The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5(H) and D6(H) are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system

Hyb:A bioinformatics pipeline for the analysis of CLASH (crosslinking, ligation and sequencing of hybrids) data

Peer reviewedPublisher PD

Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. “Click” chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ¿PEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.We are grateful to Stephen J. Gould (Johns Hopkins University, Baltimore), Nancy E. Braverman (McGill University, Montreal), Wolfgang Schliebs (Ruhr University, Bochum) and Daniel Passon (EMBL, Hamburg) for providing plasmids and antibodies. Work in J.E.A. lab is funded by FEDER (Fundo Europeu de Desenvolvimento Regional), through COMPETE 2020 – Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and “The molecular mechanisms of peroxisome biogenesis” (PTDC/BEXBCM/2311/2014), and through Norte 2020 – Programa Operacional Regional do Norte, under the application of the “Porto Neurosciences and Neurologic Disease Research Initiative at i3S” (NORTE-01-0145-FEDER-000008). We acknowledge support by the Open Access Publishing Fund of the University of Tübingen and the Deutsche Forschungsgemeinschaft for publishing costs

The effect of genotype and zeranol implantation on performance of steers grazing Mitchell grass pastures in north-west Queensland

[Summary]: Steers of seven different genotypes were purchased from six locations in north-west Queensland and their liveweight performance was compared while grazing Mitchell grass pastures. Only small differences were recorded in liveweight gain between steers containing the approximate breed components of Africander 3 Shorthorn 5, Sahiwal 3 Shorthorn 5, Santa Gertrudis, Brahman 4 British 4, and Brahman 6 British 2. The Bos indicus infused steers had annual liveweight gains of 135 compared with 110kg / head / year in the comparable age Hereford group. Zeranol implantation gave 6% advantage in liveweight gain durin the first 84 d post implantation. This response was consisten across genotype groups. Final liveweight of treated steers was 3% heavier than of untreated steers, although zeranol treatment was given 444 d before slaughter

Resolution of Ultramicroscopy and Field of View Analysis

In a recent publication we described a microscopical technique called Ultramicroscopy, combined with a histological procedure that makes biological samples transparent. With this combination we can gather three-dimensional image data of large biological samples. Here we present the theoretical analysis of the z-resolution. By analyzing the cross-section of the illuminating sheet of light we derive resolution values according to the Rayleigh-criterion. Next we investigate the resolution adjacent to the focal point of the illumination beam, analyze throughout what extend the illumination beam is of acceptable sharpness and investigate the resolution improvements caused by the objective lens. Finally we conclude with a useful rule for the sampling rates. These findings are of practical importance for researchers working with Ultramicroscopy to decide on adequate sampling rates. They are also necessary to modify deconvolution techniques to gain further image improvements

No Elevated Plasma Catecholamine Levels during Sleep in Newly Diagnosed, Untreated Hypertensives

The sympatho-adrenergic system is highly involved in regulating sleep, wake and arousal states, and abnormalities in this system are regarded as a key factor in the development and progression of arterial hypertension. While hypertension is associated with a hyperadrenergic state during wakefulness, the effect of hypertension on plasma-catecholamine levels during sleep is not yet known. Twelve young participants with newly diagnosed, untreated hypertension and twelve healthy controls slept for 7 hours in the sleep laboratory. Before and after sleep, subjects rested in a supine position for 3-h periods of wakefulness. We sampled blood at a fast rate (1/10 min) and monitored blood pressure and heart rate continuously. We show that plasma NE and E levels did not differ between hypertensives and normotensive during sleep as well as before and after sleep. Blood pressure was higher in hypertensives, reaching the largest group difference in the morning after sleep. Unlike in the normotensives, in the hypertensive participants the morning rise in blood pressure did not correlate with the rise in catecholamine levels at awakening. Our results suggest that hypertension in its early stages is not associated with a strong hyperadrenergic state during sleep. In showing a diminished control of blood pressure through sympatho-adrenergic signals in hypertensive participants, our data point towards a possible involvement of dysfunctional sleep-related blood pressure regulation in the development of hypertension

Voxel-wise comparisons of cellular microstructure and diffusion-MRI in mouse hippocampus using 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND)

A key challenge in medical imaging is determining a precise correspondence between image properties and tissue microstructure. This comparison is hindered by disparate scales and resolutions between medical imaging and histology. We present a new technique, 3D Bridging of Optically-clear histology with Neuroimaging Data (3D-BOND), for registering medical images with 3D histology to overcome these limitations. Ex vivo 120 × 120 × 200 μm resolution diffusion-MRI (dMRI) data was acquired at 7 T from adult C57Bl/6 mouse hippocampus. Tissue was then optically cleared using CLARITY and stained with cellular markers and confocal microscopy used to produce high-resolution images of the 3D-tissue microstructure. For each sample, a dense array of hippocampal landmarks was used to drive registration between upsampled dMRI data and the corresponding confocal images. The cell population in each MRI voxel was determined within hippocampal subregions and compared to MRI-derived metrics. 3D-BOND provided robust voxel-wise, cellular correlates of dMRI data. CA1 pyramidal and dentate gyrus granular layers had significantly different mean diffusivity (p > 0.001), which was related to microstructural features. Overall, mean and radial diffusivity correlated with cell and axon density and fractional anisotropy with astrocyte density, while apparent fibre density correlated negatively with axon density. Astrocytes, axons and blood vessels correlated to tensor orientation

Whole-brain profiling of cells and circuits in mammals by tissue clearing and light-sheet microscopy

Tissue clearing and light-sheet microscopy have a 100-year-plus history, yet these fields have been combined only recently to facilitate novel experiments and measurements in neuroscience. Since tissue-clearing methods were first combined with modernized light-sheet microscopy a decade ago, the performance of both technologies has rapidly improved, broadening their applications. Here, we review the state of the art of tissue-clearing methods and light-sheet microscopy and discuss applications of these techniques in profiling cells and circuits in mice. We examine outstanding challenges and future opportunities for expanding these techniques to achieve brain-wide profiling of cells and circuits in primates and humans. Such integration will help provide a systems-level understanding of the physiology and pathology of our central nervous system.P 28338 - Austrian Science Fund FWF; U01 MH105971 - NIMH NIH HHS; U01 MH114824 - NIMH NIH HHS; Howard Hughes Medical InstituteAccepted manuscrip