BCAA catabolism in brown fat controls energy homeostasis through SLC25A44.

Branched-chain amino acid (BCAA; valine, leucine and isoleucine) supplementation is often beneficial to energy expenditure; however, increased circulating levels of BCAA are linked to obesity and diabetes. The mechanisms of this paradox remain unclear. Here we report that, on cold exposure, brown adipose tissue (BAT) actively utilizes BCAA in the mitochondria for thermogenesis and promotes systemic BCAA clearance in mice and humans. In turn, a BAT-specific defect in BCAA catabolism attenuates systemic BCAA clearance, BAT fuel oxidation and thermogenesis, leading to diet-induced obesity and glucose intolerance. Mechanistically, active BCAA catabolism in BAT is mediated by SLC25A44, which transports BCAAs into mitochondria. Our results suggest that BAT serves as a key metabolic filter that controls BCAA clearance via SLC25A44, thereby contributing to the improvement of metabolic health

First-line therapy in atypical hemolytic uremic syndrome: consideration on infants with a poor prognosis.

BackgroundAtypical hemolytic uremic syndrome (aHUS) is a rare and heterogeneous disorder. The first line treatment of aHUS is plasma therapy, but in the past few years, the recommendations have changed greatly with the advent of eculizumab, a humanized monoclonal anti C5-antibody. Although recent recommendations suggest using it as a primary treatment for aHUS, important questions have arisen about the necessity of immediate use of eculizumab in all cases. We aimed to draw attention to a specific subgroup of aHUS patients with rapid disease progression and high mortality, in whom plasma therapy may not be feasible.MethodsWe present three pediatric patients of acute complement-mediated HUS with a fatal outcome. Classical and alternative complement pathway activity, levels of complement factors C3, C4, H, B and I, as well as of anti-factor H autoantibody and of ADAMTS13 activity were determined. The coding regions of CFH, CFI, CD46, THBD, CFB and C3 genes were sequenced and the copy number of CFI, CD46, CFH and related genes were analyzed.ResultsWe found severe activation and consumption of complement components in these patients, furthermore, in one patient we identified a previously not reported mutation in CFH (Ser722Stop), supporting the diagnosis of complement-mediated HUS. These patients were not responsive to the FFP therapy, and all cases had fatal outcome.ConclusionTaking the heterogeneity and the variable prognosis of atypical HUS into account, we suggest that the immediate use of eculizumab should be considered as first-line therapy in certain small children with complement dysregulation

Extensions of MADM (Mosaic Analysis with Double Markers) in Mice

Mosaic Analysis with Double Markers (MADM) is a method for generating genetically mosaic mice, in which sibling mutant and wild-type cells are labeled with different fluorescent markers. It is a powerful tool that enables analysis of gene function at the single cell level in vivo. It requires transgenic cassettes to be located between the centromere and the mutation in the gene of interest on the same chromosome. Here we compare procedures for introduction of MADM cassettes into new loci in the mouse genome, and describe new approaches for expanding the utility of MADM. We show that: 1) Targeted homologous recombination outperforms random transgenesis in generation of reliably expressed MADM cassettes, 2) MADM cassettes in new genomic loci need to be validated for biallelic and ubiquitous expression, 3) Recombination between MADM cassettes on different chromosomes can be used to study reciprocal chromosomal deletions/duplications, and 4) MADM can be modified to permit transgene expression by combining it with a binary expression system. The advances described in this study expand current, and enable new and more versatile applications of MADM

DLG5 variants are associated with multiple congenital anomalies including ciliopathy phenotypes

BackgroundCilia are dynamic cellular extensions that generate and sense signals to orchestrate proper development and tissue homeostasis. They rely on the underlying polarisation of cells to participate in signalling. Cilia dysfunction is a well-known cause of several diseases that affect multiple organ systems including the kidneys, brain, heart, respiratory tract, skeleton and retina.MethodsAmong individuals from four unrelated families, we identified variants in discs large 5 (DLG5) that manifested in a variety of pathologies. In our proband, we also examined patient tissues. We depleted dlg5 in Xenopus tropicalis frog embryos to generate a loss-of-function model. Finally, we tested the pathogenicity of DLG5 patient variants through rescue experiments in the frog model.ResultsPatients with variants of DLG5 were found to have a variety of phenotypes including cystic kidneys, nephrotic syndrome, hydrocephalus, limb abnormalities, congenital heart disease and craniofacial malformations. We also observed a loss of cilia in cystic kidney tissue of our proband. Knockdown of dlg5 in Xenopus embryos recapitulated many of these phenotypes and resulted in a loss of cilia in multiple tissues. Unlike introduction of wildtype DLG5 in frog embryos depleted of dlg5, introduction of DLG5 patient variants was largely ineffective in restoring proper ciliation and tissue morphology in the kidney and brain suggesting that the variants were indeed detrimental to function.ConclusionThese findings in both patient tissues and Xenopus shed light on how mutations in DLG5 may lead to tissue-specific manifestations of disease. DLG5 is essential for cilia and many of the patient phenotypes are in the ciliopathy spectrum.</jats:sec

Crowding Alone Cannot Account for Cosolute Effect on Amyloid Aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed “macromolecular crowding”. Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation

Clinical and Functional Characterization of URAT1 Variants

Idiopathic renal hypouricaemia is an inherited form of hypouricaemia, associated with abnormal renal handling of uric acid. There is excessive urinary wasting of uric acid resulting in hypouricaemia. Patients may be asymptomatic, but the persistent urinary abnormalities may manifest as renal stone disease, and hypouricaemia may manifest as exercise induced acute kidney injury. Here we have identified Macedonian and British patients with hypouricaemia, who presented with a variety of renal symptoms and signs including renal stone disease, hematuria, pyelonephritis and nephrocalcinosis. We have identified heterozygous missense mutations in SLC22A12 encoding the urate transporter protein URAT1 and correlate these genetic findings with functional characterization. Urate handling was determined using uptake experiments in HEK293 cells. This data highlights the importance of the URAT1 renal urate transporter in determining serum urate concentrations and the clinical phenotypes, including nephrolithiasis, that should prompt the clinician to suspect an inherited form of renal hypouricaemia

A tau homeostasis signature is linked with the cellular and regional vulnerability of excitatory neurons to tau pathology.

Excitatory neurons are preferentially impaired in early Alzheimer's disease but the pathways contributing to their relative vulnerability remain largely unknown. Here we report that pathological tau accumulation takes place predominantly in excitatory neurons compared to inhibitory neurons, not only in the entorhinal cortex, a brain region affected in early Alzheimer's disease, but also in areas affected later by the disease. By analyzing RNA transcripts from single-nucleus RNA datasets, we identified a specific tau homeostasis signature of genes differentially expressed in excitatory compared to inhibitory neurons. One of the genes, BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, was identified as a hub, or master regulator, gene. We verified that reducing BAG3 levels in primary neurons exacerbated pathological tau accumulation, whereas BAG3 overexpression attenuated it. These results define a tau homeostasis signature that underlies the cellular and regional vulnerability of excitatory neurons to tau pathology

Fortunella margarita Transcriptional Reprogramming Triggered by Xanthomonas citri subsp. citri

<p>Abstract</p> <p>Background</p> <p>Citrus canker disease caused by the bacterial pathogen <it>Xanthomonas citri </it>subsp. <it>citri (</it>Xcc) <it>has </it>become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative <it>Fortunella margarita </it>Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in <it>Fortunella </it>and citrus at the molecular level.</p> <p>Results</p> <p><b>cDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray</b>. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus.</p> <p>Conclusion</p> <p>Functional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases.</p

Genetic drivers of kidney defects in the digeorge syndrome

BACKGROUND The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. METHODS We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. RESULTS We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P = 4.5×1014). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-Altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. CONCLUSIONS We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver