Reassortant DS‐1‐like G1P[4] Rotavirus A strains generated from co‐circulating strains in Vietnam, 2012/2013

One major mechanism by which Rotavirus A (RVA) evolves is genetic reassortment between strains with different genotype constellations. However, the parental strains of the reassortants generated have seldom been identified. Here, the whole genome of two suspected reassortants, RVA/Human‐wt/VNM/SP127/2013/G1P[4] and RVA/Human‐wt/VNM/SP193/2013/G1P[4], with short RNA electropherotypes were examined by Illumina MiSeq sequencing and their ancestral phylogenies reconstructed. Their genotype constellation, G1‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2, indicated that they were G1 VP7 mono‐reassortants possessing DS‐1‐like genetic backbones. The two strains were ≧99.7% identical across the genome. While their VP7 genes were ≧99.7 identical to that of a Wa‐like strain RVA/Human‐wt/VNM/SP110/2012/G1P[8] which co‐circulated during the 2012/2013 season, 10 genes were ≧99.8% identical to that of the DS‐1‐like strains RVA/Human‐wt/VNM/SP015/2012/G2P[4] (and SP108) that co‐circulated during the season. The identities were consistent with the phylogenetic relationships observed between the genes of the reassortants and those of the afore‐mentioned strains. Consequently, the G1P[4] strains appear to have been generated by genetic reassortment between SP110‐like and SP015‐like strains. In conclusion, this study provides robust molecular evidence for the first time that G1P[4] strains detected in Hanoi Vietnam were generated by inter‐genogroup reassortment between co‐circulating G1P[8] and G2P[4] strains within the same place and season.

     

Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs

VP7 of group A rotavirus(RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector,three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains.Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6 F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.     

A Nck‐associated protein 1‐like protein affects drought sensitivity by its involvement in leaf epidermal development and stomatal closure in rice

Water deficit is a major environmental threat affecting crop yields worldwide. In this study, a drought stress‐sensitive mutant drought sensitive 8 (ds8) was identified in rice (Oryza sativa L.). The DS8 gene was cloned using a map‐based approach. Further analysis revealed that DS8 encoded a Nck‐associated protein 1 (NAP1)‐like protein, a component of the SCAR/WAVE complex, which played a vital role in actin filament nucleation activity. The mutant exhibited changes in leaf cuticle development. Functional analysis revealed that the mutation of DS8 increased stomatal density and impaired stomatal closure activity. The distorted actin filaments in the mutant led to a defect in abscisic acid (ABA)‐mediated stomatal closure and increased ABA accumulation. All these resulted in excessive water loss in ds8 leaves. Notably, antisense transgenic lines also exhibited increased drought sensitivity, along with impaired stomatal closure and elevated ABA levels. These findings suggest that DS8 affects drought sensitivity by influencing actin filament activity.     

Optimization of norovirus virus‐like particle production in Pichia pastoris using a real‐time near‐infrared bioprocess monitor

The production of norovirus virus‐like particles (NoV VLPs) displaying NY‐ESO‐1 cancer testis antigen in Pichia pastoris BG11 Mut⁺ has been enhanced through feed‐strategy optimization using a near‐infrared bioprocess monitor (RTBio^® Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real‐time. The production of NoV VLPs displaying NY‐ESO‐1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two‐fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP‐NY‐ESO‐1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L^?1 during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1‐NY‐ESO‐1 yield of 0.85 g L^?1. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518–526, 2016     

Distribution and Expression of Elicitin‐like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin‐like proteins (POD‐1, POD‐2, POS‐1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D‐type isolate containing POD‐1 and POD‐2, and the S‐type isolate containing POS‐1. We identified the genes encoding these elicitin‐like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D‐type isolate MMR2 was constructed and genomic regions containing the elicitin‐like protein genes were identified. Southern blot analyses with probes derived from pod‐1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D‐type isolates carried pod‐1, pod‐2 and two oligandrin genes, termed oli‐d1 and oli‐d2, while the S‐type isolates carried pos‐1 and one oligandrin gene termed oli‐s1. Phylogenetic analysis of POD‐1, POD‐2, POS‐1, Oli‐D1, Oli‐D2 and Oli‐S1 with the previously defined elicitins and elicitin‐like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT‐PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D‐type and the S‐type isolates, physical maps of the flanking regions around pod‐1, pod‐2, pos‐1 and the oligandrin genes were constructed. The maps suggest that the D‐type isolates may be derived from the S‐type isolates due to gene duplication and deletion events.     

Identification of quantitative trait loci and candidate genes for an anxiolytic‐like response to ethanol in BXD recombinant inbred strains

Genetic differences in acute behavioral responses to ethanol contribute to the susceptibility to alcohol use disorder and the reduction of anxiety is a commonly reported motive underlying ethanol consumption among alcoholics. Therefore, we studied the genetic variance in anxiolytic‐like responses to ethanol across the BXD recombinant inbred (RI) mouse panel using the light–dark transition model of anxiety. Strain‐mean genetic mapping and a mixed‐model quantitative trait loci (QTL) analysis replicated several previously published QTL for locomotor activity and identified several novel anxiety‐related loci. Significant loci included a chromosome 11 saline anxiety‐like QTL (Salanq1) and a chromosome 12 locus (Etanq1) influencing the anxiolytic‐like response to ethanol. Etanq1 was successfully validated by studies with BXD advanced intercross strains and fine‐mapped to a region comprising less than 3.5 Mb. Through integration of genome‐wide mRNA expression profiles of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens and ventral midbrain) across the BXD RI panel, we identified high priority candidate genes within Etanq1, the strongest of which was Ninein (Nin), a Gsk3β‐interacting protein that is highly expressed in the brain.     

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

Reassortment of Human and Animal Rotavirus Gene Segments in Emerging DS-1-Like G1P[8] Rotavirus Strains

The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.     

Effect of the DnaK chaperone on the conformational quality of JCV VP1 virus‐like particles produced in Escherichia coli

Protein nanoparticles such as virus‐like particles (VLPs) can be obtained by recombinant protein production of viral capsid proteins and spontaneous self‐assembling in cell factories. Contrarily to infective viral particles, VLPs lack infective viral genome while retaining important viral properties like cellular tropism and intracellular delivery of internalized molecules. These properties make VLPs promising and fully biocompatible nanovehicles for drug delivery. VLPs of human JC virus (hJCV) VP1 capsid protein produced in Escherichia coli elicit variable hemagglutination properties when incubated at different NaCl concentrations and pH conditions, being optimal at 200 mM NaCl and at pH range between 5.8 and 7.5. In addition, the presence or absence of chaperone DnaK in E. coli cells influence the solubility of recombinant VP1 and the conformational quality of this protein in the VLPs. The hemagglutination ability of hJCV VP1 VLPs contained in E. coli cell extracts can be modulated by buffer composition in the hemagglutination assay. It has been also determined that the production of recombinant hJCV VP1 in E. coli is favored by the absence of chaperone DnaK as observed by Western Blot analysis in different E. coli genetic backgrounds, indicating a proteolysis targeting role for DnaK. However, solubility is highly compromised in a DnaK^? E. coli strain suggesting an important role of this chaperone in reduction of protein aggregates. Finally, hemagglutination efficiency of recombinant VP1 is directly related to the presence of DnaK in the producing cells. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:744–748, 2014     

Insert engineering and solubility screening improves recovery of virus‐like particle subunits displaying hydrophobic epitopes

The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface‐exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface‐exposed loops of VP1, and expressed in Escherichia coli. VP1 pentamers, also known as capsomeres, were purified via a glutathione‐S‐transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high‐throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l ‐arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus‐like particles.     

CRISPR/dCas9‐mediated activation of multiple endogenous target genes directly converts human foreskin fibroblasts into Leydig‐like cells

Recently, Leydig cell (LC) transplantation has been revealed as a promising strategy for treating male hypogonadism; however, the key problem restricting the application of LC transplantation is a severe lack of seed cells. It seems that targeted activation of endogenous genes may provide a potential alternative. Therefore, the aim of this study was to determine whether targeted activation of Nr5a1, Gata4 and Dmrt1 (NGD) via the CRISPR/dCas9 synergistic activation mediator system could convert human foreskin fibroblasts (HFFs) into functional Leydig‐like cells. We first constructed the stable Hsd3b‐dCas9‐MPH‐HFF cell line using the Hsd3b‐EGFP, dCas9‐VP64 and MS2‐P65‐HSF1 lentiviral vectors and then infected it with single guide RNAs. Next, we evaluated the reprogrammed cells for their reprogramming efficiency, testosterone production characteristics and expression levels of Leydig steroidogenic markers by quantitative real‐time polymerase chain reaction or Western blotting. Our results showed that the reprogramming efficiency was close to 10% and that the reprogrammed Leydig‐like cells secreted testosterone rapidly and, more importantly, responded effectively to stimulation with human chorionic gonadotropin and expressed Leydig steroidogenic markers. Our findings demonstrate that simultaneous targeted activation of the endogenous NGD genes directly reprograms HFFs into functional Leydig‐like cells, providing an innovative technology that may have promising potential for the treatment of male androgen deficiency diseases.     

Killer immunoglobulin‐like receptor repertoire analysis in a Caucasian Spanish cohort with inflammatory bowel disease

Immunological molecules are implicated in inflammatory disorders, including inflammatory bowel disease (IBD; Crohn disease [CD] and ulcerative colitis [UC]). Killer cell immunoglobulin‐like receptors (KIRs) are also genetically variable proteins involved in immune function. They are expressed by NK cells and certain T lymphocytes, regulate specificity and function by interaction with HLA Class I molecules, may be either inhibitory or activating and are polymorphic both in terms of alleles and haplotype gene content. Genetic associations between activating KIRs and certain autoimmune and inflammatory diseases have been reported; however, a possible association between KIR and IBD remains unclear. The aim of this study was to determine the relationship between KIR repertoire and IBD pathologies in a Spanish cohort. KIR variability was analyzed using PCR–sequence specific oligonucleotide probes (SSOP). Inhibitory KIR2DL5 was found more frequently in UC and IBD patient groups than in healthy controls (P = 0.028 and P = 0.01, respectively), as was activating KIR2DS1 (P = 0.02, Pc > 0.05, UC vs. Controls; P = 0.001, Pc = 0.01, IBD vs Controls; P = 0.01, Pc > 0.05, Controls vs CR), KIR2DS5 (P = 0.0028, Pc = 0.04, Controls vs UC; P = 0.0001, Pc = 0.0017, Controls vs IBD; P = 0.01, Pc > 0.05, Controls vs CD) and KIR3DS1 (P = 0.012, Pc > 0.05, Controls vs IBD). Our data suggest that imbalance between activating and inhibitory KIR may partially explain the different pathogeneses of these IBDs and that there is a hypothetical role for the telomeric B region (which contains both KIR2DS5 and KIR2DS1) in these diseases.     

Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus‐like particle

Virus‐like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co‐expression of unmodified VP1 and modular VP1‐RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs.     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.     

Potential for plant growth promotion by a consortium of stress‐tolerant 2,4‐dinitrotoluene‐degrading bacteria: isolation and characterization of a military soil

The presence of explosives in soils and the interaction with drought stress and nutrient limitation are among the environmental factors that severely affect plant growth on military soils. In this study, we seek to isolate and identify the cultivable bacteria of a 2,4‐dinitrotoluene (DNT) contaminated soil (DS) and an adjacent grassland soil (GS) of a military training area aiming to isolate new plant growth‐promoting (PGP) and 2,4‐DNT‐degrading strains. Metabolic profiling revealed disturbances in Ecocarbon use in the bare DS; isolation of cultivable strains revealed a lower colony‐forming‐unit count and a less diverse community associated with DS in comparison with GS. New 2,4‐DNT‐tolerant strains were identified by selective enrichments, which were further characterized by auxanography for 2,4‐DNT use, resistance to drought stress, cold, nutrient starvation and PGP features. By selecting multiple beneficial PGP and abiotic stress‐resistant strains, efficient 2,4‐DNT‐degrading consortia were composed. After inoculation, consortium UHasselt Sofie 3 with seven members belonging to Burkholderia, Variovorax, Bacillus, Pseudomonas and Ralstonia species was capable to successfully enhance root length of Arabidopsis under 2,4‐DNT stress. After 9 days, doubling of main root length was observed. Our results indicate that beneficial bacteria inhabiting a disturbed environment have the potential to improve plant growth and alleviate 2,4‐DNT stress.     

Structure,function and evolution of the hemerythrin‐like domain superfamily

Hemerythrin‐like proteins have generally been studied for their ability to reversibly bind oxygen through their binuclear nonheme iron centers. However, in recent years, it has become increasingly evident that some members of the hemerythrin‐like superfamily also participate in many other biological processes. For instance, the binuclear nonheme iron site of YtfE, a hemerythrin‐like protein involved in the repair of iron centers in Escherichia coli, catalyzes the reduction of nitric oxide to nitrous oxide, and the human F‐box/LRR‐repeat protein 5, which contains a hemerythrin‐like domain, is involved in intracellular iron homeostasis. Furthermore, structural data on hemerythrin‐like domains from two proteins of unknown function, PF0695 from Pyrococcus furiosus and NMB1532 from Neisseria meningitidis, show that the cation‐binding sites, typical of hemerythrin, can be absent or be occupied by metal ions other than iron. To systematically investigate this functional and structural diversity of the hemerythrin‐like superfamily, we have collected hemerythrin‐like sequences from a database comprising fully sequenced proteomes and generated a cluster map based on their all‐against‐all pairwise sequence similarity. Our results show that the hemerythrin‐like superfamily comprises a large number of protein families which can be classified into three broad groups on the basis of their cation‐coordinating residues: (a) signal‐transduction and oxygen‐carrier hemerythrins (H‐HxxxE‐HxxxH‐HxxxxD); (b) hemerythrin‐like (H‐HxxxE‐H‐HxxxE); and, (c) metazoan F‐box proteins (H‐HExxE‐H‐HxxxE). Interestingly, all but two hemerythrin‐like families exhibit internal sequence and structural symmetry, suggesting that a duplication event may have led to the origin of the hemerythrin domain.