A massive quiescent galaxy at redshift 4.658.

The extremely rapid assembly of the earliest galaxies during the first billion years of cosmic history is a major challenge for our understanding of galaxy formation physics1-5. The advent of the James Webb Space Telescope (JWST) has exacerbated this issue by confirming the existence of galaxies in substantial numbers as early as the first few hundred million years6-8. Perhaps even more surprisingly, in some galaxies, this initial highly efficient star formation rapidly shuts down, or quenches, giving rise to massive quiescent galaxies as little as 1.5 billion years after the Big Bang9,10. However, due to their faintness and red colour, it has proven extremely challenging to learn about these extreme quiescent galaxies, or to confirm whether any existed at earlier times. Here we report the spectroscopic confirmation of a massive quiescent galaxy, GS-9209, at redshift, z = 4.658, just 1.25 billion years after the Big Bang, using the JWST Near-Infrared Spectrograph (NIRSpec). From these data we infer a stellar mass of M* = 3.8 ± 0.2 × 1010 M⊙, which formed over a roughly 200 Myr period before this galaxy quenched its star-formation activity at [Formula: see text], when the Universe was approximately 800 Myr old. This galaxy is both a likely descendent of the highest-redshift submillimetre galaxies and quasars, and a likely progenitor for the dense, ancient cores of the most massive local galaxies.

Confirmation and refutation of very luminous galaxies in the early Universe.

During the first 500 million years of cosmic history, the first stars and galaxies formed, seeding the Universe with heavy elements and eventually reionizing the intergalactic medium1-3. Observations with the James Webb Space Telescope (JWST) have uncovered a surprisingly high abundance of candidates for early star-forming galaxies, with distances (redshifts, z), estimated from multiband photometry, as large as z ≈ 16, far beyond pre-JWST limits4-9. Although such photometric redshifts are generally robust, they can suffer from degeneracies and occasionally catastrophic errors. Spectroscopic measurements are required to validate these sources and to reliably quantify physical properties that can constrain galaxy formation models and cosmology10. Here we present JWST spectroscopy that confirms redshifts for two very luminous galaxies with z > 11, and also demonstrates that another candidate with suggested z ≈ 16 instead has z = 4.9, with an unusual combination of nebular line emission and dust reddening that mimics the colours expected for much more distant objects. These results reinforce evidence for the early, rapid formation of remarkably luminous galaxies while also highlighting the necessity of spectroscopic verification. The large abundance of bright, early galaxies may indicate shortcomings in current galaxy formation models or deviations from physical properties (such as the stellar initial mass function) that are generally believed to hold at later times.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Implementing prescribing safety indicators in prisons: A mixed methods study.

AIMS: To examine the prevalence of potentially hazardous prescribing in the prison setting using prescribing safety indicators (PSIs) and explore their implementation and use in practice. METHODS: PSIs were identified and reviewed by the project team following a literature review and a nominal group discussion. Pharmacists at 2 prison sites deployed the PSIs using search protocols within their electronic health record. Prevalence rates and 95% confidence intervals (CIs) were generated for each indicator. Semi-structured interviews with 20 prison healthcare staff across England and Wales were conducted to explore the feasibility of deploying and using PSIs in prison settings. RESULTS: Thirteen PSIs were successfully deployed mostly comprising drug-drug interactions (n = 9). Five yielded elevated prevalence rates: use of anticholinergics if aged ≥65 years (Site B: 25.8% [95%CI: 10.4-41.2%]), lack of antipsychotic monitoring for >12 months (Site A: 39.1% [95%CI: 27.1-52.1%]; Site B: 28.6% [95%CI: 17.9-41.4%]), prolonged use of hypnotics (Site B: 46.3% [95%CI: 35.6-57.1%]), antiplatelets prescribed with nonsteroidal anti-inflammatory drugs without gastrointestinal protection (Site A: 12.5% [95%CI: 0.0-35.4%]; Site B: 16.7% [95%CI: 0.4-64.1%]), and selective serotonin/norepinephrine reuptake inhibitors prescribed with nonsteroidal anti-inflammatory drugs/antiplatelets without gastrointestinal protection (Site A: 39.6% [95%CI: 31.2-48.4%]; Site B: 33.3% [95%CI: 20.8-47.9%]). Prison healthcare staff supported the use of PSIs and identified key considerations to guide its successful implementation, including staff engagement and PSI 'champions'. To respond to PSI searches, stakeholders suggested contextualised patient support through intraprofessional collaboration. CONCLUSION: We successfully implemented a suite of PSIs into 2 prisons, identifying those with higher prevalence values as intervention targets. When appropriately resourced and integrated into staff workflow, PSI searches may support prescribing safety in prisons.

Implications of Foraging and Interspecies Interactions of Birds for Carriage of Escherichia coli Strains Resistant to Critically Important Antimicrobials.

Globally, gulls have been associated with carriage of high levels of Escherichia coli strains resistant to critically important antimicrobials (CIAs), a major concern, as these antimicrobials are the sole alternative or one among only a few alternatives available to treat severe life-threatening infections in humans. Previous studies of Australian silver gulls demonstrated high levels of resistance to CIAs, particularly fluoroquinolone and extended-spectrum cephalosporins, among E. coli strains (carriage at 24% and 22%, respectively). This study aimed to identify and characterize strains from four distinct bird species inhabiting a common coastal environment, determine the frequency of carriage of CIA-resistant E. coli strains, and examine if these resistant clones and their resistance-encoding mobile genetic elements (MGEs) could be transmitted between species. CIA-resistant E. coli was detected in silver gulls (53%), little penguins (11%), and feral pigeons (10%), but not in bridled terns. In total, 37 different sequence types (STs) were identified, including clinically significant human-associated lineages, such as ST131, ST95, ST648, ST69, ST540, ST93, ST450, and ST10. Five main mobile genetic elements associated with blaCTX-M-positive E. coli strains isolated from three bird species were detected. Examination of clonal lineages and MGEs provided indirect evidence of transfer of resistance between bird species. The carriage of CIA-resistant E. coli by gulls and pigeons with proximity to humans, and in some instances food-producing animals, increases the likelihood of further bidirectional dissemination.IMPORTANCE It has been shown that 20% of Australian silver gulls carry drug-resistant Escherichia coli strains of anthropogenic origin associated with severe diseases, such as sepsis and urinary tract infections, in humans. To further characterize the dynamics of drug-resistant E. coli in wildlife populations, we investigated the carriage of critically important antimicrobial (CIA) drug-resistant E. coli in four bird species in a common environment. Our results indicated that gulls, pigeons, and penguins carried drug-resistant E. coli strains, and analysis of mobile genetic elements associated with resistance genes indicated interspecies resistance transfer. Terns, representing a bird species that forages on natural food sources at sea and distant from humans, did not test positive for drug-resistant E. coli This study demonstrates carriage of CIA-resistant bacteria in multiple bird species living in areas commonly inhabited by humans and provides further evidence for a leapfrog effect of resistance in wildlife, facilitated by feeding habits.

Immobilization by Surface Conjugation of Cyclic Peptides for Effective Mimicry of the HCV-Envelope E2 Protein as a Strategy toward Synthetic Vaccines.

Mimicry of the binding interface of antibody-antigen interactions using peptide-based modulators (i.e., epitope mimics) has promising applications for vaccine design. These epitope mimics can be synthesized in a streamlined and straightforward fashion, thereby allowing for high-throughput analysis. The design of epitope mimics is highly influenced by their spatial configuration and structural conformation. It is widely assumed that for proper mimicry sufficient conformational constraints have to be implemented. This paper describes the synthesis of bromide derivatives functionalized with a flexible TEG linker equipped with a thiol-moiety that could be used to support cyclic or linear peptides. The cyclic and linear epitope mimics were covalently conjugated via the free thiol-moiety on maleimide-activated plate surfaces. The resulting covalent, uniform, and oriented coated surface of cyclic or linear epitope mimics were subjected to an ELISA to investigate the effect of peptide cyclization with respect to mimicry of an antigen-antibody interaction of the HCV E2 glycoprotein. To the best of our knowledge, the benefit of cyclized peptides over linear peptides has been clearly demonstrated here for the first time. Cyclic epitope mimics, and not the linear epitope mimics, demonstrated specificity toward their monoclonal antibodies HC84.1 and V3.2, respectively. The described strategy for the construction of epitope mimics shows potential for high-throughput screening of key binding residues by simply changing the amino acid sequences within synthetic peptides. In this way, leucine-438 has been identified as a key binding residue for binding monoclonal antibody V3.2.

Role of Conserved E2 Residue W420 in Receptor Binding and Hepatitis C Virus Infection.

UNLABELLED: Hepatitis C virus (HCV) enters cells via interactions with several host factors, a key one being that between the viral E2 envelope glycoprotein and the CD81 receptor. We previously identified E2 tryptophan residue 420 (W420) as an essential CD81-binding residue. However, the importance of W420 in the context of the native virion is unknown, as those previous studies predate the infectious HCV cell culture (cell culture-derived HCV [HCVcc]) system. Here, we introduced four separate mutations (F, Y, A, or R) at position 420 within the infectious HCVcc JFH-1 genome and characterized their effects on the viral life cycle. While all mutations reduced E2-CD81 binding, only two (W420A and W420R) reduced HCVcc infectivity. Further analyses of mutants with hydrophobic residues (F or Y) found that interactions with the receptors SR-BI and CD81 were modulated, which in turn determined the viral uptake route. Both mutant viruses were significantly less dependent on SR-BI, and its lipid transfer activity, for virus entry. Furthermore, these viruses were resistant to the drug erlotinib, which targets epidermal growth factor receptor (EGFR) (a host cofactor for HCV entry) and also blocks SR-BI-dependent high-density lipoprotein (HDL)-mediated enhancement of virus entry. Together, our data indicate a model where an alteration at position 420 causes a subtle change in the E2 conformation that prevents interaction with SR-BI and increases accessibility to the CD81-binding site, in turn favoring a particular internalization route. These results further show that a hydrophobic residue with a strong preference for tryptophan at position 420 is important, both functionally and structurally, to provide an additional hydrophobic anchor to stabilize the E2-CD81 interaction. IMPORTANCE: Hepatitis C virus (HCV) is a leading cause of liver disease, causing up to 500,000 deaths annually. The first step in the viral life cycle is the entry process. This study investigates the role of a highly conserved residue, tryptophan residue 420, of the viral glycoprotein E2 in this process. We analyzed the effect of changing this residue in the virus and confirmed that this region is important for binding to the CD81 receptor. Furthermore, alteration of this residue modulated interactions with the SR-BI receptor, and changes to these key interactions were found to affect the virus internalization route involving the host cofactor EGFR. Our results also show that the nature of the amino acid at this position is important functionally and structurally to provide an anchor point to stabilize the E2-CD81 interaction.

Early star-forming galaxies and the reionization of the Universe.

Star-forming galaxies trace cosmic history. Recent observational progress with the NASA Hubble Space Telescope has led to the discovery and study of the earliest known galaxies, which correspond to a period when the Universe was only ∼800 million years old. Intense ultraviolet radiation from these early galaxies probably induced a major event in cosmic history: the reionization of intergalactic hydrogen.

Improvement in peripheral blood disease burden in patients with Sézary syndrome and leukemic mycosis fungoides after total skin electron beam therapy.

BACKGROUND: There is a paucity of effective therapies for patients with Sézary syndrome and advanced mycosis fungoides with peripheral blood involvement. Total skin electron beam (TSEB) radiation therapy is an extremely effective skin-directed therapy for these patients, but, until recently, it was thought not to signifcantly affect the peripheral blood malignant T-cell population. OBJECTIVE: We conducted this study to determine if TSEB has therapeutic effect on the peripheral blood in patients with advanced mycosis fungoides and Sézary syndrome. METHODS: All patients on stable medication regimens seen in our photopheresis facility who received TSEB therapy between January 2008 and October 2011 at Temple University Hospital, Philadelphia, PA, were analyzed retrospectively for improvement in the peripheral blood, as documented by flow cytometry. RESULTS: Six of 11 patients achieved 50% or greater decrease in their peripheral blood malignant T-cell population after TSEB therapy, for an overall response rate of 55%. Within the group of patients who had a response in the skin, 67% also had a response in the peripheral blood. LIMITATIONS: This analysis is limited in 3 ways. First, the sample described is small. Second, the results may be confounded by the fact that each patient was on other systemic therapies in addition to TSEB, albeit stable pre-existing regimens. The time interval between completion of TSEB therapy and repetition of flow cytometry was not standardized among patients, which may result in an underestimation of the overall response to TSEB therapy. CONCLUSION: In patients with advanced mycosis fungoides and Sézary syndrome, the peripheral blood tumor burden may improve after treatment with TSEB.

Immunogenicity of a Candidate Live Attenuated Vaccine for Rift Valley Fever Virus with a Two-Segmented Genome.

Rift Valley fever virus (RVFV) is an emerging arbovirus that affects both ruminants and humans. RVFV causes severe and recurrent outbreaks in Africa and the Arabian Peninsula with a significant risk for emergence into new locations. Although there are a variety of RVFV veterinary vaccines for use in endemic areas, there is currently no licensed vaccine for human use; therefore, there is a need to develop and assess new vaccines. Herein, we report a live-attenuated recombinant vaccine candidate for RVFV, based on the previously described genomic reconfiguration of the conditionally licensed MP12 vaccine. There are two general strategies used to develop live-attenuated RVFV vaccines, one being serial passage of wild-type RVFV strains to select attenuated mutants such as Smithburn, Clone 13, and MP12 vaccine strains. The second strategy has utilized reverse genetics to attenuate RVFV strains by introducing deletions or insertions within the viral genome. The novel candidate vaccine characterized in this report contains a two-segmented genome that lacks the medium viral segment (M) and two virulence genes (nonstructural small and nonstructural medium). The vaccine candidate, named r2segMP12, was evaluated for the production of neutralizing antibodies to RVFV in outbred CD-1 mice. The immune response induced by the r2segMP12 vaccine candidate was directly compared to the immune response induced by the rMP12 parental strain vaccine. Our study demonstrated that a single immunization with the r2segMP12 vaccine candidate at 105 plaque-forming units elicited a higher neutralizing antibody response than the rMP12 vaccine at the same vaccination titer without the need for a booster.

Comparison of Immunogenicity Between a Candidate Live Attenuated Vaccine and an Inactivated Vaccine for Cache Valley Virus.

Cache Valley virus (CVV) is a mosquito-borne bunyavirus that is enzootic throughout the new world. Although CVV is known as an important agricultural pathogen, primarily associated with embryonic lethality and abortions in ruminants, it has recently been recognized for its expansion as a zoonotic pathogen. With the increased emergence of bunyaviruses with human and veterinary importance, there have been significant efforts dedicated to the development of bunyavirus vaccines. In this study, the immunogenicity of a candidate live-attenuated vaccine (LAV) for CVV, which contains the deletion of the nonstructural small (NSs) and nonstructural medium (NSm) genes (2delCVV), was evaluated and compared with an autogenous candidate vaccine created through the inactivation of CVV using binary ethylenimine (BEI) with an aluminum hydroxide adjuvant (BEI-CVV) in sheep. Both 2delCVV and BEI-CVV produced a neutralizing antibody response that exceeds the correlate of protection, that is, plaque reduction neutralization test titer >10. However, on day 63 postinitial immunization, 2delCVV was more immunogenic than BEI-CVV. These results warrant further development of 2delCVV as a candidate LAV and demonstrate that the double deletion of the NSs and NSm genes can be applied to the development of vaccines and as a common attenuation strategy for orthobunyaviruses.

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping.

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.

The Neutralizing Antibody Responses of Individuals That Spontaneously Resolve Hepatitis C Virus Infection.

Hepatitis C virus (HCV) infection is a major global health problem. In the majority of cases the virus is not cleared by the host immune response and progresses to chronic infection. Studies of the neutralizing antibody responses in individuals that naturally clear infection are limited. Understanding what constitutes a successful antibody response versus one that has 'failed' and resulted in chronic infection is important to understand what type of antibody response would need to be elicited by a protective vaccine. Samples from spontaneous clearers are difficult to obtain therefore studies are often limited. In our study through HCV Research UK, we had access to a cohort of over 200 samples. We identified the samples that contained HCV neutralizing antibodies using ELISA and HCV pseudoparticle (HCVpp) assays. We then utilised mutagenesis and cross-competition analysis to determine the profile of the neutralizing antibody responses. In addition, we analysed a cohort of samples from chronic infection using the same techniques to enable direct comparison of the antibody profiles observed in both cohorts. We conclude that similar profiles are present in both cohorts indicating that it is not the neutralizing antibody response per se that determines the outcome of infection. These data will provide useful information for future HCV vaccine design.

Exploring the challenges to safer prescribing and medication monitoring in prisons: A qualitative study with health care staff.

INTRODUCTION: Research suggests that patients who are prisoners experience greater morbidity, increased health inequalities and frequent preventable harm, compared to the general population. Little is known about the process and influencing factors for safe prescribing in the unique prison environment, which may limit the development efforts to improve the quality of care in prisons. This study aimed to understand the process and challenges associated with prescribing in prisons, explore the causes and impact of these challenges, and explore approaches to improve prescribing safety in prisons. METHODS: Grounded theory informed data collection and analysis of a nominal group discussion by seven participants and semi-structured telephone interviews with twenty prison healthcare staff, including GPs, pharmacists, psychiatrists and nurses. FINDINGS: The underlying complexity of prescribing in prison settings increased the level of challenge and influenced the safety of this process. Multiple contributors to the challenges of safe prescribing were identified (comprising governance and policy; the prison structure; staff retention, training and skill mix; IT systems and interface; polypharmacy and co-morbidity; tradability and patient behaviour) with overarching constructs of variations in practice/policy and the influence of prison culture. Participants identified measures to address these challenges through multi-disciplinary collaborative working, increased consistency in processes, and the need for more innovation and education/training. CONCLUSIONS: Our study highlighted that healthcare provision in prisons is unique and needs to tailor the care provided to patients without enforcing a model focused on primary, secondary or tertiary care. Participants emphasised a necessary shift in workplace culture and behaviour change to support improvements. The COM-B model of behaviour change may be effectively applied to develop interventions in organisations that have in-depth understanding of their own unique challenges.

Replication Kinetics of a Candidate Live-Attenuated Vaccine for Cache Valley Virus in Aedes albopictus.

Background: The emergence or re-emergence of several orthobunyaviruses (order: Bunyavirales; family: Peribunyaviridae), including Cache Valley virus (CVV) and Oropouche virus, warrants the development and evaluation of candidate live-attenuated vaccines (LAVs). Ideally, these vaccines would elicit long-lasting immunity with one single immunization. Materials and Methods: Since the deletion of two virulence factors, NSs and NSm, has been shown to attenuate the virulence phenotype of orthobunyaviruses, phleboviruses, and nairoviruses, genetic manipulation of the viral genome is considered an effective strategy for the rational design of candidate LAVs for bunyaviruses across multiple families. In addition, the deletion of Rift Valley fever virus NSs and NSm genes has been shown to reduce transmission by mosquitoes. Results: In this study, the ability of a CVV mutant lacking the NSs and NSm genes (2delCVV) to replicate in intrathoracically injected Aedes albopictus was compared with the parental wild-type CVV (wtCVV) 6V633 strain. In contrast to the robust replication of wtCVV in injected mosquitoes, the multiplication kinetics of the 2delCVV mutant was reduced by more than a 100-fold. Conclusion: These results suggest that the deletion of NSm and NSs genes is a feasible approach to rationally design candidate orthobunyavirus LAVs that are highly attenuated in mosquitoes and, therefore, pose little risk of reversion to virulence and transmission.

Exploration of immunological responses underpinning severe fever with thrombocytopenia syndrome virus infection reveals IL-6 as a therapeutic target in an immunocompromised mouse model.

Dabie bandavirus (previously severe fever with thrombocytopenia syndrome virus; SFTSV), is an emerging tick-borne bunyavirus responsible for severe fever with thrombocytopenia syndrome (SFTS), a disease with high case fatality that is characterized by high fever, thrombocytopenia, and potentially lethal hemorrhagic manifestations. Currently, neither effective therapeutic strategies nor approved vaccines exist for SFTS. Therefore, there remains a pressing need to better understand the pathogenesis of the disease and to identify therapeutic strategies to ameliorate SFTS outcomes. Using a type I interferon (IFN)-deficient mouse model, we investigated the viral tropism, disease kinetics, and the role of the virulence factor nonstructural protein (NSs) in SFTS. Ly6C+ MHCII+ cells in the lymphatic tissues were identified as an important target cell for SFTSV. Advanced SFTS was characterized by significant migration of inflammatory leukocytes, notably neutrophils, into the lymph node and spleen, however, these cells were not required to orchestrate the disease phenotype. The development of SFTS was associated with significant upregulation of proinflammatory cytokines, including high levels of IFN-γ and IL-6 in the serum, lymph node, and spleen. Humoral immunity generated by inoculation with delNSs SFTSV was 100% protective. Importantly, NSs was critical to the inhibition of the host IFNɣ response or downstream IFN-stimulated gene production and allowed for the establishment of severe disease. Finally, therapeutic but not prophylactic use of anti-IL-6 antibodies significantly increased the survival of mice following SFTSV infection and, therefore, this treatment modality presents a novel therapeutic strategy for treating severe SFTS.

Using foresight to prepare animal health today for tomorrow's challenges.

Foresight has been introduced in Canada in the area of animal health as a process to broaden thinking about the future and inform policy development. Its initial use and evolution through projects and studies over the past decade are described, demonstrating real action in animal health. Despite positive, continuing, and exciting results in animal health policy development, foresight's widespread acceptance and use thus far have been limited. Critical components for success, drawn from the Canadian experience, are described, and recommendations are offered for further action in animal health.

The star-formation history of the Universe from the stellar populations of nearby galaxies.

The determination of the star-formation history of the Universe is a key goal of modern cosmology, as it is crucial to our understanding of how galactic structures form and evolve. Observations of young stars in distant galaxies at different times in the past have indicated that the stellar birthrate peaked some eight billion years ago before declining by a factor of around ten to its present value. Here we report an analysis of the 'fossil record' of the current stellar populations of 96,545 nearby galaxies, from which we obtained a complete star-formation history. Our results broadly support those derived from high-redshift galaxies. We find, however, that the peak of star formation was more recent--around five billion years ago. We also show that the bigger the stellar mass of the galaxy, the earlier the stars were formed, which indicates that high- and low-mass galaxies have very different histories.

A cytoplasmic region determines single-channel conductance in 5-HT3 receptors.

5-hydroxytryptamine type 3 (5-HT3) receptors are cation-selective transmitter-gated ion channels of the Cys-loop superfamily. The single-channel conductance of human recombinant 5-HT3 receptors assembled as homomers of 5-HT3A subunits, or heteromers of 5-HT3A and 5-HT3B subunits, are markedly different, being 0.4 pS (refs 6, 9) and 16 pS (ref. 7), respectively. Paradoxically, the channel-lining M2 domain of the 5-HT3A subunit would be predicted to promote cation conduction, whereas that of the 5-HT3B subunit would not. Here we describe a determinant of single-channel conductance that can explain these observations. By constructing chimaeric 5-HT3A and 5-HT3B subunits we identified a region (the 'HA-stretch') within the large cytoplasmic loop of the receptor that markedly influences channel conductance. Replacement of three arginine residues unique to the HA-stretch of the 5-HT3A subunit by their 5-HT3B subunit counterparts increased single-channel conductance 28-fold. Significantly, ultrastructural studies of the Torpedo nicotinic acetylcholine receptor indicate that the key residues might frame narrow openings that contribute to the permeation pathway. Our findings solve the conundrum of the anomalously low conductance of homomeric 5-HT3A receptors and indicate an important function for the HA-stretch in Cys-loop transmitter-gated ion channels.