Actin-mediated endocytosis in the E-YSL helps drive epiboly in zebrafish.

In zebrafish, a punctate band of F-actin is reported to develop in the external yolk syncytial layer (E-YSL) during the latter part of epiboly in zebrafish embryos. Here, electron microscopy (EM) and fluorescence confocal microscopy were conducted to investigate dynamic changes in the E-YSL membrane during epiboly. Using scanning EM, we report that the surface of the E-YSL is highly convoluted, consisting of a complex interwoven network of branching membrane surface microvilli-like protrusions. The region of membrane surface protrusions was relatively wide at 30% epiboly but narrowed as epiboly progressed. This narrowing was coincident with the formation of the punctate actin band. We also demonstrated using immunogold transmission EM that actin clusters were localized at the membrane surface mainly within the protrusions as well as in deeper locations of the E-YSL. Furthermore, during the latter part of epiboly, the punctate actin band was coincident with a region of highly dynamic endocytosis. Treatment with cytochalasin B led to the disruption of the punctate actin band and the membrane surface protrusions, as well as the attenuation of endocytosis. Together, our data suggest that, in the E-YSL, the region encompassing the membrane surface protrusions and its associated punctate actin band are likely to be an integral part of the localized endocytosis, which is important for the progression of epiboly in zebrafish embryos.

Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody.

Amplification and overexpression of HER2 (human epidermal growth factor receptor 2), an ErbB2 receptor tyrosine kinase, have been implicated in human cancer and metastasis. A bispecific tetravalent anti-HER2 antibody (anti-HER2-Bs), targeting two non-overlapping epitopes on HER2 in domain IV (trastuzumab) and domain II (39S), has been reported to induce rapid internalization and efficient degradation of HER2 receptors. In this study, we investigated the molecular mechanism of this antibody-induced rapid HER2 internalization and intracellular trafficking. Using quantitative fluorescent imaging, we compared the internalization kinetics of anti-HER2-Bs and its parental arm antibodies, alone or in combinations and under various internalization-promoting conditions. The results demonstrated that concurrent engagement of both epitopes was necessary for rapid anti-HER2-Bs internalization. Cellular uptake of anti-HER2-Bs and parental arm antibodies occurred via clathrin-dependent endocytosis; however, inside the cells antibodies directed different trafficking pathways. Trastuzumab dissociated from HER2 in 2 h, enabling the receptor to recycle, whereas anti-HER2-Bs stayed associated with the receptor throughout the entire endocytic pathway, promoting receptor ubiquitination, trafficking to the lysosomes, and efficient degradation. Consistent with routing HER2 to degradation, anti-HER2-Bs significantly reduced HER2 shedding and altered its exosomal export. Collectively, these results enable a better understanding of the mechanism of action of anti-Her2-Bs and can guide the rational design of anti-HER2 therapeutics as well as other bispecific molecules.

Impact of IL28B and PNPLA3 polymorphisms on treatment outcomes in patients infected with genotype 6 hepatitis C virus.

BACKGROUND/AIMS: Interleukin-28B (IL28B) and patatin-like phospholipase domain containing 3 (PNPLA3) gene polymorphisms are associated with hepatitis C virus (HCV) clearance and fatty liver, respectively. We aimed to test if their polymorphisms are associated with virologic responses in Chinese chronic hepatitis C (CHC) patients. METHODS: This was a retrospective-prospective cohort study. Consecutive patients infected by genotype 1 and 6 HCV received antiviral therapy were included. Host IL-28B rs12979860/rs8099917 and PNPLA3 rs738409 genotype were tested. The primary outcome was sustained virologic response (sustained virologic response [SVR]: undetectable HCV RNA 24 weeks post-treatment). RESULTS: From 305 patients had positive antibody to HCV, 52 and 31 patients infected by genotype 1 and 6 HCV, respectively were recruited. Mean age was 58 ± 11 years; 70% were male. Mean baseline HCV RNA was 6.8 ± 2.7 log IU/ml. The SVR for patients infected by genotype 1 and 6 HCV was 67.3% and 90.3%, respectively. The proportions of IL28B genotypes were 78%, 21%, and 1% for TT/TG/GG at rs8099917, and 81%, 18%, and 1% for CC/TC/TT at rs12979860, respectively. The proportions of PNPLA3 rs738409 genotypes were 16%, 36%, and 48% for GG/GC/CC. IL28B genotype was significantly associated with SVR in patients infected by genotype 1 but not genotype 6 HCV, with 80% versus 38% of patients infected by genotype 1 achieved SVR carried TT versus TG/GG at rs8099917, respectively (P=0.003). PNPLA3 genotype was not associated with SVR. CONCLUSIONS: IL28B gene with rs8099917 T allele as an independent predictor of SVR in Chinese CHC patients infected by genotype 1 but not genotype 6 HCV.

Quantitative measurement of the target-mediated internalization kinetics of biopharmaceuticals.

PURPOSE: Measurement of internalization of biopharmaceuticals targeting cell surface proteins can greatly facilitate drug development. The objective of this study was to develop a reliable method for determination of internalization rate constant (kint) and to demonstrate its utility. METHODS: This method utilized confocal imaging to record the internalization kinetics of fluorescence-tagged biopharmaceuticals in live-cells and a quantitative image-analysis algorithm for kint determination. Kint was incorporated into a pharmacokinetic-pharmacodynamic (PK-PD) model for simulation of the drug PK profiles, target occupancy and the displacement of endogenous ligand. RESULTS: The method was highly sensitive, allowing kint determination in cells expressing as low as 5,000 receptors/cell, and was amenable to adherent and suspension cells. Its feasibility in a mixed cell population, such as whole blood, was also demonstrated. Accurate assessment of the kint was largely attributed to continuous monitoring of internalization in live cells, rapid confocal image acquisition and quantitative image-analysis algorithm. Translational PK-PD simulations demonstrated that kint is a major determinant of the drug PK profiles, target occupancy, and the displacement of endogenous ligand. CONCLUSIONS: The developed method is robust for broad cell types. Reliable kint assessment can greatly expedite biopharmaceutical development by facilitating target evaluation, drug affinity goal setting, and clinical dose projection.

SH3 interactome conserves general function over specific form.

Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.

Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis.

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

Meta-regression analysis using latitude as moderator of paternal age related schizophrenia risk: high ambient temperature induced de novo mutations or is it related to the cold?

While the season of birth, latitude and first admission effects suggest higher risk of schizophrenia with cold climate, the high ambient temperature induced de novo mutation hypothesis suggests the opposite. We conducted a systematic review and meta-analysis (4 case-control studies and 5 cohort studies). We used annual mean daily temperature and latitude of study sites as direct and indirect measures of ambient temperature respectively. Using case-control studies conducted in the Northern hemisphere for meta-regression, high latitude and low ambient temperature were found to increase paternal age related schizophrenia risk significantly. More research is needed to support the de novo mutation hypothesis.

Prevalence and biopsychosocial correlates of erectile dysfunction in Hong Kong: a population-based study.

OBJECTIVES: To describe the prevalence of erectile dysfunction (ED) in Hong Kong and identify the biopsychosocial correlates of ED. METHODS: This was a descriptive and analytic population-representative cross-sectional study of ED in Hong Kong. The study subjects were 1506 men aged 26 to 70 years, recruited by two-stage stratified random sampling, and interviewed face-to-face by trained interviewers with structured questionnaires. RESULTS: The overall prevalence of ED was 36.7% (95% confidence interval [CI] 33.7 to 39.7). The age-specific prevalence rate was 18.3% (95% CI 11.1% to 25.4%), 28.6% (95% CI 23.5% to 33.6%), 37.9% (95% CI 32.3% to 43.5%), 47.3% (95% CI 40.1% to 54.5%), and 61.1% (95% CI 51.1% to 71.0%) for the age groups 26 to 30, 31 to 40, 41 to 50, 51 to 60, and 61 to 70 years, respectively. The severity of ED increased with age (P <0.01), and sexual satisfaction decreased with age (P = 0.01). Age (odds ratio [OR] 1.26, P <0.01), living on Hong Kong Island (OR 0.71, P = 0.04), General Health Questionnaire score (OR 1.03, P <0.01), current smokers of 30 or more cigarettes per day (OR 2.11, P = 0.05), and hours spent on work, housework, and self-study (OR 0.945, P = 0.03) were independently associated with ED. CONCLUSIONS: To our knowledge, this is the first population-representative study of ED in Hong Kong. The prevalence and severity of ED increased with age, and we found biological (age), psychological (General Health Questionnaire), and social (smoking, Hong Kong district, "hours spent on work") factors to have independent influences on ED. The negative association between "hours spent on work" and ED is a novel finding. The results of this study have shown that Hong Kong has a high prevalence of ED compared with Western populations.

Efficacy and safety of atomoxetine for attention-deficit/hyperactivity disorder in children and adolescents-meta-analysis and meta-regression analysis.

OBJECTIVES: The objective of this study was to evaluate the efficacy and safety of atomoxetine in children and adolescents. MATERIALS AND METHODS: We searched for studies published between 1985 and 2006 through Medline, PubMed, PsychInfo and Cochrane Central Register of Controlled Trials (CENTRAL 2006 Issue 3) using keywords related to atomoxetine and attention-deficit/hyperactivity disorder (ADHD) and scanned though reference lists. We included nine randomized placebo-controlled trials (atomoxetine:placebo = 1,150:678). RESULTS: Atomoxetine was superior (p < 0.01) to placebo in reducing ADHD symptoms across different scales (Attention-Deficit/Hyperactivity Disorder Rating Scale-IV, Conners' Parent and Teacher Rating Scales-Revised:Short Form, Clinical Global Impression-Severity) rated by different raters (parent, teacher, clinician). The number-needed-to-treat (NNTs) for treatment response and relapse prevention were 3.43 (95% CI, 2.79-4.45) and 10.30 (95% CI, 5.89-40.62), respectively. High baseline ADHD symptoms (p = 0.02) was associated with greater reduction in ADHD symptoms, whereas male gender (p = 0.02), comorbid oppositional defiant disorder (ODD) status (p = 0.01) and ADHD hyperactive/impulsive subtype (p = 0.01) were associated with smaller reductions. The commonest adverse events were gastrointestinal [appetite decrease, number-needed-to-harm (NNH) = 8.81; abdominal pain, NNH = 22.48; vomiting, NNH = 29.96; dyspepsia, NNH = 49.38] and sleep related (somnolence, NNH = 19.41). Young age (p = 0.03) and high baseline hyperactive/impulsive symptoms (p < 0.01) were associated with more adverse events, whereas ADHD inattentive subtype (p = 0.04) was associated with less adverse events. Quality of life using Child Health Questionnaire (CHQ) improved (p < 0.01) with atomoxetine treatment. Both ADHD and ODD symptoms (p < 0.01) were reduced in comorbid ADHD+ODD, and ODD status was not associated with more adverse events. Efficacy and side effects were not altered by comorbid general anxiety disorder or major depression. CONCLUSIONS: Atomoxetine is efficacious in reducing ADHD symptoms. It may have a role in treating comorbid ODD or depression, and probably in comorbid anxiety.

Depressive symptomatology and male sexual functions in late life.

BACKGROUND: We aimed to investigate the association between depressive symptoms and various male sexual functions, and to identify which depressive symptoms are most predictive of erectile dysfunction (ED). METHODS: This was an analytic cross-sectional study with 160 sexually active men aged 50 or above recruited from a large primary care treatment centre. The 5 domains (erectile function, EF; intercourse satisfaction, IS; orgasmic function, OF; sexual desire, SD; overall satisfaction, OS) of the International Index of Erectile Function (IIEF-15) were used to assess various sexual functions. Depressive symptomatology was measured by Geriatric Depression Scale and reconfirmed with General Health Questionnaire. RESULTS: The level of depressive symptoms was negatively associated with erectile function (p<0.01), orgasmic function (p=0.02), intercourse satisfaction (p=0.04) and overall satisfaction (p<0.01), and was independent of age, education and number of health conditions, but was not associated with sexual desire (p=0.97). Erectile dysfunction was significantly associated with age (OR=1.12; 95% CI 1.05-1.19; p<0.01) and level of depressive symptoms (OR=1.39; 95% CI 1.05-1.85; p=0.02) after multivariate adjustment. In particular, only "low mood" (p=0.03) and "worthlessness" (p=0.03) remained positively associated with ED after multivariate adjustments. LIMITATIONS: Cross-sectional design cannot demonstrate direction of causality. CONCLUSIONS: We are the first to implicate "low mood" and "worthlessness" in the association between depressive symptoms and ED, and this is the first study to investigate the association in Chinese.

Organization and function of microfilaments during late epiboly in zebrafish embryos.

We report that, during epiboly in zebrafish, three F-actin--based structures appear only after the blastoderm migrates past the embryonic equator. They are composed of two ring-like F-actin structures that form at the deep cell and enveloping layer margins of the blastoderm and a punctate actin band that develops in the external yolk syncytial layer. Treatment with cytochalasin B or the calcium chelator dibromo-BAPTA results in the disruption of all three of these actin-based structures, leading to the slowing or immediate arrest of epiboly, respectively, followed by a failure of yolk cell occlusion and the eventual lysis of the embryo through the vegetal pole region. We suggest, therefore, that these structures function in the occlusion of the vegetal portion of the yolk cell during the latter stages of epiboly. Possible roles for these new structures, their modulation by Ca2+, as well as the functions of other previously described F-actin--based structures observed throughout epiboly, are discussed.