Morphological change caused by loss of the taxon-specific polyalanine tract in Hoxd-13

Sequence comparison of Hoxd-13 among vertebrates revealed the presence of taxon-specific polyalanine tracts in amniotes. To investigate their function at the organismal level, we replaced the wild-type Hoxd-13 gene with one lacking the 15-residue polyalanine tract by using homologous recombination. Sesamoid bone formation in knock-in mice was different from that in the wild type; this was observed not only in the homozygotes but also in the heterozygotes. The present study provides the first direct evidence that taxon-specific homopolymeric amino acid repeats are involved in phenotypic diversification at the organismal level.     

Indian-Ink Perfusion Based Method for Reconstructing Continuous Vascular Networks in Whole Mouse Brain

The topology of the cerebral vasculature, which is the energy transport corridor of the brain, can be used to study cerebral circulatory pathways. Limited by the restrictions of the vascular markers and imaging methods, studies on cerebral vascular structure now mainly focus on either observation of the macro vessels in a whole brain or imaging of the micro vessels in a small region. Simultaneous vascular studies of arteries, veins and capillaries have not been achieved in the whole brain of mammals. Here, we have combined the improved gelatin-Indian ink vessel perfusion process with Micro-Optical Sectioning Tomography for imaging the vessel network of an entire mouse brain. With 17 days of work, an integral dataset for the entire cerebral vessels was acquired. The voxel resolution is 0.35×0.4×2.0 µm³ for the whole brain. Besides the observations of fine and complex vascular networks in the reconstructed slices and entire brain views, a representative continuous vascular tracking has been demonstrated in the deep thalamus. This study provided an effective method for studying the entire macro and micro vascular networks of mouse brain simultaneously.     

An Automated Three-Dimensional Detection and Segmentation Method for Touching Cells by Integrating Concave Points Clustering and Random Walker Algorithm

Characterizing cytoarchitecture is crucial for understanding brain functions and neural diseases. In neuroanatomy, it is an important task to accurately extract cell populations'' centroids and contours. Recent advances have permitted imaging at single cell resolution for an entire mouse brain using the Nissl staining method. However, it is difficult to precisely segment numerous cells, especially those cells touching each other. As presented herein, we have developed an automated three-dimensional detection and segmentation method applied to the Nissl staining data, with the following two key steps: 1) concave points clustering to determine the seed points of touching cells; and 2) random walker segmentation to obtain cell contours. Also, we have evaluated the performance of our proposed method with several mouse brain datasets, which were captured with the micro-optical sectioning tomography imaging system, and the datasets include closely touching cells. Comparing with traditional detection and segmentation methods, our approach shows promising detection accuracy and high robustness.     

Microbial diversity of cellulolytic community of the sandy mat from lake Zun-Torey (Southern Transbaikalia)

The diversity of the microbial community from the sandy mat of the soda lake Zun-Torey (Transbaikalia) has been studied using enrichment cultures. The use of classical microbiological and molecular genetic techniques allows one to assess the numbers and diversity of species of decomposer bacteria from the cellulolytic communities formed in different horizons of the sandy mat.     

Derivation and validation of a clinical model to predict death or cardiac hospitalizations while on the cardiac surgery waitlist

Background:Waitlist management is a global challenge. For patients with severe cardiovascular diseases awaiting cardiac surgery, prolonged wait times are associated with unplanned hospitalizations. To facilitate evidence-based resource allocation, we derived and validated a clinical risk model to predict the composite outcome of death and cardiac hospitalization of patients on the waitlist for cardiac surgery.Methods:We used the CorHealth Ontario Registry and linked ICES health care administrative databases, which have information on all Ontario residents. We included patients 18 years or older who waited at home for coronary artery bypass grafting, valvular or thoracic aorta surgeries between 2008 and 2019. The primary outcome was death or an unplanned cardiac hospitalizaton, defined as nonelective admission for heart failure, myocardial infarction, unstable angina or endocarditis. We randomly divided two-thirds of these patients into derivation and one-third into validation data sets. We derived the model using a multivariable Cox proportional hazard model with backward stepwise variable selection.Results:Among 62 375 patients, 41 729 patients were part of the derivation data set and 20 583 were part of the validation data set. Of the total, 3033 (4.9%) died or had an unplanned cardiac hospitalization while waiting for surgery. The area under the curve of our model at 15, 30, 60 and 89 days was 0.85, 0.82, 0.81 and 0.80, respectively, in the derivation cohort and 0.83, 0.80, 0.78 and 0.78, respctively, in the validation cohort. The model calibrated well at all time points.Interpretation:We derived and validated a clinical risk model that provides accurate prediction of the risk of death and unplanned cardiac hospitalization for patients on the cardiac surgery waitlist. Our model could be used for quality benchmarking and data-driven decision support for managing access to cardiac surgery.

Waitlist management is an ongoing challenge for publicly funded health care systems because available resources are finite. This challenge has become more pervasive since the onset of the COVID-19 pandemic, as many nonemergent procedures have been postponed to preserve system capacity for patients with COVID-19.¹The decision to triage patients booked for cardiac surgery balances the likelihood of disease decompensation and the availability of operating room and intensive care unit resources. Our group has previously developed and validated the CardiOttawa Length of Stay Score to estimate the likelihood of high or low needs for postoperative intensive care unit resources,² and the CardiOttawa Waitlist Mortality Score to support evidence-based prioritization for cardiac surgeries. These risk models have been combined into a single triage decision support tool that is used on a daily basis at our institution (available with sign up at https://cardiottawa.ottawaheart.ca). No models are available to predict unplanned hospitalizations for patients on the waitlist for definitive surgical interventions. We therefore conducted a population-based study in Ontario, Canada to derive and validate a clinical model to predict the composite outcome of death or unplanned cardiac hospitalizations in patients on the waitlist for cardiac surgery.     

A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70-facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone.     

Calcium Triggered Lα-H2 Phase Transition Monitored by Combined Rapid Mixing and Time-Resolved Synchrotron SAXS

Background

Awad et al. [1] reported on the Ca²⁺-induced transitions of dioleoyl-phosphatidylglycerol (DOPG)/monoolein (MO) vesicles to bicontinuous cubic phases at equilibrium conditions. In the present study, the combination of rapid mixing and time-resolved synchrotron small-angle X-ray scattering (SAXS) was applied for the in-situ investigations of fast structural transitions of diluted DOPG/MO vesicles into well-ordered nanostructures by the addition of low concentrated Ca²⁺ solutions.

Methodology/Principal Findings

Under static conditions and the in absence of the divalent cations, the DOPG/MO system forms large vesicles composed of weakly correlated bilayers with a d-spacing of ∼140 Å (L_α-phase). The utilization of a stopped-flow apparatus allowed mixing these DOPG/MO vesicles with a solution of Ca²⁺ ions within 10 milliseconds (ms). In such a way the dynamics of negatively charged PG to divalent cation interactions, and the kinetics of the induced structural transitions were studied. Ca²⁺ ions have a very strong impact on the lipidic nanostructures. Intriguingly, already at low salt concentrations (DOPG/Ca²⁺>2), Ca²⁺ ions trigger the transformation from bilayers to monolayer nanotubes (inverted hexagonal phase, H₂). Our results reveal that a binding ratio of 1 Ca²⁺ per 8 DOPG is sufficient for the formation of the H₂ phase. At 50°C a direct transition from the vesicles to the H₂ phase was observed, whereas at ambient temperature (20°C) a short lived intermediate phase (possibly the cubic Pn3m phase) coexisting with the H₂ phase was detected.

Conclusions/Significance

The strong binding of the divalent cations to the negatively charged DOPG molecules enhances the negative spontaneous curvature of the monolayers and causes a rapid collapsing of the vesicles. The rapid loss of the bilayer stability and the reorganization of the lipid molecules within ms support the argument that the transition mechanism is based on a leaky fusion of the vesicles.     

Discovery of orally bioavailable cyclohexanol-based NR2B-selective NMDA receptor antagonists with analgesic activity utilizing a scaffold hopping approach

NR2B subunit containing N-methyl-d-aspartate (NMDA) receptor is an attractive target for chronic pain due to its involvement in disease states and its limited distribution in the central nervous system. Cyclohexanol-based leads 6a and 6c were identified as potent NR2B-selective NMDA antagonists utilizing a scaffold hopping approach. Further optimization of this series through replacement of the amide in the leads with an isoxazole and efforts to optimize the pharmacokinetic profiles led to the discovery of orally available brain penetrants 7k and 7l, which demonstrated analgesic activity in the mouse formalin test at early and late phases.     

Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response.

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.