Scaffoldin Modules Serving as ��Cargo�� Domains To Promote the Secretion of Heterologous Cellulosomal Cellulases by Clostridium acetobutylicum

The secretion of large heterologous cellulases by Clostridium acetobutylicum was formerly shown to be deleterious. To circumvent this issue, various scaffoldins'' modules were grafted at their N termini. Family 3a cellulose binding module combined with an X2 module(s) was found to trigger the secretion of Clostridium cellulolyticum cellulases by the solventogenic bacterium.     

Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the native endothelium.     

TLR2 signaling renders quiescent naive and memory CD4+ T cells more susceptible to productive infection with X4 and R5 HIV-type 1

It has been recently demonstrated that circulating microbial products are responsible for a systemic immune activation in individuals infected with HIV-type 1. Bacterial products carry structural conserved motifs recognized by TLRs. Some TLR members are expressed in primary human CD4+ T cells but the precise functional role played by these pattern recognition receptors is still imprecise. In this study, we report that engagement of TLR2 in quiescent naive and memory CD4+ T cells leads to the acquisition of an effector-like phenotype. Interestingly, engagement of TLR2 renders both cell subsets more susceptible to productive infection with X4 virions and a higher virus production was seen with R5 viruses. It can be proposed that exposure of resting CD4+ T cells to pathogen-derived products that can engage TLR2 induces the acquisition of an effector-like phenotype in naive and memory CD4+ T lymphocytes, a phenomenon that might result in an acceleration of virus replication, immune dysregulation, and HIV-type 1-mediated disease progression.     

Noncatalytic role of the FKBP52 peptidyl-prolyl isomerase domain in the regulation of steroid hormone signaling

Hormone-dependent transactivation by several of the steroid hormone receptors is potentiated by the Hsp90-associated cochaperone FKBP52, although not by the closely related FKBP51. Here we analyze the mechanisms of potentiation and the functional differences between FKBP51 and FKBP52. While both have peptidyl-prolyl isomerase activity, this is not required for potentiation, as mutations abolishing isomerase activity did not affect potentiation. Genetic selection in Saccharomyces cerevisiae for gain of potentiation activity in a library of randomly mutated FKBP51 genes identified a single residue at position 119 in the N-terminal FK1 domain as being a critical difference between these two proteins. In both the yeast model and mammalian cells, the FKBP51 mutation L119P, which is located in a hairpin loop overhanging the catalytic pocket and introduces the proline found in FKBP52, conferred significant potentiation activity, whereas the converse P119L mutation in FKBP52 decreased potentiation. A second residue in this loop, A116, also influences potentiation levels; in fact, the FKBP51-A116V L119P double mutant potentiated hormone signaling as well as wild-type FKBP52 did. These results suggest that the FK1 domain, and in particular the loop overhanging the catalytic pocket, is critically involved in receptor interactions and receptor activity.     

Can the biomass-ratio hypothesis predict mixed-species litter decomposition along a climatic gradient?

Background and Aims

The biomass-ratio hypothesis states that ecosystem properties are driven by the characteristics of dominant species in the community. In this study, the hypothesis was operationalized as community-weighted means (CWMs) of monoculture values and tested for predicting the decomposition of multispecies litter mixtures along an abiotic gradient in the field.

Methods

Decomposition rates (mg g⁻¹ d⁻¹) of litter from four herb species were measured using litter-bed experiments with the same soil at three sites in central France along a correlated climatic gradient of temperature and precipitation. All possible combinations from one to four species mixtures were tested over 28 weeks of incubation. Observed mixture decomposition rates were compared with those predicted by the biomass-ratio hypothesis. Variability of the prediction errors was compared with the species richness of the mixtures, across sites, and within sites over time.

Key Results

Both positive and negative prediction errors occurred. Despite this, the biomass-ratio hypothesis was true as an average claim for all sites (r = 0·91) and for each site separately, except for the climatically intermediate site, which showed mainly synergistic deviations. Variability decreased with increasing species richness and in less favourable climatic conditions for decomposition.

Conclusions

Community-weighted mean values provided good predictions of mixed-species litter decomposition, converging to the predicted values with increasing species richness and in climates less favourable to decomposition. Under a context of climate change, abiotic variability would be important to take into account when predicting ecosystem processes.     

Impact of Chronic Kidney Disease on Use of Evidence-Based Therapy in Stable Coronary Artery Disease: A Prospective Analysis of 22,272 Patients

Purpose

To assess the frequency of chronic kidney disease (CKD), define the associated demographics, and evaluate its association with use of evidence-based drug therapy in a contemporary global study of patients with stable coronary artery disease.

Methods

22,272 patients from the ProspeCtive observational LongitudinAl RegIstry oF patients with stable coronary arterY disease (CLARIFY) were included. Baseline estimated glomerular filtration rate (eGFR) was calculated (CKD-Epidemiology Collaboration formula) and patients categorised according to CKD stage: >89, 60–89, 45–59 and <45 mL/min/1.73 m².

Results

Mean (SD) age was 63.9±10.4 years, 77.3% were male, 61.8% had a history of myocardial infarction, 71.9% hypertension, 30.4% diabetes and 75.4% dyslipidaemia. Chronic kidney disease (eGFR<60 mL/min/1.73 m²) was seen in 22.1% of the cohort (6.9% with eGFR<45 mL/min/1.73 m²); lower eGFR was associated with increasing age, female sex, cardiovascular risk factors, overt vascular disease, other comorbidities and higher systolic but lower diastolic blood pressure. High use of secondary prevention was seen across all CKD stages (overall 93.4% lipid-lowering drugs, 95.3% antiplatelets, 75.9% beta-blockers). The proportion of patients taking statins was lower in patients with CKD. Antiplatelet use was significantly lower in patients with CKD whereas oral anticoagulant use was higher. Angiotensin-converting enzyme inhibitor use was lower (52.0% overall) and inversely related to declining eGFR, whereas angiotensin-receptor blockers were more frequently prescribed in patients with reduced eGFR.

Conclusions

Chronic kidney disease is common in patients with stable coronary artery disease and is associated with comorbidities. Whilst use of individual evidence-based medications for secondary prevention was high across all CKD categories, there remains an opportunity to improve the proportion who take all three classes of preventive therapies. Angiotensin-converting enzyme inhibitors were used less frequently in lower eGRF categories. Surprisingly the reverse was seen for angiotensin-receptor blockers. Further evaluation is required to fully understand these associations. The CLARIFY (ProspeCtive observational LongitudinAl RegIstry oF patients with stable coronary arterY disease) Registry is registered in the ISRCTN registry of clinical trials with the number ISRCTN43070564. http://www.controlled-trials.com/ISRCTN43070564.     

Common marmosets (Callithrix jacchus) as a nonhuman primate model to assess the virulence of eastern equine encephalitis virus strains

Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral disease in North America (NA) and is a potential biological weapon. However, genetically and antigenically distinct strains from South America (SA) have seldom been associated with human disease or mortality despite serological evidence of infection. Because mice and other small rodents do not respond differently to the NA versus SA viruses like humans, we tested common marmosets (Callithrix jacchus) by using intranasal infection and monitoring for weight loss, fever, anorexia, depression, and neurologic signs. The NA EEEV-infected animals either died or were euthanized on day 4 or 5 after infection due to anorexia and neurologic signs, but the SA EEEV-infected animals remained healthy and survived. The SA EEEV-infected animals developed peak viremia titers of 2.8 to 3.1 log(10) PFU/ml on day 2 or 4 after infection, but there was no detectable viremia in the NA EEEV-infected animals. In contrast, virus was detected in the brain, liver, and muscle of the NA EEEV-infected animals at the time of euthanasia or death. Similar to the brain lesions described for human EEE, the NA EEEV-infected animals developed meningoencephalitis in the cerebral cortex with some perivascular hemorrhages. The findings of this study identify the common marmoset as a useful model of human EEE for testing antiviral drugs and vaccine candidates and highlight their potential for corroborating epidemiological evidence that some, if not all, SA EEEV strains are attenuated for humans.     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

A dynamic leaf gas‐exchange strategy is conserved in woody plants under changing ambient CO2: evidence from carbon isotope discrimination in paleo and CO2 enrichment studies

Rising atmospheric [CO₂], c_a, is expected to affect stomatal regulation of leaf gas‐exchange of woody plants, thus influencing energy fluxes as well as carbon (C), water, and nutrient cycling of forests. Researchers have proposed various strategies for stomatal regulation of leaf gas‐exchange that include maintaining a constant leaf internal [CO₂], c_i, a constant drawdown in CO₂ (c_a ? c_i), and a constant c_i/c_a. These strategies can result in drastically different consequences for leaf gas‐exchange. The accuracy of Earth systems models depends in part on assumptions about generalizable patterns in leaf gas‐exchange responses to varying c_a. The concept of optimal stomatal behavior, exemplified by woody plants shifting along a continuum of these strategies, provides a unifying framework for understanding leaf gas‐exchange responses to c_a. To assess leaf gas‐exchange regulation strategies, we analyzed patterns in c_i inferred from studies reporting C stable isotope ratios (δ¹³C) or photosynthetic discrimination (?) in woody angiosperms and gymnosperms that grew across a range of c_a spanning at least 100 ppm. Our results suggest that much of the c_a‐induced changes in c_i/c_a occurred across c_a spanning 200 to 400 ppm. These patterns imply that c_a ? c_i will eventually approach a constant level at high c_a because assimilation rates will reach a maximum and stomatal conductance of each species should be constrained to some minimum level. These analyses are not consistent with canalization toward any single strategy, particularly maintaining a constant c_i. Rather, the results are consistent with the existence of a broadly conserved pattern of stomatal optimization in woody angiosperms and gymnosperms. This results in trees being profligate water users at low c_a, when additional water loss is small for each unit of C gain, and increasingly water‐conservative at high c_a, when photosystems are saturated and water loss is large for each unit C gain.