On the Vertical Distribution of the Metazoan Meiofauna in Shelf Break and Upper Slope Habitats of the NE Atlantic

The metazoan meiofauna of nine stations in shelf break and upper slope areas (70 to 1500 m water depth) of the N.E. Atlantic were investigated in order to assess which environmental factors are important in the control of densities and sediment profiles. Total meiofaunal densities (ranging between 368 and 1523 ind/10 cm²) were correlated with bacterial densities, an important food source for meiofauna. However, considering sediment vertical distribution profiles, the relative importance of both food and oxygen on the meiofauna became obvious. A combination of both bacterial densities and oxygen supply could explain about 95% of the variability in the vertical profiles of the meiofauna densities. Meiofauna numbers increase in proportion to food availability in the surface sediment layers, but this relationship breaks down in deeper sediment layers where the oxygen supply is often limiting, particularly in fine sediments.     

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1′-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1′-modification.     

Biogeographic consequences of shifting climate for the western massasauga (Sistrurus tergeminus)

The western massasauga (Sistrurus tergeminus) is a small pit viper with an extensive geographic range, yet observations of this species are relatively rare. They persist in patchy and isolated populations, threatened by habitat destruction and fragmentation, mortality from vehicle collisions, and deliberate extermination. Changing climates may pose an additional stressor on the survival of isolated populations. Here, we evaluate historic, modern, and future geographic projections of suitable climate for S. tergeminus to outline shifts in their potential geographic distribution and inform current and future management. We used maximum entropy modeling to build multiple models of the potential geographic distribution of S. tergeminus. We evaluated the influence of five key decisions made during the modeling process on the resulting geographic projections of the potential distribution, allowing us to identify areas of model robustness and uncertainty. We evaluated models with the area under the receiver operating curve and true skill statistic. We retained 16 models to project both in the past and future multiple general circulation models. At the last glacial maximum, the potential geographic distribution associated with S. tergeminus occurrences had a stronghold in the southern part of its current range and extended further south into Mexico, but by the mid‐Holocene, its modeled potential distribution was similar to its present‐day potential distribution. Under future model projections, the potential distribution of S. tergeminus moves north, with the strongest northward trends predicted under a climate scenario increase of 8.5 W/m². Some southern populations of S. tergeminus have likely already been extirpated and will continue to be threatened by shifting availability of suitable climate, as they are already under threat from desertification of grasslands. Land use and habitat loss at the northern edge of the species range are likely to make it challenging for this species to track suitable climates northward over time.     

Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis

Experimental cerebral malaria (ECM) resulting from Plasmodium berghei ANKA infection involves T lymphocytes. However, the mechanisms of T cell-mediated pathogenesis remain unknown. We found that, in contrast to ECM-susceptible C57BL6 mice, perforin-deficient (PFP-KO) mice were resistant to ECM in the absence of brain lesions, whereas cytoadherence of parasitized erythrocytes and massive accumulation of activated/effector CD8 lymphocytes were observed in both groups of mice. ECM is induced in PFP-KO mice after adoptive transfer of cytotoxic CD8+ cells from infected C57BL6 mice, which were directed to the brain of PFP-KO mice. This specific recruitment might involve chemokine/chemokine receptors, since their expression was up-regulated on activated CD8 cells, and susceptibility to ECM was delayed in CCR5-KO mice. Thus, lymphocyte cytotoxicity and cell trafficking are key players in ECM pathogenesis.     

Transport and survival of bacterial and viral tracers through submerged-flow constructed wetland and sand-filter system

Untreated or improperly treated wastewater has often been cited as the primary contamination source of groundwater. The use of decentralized wastewater treatment systems has applicability around the world since it obviates the need for extensive infrastructure development and expenditures. The use of a submerged flow constructed wetland (CW) and a sand filter to remove bacterial and viral pathogens from wastewater streams was evaluated in this study Salmonella sp. and a bacteriophages tracer were used in conjunction with the conservative bromide tracer to understand the fate and transport of these organisms in these treatment systems. Viral breakthrough numbers in the sand filter and CW were similar with a Spearman Rank correlation of 0.8 (P<0.05). In the CW, the virus exhibited almost a 3-log reduction, while in the sand filter, the viruses exhibited a 2-log reduction. The bacterial tracers, however, did not exhibit similar reductions. Low numbers of bacteria and viruses were still detectable in the effluent streams suggesting that disinfection of the effluent is critical. The survival of the tracer bacteria and viruses was as expected dependent on the biotic and abiotic conditions existing within the wastewater. The results suggest that the microbial removal characteristics of decentralized wastewater treatment systems can vary and depend on factors such as adsorption, desorption and inactivation which in turn depend on the design specifics such as filter media characteristics and local climatic conditions.     

Angiotensin II and its receptors in human endocardial endothelial cells: role in modulating intracellular calcium

The aims of the present study are to investigate the presence and distribution of angiotensin II (Ang II), as well as AT1 and AT2 receptors, in endocardial endothelial cells (EECs) and to determine if the effect of Ang II on intracellular calcium in these cells is mediated via the AT1 or the AT2 receptor. Immunofluorescence and 3D confocal microscopy techniques were used on 20-week-old fetal human EECs. Our results showed that Ang II and its receptors, the AT1 and the AT2 types, are present and exhibit a different distribution in human EECs. Ang II labelling is found throughout the cell with a fluorescence signal higher in the cytosol when compared with the nucleus. Like Ang II, the AT1 receptor fluorescence signal is also homogeneously distributed in human EECs but with a preferential labelling at the level of the nucleus, while the AT2 receptor labelling is solely present in the nucleus. Using fluo-3 and 3D confocal microscopy technique, superfusion of human EECs with increasing concentration of Ang II induced a dose-dependent sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of Ang II on human EEC's intracellular Ca2+ ([Ca2+]) was completely prevented by losartan, an AT1 receptor antagonist. Our results suggest that Ang II, as well as AT1 and AT2 receptors, is present but differentially distributed in EECs of 20-week-old fetal human hearts, and that the AT1 receptor mediates the effects of Ang II on [Ca2+]i in these cells.     

A conserved insertion in protein-primed DNA polymerases is involved in primer terminus stabilisation

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or alpha-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5' exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in phi29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.     

Formation and Function of the Rbl2p–β-Tubulin Complex

The yeast protein Rbl2p suppresses the deleterious effects of excess β-tubulin as efficiently as does α-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with β-tubulin that does not contain α-tubulin, thus defining a second pool of β-tubulin in the cell. Formation of the complex depends upon the conformation of β-tubulin. Newly synthesized β-tubulin can bind to Rbl2p before it binds to α-tubulin. Rbl2p can also bind β-tubulin from the α/β-tubulin heterodimer, apparently by competing with α-tubulin. The Rbl2p–β-tubulin complex has a half-life of ~2.5 h and is less stable than the α/β-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing β-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p–β-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.     

Impact of seasonal variations and nutrient inputs on nitrogen cycling and degradation of hexadecane by replicated river biofilms

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.