Sensitive detection of SARS-CoV-2 main protease 3CLpro with an engineered ribonuclease zymogen.

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID-19 pandemic caused by the etiological agent SARS-CoV-2. All common assays for infection rely on the detection of viral sub-components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID-19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false-negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS-CoV-2 main protease, 3CLpro . Zymogen activation by 3CLpro leads to a >300-fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen-detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications.

Assessing knowledge gaps and empowering Extension workers in Illinois with information on ticks and tickborne diseases through KAP surveys.

Tickborne diseases (TBDs) are increasingly prevalent in Illinois and the Upper Midwest region. People who work in occupations that require time outdoors in agricultural or natural settings, such as some Extension workers, are at risk of tick bites and TBDs. Additionally, Extension workers are often a primary source of information about ticks and TBDs in rural communities. However, there is limited information on the level of awareness about ticks and TBDs in the Extension community. The goals of this study were to sequentially i) determine the baseline awareness of Extension workers in Illinois about ticks and TBDs using a knowledge, attitudes, and practices (KAP) survey tool, ii) provide comprehensive training on ticks and TBDs to this demographic, and iii) measure the uptake of knowledge after the training intervention through a post-training survey. The study period was from June 2022 until May 2023. We received 233 pre-training and 93 paired post-training survey responses. Most survey respondents were Extension volunteers, identified as women, and were over 50 years old. Knowledge about ticks and TBDs varied. We identified several gaps in their current tick awareness, most importantly, in tick prevention measures, tick identification, and TBDs in general. TBD knowledge, attitude, and practice scores all significantly improved after training (p < 0.001), with a mean difference of 10.47, 1.49, and 2.64 points, respectively. Additionally, both Extension professionals (79.2 %) and Extension volunteers (66.7 %) were more likely to feel confident in engaging with their stakeholders on ticks and TBDs after participating in training. Poisson models revealed that higher attitude and practice scores and greater self-reported knowledge were the factors most significantly associated with higher TBD knowledge. We found that greater concern for ticks and TBD (attitudes) and adherence to science-based prevention and management methods (practices) were also associated with higher knowledge scores. To our knowledge, this is the first study in Illinois to capture Extension workers' awareness of ticks and TBDs. The results highlight Extension workers' interest in filling knowledge gaps through learning, and the importance of training Extension workers to disseminate reliable and updated information on ticks and TBDs to their constituents, a critical step in preventing TBDs.

Elucidating the Varying Impact of Necrotic Enteritis Using Performance and Health Indicators in Broiler Infection Models.

Necrotic enteritis (NE) continues to be a significant burden to the poultry industry, compounded by pressure to reduce antibiotic use. Researchers use NE-challenge models to study the infection biology of NE and as screening tools to develop potential novel interventions. Currently, data are limited comparing such models between research establishments, and few indicate which quantitative metrics provide the most accurate measure for determining the efficacy of interventions. We compared data from 10 independent NE-challenge trials incorporating six challenge models employed in four geographical regions to determine the extent of variability in bird responses and to determine, using principal component analysis (PCA), which variables discriminated most effectively between nonchallenged control (NC) and challenged control (CC) groups. Response variables related to growth performance (weight gain, feed intake, feed conversion), health (mortality, lesion scores, NE induction rate), and, in three trials only, gut integrity (tight junction protein claudin-1, claudin-2, and zonula occludens-1 expression, coccidia counts, and intestinal permeability [assessed by FITC-dextran assay]). Treatments included a CC, which varied between trials (for example, in Eimeria predisposition, Clostridium perfringens strain, and days of inoculation), and a NC. The degree of response to challenge in CC birds varied significantly among models and trials. In all trials, lesion scores 1 to 4 days postchallenge were increased in CC vs. NC birds and varied both within and among models (by 0.29-1.17 points and 0.05-2.50 points, respectively). In addition, NE-related mortality at day 28 was increased in CC vs. NC, both within and among models (by 1.79%-4.72% and 0.02%-16.70%, respectively), and final (day 35 or 42) body weight was reduced by 3.9%-14.4% and overall FCR increased by up to 27% across trials (P , 0.05). A PCA on the combined dataset including only performance indicators failed to adequately differentiate NC and CC groups. However, the combination of performance and gut integrity variables and standardization of data by trial and phase achieved greater resolution between groups. This indicated that the inclusion of both types of variables in future NE-challenge studies would enable the generation of more robust predictions about intervention efficacy from different types of infection models. A final PCA based on a subset of key indicator variables, including body weight, feed intake, feed conversion ratio, mortality, and lesion score, achieved a good level of separation between NC and CC status of birds and could, with further research, be a useful supplement to existing approaches for assessing and predicting the NE status of birds in the field.

Determinación del impacto variable de la enteritis necrótica mediante indicadores de rendimiento y salud en modelos de infección de pollos de engorde. La enteritis necrótica (EN) sigue siendo un problema importante para la industria avícola, que se ha agudizado por la presión para reducir el uso de antibióticos. Los investigadores utilizan modelos de desafío de enteritis necrótica para estudiar la biología de la infección de este problema y como herramientas de detección para desarrollar posibles intervenciones novedosas. Actualmente, los datos que comparan dichos modelos entre grupos de investigación son limitados y pocos indican qué métricas cuantitativas proporcionan la medida más precisa para determinar la eficacia de las intervenciones. Comparamos datos de 10 ensayos independientes de desafío para enteritis necrótica que incorporan seis modelos de desafío empleados en cuatro regiones geográficas para determinar el grado de variabilidad en las respuestas de las aves y determinar, utilizando el análisis de componentes principales (PCA), qué variables discriminaron más efectivamente entre el control no desafiado (NC) y grupos de control desafiados (CC). Variables de respuesta relacionadas con el rendimiento del crecimiento (aumento de peso, consumo de alimento, conversión alimenticia), salud (mortalidad, puntuaciones de lesiones, tasa de inducción de enteritis necrótica) y, en sólo tres ensayos, la integridad intestinal (proteína de unión estrecha claudina-1, claudina-2, y expresión de zonula occludens-1, recuentos de coccidias y permeabilidad intestinal [evaluada mediante ensayo FITC-dextrano]). Los tratamientos incluyeron un control desafiado, que fue variable entre los ensayos (por ejemplo, en la predisposición a Eimeria, la cepa de Clostridium perfringens y los días de inoculación) y un control no desafiado. El grado de respuesta al desafío en aves del grupo control desafiado varió significativamente entre modelos y ensayos. En todos los ensayos, las puntuaciones de lesiones de 1 a 4 días después del desafío aumentaron en las aves del grupo control desafiado en comparación con el control no desafiado y variaron tanto dentro como entre los modelos (entre 0.29 y 1.17 puntos y entre 0.05 y 2.50 puntos, respectivamente). Además, la mortalidad relacionada con enteritis necrótica en el día 28 aumentó en el control desafiado en comparación con el control no desafiado, tanto dentro como entre modelos (entre un 1.79% y un 4.72% y entre un 0.02% y un 16.70%, respectivamente), y el peso corporal final (día 35 o 42). se redujo entre un 3.9% y un 14.4% y la conversión alimenticia en general aumentó hasta un 27% en todos los ensayos (P ,0.05). El análisis de componentes principales sobre el conjunto de datos combinado que incluye solo indicadores de desempeño no logró diferenciar adecuadamente los grupos control no desafiado y desafiado. Sin embargo, la combinación de variables de rendimiento e integridad intestinal y la estandarización de los datos por ensayo y fase lograron una mayor resolución entre los grupos. Esto indicó que la inclusión de ambos tipos de variables en futuros estudios de desafío para enteritis necrótica permitiría la generación de predicciones más sólidas sobre la eficacia de la intervención a partir de diferentes tipos de modelos de infección. Un análisis de componentes principales final basado en un subconjunto de variables indicadoras clave, incluido el peso corporal, el consumo de alimento, el índice de conversión alimenticia, la mortalidad y la puntuación de las lesiones, logró un buen nivel de separación entre el estado de las aves no desafiadas y desafiadas y podría, con más investigación, ser un complemento útil a los enfoques existentes para evaluar y predecir el estado de enteritis necrótica de las aves en el campo.

A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Practical considerations of diffusion-weighted MRS with ultra-strong diffusion gradients.

Introduction: Diffusion-weighted magnetic resonance spectroscopy (DW-MRS) offers improved cellular specificity to microstructure-compared to water-based methods alone-but spatial resolution and SNR is severely reduced and slow-diffusing metabolites necessitate higher b-values to accurately characterize their diffusion properties. Ultra-strong gradients allow access to higher b-values per-unit time, higher SNR for a given b-value, and shorter diffusion times, but introduce additional challenges such as eddy-current artefacts, gradient non-uniformity, and mechanical vibrations. Methods: In this work, we present initial DW-MRS data acquired on a 3T Siemens Connectom scanner equipped with ultra-strong (300 mT/m) gradients. We explore the practical issues associated with this manner of acquisition, the steps that may be taken to mitigate their impact on the data, and the potential benefits of ultra-strong gradients for DW-MRS. An in-house DW-PRESS sequence and data processing pipeline were developed to mitigate the impact of these confounds. The interaction of TE, b-value, and maximum gradient amplitude was investigated using simulations and pilot data, whereby maximum gradient amplitude was restricted. Furthermore, two DW-MRS voxels in grey and white matter were acquired using ultra-strong gradients and high b-values. Results: Simulations suggest T2-based SNR gains that are experimentally confirmed. Ultra-strong gradient acquisitions exhibit similar artefact profiles to those of lower gradient amplitude, suggesting adequate performance of artefact mitigation strategies. Gradient field non-uniformity influenced ADC estimates by up to 4% when left uncorrected. ADC and Kurtosis estimates for tNAA, tCho, and tCr align with previously published literature. Discussion: In conclusion, we successfully implemented acquisition and data processing strategies for ultra-strong gradient DW-MRS and results indicate that confounding effects of the strong gradient system can be ameliorated, while achieving shorter diffusion times and improved metabolite SNR.

Religion, Accountability, and the Risk of Aggressive Misconduct Among Prisoners: Preliminary Evidence of Restorative Rehabilitation.

Prior research has found inverse relationship between religious involvement and misconduct among prisoners. In this study, we examined the mediating role of accountability as welcoming responsibility for one's actions with respect to the transcendent (e.g., God or a higher power) and other people. We applied structural equation modeling to analyze data from surveys with 339 individuals (108 females and 231 males) incarcerated at three facilities in Texas. We found that prisoner religiosity was positively related to transcendent accountability, which was in turn positively associated with human accountability. Human accountability was then positively associated with empathy and negatively associated with impulsivity. Via impulsivity, human accountability was indirectly and inversely related to anger, depression, and anxiety. Further, the other-directed negative emotion (anger) was positively associated with the risk of other-directed, aggressive misconduct. The results suggest that accountability as forward-looking, active responsibility may play an important role in the restorative rehabilitation of prisoners.

The impact of cumulative obstetric complications and childhood trauma on brain volume in young people with psychotic experiences.

Psychotic experiences (PEs) occur in 5-10% of the general population and are associated with exposure to childhood trauma and obstetric complications. However, the neurobiological mechanisms underlying these associations are unclear. Using the Avon Longitudinal Study of Parents and Children (ALSPAC), we studied 138 young people aged 20 with PEs (n = 49 suspected, n = 53 definite, n = 36 psychotic disorder) and 275 controls. Voxel-based morphometry assessed whether MRI measures of grey matter volume were associated with (i) PEs, (ii) cumulative childhood psychological trauma (weighted summary score of 6 trauma types), (iii) cumulative pre/peri-natal risk factors for psychosis (weighted summary score of 16 risk factors), and (iv) the interaction between PEs and cumulative trauma or pre/peri-natal risk. PEs were associated with smaller left posterior cingulate (pFWE < 0.001, Z = 4.19) and thalamus volumes (pFWE = 0.006, Z = 3.91). Cumulative pre/perinatal risk was associated with smaller left subgenual cingulate volume (pFWE < 0.001, Z = 4.54). A significant interaction between PEs and cumulative pre/perinatal risk found larger striatum (pFWE = 0.04, Z = 3.89) and smaller right insula volume extending into the supramarginal gyrus and superior temporal gyrus (pFWE = 0.002, Z = 4.79), specifically in those with definite PEs and psychotic disorder. Cumulative childhood trauma was associated with larger left dorsal striatum (pFWE = 0.002, Z = 3.65), right prefrontal cortex (pFWE < 0.001, Z = 4.63) and smaller left insula volume in all participants (pFWE = 0.03, Z = 3.60), and there was no interaction with PEs group. In summary, pre/peri-natal risk factors and childhood psychological trauma impact similar brain pathways, namely smaller insula and larger striatum volumes. The effect of pre/perinatal risk was greatest in those with more severe PEs, whereas effects of trauma were seen in all participants. In conclusion, environmental risk factors affect brain networks implicated in schizophrenia, which may increase an individual's propensity to develop later psychotic disorders.

The thymus: what's normal and what's not? Problem-solving with MRI.

Anterior mediastinal masses can be difficult to characterise on computed tomography (CT) due to the wide spectrum of normal appearances of thymic tissue as well as the challenge of differentiating between benign and malignant pathologies. Additionally, attenuation of cystic mediastinal lesions can be misinterpreted on CT due to varying attenuation values. Anecdotally, non-vascular magnetic resonance imaging (MRI) of the thorax is underutilised across radiology departments in the UK, but has been shown to improve diagnostic certainty and reduce unnecessary surgical intervention. T2-weighted MRI is useful in confirming the cystic nature of lesions, whereas chemical shift techniques can be utilised to document the presence of macroscopic and intra-cellular fat and thus help distinguish between benign and malignant pathologies. In this review article, we present a practical approach to using MRI for the characterisation of anterior mediastinal lesions based on our clinical experience in a UK district general hospital.

Bayesian Inference Elucidates the Catalytic Competency of the SARS-CoV-2 Main Protease 3CLpro.

The main protease of SARS-CoV-2, 3CLpro, is a dimeric enzyme that is indispensable to viral replication and presents an attractive opportunity for therapeutic intervention. Previous reports regarding the key properties of 3CLpro and its highly similar SARS-CoV homologue conflict dramatically. Values of the dimeric Kd and enzymic kcat/KM differ by 106- and 103-fold, respectively. Establishing a confident benchmark of the intrinsic capabilities of this enzyme is essential for combating the current pandemic as well as potential future outbreaks. Here, we use enzymatic methods to characterize the dimerization and catalytic efficiency of the authentic protease from SARS-CoV-2. Specifically, we use the rigor of Bayesian inference in a Markov Chain Monte Carlo analysis of progress curves to circumvent the limitations of traditional Michaelis-Menten initial rate analysis. We report that SARS-CoV-2 3CLpro forms a dimer at pH 7.5 that has Kd = 16 ± 4 nM and is capable of catalysis with kcat = 9.9 ± 1.5 s-1, KM = 0.23 ± 0.01 mM, and kcat/KM = (4.3 ± 0.7) × 104 M-1 s-1. We also find that enzymatic activity decreases substantially in solutions of high ionic strength, largely as a consequence of impaired dimerization. We conclude that 3CLpro is a more capable catalyst than appreciated previously, which has important implications for the design of antiviral therapeutic agents that target 3CLpro.

Body composition, gastrointestinal, and reproductive differences between broiler breeders fed using everyday or skip-a-day rearing programs.

Broiler breeder feed restriction practices have intensified as broiler feed efficiency has been improved. Skip-a-day (SAD) rearing regimen has controlled breeder growth, although this practice has become questionable for the modern breeder. We compared everyday (ED) and SAD programs and evaluated their impact on pullet growth performance, body composition, gastrointestinal tract development, and reproduction. At d 0, Ross 708 (Aviagen) pullet chicks (n = 1,778) were randomly assigned to 7 floor pens. Three pens were fed using the ED and 4 pens with SAD program through wk 21 using a chain-feeder system. ED and SAD grower diets were formulated to be isonutritious, with the only difference that ED diets had more crude fiber. Pullets (n = 44 per pen) were moved to 16 hen pens by treatment at wk 21 with 3 YP males (Aviagen) in each pen. All birds were fed common laying diets. In addition to BW data, sampled pullets and hens were scanned using dual energy X-ray absorptiometry (DEXA) to obtain body bone density and composition. Hen performance and hatchery metrics were recorded through wk 60. ED birds were heavier with similar nutrient intake from wk 10 to 45 (P ≤ 0.013). Pullet uniformity was unaffected by feeding method (P ≥ 0.443). SAD pullets had less body fat at wk 19 (P = 0.034) compared to ED pullets, likely as a metabolic consequence of intermittent feeding. SAD birds had lower bone density at wk 7, 15, and 19 (P ≤ 0.026). At 4 wk of age, SAD pullets had less intestinal villi goblet cells compared to ED pullets (P ≤ 0.050), possibly explained by the effect that feed removal has on cell migration rates. Overall egg-specific gravity (P = 0.057) and hatch of fertile % (P = 0.088) tended to be higher in eggs from ED hens. Altogether, ED feeding increased young pullet intestinal goblet cells and increased both bone density and body fat at wk 19. ED program improved pullet feed conversion (2.6% less feed) and increased eggshell quality and hatch of fertile.

Validation of personal protective equipment ensembles, incorporating powered air-purifying respirators protected from contamination, for the care of patients with high-consequence infectious diseases.

BACKGROUND: The UK High-Consequence Infectious Diseases (HCID) Network of high-level isolation units provides care for patients with contact- or airborne-transmissible highly infectious and highly dangerous diseases. In most HCID units, the healthcare workers (HCWs) wear personal protective equipment (PPE) ensembles incorporating a powered air-purifying respirator (PAPR) for head and respiratory protection. Some PAPRs have components worn outside/over other PPE, necessitating decontamination of re-usable elements. Two alternative PAPRs, with all re-usable elements worn under PPE, were trialled in this study. AIM: To undertake scenario-based testing of PAPRs and PPE to determine usability, comfort and ability to remove contaminated PPE without personal cross-contamination. METHODS: Trained healthcare volunteers (N=20) wearing PAPR/PPE ensembles were sprayed with ultraviolet fluorescent markers. They undertook exercises to mimic patient care, and subsequently, after doffing the contaminated PPE following an established protocol, any personal cross-contamination was visualized under ultraviolet light. Participants also completed a questionnaire to gauge how comfortable they found the PPE. FINDINGS AND CONCLUSIONS: The ensembles were tested under extreme 'worst case scenario' conditions, augmented by physical and manual dexterity tests. Participating volunteers considered the exercise to be beneficial in terms of training and PPE evaluation. Data obtained, including feedback from questionnaires and doffing buddy observations, supported evidence-based decisions on the PAPR/PPE ensemble to be adopted by the HCID Network. One cross-contamination event was recorded in the ensemble chosen; this could be attributed to doffing error, and could therefore be eliminated with further practice.

Impact of Hydrocodone Rescheduling on Dental Prescribing of Opioids.

INTRODUCTION: In the United States, dentists frequently prescribe hydrocodone. In October 2014, the US Drug Enforcement Administration rescheduled hydrocodone from controlled substance schedule III to II, introducing more restricted prescribing and dispensing regulations, which may have changed dental prescribing of opioids. OBJECTIVE: The study aim was to evaluate the impact of the hydrocodone rescheduling on dental prescribing of opioids in the United States. METHODS: This was a cross-sectional study of opioids prescribed by dentists between October 2012 and October 2016, using the IQVIA Longitudinal Prescription Dataset. Monthly dentist-based opioid prescribing rate (opioid prescription [Rx]/1,000 dentists) and monthly average opioid dosages per prescription (mean morphine milligram equivalent per day [MME/d]) were measured in the 24 mo before and after hydrocodone rescheduling in October 2014 (index or interruption). An interrupted time-series analysis was conducted using segmented ordinary least square regression models, with Newey-West standard errors to handle autocorrelation. RESULTS: Dentists prescribed 50,412,942 opioid prescriptions across the 49 mo. Hydrocodone was the most commonly prescribed opioid pre- and postindex (74.9% and 63.8%, respectively), followed by codeine (13.8% and 21.6%), oxycodone (8.1% and 9.5%), and tramadol (2.9% and 4.8%). At index, hydrocodone prescribing immediately decreased by -834.8 Rx/1,000 dentists (95% confidence interval [CI], -1,040.2 to -629.4), with increased prescribing of codeine (421.9; 95% CI, 369.7-474.0), oxycodone (85.3; 95% CI, 45.4-125.2), and tramadol (111.8; 95% CI, 101.4-122.3). The mean MME increased at index for all opioids except for hydrocodone, and dosages subsequently decreased during the postindex period. CONCLUSION: Following the rescheduling, dentist prescribing of hydrocodone declined while prescribing of nonhydrocodone opioids increased. Understanding the impact of this regulation informs strategies to ensure appropriate prescribing of opioids for dental pain. KNOWLEDGE TRANSFER STATEMENT: The study findings can be used by policy makers to make informed decisions in developing future risk mitigation strategies aimed to regulate opioid prescribing behaviors. Furthermore, dentist-specific resources and guidelines are needed subsequent to these policies in order to meet the dental population needs.

Degradation of polymer banknotes through handling, and effect on fingermark visualisation.

The surface structure of mint (as-issued) and handled polymer five pounds sterling banknotes was studied by atomic force microscopy and laser scanning confocal microscopy. A total of 1856 fingermarks on mint and handled banknotes from four different issuing banks (Bank of England, Bank of Scotland, Royal Bank of Scotland and Clydesdale Bank) were visualised with Vacuum Metal Deposition (VMD), Cyanoacrylate Fuming (CAF) and, on Clydesdale Bank notes, magnetic fluorescent powder. VMD was significantly more effective in developing fingermarks on handled banknotes, across all the banks studied, although effectiveness varied with issuing bank. For example, on handled Bank of England notes 45% of marks showed ridge detail with VMD development and 28% with CAF; for Bank of Scotland handled notes success rates were 17% with VMD and 1% with CAF. Microscopy of degraded banknotes showed the loss of intaglio printing and the formation of a cracked surface structure in the handled notes. These features can lead to the trapping of powder, or contaminants, increasing quantity of development agent in fingermark background between the ridges, decreasing contrast and decreasing performance of powder-based fingermark development techniques. These same features can restrict the migration of components of the fingermark, preventing fingermarks degrading through spread of material and thus reducing potential formation of empty prints, so that VMD development is not adversely affected.

Enthesis: not the same in each localisation - a molecular, histological and biomechanical study.

The interphase between tendon and bone consists of a highly specialised tissue called enthesis. Typically, the enthesis is described as a succession of four different zones: tendon, non-mineralised fibrocartilage, mineralised fibrocartilage and bone. However, the microstructure of the entheses, cellular composition and mechanical properties vary depending on their anatomical location. The present study aimed to characterise three of the most relevant sites of enthesis injury in a rat model: the patellar tendon, the Achilles tendon and the supraspinatus enthesis, in terms of biomechanics, histology and genetic expression. The patellar enthesis presented the highest ultimate load and lowest stiffness of the three, while the supraspinatus was the weakest and stiffest. The histological characterisation revealed key differences at the insertion site for each enthesis. The patellar enthesis showed a large cartilaginous area at the tendon-to-bone interphase whilst this interphase was smaller in the supraspinatus entheses samples. Furthermore, the Achilles tendon enthesis displayed a more abrupt transition from tendon to bone. Additionally, each enthesis exhibited a particular and distinct pattern of expression of tenogenic, chondrogenic and osteogenic markers. This study provided valuable insights for a better understanding of the three entheses at relevant anatomical sites. Moreover, the larger cross-sectional area of the patellar enthesis, the strong mechanical properties and the easier surgical access to this location led to the conclusion that the patellar tendon enthesis site could be most suitable for the development of a preclinical model for general enthesis regeneration studies in rats.

Development and operation of the defence COVID-19 lab as a SARS-CoV-2 diagnostic screening capability for UK military personnel.

BACKGROUND: In the face of the COVID-19 pandemic, the Defence Science and Technology Laboratory (Dstl) and Defence Pathology combined to form the Defence Clinical Lab (DCL), an accredited (ISO/IEC 17025:2017) high-throughput SARS-CoV-2 PCR screening capability for military personnel. LABORATORY STRUCTURE AND RESOURCE: The DCL was modular in organisation, with laboratory modules and supporting functions combining to provide the accredited SARS-CoV-2 (envelope (E)-gene) PCR assay. The DCL was resourced by Dstl scientists and military clinicians and biomedical scientists. LABORATORY RESULTS: Over 12 months of operation, the DCL was open on 289 days and tested over 72 000 samples. Six hundred military SARS-CoV-2-positive results were reported with a median E-gene quantitation cycle (Cq) value of 30.44. The lowest Cq value for a positive result observed was 11.20. Only 64 samples (0.09%) were voided due to assay inhibition after processing started. CONCLUSIONS: Through a sustained effort and despite various operational issues, the collaboration between Dstl scientific expertise and Defence Pathology clinical expertise provided the UK military with an accredited high-throughput SARS-CoV-2 PCR test capability at the height of the COVID-19 pandemic. The DCL helped facilitate military training and operational deployments contributing to the maintenance of UK military capability. In offering a bespoke capability, including features such as testing samples in unit batches and oversight by military consultant microbiologists, the DCL provided additional benefits to the UK Ministry of Defence that were potentially not available from other SARS-CoV-2 PCR laboratories. The links between Dstl and Defence Pathology have also been strengthened, benefitting future research activities and operational responses.

Amoebic crab disease (ACD) in edible crab Cancer pagurus from the English Channel, UK.

The genera Paramoeba and Neoparamoeba (Amoebozoa, Dactylopodida, Paramoebidae) include well-known opportunistic pathogens associated with fish (N. peruans; amoebic gill disease), lobsters, molluscs and sea urchins, but only rarely with crabs (grey crab disease of blue crabs). Following reports of elevated post-capture mortality in edible crabs Cancer pagurus captured from a site within the English Channel fishery in the UK, a novel disease (amoebic crab disease, ACD) was detected in significant proportions of the catch. We present histopathological, transmission electron microscopy and molecular phylogenetic data, showing that this disease is defined by colonization of haemolymph, connective tissues and fixed phagocytes by amoeboid cells, leading to tissue destruction and presumably death in severely diseased hosts. The pathology was strongly associated with a novel amoeba with a phylogenetic position on 18S rRNA gene trees robustly sister to Janickina pigmentifera (which groups within the current circumscription of Paramoeba/Neoparamoeba), herein described as Janickina feisti n. sp. We provide evidence that J. feisti is associated with ACD in 50% of C. pagurus sampled from the mortality event. A diversity of other paramoebid sequence types, clustering with known radiations of N. pemaquidensis and N. aestuarina and a novel N. aestuarina sequence type, was detected by PCR in most of the crabs investigated, but their detection was much less strongly associated with clinical signs of disease. The discovery of ACD in edible crabs from the UK is discussed relative to published historical health surveys for this species.

Prediction of QcrB Inhibition as a Measure of Antitubercular Activity with Machine Learning Protocols.

It has always been a challenge to develop interventional therapies for Mycobacterium tuberculosis. Over the years, several attempts at developing such therapies have hit a dead-end owing to rapid mutation rates of the tubercular bacilli and their ability to lay dormant for years. Recently, cytochrome bcc complex (QcrB) has shown some promise as a novel target against the tubercular bacilli, with Q203 being the first molecule acting on this target. In this paper, we report the deployment of several ML-based approaches to design molecules against QcrB. Machine learning (ML) models were developed based on a data set of 350 molecules using three different sets of molecular features, i.e., MACCS keys, ECFP6 fingerprints, and Mordred descriptors. Each feature set was trained on eight ML classifier algorithms and optimized to classify molecules accurately. The support vector machine-based classifier using the ECFP6 feature set was found to be the best classifier in this study. Further, screening of the known imidazopyridine amide inhibitors demonstrated that the model correctly classified the most potent molecules as actives, hence validating the model for future applications.