Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Duration of Plague (Yersinia pestis) Outbreaks in Black-Tailed Prairie Dog (Cynomys ludovicianus) Colonies of Northern Colorado

Plague, caused by the bacterium Yersinia pestis, triggers die-offs in colonies of black-tailed prairie dogs (Cynomys ludovicianus), but the time-frame of plague activity is not well understood. We document plague activity in fleas from prairie dogs and their burrows on three prairie dog colonies that suffered die-offs. We demonstrate that Y. pestis transmission occurs over periods from several months to over a year in prairie dog populations before observed die-offs.     

Functional characterization of naturally occurring genetic variations of the human guanine-rich RNA sequence binding factor 1 (GRSF1)

The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D–models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.     

Pig Manure Contamination Marker Selection Based on the Influence of Biological Treatment on the Dominant Fecal Microbial Groups

The objective of this study was to identify a microbial marker for pig manure contamination. We quantified the persistence of four dominant bacterial groups from the pig intestinal tract throughout manure handling at 10 livestock operations (including aerobic digestion) by using molecular typing. The partial 16S rRNA genes of Bacteroides-Prevotella, Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium group isolates were amplified and analyzed by capillary electrophoresis single-strand conformation polymorphism. The most dominant bacterial populations were identified by cloning and sequencing their 16S rRNA genes. The results showed that Bifidobacterium spp. and, to a lesser extent, members of the BSL group, were less affected by the aerobic treatment than either Eubacterium-Clostridiaceae or Bacteroides-Prevotella. Two Bifidobacterium species found in raw manure were still present in manure during land application, suggesting that they can survive outside the pig intestinal tract and also survive aerobic treatment. The 16S-23S rRNA internal transcribed spacer of one species, Bifidobacterium thermacidophilum subsp. porcinum, was sequenced, and a specific pair of primers was designed for its detection in the environment. With this nested PCR assay, this potential marker was not detected in samples from 30 bovine, 30 poultry, and 28 human fecal samples or in 15 urban wastewater effluents. As it was detected in runoff waters after spreading of pig manure, we propose this marker as a suitable microbial indicator of pig manure contamination.Brittany represents only 7% of France but is the main pig production area and hosts approximately 14 million fatteners per year. This high concentration of confined pig feeding has led to the overapplication of manure to soil, which contributes to water pollution. Physical and biological manure treatment processes have been developed to limit nitrogen and phosphorus pollution (5). As these treatments were not designed to eliminate microbial pollution, even treated manure can contain pathogenic microorganisms (27) and agricultural soils and water systems can thus potentially still be contaminated through surface runoff and seepage. As manure application can increase the number of pathogens in the soil (18), pig feces may represent a significant risk to human health in Brittany. Currently, the monitoring of bacteria to assess fecal contamination (Escherichia coli, fecal coliforms, and enterococci) does not differentiate contamination from pig slurry from pollution by other animals or humans. It is thus important to develop analytic tools to specifically detect this source of pollution.Many studies have already proposed potential markers for the detection of host-specific fecal pollution (2, 3, 8, 12-15, 20, 37, 38, 48, 49). Much of this research has concentrated on distinguishing human and animal sources of contamination (3, 8, 20, 30, 38). Some studies have focused on identifying individual sources of animal pollution and have described molecular markers for feces from ducks (13), chickens (37), bovines (2, 3, 49), or cervids (6). Biomarkers have been proposed for porcine fecal contamination but rarely for porcine manure, the bacterial composition of which differs from that of porcine feces (9). Molecular markers have been developed to target the 16S rRNA gene sequences of dominant Eubacteria (2, 14, 43, 48) or methanogenic Archaebacteria (54) of the pig intestinal tract, whereas Khatib et al. (29) targeted the STII toxin gene from enterotoxigenic E. coli. Among the dominant groups of pig fecal Eubacteria, which include Bacteroides-Prevotella, Eubacterium-Clostridiacea, Lactobacillus-Streptococcus (34, 45, 51, 58), and to a lesser extent Bifidobacterium (40), the Bacteroides-Prevotella group has been particularly well studied (14, 22, 44). This marker of pig feces was described by Okabe et al. (44), but their work was based on feces sampled from only two farms and the number of clones analyzed was low. Gourmelon et al. (22) also detected the presence of a specific marker of pig feces belonging to the Bacteroides-Prevotella group in five stored manure samples. Although these studies revealed the presence of specific markers in fecal samples and in the subsequent pig manure samples, they did not address the possible disappearance of these anaerobic bacteria during the storage or biological treatment of the manure.Due to the lack of data concerning the bacterial flora of manure, the aims of this study were (i) to compare the monitoring of the Bacteroides-Prevotella group with that of Eubacterium-Clostridiaceae, Bacillus-Streptococcus-Lactobacillus (BSL), and Bifidobacterium throughout the biological manure treatment process and (ii) to search for a molecular marker among these groups of bacteria that was consistently present in the manure intended for land application. In the first part of this study, the persistence of the dominant bacteria throughout treatment was studied by using molecular typing, capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) (45) based on the analysis of the 16S rRNA genes. CE-SSCP is a fingerprinting technique in which single-stranded DNA fragments of the same length are separated based on the conformation of their secondary structure (23). The major advantages of this technique are its reproducibility between runs and its high resolution power with fewer false results than with denaturing gradient gel electrophoresis (25, 26).The second part of this article describes the relevance of the potential marker of pig manure (Bifidobacterium thermacidophilum subsp. porcinum) selected according to the results of the CE-SSCP profiles and the subsequent identification of dominant peaks of the CE-SSCP profiles. The specificity of this pig marker was then tested by assessing the host distribution in a selection of fecal, manure, and wastewater samples.     

HSV‐1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin‐Dependent Endocytosis

Herpes simplex virus‐1 (HSV‐1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin‐dependent endocytosis plays a major role in this process. Dominant‐negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin‐dependent and ‐independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non‐infectious HSV‐1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein‐sorting event during HSV‐1 envelopment.      

A missense mutation in the agouti signaling protein gene (ASIP) is associated with the no light points coat phenotype in donkeys

Background

Seven donkey breeds are recognized by the French studbook and are characterized by a black, bay or grey coat colour including light cream-to-white points (LP). Occasionally, Normand bay donkeys give birth to dark foals that lack LP and display the no light points (NLP) pattern. This pattern is more frequent and officially recognized in American miniature donkeys. The LP (or pangare) phenotype resembles that of the light bellied agouti pattern in mouse, while the NLP pattern resembles that of the mammalian recessive black phenotype; both phenotypes are associated with the agouti signaling protein gene (ASIP).

Findings

We used a panel of 127 donkeys to identify a recessive missense c.349 T > C variant in ASIP that was shown to be in complete association with the NLP phenotype. This variant results in a cysteine to arginine substitution at position 117 in the ASIP protein. This cysteine is highly-conserved among vertebrate ASIP proteins and was previously shown by mutagenesis experiments to lie within a functional site. Altogether, our results strongly support that the identified mutation is causative of the NLP phenotype.

Conclusions

Thus, we propose to name the c.[349 T > C] allele in donkeys, the a^nlp allele, which enlarges the panel of coat colour alleles in donkeys and ASIP recessive loss-of-function alleles in animals.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0112-x) contains supplementary material, which is available to authorized users.     

Functional consequences of neuromyelitis optica-IgG astrocyte interactions on blood-brain barrier permeability and granulocyte recruitment

Autoantibody neuromyelitis optica-IgG (NMO-IgG) recognizing aquaporin-4 (AQP4) is implicated as playing a central role in the physiopathology of NMO. The aim of this in vitro-based study was to characterize functional consequences of interaction between NMO-IgG and cells of the neurovascular unit (astrocytes and brain endothelium) that would provide insight into recognized features of NMO, namely altered blood-brain barrier (BBB) permeability and granulocyte recruitment. We used sera from NMO and longitudinally extensive transverse myelitis cases shown to bind in a characteristic perivascular pattern to primate cerebellar slices. Using flow cytometry, we found that sera from NMO-IgG-positive patients reacted with CNS-derived human fetal astrocytes, whereas sera from multiple sclerosis patients did not. We demonstrated that NMO-IgG binding to astrocytes alters aquaporin-4 polarized expression and increases permeability of a human BBB endothelium/astrocyte barrier. We further demonstrated that NMO-IgG binding to human fetal astrocytes can result in NK cell degranulation, astrocyte killing by Ab-dependent cellular cytotoxicity and complement-dependent granulocyte attraction through the BBB model. Our study highlights important functional roles for NMO-IgG that could account for pathological lesions and BBB dysfunction observed in NMO.     

Dephytinisation with Intrinsic Wheat Phytase and Iron Fortification Significantly Increase Iron Absorption from Fonio (Digitaria exilis) Meals in West African Women

Low iron and high phytic acid content make fonio based meals a poor source of bioavailable iron. Phytic acid degradation in fonio porridge using whole grain cereals as phytase source and effect on iron bioavailability when added to iron fortified fonio meals were investigated. Grains, nuts and seeds collected in Mali markets were screened for phytic acid and phytase activity. We performed an iron absorption study in Beninese women (n = 16), using non-dephytinised fonio porridge (FFP) and dephytinised fonio porridge (FWFP; 75% fonio-25% wheat), each fortified with ⁵⁷Fe or ⁵⁸Fe labeled FeSO₄. Iron absorption was quantified by measuring the erythrocyte incorporation of stable iron isotopes. Phytic acid varied from 0.39 (bambara nut) to 4.26 g/100 g DM (pumpkin seed), with oilseeds values higher than grains and nuts. Phytase activity ranged from 0.17±1.61 (fonio) to 2.9±1.3 phytase unit (PU) per g (whole wheat). Phytic acid was almost completely degraded in FWFP after 60 min of incubation (pH≈5.0, 50°C). Phytate∶iron molar ratios decreased from 23.7∶1 in FFP to 2.7∶1 in FWFP. Iron fortification further reduced phytate∶iron molar ratio to 1.9∶1 in FFP and 0.3∶1 in FWFP, respectively. Geometric mean (95% CI) iron absorption significantly increased from 2.6% (0.8–7.8) in FFP to 8.3% (3.8–17.9) in FWFP (P<0.0001). Dephytinisation of fonio porridge with intrinsic wheat phytase increased fractional iron absorption 3.2 times, suggesting it could be a possible strategy to decrease PA in cereal-based porridges.     

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.     

Parasite-induced suppression of aggregation under predation risk in a freshwater amphipod: Sociality of infected amphipods

Recent findings suggest that grouping with conspecifics is part of the behavioural defences developed by amphipod crustaceans to face predation risk by fish. Amphipods commonly serve as intermediate hosts for trophically transmitted parasites. These parasites are known for their ability to alter intermediate host phenotype in a way that promotes predation by definitive hosts, where they reproduce. If aggregation in amphipods dilutes the risk to be preyed on by fish, then it may dilute the probability of transmission for the parasite using fish as definitive hosts. Using experimental infections, we tested whether infection with the fish acanthocephalan Pomphorhynchus laevis alters attraction to conspecifics in the amphipod intermediate host Gammarus pulex. We also measured G. pulex's activity and reaction to light to detect potential links between changes in aggregation and changes in other behaviours. The attraction to conspecifics in the presence of predator cue, a behaviour found in uninfected gammarids, was cancelled by the infection, while phototaxis was reversed and activity unchanged. We found no correlation between the three behaviours in infected amphipods, while activity and aggregation were negatively correlated in uninfected individuals after the detection of predation cue. The physiological causes and the adaptive value of aggregation suppression are discussed in the context of a multidimensional manipulation.