Molecular monitoring of the Leu-164 mutation of dihydrofolate reductase in a highly sulfadoxine/pyrimethamine-resistant area in Africa

Background

Procalcitonin (PCT) is closely correlated with parasite burden and clinical outcome in falciparum malaria. The role of PCT in tertian malaria has not previously been investigated.

Patients and methods

PCT serum levels in 37 patients with tertian malaria were analysed. Clinical and laboratory parameters were assessed and statistically correlated both to the initial PCT levels and during the course of the disease.

Results

PCT levels rose for one day after commencing treatment and declined thereafter. However, there was no significant correlation with parasite burden, clinical parameters, laboratory values, or the presence of semi-immunity. Before treatment, the majority of patients showed normal or slightly elevated PCT levels (< 2.5 ng/ml), but PCT was markedly elevated (4.8 – 47 ng/ml) in one third of the population. The two groups did not differ by any other of the assessed parameters. Thus, while the post-treatment course of PCT resembles falciparum malaria, the lack of correlation between disease severity and even high PCT levels in a large proportion of patients is intriguing.

Conclusions

There is a fundamental difference in the relationship of PCT with tertian malaria not seen in other infectious diseases in which elevated PCT levels have been observed. This suggests distinct pathophysiological pathways in malaria.     

The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.     

Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells

The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.     

Cellular localization of the cystic fibrosis transmembrane conductance regulator in mouse intestinal tract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and cGMP-regulated chloride channel critical to the regulation of intestinal fluid, chloride, and bicarbonate secretion. In cystic fibrosis (CF), mutations in CFTR result in downregulation of CFTR function and small intestinal obstruction. Unlike the human CF intestine, severe gastrointestinal disease and lethal obstruction is common in transgenic mice deficient in CFTR. The relevance of the physiology of CFTR and pathophysiology of CF in genetically altered mice to that of human CF disease remains incompletely understood. We hypothesized that the expression and distribution of CFTR in mouse intestine may differ from that of human and may contribute to the variation in disease expression between the two species. Using immunocytochemical and immunoblot techniques and well-characterized anti-rodent anti-CFTR antibodies, we examined the cellular distribution of CFTR in the mouse intestinal tract. We identified significant differences in villus distribution for CFTR in the mouse proximal small intestine compared to those previously reported for human and rat. These observations are important to the understanding of CFTR pathophysiology in transgenic CF mouse model systems and bear relevance to the different phenotypic expression of disease in mice compared to human.     

ROS Production via P2Y1-PKC-NOX2 Is Triggered by Extracellular ATP after Electrical Stimulation of Skeletal Muscle Cells

During exercise, skeletal muscle produces reactive oxygen species (ROS) via NADPH oxidase (NOX2) while inducing cellular adaptations associated with contractile activity. The signals involved in this mechanism are still a matter of study. ATP is released from skeletal muscle during electrical stimulation and can autocrinely signal through purinergic receptors; we searched for an influence of this signal in ROS production. The aim of this work was to characterize ROS production induced by electrical stimulation and extracellular ATP. ROS production was measured using two alternative probes; chloromethyl-2,7- dichlorodihydrofluorescein diacetate or electroporation to express the hydrogen peroxide-sensitive protein Hyper. Electrical stimulation (ES) triggered a transient ROS increase in muscle fibers which was mimicked by extracellular ATP and was prevented by both carbenoxolone and suramin; antagonists of pannexin channel and purinergic receptors respectively. In addition, transient ROS increase was prevented by apyrase, an ecto-nucleotidase. MRS2365, a P2Y₁ receptor agonist, induced a large signal while UTPyS (P2Y₂ agonist) elicited a much smaller signal, similar to the one seen when using ATP plus MRS2179, an antagonist of P2Y₁. Protein kinase C (PKC) inhibitors also blocked ES-induced ROS production. Our results indicate that physiological levels of electrical stimulation induce ROS production in skeletal muscle cells through release of extracellular ATP and activation of P2Y1 receptors. Use of selective NOX2 and PKC inhibitors suggests that ROS production induced by ES or extracellular ATP is mediated by NOX2 activated by PKC.     

Triglyceride-Increasing Alleles Associated with Protection against Type-2 Diabetes

Elevated plasma triglyceride (TG) levels are an established risk factor for type-2 diabetes (T2D). However, recent studies have hinted at the possibility that genetic risk for TG may paradoxically protect against T2D. In this study, we examined the association of genetic risk for TG with incident T2D, and the interaction of baseline TG with TG genetic risk on incident T2D in 13,247 European-Americans (EA) and 3,238 African-Americans (AA) from three prospective cohort studies. A TG genetic risk score (GRS) was calculated based on 31 validated single nucleotide polymorphisms (SNPs). We considered several baseline covariates, including body- mass index (BMI) and lipid traits. Among EA and AA, we find, as expected, that baseline levels of TG are strongly positively associated with incident T2D (p<2 x 10^-10). However, the TG GRS is negatively associated with T2D (p=0.013), upon adjusting for only race, in the full dataset. Upon additionally adjusting for age, sex, BMI, high-density lipoprotein cholesterol and TG, the TG GRS is significantly and negatively associated with T2D incidence (p=7.0 x 10^-8), with similar trends among both EA and AA. No single SNP appears to be driving this association. We also find a significant statistical interaction of the TG GRS with TG (p_interaction=3.3 x 10^-4), whereby the association of TG with incident T2D is strongest among those with low genetic risk for TG. Further research is needed to understand the likely pleiotropic mechanisms underlying these findings, and to clarify the causal relationship between T2D and TG.     

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA_257–264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific K^b:OVA_257–264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA_257–264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.     

The Use of Nanotrap Particles in the Enhanced Detection of Rift Valley Fever Virus Nucleoprotein

BackgroundRift Valley fever virus (RVFV) is a highly pathogenic arthropod-borne virus that has a detrimental effect on both livestock and human populations. While there are several diagnostic methodologies available for RVFV detection, many are not sensitive enough to diagnose early infections. Furthermore, detection may be hindered by high abundant proteins such as albumin. Previous findings have shown that Nanotrap particles can be used to significantly enhance detection of various small analytes of low abundance. We have expanded upon this repertoire to show that this simple and efficient sample preparation technology can drastically improve the detection of the RVFV nucleoprotein (NP), the most abundant and widely used viral protein for RVFV diagnostics.ResultsAfter screening multiple Nanotrap particle architectures, we found that one particle, NT45, was optimal for RVFV NP capture, as demonstrated by western blotting. NT45 significantly enhanced detection of the NP at levels undetectable without the technology. Importantly, we demonstrated that Nanotrap particles are capable of concentrating NP in a number of matrices, including infected cell lysates, viral supernatants, and animal sera. Specifically, NT45 enhanced detection of NP at various viral titers, multiplicity of infections, and time points. Our most dramatic results were observed in spiked serum samples, where high abundance serum proteins hindered detection of NP without Nanotrap particles. Nanotrap particles allowed for sample cleanup and subsequent detection of RVFV NP. Finally, we demonstrated that incubation of our samples with Nanotrap particles protects the NP from degradation over extended periods of time (up to 120 hours) and at elevated temperatures (at 37ºC).ConclusionThis study demonstrates that Nanotrap particles are capable of drastically lowering the limit of detection for RVFV NP by capturing, concentrating, and preserving RVFV NP in clinically relevant matrices. These studies can be extended to a wide range of pathogens and their analytes of diagnostic interest.     

Oxidative Stress Induces Caveolin 1 Degradation and Impairs Caveolae Functions in Skeletal Muscle Cells

Increased level of oxidative stress, a major actor of cellular aging, impairs the regenerative capacity of skeletal muscle and leads to the reduction in the number and size of muscle fibers causing sarcopenia. Caveolin 1 is the major component of caveolae, small membrane invaginations involved in signaling and endocytic trafficking. Their role has recently expanded to mechanosensing and to the regulation of oxidative stress-induced pathways. Here, we increased the amount of reactive oxidative species in myoblasts by addition of hydrogen peroxide (H₂O₂) at non-toxic concentrations. The expression level of caveolin 1 was significantly decreased as early as 10 min after 500 μM H₂O₂ treatment. This reduction was not observed in the presence of a proteasome inhibitor, suggesting that caveolin 1 was rapidly degraded by the proteasome. In spite of caveolin 1 decrease, caveolae were still able to assemble at the plasma membrane. Their functions however were significantly perturbed by oxidative stress. Endocytosis of a ceramide analog monitored by flow cytometry was significantly diminished after H₂O₂ treatment, indicating that oxidative stress impaired its selective internalization via caveolae. The contribution of caveolae to the plasma membrane reservoir has been monitored after osmotic cell swelling. H₂O₂ treatment increased membrane fragility revealing that treated cells were more sensitive to an acute mechanical stress. Altogether, our results indicate that H₂O₂ decreased caveolin 1 expression and impaired caveolae functions. These data give new insights on age-related deficiencies in skeletal muscle.