Phylogenetic analysis and secondary structure of the Bacillus subtilis bacteriophage RNA required for DNA packaging

An unusual RNA molecule encoded by the Bacillus subtilis bacteriophage phi 29 is a structural component of the viral prohead and is required for the ATP-dependent packaging of DNA. Here we report a model of secondary structure for this prohead RNA developed from a phylogenetic analysis of the primary sequences of prohead RNAs of related phages. Twenty-nine phages related to phi 29 were found to produce prohead RNAs. These RNAs were analyzed by their ability to replace phi 29 RNA in in vitro phage assembly, by Northern blot hybridization with a probe complementary to phi 29 RNA, and by partial and complete sequence analyses. These analyses revealed four quite different sequences ranging in length from 161 to 174 residues. The secondary structure deduced from these sequences, in agreement with earlier observations, indicated that prohead RNA is organized into two domains. The larger 5'-domain (Domain I) is composed of 113-117 residues and contains four helices. Three of these helices appear to be organized into a central stem that is interrupted by two unpaired loops and the fourth helix and loop. The smaller 3'-domain (Domain II) is composed of 40-44 residues and consists of two helices. Domains I and II are separated by 8-13 unpaired residues. Nuclease cleavage occurs readily in this single-stranded joining region, and this cleavage allows the subsequent separation of the two RNA domains. The separated Domain I is fully active in DNA packaging in vitro. The functional significance and biological role of Domain II are unknown. The phylogenetic secondary structure model provides a basis for further analysis of the role of this RNA in bacteriophage morphogenesis.     

Validation of radiocarpal joint contact models based on images from a clinical MRI scanner

This study was undertaken to assess magnetic resonance imaging (MRI)-based radiocarpal surface contact models of functional loading in a clinical MRI scanner for future in vivo studies, by comparison with experimental measures from three cadaver forearm specimens. Experimental data were acquired using a Tekscan sensor during simulated light grasp. Magnetic resonance (MR) images were used to obtain model geometry and kinematics (image registration). Peak contact pressures (PPs) and average contact pressures (APs), contact forces and contact areas were determined in the radiolunate and radioscaphoid joints. Contact area was also measured directly from MR images acquired with load and compared with model data. Based on the validation criteria (within 25% of experimental data), out of the six articulations (three specimens with two articulations each), two met the criterion for AP (0%, 14%); one for peak pressure (20%); one for contact force (5%); four for contact area with respect to experiment (8%, 13%, 19% and 23%), and three contact areas met the criterion with respect to direct measurements (14%, 21% and 21%). Absolute differences between model and experimental PPs were reasonably low (within 2.5 MPa). Overall, the results indicate that MRI-based models generated from 3T clinical MR scanner appear sufficient to obtain clinically relevant data.     

Wild Type Mesenchymal Cells Contribute to the Lung Pathology of Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease.     

On Extrapolating Past the Range of Observed Data When Making Statistical Predictions in Ecology

Ecologists are increasingly using statistical models to predict animal abundance and occurrence in unsampled locations. The reliability of such predictions depends on a number of factors, including sample size, how far prediction locations are from the observed data, and similarity of predictive covariates in locations where data are gathered to locations where predictions are desired. In this paper, we propose extending Cook’s notion of an independent variable hull (IVH), developed originally for application with linear regression models, to generalized regression models as a way to help assess the potential reliability of predictions in unsampled areas. Predictions occurring inside the generalized independent variable hull (gIVH) can be regarded as interpolations, while predictions occurring outside the gIVH can be regarded as extrapolations worthy of additional investigation or skepticism. We conduct a simulation study to demonstrate the usefulness of this metric for limiting the scope of spatial inference when conducting model-based abundance estimation from survey counts. In this case, limiting inference to the gIVH substantially reduces bias, especially when survey designs are spatially imbalanced. We also demonstrate the utility of the gIVH in diagnosing problematic extrapolations when estimating the relative abundance of ribbon seals in the Bering Sea as a function of predictive covariates. We suggest that ecologists routinely use diagnostics such as the gIVH to help gauge the reliability of predictions from statistical models (such as generalized linear, generalized additive, and spatio-temporal regression models).     

Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

The lymphatic system plays an important role in cancer metastasis and inhibition of lymphangiogenesis could be valuable in fighting cancer dissemination. Podoplanin (Pdpn) is a small, transmembrane glycoprotein expressed on the surface of lymphatic endothelial cells (LEC). During mouse development, binding of Pdpn to the C-type lectin-like receptor 2 (CLEC-2) on platelets is critical for the separation of the lymphatic and blood vascular systems. Competitive inhibition of Pdpn functions with a soluble form of the protein, Pdpn-Fc, leads to reduced lymphangiogenesis in vitro and in vivo. However, the transgenic overexpression of human Pdpn-Fc in mouse skin causes disseminated intravascular coagulation due to platelet activation via CLEC-2. In the present study, we produced and characterized a mutant form of mouse Pdpn-Fc, in which threonine 34, which is considered essential for CLEC-2 binding, was mutated to alanine (PdpnT34A-Fc). Indeed, PdpnT34A-Fc displayed a 30-fold reduced binding affinity for CLEC-2 compared with Pdpn-Fc. This also translated into fewer side effects due to platelet activation in vivo. Mice showed less prolonged bleeding time and fewer embolized vessels in the liver, when PdpnT34A-Fc was injected intravenously. However, PdpnT34A-Fc was still as active as wild-type Pdpn-Fc in inhibiting lymphangiogenesis in vitro and also inhibited lymphangiogenesis in vivo. These data suggest that the function of Pdpn in lymphangiogenesis does not depend on threonine 34 in the CLEC-2 binding domain and that PdpnT34A-Fc might be an improved inhibitor of lymphangiogenesis with fewer toxic side effects.     

Wireless Tri-Axial Trunk Accelerometry Detects Deviations in Dynamic Center of Mass Motion Due to Running-Induced Fatigue

Small wireless trunk accelerometers have become a popular approach to unobtrusively quantify human locomotion and provide insights into both gait rehabilitation and sports performance. However, limited evidence exists as to which trunk accelerometry measures are suitable for the purpose of detecting movement compensations while running, and specifically in response to fatigue. The aim of this study was therefore to detect deviations in the dynamic center of mass (CoM) motion due to running-induced fatigue using tri-axial trunk accelerometry. Twenty runners aged 18–25 years completed an indoor treadmill running protocol to volitional exhaustion at speeds equivalent to their 3.2 km time trial performance. The following dependent measures were extracted from tri-axial trunk accelerations of 20 running steps before and after the treadmill fatigue protocol: the tri-axial ratio of acceleration root mean square (RMS) to the resultant vector RMS, step and stride regularity (autocorrelation procedure), and sample entropy. Running-induced fatigue increased mediolateral and anteroposterior ratios of acceleration RMS (p < .05), decreased the anteroposterior step regularity (p < .05), and increased the anteroposterior sample entropy (p < .05) of trunk accelerometry patterns. Our findings indicate that treadmill running-induced fatigue might reveal itself in a greater contribution of variability in horizontal plane trunk accelerations, with anteroposterior trunk accelerations that are less regular from step-to-step and are less predictable. It appears that trunk accelerometry parameters can be used to detect deviations in dynamic CoM motion induced by treadmill running fatigue, yet it is unknown how robust or generalizable these parameters are to outdoor running environments.     

Testing the Role of Climate Change in Species Decline: Is the Eastern Quoll a Victim of a Change in the Weather?

To conserve a declining species we first need to diagnose the causes of decline. This is one of the most challenging tasks faced by conservation practitioners. In this study, we used temporally explicit species distribution models (SDMs) to test whether shifting weather can explain the recent decline of a marsupial carnivore, the eastern quoll (Dasyurus viverrinus). We developed an SDM using weather variables matched to occurrence records of the eastern quoll over the last 60 years, and used the model to reconstruct variation through time in the distribution of climatically suitable range for the species. The weather model produced a meaningful prediction of the known distribution of the species. Abundance of quolls, indexed by transect counts, was positively related to the modelled area of suitable habitat between 1990 and 2004. In particular, a sharp decline in abundance from 2001 to 2003 coincided with a sustained period of unsuitable weather over much of the species’ distribution. Since 2004, abundance has not recovered despite a return to suitable weather conditions, and abundance and area of suitable habitat have been uncorrelated. We suggest that fluctuations in weather account for the species’ recent decline, but other unrelated factors have suppressed recovery.     

Rapid glutamate decarboxylase assay for detection of Escherichia coli.

A rapid test procedure for the enzyme glutamate decarboxylase was developed for detection of Escherichia coli. The assay procedure was able to confirm the presence of E. coli in enteric broth cultures with 95% specificity for both pure cultures and environmental samples. The procedure was capable of detecting survivors among chlorine-exposed cells.