The Sec1/Munc18 protein Vps45 regulates cellular levels of its SNARE binding partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic

Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c <i>In Vitro</i>

Background: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes. Methodology/Principal Findings Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex. Conclusion/Significance Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion

The role of the yeast Sec1/Munc18 protein, Vps45p, in the assembly of its cognate snare complex

Members of the SNARE (soluble NSF attachment protein receptor) superfamily of proteins are indispensable players in all intracellular transport pathways. Sec1/Munc18 (SM) proteins are also essential for membrane trafficking, but a model encompassing universal function(s) for SM proteins has been difficult to formulate. The yeast SM protein Vps45p regulates traffic from the trans-Golgi network (TGN) to late endosomes and facilitates the assembly of its cognate SNARE proteins (Tlg2p, Tlg1p, Vti1p, and Snc2p) into complexes. The first section of this work addresses the interaction(s) between Vps45p and its cognate SNARE partners. The second area of investigation in this work addresses the functional role(s) of these interactions. Analysis of one mode of interaction between Vps45p and its cognate syntaxin, Tlg2p, reveals that this mode of interaction is dispensable for Vps45p function. A dominant negative version of Vps45p is exploited to uncover an interaction between Vps45p and SNARE complexes that is mediated via a second, distinct mode. An investigation of the ability of Vps45p to interact with each of its individual cognate SNARE proteins also uncovers an interaction between Vps45p and its cognate v-SNARE, Snc2p. Collectively, these results suggest that Vps45p executes its function(s) through multiple modes of interaction with its cognate SNARE proteins, and support a model in which Vps45p undertakes a series of distinct interactions with specific intermediates of the SNARE cycle. Such a model would allow Vps45p to play multiple roles in the SNARE cycle and would also help contribute to a unifying hypothesis to describe SM protein function

The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p–Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle

Weighing the Evidence of Efficacy of Oral PrEP for HIV Prevention in Women in Southern Africa

As oral tenofovir-based regimens for preexposure prophylaxis (PrEP) are adopted as standard of care for HIV prevention, their utilization in clinical trials among women in southern Africa will require an accurate estimate of oral PrEP efficacy in this population. This information is critical for women in choosing this prevention strategy, and in public health policy making. Estimates of the efficacy of oral PrEP regimens containing tenofovir have varied widely across trials that enrolled women, with some studies reporting high efficacy and others reporting no efficacy. Although poor adherence is strongly associated with lack of efficacy, other factors, such as mode of transmission (sexual vs. parenteral), predominant HIV subtype (C vs. non-C), intensity of exposure, and percentage of stable serodiscordant couples, may also contribute to the variation in efficacy estimates. In this article, we evaluate the evidence for PrEP efficacy in women and propose potential explanations for the observed differences in efficacy among studies. Our review emphasizes the need to continue to refine estimates of efficacy and effectiveness of tenofovir-based oral PrEP so as to best develop the next generation of HIV prevention tools, and to inform public policies directed toward HIV prevention

Orchestrating single-cell analysis with Bioconductor

Recent technological advancements have enabled the profiling of a large number of genome-wide features in individual cells. However, single-cell data present unique challenges that require the development of specialized methods and software infrastructure to successfully derive biological insights. The Bioconductor project has rapidly grown to meet these demands, hosting community-developed open-source software distributed as R packages. Featuring state-of-the-art computational methods, standardized data infrastructure and interactive data visualization tools, we present an overview and online book (https://osca.bioconductor.org) of single-cell methods for prospective users

HAI and NAI titer correlates of inactivated and live attenuated influenza vaccine efficacy

Abstract Background High hemagglutination inhibition (HAI) and neuraminidase inhibition (NAI) titers are generally associated with reduced influenza risk. While repeated influenza vaccination reduces seroresponse, vaccine effectiveness is not always reduced. Methods During the 2007-2008 influenza season, a randomized, placebo-controlled trial (FLUVACS) evaluated the efficacies of live-attenuated (LAIV) and inactivated influenza vaccines (IIV) among healthy adults aged 18-49 in Michigan; IIV vaccine efficacy (VE) and LAIV VE against influenza disease were estimated at 68% and 36%. Using the principal stratification/VE moderation framework, we analyzed data from this trial to assess how each VE varied by HAI or NAI responses to vaccination observed for vaccinated individuals and predicted counterfactually for placebo recipients. We also assessed how each VE varied with pre-vaccination/baseline variables including HAI titer, NAI titer, and vaccination history. Results IIV VE appeared to increase with Day 30 post-vaccination HAI titer, albeit not significantly (p=0.20 and estimated VE 14.4%, 70.5%, and 85.5% at titer below the assay lower quantification limit, 512, and 4096 (maximum)). Moreover, IIV VE increased significantly with Day 30 post-vaccination NAI titer (p=0.040), with estimated VE zero at titer 10 and 92.2% at highest titer 640. There was no evidence that fold-change in post-vaccination HAI or NAI titer associated with IIV VE (p=0.76, 0.38). For LAIV, there was no evidence that VE associated with post-vaccination or fold-rise HAI or NAI titers (p-values >0.40). For IIV, VE increased with increasing baseline NAI titer in those previously vaccinated, but VE decreased with increasing baseline NAI titer in those previously unvaccinated. In contrast, for LAIV, VE did not depend on previous vaccination or baseline HAI or NAI titer. Conclusions: Future efficacy trials should measure baseline and post-vaccination antibody titers in both vaccine and control/placebo recipients, enabling analyses to better elucidate correlates of vaccine- and natural-protection. Trial registration: ClinicalTrials.gov NCT00538512. October 1, 2007.https://deepblue.lib.umich.edu/bitstream/2027.42/149182/1/12879_2019_Article_4049.pd

Fracción de carbono en la biomasa de Prosopis affinis sprengel (Fabaceae) en un bosque nativo del espinal (Argentina)

Sione, Silvana M. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Ledesma, Silvia G. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Rosenberger, Leandro J.. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Oszust, José D. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Carpp, Ignacio A. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Wilson, Marcelo Germán. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Paraná (EEA Paraná). Oro Verde, Entre Ríos, Argentina.Andrade Castañeda, Hernán J. Universidad del Tolima. Facultad de Ingeniería Agronómica. Ibagué, Colombia.Sasal, María Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Paraná (EEA Paraná). Oro Verde, Entre Ríos, Argentina.6-15La estimación del carbono (C) almacenado en la biomasa forestal requiere información precisa acerca de la fracción de C por especie y por componente de la biomasa. Nuestro objetivo fue estimar la fracción de C en los diferentes componentes aéreos de la biomasa del ñandubay (Prosopis affinis) y su variación por clase diamétrica en un bosque nativo del Espinal (Entre Ríos, Argentina). En 30 individuos de cinco clases diamétricas diferentes, se estimó la fracción de C en tres componentes: fustes (F), ramas grandes (RG), y ramas pequeñas+hojas+flores+frutos (RPHFF). Las fracciones medias de C evidenciaron diferencias altamente significativas (p menor 0,01) entre componentes, correspondiendo el menor valor a RPHFF (0,457). El mayor contenido de C correspondió a los F, el que no difirió estadísticamente de las RG (0,485 y 0,482, respectivamente). El valor medio de la fracción de C fue de 0,474 ± 0,023. Considerando la distribución de la biomasa individual en los diferentes componentes y la fracción de C de cada uno, se obtuvo una fracción promedio ponderada de 0,472 ± 0,013. Las fracciones de C mostraron diferencias significativas entre las diferentes clases diamétricas (p menor a 0,05) solamente en los F, correspondiendo los mayores valores a los individuos con diámetros superiores a 20 cm. Los valores obtenidos en este trabajo resultan inferiores al valor por defecto de 0,50 sugerido por el IPCC (Intergovernmental Panel on Climate Change). La información generada constituye una herramienta que aporta precisión a las estimaciones del C almacenado y de las emisiones de CO2 causadas por la deforestación y degradación de los bosques nativos del Espinal entrerriano

Fracción de carbono en la biomasa de Prosopis affinis sprengel (Fabaceae) en un bosque nativo del espinal (Argentina)

Sione, Silvana M. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Ledesma, Silvia G. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Rosenberger, Leandro J.. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Oszust, José D. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Carpp, Ignacio A. Universidad Nacional de Entre Ríos. Facultad de Ciencias Agropecuarias.Verde, Entre Ríos, Argentina.Wilson, Marcelo Germán. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Paraná (EEA Paraná). Oro Verde, Entre Ríos, Argentina.Andrade Castañeda, Hernán J. Universidad del Tolima. Facultad de Ingeniería Agronómica. Ibagué, Colombia.Sasal, María Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Paraná (EEA Paraná). Oro Verde, Entre Ríos, Argentina.6-15La estimación del carbono (C) almacenado en la biomasa forestal requiere información precisa acerca de la fracción de C por especie y por componente de la biomasa. Nuestro objetivo fue estimar la fracción de C en los diferentes componentes aéreos de la biomasa del ñandubay (Prosopis affinis) y su variación por clase diamétrica en un bosque nativo del Espinal (Entre Ríos, Argentina). En 30 individuos de cinco clases diamétricas diferentes, se estimó la fracción de C en tres componentes: fustes (F), ramas grandes (RG), y ramas pequeñas+hojas+flores+frutos (RPHFF). Las fracciones medias de C evidenciaron diferencias altamente significativas (p menor 0,01) entre componentes, correspondiendo el menor valor a RPHFF (0,457). El mayor contenido de C correspondió a los F, el que no difirió estadísticamente de las RG (0,485 y 0,482, respectivamente). El valor medio de la fracción de C fue de 0,474 ± 0,023. Considerando la distribución de la biomasa individual en los diferentes componentes y la fracción de C de cada uno, se obtuvo una fracción promedio ponderada de 0,472 ± 0,013. Las fracciones de C mostraron diferencias significativas entre las diferentes clases diamétricas (p menor a 0,05) solamente en los F, correspondiendo los mayores valores a los individuos con diámetros superiores a 20 cm. Los valores obtenidos en este trabajo resultan inferiores al valor por defecto de 0,50 sugerido por el IPCC (Intergovernmental Panel on Climate Change). La información generada constituye una herramienta que aporta precisión a las estimaciones del C almacenado y de las emisiones de CO2 causadas por la deforestación y degradación de los bosques nativos del Espinal entrerriano