Crystallographic Analyses of Ion Channels: Lessons and Challenges

Membrane proteins fascinate at many levels, from their central functional roles in transport, energy transduction, and signal transduction processes to structural questions concerning how they fold and operate in the exotic environments of the membrane bilayer and the water-bilayer interface and to methodological issues associated with studying membrane proteins either in situ or extracted from the membrane. This interplay is beautifully exemplified by ion channels, a collection of integral membrane proteins that mediate the transmembrane passage of ions down their electrochemical potential gradient (for general reviews, see Refs. 1 and 2). Ion channels are key elements of signaling and sensing pathways, including nerve cell conduction, hormone response, and mechanosensation. The characteristic properties of ion channels reflect their conductance, ion selectivity, and gating. Ion channels are often specific for a particular type of ion (such as potassium or chloride) or a class of ions (such as anions) and are typically regulated by conformational switching of the protein structure between "open" and "closed" states. This conformational switching may be gated in response to changes in membrane potential, ligand binding, or application of mechanical forces. Detailed functional characterizations of channels and their gating mechanisms have been achieved, reflecting exquisite methodological advances such as patch clamp methods that can monitor the activities of individual channels (3). Until recently, corresponding information about the three-dimensional structures of channels was not available, reflecting difficulties in obtaining sufficient quantities of membrane proteins for crystallization trials. Happily, this situation has started to change with the structure determinations of the Streptomyces lividans K+ channel (KcsA (4)) and the Mycobacterium tuberculosis mechanosensitive channel (MscL (5)). A variety of reviews (6-12) have appeared recently that discuss functional implications of these channel structures. This review discusses these developments from a complementary perspective, by considering the implications of these structures from within the larger framework of membrane protein structure and function. Because of space restrictions, this review necessarily emphasizes membrane proteins that are composed primarily of alpha-helical bundles, such as KcsA and MscL, rather than beta-barrel proteins, such as porins, typically found in bacterial outer membranes

BATSE Observations of Gamma-Ray Burst Spectra. IV. Time-Resolved High-Energy Spectroscopy

We report on the temporal behavior of the high-energy power law continuum component of gamma-ray burst spectra with data obtained by the Burst and Transient Source Experiment. We have selected 126 high fluence and high flux bursts from the beginning of the mission up until the present. Much of the data were obtained with the Large Area Detectors, which have nearly all-sky coverage, excellent sensitivity over two decades of energy and moderate energy resolution, ideal for continuum spectra studies of a large sample of bursts at high time resolution. At least 8 spectra from each burst were fitted with a spectral form that consisted of a low-energy power law, a spectral break at middle energies and a high-energy continuum. In most bursts (122), the high-energy continuum was consistent with a power law. The evolution of the fitted high-energy power-law index over the selected spectra for each burst is inconsistent with a constant for 34% of the total sample. The sample distribution of the average value for the index from each burst is fairly narrow, centered on -2.12. A linear trend in time is ruled out for only 20% of the bursts, with hard-to-soft evolution dominating the sample (100 events). The distribution for the total change in the power-law index over the duration of a burst peaks at the value -0.37, and is characterized by a median absolute deviation of 0.39, arguing that a single physical process is involved. We present analyses of the correlation of the power-law index with time, burst intensity and low-energy time evolution. In general, we confirm the general hard-to-soft spectral evolution observed in the low-energy component of the continuum, while presenting evidence that this evolution is different in nature from that of the rest of the continuum.Comment: 30 pages, with 2 tables and 9 figures To appear in The Astrophysical Journal, April 1, 199

Gut bacteria responding to dietary change encode sialidases that exhibit preference for red meat-associated carbohydrates.

Dietary habits have been associated with alterations of the human gut resident microorganisms contributing to obesity, diabetes and cancer1. In Western diets, red meat is a frequently eaten food2, but long-term consumption has been associated with increased risk of disease3,4. Red meat is enriched in N-glycolylneuraminic acid (Neu5Gc) that cannot be synthesized by humans5. However, consumption can cause Neu5Gc incorporation into cell surface glycans6, especially in carcinomas4,7. As a consequence, an inflammatory response is triggered when Neu5Gc-containing glycans encounter circulating anti-Neu5Gc antibodies8,9. Although bacteria can use free sialic acids as a nutrient source10-12, it is currently unknown if gut microorganisms contribute to releasing Neu5Gc from food. We found that a Neu5Gc-rich diet induces changes in the gut microbiota, with Bacteroidales and Clostridiales responding the most. Genome assembling of mouse and human shotgun metagenomic sequencing identified bacterial sialidases with previously unobserved substrate preference for Neu5Gc-containing glycans. X-ray crystallography revealed key amino acids potentially contributing to substrate preference. Additionally, we verified that mouse and human sialidases were able to release Neu5Gc from red meat. The release of Neu5Gc from red meat using bacterial sialidases could reduce the risk of inflammatory diseases associated with red meat consumption, including colorectal cancer4 and atherosclerosis13

Jet-Induced Emission-Line Nebulosity and Star Formation in the High-Redshift Radio Galaxy 4C41.17

The high redshift radio galaxy 4C41.17 consists of a powerful radio source in which previous work has shown that there is strong evidence for jet-induced star formation along the radio axis. We argue that nuclear photoionization is not responsible for the excitation of the emission line clouds and we construct a jet-cloud interaction model to explain the major features revealed by the data. The interaction of a high-powered jet with a dense cloud in the halo of 4C41.17 produces shock-excited emission-line nebulosity through ~1000 km/s shocks and induces star formation. The CIII to CIV line ratio and the CIV luminosity emanating from the shock, imply that the pre-shock density in the line-emitting cloud is high enough (~1-10 cm^-3) that shock initiated star formation could proceed on a timescale of order a few x 10^6 yrs, well within the estimated dynamical age of the radio source. Broad (FWHM ~ 100 - 1400 km/s) emission lines are attributed to the disturbance of the gas cloud by a partial bow--shock and narrow emission lines (FWHM ~ 500 - 650 km/s) (in particular CIV) arise in precursor emission in relatively low metallicity gas. The implied baryonic mass ~ 8 \times 10^{10} solar masses of the cloud is high and implies that Milky Way size condensations existed in the environments of forming radio galaxies at a redshift of 3.8. Our interpretation of the data provides a physical basis for the alignment of the radio, emission-line and UV continuum images in some of the highest redshift radio galaxies and the analysis presented here may form a basis for the calculation of densities and cloud masses in other high redshift radio galaxies.Comment: 18 pages, 5 figures; uses astrobib.sty and aaspp4.sty. Better versions of figures available via anonymous from ftp://mso.anu.edu.au:pub/pub/geoff/4C41.1

Cloning and Characterisation of Schistosoma japonicum Insulin Receptors

Background: Schistosomes depend for growth and development on host hormonal signals, which may include the insulin signalling pathway. We cloned and assessed the function of two insulin receptors from Schistosoma japonicum in order to shed light on their role in schistosome biology

General practitioner workforce sustainability to maximise effective and equitable patient care: a realist review protocol

Introduction There are not enough general practitioners (GPs) in the UK National Health Service. This problem is worse in areas of the country where poverty and underinvestment in health and social care mean patients experience poorer health compared with wealthier regions. Encouraging more doctors to choose and continue in a GP career is a government priority. This review will examine which aspects of the healthcare system affect GP workforce sustainability, how, why and for whom. Methods and analysis A realist review is a theory-driven interpretive approach to evidence synthesis, that brings together qualitative, quantitative, mixed-methods research and grey literature. We will use a realist approach to synthesise data from the available published literature to refine an evidence-based programme theory that will identify the important contextual factors and underlying mechanisms that underpin observed outcomes relating to GP workforce sustainability. Our review will follow Pawson’s five iterative stages: (1) finding existing theories, (2) searching for evidence, (3) article selection, (4) data extraction and (5) synthesising evidence and drawing conclusions. We will work closely with key stakeholders and embed patient and public involvement throughout the review process to refine the focus of the review and enhance the impact and relevance of our research. Ethics and dissemination This review does not require formal ethical approval as it draws on secondary data from published articles and grey literature. Findings will be disseminated through multiple channels, including publication in peer-reviewed journals, at national and international conferences, and other digital scholarly communication tools such as video summaries, X and blog posts. PROSPERO registration number CRD42023395583

Long-Term Health Risks for Children and Young Adults after Infective Gastroenteritis

TOC Summary: Prior episodes increase risk for long-term adverse health effects

Increased Microerythrocyte Count in Homozygous α+-Thalassaemia Contributes to Protection against Severe Malarial Anaemia

Karen Day and colleagues show that increased microcytic erythrocyte count may contribute substantially to the protection of α+-thalassaemia-homozygous children against severe malaria anaemia

The Impact of Delayed Treatment of Uncomplicated \u3ci\u3eP. falciparum\u3c/i\u3e Malaria on Progression to Severe Malaria: A Systematic Review and a Pooled Multicentre Individual-Patient Meta-Analysis

BACKGROUND: Delay in receiving treatment for uncomplicated malaria (UM) is often reported to increase the risk of developing severe malaria (SM), but access to treatment remains low in most high-burden areas. Understanding the contribution of treatment delay on progression to severe disease is critical to determine how quickly patients need to receive treatment and to quantify the impact of widely implemented treatment interventions, such as \u27test-and-treat\u27 policies administered by community health workers (CHWs). We conducted a pooled individual-participant meta-analysis to estimate the association between treatment delay and presenting with SM. METHODS AND FINDINGS: A search using Ovid MEDLINE and Embase was initially conducted to identify studies on severe Plasmodium falciparum malaria that included information on treatment delay, such as fever duration (inception to 22nd September 2017). Studies identified included 5 case-control and 8 other observational clinical studies of SM and UM cases. Risk of bias was assessed using the Newcastle-Ottawa scale, and all studies were ranked as \u27Good\u27, scoring ≥7/10. Individual-patient data (IPD) were pooled from 13 studies of 3,989 (94.1% aged \u3c15 years) SM patients and 5,780 (79.6% aged \u3c15 years) UM cases in Benin, Malaysia, Mozambique, Tanzania, The Gambia, Uganda, Yemen, and Zambia. Definitions of SM were standardised across studies to compare treatment delay in patients with UM and different SM phenotypes using age-adjusted mixed-effects regression. The odds of any SM phenotype were significantly higher in children with longer delays between initial symptoms and arrival at the health facility (odds ratio [OR] = 1.33, 95% CI: 1.07-1.64 for a delay of \u3e24 hours versus ≤24 hours; p = 0.009). Reported illness duration was a strong predictor of presenting with severe malarial anaemia (SMA) in children, with an OR of 2.79 (95% CI:1.92-4.06; p \u3c 0.001) for a delay of 2-3 days and 5.46 (95% CI: 3.49-8.53; p \u3c 0.001) for a delay of \u3e7 days, compared with receiving treatment within 24 hours from symptom onset. We estimate that 42.8% of childhood SMA cases and 48.5% of adult SMA cases in the study areas would have been averted if all individuals were able to access treatment within the first day of symptom onset, if the association is fully causal. In studies specifically recording onset of nonsevere symptoms, long treatment delay was moderately associated with other SM phenotypes (OR [95% CI] \u3e3 to ≤4 days versus ≤24 hours: cerebral malaria [CM] = 2.42 [1.24-4.72], p = 0.01; respiratory distress syndrome [RDS] = 4.09 [1.70-9.82], p = 0.002). In addition to unmeasured confounding, which is commonly present in observational studies, a key limitation is that many severe cases and deaths occur outside healthcare facilities in endemic countries, where the effect of delayed or no treatment is difficult to quantify. CONCLUSIONS: Our results quantify the relationship between rapid access to treatment and reduced risk of severe disease, which was particularly strong for SMA. There was some evidence to suggest that progression to other severe phenotypes may also be prevented by prompt treatment, though the association was not as strong, which may be explained by potential selection bias, sample size issues, or a difference in underlying pathology. These findings may help assess the impact of interventions that improve access to treatment

Somatic Variants in SVIL in Cerebral Aneurysms

Publisher Copyright: © American Academy of Neurology.Background and ObjectivesWhile somatic mutations have been well-studied in cancer, their roles in other complex traits are much less understood. Our goal is to identify somatic variants that may contribute to the formation of saccular cerebral aneurysms.MethodsWe performed whole-exome sequencing on aneurysm tissues and paired peripheral blood. RNA sequencing and the CRISPR/Cas9 system were then used to perform functional validation of our results.ResultsSomatic variants involved in supervillin (SVIL) or its regulation were found in 17% of aneurysm tissues. In the presence of a mutation in the SVIL gene, the expression level of SVIL was downregulated in the aneurysm tissue compared with normal control vessels. Downstream signaling pathways that were induced by knockdown of SVIL via the CRISPR/Cas9 system in vascular smooth muscle cells (vSMCs) were determined by evaluating changes in gene expression and protein kinase phosphorylation. We found that SVIL regulated the phenotypic modulation of vSMCs to the synthetic phenotype via Krüppel-like factor 4 and platelet-derived growth factor and affected cell migration of vSMCs via the RhoA/ROCK pathway.DiscussionWe propose that somatic variants form a novel mechanism for the development of cerebral aneurysms. Specifically, somatic variants in SVIL result in the phenotypic modulation of vSMCs, which increases the susceptibility to aneurysm formation. This finding suggests a new avenue for the therapeutic intervention and prevention of cerebral aneurysms.Peer reviewe