomes showed complete separation of the planktonic from the adhering oral microbiome. Next, adhesion forces of corresponding bacterial strains with saliva-coated enamel surfaces were measured by atomic force microscopy. Species that were found predominantly in the adhering microbiome had significantly higher adhesion forces to saliva-coated enamel (-0.60 to -1.05 nN) than did species mostly present in the planktonic microbiome (-0.40 to -0.55 nN). It is concluded that differences in composition of the planktonic and the adhering oral microbiome are due to small differences in the forces by which strains adhere to saliva-coated enamel, providing an important step in understanding site- and material-specific differences in the composition of biofilms in the oral cavity.

Next-generation sequencing of endoscopic biopsies identifies ARID1A as a tumor-suppressor gene in Barrett's esophagus.

The incidence of Barrett's esophagus (BE)-associated esophageal adenocarcinoma (EAC) is increasing. Next-generation sequencing (NGS) provides an unprecedented opportunity to uncover genomic alterations during BE pathogenesis and progression to EAC, but treatment-naive surgical specimens are scarce. The objective of this study was to establish the feasibility of using widely available endoscopic mucosal biopsies for successful NGS, using samples obtained from a BE 'progressor'. Paired-end whole-genome NGS was performed on the Illumina platform using libraries generated from mucosal biopsies of normal squamous epithelium (NSE), BE and EAC obtained from a patient who progressed to adenocarcinoma during endoscopic surveillance. Selective validation studies, including Sanger sequencing, immunohistochemistry and functional assays, were performed to confirm the NGS findings. NGS identified somatic nonsense mutations of AT-rich interactive domain 1A (SWI like) (ARID1A) and PPIE and an additional 37 missense mutations in BE and/or EAC, which were confirmed by Sanger sequencing. ARID1A mutations were detected in 15% (3/20) high-grade dysplasia (HGD)/EAC patients. Immunohistochemistry performed on an independent archival cohort demonstrated ARID1A protein loss in 0% (0/76), 4.9% (2/40), 14.3% (4/28), 16.0% (8/50) and 12.2% (12/98) of NSE, BE, low-grade dysplasia, HGD and EAC tissues, respectively, and was inversely associated with nuclear p53 accumulation (P=0.028). Enhanced cell growth, proliferation and invasion were observed on ARID1A knockdown in EAC cells. In addition, genes downstream of ARID1A that potentially contribute to the ARID1A knockdown phenotype were identified. Our studies establish the feasibility of using mucosal biopsies for NGS, which should enable the comparative analysis of larger 'progressor' versus 'non-progressor' cohorts. Further, we identify ARID1A as a novel tumor-suppressor gene in BE pathogenesis, reiterating the importance of aberrant chromatin in the metaplasia-dysplasia sequence.

New advances in the pathogenesis and progression of barrett's esophagus.

Barrett's esophagus (BE) is a premalignant condition in the esophagus, with a rising incidence rate among Caucasians, and an established risk factor for the subsequent progression to esophageal adenocarcinoma (EAC). In contrast to the stratified squamous epithelium that normally lines the distal esophagus, BE is characterized by columnar epithelium that to some extent resembles the mucosa of the lower intestinal tract. The mechanism of intestinalization of the esophagus is still uncertain. For many years, it was postulated that either abnormal differentiation of resident progenitor cells in the esophagus, or transdifferentiation of mature esophageal keratinocytes provoked by reflux-induced genetic alterations, resulted in the BE phenotype. However, more recent studies suggest that indigenous progenitor cells at the gastro-esophageal junction might, under unfavorable conditions such as TP63 loss or an activated inflammatory response, migrate to the esophagus and initiate columnar cell differentiation. In this review, we discuss the competing theories of the origins of BE, as well as the role of developmental signaling pathways such as Notch, Hedgehog, and Wnt/ß-catenin signaling that have been implicated in the molecular pathogenesis of BE and EAC. Additionally, we provide an overview of the mutational landscapes of BE and EAC, derived from the results of recently published next generation sequencing (NGS) studies. Future research should elucidate whether NGS on endoscopic mucosal biopsies can help in identifying BE patients at highest risk for EAC development, and whether some of the prevalent mutations are "actionable", leading to improvements in current therapeutic strategies for BE and EAC.

Designing, optimization and validation of tetra-primer ARMS PCR protocol for genotyping mutations in caprine Fec genes.

New, quick, and inexpensive methods for genotyping novel caprine Fec gene polymorphisms through tetra-primer ARMS PCR were developed in the present investigation. Single nucleotide polymorphism (SNP) genotyping needs to be attempted to establish association between the identified mutations and traits of economic importance. In the current study, we have successfully genotyped three new SNPs identified in caprine fecundity genes viz. T(-242)C (BMPR1B), G1189A (GDF9) and G735A (BMP15). Tetra-primer ARMS PCR protocol was optimized and validated for these SNPs with short turn-around time and costs. The optimized techniques were tested on 158 random samples of Black Bengal goat breed. Samples with known genotypes for the described genes, previously tested in duplicate using the sequencing methods, were employed for validation of the assay. Upon validation, complete concordance was observed between the tetra-primer ARMS PCR assays and the sequencing results. These results highlight the ability of tetra-primer ARMS PCR in genotyping of mutations in Fec genes. Any associated SNP could be used to accelerate the improvement of goat reproductive traits by identifying high prolific animals at an early stage of life. Our results provide direct evidence that tetra-primer ARMS-PCR is a rapid, reliable, and cost-effective method for SNP genotyping of mutations in caprine Fec genes.

Polymorphism of BMP4 gene in Indian goat breeds differing in prolificacy.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß (transforming growth factor-beta) superfamily, of which BMP4 is the most important due to its crucial role in follicular growth and differentiation, cumulus expansion and ovulation. Reproduction is a crucial trait in goat breeding and based on the important role of BMP4 gene in reproduction it was considered as a possible candidate gene for the prolificacy of goats. The objective of the present study was to detect polymorphism in intronic, exonic and 3' un-translated regions of BMP4 gene in Indian goats. Nine different goat breeds (Barbari, Beetal, Black Bengal, Malabari, Jakhrana (Twinning>40%), Osmanabadi, Sangamneri (Twinning 20-30%), Sirohi and Ganjam (Twinning<10%)) differing in prolificacy and geographic distribution were employed for polymorphism scanning. Cattle sequence (AC_000167.1) was used to design primers for the amplification of a targeted region followed by direct DNA sequencing to identify the genetic variations. Single nucleotide polymorphisms (SNPs) were not detected in exon 3, the intronic region and the 3' flanking region. A SNP (G1534A) was identified in exon 2. It was a non-synonymous mutation resulting in an arginine to lysine change in a corresponding protein sequence. G to A transition at the 1534 locus revealed two genotypes GG and GA in the nine investigated goat breeds. The GG genotype was predominant with a genotype frequency of 0.98. The GA genotype was present in the Black Bengal as well as Jakhrana breed with a genotype frequency of 0.02. A microsatellite was identified in the 3' flanking region, only 20 nucleotides downstream from the termination site of the coding region, as a short sequence with more than nineteen continuous and repeated CA dinucleotides. Since the gene is highly evolutionarily conserved, identification of a non-synonymous SNP (G1534A) in the coding region gains further importance. To our knowledge, this is the first report of a mutation in the coding region of the caprine BMP4 gene. But whether the reproduction trait of goat is associated with the BMP4 polymorphism, needs to be further defined by association studies in more populations so as to delineate an effect on it.

Single nucleotide polymorphisms in caprine calpastatin gene.

The calpains and calpastatin (CAST) make up a major cytosolic proteolytic system, the calpain-calpastatin system, found in mammalian tissues. The relative levels of the components of the calpain-calpastatin system determine the extent of meat tenderization during postmortem storage. Calpastatin (CAST) is a protein inhibitor of the ubiquitous calcium-dependent proteases-micro-calpain and m-calpain. Polymorphisms in the bovine, ovine and pig CAST gene have been associated with meat tenderness but little is known about how caprine CAST gene may affect goat meat quality traits. In this study we selected different parts of the CAST gene: 1) that have been previously reported to be polymorphic, intron 5 and 12 and 3'UTR; 2) first time explored (exon 3, 7 and 8 and part of intron 7 and 8) to investigate polymorphic status of caprine CAST gene. Using comparative sequencing ten novel SN Ps located in exon 3 and intron 5, 7 and 8 were identified. Previously reported SNPs in intron 5, 3'UTR and intron 12 were absent. Sequence analysis revealed a non synonymous amino acid variation in exon 3, which would result in Lys/Arg substitution in the corresponding protein sequence. Considerable variation was detected in intronic regions. Twenty-four InDel were also recognized in intronic regions (15) and 3'UTR (9). All the sequences shared high homology with published bovine and ovine sequences. Three PCR-RFLP loci have been established for further analyzing genetic polymorphism in indigenous goats.