Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in the brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated brain-wide conditional LRRC8A knockout mice (LRRC8A bKO) using NestinCre -driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

Neuronal-driven glioma growth requires Gαi1 and Gαi3.

Neuroligin-3 (NLGN3) is necessary and sufficient to promote glioma cell growth. The recruitment of Gαi1/3 to the ligand-activated receptor tyrosine kinases (RTKs) is essential for mediating oncogenic signaling. Methods: Various genetic strategies were utilized to examine the requirement of Gαi1/3 in NLGN3-driven glioma cell growth. Results: NLGN3-induced Akt-mTORC1 and Erk activation was inhibited by decreasing Gαi1/3 expression. In contrast ectopic Gαi1/3 overexpression enhanced NLGN3-induced signaling. In glioma cells, NLGN3-induced cell growth, proliferation and migration were attenuated by Gαi1/3 depletion with shRNA, but facilitated with Gαi1/3 overexpression. Significantly, Gαi1/3 silencing inhibited orthotopic growth of patient-derived glioma xenografts in mouse brain, whereas forced Gαi1/3-overexpression in primary glioma xenografts significantly enhanced growth. The growth of brain-metastatic human lung cancer cells in mouse brain was largely inhibited with Gαi1/3 silencing. It was however expedited with ectopic Gαi1/3 overexpression. In human glioma Gαi3 upregulation was detected, correlating with poor prognosis. Conclusion: Gαi1/3 mediation of NLGN3-induced signaling is essential for neuronal-driven glioma growth.

Selective effects of protein 4.1N deficiency on neuroendocrine and reproductive systems.

Protein 4.1N, a member of the protein 4.1 family, is highly expressed in the brain. But its function remains to be fully defined. Using 4.1N-/- mice, we explored the function of 4.1N in vivo. We show that 4.1N-/- mice were born at a significantly reduced Mendelian ratio and exhibited high mortality between 3 to 5 weeks of age. Live 4.1N-/- mice were smaller than 4.1N+/+ mice. Notably, while there were no significant differences in organ/body weight ratio for most of the organs, the testis/body and ovary/body ratio were dramatically decreased in 4.1N-/- mice, demonstrating selective effects of 4.1N deficiency on the development of the reproductive systems. Histopathology of the reproductive organs showed atrophy of both testis and ovary. Specifically, in the testis there is a lack of spermatogenesis, lack of leydig cells and lack of mature sperm. Similarly, in the ovary there is a lack of follicular development and lack of corpora lutea formation, as well as lack of secretory changes in the endometrium. Examination of pituitary glands revealed that the secretory granules were significantly decreased in pituitary glands of 4.1N-/- compared to 4.1N+/+. Moreover, while GnRH was expressed in both neuronal cell body and axons in the hypothalamus of 4.1N+/+ mice, it was only expressed in the cell body but not the axons of 4.1N-/- mice. Our findings uncover a novel role for 4.1N in the axis of hypothalamus-pituitary gland-reproductive system.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

The contribution of beta-2 adrenergic, muscarinic and histamine (H1) receptors, calcium and potassium channels and cyclooxygenase pathway in the relaxant effect of Allium cepa L. on the tracheal smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: There are report regarding therapeutic effects for Allium cepa L. (A. cepa) in Iranian traditional medicine and the plant has showed anti-inflammatory, anti-allergic, anti-hyperglycemic, antioxidant, anti-cancer, anti-hypertension, anti-hypercholesterolemia and anti-asthmatic activities in previous studies. AIM OF THE STUDY: In this study, the contribution of ß2 adrenergic, muscarinic and histamine (H1) receptors, calcium and potassium channels, and cyclooxygenase pathway in the relaxant effect of A. cepa extract on tracheal smooth muscle (TSM) was assessed. MATERIALS AND METHODS: TSM was contracted by KCl (60 mM) or methacholine (10 µM) for 5 min and cumulative concentrations of A. cepa extract (2, 4, 8, 16, 32 and 64 mg/ml) were added to organ bath every 5 min. Theophylline (0.2, 0.4, 0.6 and 0.8 mM) as positive control, and saline (1 ml) as negative control were also examined in non-incubated tissues. The relaxant effect of A. cepa extract was examined on non-incubated and incubated TSM with propranolol, chlorpheniramine, diltiazem, atropine, glibenclamide and indomethacin. RESULTS: A. cepa showed concentration-dependent relaxant effects on non-incubated TSM contracted by KCl (60 mM) or methacholine (10 µM), (P < 0.01 to p < 0.001). There was no significant difference in the relaxant effects of A. cepa between non-incubated and incubated tissues with glibenclamide, atropine, chlorpheniramine and indomethacin. The plant extract showed significant lower relaxant effects in incubated TSM with propranolol and diltiazem compared to non-incubated tissues. EC50 values of A. cepa extract in incubated TSM with propranolol and diltiazem were significantly lower than non-incubated tissues (p < 0.001 and p < 0.05, respectively). The relaxant effects of different concentrations of the extract of A. cepa were not significantly different with those of theophylline. The concentrations of A. cepa extract and theophylline were significant correlated with their relaxant effects (p < 0.05 to p < 0.001). In incubated TSM with propranolol and diltiazem, concentration ratio minus one (CR-1) values was positive (2.65 ±â€¯0.63 and 1.28 ±â€¯0.43 respectively). CONCLUSION: The A. cepa extract showed relatively potent relaxant effect on TSM which was comparable to the effect of theophylline. The results showed that ß2-adrenergic stimulatory and/or calcium channel blockade are the possible mechanisms for the relaxant effects of the plant.

The Brain Network in a Model of Thalamocortical Dysrhythmia.

Sensory information processing and higher cognitive functions rely on the interactions between thalamus and cortex. Many types of neurological and psychiatric disorders are accompanied or driven by alterations in the brain connectivity. In this study, putative changes in functional and effective corticocortical (CC), thalamocortical (TC), and corticothalamic (CT) connectivity during wakefulness and slow-wave sleep (SWS) in a model of thalamocortical dysrhythmia, TRIP8b-/- mice, and in control (wild-type or WT) mice are described. Coherence and nonlinear Granger causality (GC) were calculated for twenty 10 s length epochs of SWS and active wakefulness (AW) of each animal. Coherence was reduced between 4 and ca 20 Hz in the cortex and between cortex and thalamus during SWS compared with AW in WT but not in TRIP8b-/- mice. Moreover, TRIP8b-/- mice showed lower CT coherence during AW compared with WT mice; these differences were no longer present during SWS. Unconditional GC analysis also showed sleep-related reductions in TC and CT couplings in WT mice, while TRIP8b-/- mice showed diminished wake and enhanced sleep CC coupling and rather strong CT-directed coupling during wake and sleep, although smaller during sleep. Conditional GC coupling analysis confirmed the diminished CC and enhanced CT coupling in TRIP8b-/- mice. Our findings indicate that altered properties of hyperpolarization-activated cyclic nucleotide-gated cation channels, characterizing TRIP8b-/- mice, have clear effects on CC, TC, and CT networks. A more complete understanding of the function of the altered communication within these networks awaits detailed phenotyping of TRIP8b-/- mice aimed at specifics of sensory and attentional processes.

In Vivo Electrocochleography in Hybrid Cochlear Implant Users Implicates TMPRSS3 in Spiral Ganglion Function.

Cochlear implantation, a surgical method to bypass cochlear hair cells and directly stimulate the spiral ganglion, is the standard treatment for severe-to-profound hearing loss. Changes in cochlear implant electrode array design and surgical approach now allow for preservation of acoustic hearing in the implanted ear. Electrocochleography (ECochG) was performed in eight hearing preservation subjects to assess hair cell and neural function and elucidate underlying genetic hearing loss. Three subjects had pathogenic variants in TMPRSS3 and five had pathogenic variants in genes known to affect the cochlear sensory partition. The mechanism by which variants in TMPRSS3 cause genetic hearing loss is unknown. We used a 500-Hz tone burst to record ECochG responses from an intracochlear electrode. Responses consist of a cochlear microphonic (hair cell) and an auditory nerve neurophonic. Cochlear microphonics did not differ between groups. Auditory nerve neurophonics were smaller, on average, in subjects with TMPRSS3 deafness. Results of this proof-of-concept study provide evidence that pathogenic variants in TMPRSS3 may impact function of the spiral ganglion. While ECochG as a clinical and research tool has been around for decades, this study illustrates a new application of ECochG in the study of genetic hearing and deafness in vivo.

Sushi repeat-containing protein X-linked 2: A novel phylogenetically conserved hypothalamo-pituitary protein.

Sushi repeat-containing protein X-linked 2 (SRPX2) is a novel protein associated with language development, synaptic plasticity, tissue remodeling, and angiogenesis. We investigated the expression and spatial localization of SRPX2 in normal mouse, rat, monkey, and human brain using in situ hybridization and immunohistochemistry. Antibody specificity was determined using in vitro siRNA based silencing of SRPX2. Cell type-specific expression was verified by double-labeling with oxytocin or vasopressin. Western blot was used to detect SRPX2 protein in rat and human plasma and cerebrospinal fluid. Unexpectedly, SRPX2 mRNA expression levels were strikingly higher in the hypothalamus as compared to the cortex. All SRPX2 immunoreactive (ir) neurons were localized in the hypothalamic paraventricular, periventricular, and supraoptic nuclei in mouse, rat, monkey, and human brain. SRPX2 colocalized with vasopressin or oxytocin in paraventricular and supraoptic neurons. Hypothalamic SRPX2-ir positive neurons gave origin to dense projections traveling ventrally and caudally toward the hypophysis. Intense axonal varicosities and terminal arborizations were identified in the rat and human neurohypophysis. SRPX2-ir cells were also found in the adenohypophysis. Light SRPX2-ir projections were observed in the dorsal and ventral raphe, locus coeruleus, and the nucleus of the solitary tract in mouse, rat and monkey. SRPX2 protein was also detected in plasma and CSF. Our data revealed intense phylogenetically conserved expression of SRPX2 protein in distinct hypothalamic nuclei and the hypophysis, suggesting its active role in the hypothalamo-pituitary axis. The presence of SRPX2 protein in the plasma and CSF suggests that some of its functions depend on secretion into body fluids.

Phytoceuticals in Fenugreek Ameliorate VLDL Overproduction and Insulin Resistance via the Insig Signaling Pathway.

SCOPE: This study aims to characterize the effect of fenugreek (Trigonella foenum-graecum) seed and its phytoceutical trigonelline in antimetabolic inflammation and ameliorating overproduction of very low density lipoprotein (VLDL) in insulin resistance. METHODS AND RESULTS: Two groups of genetic hyperlipidemic mice generated by depletion of cAMP responsive element binding protein H (CREBH) are fed either a chow containing 2% fenugreek seed or vehicle for 7 weeks. Q-RT-PCR and immunoblotting analysis demonstrated that fenugreek seed containing diet inhibits hepatic SREBP-1c activation and the subsequent de novo lipogenesis by enhancing expression of insulin-inducible gene-1 (Insig-1) and gene-2 (Insig-2). mRNA expression of PPARα and its target genes that are involved in fatty acid ß-oxidation are also upregulated in the fenugreek seed fed-mice which is accompanied by significantly reduced hepatic lipid accumulation and VLDL secretion, improved endoplasmic reticulum (ER) stress, and ameliorated metabolic inflammation. These actions enhance insulin sensitivity and improve hyperlipidemia. In vitro, treating a rat hepatoma cell line, McA-RH7777 (McA), with trigonelline is able to recapitulate the results observed in vivo. CONCLUSIONS: This study unveils a novel mechanism of fenugreek seed and trigonelline in countering hepatic VLDL overproduction and insulin resistance by enhancing the Insig signaling pathways and ameliorating metabolic inflammatory stress in the liver.

The role of TMPRSS6 and HFE variants in iron deficiency anemia in celiac disease.

We investigated the role of HFE C282Y, H63D, and TMPRSS6 A736V variants in the pathogenesis of iron deficiency anemia (IDA) in celiac disease (CD) patients, at diagnosis and after 1 year of gluten-free diet (GFD). Demographic and clinical features were prospectively recorded for all CD patients between 2013 and 2017. C282Y, H63D, and A736V variants were evaluated for CD patients and controls. Finally, 505 consecutive CD patients and 539 age-matched control subjects were enrolled. At diagnosis, 229 CD subjects had IDA (45.3%), with a subgroup of anemic patients (45.4%) presented persistent IDA at follow-up. C282Y allele frequency was significantly increased in CD compared with controls (1.1% vs 0.2%, P = .001), whereas H63D and A736V allele frequencies were similar among patients and controls (P = .92 and .84, respectively). At diagnosis, C282Y variant in anemic CD patients was significantly increased compared to nonanemic group (2% and 0.5%, P = .04). At follow-up, A736V was significantly increased in IDA persistent than in IDA not persistent (57.7% vs 35.2%, P < .0001). CD patients with H63D mutation showed higher Hb, MCV, serum iron, and ferritin levels than subjects without HFE mutations. Decreased hepcidin values were observed in anemic compared to nonanemic subjects at follow-up (1.22 ± 1.14 vs 2.08 ± 2.15, P < .001). This study suggests a protective role of HFE in IDA CD patients and confirms the role of TMPRSS6 in predicting oral iron response modulating hepcidin action on iron absorption. Iron supplementation therapeutic management in CD could depend on TMPRSS6 genotype that could predict persistent IDA despite iron supplementation and GFD.

N-3 polyunsaturated fatty acids increase hepatic fibroblast growth factor 21 sensitivity via a PPAR-γ-ß-klotho pathway.

SCOPE: Fibroblast growth factor 21 (FGF21) participated in fish oil-mediated hepatic lipid-regulation and anti-inflammatory effects in high-fat diet fed-mice. However, fish oil supplementation did not significantly increase FGF21 protein levels. Whether fish oil-induced benefits in the liver are related to hepatic FGF21 sensitivity remains unclear. METHODS AND RESULTS: Male C57BL/6J mice were fed a low-fat diet (LFD), a high-fat diet (HFD) or a fish oil-supplemented high-fat diet (FOD) for 12 weeks. Fish oil improved HFD-induced hepatic steatosis and inflammation, while it exerted no obvious effect on FGF21 protein expression. FGF21 knockout studies demonstrated FGF21 participated in fish oil-induced metabolic benefits. In vivo and in vitro experiments showed n-3 PUFAs, DHA and EPA, enhanced hepatic FGF21 sensitivity by increased hepatic ß-klotho expression. PPAR-γ siRNA knockdown and PPAR-γ antagonist (GW9662) treatment blocked the effects of DHA to enhance ß-klotho expression and FGF21 sensitivity. In addition, PPAR-γ activation enhanced ß-klotho expression and FGF21 signaling response, illustrating PPAR-γ participated in DHA-regulated ß-klotho expression and FGF21 sensitivity. CONCLUSION: Our data indicate n-3 PUFAs increase hepatic FGF21 sensitivity and ß-klotho expression probably through a PPAR-γ-dependent mechanism, and may thereby exert hepatic beneficial effects on lipid metabolism.

VMP1-related autophagy induced by a fructose-rich diet in ß-cells: its prevention by incretins.

The aim of the present study was to demonstrate the role of autophagy and incretins in the fructose-induced alteration of ß-cell mass and function. Normal Wistar rats were fed (3 weeks) with a commercial diet without (C) or with 10% fructose in drinking water (F) alone or plus sitagliptin (CS and FS) or exendin-4 (CE and FE). Serum levels of metabolic/endocrine parameters, ß-cell mass, morphology/ultrastructure and apoptosis, vacuole membrane protein 1 (VMP1) expression and glucose-stimulated insulin secretion (GSIS) were studied. Complementary to this, islets isolated from normal rats were cultured (3 days) without (C) or with F and F + exendin-4 or chloroquine. Expression of autophagy-related proteins [VMP1 and microtubule-associated protein light chain 3 (LC3)], apoptotic/antiapoptotic markers (caspase-3 and Bcl-2), GSIS and insulin mRNA levels were measured. F rats developed impaired glucose tolerance (IGT) and a significant increase in plasma triacylglycerols, thiobarbituric acid-reactive substances, insulin levels, homoeostasis model assessment (HOMA) for insulin resistance (HOMA-IR) and ß-cell function (HOMA-ß) indices. A significant reduction in ß-cell mass was associated with an increased apoptotic rate and morphological/ultrastructural changes indicative of autophagic activity. All these changes were prevented by either sitagliptin or exendin-4. In cultured islets, F significantly enhanced insulin mRNA and GSIS, decreased Bcl-2 mRNA levels and increased caspase-3 expression. Chloroquine reduced these changes, suggesting the participation of autophagy in this process. Indeed, F induced the increase of both VMP1 expression and LC3-II, suggesting that VMP1-related autophagy is activated in injured ß-cells. Exendin-4 prevented islet-cell damage and autophagy development. VMP1-related autophagy is a reactive process against F-induced islet dysfunction, being prevented by exendin-4 treatment. This knowledge could help in the use of autophagy as a potential target for preventing progression from IGT to type 2 diabetes mellitus.

Precise Somatotopic Thalamocortical Axon Guidance Depends on LPA-Mediated PRG-2/Radixin Signaling.

Precise connection of thalamic barreloids with their corresponding cortical barrels is critical for processing of vibrissal sensory information. Here, we show that PRG-2, a phospholipid-interacting molecule, is important for thalamocortical axon guidance. Developing thalamocortical fibers both in PRG-2 full knockout (KO) and in thalamus-specific KO mice prematurely entered the cortical plate, eventually innervating non-corresponding barrels. This misrouting relied on lost axonal sensitivity toward lysophosphatidic acid (LPA), which failed to repel PRG-2-deficient thalamocortical fibers. PRG-2 electroporation in the PRG-2-/- thalamus restored the aberrant cortical innervation. We identified radixin as a PRG-2 interaction partner and showed that radixin accumulation in growth cones and its LPA-dependent phosphorylation depend on its binding to specific regions within the C-terminal region of PRG-2. In vivo recordings and whisker-specific behavioral tests demonstrated sensory discrimination deficits in PRG-2-/- animals. Our data show that bioactive phospholipids and PRG-2 are critical for guiding thalamic axons to their proper cortical targets.

Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function.

Impaired signaling through the Fms-like tyrosine kinase 3 receptor increases osteoclast formation and bone damage in arthritis.

Osteoclasts are bone-resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms-like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms-like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms-like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms-like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms-like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin-induced arthritis using mice that lack the gene for Flt3l (Flt3L(-/-)) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L(-/-) mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L(-/-) mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L(-/-) mice with Fms-like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L(-/-) mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms-like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

[Stimulating Type I interferon response with small molecules: revival of an old idea]. / Stimuler la réponse interféron de type I avec des petites molécules : le renouveau d'une vieille idée.

Type I interferons play a central role in the establishment of an innate immune response against viral infections and tumor cells. Shortly after their discovery in 1957, several groups have looked for small molecules capable of inducing the expression of these cytokines with therapeutic applications in mind. A set of active compounds in mice were identified, but because of their relative inefficiency in humans for reasons not understood at the time, these studies fell into oblivion. In recent years, the characterization of pathogen recognition receptors and the signaling pathways they activate, together with the discovery of plasmacytoid dendritic cells, have revolutionized our understanding of innate immunity. These discoveries and the popularization of high-throughput screening technologies have renewed the interest for small molecules that can induce type I interferons. Proofs about their therapeutic potency in humans are expected very soon.

Functional reconstitution of Drosophila melanogaster NMJ glutamate receptors.

The Drosophila larval neuromuscular junction (NMJ), at which glutamate acts as the excitatory neurotransmitter, is a widely used model for genetic analysis of synapse function and development. Despite decades of study, the inability to reconstitute NMJ glutamate receptor function using heterologous expression systems has complicated the analysis of receptor function, such that it is difficult to resolve the molecular basis for compound phenotypes observed in mutant flies. We find that Drosophila Neto functions as an essential component required for the function of NMJ glutamate receptors, permitting analysis of glutamate receptor responses in Xenopus oocytes. In combination with a crystallographic analysis of the GluRIIB ligand binding domain, we use this system to characterize the subunit dependence of assembly, channel block, and ligand selectivity for Drosophila NMJ glutamate receptors.

The Ras/MAPK pathway and hepatocarcinoma: pathogenesis and therapeutic implications.

BACKGROUND: Hepatocellular carcinoma (HCC) is still a major health problem, often diagnosed at an advanced stage. The multikinase inhibitor sorafenib is to date the sole approved systemic therapy. Several signalling pathways are implicated in tumour development and progression. Among these pathways, the Ras/MAPK pathway is activated in 50-100% of human HCCs and is correlated with a poor prognosis. The aim of this work was to review the main intracellular mechanisms leading to aberrant Ras pathway activation in HCC and the potential therapeutic implications. MATERIALS AND METHODS: This review is based on the material found on PubMed up to December 2014. 'Ras signaling, Ras dysregulation, Ras inhibition, MAPK pathway, cancer, hepatocarcinoma and liver cancer' alone or in combination were the main terms used for online research. RESULTS: Multiple mechanisms lead to the deregulation of the Ras pathway in liver cancer. Ras and Raf gene mutations are rare events in human hepatocarcinogenesis in contrast to experimental models in rodents. Downregulation of several Ras/MAPK pathway inhibitors such as GAPs, RASSF proteins, DUSP1, Sprouty and Spred proteins is largely implicated in the aberrant activation of this pathway in the context of wild-type Ras and Raf genes. Epigenetic or post-transcriptional mechanisms lead to the downregulation of these tumour suppressor genes. CONCLUSION: Ras/MAPK pathway effectors may be considered as potential therapeutic targets in the field of HCC. In particular after the arrival of sorafenib, more Ras/MAPK inhibitors have emerged and are still in preclinical or clinical investigation for HCC therapy.

Progesterone and HMOX-1 promote fetal growth by CD8+ T cell modulation.

Intrauterine growth restriction (IUGR) affects up to 10% of pregnancies in Western societies. IUGR is a strong predictor of reduced short-term neonatal survival and impairs long-term health in children. Placental insufficiency is often associated with IUGR; however, the molecular mechanisms involved in the pathogenesis of placental insufficiency and IUGR are largely unknown. Here, we developed a mouse model of fetal-growth restriction and placental insufficiency that is induced by a midgestational stress challenge. Compared with control animals, pregnant dams subjected to gestational stress exhibited reduced progesterone levels and placental heme oxygenase 1 (Hmox1) expression and increased methylation at distinct regions of the placental Hmox1 promoter. These stress-triggered changes were accompanied by an altered CD8+ T cell response, as evidenced by a reduction of tolerogenic CD8+CD122+ T cells and an increase of cytotoxic CD8+ T cells. Using progesterone receptor- or Hmox1-deficient mice, we identified progesterone as an upstream modulator of placental Hmox1 expression. Supplementation of progesterone or depletion of CD8+ T cells revealed that progesterone suppresses CD8+ T cell cytotoxicity, whereas the generation of CD8+CD122+ T cells is supported by Hmox1 and ameliorates fetal-growth restriction in Hmox1 deficiency. These observations in mice could promote the identification of pregnancies at risk for IUGR and the generation of clinical interventional strategies.

NPC1L1 is a key regulator of intestinal vitamin K absorption and a modulator of warfarin therapy.

Vitamin K (VK) is a micronutrient that facilitates blood coagulation. VK antagonists, such as warfarin, are used in the clinic to prevent thromboembolism. Because VK is not synthesized in the body, its intestinal absorption is crucial for maintaining whole-body VK levels. However, the molecular mechanism of this absorption is unclear. We demonstrate that Niemann-Pick C1-like 1 (NPC1L1) protein, a cholesterol transporter, plays a central role in intestinal VK uptake and modulates the anticoagulant effect of warfarin. In vitro studies using NPC1L1-overexpressing intestinal cells and in vivo studies with Npc1l1-knockout mice revealed that intestinal VK absorption is NPC1L1-dependent and inhibited by ezetimibe, an NPC1L1-selective inhibitor clinically used for dyslipidemia. In addition, in vivo pharmacological studies demonstrated that the coadministration of ezetimibe and warfarin caused a reduction in hepatic VK levels and enhanced the pharmacological effect of warfarin. Adverse events caused by the coadministration of ezetimibe and warfarin were rescued by oral VK supplementation, suggesting that the drug-drug interaction effects observed were the consequence of ezetimibe-mediated VK malabsorption. This mechanism was supported by a retrospective evaluation of clinical data showing that, in more than 85% of warfarin-treated patients, the anticoagulant activity was enhanced by cotreatment with ezetimibe. Our findings provide insight into the molecular mechanism of VK absorption. This new drug-drug interaction mechanism between ezetimibe (a cholesterol transport inhibitor) and warfarin (a VK antagonist and anticoagulant) could inform clinical care of patients on these medications, such as by altering the kinetics of essential, fat-soluble vitamins.