Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol

Biofuels provide a potential route to avoiding the global political instability and environmental issues that arise from reliance on petroleum. Currently, most biofuel is in the form of ethanol generated from starch or sugar, but this can meet only a limited fraction of global fuel requirements. Conversion of cellulosic biomass, which is both abundant and renewable, is a promising alternative. However, the cellulases and pretreatment processes involved are very expensive. Genetically engineering plants to produce cellulases and hemicellulases, and to reduce the need for pretreatment processes through lignin modification, are promising paths to solving this problem, together with other strategies, such as increasing plant polysaccharide content and overall biomass.     

Sequence and Gene Expression Analyses of Plasmid pHPM8 from Helicobacter pylori Reveal the Presence of Two Operons with Putative Roles in Plasmid Replication and Antibiotic Activity

The DNA sequence of a 7.8-kb Helicobacter pylori plasmid, pHPM8, was determined. Six open reading frames (ORFs) were present. Ribonuclease protection studies showed that ORF1/ORF2 and ORF3/ORF4 genes are organized in operons possibly involved in plasmid replication and in production of a peptide with antibiotic activity, respectively. Finding areas of pHPM8 with a high level of identity to H. pylori chromosomal DNA supported the hypothesis that recombination occurs between plasmids and the chromosome of H. pylori.     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

MyD88 oligomer size functions as a physical threshold to trigger IL1R Myddosome signaling

A recurring feature of innate immune receptor signaling is the self-assembly of signaling proteins into oligomeric complexes. The Myddosome is an oligomeric complex that is required to transmit inflammatory signals from TLR/IL1Rs and consists of MyD88 and IRAK family kinases. However, the molecular basis for how Myddosome proteins self-assemble and regulate intracellular signaling remains poorly understood. Here, we developed a novel assay to analyze the spatiotemporal dynamics of IL1R and Myddosome signaling in live cells. We found that MyD88 oligomerization is inducible and initially reversible. Moreover, the formation of larger, stable oligomers consisting of more than four MyD88s triggers the sequential recruitment of IRAK4 and IRAK1. Notably, genetic knockout of IRAK4 enhanced MyD88 oligomerization, indicating that IRAK4 controls MyD88 oligomer size and growth. MyD88 oligomer size thus functions as a physical threshold to trigger downstream signaling. These results provide a mechanistic basis for how protein oligomerization might function in cell signaling pathways.     

Structure of the gene of tum- transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. We found that the gene conferring expression of tum- antigen P91A contains 12 exons, encoding a 60 kd protein lacking a typical N-terminal signal sequence. The sequence shows no significant similarity with sequences in current data bases. A mutation that causes expression of the antigen is located in exon 4; it is the only apparent difference between the normal and the antigenic alleles. A short synthetic peptide corresponding to a region of exon 4 located around this mutation makes P815 cells sensitive to lysis by anti-P91A cytolytic T cells. The mutation creates a strong aggretope enabling the peptide to bind the H-2 Ld molecule. Several secondary tumor cell variants that no longer express tum- antigen P91A were found to carry deletions in the gene.     

Creatine–facilitated protection of stress caused by disrupted circadian rhythm

Disturbances in natural Circadian rhythm are well-known stress factors, affecting a range of metabolic pathways in the living body including the brain. Hence, discovery of natural compounds that could help to prevent and cure of adverse changes is very important. One of the recently discussed substances is creatine, that is believed to have anti-stressor properties. Recent paper describes the impact of intraperitoneally injected creatine (140 mg/kg) into rats with a disturbed natural circadian rhythm for an extended period of time (30 days). Markedly, creatine-treated animals show positive changes in open-field behavioral parameters, and an increase in certain antioxidant enzymes’ (SOD, catalase) activity in the hippocampus, whereas the concentration of nitric oxide, H₂O₂, and Ca²⁺ are approximated to the control value. Similar findings were also observed in case of Na⁺/K⁺- and Ca²⁺-ATPases. To sum up, the recent findings allow the conclusion that oxidative stress induced by long-term disturbances in natural circadian rhythm is accompanied and likely provoked by an increase in Ca²⁺-cytotoxicity, which is supposedly normalized by the creatine’s indirect action on the NMDA receptor. Therefore, impact on energy mediating pathways has a positive effect on stabilization of antioxidant and various metabolic systems and protecting hippocampal cells from stress.     

Streamlined production of genetically modified T cells with activation,transduction and expansion in closed-system G-Rex bioreactors

BackgroundGas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention.MethodsPeripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes.ResultsThe simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically.DiscussionThis study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.     

Liquid chromatographic and spectrofluorimetric assays of empagliflozin: Applied to degradation kinetic study and content uniformity testing

Stability‐indicating high‐performance liquid chromatography (HPLC) and spectrofluorimetric methods were developed for determination of empagliflozin (EGF). EGF was subjected to oxidation, wet heat, photo‐degradation, acid hydrolysis and alkali hydrolysis. The alkaline degradation pathway was subjected to a kinetics study as the major product obtained after stress conditions. Arrhenius plots were constructed and the activation energies of the degradation process were calculated. HPLC was used for the kinetic study as it enabled simultaneous determination of EGF and the degradation product while the spectrofluorimetric assay was applied to content uniformity testing due to its higher sensitivity and lower limit of detection (LOD). Isocratic chromatographic elution was attained for HPLC on a Intersil® C₁₈ column (150 mm × 4 mm, 5 μm), using a mobile phase of acetonitrile–potassium dihydrogen phosphate buffer pH 4, (50:50, v/v) at a flow rate of 1 ml/min with ultraviolet (UV) detection at 225 nm. The relative fluorescence intensity was recorded by spectrofluorimeter applying synchronous mode using ?λ = 70 nm at 297.6 nm. Linearity ranges were found to be 5–50 μg/ml and 50–1000 ng/ml for HPLC and spectrofluorimetric methods, respectively.     

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.