Lipids driving protein structure? Evolutionary adaptations in Kir channels

Many eukaryotic channels, transporters and receptors are activated by phosphatidyl inositol bisphosphate (PIP(2)) in the membrane, and every member of the eukaryotic inward rectifier potassium (Kir) channel family requires membrane PIP(2) for activity. In contrast, a bacterial homolog (KirBac1.1) is specifically inhibited by PIP(2). We speculate that a key evolutionary adaptation in eukaryotic channels is the insertion of additional linkers between trans-membrane and cytoplasmic domains, revealed by new crystal structures, that convert PIP(2) inhibition to activation. Such an adaptation may reflect a novel evolutionary drive to protein structure,; one that was necessary to permit channel function within the highly negatively charged membranes that evolved in the eukaryotic lineage

Site-specific effects of neurosteroids on GABA(A) receptor activation and desensitization

This study examines how site-specific binding to three identified neurosteroid-binding sites in the

Recidivism, health and social functioning following release to the community of NSW prisoners with problematic drug use: Study protocol of the population-based retrospective cohort study on the evaluation of the Connections Program

Introduction The rising rate of incarceration in Australia, driven by high reoffending, is a major public health problem. Problematic drug use is associated with increasing rates of reoffending and return to custody of individuals. Throughcare provides support to individuals during imprisonment through to post-release, improving both the transition to community and health outcomes post-incarceration. The aim of this study is to evaluate the Connections Programme (CP) that utilises a throughcare approach for release planning of people in prison with a history of problematic drug use. The study protocol is described. Methods and analysis Population-based retrospective cohort study. The study will use record linkage of the Connections dataset with 10 other New South Wales (NSW) population datasets on offending, health service utilisation, opioid substitution therapy, pregnancy, birth and mortality. The study includes all patients who were eligible to participate in the CP between January 2008 and December 2015 stratified by patients who were offered CP and eligible patients who were not offered the programme (non-CP (NCP)). Propensity-score matching will be used to appropriately adjust for the observable differences between CP and NCP. The differences between two groups will be examined using appropriate univariate and multivariate analyses. A generalised estimating equation approach, which can deal with repeat outcomes for individuals will be used to examine recidivism, mortality and other health outcomes, including perinatal and infant outcomes. Survival analysis techniques will be used to examine the effect of the CP by sex and Indigenous status on the ‘time-to’ health-related outcomes after adjusting for potential confounders

Evolution of Neuronal and Endothelial Transcriptomes in Primates

The study of gene expression evolution in vertebrates has hitherto focused on the analysis of transcriptomes in tissues of different species. However, because a tissue is made up of different cell types, and cell types differ with respect to their transcriptomes, the analysis of tissues offers a composite picture of transcriptome evolution. The isolation of individual cells from tissue sections opens up the opportunity to study gene expression evolution at the cell type level. We have stained neurons and endothelial cells in human brains by antibodies against cell type-specific marker proteins, isolated the cells using laser capture microdissection, and identified genes preferentially expressed in the two cell types. We analyze these two classes of genes with respect to their expression in 62 different human tissues, with respect to their expression in 44 human “postmortem” brains from different developmental stages and with respect to between-species brain expression differences. We find that genes preferentially expressed in neurons differ less across tissues and developmental stages than genes preferentially expressed in endothelial cells. We also observe less expression differences within primate species for neuronal transcriptomes. In stark contrast, we see more gene expression differences between humans, chimpanzees, and rhesus macaques relative to within-species differences in genes expressed preferentially in neurons than in genes expressed in endothelial cells. This suggests that neuronal and endothelial transcriptomes evolve at different rates within brain tissue

KirBac1.1: It's an Inward Rectifying Potassium Channel

KirBac1.1 is a prokaryotic homologue of eukaryotic inward rectifier potassium (Kir) channels. The crystal structure of KirBac1.1 and related KirBac3.1 have now been used extensively to generate in silico models of eukaryotic Kir channels, but functional analysis has been limited to 86Rb+ flux experiments and bacteria or yeast complementation screens, and no voltage clamp analysis has been available. We have expressed pure full-length His-tagged KirBac1.1 protein in Escherichia coli and obtained voltage clamp recordings of recombinant channel activity in excised membrane patches from giant liposomes. Macroscopic currents of wild-type KirBac1.1 are K+ selective and spermine insensitive, but blocked by Ba2+, similar to “weakly rectifying” eukaryotic Kir1.1 and Kir6.2 channels. The introduction of a negative charge at a pore-lining residue, I138D, generates high spermine sensitivity, similar to that resulting from the introduction of a negative charge at the equivalent position in Kir1.1 or Kir6.2. KirBac1.1 currents are also inhibited by PIP2, consistent with 86Rb+ flux experiments, and reversibly inhibited by short-chain di-c8-PIP2. At the single-channel level, KirBac1.1 channels show numerous conductance states with two predominant conductances (15 pS and 32 pS at −100 mV) and marked variability in gating kinetics, similar to the behavior of KcsA in recombinant liposomes. The successful patch clamping of KirBac1.1 confirms that this prokaryotic channel behaves as a bona fide Kir channel and opens the way for combined biochemical, structural, and electrophysiological analysis of a tractable model Kir channel, as has been successfully achieved for the archetypal K+ channel KcsA

A Sound Approach to Language Matters: In Honor of Ocke-Schwen Bohn

The contributions in this Festschrift were written by Ocke’s current and former PhD-students, colleagues and research collaborators. The Festschrift is divided into six sections, moving from the smallest building blocks of language, through gradually expanding objects of linguistic inquiry to the highest levels of description - all of which have formed a part of Ocke’s career, in connection with his teaching and/or his academic productions: “Segments”, “Perception of Accent”, “Between Sounds and Graphemes”, “Prosody”, “Morphology and Syntax” and “Second Language Acquisition”. Each one of these illustrates a sound approach to language matters

Line-Scanning Particle Image Velocimetry: An Optical Approach for Quantifying a Wide Range of Blood Flow Speeds in Live Animals

The ability to measure blood velocities is critical for studying vascular development, physiology, and pathology. A key challenge is to quantify a wide range of blood velocities in vessels deep within living specimens with concurrent diffraction-limited resolution imaging of vascular cells. Two-photon laser scanning microscopy (TPLSM) has shown tremendous promise in analyzing blood velocities hundreds of micrometers deep in animals with cellular resolution. However, current analysis of TPLSM-based data is limited to the lower range of blood velocities and is not adequate to study faster velocities in many normal or disease conditions.We developed line-scanning particle image velocimetry (LS-PIV), which used TPLSM data to quantify peak blood velocities up to 84 mm/s in live mice harboring brain arteriovenous malformation, a disease characterized by high flow. With this method, we were able to accurately detect the elevated blood velocities and exaggerated pulsatility along the abnormal vascular network in these animals. LS-PIV robustly analyzed noisy data from vessels as deep as 850 µm below the brain surface. In addition to analyzing in vivo data, we validated the accuracy of LS-PIV up to 800 mm/s using simulations with known velocity and noise parameters.To our knowledge, these blood velocity measurements are the fastest recorded with TPLSM. Partnered with transgenic mice carrying cell-specific fluorescent reporters, LS-PIV will also enable the direct in vivo correlation of cellular, biochemical, and hemodynamic parameters in high flow vascular development and diseases such as atherogenesis, arteriogenesis, and vascular anomalies

The Cerebral Microvasculature in Schizophrenia: A Laser Capture Microdissection Study

BACKGROUND: Previous studies of brain and peripheral tissues in schizophrenia patients have indicated impaired energy supply to the brain. A number of studies have also demonstrated dysfunction of the microvasculature in schizophrenia patients. Together these findings are consistent with a hypothesis of blood-brain barrier dysfunction in schizophrenia. In this study, we have investigated the cerebral vascular endothelium of schizophrenia patients at the level of transcriptomics. METHODOLOGY/PRINCIPAL FINDINGS: We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from schizophrenia patients and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using two independent microarray platforms, Affymetrix HG133plus2.0 GeneChips and CodeLink Whole Human Genome arrays. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. We then compared neuronal and endothelial data separately between schizophrenic subjects and controls. Analysis of the endothelial samples showed differences in gene expression between schizophrenics and controls which were reproducible in a second microarray platform. Functional profiling revealed that these changes were primarily found in genes relating to inflammatory processes. CONCLUSIONS/SIGNIFICANCE: This study provides preliminary evidence of molecular alterations of the cerebral microvasculature in schizophrenia patients, suggestive of a hypo-inflammatory state in this tissue type. Further investigation of the blood-brain barrier in schizophrenia is warranted