Daily Brief Heat Therapy Reduces Seizures in A350V IQSEC2 Mice and Is Associated with Correction of AMPA Receptor-Mediated Synaptic Dysfunction.

Purposeful induction of fever for healing, including the treatment of epilepsy, was used over 2000 years ago by Hippocrates. More recently, fever has been demonstrated to rescue behavioral abnormalities in children with autism. However, the mechanism of fever benefit has remained elusive due in large part to the lack of appropriate human disease models recapitulating the fever effect. Pathological mutations in the IQSEC2 gene are frequently seen in children presenting with intellectual disability, autism and epilepsy. We recently described a murine A350V IQSEC2 disease model, which recapitulates important aspects of the human A350V IQSEC2 disease phenotype and the favorable response to a prolonged and sustained rise in body core temperature in a child with the mutation. Our goal has been to use this system to understand the mechanism of fever benefit and then develop drugs that can mimic this effect and reduce IQSEC2-associated morbidity. In this study, we first demonstrate a reduction in seizures in the mouse model following brief periods of heat therapy, similar to what was observed in a child with the mutation. We then show that brief heat therapy is associated with the correction of synaptic dysfunction in neuronal cultures of A350V mice, likely mediated by Arf6-GTP.

Nimbin analog N2 alleviates high testosterone induced oxidative stress in CHO cells and alters the expression of Tox3 and Dennd1a signal transduction pathway involved in the PCOS zebrafish.

Polycystic ovarian syndrome (PCOS) is a hormonal disorder that causes enlargement of ovaries and follicular maturation arrest, which lacks efficient treatment. N2, a semi-natural triterpenoid from the neem family, was already reported to have antioxidant and antiinflammatory properties in our previous report. This study investigated the anti-androgenic property of N2 on testosterone-induced oxidative stress in Chinese Hamster Ovarian cells (CHO) and PCOS zebrafish model. The testosterone exposure disrupted the antioxidant enzymes and ROS level and enhanced the apoptosis in both CHO cells and PCOS zebrafish. However, N2 significantly protected the CHO cells from ROS and apoptosis. N2 improved the Gonado somatic index (GSI) and upregulated the expression of the SOD enzyme in zebrafish ovaries. Moreover, the testosterone-induced follicular maturation arrest was normalized by N2 treatment in histopathology studies. In addition, the gene expression studies of Tox3 and Denndla in zebrafish demonstrated that N2 could impair PCOS condition. Furthermore, to confirm the N2 activity, the in-silico studies were performed against PCOS susceptible genes Tox3 and Dennd1a using molecular docking and molecular dynamic simulations. The results suggested that N2 alleviated the oxidative stress and apoptosis in-vitro and in-vivo and altered the expression of PCOS key genes.

DOCK2 and phosphoinositide-3 kinase δ mediate two complementary signaling pathways for CXCR5-dependent B cell migration.

Naive B cells use the chemokine receptor CXCR5 to enter B cell follicles, where they scan CXCL13-expressing ICAM-1+ VCAM-1+ follicular dendritic cells (FDCs) for the presence of antigen. CXCL13-CXCR5-mediated motility is mainly driven by the Rac guanine exchange factor DOCK2, which contains a binding domain for phosphoinositide-3,4,5-triphosphate (PIP3) and other phospholipids. While p110δ, the catalytic subunit of the class IA phosphoinositide-3-kinase (PI3K) δ, contributes to CXCR5-mediated B cell migration, the precise interdependency of DOCK2, p110δ, or other PI3K family members during this process remains incompletely understood. Here, we combined in vitro chemotaxis assays and in vivo imaging to examine the contribution of these two factors during murine naïve B cell migration to CXCL13. Our data confirm that p110δ is the main catalytic subunit mediating PI3K-dependent migration downstream CXCR5, whereas it does not contribute to chemotaxis triggered by CXCR4 or CCR7, two other chemokine receptors expressed on naïve B cells. The contribution of p110δ activity to CXCR5-driven migration was complementary to that of DOCK2, and pharmacological or genetic interference with both pathways completely abrogated B cell chemotaxis to CXCL13. Intravital microscopy of control and gene-deficient B cells migrating on FDCs confirmed that lack of DOCK2 caused a profound migration defect, whereas p110δ contributed to cell speed and directionality. B cells lacking active p110δ also displayed defective adhesion to ICAM-1; yet, their migration impairment was maintained on ICAM-1-deficient FDCs. In sum, our data uncover two complementary signaling pathways mediated by DOCK2 and p110δ, which enable CXCR5-driven naïve B cell examination of FDCs.

Electroacupuncture Attenuates Post-Inflammatory IBS-Associated Visceral and Somatic Hypersensitivity and Correlates With the Regulatory Mechanism of Epac1-Piezo2 Axis.

Electroacupuncture (EA) is considered to have a therapeutic effect in the relief of irritable bowel syndrome (IBS)-associated visceral hypersensitivity via the reduction of the level of 5-hydroxytryptamine (5-HT) and 5-HT3 receptors (5-HT3R). However, whether Epac1/Piezo2, as the upstream of 5-HT, is involved in this process remains unclear. We investigated whether EA at the ST36 and ST37 acupoints alleviated visceral and somatic hypersensitivity in a post-inflammatory IBS (PI-IBS) model mice via the Epac1-Piezo2 axis. In this study, we used 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced PI-IBS as a mouse model. Visceral sensitivity was assessed by the abdominal withdrawal reflex test. Somatic sensitivity was evaluated by the hind paw withdrawal threshold. Quantitative real-time PCR, immunofluorescence staining, ELISA, and Western blotting were performed to examine the expressions of Epac1, Piezo2, 5-HT, and 5-HT3R from the mouse distal colon/L5-S2 dorsal root ganglia (DRG). Our results showed that EA improved the increased visceral sensation and peripheral mechanical hyperalgesia in PI-IBS model mice, and the effects of EA were superior to the sham EA. EA significantly decreased the protein and mRNA levels of Epac1 and Piezo2, and reduced 5-HT and 5-HT3R expressions in the distal colon. Knockdown of colonic Piezo2 eliminated the effect of EA on somatic hypersensitivity. Combined knockdown of colonic Epac1 and Piezo2 synergized with EA in relieving visceral hypersensitivity and blocked the effect of EA on somatic hypersensitivity. Additionally, protein levels of Epac1 and Piezo2 were also found to be decreased in the L5-S2 DRGs after EA treatment. Taken together, our study suggested that EA at ST36 and ST37 can alleviate visceral and somatic hypersensitivity in PI-IBS model mice, which is closely related to the regulation of the Epac1-Piezo2 axis.

Clinical, pathological and genetic features and follow-up of 110 patients with late-onset MADD: a single-center retrospective study.

To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy.

Proteomic and molecular evidences of Il1rl2, Ric8a, Krt18 and Hsp90b1 modulation during experimental hepatic fibrosis and pomegranate supplementation.

The inspection of variations in the proteomic aspects conspire the biomarker discovery in diagnostics of peculiar diseases. Recent developments in high-throughput proteomic techniques have provided leverage in the discovery of biomarkers during the etiology of various diseases. We identified potential biomarkers by utilizing proteomics, bioinformatics and gene expression studies. Meticulous assessment of collagen and hydroxyproline levels along with the glycogen and protein carbonyl levels exhibited deterioration in the N' - Nitrosodiethylamine (NDEA) administered rat livers and subsequent salubrious effect of pomegranate juice. The immunohistochemical inspection of iNOS and nitrite estimation indicated the peccant fibrotic alterations. 2D proteome profiling and MALDI-TOF MS/MS furthered the significant biomarkers to be analyzed for the gene ontology by PANTHER, cluster analysis by DAVID and network simulation by STRING 10.0. Several genes found relevant after MALDI analysis were evaluated by real-time PCR (RTPCR). Our data revealed CYP2b15, HSP70, TRFE, HPT, Il1rl2, Ric8a, Krt18, Hsp90b1 and iNOS as novel biomarkers for the mechanism of pomegranate against liver fibrosis. It can be inferred that NDEA-induced liver fibrosis actuates various biological pathways by the identified biomarkers and pomegranate juice modifies them.

Analysis of gene variants in the GASH/Sal model of epilepsy.

Epilepsy is a complex neurological disorder characterized by sudden and recurrent seizures, which are caused by various factors, including genetic abnormalities. Several animal models of epilepsy mimic the different symptoms of this disorder. In particular, the genetic audiogenic seizure hamster from Salamanca (GASH/Sal) animals exhibit sound-induced seizures similar to the generalized tonic seizures observed in epileptic patients. However, the genetic alterations underlying the audiogenic seizure susceptibility of the GASH/Sal model remain unknown. In addition, gene variations in the GASH/Sal might have a close resemblance with those described in humans with epilepsy, which is a prerequisite for any new preclinical studies that target genetic abnormalities. Here, we performed whole exome sequencing (WES) in GASH/Sal animals and their corresponding controls to identify and characterize the mutational landscape of the GASH/Sal strain. After filtering the results, moderate- and high-impact variants were validated by Sanger sequencing, assessing the possible impact of the mutations by "in silico" reconstruction of the encoded proteins and analyzing their corresponding biological pathways. Lastly, we quantified gene expression levels by RT-qPCR. In the GASH/Sal model, WES showed the presence of 342 variations, in which 21 were classified as high-impact mutations. After a full bioinformatics analysis to highlight the high quality and reliable variants, the presence of 3 high-impact and 15 moderate-impact variants were identified. Gene expression analysis of the high-impact variants of Asb14 (ankyrin repeat and SOCS Box Containing 14), Msh3 (MutS Homolog 3) and Arhgef38 (Rho Guanine Nucleotide Exchange Factor 38) genes showed a higher expression in the GASH/Sal than in control hamsters. In silico analysis of the functional consequences indicated that those mutations in the three encoded proteins would have severe functional alterations. By functional analysis of the variants, we detected 44 significantly enriched pathways, including the glutamatergic synapse pathway. The data show three high-impact mutations with a major impact on the function of the proteins encoded by these genes, although no mutation in these three genes has been associated with some type of epilepsy until now. Furthermore, GASH/Sal animals also showed gene variants associated with different types of epilepsy that has been extensively documented, as well as mutations in other genes that encode proteins with functions related to neuronal excitability, which could be implied in the phenotype of the GASH/Sal. Our findings provide valuable genetic and biological pathway data associated to the genetic burden of the audiogenic seizure susceptibility and reinforce the need to validate the role of each key mutation in the phenotype of the GASH/Sal model.

Glucose stimulates somatostatin secretion in pancreatic δ-cells by cAMP-dependent intracellular Ca2+ release.

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+ , increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell.

Exchange-protein activated by cAMP (EPAC) regulates L-type calcium channel in atrial fibrillation of heart failure model.

OBJECTIVE: Both atrial fibrillation (AF) and heart failure (HF) are increasingly prevalent and related to high hospitalization rate and mortality. AF is a cause as well as a consequence of HF, with complicated interactions resulting in impairment of cardiac systolic and diastolic function. Conversely, the complex structural and neurohormonal alterations in HF contribute to the occurrence and development of AF. However, the molecular mechanism remains unclear. This study aims to explore the effect of Exchange-protein activated by cAMP 1 (EPAC1) on AF in isoproterenol (ISO)-induced HF and the potential molecular mechanism. MATERIALS AND METHODS: Mice and cultured isolated adult cardiomyocytes were treated with ISO and or not EPAC1 inhibitor CE3F4. Programmed electrical stimulation (PES) was performed to induce AF. EPAC1 expression was determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Western blot. Cellular electrophysiology was examined by whole cell patch clamp. RESULTS: Both mRNA and protein levels of EPAC1 were upregulated in HF mice. ISO increased the AF susceptibility, and the negative effect was deteriorated by CE3F4. ISO mediated high AF susceptibility of HF via prolonging action potential and exciting L-type calcium channel (LTCC). These could also be reversed by CE3F4 treatment. CONCLUSIONS: EPAC1 increased the AF susceptibility in ISO-induced HF mouse model via alternating LTCC.

Differential mechanisms of adenosine- and ATPγS-induced microvascular endothelial barrier strengthening.

Maintenance of the endothelial cell (EC) barrier is critical to vascular homeostasis and a loss of barrier integrity results in increased vascular permeability. While the mechanisms that govern increased EC permeability have been under intense investigation over the past several decades, the processes regulating the preservation/restoration of the EC barrier remain poorly understood. Herein we show that the extracellular purines, adenosine (Ado) and adenosine 5'-[γ-thio]-triphosphate (ATPγS) can strengthen the barrier function of human lung microvascular EC (HLMVEC). This ability involves protein kinase A (PKA) activation and decreases in myosin light chain 20 (MLC20) phosphorylation secondary to the involvement of MLC phosphatase (MLCP). In contrast to Ado, ATPγS-induced PKA activation is accompanied by a modest, but significant decrease in cyclic adenosine monophosphate (cAMP) levels supporting the existence of an unconventional cAMP-independent pathway of PKA activation. Furthermore, ATPγS-induced EC barrier strengthening does not involve the Rap guanine nucleotide exchange factor 3 (EPAC1) which is directly activated by cAMP but is instead dependent upon PKA-anchor protein 2 (AKAP2) expression. We also found that AKAP2 can directly interact with the myosin phosphatase-targeting protein MYPT1 and that depletion of AKAP2 abolished ATPγS-induced increases in transendothelial electrical resistance. Ado-induced strengthening of the HLMVEC barrier required the coordinated activation of PKA and EPAC1 in a cAMP-dependent manner. In summary, ATPγS-induced enhancement of the EC barrier is EPAC1-independent and is instead mediated by activation of PKA which is then guided by AKAP2, in a cAMP-independent mechanism, to activate MLCP which dephosphorylates MLC20 resulting in reduced EC contraction and preservation.

Trio GEF mediates RhoA activation downstream of Slit2 and coordinates telencephalic wiring.

Trio, a member of the Dbl family of guanine nucleotide exchange factors, activates Rac1 downstream of netrin 1/DCC signalling in axon outgrowth and guidance. Although it has been proposed that Trio also activates RhoA, the putative upstream factors remain unknown. Here, we show that Slit2 induces Trio-dependent RhoA activation, revealing a crosstalk between Slit and Trio/RhoA signalling. Consistently, we found that RhoA activity is hindered in vivo in Trio mutant mouse embryos. We next studied the development of the ventral telencephalon and thalamocortical axons, which have been previously shown to be controlled by Slit2. Remarkably, this analysis revealed that Trio knockout (KO) mice show phenotypes that bear strong similarities to the ones that have been reported in Slit2 KO mice in both guidepost corridor cells and thalamocortical axon pathfinding in the ventral telencephalon. Taken together, our results show that Trio induces RhoA activation downstream of Slit2, and support a functional role in ensuring the proper positioning of both guidepost cells and a major axonal tract. Our study indicates a novel role for Trio in Slit2 signalling and forebrain wiring, highlighting its role in multiple guidance pathways as well as in biological functions of importance for a factor involved in human brain disorders.

AtBET5 is essential for exine pattern formation and apical meristem organization in Arabidopsis.

BET5 is a component of trafficking protein particle (TRAPP) which has been studied extensively in non-plant organisms where they are involved in membrane trafficking within Golgi and between Golgi and early endosomes. Recent analysis of TRAPP in different classes of organisms indicates that TRAPP function might exhibit differences among organisms. A single copy of the BET5 gene named AtBET5 was found in the Arabidopsis genome based on sequence similarity. Developmental phenotype and the underlying mechanisms have been characterized upon transcriptional knock-down lines generated by both T-DNA insertion and RNAi. Pollen grains of the T-DNA insertional line present reduced fertility and pilate exine instead of tectate exine. Perturbation of the AtBET5 expression by RNAi leads to apical meristematic organization defects and reduced fertility as well. The reduced fertility was due to the pollination barrier caused by an altered composition and structure of pollen walls. Auxin response in root tip cells is altered and there is a severe disruption in polar localization of PIN1-GFP, but to a less extent of PIN2-GFP in the root tips, which causes the apical meristematic organization defects and might also be responsible for the secretion of sporopollenin precursor or polar targeting of sporopollenin precursor transporters.

Folic acid supplementation alters the DNA methylation profile and improves insulin resistance in high-fat-diet-fed mice.

Folic acid (FA) supplementation may protect from obesity and insulin resistance, the effects and mechanism of FA on chronic high-fat-diet-induced obesity-related metabolic disorders are not well elucidated. We adopted a genome-wide approach to directly examine whether FA supplementation affects the DNA methylation profile of mouse adipose tissue and identify the functional consequences of these changes. Mice were fed a high-fat diet (HFD), normal diet (ND) or an HFD supplemented with folic acid (20 µg/ml in drinking water) for 10 weeks, epididymal fat was harvested, and genome-wide DNA methylation analyses were performed using methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mice exposed to the HFD expanded their adipose mass, which was accompanied by a significant increase in circulating glucose and insulin levels. FA supplementation reduced the fat mass and serum glucose levels and improved insulin resistance in HFD-fed mice. MeDIP-seq revealed distribution of differentially methylated regions (DMRs) throughout the adipocyte genome, with more hypermethylated regions in HFD mice. Methylome profiling identified DMRs associated with 3787 annotated genes from HFD mice in response to FA supplementation. Pathway analyses showed novel DNA methylation changes in adipose genes associated with insulin secretion, pancreatic secretion and type 2 diabetes. The differential DNA methylation corresponded to changes in the adipose tissue gene expression of Adcy3 and Rapgef4 in mice exposed to a diet containing FA. FA supplementation improved insulin resistance, decreased the fat mass, and induced DNA methylation and gene expression changes in genes associated with obesity and insulin secretion in obese mice fed a HFD.

Epac2a-knockout mice are resistant to dexamethasone-induced skeletal muscle atrophy and short-term cold stress.

Exchange protein directly activated by cAMP (Epac) 2a-knockout (KO) mice exhibit accelerated diet-induced obesity and are resistant to leptin-mediated adipostatic signaling from the hypothalamus to adipose tissue, with sustained food intake. However, the impact of Epac2a deficiency on hypothalamic regulation of sympathetic nervous activity (SNA) has not been elucidated. This study was performed to elucidate the response of Epac2a-KO mice to dexamethasone-induced muscle atrophy and acute cold stress. Compared to age-matched wild-type mice, Epac2a-KO mice showed higher energy expenditures and expression of myogenin and uncoupling protein-1 in skeletal muscle (SM) and brown adipose tissue (BAT), respectively. Epac2a-KO mice exhibited greater endurance to dexamethasone and cold stress. In wild-type mice, exogenous leptin mimicked the responses observed in Epac2a-KO mice. This suggests that leptin-mediated hypothalamic signaling toward SNA appears to be intact in these mice. Hence, the potentiated responses of SM and BAT may be due to their high plasma leptin levels. [BMB Reports 2018; 51(1): 39-44].

MiR 376c inhibits osteoblastogenesis by targeting Wnt3 and ARF-GEF-1 -facilitated augmentation of beta-catenin transactivation.

Wnt signaling pathway plays important role in all aspects of skeletal development which include chondrogenesis, osteoblastogenesis, and osteoclastogenesis. Induction of the Wnt-3 signaling pathway promotes bone formation while inactivation of the pathway leads to bone related disorders like osteoporosis. Wnt signaling thus has become a desired target to treat osteogenic disorders. MicroRNAs (miRNAs) represent an important category of elements that interact with Wnt signaling molecules to regulate osteogenesis. Here, we show that miR-376c, a well-characterized tumor suppressor which inhibits cell proliferation and invasion in osteosarcoma by targeting to transforming growth factor-alpha, suppresses osteoblast proliferation, and differentiation. Over-expression of miR-376c inhibited osteoblast differentiation, whereas inhibition of miR-376c function by antimiR-376c promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Target prediction analysis tools and experimental validation by luciferase 3' UTR reporter assay along with qRT-PCR identified Wnt-3 and ARF-GEF-1 as direct targets of miR-376c. It was seen that over-expression of miR-376c leads to repression of canonical Wnt/ß-catenin signaling. Our overall results suggest that miR-376c targets Wnt-3 and ARF-GEF-1 suppresses ARF-6 activation which prevents the release of ß-catenin and its transactivation thereby inhibiting osteoblast differentiation. Although miR-376c is known to be a tumor repressor; we have identified a second complementary function of miR-376c where it inhibits Wnt-3-mediated osteogenesis and promotes bone loss.

Characterization of Zebrafish Models of Marinesco-Sjögren Syndrome.

SIL1 is a nucleotide exchange factor for the endoplasmic reticulum chaperone, BiP. Mutations in the SIL1 gene cause Marinesco-Sjögren syndrome (MSS), an autosomal recessive disease characterized by cerebellar ataxia, mental retardation, congenital cataracts, and myopathy. To create novel zebrafish models of MSS for therapeutic drug screening, we analyzed phenotypes in sil1 knock down fish by two different antisense oligo morpholinos. Both sil1 morphants had abnormal formation of muscle fibers and irregularity of the myosepta. Moreover, they showed smaller-sized eyes and loss of purkinje cells in cerebellar area compared to controls. Immunoblotting analysis revealed increased protein amounts of BiP, lipidated LC3, and caspase 3. These data supported that the sil1 morphants can represent mimicking phenotypes of human MSS. The sil1 morphants phenocopy the human MSS disease pathology and are a good animal model for therapeutic studies.

Precise and selective sensing of DNA-DNA hybridization by graphene/Si-nanowires diode-type biosensors.

Single-Si-nanowire (NW)-based DNA sensors have been recently developed, but their sensitivity is very limited because of high noise signals, originating from small source-drain current of the single Si NW. Here, we demonstrate that chemical-vapor-deposition-grown large-scale graphene/surface-modified vertical-Si-NW-arrays junctions can be utilized as diode-type biosensors for highly-sensitive and -selective detection of specific oligonucleotides. For this, a twenty-seven-base-long synthetic oligonucleotide, which is a fragment of human DENND2D promoter sequence, is first decorated as a probe on the surface of vertical Si-NW arrays, and then the complementary oligonucleotide is hybridized to the probe. This hybridization gives rise to a doping effect on the surface of Si NWs, resulting in the increase of the current in the biosensor. The current of the biosensor increases from 19 to 120% as the concentration of the target DNA varies from 0.1 to 500 nM. In contrast, such biosensing does not come into play by the use of the oligonucleotide with incompatible or mismatched sequences. Similar results are observed from photoluminescence microscopic images and spectra. The biosensors show very-uniform current changes with standard deviations ranging ~1 to ~10% by ten-times endurance tests. These results are very promising for their applications in accurate, selective, and stable biosensing.

Arabidopsis RhoGDIs Are Critical for Cellular Homeostasis of Pollen Tubes.

Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth.

Influenza virus-host interactome screen as a platform for antiviral drug development.

Host factors required for viral replication are ideal drug targets because they are less likely than viral proteins to mutate under drug-mediated selective pressure. Although genome-wide screens have identified host proteins involved in influenza virus replication, limited mechanistic understanding of how these factors affect influenza has hindered potential drug development. We conducted a systematic analysis to identify and validate host factors that associate with influenza virus proteins and affect viral replication. After identifying over 1,000 host factors that coimmunoprecipitate with specific viral proteins, we generated a network of virus-host protein interactions based on the stage of the viral life cycle affected upon host factor downregulation. Using compounds that inhibit these host factors, we validated several proteins, notably Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) and JAK1, as potential antiviral drug targets. Thus, virus-host interactome screens are powerful strategies to identify targetable host factors and guide antiviral drug development.

Nerve growth factor mediates a switch in intracellular signaling for PGE2-induced sensitization of sensory neurons from protein kinase A to Epac.

We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund's adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation.