Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production

Overcoming the recalcitrance in lignocellulosic biomass for efficient hydrolysis of the polysaccharides cellulose and hemicellulose to fermentable sugars is a research priority for the transition from a fossilfuel-based economy to a renewable carbohydrate economy. Methylglucuronoxylans (MeGXn) are the major components of hemicellulose in woody biofuel crops. Here, we describe efficient production of the GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants and demonstrate exceptional stability and catalytic activities of the in planta produced enzyme. Fully expanded leaves from homotransplastomic plants contained enzymatically active Xyl10B at a level of 11-15% of their total soluble protein. Transplastomic plants and their seed progeny were morphologically indistinguishable from non-transgenic plants. Catalytic activity of in planta produced Xyl10B was detected with poplar, sweetgum and birchwood xylan substrates following incubation between 40 and 90 °C and was also stable in dry and stored leaves. Optimal yields of Xyl10B were obtained from dry leaves if crude protein extraction was performed at 85 °C. The transplastomic plant derived Xyl10B showed exceptional catalytic activity and enabled the complete hydrolysis of MeGXn to fermentable sugars with the help of a single accessory enzyme (α-glucuronidase) as revealed by the sugar release assay. Even without this accessory enzyme, the majority of MeGXn was hydrolyzed by the transplastomic plant-derived Xyl10B to fermentable xylose and xylobiose.     

Posture shifting after spinal cord injury using functional neuromuscular stimulation--a computer simulation study

The ability for individuals with spinal cord injury (SCI) to affect changes in standing posture with functional neuromuscular stimulation (FNS) was explored using an anatomically inspired musculoskeletal model of the trunk, pelvis and lower extremities (LE). The model tracked trajectories for anteriorly and laterally shifting movements away from erect stance. Forces were applied to both shoulders to represent upper extremity (UE) interaction with an assistive device (e.g., a walker). The muscle excitations required to execute shifting maneuvers with UE forces <10% body-weight (BW) were determined via dynamic optimization. Nine muscle sets were examined to maximize control of shifting posture. Inclusion of the Psoas and External Obliques bilaterally resulted in the least relative UE effort (0.119, mean UE effort = 45.3N ≡ 5.4% BW) for anterior shifting. For lateral shifting, the set including the Psoas and Latissimus Dorsi bilaterally yielded the best performance (0.025, mean UE effort = 27.8 N ≡ 3.3% BW). However, adding the Psoas alone bilaterally competed favorably in overall best performance across both maneuvers. This study suggests suitable activation to specific muscles of the trunk and LE can enable individuals with SCI to alter their standing postures with minimal upper-body effort and subsequently increase reach and standing work volume.     

Role of 677C→T polymorphism a single substitution in methylenetetrahydrofolate reductase (MTHFR) gene in North Indian infertile men

Failure or severe difficulty in conceiving a child is surprisingly common, worldwide problem. Half of these cases are due to male factors with defects in sperm (1 in 15 men) being the single most common cause. Also about 60–75 % of male infertility cases are idiopathic, since the molecular mechanisms underlying the defects remain unknown. DNA methylation is crucial for spermatogenesis and high methylenetetrahydrofolate reductase (MTHFR) activity in adult testis than other organs in mouse, signifies its critical role in spermatogenesis. According to recent findings there is a correlation of epigenetic regulation of several imprinted genes with disturbed spermatogenesis and fertility. Consequently any change in the MTHFR gene sequence can modify the spermatogenesis including transmission of infertility to the carriers. The aim of the study is to analyze the distribution of the single nucleotide polymorphism C677T in the MTHFR gene in 637 North Indian infertile patients and 364 fertile North Indian men as controls by using PCR–RFLP technique and Chi Square test for statistical analysis. The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total infertile men were 70.17, 24.17, 5.65 % in infertile men and 75.27, 21.7, 2.74 % in controls, respectively. The average frequency of the MTHFR 677T allele was 17.73 % in infertile men as compared to 13.59 % in controls. The statistical difference was significant. Disease risk was found 2.27-folds increased in patients who were carrying T allele. We found an association of C677T polymorphism with male infertility and that it may be a genetic risk factor for male infertility in North Indian population.     

Determination of plasma microRNA for early detection of gastric cancer

Gastric cancer is the fourth most prevalent malignancy worldwide and remains the second most common cause of cancer-related death globally. Understanding the molecular structure of gastric carcinogenesis might identify new diagnostic and therapeutic strategies for this disease. Thus, early detection of gastric cancer is a key measure to reduce the mortality and improve the prognosis of gastric cancer. There have recently been several reports that microRNAs (miRNAs) circulate in highly stable, cell-free forms in blood. Because serum and plasma miRNAs are relatively easy to access, circulating miRNAs also have great potential to serve as non-invasive biomarkers. Although a number of miRNAs associated with gastric cancer have been identified, the underlying mechanism of these miRNAs in tumorigenesis and tumor progression remains to be investigated. The purpose of this study is to identify the potential of serum miRNAs as biomarkers for early detection of gastric cancer patients. RNA was isolated using the High Pure miRNA Isolation Kit (Roche) following the manufacturer’s protocol. cDNA and preamplification protocols were obtained from the isolated plasma miRNAs. The BioMark? 96.96 Dynamic Array (Fluidigm Corporation) for real-time qPCR was used to simultaneously quantite the expression of 740 miRNAs. All statistical analyses were performed using the Biogazelle’s qbase PLUS 2.0 software. In this study, among 740 miRNAs that we analyzed only miR-195-5p was significantly (p < 0.05, fold changes = 13, 3) down-regulated in gastric cancer patients compared with control. We demonstrated that miR-195-5p is a novel tumor suppressor miRNA and may contribute to gastric carcinogenesis. The miRNA expression profile described in this study should contribute to future studies on the role of miRNAs in gastric cancer.     

Characterization of oxidative stress in blood from diabetic vs. hypercholesterolaemic patients,using a novel synthesized marker

In the present study, we extend our novel concept of designing and using exogenous markers for the characterization of oxidative stress (OS) and OS-associated diseases. The aim was to use such a synthetic compound as a tool for studying OS in blood from diabetic and hypercholesterolaemic (Hc) patients. The marker used N-linoleoyl tyrosine (LT) was constructed from tyrosine and linoleic acid (LA); both components are known to be easily oxidized upon exposure to different types of reactive oxygen/nitrogen species (ROS/RNS), and to generate specific oxidized products, depending on the type of oxidants present in vivo. Using the LT probe, we showed that the ratios of oxidized LT to total LT (Ox-LT/LT) is significantly higher in blood samples obtained from diabetic patients, than in Hc patients or healthy control subjects. LC/MS analysis revealed that blood from diabetic patients oxidizes the marker with predominant formation of Ox-LT hydroperoxide (LT-OOH) and epoxide (epoxy-LT), where the LA moiety is oxidized to hydroperoxide and to epoxide, respectively. Analysis of oxysterol levels in these samples (GC/MS) revealed that the blood of both diabetic and Hc patients contained significantly more oxysterols than blood of control subjects. Consumption of pomegranate juice by diabetic patients for 3 months suppressed their blood capacity to oxidize the LT and similarly also reduced their blood oxysterol/total cholesterol ratio by 93%. The use of an exogenous marker to characterize OS in blood samples yields important information on the extent of OS, and can provide a fingerprint for the early identification of different pathological conditions associated with OS.     

Single nucleotide polymorphisms in <Emphasis Type="Italic">TaER</Emphasis> genes and their association with carbon isotope discrimination in wheat genotypes under drought

Candidate gene association studies implicate the detection of contributing single nucleotide polymorphism (SNP) for the target traits and have been recommended as a promising technique to anatomize the complex characters in plants. The ERECTA gene in plants controls different physiological functions. In this study, we identified SNPs in 1.1 kb partial sequences of TaER-1 and TaER-2 of wheat (Triticum aestivum L.). Thirty-nine SNPs were identified in the coding regions of TaER-1 gene in 33 wheat genotypes, of which 20 SNPs caused non-synonymous mutations while 19 SNPs produced synonymous mutations; 31 SNPs were located in the coding regions of TaER-2 gene in 26 genotypes, of which 18 SNPs caused non-synonymous mutations and 13 SNPs caused synonymous mutations. In addition, 32 SNPs in TaER-1 and 9 SNPs in TaER-2 were also identified in the non-coding regions. Moreover, the significant genetic associations of SNPs of TaER-1 and TaER-2 genes with carbon isotope discrimination, stomatal conductance, photosynthetic rate, transpiration rate, intrinsic water use efficiency (iWUE), leaf length, leaf width, stomatal density, epidermal cell density, and stomatal index were noted in wheat genotypes. This study confirms the importance of TaER-1 and TaER-2 genes which could improve iWUE of wheat by regulating leaf gas exchange and leaf structural traits. These identified SNPs may play a critical role in molecular breeding by means of marker-assisted selection.     

Cloning, expression, and purification of recombinant protein from a single synthetic multivalent construct of Mycobacterium tuberculosis

Tuberculosis remains a major infectious disease with over 8 million new cases and 2 million deaths annually. Therefore, a vaccine more potent than BCG is desperately needed. In this regard, an approximately 800 bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and four immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6 x His-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine.