BACKGROUND:Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking.RESULTS:To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised.CONCLUSIONS:Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

New epigenetic technologies may uncover etiopathogenic mechanisms of major psychosis. In this study, we applied padlock probe-based ultra-deep bisulfite sequencing for fine mapping of modified cytosines of the HLA complex group 9 (nonprotein coding) gene in the postmortem brains of individuals affected with schizophrenia or bipolar disorder and unaffected controls. Significant differences between patients and controls were detected in both CpG and CpH modifications. In addition, we identified epigenetic age effects, DNA modification differences between sense and anti-sense strands, and demonstrated how DNA modification data can be used in clustering of patient populations. Our findings revealed new epigenetic complexities but also highlighted the potential of DNA modification approaches in the search of heterogeneous causes of major psychiatric disease.

Amyotrophic lateral sclerosis (ALS) is characterised by a loss of motor neurons, leading to paralysis. Several autosomal dominant genes were implicated in ALS pathogenesis, such as SOD1 , with over 180 reported mutations (http://alsod.iop.kcl.ac.uk/), including a p.T137A substitution.1 ,2 Recently, ARHGEF28 , encoding an RNA binding protein involved in the aggregation of light neurofilaments in ALS, was suggested as a novel ALS gene (a heterozygous K280M>fs40X mutation was detected in three patients).3 ,4 Risk of ALS may be modulated by environmental factors, sex and ageing, which could be linked to epigenetic events (eg, DNA methylation). Monozygotic (MZ) twins provide the best opportunity to investigate environmental/epigenetic factors in disease development.5 Hence, we studied ALS-discordant MZ twins, including the evaluation of DNA methylation (DNAm) age, which is an accurate predictor of chronological age across different tissues.6 It is possible that DNAm age better reflects biological age than chronological age does, since age acceleration (DNAm age minus chronological age) was associated with several disorders and mortality. Participants were recruited from the ALS Clinic at the Sunnybrook Health Sciences Centre. Genetic and epigenetic analyses were conducted as described in the online supplementary methods, including mutation analysis of SOD1 and C9orf72 , NeuroX array, reverse transcription PCR (RT-PCR), whole genome DNA methylation array (HumanMethylation450 BeadChip) and bisulfite pyrosequencing. ### Supplementary data [jnnp-2016-313592supp.pdf] The MZ twins from a Canadian PED24 family of Italian origin were 52 years old at last examination and ALS-discordant for 17 years, with the second-born twin (9377) diagnosed with ALS at 35 years (figure 1 …

Background: In thalassemia patients blood transfusion is the main mainstay of therapy.Blood transfusion is associated with oxidative stress. Oxidative stre s has its influence on plasma proteins. The presen t study was carried out to explore the level of carbo nylation of serum protein content and oxidative changes in serum proteins of thalassemia subjects o n regular blood transfusion. Method:Blood was collected from thalassemia patients recei ving long duration of blood transfusion and from newly diagnosed cases of thalassemia who never receive blood transfusion and from carriers or traits of thalassemia as control.The extent of carb onylation of serum protein was estimated by thiobarbuturic acid method .The effects of in vitro treatment of serum protein H2O2 on the above mentioned parameters were observed. Observation:Carbonylation of serum protein was significantly hi gher thalassemia subjects on regular blood transfusion.Enhanced carbonylation of serum p rotein by in vitro H2O2 treatment suggest that oxidative stress can cause carbonylation of serum p roteins. Conclusion:carbonylation of serum protein can be used as indic ator of duration of transfusion therapy.

BACKGROUND:Aminophylline can trigger seizures in patients without known underlying epilepsy or added risk factor for seizure exacerbation in epilepsy. Most of these seizures are difficult to control and are underappreciated compared to other drug toxicities. Despite a long clinical history of aminophylline-induced seizures, relatively little is known about the underlying molecular mechanisms that contribute to methylxanthine-induced seizure generation.OBJECTIVE:The present study evaluated the possible involvement of free radicals in aminophylline induced seizures in rat.METHOD:The rats were divided into two groups. The first group graded single doses of aminophylline from 100 to 300 mg/kg were administered intraperitoneally. On the basis of the results Aminophylline, a dose (300 mg/kg) producing tonic-clonic seizures and mortality in 100% animals was selected as control in the study. The second group were subjected to single antioxidant (Vitamin E or Vitamin C) or in combination for 45 days then single doses of aminophylline 300 mg/kg administered intraperitoneally to rats.RESULT:Aminophylline induced convulsions in rats in a dose-dependent manner, and both incidence of seizure and mortality were maximum at 300 mg/kg and there was significant increase of free radical generation. But though pre-treatment with antioxidants showed differential attenuating effects on aminophylline induced free radical generation as we all known but they were very much ineffective in antagonizing aminophylline induced seizures and post-seizure mortality by any appreciable extent.CONCLUSION:Though Aminophylline induces oxidative stress the results are suggestive that at least free radicals is not only cause of convulsiogenic effects and post-seizure mortality of aminophylline.

BACKGROUND:Hypovitaminosis D appears to parallel several cardiovascular and pulmonary diseases. However, previous findings cannot be considered conclusive, since the association may have been confounded by different anthropometric variables that were not accounted for. The present cross-sectional study was conducted to investigate the association between important explanatory variables and circulation levels of vitamin D.METHODS:A total of 553 individuals attending the metabolic and medical lifestyle management clinic of the Burdwan district of India were selected from 1289 people by simple random sampling, and information regarding relevant variables and their blood was obtained. Serum 25-hydroxyvitamin (OH) D level and forced expiratory volume in 1 second were measured. Associations between different explanatory variables and circulatory 25(OH) D were analysed in linear regression models.RESULTS:Of the population studied, 53% had insufficient vitamin D levels, while approximately 9% were vitamin D deficient. Variables that significantly associated with lower 25(OH)D levels were obesity, current smoking, forced expiratory volume 1 and depression.CONCLUSION:Lower serum levels of vitamin D were associated with different variables that should be explored in several diseases before a conclusion of hypovitaminosis D is drawn.

Background: Doxorubicin is a potent chemotherapeutic drug The clinical usefulness of doxorubicin has been limited largely by the risk of cardiomyopathy and life-threatening heart failure. Cellular changes leading to this toxicity are suggested to be mediated through a druginduced increase oxidative stress. Grapeseed Proanthocyanidin [GSP] seeds possessing a potent antioxidant properties Aim: So, the present study was conducted to investigate possible protective effects of Grape seed and skin extract in doxorubicin-induced cardiotoxicity in rat model. Method: The male Wister rats (n = 34) were randomly selected and divided into three groups. The control group (n = 12) received distilled water [1ml/kg body weight] orally for 15 days, second group (n = 10) rats were injected intra peritoneal with a single dose of Doxorubicin (10mg/kg) in normal saline and in third group Doxorubicin (10mg/kg) was administered intraperitoneally and pretreatment with Grape seed and skin extract extract (200mg/kg body weight) before one hour DOX treatment by oral gavage for 15 consecutive days. Result: It was revealed by elevated serum cardiac biomarkers in comparison of control and associated with increasing levels of myocardial malondialdehyde [MDA] with simultaneously increase in the level of Superoxide dismutase. On daily oral administration of aqueous suspension of GSP seeds extract in the dose of (200 mg\kg ) for 15 days produced normalization in the serum levels of heart marker enzymes. Conclusion: The present study has demonstrated that Grape seed and its skin has got definite potency to ameliorate oxidative stress and Doxorubicin induced cardiotoxicity.

BACKGROUND:Major depressive disorder (MDD) exhibits numerous clinical and molecular features that are consistent with putative epigenetic misregulation. Despite growing interest in epigenetic studies of psychiatric diseases, the methodologies guiding such studies have not been well defined.METHODS:We performed DNA modification analysis in white blood cells from monozygotic twins discordant for MDD, in brain prefrontal cortex, and germline (sperm) samples from affected individuals and control subjects (total N = 304) using 8.1K CpG island microarrays and fine mapping. In addition to the traditional locus-by-locus comparisons, we explored the potential of new analytical approaches in epigenomic studies.RESULTS:In the microarray experiment, we detected a number of nominally significant DNA modification differences in MDD and validated selected targets using bisulfite pyrosequencing. Some MDD epigenetic changes, however, overlapped across brain, blood, and sperm more often than expected by chance. We also demonstrated that stratification for disease severity and age may increase the statistical power of epimutation detection. Finally, a series of new analytical approaches, such as DNA modification networks and machine-learning algorithms using binary and quantitative depression phenotypes, provided additional insights on the epigenetic contributions to MDD.CONCLUSIONS:Mapping epigenetic differences in MDD (and other psychiatric diseases) is a complex task. However, combining traditional and innovative analytical strategies may lead to identification of disease-specific etiopathogenic epimutations.

In thalassemia patients blood transfusion is the mainstay of therapy. Blood transfusion is associated with oxidative stress. Oxidative stress has its influence on plasma proteins. The present study was carried out to explore the level of sialic acid content and oxidative changes in serum proteins of thalassemia subjects on regular blood transfusion. Blood was collected from thalassemia patients receiving long duration of blood transfusion and from newly diagnosed cases of thalassemia who never receive blood transfusion and from carriers or traits of thalassemia as control. The extent of carbonylation and desialylation of serum protein was estimated by dinitrophenylhydrazine and thiobarbuturic acid method respectively. The effects of in vitro treatment of serum protein H2O2 on the above mentioned parameters were observed. Carbonylation of serum protein was significantly higher and sialic acid content of serum protein significantly lower in thalassemia subjects on regular blood transfusion. A significant negative correlation exists between carbonylation and sialic acid content of serum protein. Enhanced desialylation and carbonylation of serum protein by in vitro H2O2 treatment suggest that oxidative stress can cause desialylation of serum proteins.

Epigenetic studies of DNA and histone modifications represent a new and important activity in molecular investigations of human disease. Our previous epigenome-wide scan identified numerous DNA methylation differences in post-mortem brain samples from individuals affected with major psychosis. In this article, we present the results of fine mapping DNA methylation differences at the human leukocyte antigen (HLA) complex group 9 gene (HCG9) in bipolar disorder (BPD). Sodium bisulfite conversion coupled with pyrosequencing was used to interrogate 28 CpGs spanning ∼700 bp region of HCG9 in 1402 DNA samples from post-mortem brains, peripheral blood cells and germline (sperm) of bipolar disease patients and controls. The analysis of nearly 40 000 CpGs revealed complex relationships between DNA methylation and age, medication as well as DNA sequence variation (rs1128306). Two brain tissue cohorts exhibited lower DNA methylation in bipolar disease patients compared with controls at an extended HCG9 region (P=0.026). Logistic regression modeling of BPD as a function of rs1128306 genotype, age and DNA methylation uncovered an independent effect of DNA methylation in white blood cells (odds ratio (OR)=1.08, P=0.0077) and the overall sample (OR=1.24, P=0.0011). Receiver operating characteristic curve A prime statistics estimated a 69–72% probability of correct BPD prediction from a case vs control pool. Finally, sperm DNA demonstrated a significant association (P=0.018) with BPD at one of the regions demonstrating epigenetic changes in the post-mortem brain and peripheral blood samples. The consistent multi-tissue epigenetic differences at HCG9 argue for a causal association with BPD.

The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. Here we characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent data sets and with single-molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.

Bipolar disease (BPD) is a complex major psychiatric disorder that affects between 1% and 2% of the population and exhibits ?85% heritability. This has made BPD an appealing target for genetic studies yet, despite numerous attempts, the genetic basis of this disease remains elusive. Recently, it has come to light that epigenetic factors may also influence the development of BPD. These factors act via stable but reversible modifications of DNA and chromatin structure. In this chapter, we revisit the epidemiological, clinical, and molecular findings in BPD and reanalyze them from the perspective of inherited and acquired epigenetic misregulation. Epigenetic research has great potential to enhance our understanding of the molecular basis of BPD.

The progress of fracture union requires close monitoring. Whereas, clinical examination and radiographic studies assess the outcome, biochemical markers like serum alkaline phosphatase and urinary hydroxyproline reflect the actual status of bone resorption and bone formation over a short time frame. 36 patients of long bone fracture were randomly allocated for the study. When the patient reported to the Department of Orthopedics after fracture, serum and urinary samples were collected and X-ray of the affected part were taken. Subsequent samples were collected and X-ray taken just after management (either operative or conservative), after 3rd, 5th, 8th and 12th week, respectively after onset of fracture. According to the course of callus formation the patients were divided into two groups that progressed to proper union or malunion. The levels of serum alkaline phosphatase, urinary total and free hydroxyproline levels were measured and statistically analysed and compared. A statistically significant positive correlation between total urinary hydroxyproline excretion and serum alkaline phosphatase indicate progress towards satisfactory union. Thus, serial monitoring of biochemical markers of bone turnover can be used as an adjunct to clinical and radiological evidence of fracture healing.