IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to?cause programmed cell death. We discovered that?thioredoxin-interacting protein (TXNIP) is?a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule?IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.     

Effect of nitric oxide signaling in bacterial-treated soybean plant under salt stress

To understand protective roles of nitric oxide against salt stress, the effects of exogenous sodium nitroprusside on activities of lipoxygenase, peroxidase, phenylalanine ammonialyase, catalase, superoxide dismutase enzymes, proline accumulation, and distribution of sodium in soybean plants under salt were determined. Application of sodium nitroprusside + bacterium enhanced plant growth-promotion characteristics, activities of different enzymes, and proline accumulation in the presence of sodium nitroprusside under salt stress. Treatment with NaCl at 200 mM and sodium nitroprusside (0.1 mM) reduced Na⁺ levels but increased K⁺ levels in leaves in comparison with the NaCl-treated plants. Correspondingly, the plants treated with exogenous sodium nitroprusside and NaCl maintained a lower ratio of [Na⁺]/[K⁺] in NaCl-stressed plants.     

Aspergillus terreus NADP-glutamate dehydrogenase is kinetically distinct from the allosteric enzyme of other Aspergilli

NADP-Glutamate dehydrogenase (NADP-GDH) located at the interface of carbon and nitrogen metabolism has the potential to dictate fungal carbon flux. NADP-GDH from Aspergillus terreus, itaconate producer and an opportunistic pathogen, was purified to homogeneity using novel reactive dye-affinity resins. The pure enzyme was extensively characterized for its biochemical and kinetic properties and compared with its well studied Aspergillus niger counterpart. The A. terreus NADP-GDH was more stable and showed non-competitive ammonium inhibition with respect to glutamate. It exhibited hyperbolic 2-oxoglutarate saturation albeit with a weak substrate inhibition. This is in contrast to the allosteric nature of the enzyme from other Aspergilli. Differential susceptibility to chymotrypsin is also consistent with the absence of substrate cooperativity and conformational changes associated with A. terreus NADP-GDH. The non-allosteric nature of A. terreus NADP-GDH provides a unique opportunity to assess the contribution of allostery in metabolic regulation.     

Human embryonic stem cell proliferation and differentiation as parameters to evaluate developmental toxicity

In vitro models based on embryonic stem cells (ESC) are highly promising for improvement of predictive toxicology screening in humans. After the successful validation of embryonic stem cell test (EST) in 2001; concerns have been raised on the usage of mouse ESC and also the morphological evaluation of beating cell clusters. This requires specialized skill-sets and is highly prone to misjudgement and false positive results. To overcome these limitations, we undertook the present study incorporating improvisations over the conventional EST. Here, we explored the potential of a human ESC (hESC)-based assay to evaluate the potential toxicity of penicillin-G, caffeine, and hydroxyurea. Drug treatment inhibited hESC adhesion and substantially altered the morphology and viability (～ 50%) of embryoid bodies (EBs). Flow cytometry analysis not only showed a significant increase of apoptotic cells in the highest doses but also induced a diverse pattern in DNA content and cell cycle distribution relative to control. Both semi-quantitative and quantitative RT-PCR studies revealed a selective down regulation of markers associated with stemness (Nanog, Rex1, SOX-2, and hTERT); cardiac mesoderm (Cripto1, MEF-2C, and Brachyury); hepatic endoderm (AFP, HNF-3β, HNF-4α, GATA-4, and SOX-17); and neuroectoderm (Nestin, SOX-1, NURR1, NEFH, Synaptophysin, TH, and Olig2) in a drug as well as dose dependent manner indicating abnormal differentiation. Furthermore, a decrease in the expression of AFP and GFAP proteins followed by a dose-dependent reduction in the levels of hCG-β, progesterone-II, and estradiol hormones was demonstrated by immunocytochemistry and ECLIA, respectively. This new and unique approach comprising of DNA cell cycle analysis, germ layer-specific marker expression and hormone levels as endpoints might offer a clinically relevant and commercially viable alternative for predicting in vivo developmental toxicity.     

Dysfunction of the ubiquitin ligase ube3a may be associated with synaptic pathophysiology in a mouse model of huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.     

Evaluation of <Emphasis Type="Italic">in silico</Emphasis> algorithms for use with ACMG/AMP clinical variant interpretation guidelines

Background

The American College of Medical Genetics and American College of Pathologists (ACMG/AMP) variant classification guidelines for clinical reporting are widely used in diagnostic laboratories for variant interpretation. The ACMG/AMP guidelines recommend complete concordance of predictions among all in silico algorithms used without specifying the number or types of algorithms. The subjective nature of this recommendation contributes to discordance of variant classification among clinical laboratories and prevents definitive classification of variants.

Results

Using 14,819 benign or pathogenic missense variants from the ClinVar database, we compared performance of 25 algorithms across datasets differing in distinct biological and technical variables. There was wide variability in concordance among different combinations of algorithms with particularly low concordance for benign variants. We also identify a previously unreported source of error in variant interpretation (false concordance) where concordant in silico predictions are opposite to the evidence provided by other sources. We identified recently developed algorithms with high predictive power and robust to variables such as disease mechanism, gene constraint, and mode of inheritance, although poorer performing algorithms are more frequently used based on review of the clinical genetics literature (2011–2017).

Conclusions

Our analyses identify algorithms with high performance characteristics independent of underlying disease mechanisms. We describe combinations of algorithms with increased concordance that should improve in silico algorithm usage during assessment of clinically relevant variants using the ACMG/AMP guidelines.

     

Caenorhabditis elegans Recognizes a Bacterial Quorum-sensing Signal Molecule through the AWCON Neuron

In a process known as quorum sensing, bacteria use chemicals called autoinducers for cell-cell communication. Population-wide detection of autoinducers enables bacteria to orchestrate collective behaviors. In the animal kingdom detection of chemicals is vital for success in locating food, finding hosts, and avoiding predators. This behavior, termed chemotaxis, is especially well studied in the nematode Caenorhabditis elegans. Here we demonstrate that the Vibrio cholerae autoinducer (S)-3-hydroxytridecan-4-one, termed CAI-1, influences chemotaxis in C. elegans. C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant defective for CAI-1 production. The position of the CAI-1 ketone moiety is the key feature driving CAI-1-directed nematode behavior. CAI-1 is detected by the C. elegans amphid sensory neuron AWC^ON. Laser ablation of the AWC^ON cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. These analyses define the structural features of a bacterial-produced signal and the nematode chemosensory neuron that permit cross-kingdom interaction.