Ubiquitin Hydrolase UCH-L1 Destabilizes mTOR Complex 1 by Antagonizing DDB1-CUL4-Mediated Ubiquitination of Raptor

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates processes including mRNA translation, proliferation, and survival. By assembling with different cofactors, mTOR forms two complexes with distinct biological functions. Raptor-bound mTOR (mTORC1) governs cap-dependent mRNA translation, whereas mTOR, rictor, and mSin1 (mTORC2) activate the survival and proliferative kinase Akt. How the balance between the competing needs for mTORC1 and -2 is controlled in normal cells and deregulated in disease is poorly understood. Here, we show that the ubiquitin hydrolase UCH-L1 regulates the balance of mTOR signaling by disrupting mTORC1. We find that UCH-L1 impairs mTORC1 activity toward S6 kinase and 4EBP1 while increasing mTORC2 activity toward Akt. These effects are directly attributable to a dramatic rearrangement in mTOR complex assembly. UCH-L1 disrupts a complex between the DDB1-CUL4 ubiquitin ligase complex and raptor and counteracts DDB1-CUL4-mediated raptor ubiquitination. These events lead to mTORC1 dissolution and a secondary increase in mTORC2. Experiments in Uchl1-deficient and transgenic mice suggest that the balance between these pathways is important for preventing neurodegeneration and the development of malignancy. These data establish UCH-L1 as a key regulator of the dichotomy between mTORC1 and mTORC2 signaling.     

Physical and chemical mutagens improved Sporotrichum thermophile,strain ST20 for enhanced Phytase activity

ObjectivePhosphorous is an essential micronutrient of plants and involved in critical biological functions. In nature, phosphorous is mostly present in immobilized inorganic mineral and in the fixed organic form including phytic acid and phosphoesteric compounds. However, the bioavailability of bound phosphorous could be enhanced by the use of phosphate solubilizing microorganisms such as bacteria and fungi. The phytases are widespread in an environment and have been isolated from different sources comprising bacteria and fungi.MethodologyIn current studies, we show the successful use of gamma rays and EMS (Ethyl Methane Sulphonate) mutagenesis for enhanced activity of phytases in a fungal strain Sporotrichum thermophile.ResultsWe report an improved strain ST2 that could produce a clear halo zone around the colony, up to 24 mm. The maximum enzymatic activity was found of 382 U/mL on pH 5.5. However, the phytase activity was improved to 387 U/ml at 45 °C. We also report that the mutants produced through EMS showed the greater potential for phytase production.ConclusionThe current study highlights the potential of EMS mutagenesis for strain improvement over physical mutagens.     

The genetic association study of TP53 polymorphisms in Saudi obese patients

Obesity is a multifactorial metabolic disorder characterized by low grade chronic inflammation. Rare and novel mutations in genes which are vital in several key pathways have been reported to alter the energy expenditure which regulates body weight. The TP53 or p53 gene plays a prominent role in regulating various metabolic activities such as glycolysis, lipolysis, and glycogen synthesis. Recent genome-wide association studies reported that tumor suppressor gene p53 variants play a critical role in the predisposition of type 2 diabetes and obesity. Till date, no reports are available from the Arabian population; hence the present study was intended to assess the association between p53 variants with risk of obesity development in the Saudi population. We have selected three p53 polymorphisms, rs1642785 (C > G), and rs9894946 (A > G), and rs1042522 (Pro72Arg; C > G) and assessed their association with obesity risk in the Saudi population. Phenotypic and biochemical parameters were also evaluated to check their association with p53 genotypes and obesity. Genotyping was carried out on 136 obese and 122 normal samples. We observed that there is significantly increased prevalence p52 Pro72Arg (rs1042522) polymorphism in obese persons when compared to controls at GG genotype in overall comparison (OR: 2.169, 95% CI: 1.086-4.334, p = 0.02716). Male obese subjects showed three-fold higher risk at GG genotype (OR: 3.275, 95% CI: 1.230-8.716, p = 0.01560) and two-fold risk at G allele (OR: 1.827, 95% CI: 1.128-2.958, p = 0.01388) of p53 variant Pro72Arg respectively. This variant has also shown significant influence on cholesterol, LDL level, and random insulin levels in obese subjects (p ≤ 0.05). In conclusion, p53 Pro72Arg variant is highly prevalent among obese individuals and may act as a genetic modifier for obesity development among Saudis.     

A comparative study of bacterial diversity based on culturable and culture-independent techniques in the rhizosphere of maize (Zea mays L.)

ObjectiveMaize is an important crop for fodder, food and feed industry. The present study explores the plant-microbe interactions as alternative eco-friendly sustainable strategies to enhance the crop yield.MethodologyBacterial diversity was studied in the rhizosphere of maize by culture-dependent and culture-independent techniques by soil sampling, extraction of DNA, amplification of gene of interest, cloning of desired fragment and library construction.ResultsCulturable bacteria were identified as Achromobacter, Agrobacterium, Azospirillum, Bacillus, Brevibacillus, Bosea, Enterobacter, Microbacterium, Pseudomonas, Rhodococcus, Stenotrophomonas and Xanthomonas genera. For culture-independent approach, clone library of 16S ribosomal RNA gene was assembled and 100 randomly selected clones were sequenced. Majority of the sequences were related to Firmicutes (17%), Acidobacteria (16%), Actinobacteria (17%), Alpha-Proteobacteria (7%), Delta-proteobacteria (4.2%) and Gemmatimonadetes (4.2%) However, some of the sequences (30%) were novel that showed no homologies to phyla of cultured bacteria in the database. Diversity of diazotrophic bacteria in the rhizosphere investigated by analysis of PCR-amplified nifH gene sequence that revealed abundance of sequences belonging to genera Azoarcus (25%), Aeromonas (10%), Pseudomonas (10%). The diazotrophic genera Azotobacter, Agrobacterium and Zoogloea related nifH sequences were also detected but no sequence related to Azospirillum was found showing biasness of the growth medium rather than relative abundance of diazotrophs in the rhizosphere.ConclusionThe study provides a foundation for future research on focussed isolation of the Azoarcus and other diazotrophs found in higher abundance in the rhizosphere.     

Phytoaccumulation of Heavy Metals in Natural Plants Thriving on Wastewater Effluent at Hattar Industrial Estate,Pakistan

The objective of this research was to compare the potential of native plants for the phytoaccumulation of heavy metals (HM). Thirteen predominant plant species (including trees, bushes and grasses) namely Ricinus communis, Ipomoea carnea, Cannabis sativa, Parthenium hysterophorus, Acacia nilotica, Dalbergia sissoo, Acacia modesta, Solanum nigrum, Xanthium stromarium, Chenopodium album, Cynodon dactylon, Eleusine indica, and Dactyloctenium aegyptium were collected from the wastewater originated from Hattar industrial estate of Pakistan, Plants shoots and roots were analyzed for heavy metals / metalloid: Pb, Cr, Cd, Zn, Fe, Ni, and As. Among plant species, the accumulation potential for HM varied depending on the type of element. Regardless of the plant species, HM concentrations varied in the order of Fe > Zn > Cr > Pb > Ni > Cd > As. Tree species of R. communis, A. nilotica, A. modesta, and D. sissoo exhibited an enhanced concentrations of metals. Accumulation pattern of Fe, Pb, Cd, and As in plants could be related to the HM composition of soil and wastewater. Most of the species exhibited higher HM composition in the root as compared to shoot. The species that found with greater ability to absorb HM in the root, got higher HM concentrations in its shoot. Shoot tissue concentrations of HM were attained by the species as D. sissoo > A. modesta > A. nilotica > R. communis > I. carnea > C. album > E. indica > P. hysterophorus > S. nigrum > C. sativa > D. aegyptium > X. strumarium > C. dactylon. Based on results, tree plants were noticed as higher accumulators of HM in polluted soils.     

Formulation,Characterisation and Stabilisation of Buccal Films for Paediatric Drug Delivery of Omeprazole

This study aimed to develop films for potential delivery of omeprazole (OME) via the buccal mucosa of paediatric patients. Films were prepared using hydroxypropylmethylcellulose (HPMC), methylcellulose (MC), sodium alginate (SA), carrageenan (CA) and metolose (MET) with polyethylene glycol (PEG 400) as plasticiser, OME (model drug) and L-arg (stabiliser). Gels (1% w/w) were prepared at 40°C using water and ethanol with PEG 400 (0–1% w/w) and dried in an oven (40°C). Optimised formulations containing OME and L-arg (1:1, 1:2 and 1:3) were prepared to investigate the stabilisation of the drug. Tensile properties (Texture analysis, TA), physical form (differential scanning calorimetry, DSC; X-ray diffraction, XRD; thermogravimetric analysis, TGA) and surface topography (scanning electron microscopy, SEM) were investigated. Based on the TA results, SA and MET films were chosen for OME loading and stabilisation studies as they showed a good balance between flexibility and toughness. Plasticised MET films were uniform and smooth whilst unplasticised films demonstrated rough lumpy surfaces. SA films prepared from aqueous gels showed some lumps on the surface, whereas SA films prepared from ethanolic gels were smooth and uniform. Drug-loaded gels showed that OME was unstable and therefore required addition of L-arg. The DSC and XRD suggested molecular dispersion of drug within the polymeric matrix. Plasticised (0.5% w/w PEG 400) MET films prepared from ethanolic (20% v/v) gels and containing OME: L-arg 1:2 showed the most ideal characteristics (transparency, ease of peeling and flexibility) and was selected for further investigation.KEY WORDS: buccal drug delivery, omeprazole, oral films, paediatric, plasticiser     

Cultivation-Based and Molecular Assessment of Bacterial Diversity in the Rhizosheath of Wheat under Different Crop Rotations

A field study was conducted to compare the formationand bacterial communities of rhizosheaths of wheat grown under wheat-cotton and wheat-rice rotation and to study the effects of bacterial inoculation on plant growth. Inoculation of Azospirillum sp. WS-1 and Bacillus sp. T-34 to wheat plants increased root length, root and shoot dry weight and dry weight of rhizosheathsoil when compared to non-inoculated control plants, and under both crop rotations. Comparing both crop rotations, root length, root and shoot dry weight and dry weight of soil attached with roots were higher under wheat-cotton rotation. Organic acids (citric acid, malic acid, acetic acid and oxalic acid) were detected in rhizosheaths from both rotations, with malic acid being most abundant with 24.8±2 and 21.3±1.5 μg g^-1 dry soil in wheat-cotton and wheat-rice rotation, respectively. Two sugars (sucrose, glucose) were detected in wheat rhizosheath under both rotations, with highest concentrations of sucrose (4.08±0.5 μg g^-1and 7.36±1.0 μg g^-1) and glucose (3.12±0.5 μg g^-1 and 3.01± μg g^-1) being detected in rhizosheaths of non-inoculated control plants under both rotations. Diversity of rhizosheath-associated bacteria was evaluated by cultivation, as well as by 454-pyrosequencing of PCR-tagged 16S rRNA gene amplicons. A total of 14 and 12 bacterial isolates predominantly belonging to the genera Arthrobacter, Azospirillum, Bacillus, Enterobacter and Pseudomonaswere obtained from the rhizosheath of wheat grown under wheat-cotton and wheat-rice rotation, respectively. Analysis of pyrosequencing data revealed Proteobacteria, Bacteriodetes and Verrucomicrobia as the most abundant phyla in wheat-rice rotation, whereas Actinobacteria, Firmicutes, Chloroflexi, Acidobacteria, Planctomycetes and Cyanobacteria were predominant in wheat-cotton rotation. From a total of 46,971 sequences, 10.9% showed ≥97% similarity with 16S rRNA genes of 32 genera previously shown to include isolates with plant growth promoting activity (nitrogen fixation, phosphate-solubilization, IAA production). Among these, the most predominant genera were Arthrobacter, Azoarcus, Azospirillum, Bacillus, Cyanobacterium, Paenibacillus, Pseudomonas and Rhizobium.     

Influence of Environmental Factors,Cultural Practices,and Herbicide Application on Seed Germination and Emergence Ecology of Ischaemum rugosum Salisb

Ischaemum rugosum Salisb. (Saramolla grass) is a noxious weed of rice that is difficult to control by chemical or mechanical means once established. A study was conducted to determine the effect of light, temperature, salt, drought, flooding, rice residue mulch, burial depth, and pre-emergence herbicides on seed germination and emergence of I. rugosum. Germination was stimulated by light and inhibited under complete darkness. Optimum temperature for germination was 30/20°C (97.5% germination). Germination reduced from 31 to 3.5% when the osmotic potential of the growing medium decreased from -0.1 to -0.6 MPa and no germination occurred at -0.8 MPa. Germination was 18 and 0.5% at 50 and 100 mM NaCl concentrations, respectively, but was completely inhibited at 150 mM or higher. Residue application at 1–6 t ha^-1 reduced weed emergence by 35–88% and shoot biomass by 55–95%. The efficacy of pre-emergence herbicides increased with increasing application rates and decreased with increasing rice residue mulching. The efficacy of herbicides was in the order of oxadiazon> pendimethalin> pretilachlor. At 6 t ha^-1, all herbicides, regardless of rates, did not differ from the control treatment. I. rugosum seeds buried at 2 cm or deeper did not emerge; however, they emerged by 4.5 and 0.5% at 0.5 and 1 cm depths, respectively, compared to the 39% germination for soil surface seeding. Flooding at 4 DAS or earlier reduced seedling emergence and shoot biomass while flooding at 8 DAS reduced only seedling emergence. The depth and timing of flooding independently reduced root biomass. Flooding at 4 and 6 cm depths reduced the root biomass. Relative to flooding on the day of sowing, flooding at 8 DAS increased root biomass by 89%. Similarly, flooding on the day of sowing and at 2 DAS reduced the root–shoot biomass ratio. Under the no-flood treatment, increasing rates of pretilachlor from 0.075 to 0.3 kg ai ha^-1 reduced weed emergence by 61–79%. At the flooding depth of 2–4 cm, pretilachlor reduced weed emergence and shoot and root biomass, but the differences across rates were non-significant. Information generated in this study will be helpful in developing integrated weed management strategies for managing this weed.     

Determination of potential role of antioxidative status and circulating biochemical markers in the pathogenesis of ethambutol induced toxic optic neuropathy among diabetic and non-diabetic patients

The present study was designed to explore the antioxidative status and circulating biochemical markers having a potential role in the pathogenesis of ethambutol (EMB) induced toxic optic neuropathy (TON) among diabetic and non-diabetic patients.Fifty patients under complete therapy of EMB for tuberculosis were included in the present study. Inclusion criteria for patients were to receive EMB everyday during treatment, a dose of 25 mg/kg for initial 2 months and 15 mg/kg during the rest of therapy period. We conducted color vision and visual acuity test for all patients.Fifteen out of fifty EMB induced TON patients, were found to be diabetic. Color vision and visual acuity test results were evaluated for diabetic and non-diabetic as well as twenty age matched controls. The results demonstrated a significant pattern of circulating biochemical markers between the studied groups. Data regarding hematological (RBC, p value = 0.02; Hemoglobin, p value = 0.02), hepatic (total bilirubin, p value = 0.01), renal (urea, p value = 0.03; creatinine, p value = 0.007), lipid (total cholesterol, p value = 0.01; total triglycerides, p value = 0.03) and antioxidative (superoxide dismutase, p value = 0.005; glutathione, p value = 0.02; catalase, p value = 0.02) profile showed a highly significant difference among the studied groups specially patients with diabetes. Malondialdehyde (MDA) level had gone significantly up in diabetic TON patients (p value = 0.02), in comparison to other antioxidants and vitamins (Vit). Vit-A, E, B₁, B₁₂ and Zinc seem to be playing a major role in the pathogenesis of TON, specially Vit-E and B₁ surpassed all the antioxidants as having highly significant inverse relationships with MDA (MDA vs Vit-E, r = −0.676^** and MDA vs Vit-B₁, r = −0.724^** respectively).We conclude that during the ethambutol therapy the decreased levels of Vit-E and Vit-B₁ possibly play a role in the development of TON and may be used as therapeutic agents to lessen the deleterious effects of ethambutol.