Identification and functional analysis of a biflavone as a novel inhibitor of transient receptor potential vanilloid 4-dependent atherogenic processes.

Atherosclerosis, a chronic inflammatory disease of large arteries, is the major contributor to the growing burden of cardiovascular disease-related mortality and morbidity. During early atherogenesis, as a result of inflammation and endothelial dysfunction, monocytes transmigrate into the aortic intimal areas, and differentiate into lipid-laden foam cells, a critical process in atherosclerosis. Numerous natural compounds such as flavonoids and polyphenols are known to have anti-inflammatory and anti-atherogenic properties. Herein, using a fluorometric imaging plate reader-supported Ca2+ influx assay, we report semi high-throughput screening-based identification of ginkgetin, a biflavone, as a novel inhibitor of transient receptor potential vanilloid 4 (TRPV4)-dependent proatherogenic and inflammatory processes in macrophages. We found that ginkgetin (1) blocks TRPV4-elicited Ca2+ influx into macrophages, (2) inhibits oxidized low-density lipoprotein (oxLDL)-induced foam cell formation by suppressing the uptake but not the binding of oxLDL in macrophages, and (3) attenuates oxLDL-induced phosphorylation of JNK2, expression of TRPV4 proteins, and induction of inflammatory mRNAs. Considered all together, the results of this study show that ginkgetin inhibits proatherogenic/inflammatory macrophage function in a TRPV4-dependent manner, thus strengthening the rationale for the use of natural compounds for developing therapeutic and/or chemopreventive molecules.

A functional genomics screen identifying blood cell development genes in Drosophila by undergraduates participating in a course-based research experience.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Imperatorin ameliorates learning and memory deficits through BDNF/TrkB and ERK/CaMKIIα/CREB signaling in prenatally-stressed female offspring.

Prenatal stress (PS) can lead to impaired spatial learning and memory in offspring. Imperatorin (IMP) is a naturally occurring furanocoumarin with many pharmacological properties. However, the effects of IMP on cognitive impairment induced by PS and the underlying molecular mechanisms remain unclear. We investigated the protective effect of IMP treatment after PS on learning and memory deficits in female offspring at postnatal 60 days. After treating prenatally-stressed offspring with IMP (15 and 30 mg/kg) for 28 days, we found that IMP increased body weight and ameliorated spatial learning and memory and working memory deficits in female offspring rats. Meanwhile, hippocampal Glu and serum corticosterone levels in prenatally-stressed offspring were significantly decreased after IMP administration. Additionally, IMP treatment significantly increased BDNF, TrkB, CaMKII, and CREB mRNA expression in the hippocampus of offspring rats. Furthermore, PS-mediated induction of RKIP protein and mRNA expression and glucocorticoid receptor protein expression in the hippocampus of offspring rats were significantly decreased by IMP treatment, and the protein expression of BDNF and TrkB and relative levels of p-EKR/ERK, p-CaMKIIα/CaMKIIα, and p-CREB/CREB were remarkably increased after IMP treatment. Taken together, IMP can ameliorate PS-induced learning and memory deficits through BDNF/TrkB and ERK/CaMKIIα/CREB signaling pathway and hypothalamic-pituitary-adrenal axis.

Correlations of neck circumference with body composition and cardiometabolic risk factors in Arab women.

BACKGROUND: Neck circumference (NC) is a relatively unused index of upper body adiposity. The present study aims to analyze the associations of NC with anthropometric measures of obesity, as well as cardiovascular and metabolic risks in Arab women. METHODS: This cross-sectional study included 623 women (aged 18-70 years) recruited from different primary care centers in Riyadh, Saudi Arabia. NC, waist circumference (WC), body mass index (BMI), blood pressure, and metabolic and serological markers were measured in all participants. Covariance and regression analyses were used to evaluate the associations between NC and cardiometabolic risk factors. RESULTS: The correlation coefficients of NC and WC with the clinical indices were highly significant (p < 0.01). Overall, the NC was positively correlated with all cardiometabolic markers except total cholesterol and LDLc (p < 0.001). Interestingly, NC was associated with cardiometabolic risk factors independent of other anthropometric indices. CONCLUSION: NC is significantly and independently associated with cardiometabolic risk factors in Arab women. LEVEL OF EVIDENCE: V, cross-sectional descriptive study.

Survival outcomes following primary tumor resection for patients with incurable metastatic colorectal carcinoma: Experience from a single institution.

OBJECTIVE: Palliative primary tumor resection (PTR) has been used for preventing and treating tumor-related complications. We aimed to determine whether PTR can increase overall survival (OS) in patients with unresectable metastatic colorectal cancer (CRC). METHODS: A retrospective review of a prospectively collected database in a single center was performed. Patients diagnosed with metastatic CRC from January 2004 to December 2014 were included. Patients who had attained curative resection or had disease recurrence were excluded. All patients were discussed at a multidisciplinary tumor board where subsequent treatment decisions were made. RESULTS: Altogether 408 patients were analyzed. Of these 145 received PTR with palliative chemotherapy (PC; group A), 110 received PC only (group B), 52 received PTR only (group C), while 101 received neither PTR nor PC (group D). Undergoing PTR led to statistically significant improvement in OS (22.7 months vs 12.1 months vs 6.9 months vs 2.7 months, P < 0.001). We performed subgroup analyses to control for potential confounders and found that the influence of PTR on OS persisted. With multivariate analysis, the predictors of poor OS were no PTR (hazards ratio [HR] 2.32, 95% confidence interval [CI] 1.82-2.96, P < 0.001), no PC (HR 4.25, 95% CI 3.27-5.33, P < 0.001) and the presence of peritoneal metastases (HR 1.37, 95% CI 1.06-1.78, P = 0.018). Diversion surgery did not lead to a statistical difference in OS. CONCLUSIONS: The absences of PTR and PC, and peritoneal metastases are independently associated with decreased OS in patients with unresectable metastatic CRC. Randomized controlled trials are needed to verify this observation.

The small molecule drug diminazene aceturate inhibits liver injury and biliary fibrosis in mice.

There is no established medical therapy to treat biliary fibrosis resulting from chronic inflammation in the biliary tree. We have recently shown that liver-specific over-expression of angiotensin converting enzyme 2 (ACE2) of the renin angiotensin system (RAS) ameliorated liver fibrosis in mice. Diminazene aceturate (DIZE), a small molecule drug approved by the US Food and Drug Administration, which is used to treat human trypanosomiasis, has been shown to have antifibrotic properties by enhancing ACE2 activity. In this study we sought to determine the therapeutic potential of DIZE in biliary fibrosis using bile duct ligated and multiple drug resistant gene-2 knockout mice. Additionally, human hepatic stellate (LX-2) and mouse Kupffer (KUP5) cell lines were used to delineate intracellular pathways. DIZE treatment, both in vivo and in vitro, markedly inhibited the activation of fibroblastic stellate cells which was associated with a reduced activation of Kupffer cells. Moreover, DIZE-inhibited NOX enzyme assembly and ROS generation, activation of profibrotic transcription factors including p38, Erk1/2 and Smad2/3 proteins and proinflammatory and profibrotic cytokine release. These changes led to a major reduction in biliary fibrosis in both models without affecting liver ACE2 activity. We conclude that DIZE has a potential to treat biliary fibrosis.

Histological findings in protocol biopsies following pediatric liver transplant: Low incidence of abnormalities at 5 years.

Histological abnormalities, including chronic hepatitis, fibrosis, and steatosis, are increasingly reported in liver biopsies of children after LT. These changes may be progressive and represent a form of rejection. Liver biochemistry is often initially normal. Our LT program began in 2002, utilizing tacrolimus and low-dose steroids for the first year post-LT. Patients undergo a protocol biopsy at 1 year post-LT prior to stopping steroids, then at 5 years and every 5 years thereafter. Target tacrolimus levels are 5-8 µg/L and 3-5 µg/L after 3 and 12 months, respectively. Between 2002 and 2009, 51 LT were performed; 50 (98%) and 49 (96%) patients survived for 1 and 5 years, respectively. A total of 43 patients (median age at LT 2.3 years) underwent a protocol biopsy at 1 year (16 male; median time post-LT 12.5 months), and 44 (20 male; median time post-LT 5.1 years) at 5 years. By 5 years, 3 had transferred to adult services; 1 was re-transplanted for graft failure and 1 moved overseas. Biopsies were reviewed by 2 pathologists. Most patients (31/44) were on tacrolimus monotherapy at 5 years. At 1 and 5 years, 29 of 43 (67.5%) and 31 of 44 (71%) biopsies were normal, respectively. Two of 44 had chronic allograft hepatitis at 5 years. Two of 43 and 1 of 44 had isolated fibrosis, 3 of 43 and 3 of 44 steatosis, and 3 of 43 and 4 of 44 acute rejection at 1 and 5 years, respectively. Other findings included predominantly biliary changes (6/43 & 3/44 at 1 and 5 years, respectively). Tacrolimus levels at 5 years were slightly higher than anticipated (median trough level 5.8 µg/L). With an immunosuppressive regimen of tacrolimus and low-dose steroids for 1 year followed by tacrolimus monotherapy thereafter, the majority of PLB were normal and no progressive changes were observed at 5 years. Compared to other LT programs, we have lower rates of chronic allograft hepatitis, steatosis, and fibrosis at 5 years. However, the tacrolimus levels at 5 years were higher than planned and this may have played a role. Further evaluation is also required to determine the potential long-term adverse effects of corticosteroid use on linear growth and bone mineral density.

Juvenile migration of the exclusively pelagic cyprinid, Gnathopogon caerulescens (Honmoroko) in Lake Biwa, Central Japan.

Migration of wild and cultivated juvenile honmoroko Gnathopogon caerulescens of from the spawning and nursery areas in Lake Biwa were investigated, both in the Ibanaiko Lagoon and its outlet to Daido River, using beam-trawl surveys in 2013 and 2014. The study demonstrated migration of G. caerulescens from a nursery lagoon toward Lake Biwa after the juvenile stage. These findings appear to be the first direct evidence for migration of an exclusively pelagic cyprinid species from a littoral nursery to a pelagic adult habitat in a large deep lake.

TRPV4 ion channel is a novel regulator of dermal myofibroblast differentiation.

Scleroderma is a multisystem fibroproliferative disease with no effective medical treatment. Myofibroblasts are critical to the fibrogenic tissue repair process in the skin and many internal organs. Emerging data support a role for both matrix stiffness, and transforming growth factor ß1 (TGFß1), in myofibroblast differentiation. Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive ion channel activated by both mechanical and biochemical stimuli. The objective of this study was to determine the role of TRPV4 in TGFß1- and matrix stiffness-induced differentiation of dermal fibroblasts. We found that TRPV4 channels are expressed and functional in both human (HDF) and mouse (MDF) dermal fibroblasts. TRPV4 activity (agonist-induced Ca2+ influx) was induced by both matrix stiffness and TGFß1 in dermal fibroblasts. TGFß1 induced expression of TRPV4 proteins in a dose-dependent manner. Genetic ablation or pharmacological antagonism of TRPV4 channel abrogated Ca2+ influx and both TGFß1-induced and matrix stiffness-induced myofibroblast differentiation as assessed by 1) α-smooth muscle actin expression/incorporation into stress fibers, 2) generation of polymerized actin, and 3) expression of collagen-1. We found that TRPV4 inhibition abrogated TGFß1-induced activation of AKT but not of Smad2/3, suggesting that the mechanism by which profibrotic TGFß1 signaling in dermal fibroblasts is modified by TRPV4 may be through non-Smad pathways. Altogether, these data identify a novel reciprocal functional link between TRPV4 activation and TGFß1 signals regulating dermal myofibroblast differentiation. These findings suggest that therapeutic inhibition of TRPV4 activity may provide a targeted approach to the treatment of scleroderma.

The Adeno-associated Virus - A Safe and Promising Vehicle for Liverspecific Gene Therapy of Inherited and Non-inherited Disorders.

INTRODUCTION: The first human adeno-associated virus (AAV) was originally discovered in 1960s as a contaminant of adenovirus stock preparation and thus it had not been of medical interest. Throughout the last three decades AAV has gained popularity to be used in gene therapy, mainly due to its replicative defectiveness and lack of pathogenicity in human. In addition, its ability to mediate stable and long-term expression in both non-dividing and dividing cells with specific tissue tropism makes AAV one of the most promising candidates for therapeutic gene transfer to treat many inherited as well as non-inherited disorders. Moreover, the use of AAV is not only restricted to overexpression of recombinant transgene, but also to over-express short hairpin RNA and microRNA to knockdown the expression of genes in targeted tissues. DISCUSSION AND CONCLUSION: This review is organized into four parts. In the first part of the review, we discuss about the discovery and history of AAV, followed by detailed AAV biology such as virus genome, virus structure and its life cycle. In the second part of the review, the discussion is centred on the molecular mechanisms of AAV and tissue transduction, including receptor recognition and cell binding, endosomal entry, virus uncoating, nuclear entry and genome replication. Advantages and limitations of using AAV as a safe vehicle for gene delivery is also discussed. In the third part of the review, we discuss about the most commonly used AAV serotypes and variants isolated from human and non-human primates, focusing on their diverse tissue tropisms, transduction efficiency, immunological profiles and their applications in animal studies. Final part of the review focuses on the recent progress of in-vivo gene transfer using AAV for inherited and non-inherited diseases in both preclinical and clinical settings with a special emphasis on potential clinical applications of AAV in the field of liver disease.

Genetic diversity and population structure of Hyporhamphus sajori (Beloniformes: Hemiramphidae) inferred from mtDNA control region and msDNA markers.

This paper presents preliminary data on the genetic diversity and population structure of Hyporhamphus sajori by analysing a 510 bp sequence in the mitochondrial DNA (mtDNA) control region and eight polymorphic microsatellite DNA loci. The H. sajori individuals from different locations were indistinguishable from one another based on mtDNA variation, as demonstrated with a neighbour-joining tree and minimum spanning network analysis. Low level of genetic diversity and the absence of population structure in H. sajori from the north-west Pacific Ocean, combined with negative indices for neutral evolution in these populations, suggest that H. sajori underwent a population expansion after a recent bottleneck. The Structure analysis, discriminant analysis of principal components (DAPC) and the pair-wise ΦST values after Bonferroni correction using eight microsatellite loci provided no clear inference on the genetic differentiation and thus no evidence of population structure of H. sajori. The genetic connectivity among locations might be due to fairly high gene flow via transport of eggs and larvae by the Kuroshio and Tsushima warm current. This study revealed low levels of genetic diversity and suggested high level of contemporary gene flow among populations of H. sajori in the East (Japan) Sea and the Pacific Ocean.

Predicting later life health status and mortality using state-level socioeconomic characteristics in early life.

Studies extending across multiple life stages promote an understanding of factors influencing health across the life span. Existing work has largely focused on individual-level rather than area-level early life determinants of health. In this study, we linked multiple data sets to examine whether early life state-level characteristics were predictive of health and mortality decades later. The sample included 143,755 U.S. employees, for whom work life claims and administrative data were linked with early life state-of-residence and mortality. We first created a "state health risk score" (SHRS) and "state mortality risk score" (SMRS) by modeling state-level contextual characteristics with health status and mortality in a randomly selected 30% of the sample (the "training set"). We then examined the association of these scores with objective health status and mortality in later life in the remaining 70% of the sample (the "test set") using multivariate linear and Cox regressions, respectively. The association between the SHRS and adult health status was ß=0.14 (95%CI: 0.084, 0.20), while the hazard ratio for the SMRS was 0.96 (95%CI: 0.93, 1.00). The association between the SHRS and health was not statistically significant in older age groups at a p-level of 0.05, and there was a statistically significantly different association for health status among movers compared to stayers. This study uses a life course perspective and supports the idea of "sensitive periods" in early life that have enduring impacts on health. It adds to the literature examining populations in the U.S. where large linked data sets are infrequently available.

An Electronic Wellness Program to Improve Diet and Exercise in College Students: A Pilot Study.

BACKGROUND: In transitioning from adolescence to adulthood, college students are faced with significant challenges to their health habits. Independence, stress, and perceived lack of time by college students have been known to result in poor eating and exercise habits, which can lead to increased disease risk. OBJECTIVE: To assess the feasibility and to determine preliminary efficacy of an electronic wellness program in improving diet and physical activity in college students. METHODS: A 24-week diet and physical activity program was delivered via email to 148 college students. The intervention involved weekly, tailored, and interactive diet and physical activity goals. The control group received nondiet and nonexercise-related health fact sheets. Anthropometric and blood pressure measurements, as well as food frequency and physical activity surveys were conducted at baseline, week 12, and week 24. Students' choice of fruit as a snack was also monitored at study visits. RESULTS: Students were 18-20 years old, 69% female, and from a diverse college campus (46% Caucasian, 23% Asian, 20% African American, 11% other). At week 24, 84% of students reported reading at least half of all emails. Mean change (standard error [SE]) from baseline of saturated fat intake was marginally significant between the treatment groups at week 24, 0.7 (SE 0.42) % kcal for control and -0.3 (SE 0.30) % kcal for intervention (P=0.048). A significant difference in percent of snacks chosen that were fruit (χ(2)1, N=221 = 11.7, P<0.001) was detected between the intervention and control group at week 24. CONCLUSIONS: Use of an electronic wellness program is feasible in college students and resulted in a decrease in saturated fat intake and an increase in observed fruit intake compared to a control group.

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.

Arcuate Na+,K+-ATPase senses systemic energy states and regulates feeding behavior through glucose-inhibited neurons.

Feeding is regulated by perception in the hypothalamus, particularly the first-order arcuate nucleus (ARC) neurons, of the body's energy state. However, the cellular device for converting energy states to the activity of critical neurons in ARC is less defined. We here show that Na(+),K(+)-ATPase (NKA) in ARC senses energy states to regulate feeding. Fasting-induced systemic ghrelin rise and glucose lowering reduced ATP-hydrolyzing activity of NKA and its substrate ATP level, respectively, preferentially in ARC. Lowering glucose concentration (LG), which mimics fasting, decreased intracellular NAD(P)H and increased Na(+) concentration in single ARC neurons that subsequently exhibited [Ca(2+)]i responses to LG, showing that they were glucose-inhibited (GI) neurons. Third ventricular injection of the NKA inhibitor ouabain induced c-Fos expression in agouti-related protein (AgRP) neurons in ARC and evoked neuropeptide Y (NPY)-dependent feeding. When injected focally into ARC, ouabain stimulated feeding and mRNA expressions for NPY and AgRP. Ouabain increased [Ca(2+)]i in single NPY/AgRP neurons with greater amplitude than in proopiomelanocortin neurons in ARC. Conversely, the specific NKA activator SSA412 suppressed fasting-induced feeding and LG-induced [Ca(2+)]i increases in ARC GI neurons. NPY/AgRP neurons highly expressed NKAα3, whose knockdown impaired feeding behavior. These results demonstrate that fasting, via ghrelin rise and LG, suppresses NKA enzyme/pump activity in ARC and thereby promotes the activation of GI neurons and NPY/AgRP-dependent feeding. This study identifies ARC NKA as a hypothalamic sensor and converter of metabolic states to key neuronal activity and feeding behaviour, providing a new target to treat hyperphagic obesity and diabetes.

ACE2 Therapy Using Adeno-associated Viral Vector Inhibits Liver Fibrosis in Mice.

Angiotensin converting enzyme 2 (ACE2) which breaks down profibrotic peptide angiotensin II to antifibrotic peptide angiotensin-(1-7) is a potential therapeutic target in liver fibrosis. We therefore investigated the long-term therapeutic effect of recombinant ACE2 using a liver-specific adeno-associated viral genome 2 serotype 8 vector (rAAV2/8-ACE2) with a liver-specific promoter in three murine models of chronic liver disease, including carbon tetrachloride-induced toxic injury, bile duct ligation-induced cholestatic injury, and methionine- and choline-deficient diet-induced steatotic injury. A single injection of rAAV2/8-ACE2 was administered after liver disease has established. Hepatic fibrosis, gene and protein expression, and the mechanisms that rAAV2/8-ACE2 therapy associated reduction in liver fibrosis were analyzed. Compared with control group, rAAV2/8-ACE2 therapy produced rapid and sustained upregulation of hepatic ACE2, resulting in a profound reduction in fibrosis and profibrotic markers in all diseased models. These changes were accompanied by reduction in hepatic angiotensin II levels with concomitant increases in hepatic angiotensin-(1-7) levels, resulting in significant reductions of NADPH oxidase assembly, oxidative stress and ERK1/2 and p38 phosphorylation. Moreover, rAAV2/8-ACE2 therapy normalized increased intrahepatic vascular tone in fibrotic livers. We conclude that rAAV2/8-ACE2 is an effective liver-targeted, long-term therapy for liver fibrosis and its complications without producing unwanted systemic effects.

The feasibility of an e-mail-delivered intervention to improve nutrition and physical activity behaviors in African American college students.

OBJECTIVES: To evaluate the feasibility and acceptability of an e-mail-delivered program to promote nutrition and physical activity in African American college students. PARTICIPANTS: Forty-seven students (76% female, aged 18-20 years). METHODS: Students participated in a 24-week randomized controlled trial, receiving either general health information or the intervention focused on diet and physical activity. RESULTS: At baseline, 80.9% and 76.0% of participants reported interest in improving diet and physical activity, respectively. Participants evidenced poor nutrition behaviors and 46% were overweight or obese. At 24 weeks, most participants (70% control, 84% intervention) were "somewhat" or "very" satisfied with the program. The program was feasible to administrate, with the exception of measurement of physical activity using accelerometers. CONCLUSIONS: An innovative e-mail-delivered program promoting positive health behaviors appears to be feasible and acceptable in African American college students. Further research is needed to evaluate program efficacy in this population, including prevention of excess weight gain.