Meta-QTLs, ortho-meta QTLs and related candidate genes for yield and its component traits under water stress in wheat (Triticum aestivum L.).

Meta-QTLs (MQTLs), ortho-MQTLs, and related candidate genes (CGs) for yield and its seven component traits evaluated under water deficit conditions were identified in wheat. For this purpose, a high density consensus map and 318 known QTLs were used for identification of 56 MQTLs. Confidence intervals (CIs) of the MQTLs were narrower (0.7-21 cM; mean = 5.95 cM) than the CIs of the known QTLs (0.4-66.6 cM; mean = 12.72 cM). Forty-seven MQTLs were co-located with marker trait associations reported in previous genome-wide association studies. Nine selected MQTLs were declared as 'breeders MQTLs' for use in marker-assisted breeding (MAB). Utilizing known MQTLs and synteny/collinearity among wheat, rice and maize, 12 ortho-MQTLs were also identified. A total of 1497 CGs underlying MQTLs were also identified, which were subjected to in-silico expression analysis, leading to identification of 64 differentially expressed CGs (DECGs) under normal and water deficit conditions. These DECGs encoded a variety of proteins, including the following: zinc finger, cytochrome P450, AP2/ERF domain-containing proteins, plant peroxidase, glycosyl transferase, glycoside hydrolase. The expression of 12 CGs at seedling stage (3 h stress) was validated using qRT-PCR in two wheat genotypes, namely Excalibur (drought tolerant) and PBW343 (drought sensitive). Nine of the 12 CGs were up-regulated and three down-regulated in Excalibur. The results of the present study should prove useful for MAB, for fine mapping of promising MQTLs and for cloning of genes across the three cereals studied. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01301-z.

Leaf rust responsive miRNA and their target genes in wheat.

Small RNA sequencing (sRNA-seq) and degradome analysis were used for the identification of miRNAs and their target host genes in a pair of near-isogenic lines (NILs), which differed for the presence of leaf rust resistance gene Lr28. The study led to identification of (i) 506 known and 346 novel miRNAs; and (ii) 5054 target genes including 4557 in silico predicted and 497 degradome-based genes using 105 differentially expressed (DE) miRNAs. A subset of 128 targets (67 in silico + 61 degradome-based) was differentially expressed in RNA-seq data that was generated by us earlier using the same pair of NILs; among these 128 targets, 58 target genes exhibited an inverse relationship with the DE miRNAs (expression of miRNAs and activation/suppression of target genes). Eight miRNAs which belonged to the conserved miRNA families and were known to be induced in response to fungal diseases in plants included the following: miR156, miR158, miR159, miR168, miR169, miR172, miR319, miR396. The target genes belonged to the following classes of genes known to be involved in downstream disease resistance pathways; peroxidases, sugar transporters, auxin response signaling, oxidation-reduction, etc. It was also noticed that although a majority of miRNAs and target genes followed the above classical inverse relationship, there were also examples, where no such relationship was observed. Among the target genes, there were also 51 genes that were not only regulated by miRNAs, but were also differentially methylated at sequences including the following segments: promotors, introns, TSS, exons. The results of the present study suggest a complex interplay among miRNA genes, target genes, and various epigenetic controls, which regulate the expression of genes involved in downstream pathways for disease resistance.

Meta-QTLs, ortho-meta-QTLs and candidate genes for grain yield and associated traits in wheat (Triticum aestivum L.).

KEY MESSAGE: In wheat, 2852 major QTLs of 8998 QTLs available for yield and related traits were used for meta-analysis; 141 meta-QTLs were identified, which included 13 breeder's MQTLs and 24 ortho-MQTLs; 1202 candidate genes and 50 homologues of genes for yield from other cereals were also identified. Meta-QTL analysis was conducted using 2852 of the 8998 known QTLs, retrieved from 230 reports published during 1999-2020 (including 19 studies on tetraploid wheat) for grain yield (GY) and the following ten component traits: (i) grain weight (GWei), (ii) grain morphology-related traits (GMRTs), (iii) grain number (GN), (iv) spikes-related traits (SRTs), (v) plant height (PH), (vi) tiller number (TN), (vii) harvest index (HI), (viii) biomass yield (BY), (ix) days to heading/flowering and maturity (DTH/F/M), and (x) grain filling duration (GFD). The study resulted in the identification of 141 meta-QTLs (MQTLs), with an average confidence interval (CI) of 1.4 cM as against a CI of > 12.1 cM (8.8 fold reduction) in the QTLs that were used. The corresponding physical length of CI ranged from 0.01 Mb to 661.9 Mb (mean, 31.5 Mb). Seventy-seven (77) of these 141 MQTLs overlapped marker-trait associations (MTAs) reported in genome-wide association studies. Also, 63 MQTLs (each based on at least 10 QTLs) were considered stable and robust, with 13 MQTLs described as breeder's MQTLs (selected based on small CI, large LOD, and high level of phenotypic variation explained). Thirty-five yield-related genes from rice, barley, and maize were also utilized to identify 50 wheat homologues in MQTLs. Further, the use of synteny and collinearity allowed the identification of 24 ortho-MQTLs which were common among the wheat, barley, rice, and maize. The results of the present study should prove useful for wheat breeding and future basic research in cereals including wheat, barley, rice, and maize.

Meta-QTLs for multiple disease resistance involving three rusts in common wheat (Triticum aestivum L.).

KEY MESSAGE: In wheat, multiple disease resistance meta-QTLs (MDR-MQTLs) and underlying candidate genes for the three rusts were identified which may prove useful for development of resistant cultivars. Rust diseases in wheat are a major threat to global food security. Therefore, development of multiple disease-resistant cultivars (resistant to all three rusts) is a major goal in all wheat breeding programs worldwide. In the present study, meta-QTLs and candidate genes for multiple disease resistance (MDR) involving all three rusts were identified using 152 individual QTL mapping studies for resistance to leaf rust (LR), stem rust (SR), and yellow rust (YR). From these 152 studies, a total of 1,146 QTLs for resistance to three rusts were retrieved, which included 368 QTLs for LR, 291 QTLs for SR, and 487 QTLs for YR. Of these 1,146 QTLs, only 718 QTLs could be projected onto the consensus map saturated with 2, 34,619 markers. Meta-analysis of the projected QTLs resulted in the identification of 86 MQTLs, which included 71 MDR-MQTLs. Ten of these MDR-MQTLs were referred to as the 'Breeders' MQTLs'. Seventy-eight of the 86 MQTLs could also be anchored to the physical map of the wheat genome, and 54 MQTLs were validated by marker-trait associations identified during earlier genome-wide association studies. Twenty MQTLs (including 17 MDR-MQTLs) identified in the present study were co-localized with 44 known R genes. In silico expression analysis allowed identification of several differentially expressed candidate genes (DECGs) encoding proteins carrying different domains including the following: NBS-LRR, WRKY domains, F-box domains, sugar transporters, transferases, etc. The introgression of these MDR loci into high-yielding cultivars should prove useful for developing high yielding cultivars with resistance to all the three rusts.

COVID-WideNet-A capsule network for COVID-19 detection.

Ever since the outbreak of COVID-19, the entire world is grappling with panic over its rapid spread. Consequently, it is of utmost importance to detect its presence. Timely diagnostic testing leads to the quick identification, treatment and isolation of infected people. A number of deep learning classifiers have been proved to provide encouraging results with higher accuracy as compared to the conventional method of RT-PCR testing. Chest radiography, particularly using X-ray images, is a prime imaging modality for detecting the suspected COVID-19 patients. However, the performance of these approaches still needs to be improved. In this paper, we propose a capsule network called COVID-WideNet for diagnosing COVID-19 cases using Chest X-ray (CXR) images. Experimental results have demonstrated that a discriminative trained, multi-layer capsule network achieves state-of-the-art performance on the COVIDx dataset. In particular, COVID-WideNet performs better than any other CNN based approaches for diagnosis of COVID-19 infected patients. Further, the proposed COVID-WideNet has the number of trainable parameters that is 20 times less than that of other CNN based models. This results in fast and efficient diagnosing COVID-19 symptoms and with achieving the 0.95 of Area Under Curve (AUC), 91% of accuracy, sensitivity and specificity respectively. This may also assist radiologists to detect COVID and its variant like delta.

A novel bicistronic DNA vaccine with enhanced protective immune response against Bacillus anthracis through DNA prime-protein boost vaccination approach.

Anthrax, by Bacillus anthracis, remains a dreadful fatal hazard worldwide. The currently used anthrax vaccines are plagued by numerous issues that limit their widespread use. As an immunization approach targeting both extracellular antigens and toxins of B. anthracis may achieve better sterile immunity, the present investigation designed a bicistronic secretory anti-anthrax DNA vaccine targeting immune response against toxin and cells. The efficacy of the vaccine was compared with monocistronic DNA vaccines and the currently used anthrax vaccine. For this, mice were immunized with the developed vaccine containing pag (encoding protective antigen to block toxin) and eag genes (encoding EA1 to target cells) of B. anthracis through DNA-prime/Protein-boost (D/P) and DNA prime/DNA-boost (D/D) approaches. There was a >2 and > 5 fold increase in specific antibody level by D/D and D/P approaches respectively, on 42nd days post-immunization (dpi). Serum cytokine profiling showed that both Th1 and Th2 immune responses were elicited, with more Th2 responses in D/P strategy. More importantly, challenge with 100 times LD50 of B. anthracis at 42nd dpi exhibited maximum cumulative survival (83.33 %) by bicistronic D/P approach. Remarkably, immunization with EA1 delayed mortality onset in infection. The study forms the first report on complement-dependent bactericidal activity of antiEA1 antibodies. In short, co-immunization of PA and EA1 through the developed bicistronic DNA vaccine would be an effective immunization approach in anthrax vaccination. Further, D/P strategy could enhance vaccine-induced immunity against B. anthracis. Altogether, the study generates certain critical insights having direct applications in next-generation vaccine development against anthrax.

Biofortification and bioavailability of Zn, Fe and Se in wheat: present status and future prospects.

KEY MESSAGE: Knowledge of genetic variation, genetics, physiology/molecular basis and breeding (including biotechnological approaches) for biofortification and bioavailability for Zn, Fe and Se will help in developing nutritionally improved wheat. Biofortification of wheat cultivars for micronutrients is a priority research area for wheat geneticists and breeders. It is known that during breeding of wheat cultivars for productivity and quality, a loss of grain micronutrient contents occurred, leading to decline in nutritional quality of wheat grain. Keeping this in view, major efforts have been made during the last two decades for achieving biofortification and bioavailability of wheat grain for micronutrients including Zn, Fe and Se. The studies conducted so far included evaluation of gene pools for contents of not only grain micronutrients as above, but also for phytic acid (PA) or phytate and phytase, so that, while breeding for the micronutrients, bioavailability is also improved. For this purpose, QTL interval mapping and GWAS were carried out to identify QTLs/genes and associated markers that were subsequently used for marker-assisted selection (MAS) during breeding for biofortification. Studies have also been conducted to understand the physiology and molecular basis of biofortification, which also allowed identification of genes for uptake, transport and storage of micronutrients. Transgenics using transgenes have also been produced. The breeding efforts led to the development of at least a dozen cultivars with improved contents of grain micronutrients, although land area occupied by these biofortified cultivars is still marginal. In this review, the available information on different aspects of biofortification and bioavailability of micronutrients including Zn, Fe and Se in wheat has been reviewed for the benefit of those, who plan to start work or already conducting research in this area.

Development of a visible loop mediated isothermal amplification assay for rapid detection of Bacillus anthracis.

Distressing effects on animal and human health with lethal progression, being used as bioweapon and shared features with non-pathogenic bacteria demands sensitive, specific, safe, cost effective and rapid detection methods for anthrax causing organisms. Conventional microbiology based diagnostics for anthrax are time consuming and need sophisticated equipment, while molecular diagnostics require less time and labor. The Loop mediated isothermal amplification assay (LAMP) is rapid, sensitive and specific assay and requires no specialized equipment. In the present study, we developed a LAMP assay for rapid as well as specific detection of Bacillus anthracis. The optimized assay produced positive results with the Sterne strain and one field isolate of B. anthracis and, negative results with other bacteria of the same and different genera within 2 h. Sensitivity was 500 fg of total DNA of B. anthracis, which was 100 times more sensitive than conventional PCR. The present study also demonstrated that the simple method of total DNA extraction by repeated boiling and freezing will not adversely affect the LAMP results. In conclusion, the optimized LAMP assay is a promising tool for the specific, sensitive, less time-consuming diagnosis for anthrax causing bacteria and also, for detecting the virulence of suspected B. anthracis cultures.

Application of Deep Learning on Student Engagement in e-learning environments.

The drastic impact of COVID-19 pandemic is visible in all aspects of our lives including education. With a distinctive rise in e-learning, teaching methods are being undertaken remotely on digital platforms due to COVID-19. To reduce the effect of this pandemic on the education sector, most of the educational institutions are already conducting online classes. However, to make these digital learning sessions interactive and comparable to the traditional offline classrooms, it is essential to ensure that students are properly engaged during online classes. In this paper, we have presented novel deep learning based algorithms that monitor the student's emotions in real-time such as anger, disgust, fear, happiness, sadness, and surprise. This is done by the proposed novel state-of-the-art algorithms which compute the Mean Engagement Score (MES) by analyzing the obtained results from facial landmark detection, emotional recognition and the weights from a survey conducted on students over an hour-long class. The proposed automated approach will certainly help educational institutions in achieving an improved and innovative digital learning method.

Genome-wide analysis of H3K4me3 and H3K27me3 modifications due to Lr28 for leaf rust resistance in bread wheat (Triticum aestivum).

KEY MESSAGE: Present study revealed a complex relationship among histone H3 methylation (examined using H3K4/K27me3 marks), cytosine DNA methylation and differential gene expression during Lr28 mediated leaf rust resistance in wheat. During the present study, genome-wide histone modifications were examined in a pair of near isogenic lines (NILs) (with and without Lr28 in the background of cv. HD2329). The two histone marks used included H3K4me3 (an activation mark) and H3K27me3 (a repression mark). The results were compared with levels of expression (using RNA-seq) and DNA methylation (MeDIP) data obtained using the same pair of NILs. Some of the salient features of the present study include the following: (i) large scale differential binding sites (DBS) were available for only H3K4me3 in the susceptible cultivar, but for both H3K4me3 and H3K27me3 in its resistant NIL; (ii) DBSs for H3K27me3 mark were more abundant (> 80%) in intergenic regions, whereas DBSs for H3K4me3 were distributed in all genomic regions including exons, introns, intergenic, TTS (transcription termination sites) and promoters; (iii) fourteen (14) genes associated with DBSs showed co-localization for both the marks; (iv) only a small fraction (7% for H3K4me3 and 12% for H3K27me3) of genes associated with DBSs matched with the levels of gene expression inferred from RNA-seq data; (v) validation studies using qRT-PCR were conducted on 26 selected representative genes; results for only 11 genes could be validated. The proteins encoded by important genes involved in promoting infection included domains generally carried by R gene proteins such as Mlo like protein, protein kinases and purple acid phosphatase. Similarly, proteins encoded by genes involved in resistance included those carrying domains for lectin kinase, R gene, aspartyl protease, etc. Overall, the results suggest a very complex network of downstream genes that are expressed during compatible and incompatible interactions; some of the genes identified during the present study may be used in future validation studies involving RNAi/overexpression approaches.

Complex relationship between DNA methylation and gene expression due to Lr28 in wheat-leaf rust pathosystem.

Differential DNA methylation due to Lr28 was examined in susceptible (S) wheat cv. HD2329 and its resistant (R) near isogenic line (NIL) (HD2329+Lr28) using two approaches: methylation sensitive amplified polymorphism (MSAP) and methylated DNA immunoprecipitation (MeDIP). S/R lines each had a large number of hypomethylated genes and relatively fewer hypermethylated genes at 96 hai (hours after inoculation) relative to 0 hbi (hours before inoculation), suggesting activation of many genes during the passage of time (96 hai), although identity of genes may differ in S and R lines. When R NIL was compared with S cultivar, there were many hypermethylated and fewer hypomethylated genes in R NIL relative to S cultivar, suggesting that many genes that are active in S cultivar are silenced in R NIL, both at 0 hbi and at 96 hai. Level of methylation was generally abundant in intergenic regions followed by that in promoters, transcription termination sites (TTSs) and exons/introns. Hypermethylation in promoter and gene body regions was not always associated with inhibition of gene expression and vice-versa, indicating that more than one regulatory mechanisms may control the expression of genes due to pathogen attack in presence and absence of Lr28. MSAP analysis also showed abundance of mCG methylation in S cultivar and that of mCCG methylation in R NIL (at 96 hai), suggesting differences in methylation context in NILs with and without Lr28. The results of the present study improved our understanding of the epigenetic control of leaf rust resistance in wheat.

Application of deep learning for fast detection of COVID-19 in X-Rays using nCOVnet.

Presently, COVID-19 has posed a serious threat to researchers, scientists, health professionals, and administrations around the globe from its detection to its treatment. The whole world is witnessing a lockdown like situation because of COVID-19 pandemic. Persistent efforts are being made by the researchers to obtain the possible solutions to control this pandemic in their respective areas. One of the most common and effective methods applied by the researchers is the use of CT-Scans and X-rays to analyze the images of lungs for COVID-19. However, it requires several radiology specialists and time to manually inspect each report which is one of the challenging tasks in a pandemic. In this paper, we have proposed a deep learning neural network-based method nCOVnet, an alternative fast screening method that can be used for detecting the COVID-19 by analyzing the X-rays of patients which will look for visual indicators found in the chest radiography imaging of COVID-19 patients.

A deep learning and grad-CAM based color visualization approach for fast detection of COVID-19 cases using chest X-ray and CT-Scan images.

The world is suffering from an existential global health crisis known as the COVID-19 pandemic. Countries like India, Bangladesh, and other developing countries are still having a slow pace in the detection of COVID-19 cases. Therefore, there is an urgent need for fast detection with clear visualization of infection is required using which a suspected patient of COVID-19 could be saved. In the recent technological advancements, the fusion of deep learning classifiers and medical images provides more promising results corresponding to traditional RT-PCR testing while making detection and predictions about COVID-19 cases with increased accuracy. In this paper, we have proposed a deep transfer learning algorithm that accelerates the detection of COVID-19 cases by using X-ray and CT-Scan images of the chest. It is because, in COVID-19, initial screening of chest X-ray (CXR) may provide significant information in the detection of suspected COVID-19 cases. We have considered three datasets known as 1) COVID-chest X-ray, 2) SARS-COV-2 CT-scan, and 3) Chest X-Ray Images (Pneumonia). In the obtained results, the proposed deep learning model can detect the COVID-19 positive cases in  ≤  2 seconds which is faster than RT-PCR tests currently being used for detection of COVID-19 cases. We have also established a relationship between COVID-19 patients along with the Pneumonia patients which explores the pattern between Pneumonia and COVID-19 radiology images. In all the experiments, we have used the Grad-CAM based color visualization approach in order to clearly interpretate the detection of radiology images and taking further course of action.

Identification of modifiers of the plant height in wheat using an induced dwarf mutant controlled by RhtB4c allele.

In wheat, 25 Rht genes for dwarfness are known, which include both, GA-insensitive and GA-responsive genes. The GA-insensitive Rht genes have been widely used, although, their suitability under abiotic stress conditions has been questioned. This necessitated a search for alternative GA-responsive, spontaneous and induced dwarfing genes. We earlier reported an induced dwarf mutant ('dwarf mutant-3'; 44 cm), and the mutant allele was named Rht4c allele (2BL). This dwarf mutant was not suitable for cultivation due to its extra dwarf nature. Therefore, we searched for naturally occurring QTLs, which would modify the phenotype of 'dwarf-mutant-3' using 'mutant-assisted gene identification and characterization' (MAGIC) approach. For this purpose, the 'dwarf mutant-3' was crossed with a tall wheat cv. NP114 and homozygous mutant F2 plants (~ 25% of the progeny) were selected, which were phenotyped for plant height and genotyped using SSR markers. The data were utilized for QTL analysis and plant height. Six modifier QTLs were identified on chromosomes 2A, 2B and 4A. Two QTLs each on 2A and 2B were responsible for increase in plant height (described as 'enhancer modifiers'), whereas the remaining two QTLs located on 4A were responsible for reducing the plant height (described as 'suppressor modifiers'). It was hypothesized that the enhancer QTLs could be exploited for the development of semi-dwarf high yielding genotypes containing Rht4c allele. This is the first study of its kind in wheat demontsrating that the MAGIC approach could be used for identification of modifiers of the mutant phenotypes of other traits for wheat improvement.

Meta-QTL analysis and identification of candidate genes for drought tolerance in bread wheat (Triticum aestivum L.).

Meta-QTL (MQTL) analysis for drought tolerance was undertaken in bread wheat to identify consensus and robust MQTLs using 340 known QTLs from 11 earlier studies; 13 MQTLs located on 6 chromosomes (1D, 3B, 5A, 6D, 7A and 7D) were identified, with maximum of 4 MQTLs on chromosome 5A. Mean confidence intervals for MQTLs were much narrower (mean, 6.01 cM; range 2.07-19.46 cM), relative to those in original QTLs (mean, 13.6 cM; range, 1.0-119.1 cM). Two MQTLs, namely MQTL4 and MQTL12, were major MQTLs with potential for use in marker-assisting breeding. As many as 228 candidate genes (CGs) were also identified using 6 of the 13 MQTLs. In-silico expression analysis of these 228 CGs allowed identification of 14 important CGs, with + 3 to - 8 fold change in expression under drought (relative to normal conditions) in a tolerant cv. named TAM107. These CGs encoded proteins belonging to the following families: NAD-dependent epimerase/dehydratase, protein kinase, NAD(P)-binding domain protein, heat shock protein 70 (Hsp70), glycosyltransferase 2-like, etc. Important MQTLs and CGs identified in the present study should prove useful for future molecular breeding and for the study of molecular basis of drought tolerance in cereals in general and wheat in particular.

H3K4/K9 acetylation and Lr28-mediated expression of six leaf rust responsive genes in wheat (Triticum aestivum).

Development of leaf rust-resistant cultivars is a priority during wheat breeding, since leaf rust causes major losses in yield. Resistance against leaf rust due to Lr genes is partly controlled by epigenetic modifications including histone acetylation that is known to respond to biotic/abiotic stresses. In the present study, enrichment of H3K4ac and H3K9ac in promoters of six defense responsive genes (N-acetyltransferase, WRKY 40, WRKY 70, ASR1, Peroxidase 12 and Sarcosine oxidase) was compared with their expression in a pair of near-isogenic lines (NILs) for the gene Lr28 following inoculation with leaf rust pathotype '77-5'; ChIP-qPCR was used for this purpose. The proximal and distal promoters of these genes contained a number of motifs that are known to respond to biotic stresses. The enrichment of two acetylation marks changed with passage of time; changes in expression of two of the six genes (N-acetyltransferase and peroxidase12), largely matched with changes in H3K4/H3K9 acetylation patterns of the two promoter regions. For example, enrichment of both the marks matched with higher expression of N-acetyltransferase gene in susceptible NIL and the deacetylation (H3K4ac) largely matched with reduced gene expression in resistant NIL. In peroxidase12, enrichment of H3K4ac and H3K9ac largely matched with higher expression in both the NILs. In the remaining four genes, changes in H3 acetylation did not always match with gene expression levels. This indicated complexity in the regulation of the expression of these remaining four genes, which may be controlled by other epigenetic/genetic regulatory mechanisms that need further analysis.

Further studies on sugar transporter (SWEET) genes in wheat (Triticum aestivum L.).

SWEET proteins represent one of the largest sugar transporter family in the plant kingdom and play crucial roles in plant development and stress responses. In the present study, a total of 108 TaSWEET genes distributed on all the 21 wheat chromosomes were identified using the latest whole genome sequence (as against 59 genes reported in an earlier report). These 108 genes included 14 of the 17 types reported in Arabidopsis and also included three novel types. Tandem duplications (22) and segmental duplications (5) played a significant role in the expansion of TaSWEET family. A number of cis-elements were also identified in the promoter regions of TaSWEET genes, indicating response of TaSWEET genes during development and also during biotic/abiotic stresses. The TaSWEET proteins carried 4-7 trans-membrane helices (TMHs) showing diversity in structure. Phylogenetic analysis using SWEET proteins of wheat and 8 other species gave four well-known clusters. Expression analysis involving both in silico and in planta indicated relatively higher expression of TaSWEET genes in water/heat sensitive and leaf rust resistant genotypes. The results provided insights into the functional role of TaSWEETs in biotic and abiotic stresses, which may further help in planning strategies to develop high yielding wheat varieties tolerant to environmental stresses.

Genetic epidemiology of malignant hyperthermia in the UK.

BACKGROUND: Gaps in our understanding of genetic susceptibility to malignant hyperthermia (MH) limit the application and interpretation of genetic diagnosis of the condition. Our aim was to define the prevalence and role of variants in the three genes implicated in MH susceptibility in the largest comprehensively phenotyped MH cohort worldwide. METHODS: We initially included one individual from each positive family tested in the UK MH Unit since 1971 to detect variants in RYR1, CACNA1S, or STAC3. Screening for genetic variants has been ongoing since 1991 and has involved a range of techniques, most recently next generation sequencing. We assessed the pathogenicity of variants using standard guidelines, including family segregation studies. The prevalence of recurrent variants of unknown significance was compared with the prevalence reported in a large database of sequence variants in low-risk populations. RESULTS: We have confirmed MH susceptibility in 795 independent families, for 722 of which we have a DNA sample. Potentially pathogenic variants were found in 555 families, with 25 RYR1 and one CACNA1S variants previously unclassified recurrent variants significantly over-represented (P<1×10-7) in our cohort compared with the Exome Aggregation Consortium database. There was genotype-phenotype discordance in 86 of 328 families suitable for segregation analysis. We estimate non-RYR1/CACNA1S/STAC3 susceptibility occurs in 14-23% of MH families. CONCLUSIONS: Our data provide current estimates of the role of variants in RYR1, CACNA1S, and STAC3 in susceptibility to MH in a predominantly white European population.

Development of real-time PCR assay for the detection of Mycoplasma bovis.

Mycoplasma bovis is one of the important bovine mycoplasma involved in economically important clinical conditions like respiratory diseases, otitis media, and mastitis. The present study was undertaken with the objective of developing a SYBR Green dye-based real-time PCR assay targeting uvrC gene for the diagnosis of M. bovis. The analytical sensitivity and specificity of the assay were evaluated. The test showed 103-fold more sensitivity than conventional PCR and detected down to 100 fg level of DNA. It was found to be specific, as no cross reactivity was shown with other related bacteria and Mycoplasma species. The developed assay was able to detect down to 40 copies of uvrC gene from spiked bovine milk samples. At present, this developed assay may be used as a valuable diagnostic tool for the detection of Mycoplasma bovis.

Differential Hepatotoxic Effects of Sodium Valproate at Different Doses in Albino Rats.

Background Liver plays an essential role for transforming and clearing chemicals that may cause harmful effects to it. Sodium Valproate, renowned to be a potent antiepileptic drug, when taken in overdose may cause toxic effects to liver and other organs as well. Liver damage can be assessed with histological changes and measurement of enzymes produced by it. Objective To investigate the histological changes induced by different doses of Sodium Valproate ranging from 100-500 mg/kg/day and observe its correlation with liver enzymes level in serum. Method Three-months old albino rats were divided into six groups, five in each. Control group was treated with normal saline and rest five groups with Sodium Valproate in different doses 100, 200, 300, 400 and 500 mg/kg/day respectively. Then, liver of those experimented rats were examined histologically under the light microscope. Furthermore, Liver enzymes; Alanine Transaminase and Aspartate transaminase were measured to assess the micro-anatomical changes in liver. Result Distorted hepatic lobular architecture with aggregations of nuclei at certain interval was observed in the groups of higher doses; 300 mg/kg/day and above. However, accumulation of adipocytes was observed in all the Sodium Valproate treated rats unlike the control group. When compared the enzyme levels among the groups, it was found to be significantly increased in dose dependent manner. Besides, it also showed skin lesions in all rats treated with the dose 400 mg/kg/day and above. Conclusion Higher doses of Sodium Valproate; 300 mg/kg/day and above induces hepatotoxicity and skin lesions in adult albino rats.