Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing

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Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells

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A Model of Alcohol Drinking under an Intermittent Access Schedule Using Group-Housed Mice

Here, we describe a new model of voluntary alcohol drinking by group-housed mice. The model employs sensor-equipped cages that track the behaviors of the individual animals via implanted radio chips. After the animals were allowed intermittent access to alcohol (three 24 h intervals every week) for 4 weeks, the proportions of licks directed toward bottles containing alcohol were 50.9% and 39.6% for the male and female mice, respectively. We used three approaches (i.e., quinine adulteration, a progressive ratio schedule and a schedule involving a risk of punishment) to test for symptoms of compulsive alcohol drinking. The addition of 0.01% quinine to the alcohol solution did not significantly affect intake, but 0.03% quinine induced a greater than 5-fold reduction in the number of licks on the alcohol bottles. When the animals were required to perform increasing numbers of instrumental responses to obtain access to the bottle with alcohol (i.e., a progressive ratio schedule), they frequently reached a maximum of 21 responses irrespective of the available reward. Although the mice rarely achieved higher response criteria, the number of attempts was ∼10 times greater in case of alcohol than water. We have developed an approach for mapping social interactions among animals that is based on analysis of the sequences of entries into the cage corners. This approach allowed us to identify the mice that followed other animals in non-random fashions. Approximately half of the mice displayed at least one interaction of this type. We have not yet found a clear correlation between imitative behavior and relative alcohol preference. In conclusion, the model we describe avoids the limitations associated with testing isolated animals and reliably leads to stable alcohol drinking. Therefore, this model may be well suited to screening for the effects of genetic mutations or pharmacological treatments on alcohol-induced behaviors.     

Too wet and too dry? Uncertainty of DEM as a potential source of significant errors in a model-based water level assessment in riparian and mire ecosystems

Modelling groundwater depths in floodplains and peatlands remains a basic approach to assessing hydrological conditions of habitats. Groundwater flow models used to compute groundwater heads are known for their uncertainties, and the calibration of these models and the uncertainty assessments of parameters remain fundamental steps in providing reliable data. However, the elevation data used to determine the geometry of model domains are frequently considered deterministic and hence are seldom considered a source of uncertainty in model-based groundwater level estimations. Knowing that even the cutting-edge laser-scanning-based digital elevation models have errors due to vegetation effects and scanning procedure failures, we provide an assessment of uncertainty of water level estimations that remain basic data for wetland ecosystem assessment and management. We found that the uncertainty of the digital elevation model (DEM) significantly influenced the results of the assessment of the habitat’s hydrological conditions expressed as groundwater depths. In extreme cases, although the average habitat suitability index (HSI) assessed in a deterministic manner was defined as ‘unsuitable’, in a probabilistic approach (grid-cell-scale estimation), it reached a value of 40% probability, signifying ‘optimum’ or ‘tolerant’. For the 24 habitats analysed, we revealed vast differences between HSI scores calculated for individual grid cells of the model and HSI scores computed as average values from the set of grid cells located within the habitat patches. We conclude that groundwater-modelling-based decision support approaches to wetland assessment can result in incorrect management if the quality of DEM has not been addressed in studies referring to groundwater depths.     

Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

Background

Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E.

Methodology/Principal Findings

We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant.

Conclusions/Significance

Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.     

Structural behaviour and strain distribution of the long bones of the human lower limbs

Although stiffness and strength of lower limb bones have been investigated in the past, information is not complete. While the femur has been extensively investigated, little information is available about the strain distribution in the tibia, and the fibula has not been tested in vitro. This study aimed at improving the understanding of the biomechanics of lower limb bones by: (i) measuring the stiffness and strain distributions of the different low limb bones; (ii) assessing the effect of viscoelasticity in whole bones within a physiological range of strain-rates; (iii) assessing the difference in the behaviour in relation to opposite directions of bending and torsion. The structural stiffness and strain distribution of paired femurs, tibias and fibulas from two donors were measured. Each region investigated of each bone was instrumented with 8–16 triaxial strain gauges (over 600 grids in total). Each bone was subjected to 6–12 different loading configurations. Tests were replicated at two different loading speeds covering the physiological range of strain-rates. Viscoelasticity did not have any pronounced effect on the structural stiffness and strain distribution, in the physiological range of loading rates explored in this study. The stiffness and strain distribution varied greatly between bone segments, but also between directions of loading. Different stiffness and strain distributions were observed when opposite directions of torque or opposite directions of bending (in the same plane) were applied. To our knowledge, this study represents the most extensive collection of whole-bone biomechanical properties of lower limb bones.     

Mammalian mitochondrial nitric oxide synthase: characterization of a novel candidate

Recently a novel family of putative nitric oxide synthases, with AtNOS1, the plant member implicated in NO production, has been described. Here we present experimental evidence that a mammalian ortholog of AtNOS1 protein functions in the cellular context of mitochondria. The expression data suggest that a candidate for mammalian mitochondrial nitric oxide synthase contributes to multiple physiological processes during embryogenesis, which may include roles in liver haematopoesis and bone development.