PCR Based Genotyping of Lulu Cattle of Nepal for A1, A2 Type Beta-caseins

Lulu is an indigenous breed of cattle (Bos taurus) found in high altitude regions of western Nepal. Population of Lulu cattle has been declining due to introgression with other exotic breeds to increase milk productivity. Here we aimed at finding potential approach for conserving Lulu cattle and its assets by studying the milk contents and investigating which variant of beta-casein protein is present in this breed. Beta caseins are an abundant protein in cow milk with A1 and A2 being the most common genetic variants of this protein. Consumption of A1 type of milk has numerous health-related complications whereas A2 type of milk has numerous human health promoting factors. We used restriction fragment length polymorphism (RFLP) for determining the A1 and A2 variant of beta casein in Lulu cattle. For performing DNA extraction, we collected (n = 18) blood samples of Lulu from Mustang and (n=17) Nepal Agriculture research council farm. The amplified fragments in 3% agarose at 251bp and 213bp respectively confirmed the presence of both A1 and A2 gene in Lulu; however, A2 was of greater abundance. Our study indicated that Lulu has A2 variant of beta-casein predominantly. The gene frequency of A1A1 is 0, A1A2 is 0.06 and A2A2 is 0.94. We further found that the allele frequency of A1 and A2 is 0.03 and 0.97 respectively. We designed special primer for sequencing CSN2 genes since A2 type beta casein gene was predominantly seen on Lulu. The sequencing result further supports our RFLP result as most of our samples have “C” nucleotide SNP in amplified CSN2 gene sequence. The Chi-square value of the current study is 0.04 which supports Hardy-Weinberg equilibrium inferring that Lulu cattle are still in the pure state, where there is no genetic introgression with the exotic breed for the sake of improvement of productivity

Vegetable Cultivation under Hydroponics in Himalayas : Challenges and Opportunities

Defence Institute of Bio-Energy Research, Haldwani has successfully developed a hydroponics system by clubbing the soil less cultivation with rain water harvesting technology. The institute has also developed a suitable nutrient composition suitable for wide range of vegetables. Among the various varieties/hybrids grown in hydroponics, cucumber line PC 4C-8 exhibited the maximum fruit yield (448.3 q/ha). Among the various tomato hybrids, Avinash-2 exhibited highest yield (1052 q/ha) and number of fruits (154 per sq m) whereas TSS, total sugars, crude protein and minerals content were the maximum in tomato hybrid DARL-304. Interestingly, hydroponics system prolonged the harvesting period by 45 days in tomato. Plant population density of 6 plants/m2 was found optimum with the highest fruit yield in tomato and cucumber. Tomato hybrid DARL-304 exhibited higher fruit yield (6.5 kg/plant). Hydroponics system have been found successful in Auli (9000’ MSL), Joshimath with fruit yield of 2.25 kg/plant to 3.79 kg/plant in tomato. The yield was higher in rain water as comparison to river water. The article deals with the hydroponics technology in detail vis-à-vis efforts made at DIBER for standardization of hydroponics technology. It is envisaged that experience gained in successful cultivation of various crops in hydroponics using single nutrient solution at various altitudes through research stations of the institute

Novel truncating mutations in CTNND1 cause a dominant craniofacial and cardiac syndrome.

CTNND1 encodes the p120-catenin (p120) protein, which has a wide range of functions, including the maintenance of cell-cell junctions, regulation of the epithelial-mesenchymal transition and transcriptional signalling. Due to advances in next-generation sequencing, CTNND1 has been implicated in human diseases including cleft palate and blepharocheilodontic (BCD) syndrome albeit only recently. In this study, we identify eight novel protein-truncating variants, six de novo, in 13 participants from nine families presenting with craniofacial dysmorphisms including cleft palate and hypodontia, as well as congenital cardiac anomalies, limb dysmorphologies and neurodevelopmental disorders. Using conditional deletions in mice as well as CRISPR/Cas9 approaches to target CTNND1 in Xenopus, we identified a subset of phenotypes that can be linked to p120-catenin in epithelial integrity and turnover, and additional phenotypes that suggest mesenchymal roles of CTNND1. We propose that CTNND1 variants have a wider developmental role than previously described and that variations in this gene underlie not only cleft palate and BCD but may be expanded to a broader velocardiofacial-like syndrome

Gene domain-specific DNA methylation episignatures highlight distinct molecular entities of ADNP syndrome.

BACKGROUND:ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome. RESULTS: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The epi-ADNP-1 episignature included ~ 6000 mostly hypomethylated CpGs, and the epi-ADNP-2 episignature included ~ 1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N- and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model. CONCLUSIONS: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome

Brain Abnormalities in Patients with Germline Variants in H3F3: Novel Imaging Findings and Neurologic Symptoms Beyond Somatic Variants and Brain Tumors

BACKGROUND AND PURPOSE: Pathogenic somatic variants affecting the genes Histone 3 Family 3A and 3B (H3F3) are extensively linked to the process of oncogenesis, in particular related to central nervous system tumors in children. Recently, H3F3 germline missense variants were described as the cause of a novel pediatric neurodevelopmental disorder. We aimed to investigate patterns of brain MR imaging of individuals carrying H3F3 germline variants. MATERIALS AND METHODS: In this retrospective study, we included individuals with proved H3F3 causative genetic variants and available brain MR imaging scans. Clinical and demographic data were retrieved from available medical records. Molecular genetic testing results were classified using the American College of Medical Genetics criteria for variant curation. Brain MR imaging abnormalities were analyzed according to their location, signal intensity, and associated clinical symptoms. Numeric variables were described according to their distribution, with median and interquartile range. RESULTS: Eighteen individuals (10 males, 56%) with H3F3 germline variants were included. Thirteen of 18 individuals (72%) presented with a small posterior fossa. Six individuals (33%) presented with reduced size and an internal rotational appearance of the heads of the caudate nuclei along with an enlarged and squared appearance of the frontal horns of the lateral ventricles. Five individuals (28%) presented with dysgenesis of the splenium of the corpus callosum. Cortical developmental abnormalities were noted in 8 individuals (44%), with dysgyria and hypoplastic temporal poles being the most frequent presentation. CONCLUSIONS: Imaging phenotypes in germline H3F3-affected individuals are related to brain features, including a small posterior fossa as well as dysgenesis of the corpus callosum, cortical developmental abnormalities, and deformity of lateral ventricles

Combining Independent, Weighted P-Values: Achieving Computational Stability by a Systematic Expansion with Controllable Accuracy

Given the expanding availability of scientific data and tools to analyze them, combining different assessments of the same piece of information has become increasingly important for social, biological, and even physical sciences. This task demands, to begin with, a method-independent standard, such as the -value, that can be used to assess the reliability of a piece of information. Good's formula and Fisher's method combine independent -values with respectively unequal and equal weights. Both approaches may be regarded as limiting instances of a general case of combining -values from groups; -values within each group are weighted equally, while weight varies by group. When some of the weights become nearly degenerate, as cautioned by Good, numeric instability occurs in computation of the combined -values. We deal explicitly with this difficulty by deriving a controlled expansion, in powers of differences in inverse weights, that provides both accurate statistics and stable numerics. We illustrate the utility of this systematic approach with a few examples. In addition, we also provide here an alternative derivation for the probability distribution function of the general case and show how the analytic formula obtained reduces to both Good's and Fisher's methods as special cases. A C++ program, which computes the combined -values with equal numerical stability regardless of whether weights are (nearly) degenerate or not, is available for download at our group website http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads/CoinedPValues.html

TLR4 Asp299Gly and Thr399Ile Polymorphisms: No Impact on Human Immune Responsiveness to LPS or Respiratory Syncytial Virus

A broad variety of natural environmental stimuli, genotypic influences and timing all contribute to expression of protective versus maladaptive immune responses and the resulting clinical outcomes in humans. The role of commonly co-segregating Toll-like receptor 4 (TLR4) non-synonymous single nucleotide polymorphisms Asp299Gly and Thr399Ile in this process remains highly controversial. Moreover, what differential impact these polymorphisms might have in at risk populations with respiratory dysfunction, such as current asthma or a history of infantile bronchiolitis, has never been examined. Here we determine the importance of these polymorphisms in modulating LPS and respiratory syncytial virus (RSV)--driven cytokine responses. We focus on both healthy children and those with clinically relevant respiratory dysfunction.To elucidate the impact of TLR4 Asp299Gly and Thr399Ile on cytokine production, we assessed multiple immune parameters in over 200 pediatric subjects aged 7-9. Genotyping was followed by quantification of pro- and anti-inflammatory cytokine responses by fresh peripheral blood mononuclear cells upon acute exposure to LPS or RSV.In contrast to early reports, neither SNP influenced immune responses evoked by LPS exposure or RSV infection, as measured by the intermediate phenotype of pro- and anti-inflammatory cytokine responses to these ubiquitous agents. There is no evidence of altered sensitivity in populations with "at risk" clinical phenotypes.Genomic medicine seeks to inform clinical practice. Determination of the TLR4 Asp299Gly/Thr399Ile haplotype is of no clinical benefit in predicting the nature or intensity of cytokine production in children whether currently healthy or among specific at-risk groups characterized by prior infantile broncholitis or current asthma

Coronavirus Papain-like Proteases Negatively Regulate Antiviral Innate Immune Response through Disruption of STING-Mediated Signaling

Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV) NL63 and severe acute respiratory syndrome (SARS) CoV papain-like proteases (PLP) antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS). STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN). We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM) or SARS-CoV (PLpro-TM) inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction