An E2–F12 complex is required for intracellular enveloped virus morphogenesis during vaccinia infection

The vaccinia virus protein, F12, has been suggested to play an important role in microtubule-based transport of intracellular enveloped virus (IEV). We found that GFP-F12 is recruited to IEV moving on microtubules but is released from virus particles when they switch to actin-based motility. In the absence of F12, although the majority of IEV remain close to their peri-nuclear site of assembly, a small number of IEV still move with linear trajectories at speeds of 0.85 μm s⁻¹, consistent with microtubule transport. Using a recombinant virus expressing GST-F12, we found that the viral protein E2 interacts directly with F12. In infected cells, GFP-E2 is observed on moving IEV as well as in the Golgi region, but is not associated with actin tails. In the absence of E2L, IEV accumulate in the peri-nuclear region and F12 is not recruited. Conversely, GFP-E2 is not observed on IEV in the absence of F12. Ultra-structural analysis of ΔE2L- and ΔF12L-infected cells reveals that loss of either protein results in defects in membrane wrapping during IEV formation. We suggest that E2 and F12 function as a complex that is necessary for IEV morphogenesis prior to their microtubule-based transport towards the plasma membrane.     

F11-Mediated Inhibition of RhoA Signalling Enhances the Spread of Vaccinia Virus In Vitro and In Vivo in an Intranasal Mouse Model of Infection

The cortical actin cytoskeleton beneath the plasma membrane represents a physical barrier that vaccinia virus has to overcome during its exit from an infected cell. Previous observations using overexpression and pharmacological approaches suggest that vaccinia enhances its release by modulating the cortical actin cytoskeleton by inhibiting RhoA signalling using the viral protein F11. We have now examined the role of F11 and its ability to interact with RhoA to inhibit its downstream signalling in the spread of vaccinia infection both in vitro and in vivo. Live cell imaging over 48 hours reveals that loss of F11 or its ability to bind RhoA dramatically reduces the rate of cell-to-cell spread of the virus in a cell monolayer. Cells infected with the ΔF11L virus also maintained their cell-to-cell contacts, and did not undergo virus-induced motility as observed during wild-type infections. The ΔF11L virus is also attenuated in intranasal mouse models of infection, as it is impaired in its ability to spread from the initial sites of infection to the lungs and spleen. Loss of the ability of F11 to bind RhoA also reduces viral spread in vivo. Our results clearly establish that viral-mediated inibition of RhoA signalling can enhance the spread of infection not only in cell monolayers, but also in vivo.     

Design and total synthesis of a fluorescent phorboxazole A analog for cellular studies

To enable studies to elucidate the intracellular processing and targeting of the potent cytostatic/apoptotic anticancer natural products phorboxazoles A and B, a fluorescent derivative has been developed. This involved the total syntheses of the terminal alkyne 33-O-Me-45,46-dehydrobromophorboxazole A (MDHBPA) and a terminal vinyl iodide derivative of the blue fluorescent dye N,N,-dimethyl-7-aminocoumarin (DMC). Sonogashira coupling of these partners provided enyne DMC-MDHBPA in high yield.     

The induction of preterm labor in rhesus macaques is determined by the strength of immune response to intrauterine infection

Intrauterine infection/inflammation (IUI) is a major contributor to preterm labor (PTL). However, IUI does not invariably cause PTL. We hypothesized that quantitative and qualitative differences in immune response exist in subjects with or without PTL. To define the triggers for PTL, we developed rhesus macaque models of IUI driven by lipopolysaccharide (LPS) or live Escherichia coli. PTL did not occur in LPS challenged rhesus macaques, while E. coli–infected animals frequently delivered preterm. Although LPS and live E. coli both caused immune cell infiltration, E. coli–infected animals showed higher levels of inflammatory mediators, particularly interleukin 6 (IL-6) and prostaglandins, in the chorioamnion-decidua and amniotic fluid (AF). Neutrophil infiltration in the chorio-decidua was a common feature to both LPS and E. coli. However, neutrophilic infiltration and IL6 and PTGS2 expression in the amnion was specifically induced by live E. coli. RNA sequencing (RNA-seq) analysis of fetal membranes revealed that specific pathways involved in augmentation of inflammation including type I interferon (IFN) response, chemotaxis, sumoylation, and iron homeostasis were up-regulated in the E. coli group compared to the LPS group. Our data suggest that the intensity of the host immune response to IUI may determine susceptibility to PTL.     

Enhanced isoprene-related tolerance of heat- and light-stressed photosynthesis at low, but not high, CO2 concentrations

The principal function of isoprene biosynthesis in plants remains unclear, but emission rates are positively correlated with temperature and light, supporting a role for isoprene in maintaining photosynthesis under transient heat and light stress from sunflecks. Isoprene production is also inversely correlated with CO(2) concentrations, implying that rising CO(2) may reduce the functional importance of isoprene. To understand the importance of isoprene in maintaining photosynthesis during sunflecks, we used RNAi technology to suppress isoprene production in poplar seedlings and compared the responses of these transgenic plants to wild-type and empty-vector control plants. We grew isoprene-emitting and non-emitting trees at low (190 ppm) and high (590 ppm) CO(2) concentrations and compared their photosynthetic responses to short, transient periods of high light and temperature, as well as their photosynthetic thermal response at constant light. While there was little difference between emitting and non-emitting plants in their photosynthetic responses to simulated sunflecks at high CO(2), isoprene-emitting trees grown at low CO(2) had significantly greater photosynthetic sunfleck tolerance than non-emitting plants. Net photosynthesis at 42°C was 50% lower in non-emitters than in isoprene-emitting trees at low CO(2), but only 22% lower at high CO(2). Dark respiration rates were significantly higher in non-emitting poplar from low CO(2), but there was no difference between isoprene-emitting and non-emitting lines at high CO(2). We propose that isoprene biosynthesis may have evolved at low CO(2) concentrations, where its physiological effect is greatest, and that rising CO(2) will reduce the functional benefit of isoprene in the near future.     

Engineering cyanobacteria to generate high-value products

Although many microorganisms have been used for the bioindustrial generation of valuable metabolites, the productive potential of cyanobacterial species has remained largely unexplored. Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements, tolerance of marginal agricultural environments, rapid genetics, and carbon-neutral applications that could be leveraged to address global climate change concerns. Here, we review recent research involving the engineering of cyanobacterial species for the production of valuable bioindustrial compounds, including natural cyanobacterial products (e.g. sugars and isoprene), biofuels (e.g. alcohols, alkanes and hydrogen), and other commodity chemicals. Biological and economic obstacles to scaled cyanobacterial production are highlighted, and methods for increasing cyanobacterial production efficiencies are discussed.     

Plasma-based approach to measure target engagement for liver-targeting stearoyl-CoA desaturase 1 inhibitors

A positive correlation between stearoyl-CoA desaturase (SCD)1 expression and metabolic diseases has been reported in rodents and humans. These findings indicate that SCD1 is a promising therapeutic target for the chronic treatment of diabetes and dyslipidemia. The SCD1 enzyme is expressed at high levels in several human tissues and is required for the biosynthesis of monounsaturated fatty acids, which are involved in many biological processes. Liver-targeted SCD inhibitors were designed to pharmacologically manipulate SCD1 activity in the liver to avoid adverse events due to systemic inhibition. This article describes the development of a plasma-based SCD assay to assess the level of SCD inhibition, which is defined in this article as target engagement. Essentially, animals are dosed with an exogenous deuterated tracer (d7-stearic acid) as substrate, and the converted d7-oleic acid product is measured to monitor SCD1 inhibition. This study reveals that this plasma-based assay correlates with liver SCD1 inhibition and can thus have clinical utility.     

A local pancreatic renin-angiotensin system: endocrine and exocrine roles

The renin-angiotensin system (RAS) is classically characterized as a circulating hormonal system primarily through the production of the physiologically active product angiotensin II (Ang II) that plays a crucial role in the regulation of blood pressure, fluid and electrolyte homeostasis. In addition to this circulating RAS, numerous tissues and organs have been recently demonstrated to exhibit their own RAS products and activities. Such an intrinsic RAS can modulate the specific local functions of their respective tissues and organs, frequently in a paracrine and autocrine manner. Recent findings from our laboratories and others have made a significant contribution on the expression, localization, regulation, and potential role of a local RAS in the pancreas. Although, it is quite intriguing that components of the local pancreatic RAS are responsive to various physiological and pathophysiological conditions, the crucial role of this system in regulating the exocrine and endocrine functions and ultimately the clinical relevance to pancreatic disease is still largely equivocal. Of particular interest in this context are the actions of pancreatic RAS on the growth, anti-proliferation and free radical generation in the pancreas. The aims of the current article focus on the emerging data on the local pancreatic RAS; its involvement in exocrine acinar and endocrine islet aspects, and the clinical significance in the pancreas are particularly addressed. The target for the local pancreatic RAS may provide a new insight into future management of various clinical conditions including islet transplants, diabetes mellitus, pancreatic cancer, pancreatitis and cystic fibrosis.