Biallelic SQSTM1 mutations in early-onset, variably progressive neurodegeneration.

OBJECTIVE: To characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families. METHODS: We used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model. RESULTS: We identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years. CONCLUSIONS: This study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy

Genetic variants of HvCbf14 are statistically associated with frost tolerance in a European germplasm collection of Hordeum vulgare

Two quantitative trait loci (Fr-H1 and Fr-H2) for frost tolerance (FT) have been discovered on the long arm of chromosome 5H in barley. Two tightly linked groups of CBF genes, known to play a key role in the FT regulatory network in A. thaliana, have been found to co-segregate with Fr-H2. Here, we investigate the allelic variations of four barley CBF genes (HvCbf3, HvCbf6, HvCbf9 and HvCbf14) in a panel of European cultivars, landraces and H. spontaneum accessions. In the cultivars a reduction of nucleotide and haplotype diversities in CBFs compared with the landraces and the wild ancestor H. spontaneum, was evident. In particular, in cultivars the loss of HvCbf9 genetic variants was higher compared to other sequences. In order to verify if the pattern of CBF genetic variants correlated with the level of FT, an association procedure was adopted. The pairwise analysis of linkage disequilibrium (LD) among the genetic variants in four CBF genes was computed to evaluate the resolution of the association procedure. The pairwise plotting revealed a low level of LD in cultivated varieties, despite the tight physical linkage of CBF genes analysed. A structured association procedure based on a general liner model was implemented, including the variants in CBFs, of Vrn-H1, and of two reference genes not involved in FT (α-Amy1 and Gapdh) and considering the phenotypic data for FT. Association analysis recovered two nucleotide variants of HvCbf14 and one nucleotide variant of Vrn-H1 as statistically associated to FT

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

HIV-1 gag-specific cytotoxic T lymphocytes defined with recombinant vaccinia virus and synthetic peptides.

Current candidate vaccines fail to protect primates against challenge with human immunodeficiency virus (HIV) in the presence of antibody responses; this underlines the importance of studying cell-mediated immunity to HIV and identifying specific epitopes that stimulate cytotoxic T lymphocytes (CTL). Using a recombinant vaccinia virus to express the gag protein of HIV-1 we found HLA class-I-restricted gag-specific CTL in thirteen out of fifteen healthy HIV seropositive patients. We then used short synthetic peptides in the lysis assay to screen for gag CTL epitopes. In one patient we have identified a peptide in p24 that is recognized by CTL in association with HLA-B27. This peptide, and further peptide sequences defined by these methods, could be incorporated in vaccines designed to induce cell-mediated immunity against HIV

Defects in GLP-1 response to an oral challenge do not play a significant role in the pathogenesis of prediabetes.

CONTEXT: There has been much speculation as to whether defects in glucagon-like peptide-1 (GLP-1) secretion play a role in the pathogenesis of type 2 diabetes and the progression from normal glucose tolerance to prediabetes and diabetes. OBJECTIVE: Our objective was to determine whether fasting and postchallenge concentrations of active and total GLP-1 decrease as glucose tolerance and insulin secretion worsen across the spectrum of prediabetes. DESIGN: This was a cross-sectional study. SETTING: The study was performed in the clinical research unit of an academic medical center. PARTICIPANTS: Participants included 165 subjects with a fasting glucose below 7.0 mmol/liter and not taking medications known to affect gastrointestinal motility or glucose metabolism. INTERVENTION: Intervention included a 2-h, 75-g oral glucose tolerance test with insulin, C-peptide, glucagon, and GLP-1 measurements at seven time points. MAIN OUTCOME MEASURE: We evaluated the association of integrated, incremental active, and total GLP-1 concentrations with integrated, incremental glucose response to 75 g oral glucose. RESULTS: After accounting for covariates, there was no evidence of a relationship of incremental glucose concentrations after oral glucose tolerance test with active and total GLP-1 (r(s) = -0.16 and P = 0.14, and r(s) = 0.00 and P > 0.9, respectively). There also was no association of GLP-1 concentrations with insulin secretion and action. CONCLUSIONS: The lack of association of GLP-1 concentrations with glucose tolerance status and with insulin secretion and action in a cohort encompassing the full spectrum of prediabetes strongly argues against a significant contribution of defects in GLP-1 secretion to the pathogenesis of prediabetes

The effect of a bile Acid sequestrant on glucose metabolism in subjects with type 2 diabetes.

We designed an experiment to examine the effect of bile acid sequestration with Colesevelam on fasting and postprandial glucose metabolism in type 2 diabetes. To do so, we tested the hypothesis that Colesevelam increases the disposition index (DI), and this increase is associated with increased glucagon-like peptide-1 (GLP-1) concentrations. Thirty-eight subjects on metformin monotherapy were studied using a double-blind, placebo-controlled, parallel-group design. Subjects were studied before and after 12 weeks of Colesevelam or placebo using a labeled triple-tracer mixed meal to measure the rate of meal appearance (Meal Ra), endogenous glucose production (EGP), and glucose disappearance (Rd). Insulin sensitivity and \u3b2-cell responsivity indices were estimated using the oral minimal model and then used to calculate DI. Therapy with Colesevelam was associated with a decrease in fasting (7.0 \ub1 0.2 vs. 6.6 \ub1 0.2 mmol/L; P = 0.004) and postprandial glucose concentrations (3,145 \ub1 138 vs. 2,896 \ub1 127 mmol/6 h; P = 0.01) in the absence of a change in insulin concentrations. Minimal model-derived indices of insulin secretion and action were unchanged. Postprandial GLP-1 concentrations were not altered by Colesevelam. Although EGP and Rd were unchanged, integrated Meal Ra was decreased by Colesevelam (5,191 \ub1 204 vs. 5,817 \ub1 204 \u3bcmol/kg/6 h; P = 0.04), suggesting increased splanchnic sequestration of meal-derived glucose

Direct Effects of Exendin-(9,39) and GLP-1-(9,36)amide on Insulin Action, \u3b2-Cell Function and Glucose Metabolism in Non-Diabetic Subjects.

Exendin-(9,39) is a competitive antagonist of Glucagon-Like Peptide-1 (GLP-1) at its receptor. However, it is unclear if it has direct and unique effects of its own. We tested the hypothesis that Exendin-(9,39) and GLP-1-(9,36) amide have direct effects on hormone secretion and \u3b2-cell function as well as glucose metabolism in healthy subjects. Glucose containing [3-3H]-glucose was infused to mimic the systemic appearance of glucose after a meal. Saline (S), GLP-1-(9,36) amide (G), or Exendin-(9,39) at 30pmol/kg/min (Ex30) or 300pmol/kg/min (Ex300) were infused in random order on separate days. Integrated glucose concentrations were slightly but significantly increased by Exendin-(9,39) (365\ub143 vs. 383\ub135 vs. 492\ub149 vs. 337\ub150 mmol per 6hr, S, Ex30, Ex300 and G respectively, p=0.05). Insulin secretion did not differ amongst groups. However, insulin action was lowered by Exendin-(9,39) (25\ub14 vs. 20\ub14 vs. 18\ub13 vs. 21\ub14 10-4 dl/kg(min per \u3bcU/ml), p=0.02), resulting in a lower disposition index during Exendin-(9,39) infusion (1118\ub1118 vs. 816\ub183 vs. 725\ub1127 vs. 955\ub1166 10-14 dl/kg/min2 per pmol/l, p=0.003). Endogenous glucose production and glucose disappearance did not differ significantly amongst groups. We conclude that Exendin-(9,39), but not GLP-1-(9,36) amide, decreases insulin action and disposition index in healthy humans