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Mutations in the small heat shock proteins α-crystallins have been linked to autosomal dominant cataracts in humans. Extensive studies in vitro have revealed a spectrum of alterations to the structure and function of these proteins including shifts in the size of the oligomer, modulation of subunit exchange and modification of their affinity to client proteins. Although mouse models of these mutants were instrumental in identifying changes in cellular proliferation and lens development, a direct comparative analysis of their effects on lens proteostasis has not been performed. Here, we have transgenically expressed cataract-linked mutants of αA- and αB-crystallin in the zebrafish lens to dissect the underlying molecular changes that contribute to the loss of lens optical properties. Zebrafish lines expressing these mutants displayed a range of morphological lens defects. Phenotype penetrance and severity were dependent on the mutation even in fish lines lacking endogenous α-crystallin. The mechanistic origins of these differences were investigated by the transgenic co-expression of a destabilized human γD-crystallin mutant. We found that the R49C but not the R116C mutant of αA-crystallin drove aggregation of γD-crystallin, although both mutants have similar affinity to client proteins in vitro. Our working model attributes these differences to the propensity of R49C, located in the buried N-terminal domain of αA-crystallin, to disulfide crosslinking as previously demonstrated in vitro. Our findings complement and extend previous work in mouse models and emphasize the need of investigating chaperone/client protein interactions in appropriate cellular context. [ABSTRACT FROM AUTHOR]

Bronchopulmonary dysplasia (BPD) remains the most common and serious chronic lung disease of premature infants. Severe BPD complicated with pulmonary hypertension (PH) increases the mortality of these infants. Riociguat is an allosteric soluble guanylate cyclase stimulator and is approved by the FDA for treating PH in adults. However, it has not been approved for use in neonates due to concern for adverse effects on long bone growth. To address this concern we investigated if administration of riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without side effects on long bone growth in newborn rats. Newborn rats were randomized to normoxia (21% O2) or hyperoxia (85% O2) exposure groups within 24 hours of birth, and received riociguat or placebo by once daily intraperitoneal injections during continuous normoxia or hyperoxia exposure for 9 days. In the hyperoxia control group, radial alveolar count, mean linear intercept and vascular density were significantly decreased, the pathological hallmarks of BPD, and these were accompanied by an increased inflammatory response. There was also significantly elevated vascular muscularization of peripheral pulmonary vessels, right ventricular systolic pressure and right ventricular hypertrophy indicating PH. However, administration of riociguat significantly decreased lung inflammation, improved alveolar and vascular development, and decreased PH during hyperoxia by inducing cGMP production. Additionally, riociguat did not affect long bone growth or structure. These data indicate that riociguat is beneficial in preventing hyperoxia-induced lung injury and PH without affecting long bone growth and structure and hence, suggests riociguat may be a potential novel agent for preventing BPD and PH in neonates. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDA) is the fourth-leading cause of cancer death in the United States with a 5-year overall survival rate of 8% for all stages combined. But this decreases to 3% for the majority of patients that present with stage IV PDA at time of diagnosis. The lack of distinct early symptoms for PDA is one of the primary reasons for the late diagnosis. Common symptoms like weight loss, abdominal and back pains, and jaundice are often mistaken for symptoms of other issues and do not appear until the cancer has progressed to a late stage. Thus the development of novel imaging platforms for PDA is crucial for the early detection of the disease. MUC1 is a tumor-associated antigen (tMUC1) expressed on 80% of PDA. The goal of this study was to determine the targeting and detection capabilities of a tMUC1 specific antibody, TAB004. TAB004 antibody conjugated to a near infrared fluorescent probe was injected intraperitoneally into immune competent orthotopic and spontaneous models of PDA. Results show that fluorophore conjugated TAB004 specifically targets a) 1 week old small tumor in the pancreas in an orthotopic PDA model and b) very early pre-neoplastic lesions (PanIN lesions) that develop in the spontaneous PDA model before progression to adenocarcinoma. Thus, TAB004 is a promising antibody to deliver imaging agents directly to the pancreatic tumor microenvironment, significantly affecting early detection of PDA. [ABSTRACT FROM AUTHOR]

The success of many endosseous implants in orthopaedic and dental applications depends on the surface characteristics, as they affect osseous integration. Previous investigations indicated that a novel large-grit sand-blasted and acid-etched (SLA) titanium (denoted as SLAffinity-Ti) implant had better bone integration than that of a comparably shaped implant with a plasma-sprayed titanium surface. The purpose of the present investigation was to create a SLAffinity surface on pedicle screws and trauma screws and to compare it with the surfaces of a sand-blasted-only implant and commercial implants in terms of bone integration. The cortical bone and spine of twelve minipigs were implanted with 3 and 4 implants, respectively, and the bone integration was evaluated using micro-computed tomography (micro-CT), mechanical tests (pull-out strength and stripping torque), and histological analysis (toluidine blue and hematoxylin and eosin staining) one and three months after implantation. The micro-CT images showed that the gap between the bone and implant was consistently higher in the sand-blasted-only and commercial groups compared to that in the SLAffinity group 1 and 3 months after implantation. Moreover, the bone volume of implant inserted into bone and the percentage of implant inside bone tissue were greater in the SLAffinity screws 1 and 3 months after implantation, as compared to the sand-blasted and commercial screws. In the mechanical tests, the removal torque and pull-out strength (p < 0.05) were higher in the SLAffinity group at 1 and 3 months. The histological results were consistent with mechanical testing, showing that the SLAffinity group had the most mineralized matrix, the most bone formation around the screws, and the most bone cells in bone tissue. These findings indicate that a SLAffinity surface can effectively enhance the holding strength and integration of pedicle screws and cortical screws, promoting early healing and improving outcomes, compared to sand-blasted-only and commercial implants. [ABSTRACT FROM AUTHOR]

In the mammalian organ of Corti (OC), the stereocilia on the apical surface of hair cells (HCs) are uniformly organized in a neural to abneural axis (or medial-laterally). This organization is regulated by planar cell polarity (PCP) signaling. Mutations of PCP genes, such as Vangl2, Dvl1/2, Celsr1, and Fzd3/6, affect the formation of HC orientation to varying degrees. Prickle1 is a PCP signaling gene that belongs to the prickle / espinas / testin family. Prickle1 protein is shown to be asymmetrically localized in the HCs of the OC, and this asymmetric localization is associated with loss of PCP in Smurf mutants, implying that Prickle1 is involved in HC PCP development in the OC. A follow-up study found no PCP polarity defects after loss of Prickle1 (Prickle1-/-) in the cochlea. We show here strong Prickle1 mRNA expression in the spiral ganglion by in situ hybridization and β-Gal staining, and weak expression in the OC by β-Gal staining. Consistent with this limited expression in the OC, cochlear HC PCP is unaffected in either Prickle1C251X/C251X mice or Prickle1f/f; Pax2-cre conditional null mice. Meanwhile, type II afferents of apical spiral ganglion neurons (SGN) innervating outer hair cells (OHC) have unusual neurite growth. In addition, afferents from the apex show unusual collaterals in the cochlear nuclei that overlap with basal turn afferents. Our findings argue against the role of Prickle1 in regulating hair cell polarity in the cochlea. Instead, Prickle1 regulates the polarity-related growth of distal and central processes of apical SGNs. [ABSTRACT FROM AUTHOR]

Contexts: CD154 (commonly referred to as CD40-ligand) is a critical T cell factor that participates in the pathogenesis of autoimmune and is over-expressed in rheumatoid arthritis (RA). TNF-α blockade treatment had dramatic efficacy in RA. Objective: To investigate whether TNF-α blockade treatment can inhibit CD154 expression in RA. Methods: Blood samples were collected from 33 patients with rheumatoid arthritis before and 3 months after TNF-α blockade treatment. Clinical serological data determined by standard assays and T cell CD154 expression levels determined by flow cytometry were statistically analyzed for these two time points. Results: The percentage of CD154 expression on gated CD4+ T cells of PBMCs from RA patients after 3 months TNF-α blockade treatment was significantly lower than before treatment (2.94 ± 3.21% vs. 7.21 ± 5.64%; p = 0.0001). The disease activity and anti-CCP antibody levels were also significantly reduced after TNF-α blockade treatment. The CD154 expression levels were positively correlated with disease activity index DAS28, and CRP. The post-stimulated CD154 expression percentage of purified CD4+ T cells between baseline and after TNF-α blockade treatment was not significantly different (p = 0.221). Baseline CD154 levels were positively correlated with treatment-induced changes in DAS28 (p = 0.014; r2 = 0.187). Conclusions: TNF-α blockade treatment significantly decreased the CD154 expression on CD4+ T cells, disease activity and anti-CCP antibody simultaneously in RA patients. However TNF-α blockade did not impair T cell capacity to express CD154 after stimulation. These results suggest that decreased CD154 expression after TNF-α blockade may be due to decreased RA disease activity but not direct inhibition of CD154 responsiveness of T cells. [ABSTRACT FROM AUTHOR]

Ten reference genes were investigated for normalization of gene expression data in the shell gland of laying hens. Analyses performed with geNorm revealed that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS) were the two most stable reference genes in response to post-oviposition time alone (POT) or with nicarbazin treatment (POT+N) of laying hens. NormFinder analyses showed that the two most stable reference genes in response to POT and POT+N were 18S ribosomal RNA (18S rRNA), ribosomal protein L4 (RPL4) and HMBS, RPL4, respectively. BestKeeper analyses showed that 18S rRNA, RPL4 and HPRT1, HMBS were the two most stable reference genes for POT, and POT+N, respectively. Of the ten reference genes, all except B2M showed geNorm M <0.5, suggesting that they were stably expressed in the shell gland tissue. Consensus from these three programs suggested HPRT1 and HMBS could be used as the two most stable reference genes in the present study. Expression analyses of four candidate target genes with the two most and the two least stable genes showed that a combination of stable reference genes leads to more discriminable quantification of expression levels of target genes, while the least stable genes failed to do so. Therefore, HMBS and HPRT1 are recommended as the two most stable reference genes for the normalization of gene expression data at different stages of eggshell formation in brown-egg laying hens. Available statistical programs for reference gene ranking should include more robust analysis capability to analyse the gene expression data generated from factorial design experiments. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells (MSC) improve alveolar and vascular structures in experimental models of bronchopulmonary dysplasia (BPD). Female MSC secrete more anti-inflammatory and pro-angiogenic factors as compared to male MSC. Whether the therapeutic efficacy of MSC in attenuating lung injury in an experimental model of BPD is influenced by the sex of the donor MSC or recipient is unknown. Here we tested the hypothesis that female MSC would have greater lung regenerative properties than male MSC in experimental BPD and this benefit would be more evident in males. Objective: To determine whether intra-tracheal (IT) administration of female MSC to neonatal rats with experimental BPD has more beneficial reparative effects as compared to IT male MSC. Methods: Newborn Sprague-Dawley rats exposed to normoxia (RA) or hyperoxia (85% O2) from postnatal day (P) 2- P21 were randomly assigned to receive male or female IT bone marrow (BM)-derived green fluorescent protein (GFP+) MSC (1 x 106 cells/50 μl), or Placebo on P7. Pulmonary hypertension (PH), vascular remodeling, alveolarization, and angiogenesis were assessed at P21. PH was determined by measuring right ventricular systolic pressure (RVSP) and pulmonary vascular remodeling was evaluated by quantifying the percentage of muscularized peripheral pulmonary vessels. Alveolarization was evaluated by measuring mean linear intercept (MLI) and radial alveolar count (RAC). Angiogenesis was determined by measuring vascular density. Data are expressed as mean ± SD, and analyzed by ANOVA. Results: There were no significant differences in the RA groups. Exposure to hyperoxia resulted in a decrease in vascular density and RAC, with a significant increase in MLI, RVSP, and the percentage of partially and fully muscularized pulmonary arterioles. Administration of both male and female MSC significantly improved vascular density, alveolarization, RVSP, percent of muscularized vessels and alveolarization. Interestingly, the improvement in PH and vascular remodeling was more robust in the hyperoxic rodents who received MSC from female donors. In keeping with our hypothesis, male animals receiving female MSC, had a greater improvement in vascular remodeling. This was accompanied by a more significant decrease in lung pro-inflammatory markers and a larger increase in anti-inflammatory and pro-angiogenic markers in male rodents that received female MSC. There were no significant differences in MSC engraftment among groups. Conclusions: Female BM-derived MSC have greater therapeutic efficacy than male MSC in reducing neonatal hyperoxia-induced lung inflammation and vascular remodeling. Furthermore, the beneficial effects of female MSC were more pronounced in male animals. Together, these findings suggest that female MSC maybe the most potent BM-derived MSC population for lung repair in severe BPD complicated by PH. [ABSTRACT FROM AUTHOR]

Ischemia-reperfusion (IR)-induced acute lung injury (ALI) is implicated in several clinical conditions like lung transplantation, acute pulmonary embolism after thrombolytic therapy, re-expansion of collapsed lung from pneumothorax or pleural effusion, cardiopulmonary bypass and etc. Because mortality remains high despite advanced medical care, prevention and treatment are important clinical issues for IR-induced ALI. Vascular endothelial growth factor (VEGF) has a controversial role in ALI. We therefore conducted this study to determine the effects of anti-VEGF antibody in IR-induced ALI. In the current study, the IR-induced ALI was conducted in a rat model of isolated-perfused lung in situ in the chest. The animals were divided into the control, control + preconditioning anti-VEGF antibody (bevacizumab, 5mg/kg), IR, IR + preconditioning anti-VEGF antibody (1mg/kg), IR+ preconditioning anti-VEGF antibody (5mg/kg) and IR+ post-IR anti-VEGF antibody (5mg/kg) group. There were eight adult male Sprague-Dawley rats in each group. The IR caused significant pulmonary micro-vascular hyper-permeability, pulmonary edema, neutrophilic infiltration in lung tissues, increased tumor necrosis factor-α, and total protein concentrations in bronchoalveolar lavage fluid. VEGF and extracellular signal-regulated kinase (ERK) were increased in IR-induced ALI. Administration of preconditioning anti-VEGF antibody significantly suppressed the VEGF and ERK expressions and attenuated the IR-induced lung injury. This study demonstrates the important role of VEGF in early IR-induced ALI. The beneficial effects of preconditioning anti-VEGF antibody in IR-induced ALI include the attenuation of lung injury, pro-inflammatory cytokines, and neutrophilic infiltration into the lung tissues. [ABSTRACT FROM AUTHOR]

Background: Prostate tumor overexpressed 1 (PTOV1) has been reported to contribute to increased cancer proliferation. However, the clinical significance of PTOV1 in the development and progression of nasopharyngeal carcinoma (NPC) is unclear. Our study aimed to investigate the expression pattern of PTOV1 in NPC and its correlation with clinicopathological features of patients. Methods: Western blotting and real-time PCR were conducted to examine PTOV1 expression levels in NPC cell lines and biopsy tissues compared with normal controls. Immunohistochemistry (IHC) was performed to analyze PTOV1 protein expression in paraffin-embedded tissues from 123 patients. Statistical analyses were applied to evaluate the clinical significance of PTOV1 expression. Results: PTOV1 mRNA and protein levels were upregulated in NPC cell lines and clinical samples. IHC analyses showed that PTOV1 was highly expressed in 68 (55.3%) of 123 NPC specimens. Statistical analysis revealed that PTOV1 expression was significantly correlated with clinical stage (P < 0.001), T classification (P = 0.042) and N classification (P = 0.001). Patients with a higher PTOV1 expression had shorter overall survival compared with those with a lower PTOV1 expression level, especially in lower N stage patients. Multivariate analyses suggested that PTOV1 expression was an independent prognostic marker for survival in NPC patients. Conclusions: Our data demonstrated that PTOV1 overexpression is associated with poor survival outcomes of NPC patients, especially in N0-1 patients. Hence, PTOV1 may help to detect early lymph node metastasis of NPC patients and serve as an independent prognostic biomarker for human NPC. [ABSTRACT FROM AUTHOR]

Objective: Despite high mortality associated with serious mental illness, risk of acute myocardial infarction (AMI) remains unclear, especially for patients with bipolar disorder. The main objective was to investigate the relative risk of AMI associated with schizophrenia and bipolar disorders in a national sample. Method: Using nationwide administrative data, an 11-year historic cohort study was assembled, comprised of cases aged 18 and above who had received a diagnosis of schizophrenia or bipolar disorder, compared to a random sample of all other adults excluding those with diagnoses of serious mental illness. Incident AMI as a primary diagnosis was ascertained. Hazard ratios stratified by age and gender were calculated and Cox regression models were used to adjust for other covariates. Results: A total of 70,225 people with schizophrenia or bipolar disorder and 207,592 people without serious mental illness were compared. Hazard ratios in men adjusted for age, income and urbanization were 1.15 (95% CI 1.01~1.32) for schizophrenia and 1.37 (1.08~1.73)for bipolar disorder, and in women, 1.85 (1.58~2.18) and 1.88(1.47~2.41) respectively. Further adjustment for treated hypertension, diabetes and hyperlipidaemia attenuated the hazard ratio for men with schizophrenia but not the other comparison groups. Hazard ratios were significantly stronger in women than men and were stronger in younger compared to older age groups for both disorders; however, gender modification was only significant in people with schizophrenia, and age modification only significant in people with bipolar disorder. Conclusions: In this large national sample, schizophrenia and bipolar disorder were associated with raised risk of AMI in women and in the younger age groups although showed differences in potential confounding and modifying factors. [ABSTRACT FROM AUTHOR]

Biodiversity studies are commonly conducted using 18S rRNA genes. In this study, we compared the inter-species divergence of variable regions (V1–9) within the copepod 18S rRNA gene, and tested their taxonomic resolutions at different taxonomic levels. Our results indicate that the 18S rRNA gene is a good molecular marker for the study of copepod biodiversity, and our conclusions are as follows: 1) 18S rRNA genes are highly conserved intra-species (intra-species similarities are close to 100%); and could aid in species-level analyses, but with some limitations; 2) nearly-whole-length sequences and some partial regions (around V2, V4, and V9) of the 18S rRNA gene can be used to discriminate between samples at both the family and order levels (with a success rate of about 80%); 3) compared with other regions, V9 has a higher resolution at the genus level (with an identification success rate of about 80%); and 4) V7 is most divergent in length, and would be a good candidate marker for the phylogenetic study of Acartia species. This study also evaluated the correlation between similarity thresholds and the accuracy of using nuclear 18S rRNA genes for the classification of organisms in the subclass Copepoda. We suggest that sample identification accuracy should be considered when a molecular sequence divergence threshold is used for taxonomic identification, and that the lowest similarity threshold should be determined based on a pre-designated level of acceptable accuracy. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease such as simple steatosis, nonalcoholic steatohepatitis (NASH), cirrhosis and fibrosis. However, the molecular pathogenesis and genetic variations causing NAFLD are poorly understood. The high prevalence and incidence of NAFLD suggests that genetic variations on a large number of genes might be involved in NAFLD. To identify genetic variants causing inherited liver disease, we used zebrafish as a model system for a large-scale mutant screen, and adopted a whole genome sequencing approach for rapid identification of mutated genes found in our screen. Here, we report on a forward genetic screen of ENU mutagenized zebrafish. From 250 F2 lines of ENU mutagenized zebrafish during post-developmental stages (5 to 8 days post fertilization), we identified 19 unique mutant zebrafish lines displaying visual evidence of hepatomegaly and/or steatosis with no developmental defects. Histological analysis of mutants revealed several specific phenotypes, including common steatosis, micro/macrovesicular steatosis, hepatomegaly, ballooning, and acute hepatocellular necrosis. This work has identified multiple post-developmental mutants and establishes zebrafish as a novel animal model for post-developmental inherited liver disease. [ABSTRACT FROM AUTHOR]

CAPN5 mutations have been linked to autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV), a blinding autoimmune eye disease. Here, we link a new CAPN5 mutation to ADNIV and model the three-dimensional structure of the resulting mutant protein. In our study, a kindred with inflammatory vitreoretinopathy was evaluated by clinical eye examinations, DNA sequencing, and protein structural modeling to investigate the disease-causing mutation. Two daughters of an affected mother demonstrated symptoms of stage III ADNIV, with posterior uveitis, cystoid macular edema, intraocular fibrosis, retinal neovascularization, retinal degeneration, and cataract. The women also harbored a novel guanine to thymine (c.750G>T, p.Lys250Asn) missense mutation in exon 6 of CAPN5, a gene that encodes a calcium-activated cysteine protease, calpain-5. Modeling based on the structures of all known calpains revealed the mutation falls within a calcium-sensitive flexible gating loop that controls access to the catalytic groove. Three-dimensional modeling placed the new mutation in a region adjacent to two previously identified disease-causing mutations, all three of which likely disrupt hydrogen bonding within the gating loop, yielding a CAPN5 with altered enzymatic activity. This is the third case of a CAPN5 mutation leading to inherited uveitis and neovascular vitreoretinopathy, suggesting patients with ADNIV features should be tested for CAPN5 mutations. Structural modeling of novel variants can be used to support mechanistic consequences of the disease-causing variants. [ABSTRACT FROM AUTHOR]

Ventilator-induced lung injury is associated with inflammatory mechanism and causes high mortality. The objective of this study was to discover the role of IL-33 and its ST2 receptor in acute lung injury induced by mechanical ventilator (ventilator-induced lung injury; VILI). Male Wistar rats were intubated after tracheostomy and received ventilation at 10 cm H2O of inspiratory pressure (PC10) by a G5 ventilator for 4 hours. The hemodynamic and respiratory parameters were collected and analyzed. The morphological changes of lung injury were also assessed by histological H&E stain. The dynamic changes of lung injury markers such as TNF-α and IL-1β were measured in serum, bronchoalveolar lavage fluid (BALF), and lung tissue homogenization by ELISA assay. During VILI, the IL-33 profile change was detected in BALF, peripheral serum, and lung tissue by ELISA analysis. The Il-33 and ST2 expression were analyzed by immunohistochemistry staining and western blot analysis. The consequence of VILI by H&E stain showed inducing lung congestion and increasing the expression of pro-inflammatory cytokines such as TNF-α and IL-1β in the lung tissue homogenization, serum, and BALF, respectively. In addition, rats with VILI also exhibited high expression of IL-33 in lung tissues. Interestingly, the data showed that ST2L (membrane form) was highly accumulated in the membrane fraction of lung tissue in the PC10 group, but the ST2L in cytosol was dramatically decreased in the PC10 group. Conversely, the sST2 (soluble form) was slightly decreased both in the membrane and cytosol fractions in the PC10 group compared to the control group. In conclusion, these results demonstrated that ST2L translocation from the cytosol to the cell membranes of lung tissue and the down-expression of sST2 in both fractions can function as new biomarkers of VILI. Moreover, IL-33/ST2 signaling activated by mechanically responsive lung injury may potentially serve as a new therapy target. [ABSTRACT FROM AUTHOR]

Very late antigen-4 (VLA-4), a member of integrin superfamily, interacts with its major counter ligand vascular cell adhesion molecule-1 (VCAM-1) and plays an important role in leukocyte adhesion to vascular endothelium and immunological synapse formation. However, irregular expressions of these proteins may also lead to several autoimmune diseases and metastasis cancer. Thus, quantifying the interaction affinity of the VCAM-1/VLA-4 interaction is of fundamental importance in further understanding the nature of this interaction and drug discovery. In this study, we report an ‘in solution’ steady state organic fluorophore based quantitative fluorescence resonance energy transfer (FRET) assay to quantify this interaction in terms of the dissociation constant (Kd). We have used, in our FRET assay, the Alexa Fluor 488-VLA-4 conjugate as the donor, and Alexa Fluor 546-VCAM-1 as the acceptor. From the FRET signal analysis, Kd of this interaction was determined to be 41.82 ± 2.36 nM. To further confirm our estimation, we have employed surface plasmon resonance (SPR) technique to obtain Kd = 39.60 ± 1.78 nM, which is in good agreement with the result obtained by FRET. This is the first reported work which applies organic fluorophore based ‘in solution’ simple quantitative FRET assay to obtain the dissociation constant of the VCAM-1/VLA-4 interaction, and is also the first quantification of this interaction. Moreover, the value of Kd can serve as an indicator of abnormal protein-protein interactions; hence, this assay can potentially be further developed into a drug screening platform of VLA-4/VCAM-1 as well as other protein-ligand interactions. [ABSTRACT FROM AUTHOR]

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum. [ABSTRACT FROM AUTHOR]

The tumor microenvironment plays a crucial role during tumor development. Integrated combination of drugs that target tumor microenvironment is a promising approach to anticancer therapy. Here, we report a multifunctional combination of low-cytotoxic drugs composed of dipyridamole, bestatin and dexamethasone (DBDx) which mainly acts on the tumor microenvironment shows highly potent antitumor efficacy in vivo. In mouse hepatoma H22 model, the triple drug combination showed synergistic and highly potent antitumor efficacy. The combination indices of various combinations of the triple drugs were between 0.2 and 0.5. DBDx inhibited the growth of a panel of human tumor xenografts and showed no obvious systemic toxicity. At tolerated doses, DBDx suppressed the growth of human hepatocellular carcinoma BEL-7402, HepG2, and lung adenocarcinoma A549 xenografts by 94.5%, 93.7% and 96.9%, respectively. Clonogenic assay demonstrated that DBDx showed weak cytotoxicity. Western blot showed that Flk1 and Nos3 were down-regulated in the DBDx-treated group. Proteomic analysis showed that DBDx mainly affected the metabolic process and immune system process; in addition, the angiogenesis and VEGF signaling pathway were also affected. Conclusively, DBDx, a multifunctional drug combination of three low-cytotoxic drugs, shows synergistic and highly potent antitumor efficacy evidently mediated by the modulation of tumor microenvironment. Based on its low-cytotoxic attributes and its broad-spectrum antitumor therapeutic efficacy, this multifunctional combination might be useful in the treatment of cancers, especially those refractory to conventional chemotherapeutics. [ABSTRACT FROM AUTHOR]

Background: 5-Hydroxytryptamine (5-HT) is a powerful constrictor of coronary arteries and is considered to be involved in the pathophysiological mechanisms of coronary-artery spasm. However, the mechanism of enhancement of coronary-artery constriction to 5-HT during the development of coronary artery disease remains to be elucidated. Organ culture of intact blood-vessel segments has been suggested as a model for the phenotypic changes of smooth muscle cells in cardiovascular disease. Methodology/Principal Findings: We wished to characterize 5-HT receptor-induced vasoconstriction and quantify expression of 5-HT receptor signaling in cultured rat coronary arteries. Cumulative application of 5-HT produced a concentration-dependent vasoconstriction in fresh and 24 h-cultured rat coronary arteries without endothelia. 5-HT induced greater constriction in cultured coronary arteries than in fresh coronary arteries. U46619- and CaCl2-induced constriction in the two groups was comparable. 5-HT stimulates the 5-HT2A receptor and cascade of phospholipase C to induce coronary vasoconstriction. Calcium influx through L-type calcium channels and non-L-type calcium channels contributed to the coronary-artery constrictions induced by 5-HT. The contractions mediated by non-L-type calcium channels were significantly enhanced in cultured coronary arteries compared with fresh coronary arteries. The vasoconstriction induced by thapsigargin was also augmented in cultured coronary arteries. The decrease in Orai1 expression significantly inhibited 5-HT-evoked entry of Ca2+ in coronary artery cells. Expression of the 5-HT2A receptor, Orai1 and STIM1 were augmented in cultured coronary arteries compared with fresh coronary arteries. Conclusions: An increased contraction in response to 5-HT was mediated by the upregulation of 5-HT2A receptors and downstream signaling in cultured coronary arteries. [ABSTRACT FROM AUTHOR]

Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for progressing towards clinical symptoms of necrotic enteritis. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study was to investigate the incidence and demographic/clinical factors of alanine aminotransferase (ALT) abnormalities at the end of treatment (EOT) in chronic hepatitis C (CHC) patients with sustained virologic response (SVR). Methods and Findings: Seven hundred naïve CHC patients who underwent combination treatment between January 2003 and December 2010 were included in the study. The patients with SVR and serum ALT>upper limit of normal (ULN) at the EOT were further analyzed. The effects of clinical characteristics, treatment regimen, and virologic variables were evaluated by logistic regression. Of the 700 included patients, 488 (69.7%) achieved an SVR after treatment, and 235 (33.6%) had serum ALT levels>ULN at the EOT. Of those 488 patients, 137 (28.1%) had abnormal ALT values at the EOT. A multivariate analysis showed that the occurrence of ALT abnormalities at the EOT was significantly associated with pegylated interferon (PEG-IFN) alfa-2a (odds ratio [OR], 2.24; 95% confidence interval [CI], 1.45–3.45; P<0.001), baseline fatty liver (OR, 1.76; 95% CI, 1.16–2.76; P = 0.007), and baseline liver cirrhosis (OR, 2.35; 95% CI, 1.35–4.09; P = 0.002). Conclusions: Use of PEG-IFN-alfa-2a, fatty liver, and cirrhosis are important factors associated with EOT-ALT abnormality in CHC patients receiving combination therapy that achieve an SVR. PEG-IFN-alfa-2a-related EOT-ALT elevation will become normal at the end of follow-up, but fatty liver and cirrhosis-related ALT elevation will not be resolved. [ABSTRACT FROM AUTHOR]

We demonstrated a real-time monitoring of live cells upon laminar shear stress stimulation via surface plasmon resonance (SPR) in gold nanoslit array. A large-area gold nanostructure consisted of 500-nm-period nanoslits was fabricated on a plastic film using the thermal-annealed template-stripping method. The SPR in the gold nanoslit array provides high surface sensitivity to monitor cell adhesion changes near the sensor surface. The human non-small cell lung cancer (CL1-0), human lung fibroblast (MRC-5), and human dermal fibroblast (Hs68) were cultured on the gold nanoslits and their dynamic responses to laminar shear stress were measured under different stress magnitudes from 0 to 30 dyne/cm2. Cell adhesion was increased in CL1-0 under shear flow stimulation. No adhesion recovery was observed after stopping the flow. On the other hand, MRC-5 and Hs68 decreased adhesion and recovered from the shear stress. The degree of recovery was around 70% for MRC-5. This device provides dynamic study and early detection of cell adhesion changes under shear flow conditions. [ABSTRACT FROM AUTHOR]

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1+/− mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles. [ABSTRACT FROM AUTHOR]

Purposes: The study is to describe a new surgical technique for correcting large orbital implant exposure with extraocular muscle flaps and to propose a treatment algorithm for orbital implant exposure. Methods: In a retrospective study, seven patients with orbital implant exposure were treated with extraocular muscle flaps. All data were collected from patients in Chang Gung Memorial Hospital, Taiwan during 2007–2012. All surgeries were performed by one surgeon (Y.J.T). Patient demographics, the original etiology, details of surgical procedures, implant types, and follow-up interval were recorded. Small exposure, defined as exposure area smaller than 3 mm in diameter, was treated conservatively first with topical lubricant and prophylactic antibiotics. Larger defects were managed surgically. Results: Seven patients consisting of two males and five females were successfully treated for orbital implant exposure with extraocular muscle flaps. The average age was 36.4 (range, 3–55) years old. Five patients were referred from other hospitals. One eye was enucleated for retinoblastoma. The other six eyes were eviscerated, including one for endophthalmitis and five for trauma. Mean follow-up time of all seven patients was 19.5 (range, 2–60) months. No patient developed recurrence of exposure during follow-up. All patients were fitted with an acceptable prosthesis and had satisfactory cosmetic and functional results. Conclusions: The most common complication of orbital implant is exposure, caused by breakdown of the covering layers, leading to extrusion. Several methods were reported to manage the exposed implants. We report our experience of treating implant exposure with extraocular muscle flaps to establish a well-vascularized environment that supplies both the wrapping material and the overlying ocular surface tissue. We believe it can work as a good strategy to manage or to prevent orbital implant exposure. [ABSTRACT FROM AUTHOR]

Increasing evidence demonstrates that maternal folic acid (FA) supplementation during pregnancy reduces the risk of neural tube defects, but whether FA prevents preterm delivery and intrauterine growth restriction (IUGR) remains obscure. Previous studies showed that maternal lipopolysaccharide (LPS) exposure induces preterm delivery, fetal death and IUGR in rodent animals. The aim of this study was to investigate the effects of FA on LPS-induced preterm delivery, fetal death and IUGR in mice. Some pregnant mice were orally administered with FA (0.6, 3 or 15 mg/kg) 1 h before LPS injection. As expected, a high dose of LPS (300 μg/kg, i.p.) on gestational day 15 (GD15) caused 100% of dams to deliver before GD18 and 89.3% of fetuses dead. A low dose of LPS (75 μg/kg, i.p.) daily from GD15 to GD17 resulted in IUGR. Interestingly, pretreatment with FA prevented LPS-induced preterm delivery and fetal death. In addition, FA significantly attenuated LPS-induced IUGR. Further experiments showed that FA inhibited LPS-induced activation of nuclear factor kappa B (NF-κB) in mouse placentas. Moreover, FA suppressed LPS-induced NF-κB activation in human trophoblast cell line JEG-3. Correspondingly, FA significantly attenuated LPS-induced upregulation of cyclooxygenase (COX)-2 in mouse placentas. In addition, FA significantly reduced the levels of interleukin (IL)-6 and keratinocyte-derived cytokine (KC) in amniotic fluid of LPS-treated mice. Collectively, maternal FA supplementation during pregnancy protects against LPS-induced preterm delivery, fetal death and IUGR through its anti-inflammatory effects. [ABSTRACT FROM AUTHOR]

Hypercapnic acidosis (HCA) has protective effects in animal models of acute lung injury, but the mechanism underlying the effect of HCA is unclear. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that protects tissue from inflammation injury. We investigated whether HO-1 contributes to the protective effects of HCA in ischemia-reperfusion (IR)-induced lung injury. Typical acute lung injury in rats was successfully induced by 40 min of ischemia and 90 min of reperfusion in an isolated perfused lung model. The rat lungs were randomly assigned to the control group, IR group or IR + HCA group with or without zinc protoporphyrin IX (ZnPP), an HO-1 activity inhibitor. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lung tissues were collected to evaluate the degree of lung injury. In in vitro experiments, HO-1 siRNA transfected A549 cells were exposed to a normoxic or hypoxia-reoxygenation (H/R) environment in the presence or absence of HCA. IR caused significant increases in the pulmonary arterial pressure, lung weight to body weight and wet/dry ratios, lung weight gain, capillary filtration coefficient, lung injury scores, neutrophil infiltration, and concentrations of protein and TNF-α in the BALF. IR also induced degradation of inhibitor of nuclear factor (NF)-κB-α, increased IκB kinase (IKK)-β phosphorylation and nuclear translocation of NF-κB, and up-regulated HO-1 expression and activity. Furthermore, IR decreased Bcl-2 protein expression and increased the number of active caspase-3 stained cells. HCA treatment enhanced HO-1 expression and activity, and accordingly reduced IKK-NF-κB signaling, inhibited apoptosis, and significantly attenuated IR-induced changes. Treatment with ZnPP partially blocked the protective effect of HCA. In addition, HO-1 siRNA significantly reversed HCA-mediated inhibition of NF-κB signaling in A549 cells subjected to H/R. In conclusion, the protective effect of HCA in IR lung injury in rats was mediated in part by the anti-inflammatory and anti-apoptotic action of HO-1. [ABSTRACT FROM AUTHOR]