Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

The chemokine receptor CCR5 belongs to the class of G protein-coupled receptors. Besides its role in leukocyte trafficking, it is also the major HIV-1 coreceptor and hence a target for HIV-1 entry inhibitors. Here, we report Escherichia coli expression and a broad range of biophysical studies on E. coli-produced CCR5. After systematic screening and optimization, we obtained 10 mg of purified, detergent-solubilized, folded CCR5 from 1L culture in a triply isotope-labeled (²H/¹⁵N/¹³C) minimal medium. Thus the material is suitable for NMR spectroscopic studies. The expected α-helical secondary structure content is confirmed by circular dichroism spectroscopy. The solubilized CCR5 is monodisperse and homogeneous as judged by transmission electron microscopy. Interactions of CCR5 with its ligands, RANTES and MIP-1β were assessed by surface plasmon resonance yielding K_D values in the nanomolar range. Using size exclusion chromatography, stable monomeric CCR5 could be isolated. We show that cysteine residues affect both the yield and oligomer distribution of CCR5. HSQC spectra suggest that the transmembrane domains of CCR5 are in equilibrium between several conformations. In addition we present a model of CCR5 based on the crystal structure of CXCR4 as a starting point for protein engineering.     

Colonic Inflammation in Mice Is Improved by Cigarette Smoke through iNKT Cells Recruitment

Cigarette smoke (CS) protects against intestinal inflammation during ulcerative colitis. Immunoregulatory mechanisms sustaining this effect remain unknown. The aim of this study was to assess the effects of CS on experimental colitis and to characterize the intestinal inflammatory response at the cellular and molecular levels. Using the InExpose® System, a smoking device accurately reproducing human smoking habit, we pre-exposed C57BL/6 mice for 2 weeks to CS, and then we induced colitis by administration of dextran sodium sulfate (DSS). This system allowed us to demonstrate that CS exposure improved colonic inflammation (significant decrease in clinical score, body weight loss and weight/length colonic ratio). This improvement was associated with a significant decrease in colonic proinflammatory Th1/Th17 cytokine expression, as compared to unexposed mice (TNF (p = 0.0169), IFNγ (p<0.0001), and IL-17 (p = 0.0008)). Smoke exposure also induced an increased expression of IL-10 mRNA (p = 0.0035) and a marked recruitment of iNKT (invariant Natural Killer T; CD45+ TCRβ+ CD1d tetramer+) cells in the colon of DSS-untreated mice. Demonstration of the role of iNKT cells in CS-dependent colitis improvement was performed using two different strains of NKT cells deficient mice. Indeed, in Jα18KO and CD1dKO animals, CS exposure failed to induce significant regulation of DSS-induced colitis both at the clinical and molecular levels. Thus, our study demonstrates that iNKT cells are pivotal actors in the CS-dependent protection of the colon. These results highlight the role of intestinal iNKT lymphocytes and their responsiveness to environmental stimuli. Targeting iNKT cells would represent a new therapeutic way for inflammatory bowel diseases.     

VEGFR1 and VEGFR2 Involvement in Extracellular Galectin-1- and Galectin-3-Induced Angiogenesis

Aim

Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour progression. Thus, it is critical to identify their influence on angiogenesis.

Methods

We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay.

Results

We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and −3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis.     

Age and sex-dependent effects of landscape cover and trapping on the spatial genetic structure of the stone marten (<Emphasis Type="Italic">Martes foina</Emphasis>)

Maintenance of genetic variation is of critical importance for wild populations since low levels limit the species’ ability to respond to different threats (diseases, predators, environmental changes) in both the long and the short term. Human activities could impact the genetic variation of wild species in multiple ways, including via fragmentation and harvesting. We used an individual-based landscape genetics approach to describe the impact of landscape elements and trapping pressure on the spatial genetic structure of a large sample (n = 370) of the stone marten (Martes foina) in central-eastern France (Bresse). An analysis of isolation-by-resistance using a causal modeling approach showed an influence of landscape cover and/or trapping pressure on gene flow according to age and sex class. Overall, the connectivity in the study area is provided mainly by vegetation cover, while roads and open areas partially impede it. Unexpectedly for this “urban adapter” species, buildings could reduce gene flow. We also emphasized the sex-dependent effect of trapping on gene flow. Genetic differentiation in males was influenced by trapping pressure and landscape structure while only the latter influenced genetic differentiation in females. A stronger isolation by distance in males than in females suggested that at the scale of the study area, males are more exposed to trapping pressure, which reduces effective dispersal. Overall, the combination of both landscape and trapping costs might create an ‘ecological trap’ that could disrupt gene flow, leading to a north–south division in the study area.     

An Evolution-Guided Analysis Reveals a Multi-Signaling Regulation of Fas by Tyrosine Phosphorylation and its Implication in Human Cancers

Demonstrations of both pro-apoptotic and pro-survival abilities of Fas (TNFRSF6/CD95/APO-1) have led to a shift from the exclusive “Fas apoptosis” to “Fas multisignals” paradigm and the acceptance that Fas-related therapies face a major challenge, as it remains unclear what determines the mode of Fas signaling. Through protein evolution analysis, which reveals unconventional substitutions of Fas tyrosine during divergent evolution, evolution-guided tyrosine-phosphorylated Fas proxy, and site-specific phosphorylation detection, we show that the Fas signaling outcome is determined by the tyrosine phosphorylation status of its death domain. The phosphorylation dominantly turns off the Fas-mediated apoptotic signal, while turning on the pro-survival signal. We show that while phosphorylations at Y232 and Y291 share some common functions, their contributions to Fas signaling differ at several levels. The findings that Fas tyrosine phosphorylation is regulated by Src family kinases (SFKs) and the phosphatase SHP-1 and that Y291 phosphorylation primes clathrin-dependent Fas endocytosis, which contributes to Fas pro-survival signaling, reveals for the first time the mechanistic link between SFK/SHP-1-dependent Fas tyrosine phosphorylation, internalization route, and signaling choice. We also demonstrate that levels of phosphorylated Y232 and Y291 differ among human cancer types and differentially respond to anticancer therapy, suggesting context-dependent involvement of Fas phosphorylation in cancer. This report provides a new insight into the control of TNF receptor multisignaling by receptor phosphorylation and its implication in cancer biology, which brings us a step closer to overcoming the challenge in handling Fas signaling in treatments of cancer as well as other pathologies such as autoimmune and degenerative diseases.     

Etiology and Epidemiology of Diarrhea in Hospitalized Children from Low Income Country: A Matched Case-Control Study in Central African Republic

Background

In Sub-Saharan Africa, infectious diarrhea is a major cause of morbidity and mortality. A case-control study was conducted to identify the etiology of diarrhea and to describe its main epidemiologic risk factors among hospitalized children under five years old in Bangui, Central African Republic.

Methods

All consecutive children under five years old hospitalized for diarrhea in the Pediatric Complex of Bangui for whom a parent’s written consent was provided were included. Controls matched by age, sex and neighborhood of residence of each case were included. For both cases and controls, demographic, socio-economic and anthropometric data were recorded. Stool samples were collected to identify enteropathogens at enrollment. Clinical examination data and blood samples were collected only for cases.

Results

A total of 333 cases and 333 controls was recruited between December 2011 and November 2013. The mean age of cases was 12.9 months, and 56% were male. The mean delay between the onset of first symptoms and hospital admission was 3.7 days. Blood was detected in 5% of stool samples from cases. Cases were significantly more severely or moderately malnourished than controls. One of the sought-for pathogens was identified in 78% and 40% of cases and controls, respectively. Most attributable cases of hospitalized diarrhea were due to rotavirus, with an attributable fraction of 39%. Four other pathogens were associated with hospitalized diarrhea: Shigella/EIEC, Cryptosporidium parvum/hominis, astrovirus and norovirus with attributable fraction of 9%, 10%, 7% and 7% respectively. Giardia intestinalis was found in more controls than cases, with a protective fraction of 6%.

Conclusions

Rotavirus, norovirus, astrovirus, Shigella/EIEC, Cryptosporidium parvum/hominis were found to be positively associated with severe diarrhea: while Giardia intestinalis was found negatively associated. Most attributable episodes of severe diarrhea were associated with rotavirus, highlighting the urgent need to introduce the rotavirus vaccine within the CAR’s Expanded Program on Immunization. The development of new medicines, vaccines and rapid diagnostic tests that can be conducted at the bedside should be high priority for low-resource countries.     

High-Density Lipoprotein-Associated miR-223 Is Altered after Diet-Induced Weight Loss in Overweight and Obese Males

Background and Aims

microRNAs (miRNAs) are small, endogenous non-coding RNAs that regulate metabolic processes, including obesity. The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males.

Methods

HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet (HP, 30% of energy) with a normal protein diet (NP, 20% of energy). HDL-associated miRNAs (miR-16, miR-17, miR-126, miR-222 and miR-223) at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically.

Results

miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight (r = 0.488, p = 0.032). Changes in miR-223 levels were not associated to changes in HDL composition or size.

Conclusion

HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss.     

Personality Traits of Suicidality Are Associated with Premenstrual Syndrome and Premenstrual Dysphoric Disorder in a Suicidal Women Sample

Objective

Both Premenstrual Syndrome (PMS) and Premenstrual Dysphoric Disorder (PMDD) might increase the risk of suicidal behavior. The aim of this study was to assess the relationship between personality dimensions specifically involved in suicidal vulnerability and PMS/PMDD.

Method

We collected data from 232 women consecutively hospitalized after a suicide attempt. We examined the relationship between impulsivity, aggressiveness/hostility, hopelessness, trait anger, affect intensity, emotional lability, and PMS/PMDD. Notably, we created an algorithm from the shortened Premenstrual Assessment form in order to assess PMDD status.

Results

The proportions of PMS and PMDD among female suicide attempters were 50% and 23% respectively. Women with PMS or PMDD were more likely to endorse most of these personality traits to than those without even after controlling for potential confounders. We found an impulsive-aggressive pattern of personality in women with PMS or PMDD, independently from the time of the menstrual cycle. Interestingly, trait anger remained associated with both PMS and PMDD independently of every other personality traits. The higher the anger level, the higher the risk was to suffer from both PMS and PMDD.

Conclusions

This study demonstrates a strong, independent association between PMS/PMDD and trait anger among a representative sample of female suicide attempters. It is of major interest for clinicians in view of addressing a substantial public health problem among women of reproductive age.     

Experimental Approach to Evaluate the 11C Perfusion and Diffusion in Small Animal Tissues for HadronPET Applications

The development of a reliable dose monitoring system in hadron therapy is essential in order to control the treatment plan delivery. Positron Emission Tomography (PET) is the only method used in clinics nowadays for quality assurance. However, the accuracy of this method is limited by the loss of signal due to the biological washout processes. Up to the moment, very few studies measured the washout processes and there is no database of washout data as a function of the tissue and radioisotope. One of the main difficulties is related to the complexity of such measurements, along with the limited time slots available in hadron therapy facilities. Thus, in this work, we proposed an alternative in vivo methodology for the measurement and modeling of the biological washout parameters without any radiative devices. It consists in the implementation of a point-like radioisotope source by direct injection on the tissues of interest and its measurement by means of high-resolution preclinical PET systems. In particular, the washout of ¹¹C carbonate radioisotopes was assessed, considering that ¹¹C is is the most abundant β⁺ emitter produced by carbon beams. ¹¹C washout measurements were performed in several tissues of interest (brain, muscle and 9L tumor xenograf) in rodents (Wistar rat). Results show that the methodology presented is sensitive to the washout variations depending on the selected tissue. Finally, a first qualitative correlation between ¹¹C tumor washout properties and tumor metabolism (via ¹⁸F-FDG tracer uptake) was found.