A sensitive and specific genetically-encoded potassium ion biosensor for in vivo applications across the tree of life.

Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.

Myogenic contraction of a somatic muscle powers rhythmic flow of hemolymph through Drosophila antennae and generates brain pulsations.

Hemolymph is driven through the antennae of Drosophila melanogaster by the rhythmic contraction of muscle 16 (m16), which runs through the brain. Contraction of m16 results in the expansion of an elastic ampulla, opening ostia and filling the ampulla. Relaxation of the ampullary membrane forces hemolymph through vessels into the antennae. We show that m16 is an auto-active rhythmic somatic muscle. The activity of m16 leads to the rapid perfusion of the antenna by hemolymph. In addition, it leads to the rhythmic agitation of the brain, which could be important for clearing the interstitial space.

Non-F HBV/HDV-3 coinfection is associated with severe liver disease in Western Brazilian Amazon.

The clinical outcome of hepatitis B virus (HBV) infection may be related to host and viral genetic factors, as well as to the type of infection (monoinfection and coinfection). To analyze the distribution/combination of HBV/hepatitis D virus (HDV) genotypes and the associated clinical characteristics, 409 serum samples from patients with chronic HBV (94 of them coinfected by HDV) followed at the Viral Hepatitis Referral Center of Rio Branco, Brazil were enrolled. HBV DNA and HDV RNA were amplified, respectively, by polymerase chain reaction (PCR) and nested PCR using specific primers in the PreC/C region and the S gene, and by reverse-transcription PCR and seminested PCR using specific primers in the delta antigen region and sequenced. The proportion of women (56.1%) was significantly higher than males in this cohort ( P < 0.01). Women were significantly younger (39.8 years; 8-77 years) than males (44.7 years; 12-79 years; P < 0.01). Sixty-eight (18%) patients were infected with HBV-F genotype and 264 (69.8%) with HBV/non-F genotypes. Coinfection by HDV was detected in 23.9% (94 of 409) of this population and was more frequent in male (54.2%, 51 of 94) than in female patients (44.7%, 42 of 94; P = 0.015). HDV-3 was the most prevalent (88.9%) genotype. Almost 70% of HDV-3 coinfected patients were infected with HBV/non-F genotypes. Severe liver disease was diagnosed in 41 patients, 60.9% (25 of 41) of them coinfected with HDV. HBV/HDV coinfection was associated with male sex, age above 30 years, severe liver disease, and increased alanine aminotransferase levels. HBV/HDV-3 coinfection is associated with severe liver disease, in Rio Branco, Brazil.

Conceptualizing the health and well-being impacts of social enterprise: a UK-based study.

Social enterprises-businesses that work for social benefit rather than for the maximization of financial returns to shareholders or owners-could potentially prove to be an innovative and sustainable way of tackling 'upstream' social determinants of health. However, empirical work focusing upon how, and to what extent, social enterprise-led activity may impact upon health and well-being is still relatively scarce. This study examines how social enterprises portray their impact, and how such impacts may be considered in health and well-being terms. Through analysing evaluative reports of the work of social enterprises in Scotland (n = 17) utilizing a 'process coding' method, we investigate both the self-reported impacts of the work of social enterprises and the mechanisms by which these are said to be derived. Revisiting previous conceptualizations in the extant literature, this work allows us to present an 'empirically-informed' conceptual model of the health and well-being impacts of social enterprise-led activity, and thus presents a significant advance on previous hypothetical, theoretically-based conceptualizations. It is considered that these findings further improve our overall knowledge of ways in which social enterprise and other parts of the third sector could be considered as potentially valuable 'non-obvious' public health actors.

Protein-peptide arrays for detection of specific anti-hepatitis D virus (HDV) genotype 1, 6, and 8 antibodies among HDV-infected patients by surface plasmon resonance imaging.

Liver diseases linked to hepatitis B-hepatitis D virus co- or superinfections are more severe than those during hepatitis B virus (HBV) monoinfection. The diagnosis of hepatitis D virus (HDV) infection therefore remains crucial in monitoring patients but is often overlooked. To integrate HDV markers into high-throughput viral hepatitis diagnostics, we studied the binding of anti-HDV antibodies (Abs) using surface plasmon resonance imaging (SPRi). We focused on the ubiquitous HDV genotype 1 (HDV1) and the more uncommon African-HDV6 and HDV8 genotypes to define an array with recombinant proteins or peptides. Full-length and truncated small hepatitis D antigen (S-HDAg) recombinant proteins of HDV genotype 1 (HDV1) and 11 HDV peptides of HDV1, 6, and 8, representing various portions of the delta antigen were grafted onto biochips, allowing SPRi measurements to be made. Sixteen to 17 serum samples from patients infected with different HDV genotypes were injected onto protein and peptide chips. In all, Abs against HDV proteins and/or peptides were detected in 16 out of 17 infected patients (94.12%), although the amplitude of the SPR signal varied. The amino-terminal part of the protein was poorly immunogenic, while epitope 65-80, exposed on the viral ribonucleoprotein, may be immunodominant, as 9 patient samples led to a specific SPR signal on peptide 65 type 1 (65#1), independently of the infecting genotype. In this pilot study, we confirmed that HDV infection screening based on the reactivity of patient Abs against carefully chosen HDV peptides and/or proteins can be included in a syndrome-based viral hepatitis diagnostic assay. The preliminary results indicated that SPRi studying direct physical HDAg-anti-HDV Ab interactions was more convenient using linear peptide epitopes than full-length S-HDAg proteins, due to the regeneration process, and may represent an innovative approach for a hepatitis syndrome-viral etiology-exploring array.

HBVdb: a knowledge database for Hepatitis B Virus.

We have developed a specialized database, HBVdb (http://hbvdb.ibcp.fr), allowing the researchers to investigate the genetic variability of Hepatitis B Virus (HBV) and viral resistance to treatment. HBV is a major health problem worldwide with more than 350 million individuals being chronically infected. HBV is an enveloped DNA virus that replicates by reverse transcription of an RNA intermediate. HBV genome is optimized, being circular and encoding four overlapping reading frames. Indeed, each nucleotide of the genome takes part in the coding of at least one protein. However, HBV shows some genome variability leading to at least eight different genotypes and recombinant forms. The main drugs used to treat infected patients are nucleos(t)ides analogs (reverse transcriptase inhibitors). Unfortunately, HBV mutants resistant to these drugs may be selected and be responsible for treatment failure. HBVdb contains a collection of computer-annotated sequences based on manually annotated reference genomes. The database can be accessed through a web interface that allows static and dynamic queries and offers integrated generic sequence analysis tools and specialized analysis tools (e.g. annotation, genotyping, drug resistance profiling).

Mixed burn injury to thigh secondary to D-cell lithium battery explosion.

There is an increasing trend globally of fire incidents as a direct consequence of battery failures[1-6], but a dearth of reporting in medical literature regarding injuries associated with primary lithium cell explosions. We present the case of an electrical engineer referred to the burns team as a chemical burn secondary to a D-cell lithium battery explosion. Initial assessment revealed an entry wound on the anteromedial thigh leaking contaminated fluid. Orthogonal X-rays demonstrated the battery casing lodged within the posterior thigh compartment. The wound was managed similar to that of a ballistic injury with staged debridement, washout and delayed primary closure. This is the first reported case of a lithium-thionyl chloride battery explosion causing injury. The case highlights various issues for attending teams, including appropriate first aid for chemical burns, consideration of significant soft tissue trauma deep to seemingly innocuous wounds and safeguarding concerns surrounding domestic explosive devices.

Mathematical modeling of intracellular osmolarity and cell volume stabilization: The Donnan effect and ion transport.

The presence of impermeant molecules within a cell can lead to an increase in cell volume through the influx of water driven by osmosis. This phenomenon is known as the Donnan (or Gibbs-Donnan) effect. Animal cells actively transport ions to counteract the Donnan effect and regulate their volume, actively pumping Na+ out and K+ into their cytosol using the Na+/K+ ATPase (NKA) pump. The pump-leak equations (PLEs) are a system of algebraic-differential equations to model the membrane potential, ion (Na+, K+, and Cl-), and water flux across the cell membrane, which provide insight into how the combination of passive ions fluxes and active transport contribute to stabilizing cell volume. Our broad objective is to provide analytical insight into the PLEs through three lines of investigation: (1) we show that the provision of impermeant extracellular molecules can stabilize the volume of a passive cell; (2) we demonstrate that the mathematical form of the NKA pump is not as important as the stoichiometry for cell stabilization; and (3) we investigate the interaction between the NKA pump and cation-chloride co-transporters (CCCs) on cell stabilization, showing that NCC can destabilize a cell while NKCC and KCC can stabilize it. We incorporate extracellular impermeant molecules, NKA pump, and CCCs into the PLEs and derive the exact formula for the steady states in terms of all the parameters. This analytical expression enables us to easily explore the effect of each of the system parameters on the existence and stability of the steady states.

Identification of novel recombinants of hepatitis B virus genotypes F and G in human immunodeficiency virus-positive patients from Argentina and Brazil.

Hepatitis B virus (HBV) genotype G (HBV/G) infection is almost always detected along with a co-infecting HBV strain that can supply HBeAg, typically HBV/A2. In this study we describe, in two human immunodeficiency virus (HIV)-positive patients from Argentina and Brazil, the first report of HBV/G infection in Argentina and co-circulation of HBV/G, HBV/F and G/F recombinants in the American continent. HBV isolates carrying the 36 bp insertion of HBV/G were the most prevalent in both patients, with >99 % of colonies hybridizing to a probe specific for this insertion. Phylogenetic analyses of full-length genomes and precore/core fragments revealed that F4 and F1b were the co-infecting subgenotypes in the Brazilian and Argentinian patients, respectively. Bootscanning analysis provided evidence of recombination in several clones from both patients, with recombination breakpoints located mainly at the precore/core region. These data should encourage further investigations on the clinical implications of HBV/G recombinants in HBV/HIV co-infected patients.

Probable corticosteroid-induced reactivation of latent hepatitis B virus infection in an HIV-positive patient involving immune escape.

We describe a patient infected with human immunodeficiency virus who possessed a serological profile suggesting a previous cleared acute hepatitis B virus (HBV) infection, including high levels of antibodies against HBV surface antigen (anti-HBs). Following the administration of inhaled glucocorticosteroids combined with protease inhibitor-based antiretroviral treatment, the patient developed an unexpected severe acute hepatitis despite persistence of anti-HBs. A genotype A2 strain emerged with 2 major mutations in the S gene, sK122R and sD144E. Molecular and biological analyses strongly suggested reactivation of a latent HBV infection. The importance and the molecular basis of these 2 epitopes in immune-escape mechanisms and host-virus interactions are discussed.

The physical basis of osmosis.

Osmosis is an important force in all living organisms, yet the molecular basis of osmosis is widely misunderstood as arising from diffusion of water across a membrane separating solutions of differing osmolarities, and hence different water concentrations. In 1923, Peter Debye proposed a physical model for a semipermeable membrane emphasizing the repulsive forces between solute molecules and membrane that prevent the solute from entering the membrane. His work was hardly noticed at the time and slipped out of view. We show that Debye's analysis of van 't Hoff's law for osmotic equilibrium also provides a consistent and plausible mechanism for osmotic flow. A difference in osmolyte concentrations in solutions separated by a semipermeable membrane leads to different pressures at the two water-membrane interfaces because the total repulsive force between solute molecules and the membrane is different at the two interfaces. Water is therefore driven through the membrane for exactly the same reason that pure water flows in response to an imposed hydrostatic pressure difference. In this paper, we present the Debye model in both equilibrium and flow conditions. We point out its applicability regardless of the nature of the membrane with examples ranging from the predominantly convective flow of water through synthetic membranes and capillary walls to the purely diffusive flow of independent water molecules through a lipid bilayer and the flow of a single-file column of water molecules in narrow protein channels.

Climate change and pavement burns in the United Kingdom: a case report of two patients.

Pavement burns are more common in locations familiarised with high temperatures and a dry climate zone, but have not previously been reported in temperate climates. We present two cases of patients who suffered pavement burns in the United Kingdom during an unprecedentedly hot day in July 2022. The first case involved a 66-year-old male who suffered partial and full thickness burns requiring excision and skin grafting. The second case involved a 58-year-old female with partial thickness burns also requiring excision and skin grafting. Both patients had pre-existing co-morbidities and their pavement burns were precipitated by heat stroke. Pavement burns represent a mechanism of injury that necessitates increased operative management, length of hospital stay and cost per surface area burned when compared to flame or scald burns (Silver et al., 2015). As a result of global warming, we anticipate extreme heat events, and subsequently pavement burns, to increase in incidence in the United Kingdom. There is opportunity for education of the public and health professionals for prevention.

A method for detecting molecular transport within the cerebral ventricles of live zebrafish (Danio rerio) larvae.

The production and flow of cerebrospinal fluid performs an important role in the development and homeostasis of the central nervous system.However, these processes are difficult to study in the mammalian brain because the ventricles are situated deep within the parenchyma.In this communication we introduce the zebrafish larva as an in vivo model for studying cerebral ventricle and blood­brain barrier function. Using confocal microscopy we show that zebrafish ventricles are topologically similar to those of the mammalian brain.We describe a new method for measuring the dynamics of molecular transport within the ventricles of live zebrafish by means of the uncaging of a fluorescein derivative. Furthermore, we determine that in 5­6 days post-fertilization zebrafish, the dispersal of molecules in the ventricles is driven by a combination of ciliary motion and diffusion. The zebrafish presents a tractable system with the advantage of genetics, size and transparency for exploring ventricular physiology and for mounting large-scale high throughput experiments.

Management of minor burns during the COVID-19 pandemic: A patient-centred approach.

INTRODUCTION: The UK government introduced lockdown measures on 23 March 2020 due to the first wave of the COVID-19 pandemic. A restructuring of clinical services was necessary to accommodate mandatory changes while also maintaining the best possible standards for patient care. The present study explored the initial management, follow-up and patient-reported outcomes of burn injuries <15% total body surface area (TBSA) during the height of the COVID-19 lockdown at a tertiary burns centre. METHODS: A retrospective review of all adult patients with burns <15% TBSA during the national lockdown (23 March 2020 to 10 May 2020) was undertaken at The Queen Elizabeth Hospital Birmingham (QEHB), UK. All referrals from non-QEHB telemedicine (external) or QEHB emergency (internal) departments were reviewed for management, length of hospital stay and pattern of follow-up (ward attender, self-care, community or outreach nurses). A telephone survey based on a structured questionnaire was conducted to establish patients' satisfaction. RESULTS: A total of 84 burn patients were included in the study. The mean age was 39 years (age range = 19-91 years) and the male:female ratio was 4:1. Patients were managed non-operatively (n = 69, 82%) or operatively (n = 15, 18%). Patients attended the ward attender acute burns clinic only once (n = 36, 61%). The telephone survey captured 70% (n = 59) of the study population and 57 patients (97% of respondents) were pleased with the ongoing care and burn healing. CONCLUSION: The integration of patient led self-care, reduction in admissions, minimal clinics attendance and a telemedicine follow-up is an effective model for small burns management during the COVID-19 pandemic. A high degree of patient satisfaction was achieved with continuous and approachable communication channels with the burn multidisciplinary team. We continue to implement this effective model of burns management throughout the COVID-19 pandemic and the subsequent period. LAY SUMMARY: The lockdown measures due to the first wave of COVID-19 pandemic affected the way we manage all medical emergencies including burns. The initial management, follow-up and patient satisfaction for small burn injuries during lockdown has not been reported previously. The aim of this study is to examine the outcome in terms of small burn management, hospital stay, number of clinic reviews, healing and patient satisfaction during the lockdown period in a burn centre in the UK. This would look at the need for operations and whether patients stayed longer if they required an intervention. We reviewed adult patients with small burns during the national lockdown (23 March 2020 to 10 May 2020) at The Queen Elizabeth Hospital Birmingham (QEHB). All referrals from telemedicine, referral system (external) or QEHB (internal) were reviewed for management, length of hospital stay and pattern of follow-up. Patients were reviewed in the acute burns clinic and given advice for burn management and dressing for self-care. Follow-up was mostly via email (telemedicine) A telephone survey based on a structured questionnaire was conducted to find out patients' satisfaction. Four times more men than women had small burns during the lockdown period. The average age was 39 years. The majority were managed conservatively with dressings (82%) and a small proportion required an operation (18%). Most patients attended the acute burns clinic only once (61%) for initial assessment and management. The telephone survey captured 70% of patient and 97% of respondents were pleased with the care and burn healing. The integration of patient-led self-care, reduction in admissions, minimal clinics attendance and a telemedicine follow-up is an effective model for burns management during the COVID-19 pandemic. A high degree of patient satisfaction was achieved with continuous and approachable communication channels with burn multidisciplinary team. We continue to implement this effective model of burns management throughout the COVID-19 pandemic and the subsequent period.

A startling role for synaptic zinc.

It has long been known that the synaptic vesicles of certain glutamatergic terminals, as well as some inhibitory terminals, are richly supplied with zinc ions, yet the functional role of this pool of zinc in synaptic transmission has remained elusive. In this issue of Neuron, Hirzel et al. provide direct in vivo evidence that endogenous zinc is required for proper functioning of neuronal circuitry in the brainstem and spinal cord. They show that knockin mice carrying a point mutation which eliminates zinc potentiation of alpha1-containing glycine receptors develop severe sensorimotor deficits characteristic of impaired glycinergic neurotransmission.

Microarray for hepatitis B virus genotyping and detection of 994 mutations along the genome.

Genome analysis of hepatitis B virus (HBV) in patient sera is helpful for monitoring treatment. We developed an improved version of a DNA microarray to identify HBV genotypes and to detect mutations of interest in the S, Pol, Core, and X genes. It includes an automated software analysis of fluorescence values for simpler, more robust data interpretation. In this version, probes were added to identify genotype H, to analyze 155 additional positions, and to detect 561 additional polymorphisms. Sequences were added to the alignments to resolve hybridization problems due to natural polymorphisms in the vicinity of important codons. The duplex PCR protocol allowed whole-genome analysis in a single tube. An alternative nested-PCR protocol allowed genotyping and mutations in S and reverse transcriptase (rt) genes in patients with low viral loads, as demonstrated in patients with less than 400 HBV genome copies/ml. Reproducibility was high, with variation coefficients lower than 3%. Only 0.57% of 20,771 codons from 253 samples could not be identified. The concordance with Sanger sequencing for the identification of codons improved from 92.8% to 95.7% with the improved version. Concordance was higher than 91% for codons associated with resistance to lamivudine, emtricitabine, telbivudine, famciclovir, entecavir, and tenofovir with vaccine escape and for pre-Core mutants. Concordance was lower for adefovir resistance mutations (68.6%) and mutations in the basal core promoter (60.3%), probably because hybridization efficiency was affected by the low GC content of the probes. A concordance of 93.7% with sequencing for genotype identification was observed in 190 specimens, lower than that obtained with the first version, possibly due to mixed virus populations.

The interplay between inorganic phosphate and amino acids determines zinc solubility in brain slices.

Inorganic phosphate (Pi) is an important polyanion needed for ATP synthesis and bone formation. As it is found at millimolar levels in plasma, it is usually incorporated as a constituent of artificial CSF formulations for maintaining brain slices. In this paper, we show that Pi limits the extracellular zinc concentration by inducing metal precipitation. We present data suggesting that amino acids like histidine may counteract the Pi-induced zinc precipitation by the formation of soluble zinc complexes. We propose that the interplay between Pi and amino acids in the extracellular space may influence the availability of metals for cellular uptake.

The interaction of biological and noxious transition metals with the zinc probes FluoZin-3 and Newport Green.

Zinc-sensitive fluorescent probes have become increasingly important in the investigation of the cellular roles of zinc. There is, however, little information on how the other transition metals in cells may influence the measurement of zinc. We have characterized in vitro the interaction of the nominal zinc indicators FluoZin-3 and Newport Green with all the cationic transition metals found within cells, Cr, Mn, Fe, Co, and Cu, as well as Ni and Cd, by measuring their dissociation constants. In addition, we have shown how FluoZin-3 can be used to quantify the concentration of copper in a cell-free assay and report that the fluorescence of Newport Green is boosted by both Cu(I) and Fe(II). Furthermore, we have introduced diagnostics for detecting the interference of metals other than zinc with its measurement within cells.

Evolution of our understanding of cell volume regulation by the pump-leak mechanism.

All animal cells are surrounded by a flexible plasma membrane that is permeable to water and to small ions. Cells thus face a fundamental problem: the considerable tension that their membranes would experience if the osmotic influx of water, driven by the presence of impermeant intracellular ions, was left unopposed. The pivotal study that described the cell's remedy for this impending osmotic catastrophe-the "pump-leak mechanism" (PLM)-was published in the Journal of General Physiology by Tosteson and Hoffman in 1960. Their work revealed how the sodium pump stabilizes cell volume by eliminating the osmotic gradient. Here we describe the mechanistic basis of the PLM, trace the history of its discovery, and place it into the context of our current understanding.