Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

Tuft cells represent a fourth type of intestinal secretory cell that constitutes the primary source of endogenous intestinal opioids and are the only epithelial cell that constitutively express cyclooxygenases

Lithium carbonate in amyotrophic lateral sclerosis patients homozygous for the C-allele at SNP rs12608932 in UNC13A: protocol for a confirmatory, randomized, group-sequential, event-driven, double-blind, placebo-controlled trial

BackgroundGiven the large genetic heterogeneity in amyotrophic lateral sclerosis (ALS), it seems likely that genetic subgroups may benefit differently from treatment. An exploratory meta-analysis identified that patients homozygous for the C-allele at SNP rs12608932, a single nucleotide polymorphism in the gene UNC13A, had a statistically significant survival benefit when treated with lithium carbonate. We aim to confirm the efficacy of lithium carbonate on the time to death or respiratory insufficiency in patients with ALS homozygous for the C-allele at SNP rs12608932 in UNC13A. MethodsA randomized, group-sequential, event-driven, double-blind, placebo-controlled trial will be conducted in 15 sites across Europe and Australia. Patients will be genotyped for UNC13A; those homozygous for the C-allele at SNP rs12608932 will be eligible. Patients must have a diagnosis of ALS according to the revised El Escorial criteria, and a TRICALS risk-profile score between -6.0 and -2.0. An expected number of 1200 patients will be screened in order to enroll a target sample size of 171 patients. Patients will be randomly allocated in a 2:1 ratio to lithium carbonate or matching placebo, and treated for a maximum duration of 24 months. The primary endpoint is the time to death or respiratory insufficiency, whichever occurs first. Key secondary endpoints include functional decline, respiratory function, quality of life, tolerability, and safety. An interim analysis for futility and efficacy will be conducted after the occurrence of 41 events. DiscussionLithium carbonate has been proven to be safe and well-tolerated in patients with ALS. Given the favorable safety profile, the potential benefits are considered to outweigh the burden and risks associated with study participation. This study may provide conclusive evidence about the life-prolonging potential of lithium carbonate in a genetic ALS subgroup

A Randomized Trial of Intravenous Alteplase before Endovascular Treatment for Stroke

The value of administering intravenous alteplase before endovascular treatment (EVT) for acute ischemic stroke has not been studied extensively, particularly in non-Asian populations. METHODS We performed an open-label, multicenter, randomized trial in Europe involving patients with stroke who presented directly to a hospital that was capable of providing EVT and who were eligible for intravenous alteplase and EVT. Patients were randomly assigned in a 1:1 ratio to receive EVT alone or intravenous alteplase followed by EVT (the standard of care). The primary end point was functional outcome on the modified Rankin scale (range, 0 [no disability] to 6 [death]) at 90 days. We assessed the superiority of EVT alone over alteplase plus EVT, as well as noninferiority by a margin of 0.8 for the lower boundary of the 95% confidence interval for the odds ratio of the two trial groups. Death from any cause and symptomatic intracerebral hemorrhage were the main safety end points. RESULTS The analysis included 539 patients. The median score on the modified Rankin scale at 90 days was 3 (interquartile range, 2 to 5) with EVT alone and 2 (interquartile range, 2 to 5) with alteplase plus EVT. The adjusted common odds ratio was 0.84 (95% confidence interval [CI], 0.62 to 1.15; P=0.28), which showed neither superiority nor noninferiority of EVT alone. Mortality was 20.5% with EVT alone and 15.8% with alteplase plus EVT (adjusted odds ratio, 1.39; 95% CI, 0.84 to 2.30). Symptomatic intracerebral hemorrhage occurred in 5.9% and 5.3% of the patients in the respective groups (adjusted odds ratio, 1.30; 95% CI, 0.60 to 2.81). CONCLUSIONS In a randomized trial involving European patients, EVT alone was neither superior nor noninferior to intravenous alteplase followed by EVT with regard to disability outcome at 90 days after stroke. The incidence of symptomatic intracerebral hemorrhage was similar in the two groups

Targeted Genetic Screen in Amyotrophic Lateral Sclerosis Reveals Novel Genetic Variants with Synergistic Effect on Clinical Phenotype

Amyotrophic lateral sclerosis (ALS) is underpinned by an oligogenic rare variant architecture. Identified genetic variants of ALS include RNA-binding proteins containing prion-like domains (PrLDs). We hypothesized that screening genes encoding additional similar proteins will yield novel genetic causes of ALS. The most common genetic variant of ALS patients is a G4C2-repeat expansion within C9ORF72. We have shown that G4C2-repeat RNA sequesters RNA-binding proteins. A logical consequence of this is that loss-of-function mutations in G4C2-binding partners might contribute to ALS pathogenesis independently of and/or synergistically with C9ORF72 expansions. Targeted sequencing of genomic DNA encoding either RNA-binding proteins or known ALS genes (n = 274 genes) was performed in ALS patients to identify rare deleterious genetic variants and explore genotype-phenotype relationships. Genomic DNA was extracted from 103 ALS patients including 42 familial ALS patients and 61 young-onset (average age of onset 41 years) sporadic ALS patients; patients were chosen to maximize the probability of identifying genetic causes of ALS. Thirteen patients carried a G4C2-repeat expansion of C9ORF72. We identified 42 patients with rare deleterious variants; 6 patients carried more than one variant. Twelve mutations were discovered in known ALS genes which served as a validation of our strategy. Rare deleterious variants in RNA-binding proteins were significantly enriched in ALS patients compared to control frequencies (p = 5.31E-18). Nineteen patients featured at least one variant in a RNA-binding protein containing a PrLD. The number of variants per patient correlated with rate of disease progression (t-test, p = 0.033). We identified eighteen patients with a single variant in a G4C2-repeat binding protein. Patients with a G4C2-binding protein variant in combination with a C9ORF72 expansion had a significantly faster disease course (t-test, p = 0.025). Our data are consistent with an oligogenic model of ALS. We provide evidence for a number of entirely novel genetic variants of ALS caused by mutations in RNA-binding proteins. Moreover we show that these mutations act synergistically with each other and with C9ORF72 expansions to modify the clinical phenotype of ALS. A key finding is that this synergy is present only between functionally interacting variants. This work has significant implications for ALS therapy development

Telomere length analysis in amyotrophic lateral sclerosis using large-scale whole genome sequence data

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, leading to progressive weakness of voluntary muscles, with death following from neuromuscular respiratory failure, typically within 3 to 5 years. There is a strong genetic contribution to ALS risk. In 10% or more, a family history of ALS or frontotemporal dementia is obtained, and the Mendelian genes responsible for ALS in such families have now been identified in about 50% of cases. Only about 14% of apparently sporadic ALS is explained by known genetic variation, suggesting that other forms of genetic variation are important. Telomeres maintain DNA integrity during cellular replication, differ between sexes, and shorten naturally with age. Sex and age are risk factors for ALS and we therefore investigated telomere length in ALS. MethodsSamples were from Project MinE, an international ALS whole genome sequencing consortium that includes phenotype data. For validation we used donated brain samples from motor cortex from people with ALS and controls. Ancestry and relatedness were evaluated by principal components analysis and relationship matrices of DNA microarray data. Whole genome sequence data were from Illumina HiSeq platforms and aligned using the Isaac pipeline. TelSeq was used to quantify telomere length using whole genome sequence data. We tested the association of telomere length with ALS and ALS survival using Cox regression. ResultsThere were 6,580 whole genome sequences, reducing to 6,195 samples (4,315 from people with ALS and 1,880 controls) after quality control, and 159 brain samples (106 ALS, 53 controls). Accounting for age and sex, there was a 20% (95% CI 14%, 25%) increase of telomere length in people with ALS compared to controls (p = 1.1 x 10(-12)), validated in the brain samples (p = 0.03). Those with shorter telomeres had a 10% increase in median survival (p = 5.0x10(-7)). Although there was no difference in telomere length between sporadic ALS and familial ALS (p=0.64), telomere length in 334 people with ALS due to expanded C9orf72 repeats was shorter than in those without expanded C9orf72 repeats (p = 5.0x10(-4)). DiscussionAlthough telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS

Insulin Sensitivity Is Reflected by Characteristic Metabolic Fingerprints - A Fourier Transform Mass Spectrometric Non-Targeted Metabolomics Approach

BACKGROUND: A decline in body insulin sensitivity in apparently healthy individuals indicates a high risk to develop type 2 diabetes. Investigating the metabolic fingerprints of individuals with different whole body insulin sensitivity according to the formula of Matsuda, et al. (ISI(Matsuda)) by a non-targeted metabolomics approach we aimed a) to figure out an unsuspicious and altered metabolic pattern, b) to estimate a threshold related to these changes based on the ISI, and c) to identify the metabolic pathways responsible for the discrimination of the two patterns. METHODOLOGY AND PRINCIPAL FINDINGS: By applying infusion ion cyclotron resonance Fourier transform mass spectrometry, we analyzed plasma of 46 non-diabetic subjects exhibiting high to low insulin sensitivities. The orthogonal partial least square model revealed a cluster of 28 individuals with alterations in their metabolic fingerprints associated with a decline in insulin sensitivity. This group could be separated from 18 subjects with an unsuspicious metabolite pattern. The orthogonal signal correction score scatter plot suggests a threshold of an ISI(Matsuda) of 15 for the discrimination of these two groups. Of note, a potential subgroup represented by eight individuals (ISI(Matsuda) value between 8.5 and 15) was identified in different models. This subgroup may indicate a metabolic transition state, since it is already located within the cluster of individuals with declined insulin sensitivity but the metabolic fingerprints still show some similarities with unaffected individuals (ISI >15). Moreover, the highest number of metabolite intensity differences between unsuspicious and altered metabolic fingerprints was detected in lipid metabolic pathways (arachidonic acid metabolism, metabolism of essential fatty acids and biosynthesis of unsaturated fatty acids), steroid hormone biosyntheses and bile acid metabolism, based on data evaluation using the metabolic annotation interface MassTRIX. CONCLUSIONS: Our results suggest that altered metabolite patterns that reflect changes in insulin sensitivity respectively the ISI(Matsuda) are dominated by lipid-related pathways. Furthermore, a metabolic transition state reflected by heterogeneous metabolite fingerprints may precede severe alterations of metabolism. Our findings offer future prospects for novel insights in the pathogenesis of the pre-diabetic phase

When One Size Does Not Fit All: A Simple Statistical Method to Deal with Across-Individual Variations of Effects

In science, it is a common experience to discover that although the investigated effect is very clear in some individuals, statistical tests are not significant because the effect is null or even opposite in other individuals. Indeed, t-tests, Anovas and linear regressions compare the average effect with respect to its inter-individual variability, so that they can fail to evidence a factor that has a high effect in many individuals (with respect to the intra-individual variability). In such paradoxical situations, statistical tools are at odds with the researcher’s aim to uncover any factor that affects individual behavior, and not only those with stereotypical effects. In order to go beyond the reductive and sometimes illusory description of the average behavior, we propose a simple statistical method: applying a Kolmogorov-Smirnov test to assess whether the distribution of p-values provided by individual tests is significantly biased towards zero. Using Monte-Carlo studies, we assess the power of this two-step procedure with respect to RM Anova and multilevel mixed-effect analyses, and probe its robustness when individual data violate the assumption of normality and homoscedasticity. We find that the method is powerful and robust even with small sample sizes for which multilevel methods reach their limits. In contrast to existing methods for combining p-values, the Kolmogorov-Smirnov test has unique resistance to outlier individuals: it cannot yield significance based on a high effect in one or two exceptional individuals, which allows drawing valid population inferences. The simplicity and ease of use of our method facilitates the identification of factors that would otherwise be overlooked because they affect individual behavior in significant but variable ways, and its power and reliability with small sample sizes (<30–50 individuals) suggest it as a tool of choice in exploratory studies

Case report: mechanisms of HIV elite control in two African women

Background The majority of people living with HIV require antiretroviral therapy (ART) for controlling viral replication, however there are rare HIV controllers who spontaneously and durably control HIV in the absence of treatment. Understanding what mediates viral control in these individuals has provided us with insights into the immune mechanisms that may be important to induce for a vaccine or functional cure for HIV. To date, few African elite controllers from high incidence settings have been described. We identified virological controllers from the CAPRISA 002 cohort of HIV-1 subtype C infected women in KwaZulu Natal, South Africa, two (1%) of whom were elite controllers. We examined the genetic, clinical, immunological and virological characteristics of these two elite HIV controllers in detail, to determine whether they exhibit features of putative viral control similar to those described for elite controllers reported in the literature. Case presentation In this case report, we present clinical features, CD4+ T cell and viral load trajectories for two African women over 7 years of HIV infection. Viral load became undetectable 10 months after HIV infection in Elite Controller 1 (EC1), and after 6 weeks in Elite Controller 2 (EC2), and remained undetectable for the duration of follow-up, in the absence of ART. Both elite controllers expressed multiple HLA Class I and II haplotypes previously associated with slower disease progression (HLA-A*74:01, HLA-B*44:03, HLA-B*81:01, HLA-B*57:03, HLA-DRB1*13). Fitness assays revealed that both women were infected with replication competent viruses, and both expressed higher mRNA levels of p21, a host restriction factor associated with viral control. HIV-specific T cell responses were examined using flow cytometry. EC1 mounted high frequency HIV-specific CD8+ T cell responses, including a B*81:01-restricted Gag TL9 response. Unusually, EC2 had evidence of pre-infection HIV-specific CD4+ T cell responses. Conclusion We identified some features typical of elite controllers, including high magnitude HIV-specific responses and beneficial HLA. In addition, we made the atypical finding of pre-infection HIV-specific immunity in one elite controller, that may have contributed to very early viral control. This report highlights the importance of studying HIV controllers in high incidence settings

Genetic Control of Resistance to Trypanosoma brucei brucei Infection in Mice

Trypanosoma brucei are extracellular protozoa transmitted to mammalian host by the tsetse fly. They developed several mechanisms that subvert host's immune defenses. Therefore analysis of genes affecting host's resistance to infection can reveal critical aspects of host-parasite interactions. Trypanosoma brucei brucei infects many animal species including livestock, with particularly severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to T. b. brucei. However, genes controlling susceptibility to this parasite have not been mapped. We analyzed the genetic control of survival after T. b. brucei infection using CcS/Dem recombinant congenic (RC) strains, each of which contains a different random set of 12.5% genes of their donor parental strain STS/A on the BALB/c genetic background. The RC strain CcS-11 is even more susceptible to parasites than BALB/c or STS/A. In F2 hybrids between BALB/c and CcS-11 we detected and mapped four loci, Tbbr1-4 (Trypanosoma brucei brucei response 1–4), that control survival after T. b. brucei infection. Tbbr1 (chromosome 3) and Tbbr2 (chromosome 12) have independent effects, Tbbr3 (chromosome 7) and Tbbr4 (chromosome 19) were detected by their mutual inter-genic interaction. Tbbr2 was precision mapped to a segment of 2.15 Mb that contains 26 genes

Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

Background Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. Methodology/Principal Findings Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2–3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. Conclusions/Significance These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation