The RNA Helicase eIF4A Is Required for Sapovirus Translation

The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5′ untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5′ UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.     

Integrated algal engineering for bioenergy generation,effluent remediation,and production of high-value bioactive compounds

Increased demand for energy worldwide has resulted in increasing interest in alternative renewable sources of biofuels. Demand for improved systems of bioenergy generation, environmental remediation, and coproduction of high value bioactive compounds has led to the potential use of algae in biomass utilization. In Malaysia, palm oil industries generate high amount of solid wastes. Palm Oil Mill Effluent (POME) is estimated to be three times of the amount of crude palm oil produced. POME is a heavily polluting wastewater due to its high chemical oxygen demand (COD), high biochemical oxygen demand (BOD), and high contents of minerals such as nitrogen and phosphorus that can cause severe pollution to the environment and water resources. A combination of wastewater treatment and renewable bioenergy co-generation with recovery of high-value biochemicals would benefit the palm oil industry.     

The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.     

Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method

Background and aims Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals (^•OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to ‘fingerprint’ ^•OH-attacked polysaccharides.Methods We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during ^•OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography.Key Results Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA–pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently.Conclusions GalA–pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents (^•OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by ^•OH. The evidence shows that ^•OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). ^•OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.     

Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9

Targeted genome editing of nonrodent mammalian species has provided the potential for highly accurate interventions into gene function in humans and the generation of useful animal models of human diseases. Here we show successful clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas)-mediated gene targeting via circular plasmid injection in rabbits. The rabbit tyrosinase gene (TYR) was effectively disrupted, and we confirmed germline transmission by pronuclear injection of a circular plasmid expressing humanized Cas9 (hCas9) and single-guide RNA. Direct injection into pronuclear stage zygotes was possible following an in vitro validation assay. Neither off-target mutagenesis nor hCas9 transgenesis was detected in any of the genetically targeted pups and embryos examined. Gene targeting with this rapid and simplified strategy will help accelerate the development of translational research using other nonrodent mammalian species.     

Purification and characterization of κ-carrageenase from a novel γ-proteobacterium,<Emphasis Type="Italic">Pseudomonas elongata</Emphasis> (MTCC 5261) syn.<Emphasis Type="Italic">Microbulbifer elongatus</Emphasis> comb. Nov.

The phenotypic and carrageenolytic features of a novel halo tolerant marine bacterium, isolated from decayed red algal samples collected along the west coast of India were studied. This gram-negative strain was identified asPseudomonas elongala (MTCC 5261) syn.Microbulbifer elongalus comb. nov according to its morphological, physiological and molecular characterization. The extracellular κ-carrageenase was purified 106.54-fold by a combination of ammonium sulfate precipitation (40∼60%) and successive gel filtration chromatography. The purified protein fraction yielded significantly high activity of 426.19 units/mg protein and migrated as a single band on a sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of ∼128 kDa. For κ-carrageenase activity, optimum temperature was 40°C whereas two pH optimai.e. 5.6 and 7.7 were observed. For κ-carrageenan, the enzyme gave aK _m value of 6.66 mg/mL and aV _max value of 4 μmol/min/mg when the reaction was carried out at 40°C and pH 5.6. Isolated κ-carrageenase could successfully generate protoplasts ofKappaphycus alvarezii. This is the first report on the production of κ-carrageenase by this bacterium isolated from west coast of India. Molecular mass and various characteristics showed that the carrageenase fromP. elongata was much different from those previously reported.