The role of the microtubule cytoskeleton in poxvirus replication and pathogenesis

The subcellular space is a dense, complex environment that viral pathogens must efficiently navigate for their survival and dissemination. The prototypal poxvirus member, vaccinia virus (VACV) has been shown to subvert both host microtubule-based and actin-based transport mechanisms to this end. The utilisation of fluorescent protein technology has been at the crux of many of these discoveries, but addressing remaining questions is made difficult by the various morphological forms of VACV and the sheer number of viruses being transported at any one time in a single infected cell. We have used the development of photoswitchable fluorescent protein Dendra2 to create a cutting edge imaging system that allows us to gain insight into the dynamics of subcellular virus dissemination. We have constructed recombinant viruses expressing core protein A3 and envelope proteins B5 and F13 fused to Dendra2 in both parental and deletion strains of the Western Reserve strain and have examined the envelopment at and egress from the trans-Golgi network. Egress of wrapped virus to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope proteins A36 and F12. We have utilised a mouse-based infection model to validate the important role these transport events have during an in vivo, endemic infection. Ectromelia virus, an orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin tails, another phenotype dependent on A36 expression during VACV infection. ECTV A36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host

Characteristics of surface-water flows in the ridge and slough landscape of Everglades National Park: implications for particulate transport

Over the last one hundred years, compartmentalization and water management activities have reduced water flow to the ridge and slough landscape of the Everglades. As a result, the once corrugated landscape has become topographically and vegetationally uniform. The focus of this study was to quantify variation in surface flow in the ridge and slough landscape and to relate flow conditions to particulate transport and deposition. Over the 2002–2003 and 2003–2004 wet seasons, surface velocities and particulate accumulation were measured in upper Shark River Slough in Everglades National Park. Landscape characteristics such as elevation, plant density and biomass also were examined to determine their impact on flow characteristics and material transport. The results of this study demonstrate that the release of water during the wet season not only increases water levels, but also increased flow speeds and particulate transport and availability. Further, flow speeds were positively and significantly correlated with water level thereby enhancing particulate transport in sloughs relative to ridges especially during peak flow periods. Our results also indicate that the distribution of biomass in the water column, including floating plants and periphyton, affects velocity magnitude and shape of vertical profiles, especially in the sloughs where Utricularia spp. and periphyton mats are more abundant. Plot clearing experiments suggest that the presence of surface periphyton and Utricularia exert greater control over flow characteristics than the identity (i.e., sawgrass or spike rush) or density of emergent macrophytes, two parameters frequently incorporated into models describing flow through vegetated canopies. Based on these results, we suggest that future modeling efforts must take the presence of floating biomass, such as Utricularia, and presence of periphyton into consideration when describing particulate transport

Hydrologic measurements and implications for tree island formation within Everglades National Park

Tree islands in the Shark River Slough of the Everglades National Park (ENP), in the southern state of Florida in the United States, are part of a wetland system of densely vegetated ridges interspersed within relatively open sloughs. Human alteration of this system has had dramatic negative effects on the landscape of the region and restoration efforts will require adjusting the hydrology of the region to assure the preservation of these important ecologic features. The primary objectives of this study were to document the hydrology in the vicinity of tree islands in ENP by measuring velocities in time and space and by characterizing suspended sediments. The results of such measurements were interpreted with respect to factors that may limit tree island growth. The measurements were conducted in the vicinity of three tree islands known as Black Hammock (BH), Gumbo Limbo (GL), and an unnamed island that was named for this study as Satin Leaf (SL). Acoustical Doppler Velocity (ADV) meters were used for measuring the low velocities of the Everglades water flow. Properties of suspended sediments were characterized through measurements of particle size distribution, turbidity, concentration and particle density. Mean velocities observed at each of the tree islands varied from 0.9 to 1.4 cm/s. Slightly higher mean velocities were observed during the wet season (1.2–1.6 cm/s) versus the dry season (0.8–1.3 cm/s). Maximum velocities of more than 4 cm/s were measured in areas of Cladium jamaicense die-off and at the hardwood hammock (head) of the islands. At the island’s head, water is channelized around obstructions such as tree trunks in relatively rapid flow, which may limit the lateral extent of tree island growth. Channelization is facilitated by shade from the tree canopy, which limits the growth of underwater vegetation thereby minimizing the resistance to flow and limiting sediment deposition. Suspended sediment concentrations were low (0.5–1.5 mg/L) at all study sites and were primarily of organic origin. The mean particle size of the suspended sediments was 3 μm with a distribution that was exponential. Critical velocities needed to cause re-suspension of these particles were estimated to be above the actual velocities observed. Sediment transport within the water column appears to be at a near steady state during the conditions evaluated with low rates of sediment loss balanced by presumably the release of equivalent quantities of particles of organic origin. Existing hydrologic conditions do not appear to transport sufficient suspended sediments to result in the formation of tree islands. Of interest would be to collect hydrologic and sediment transport data during extreme hydrologic events to determine if enough sediment is transported under these conditions to promote sufficient sediment accumulations

Genetic and morphometric divergence of an invasive bird : the introduced house sparrow (Passer domesticus) in Brazil

Introduced species are interesting systems for the study of contemporary evolution in new environments because of their spatial and temporal scales. For this study we had three aims: (i) to determine how genetic diversity and genetic differentiation of introduced populations of the house sparrow (Passer domesticus) in Brazil varies with range expansion, (ii) to determine how genetic diversity and differentiation in Brazil compares to ancestral European populations; and (iii) to determine whether selection or genetic drift has been more influential on phenotypic divergence. We used six microsatellite markers to genotype six populations from Brazil and four populations from Europe. We found slightly reduced levels of genetic diversity in Brazilian compared to native European populations. However, among introduced populations of Brazil, we found no association between genetic diversity and time since introduction. Moreover, overall genetic differentiation among introduced populations was low indicating that the expansion took place from large populations in which genetic drift effects would likely have been weak. We found significant phenotypic divergence among sites in Brazil. Given the absence of a spatial genetic pattern, divergent selection and not genetic drift seems to be the main force behind most of the phenotypic divergence encountered. Unravelling whether microevolution (e.g., allele frequency change), phenotypic plasticity, or both mediated phenotypic divergence is challenging and will require experimental work (e.g., common garden experiments or breeding programs)

A36-dependent Actin Filament Nucleation Promotes Release of Vaccinia Virus

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.This work was outlined and supported by Project Grant #632785 of the National Health and Medical Research Council of Australia and The Australian Research Council Federation Discovery Project #1096623. CBW was supported by a National Health and Medical Research Council of Australia Senior Research Fellowship #571905. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Book reviews

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/45614/1/11199_2004_Article_BF00287509.pd

A prática do auxiliar de enfermagem do programa saúde da família

This study aims to evaluate the practice of nursing auxiliaries in the Family Health Program, identifying their daily activities, as well as factors that facilitate or hinder their work. A qualitative approach was chosen, using the focal group technique, with the participation of 34 nursing auxiliaries from the Federal District, Brazil. It was found that the nursing auxiliaries carry out a variety of activities, both of individual and collective nature, heavily influenced by the clinical model and with different degrees of complexity, such as: family registration, brief clinical evaluation, group activities, technical procedures, home visits, sanitary and epidemiological vigilance in day care centers. The participants related that they found it easy to bond with the clientele, but complained of the shortage of continuing education courses and imprecision as to their true assignments and lack of local coordination. The authors conclude that the program must reassess the role of this professional, reemphasizing his educational function in the community.La finalidad de este trabajo es conocer la práctica del auxiliar de enfermería que actúa en el Programa Salud de la Familia, identificando sus actividades cotidianas, así como facilidades y dificultades encontradas en su trabajo. Fue adoptada una aproximación cualitativa, utilizándose la técnica de grupo focal, con participación de 34 auxiliares de enfermería del Districto Federal, Brasil. Se constata que los auxiliares desarrollan una serie de actividades, tanto en carácter personal como en colectivo, con fuerte influencia del modelo clínico de atención y diferentes grados de complejidad, tales como: catastro de las familias, evaluación clínica sumaria, actividades de grupo, procedimientos técnicos, visitas domiciliares, vigilancia sanitaria y epidemiológica. Los profesionales dicen que encuentran facilidad en la formación de vínculo con la clientela, pero se quejan de la falta de cursos de educación continuada, indefinición de sus reales atribuciones y falta de coordinación local. La conclusión es que el programa necesita redimensionar el papel de este profesional, rescatando su función educativa delante de la comunidad.Este trabalho tem como objetivo conhecer a prática do auxiliar de enfermagem que atua no Programa Saúde da Família, identificando suas atividades cotidianas, bem como facilidades e dificuldades encontradas no seu trabalho. Optou-se pela abordagem qualitativa, utilizando-se a técnica de grupo focal, com participação de 34 auxiliares de enfermagem do Distrito Federal. Constatou-se que os auxiliares desenvolvem uma série de atividades, tanto de caráter individual como coletivo, com forte influência do modelo clínico de atenção e diferentes graus de complexidade, tais como: cadastramento das famílias, avaliação clínica sumária, atividades de grupo, procedimentos técnicos, visitas domiciliares, vigilância sanitária e epidemiológica. Os profissionais relatam que encontram facilidade na formação de vínculo com a clientela, porém se queixam da escassez de cursos de educação continuada, indefinição quanto as suas reais atribuições e falta de coordenação local. Conclui-se que o programa precisa redimensionar o papel desse profissional, resgatando sua função educativa junto à comunidade

Impacts of hurricanes on surface water flow within a wetland

s u m m a r y Between 2001 and 2005, seven category 3 or higher major hurricanes made landfall within the US. The hydrologic impacts of these distinct climatic phenomena frequently occurring in wetland watersheds, however, are not well understood. The focus of this study was to evaluate the impacts of hurricane wind and rainfall conditions on water velocity and water elevations within the study wetland, the Florida Everglades. Specifically water velocity data was measured near two tree islands (Gumbo Limbo (GL) and Satin Leaf (SL)) and wind speed, water elevation, and rainfall were obtained from nearby wind observation stations. During the direct impacts of the hurricanes (Hurricanes Katrina and Wilma), water speed, flow direction, and hydraulic gradients were altered, and the extent of variation was positively related to wind characteristics, with significant alterations in flow direction at depth during Hurricane Wilma due to higher wind speeds. After the direct impacts, the longer lasting effect of hurricanes (time scale of a few days) resulted in altered flow speeds that changed by 50% or less. These longer lasting changes in flow speeds may be due to the redistribution of emergent vegetation

Factors driving the seasonal and hourly variability of sea-spray aerosol number in the North Atlantic

Four North Atlantic Aerosol and Marine Ecosystems Study (NAAMES) field campaigns from winter 2015 through spring 2018 sampled an extensive set of oceanographic and atmospheric parameters during the annual phytoplankton bloom cycle. This unique dataset provides four seasons of open-ocean observations of wind speed, sea surface temperature (SST), seawater particle attenuation at 660 nm (cp,660, a measure of ocean particulate organic carbon), bacterial production rates, and sea-spray aerosol size distributions and number concentrations (NSSA). The NAAMES measurements show moderate to strong correlations (0.56 \u3c R \u3c 0.70) between NSSA and local wind speeds in the marine boundary layer on hourly timescales, but this relationship weakens in the campaign averages that represent each season, in part because of the reduction in range of wind speed by multiday averaging. NSSA correlates weakly with seawater cp,660 (R = 0.36, P \u3c\u3c 0.01), but the correlation with cp,660, is improved (R = 0.51, P \u3c 0.05) for periods of low wind speeds. In addition, NAAMES measurements provide observational dependence of SSA mode diameter (dm) on SST, with dm increasing to larger sizes at higher SST (R = 0.60, P \u3c\u3c 0.01) on hourly timescales. These results imply that climate models using bimodal SSA parameterizations to wind speed rather than a single SSA mode that varies with SST may overestimate SSA number concentrations (hence cloud condensation nuclei) by a factor of 4 to 7 and may underestimate SSA scattering (hence direct radiative effects) by a factor of 2 to 5, in addition to overpredicting variability in SSA scattering from wind speed by a factor of 5