Coxsackievirus B4-induced cytokine production in pancreatic cells is mediated through toll-like receptor 4

Coxsackievirus B4 (CBV4), a member of the Picornavirus genus, has long been implicated in the development of insulin-dependent diabetes mellitus (IDDM) caused by virus-induced pancreatic cell damage. The progressive destruction of pancreatic beta cells is responsible for the development of IDDM. It has recently been suggested that CBV4 infection can induce the production of proinflammatory cytokines, and these cytokines seem to be involved in the damage to the insulin-producing cells. In this study we investigated whether toll-like receptors (TLRs) are responsible for triggering the proinflammatory cytokine production in human pancreatic cells in response to CBV4. Here we demonstrate that CBV4 triggers cytokine production through a TLR4-dependent pathway. This interaction seems to be independent of virus attachment and cell entry.     

Genetic analysis of the Linnaean Ulva lactuca (Ulvales,Chlorophyta) holotype and related type specimens reveals name misapplications,unexpected origins,and new synonymies

Current usage of the name Ulva lactuca, the generitype of Ulva, remains uncertain. Genetic analyses were performed on the U. lactuca Linnaean holotype, the U. fasciata epitype, the U. fenestrata holotype, the U. lobata lectotype, and the U. stipitata lectotype. The U. lactuca holotype is nearly identical in rbcL sequence to the epitype of U. fasciata, a warm temperate to tropical species, rather than the cold temperate species to which the name U. lactuca has generally been applied. We hypothesize that the holotype specimen of U. lactuca came from the Indo‐Pacific rather than northern Europe. Our analyses indicate that U. fasciata and U. lobata are heterotypic synonyms of U. lactuca. Ulva fenestrata is the earliest name for northern hemisphere, cold temperate Atlantic and Pacific species, with U. stipitata a junior synonym. DNA sequencing of type specimens provides an unequivocal method for applying names to Ulva species.     

Linking the functions of unrelated proteins using a novel directed evolution domain insertion method

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 beta-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b(562) with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of alpha-helices and beta-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains.     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Inhibiting PHGDH with NCT-503 reroutes glucose-derived carbons into the TCA cycle,independently of its on-target effect

The small-molecule inhibitor of phosphoglycerate dehydrogenase, NCT-503, reduces incorporation of glucose-derived carbons into serine in vitro. Here we describe an off-target effect of NCT-503 in neuroblastoma cell lines expressing divergent phosphoglycerate dehydrogenase (PHGDH) levels and single-cell clones with CRISPR-Cas9-directed PHGDH knockout or their respective wildtype controls. NCT-503 treatment strongly reduced synthesis of glucose-derived citrate in all cell models investigated compared to the inactive drug control and independent of PHGDH expression level. Incorporation of glucose-derived carbons entering the TCA cycle via pyruvate carboxylase was enhanced by NCT-503 treatment. The activity of citrate synthase was not altered by NCT-503 treatment. We also detected no change in the thermal stabilisation of citrate synthase in cellular thermal shift assays from NCT-503-treated cells. Thus, the direct cause of the observed off-target effect remains enigmatic. Our findings highlight off-target potential within a metabolic assessment of carbon usage in cells treated with the small-molecule inhibitor, NCT-503.     

Nox5 mediates PDGF-induced proliferation in human aortic smooth muscle cells

The proliferation of vascular smooth muscle cells is important in the pathogenesis of many vascular diseases. Reactive oxygen species (ROS) produced by NADPH oxidases in smooth muscle cells have been shown to participate in signaling cascades regulating proliferation induced by platelet-derived growth factor (PDGF), a powerful smooth muscle mitogen. We sought to determine the role of Nox5 in the regulation of PDGF-stimulated human aortic smooth muscle cell (HASMC) proliferation. Cultured HASMC were found to express four isoforms of Nox5. When HASMC stimulated with PDGF were pretreated with N-acetyl cysteine (NAC), proliferation was significantly reduced. Proliferation induced by PDGF was also heavily dependent on JAK/STAT activation, as the JAK inhibitor, AG490, was able to completely abolish PDGF-stimulated HASMC growth. Specific knockdown of Nox5 with a siRNA strategy reduced PDGF-induced HASMC ROS production and proliferation. Additionally, siRNA to Nox5 inhibited PDGF-stimulated JAK2 and STAT3 phosphorylation. ROS produced by Nox5 play an important role in PDGF-induced JAK/STAT activation and HASMC proliferation.     

Application of Methods for Identifying Broiler Chicken Gut Bacterial Species Linked with Increased Energy Metabolism

A high-throughput microbial profiling tool based on terminal restriction fragment length polymorphism was developed to monitor the poultry gut microbiota in response to dietary manipulations. Gut microbial communities from the duodena, jejuna, ilea, and ceca of 48 birds fed either a barley control diet or barley diet supplemented with exogenous enzymes for degrading nonstarch polysaccharide were characterized by using multivariate statistical methods. Analysis of samples showed that gut microbial communities varied significantly among gut sections, except between the duodenum and jejunum. Significant diet-associated differences in gut microbial communities were detected within the ileum and cecum only. The dissimilarity in bacterial community composition between diets was 73 and 66% within the ileum and cecum, respectively. Operational taxonomic units, representing bacterial species or taxonomically related groups, contributing to diet-associated differences were identified. Several bacterial species contributed to differences between diet-related gut microbial community composition, with no individual bacterial species contributing more than 1 to 5% of the total. Using canonical analysis of principal coordinates biplots, we correlated differences in gut microbial community composition within the ileum and cecum to improved performance, as measured by apparent metabolizable energy. This is the first report that directly links differences in the composition of the gut microbial community with improved performance, which implies that the presence of specific beneficial and/or absence of specific detrimental bacterial species may contribute to the improved performance in these birds.     

Effect of climate change on breeding phenology,clutch size and chick survival of an upland bird

Upland birds are predicted to be particularly vulnerable to the effects of climate change, yet few studies have examined these effects on their breeding phenology and productivity. Laying dates of Red Grouse Lagopus lagopus scotica in the Scottish Highlands advanced by 0.5 days/year between 1992 and 2011 and were inversely correlated with pre‐laying temperature, with a near‐significant increase in temperature over this period. Earlier clutches were larger and chick survival was greater in earlier nesting attempts. However, chick survival was also higher in years with lower May temperatures and lower August temperatures in the previous year, the latter probably related to prey abundance in the subsequent breeding season. Although laying dates are advancing, climate change does not currently appear to be having an overall effect on chick survival of Red Grouse within the climate range recorded in this study.