Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

Insights into breast health issues in women's rugby.

World RugbyTM supports dedicated women's welfare, injury surveillance and medical/technical interventions, yet breast health has received limited attention. This article aims to provide insights into breast health issues in rugby, including breast impacts and injuries. We discuss how breast anatomy and position may be problematic in rugby. Breast volume relates to body size, which may be increasing in women's rugby, suggesting increased breast surface area and mass, potentially increasing injury risk. Breast health issues in rugby have been reported previously, with 58% of contact footballers (including rugby) experiencing breast injuries. There are damaging effects related to these breast health issues, with breast impacts often causing pain and swelling. Breast impacts may lead to haematomas, cysts and fat necrosis which can calcify over time making them difficult to distinguish from breast carcinoma, causing further investigation and anxiety. In sport, poor bra fit and insufficient support are associated with pain, skin strain and performance decrements. This article reports the potential implications of these breast health issues on performance in rugby. Recent breast-related projects supported by rugby communities may address recommendations identified in the literature for robust breast injury classifications, updated injury surveillance systems and prospective data collection on breast injury prevalence, severity and impact in rugby. These data should inform breast injury care pathways and intervention research, including evidence-based bra design. Understanding the implications of breast impacts on tissue properties, health and wellbeing is vital. Finally, data should inform rugby-specific breast education, raising awareness of this aspect of athlete health.

Protein Binder Toolbox for Studies of Solute Carrier Transporters.

Transporters of the solute carrier superfamily (SLCs) are responsible for the transmembrane traffic of the majority of chemical substances in cells and tissues and are therefore of fundamental biological importance. As is often the case with membrane proteins that can be heavily glycosylated, a lack of reliable high-affinity binders hinders their functional analysis. Purifying and reconstituting transmembrane proteins in their lipidic environments remains challenging and standard approaches to generate binders for multi-transmembrane proteins, such as SLCs, channels or G protein-coupled receptors (GPCRs) are lacking. While generating protein binders to 27 SLCs, we produced full length protein or cell lines as input material for binder generation by selected binder generation platforms. As a result, we obtained 525 binders for 22 SLCs. We validated the binders with a cell-based validation workflow using immunofluorescent and immunoprecipitation methods to process all obtained binders. Finally, we demonstrated the potential applications of the binders that passed our validation pipeline in structural, biochemical, and biological applications using the exemplary protein SLC12A6, an ion transporter relevant in human disease. With this work, we were able to generate easily renewable and highly specific binders against SLCs, which will greatly facilitate the study of this neglected protein family. We hope that the process will serve as blueprint for the generation of binders against the entire superfamily of SLC transporters.

Structural basis for inhibition of the lysosomal two-pore channel TPC2 by a small molecule antagonist.

Two pore channels are lysosomal cation channels with crucial roles in tumor angiogenesis and viral release from endosomes. Inhibition of the two-pore channel 2 (TPC2) has emerged as potential therapeutic strategy for the treatment of cancers and viral infections, including Ebola and COVID-19. Here, we demonstrate that antagonist SG-094, a synthetic analog of the Chinese alkaloid medicine tetrandrine with increased potency and reduced toxicity, induces asymmetrical structural changes leading to a single binding pocket at only one intersubunit interface within the asymmetrical dimer. Supported by functional characterization of mutants by Ca2+ imaging and patch clamp experiments, we identify key residues in S1 and S4 involved in compound binding to the voltage sensing domain II. SG-094 arrests IIS4 in a downward shifted state which prevents pore opening via the IIS4/S5 linker, hence resembling gating modifiers of canonical VGICs. These findings may guide the rational development of new therapeutics antagonizing TPC2 activity.

Rational design of a cyclohexanone dehydrogenase for enhanced α,ß-desaturation and substrate specificity.

The selective α,ß-desaturation of cyclic carbonyl compounds, which are found in the core of many steroid and bioactive molecules, using green chemistry is highly desirable. To achieve this task, we have for the first time described and solved the de novo structure of a member of the cyclohexanone dehydrogenase class of enzymes. The breadth of substrate specificity was investigated by assaying the cyclohexanone dehydrogenase, from Alicycliphilus denitrificans, against several cyclic ketones, lactones and lactams. To investigate substrate binding, a catalytic variant, Y195F, was generated and used to obtain a crystallographic complex with the natural substrate, cyclohexanone. This revealed substrate-active site interactions, as well as the proximity of the cofactor, flavin adenine dinucleotide, and enabled us to propose a mechanistic function to key amino acids. We then used molecular dynamic simulations to guide design to add functionality to the cyclohexanone dehydrogenase enzyme. The resulting W113A variant had overall improved enzyme activity and substrate scope, i.e., accepting the bulkier carbonyl compound, dihydrocoumarin. Structural analysis of the W113A variant revealed a broader, more open active site, which helped explain the modified substrate specificity. This work paves the way for future bespoke regioselective α,ß-desaturation in the synthesis of important bioactive molecules via rational enzyme engineering.

Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate.

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1-3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1-3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies.

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

Structural characterization of human urea transporters UT-A and UT-B and their inhibition.

In this study, we present the structures of human urea transporters UT-A and UT-B to characterize them at molecular level and to detail the mechanism of UT-B inhibition by its selective inhibitor, UTBinh-14. High-resolution structures of both transporters establish the structural basis for the inhibitor's selectivity to UT-B, and the identification of multiple binding sites for the inhibitor will aid with the development of drug lead molecules targeting both transporters. Our study also discovers phospholipids associating with the urea transporters by combining structural observations, native MS, and lipidomics analysis. These insights improve our understanding of urea transporter function at a molecular level and provide a blueprint for a structure-guided design of therapeutics targeting these transporters.

Predicting phosphorus accumulation and proposing conditions needed for an algal-based phosphorus uptake process.

Algal-based waste stabilisation ponds (WSP) are a common wastewater treatment system for small communities but have poor phosphorus removal. Under certain conditions algae in WSPs will perform 'luxury uptake' increasing their phosphorus content to over 3% (gP/gSS) by storing polyphosphate. For the first time in the literature this paper presents a systematic study which determines the conditions needed to maximise phosphorus accumulation within WSP biomass taking into account the interactions between key variables. The key variables of temperature, phosphorus concentration, light intensity, mixing intensity, organic load, and pH were evaluated in 40 batch factorial experiments using a WSP algal culture. All six variables examined had significant main effects or interactions on the phosphorus content of the biomass. These were incorporated into a regression equation which was successfully validated against independent data sets from the literature. The conditions required to maximise the phosphorus content of the biomass were predicted for both summer (high light and high temperature) and winter (low light and low temperature) scenarios. The required conditions were revealed to be high phosphorus concentration, high mixing intensity, no supplementary CO2 addition, and low organic load. Interestingly, these conditions were consistent for both summer and winter suggesting that year-round treatment is possible. Practical methods of achieving these conditions were proposed. While further work will be needed to evaluate the effect of growth and potential influence of algal species, the findings presented provide a vital step towards developing a new phosphorus removal treatment process based on an enhanced understanding of environmental biotechnology.

Biocatalytic and Chemo-Enzymatic Synthesis of Quinolines and 2-Quinolones by Monoamine Oxidase (MAO-N) and Horseradish Peroxidase (HRP) Biocatalysts.

The oxidative aromatization of aliphatic N-heterocycles is a fundamental organic transformation for the preparation of a diverse array of heteroaromatic compounds. Despite many attempts to improve the efficiency and practicality of this transformation, most synthetic methodologies still require toxic and expensive reagents as well as harsh conditions. Herein, we describe two enzymatic strategies for the oxidation of 1,2,3,4-tetrahydroquinolines (THQs) and N-cyclopropyl-N-alkylanilines into quinolines and 2-quinolones, respectively. Whole cells and purified monoamine oxidase (MAO-N) enzymes were used to effectively catalyze the biotransformation of THQs into the corresponding aromatic quinoline derivatives, while N-cyclopropyl-N-alkylanilines were converted into 2-quinolone compounds through a horseradish peroxidase (HRP)-catalyzed annulation/aromatization reaction followed by Fe-mediated oxidation.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

A cross-sectional retrospective survey of injury situation and prevalence in female recreational netball players with a focus on knee injuries.

OBJECTIVES: To examine situations of injury and injury prevalence in female adult recreational netball players with a focus on knee injuries. DESIGN: Cross sectional retrospective online survey. PARTICIPANTS: 193 female adult recreational netball players. MAIN OUTCOME MEASURES: Any injury sustained in the previous 12 months, situation of injury, any knee injuries sustained in the previous five years, the length of time unable to play netball, and knee injury management. RESULTS: In the previous 12 months, 61% of respondents sustained injury to the lower limb, and 27% to the upper limb. Lower limb injury situations were mostly landings (46%). Upper limb injury situations were mostly collisions with an opponent (27%). 46% reported sustaining a knee injury in the previous five years. Following knee injury, players were unable to play netball for 6.8 ± 7.0 months (training); and 8.2 ± 7.4 months (matches) respectively. CONCLUSIONS: Lower limb injury is more common than upper limb injury in recreational adult female adult netball players. Landing was the most common situation of injury for the lower limb including knee injuries. In the previous five years, nearly half of the players had sustained a knee injury resulting in more than six months out of the game.

Evaluation of bioelectrical impedance analysis in measuring body fat in 6-to-12-year-old boys compared with air displacement plethysmography.

Air displacement plethysmography (ADP) has been considered as the 'standard' method to determine body fat in children due to superior validity and reliability compared with bioelectrical impedance analysis (BIA). However, ADP and BIA are often used interchangeably despite few studies comparing measures of percentage body fat by ADP (%FMADP) with BIA (%FMBIA) in children with and without obesity. The objective of this study was to measure concurrent validity and reliability of %FMADP and %FMBIA in 6-to-12-year-old boys with and without obesity. Seventy-one boys (twenty-five with obesity) underwent body composition assessment. Ten boys participated in intra-day reliability analysis. %FMADP was estimated by Bodpod using sex- and age-specific equations of body density. %FMBIA was estimated by a multi-frequency, hand-to-foot device using child-specific equations based on impedance. Validity was assessed by t tests, correlation coefficients and limits of agreement (LoA); and reliability by technical error of measurement (TEM) and intraclass correlation coefficients (ICC). Compared with %FMADP, %FMBIA was significantly underestimated in the cohort (-3·4 ± 5·6 %; effect size = 0·42) and in both boys with obesity (-5·2 ± 5·5 %; ES = 0·90) and without obesity (-2·4 ± 5·5 %; ES = 0·52). A strong, significant positive correlation was found between %FMADP and %FMBIA (r = 0·80). Across the cohort, LoA were 22·3 %, and no proportional bias was detected. For reliability, TEM were 0·65 % and 0·55 %, and ICC were 0·93 and 0·95 for %FMBIA and %FMADP, respectively. Whilst both %FMADP and %FMBIA are highly reliable methods, considerable differences indicated that the devices cannot be used interchangeably in boys age 6-to-12 years.

Quality of Life in Post-Surgical Hypoparathyroidism (PoSH) in Thyroid and Parathyroid Surgery.

BACKGROUND: Post-surgical hypoparathyroidism (PoSH) is often long term, with significant associated morbidity and ongoing treatment. A recent systematic review found impaired quality of life (QoL) in patients with PoSH, despite stable treatment. Most studies did not include an appropriate control arm and further studies were recommended, taking into account underlying disease and comorbidities. This study aims to compare QoL in patients with PoSH with appropriate control groups. METHODS: This was a cross-sectional observational study using the general quality of life SF-36 tool and a hypocalcaemia symptom score (HcSS) to assess QoL in patients with PoSH and controls (who had similar surgery but without PoSH). Participants were identified from two patient groups (the Butterfly Thyroid Cancer Trust and the Association for Multiple Endocrine Neoplasia Disorders) and a single tertiary centre in the UK. RESULTS: Four hundred and thirty-nine responses (female n = 379, PoSH n = 89) were included with a median (range) age of 52 (19-92) years. Reported dates of surgery ranged from 1973 to 2019. HcSS scores showed significantly more associated symptoms in patients with PoSH than those without (p < 0.001). Although there was no overall difference in QoL between groups, patients with PoSH consistently had lower scores (p = 0.008) in the energy/fatigue subdomain of the SF-36. CONCLUSION: Patients with PoSH reported significantly more fatigue and loss of energy compared to appropriately matched controls, but overall QoL was not significantly different. Standardised QoL measures may not be sensitive enough to highlight the impact on QoL in these patients. A disease-specific tool may be required.

Can we predict the neutral breast position using the gravity-loaded breast position, age, anthropometrics and breast composition data?

BACKGROUND: This study aimed to identify the predictor variables which account for neutral breast position variance using a full independent variable dataset (the gravity-loaded breast position, age and anthropometrics, and magnetic resonance imaging breast composition data), and a simplified independent variable dataset (magnetic resonance imaging breast composition data excluded). METHODS: Breast position (three-dimensional neutral and static gravity-loaded), age, anthropometrics and magnetic resonance imaging breast composition data were collected for 80 females (bra size 32A to 38D). Correlations between the neutral breast position and the gravity-loaded breast position, age, anthropometrics, and magnetic resonance imaging breast composition data were assessed. Multiple linear and multivariate multiple regression models were utilised to predict neutral breast positions, with mean absolute differences and root mean square error comparing observed and predicted neutral breast positions. FINDINGS: Breast volume was the only breast composition variable to contribute as a predictor of the neutral breast position. While ≥69% of the variance in the anteroposterior and mediolateral neutral breast positions were accounted for utilising the gravity-loaded breast position, multivariate multiple regression modelling resulted in mean absolute differences >5 mm. INTERPRETATION: Due to the marginal contribution of breast composition data, a full independent variable dataset may be unnecessary for this application. Additionally, the gravity-loaded breast position, age, anthropometrics, and breast composition data do not successfully predict the neutral breast position. Incorporation of the neutral breast position into breast support garments may enhance bra development. However, further identification of variables which predict the neutral breast position is required.

Cryo-EM structure of the human Kv3.1 channel reveals gating control by the cytoplasmic T1 domain.

Kv3 channels have distinctive gating kinetics tailored for rapid repolarization in fast-spiking neurons. Malfunction of this process due to genetic variants in the KCNC1 gene causes severe epileptic disorders, yet the structural determinants for the unusual gating properties remain elusive. Here, we present cryo-electron microscopy structures of the human Kv3.1a channel, revealing a unique arrangement of the cytoplasmic tetramerization domain T1 which facilitates interactions with C-terminal axonal targeting motif and key components of the gating machinery. Additional interactions between S1/S2 linker and turret domain strengthen the interface between voltage sensor and pore domain. Supported by molecular dynamics simulations, electrophysiological and mutational analyses, we identify several residues in the S4/S5 linker which influence the gating kinetics and an electrostatic interaction between acidic residues in α6 of T1 and R449 in the pore-flanking S6T helices. These findings provide insights into gating control and disease mechanisms and may guide strategies for the design of pharmaceutical drugs targeting Kv3 channels.

Changes in dynamics of tumor/endothelial cell adhesive interactions depending on endothelial cell growth state and elastic properties.

Cancer cell adhesion to the endothelium is a crucial process in hematogenous metastasis, but how the integrity of the endothelial barrier and endothelial cell (EC) mechanical properties influence the adhesion between metastatic cancer cells and the endothelium remain unclear. In the present study, we have measured the adhesion between single cancer cells and two types of ECs at various growth states and their mechanical properties (elasticity) using atomic force microscopy single cell force spectroscopy. We demonstrated that the EC stiffness increased and adhesion with cancer cells decreased, as ECs grew from a single cell to a confluent state and developed cell-cell contacts, but this was reversed when confluent cells returned to a single state in a scratch assay. Our results suggest that the integrity of the endothelial barrier is an important factor in reducing the ability of the metastatic tumor cells to adhere to the vascular endothelium, extravasate and lodge in the vasculature of a distant organ where secondary metastatic tumors would develop.

FAS2FURIOUS: Moderate-Throughput Secreted Expression of Difficult Recombinant Proteins in Drosophila S2 Cells.

Recombinant protein expression in eukaryotic insect cells is a powerful approach for producing challenging targets. However, due to incompatibility with standard baculoviral platforms and existing low-throughput methodology, the use of the Drosophila melanogaster "S2" cell line lags behind more common insect cell lines such as Sf9 or High-Five™. Due to the advantages of S2 cells, particularly for secreted and secretable proteins, the lack of a simple and parallelizable S2-based platform represents a bottleneck, particularly for biochemical and biophysical laboratories. Therefore, we developed FAS2FURIOUS, a simple and rapid S2 expression pipeline built upon an existing low-throughput commercial platform. FAS2FURIOUS is comparable in effort to simple E. coli systems and allows users to clone and test up to 46 constructs in just 2 weeks. Given the ability of S2 cells to express challenging targets, including receptor ectodomains, secreted glycoproteins, and viral antigens, FAS2FURIOUS represents an attractive orthogonal approach for protein expression in eukaryotic cells.

Atomic force microscopy reveals the mechanical properties of breast cancer bone metastases.

Mechanically dependent processes are essential in cancer metastases. However, reliable mechanical characterization of metastatic cancer remains challenging whilst maintaining the tissue complexity and an intact sample. Using atomic force microscopy, we quantified the micro-mechanical properties of relatively intact metastatic breast tumours and their surrounding bone microenvironment isolated from mice, and compared with other breast cancer models both ex vivo and in vitro. A mechanical distribution of extremely low elastic modulus and viscosity was identified on metastatic tumours, which were significantly more compliant than both 2D in vitro cultured cancer cells and subcutaneous tumour explants. The presence of mechanically distinct metastatic tumour did not result in alterations of the mechanical properties of the surrounding microenvironment at meso-scale distances (>200 µm). These findings demonstrate the utility of atomic force microscopy in studies of complex tissues and provide new insights into the mechanical properties of cancer metastases in bone.