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BackgroundThe Critical Assessment of Genome Interpretation (CAGI) aims to advance the state-of-the-art for computational prediction of genetic variant impact, particularly where relevant to disease. The five complete editions of the CAGI community experiment comprised 50 challenges, in which participants made blind predictions of phenotypes from genetic data, and these were evaluated by independent assessors.ResultsPerformance was particularly strong for clinical pathogenic variants, including some difficult-to-diagnose cases, and extends to interpretation of cancer-related variants. Missense variant interpretation methods were able to estimate biochemical effects with increasing accuracy. Assessment of methods for regulatory variants and complex trait disease risk was less definitive and indicates performance potentially suitable for auxiliary use in the clinic.ConclusionsResults show that while current methods are imperfect, they have major utility for research and clinical applications. Emerging methods and increasingly large, robust datasets for training and assessment promise further progress ahead.

First trimester placental development comprises some of the most critical yet understudied events that impact fetal development. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence offspring throughout their lives. Thus, a paradigm to study early placental development is necessary, and this has spurred on the pursuit of new in vitro model systems that recapitulate specific aspects of placentation. One of the most complex and translationally valid models to arise are organoids, three-dimensional structures comprising multiple differentiated cell types that originate from a common progenitor population. Trophoblasts are the progenitor cells of the placenta, serving as the proliferative base for placental development. Recent advances have enabled the derivation of organoids from primary tissue, yet access to first trimester human samples is ethically constrained; derivation from established trophoblast stem cell lines is an alternative source. Organoids have already proven useful in generating insights into molecular events that underlie trophoblast differentiation, with the identification of new cell subtypes that are primed to differentiate down different paths. In this review, (1) we recap early pregnancy development events, (2) provide an overview of the cellular complexity of the placenta, (3) discuss the generation of organoids from tissue versus cellular sources, (4) highlight the value of translational animal models, and (5) focus on the complexities of the molecular regulation of trophoblast organoid development, differentiation, and function.

Allison Maher,1 Michelle Bennett,1 Hsin-Chia Carol Huang,1,2 Philip Gaughwin,1,3 Mary Johnson,1 Madeleine Brady,1 Kacie Patterson,1,3 Tanya Buettikofer,1,3 Jo Morris,1 Veronica Mary Rainbird,3 Imogen Mitchell,1,2 Bernie Bissett1,3 1Rehabilitation and Community Services / Respiratory Medicine, Canberra Health Services, Canberra ACT, Australia; 2College of Health and Medicine, Australian National University, Canberra ACT, Australia; 3Faculty of Health, University of Canberra, Canberra ACT, Bruce, AustraliaCorrespondence: Bernie Bissett, Email Bernie.Bissett@canberra.edu.auObjective: To describe our methodology and share the practical tools we have developed to operationalize a multidisciplinary Long COVID clinic that incorporates progressive, personalized exercise prescription as a cornerstone feature.Background: There is a lack of evidence-based guidance regarding optimal rehabilitation strategies for people with Long COVID. Existing guidelines lack precision regarding exercise dosage. As one of Australia’s few established multidisciplinary Long COVID clinics, we describe our novel approach to safely incorporating exercise of both peripheral and respiratory muscles, with essential monitoring and management of post-exertional symptom exacerbation.Methods: Working closely with primary health-care providers, our multidisciplinary team screens referrals for people aged 16 and older with Long COVID. Staff apply a three tier model of triage, dependent on the consumer’s presenting problems. Exercise-based interventions necessitate detailed monitoring for post-exertional symptom exacerbation both in the clinic and at home. Personalized exercise prescription includes resistance training at a submaximal threshold (4– 6 exercises, 3 days/week); whole-body endurance exercise titrated to the individual’s progress, at an intensity 4– 6/10 (Rate of Perceived Exertion); and for those limited by dyspnoea, high-intensity inspiratory muscle training using a threshold-based handheld device (30 repetitions per day, ≥ 50% of their maximum inspiratory pressure).Discussion: We have used these approaches for the past 2 years in 250 consumers with no serious adverse events and promising consumer feedback. Our exercise prescription is less conservative than the methods advocated in international guidelines for people with Long COVID, and these more progressive tools may be valuable in other contexts.Conclusion: In our experience, a multidisciplinary clinic-based approach to safely prescribing progressive exercise in Long COVID is feasible. Both peripheral and inspiratory muscle exercise can be effectively titrated to each individual’s symptoms, and careful monitoring for post-exertional symptom exacerbation is crucial.Plain Language Summary: Long COVID affects 5-10% of people following COVID infection. There is little specific guidance on how exercise can be safely prescribed in Long COVID. This paper is the first to provide a detailed description of an Australian multidisciplinary Long COVID clinic, including specific tools and guidance about how exercise can be prescribed while minimising post-exertional symptom exacerbation. The tools described could be valuable to other health facilities striving to optimise multidisciplinary care for people with Long COVID, incorporating safe exercise prescription.Keywords: COVID, rehabilitation, multidisciplinary care, physiotherapy, respiratory muscle training

No clear consensus has emerged from the literature on the gene expression changes that occur in human whole blood with age. In this study we compared whole blood ageing genes from the published literature with data on gene specificity for leukocyte subtypes. Surprisingly we found that highly ranked ageing genes were predominantly expressed by naïve T cells, with limited expression from more common cell types. Highly ranked ageing genes were also more likely to have decreased expression with age. Taken together, it is plausible that much of the observed gene expression changes in whole blood is reflecting the decline in abundance of naïve T cells known to occur with age, rather than changes in transcription rates in common cell types. Correct attribution of the gene expression changes that occur with age is essential for understanding the underlying mechanisms.

Functional decline with age contributes significantly to the burden of disease in developed countries. There is growing interest in the development of therapeutic interventions which slow or even reverse aging. Time and cost constraints prohibit the testing of a large number of interventions for health and lifespan extension in model organisms. Cell-based models of aging could enable high throughput testing of potential interventions. Despite extensive reports in the literature of cell properties that correlate with donor age, few are robustly observed across different laboratories. This casts doubt on the extent that aging signatures are captured in cultured cells. We tested molecular changes previously reported to correlate with donor age in peripheral blood mononuclear cells (PBMCs) and evaluated their suitability for inclusion in a panel of functional aging measures. The tested measures spanned several pathways implicated in aging including epigenetic changes, apoptosis, proteostasis, and intracellular communication. Surprisingly, only two markers correlated with donor age. DNA methylation age accurately predicted donor age confirming this is a robust aging biomarker. Additionally, the apoptotic marker CD95 correlated with donor age but only within subsets of PBMCs. To demonstrate cellular rejuvenation in response to a treatment will require integration of multiple read-outs of cell function. However, building a panel of measures to detect aging in cells is challenging and further research is needed to identify robust predictors of age in humans.

BackgroundLaser fistulectomy is a minimally invasive, sphincter-sparing procedure for treatment of anal fistula. In several studies, this method has been shown to be safe and effective, with reported success rates ranging from 40 to 88%. We hypothesized that with longer follow-up, these rates would decrease.MethodsA retrospective case analysis assessing the effectiveness of laser fistulectomy in curing fistula-in-ano tracts within a cohort of patients at a single academic institution was conducted. All patients having laser ablation between March 2016 and July 2018 were analyzed. Cure of the fistula was determined by history and postoperative physical exam, and was defined as complete closure of fistula tract with resolution of symptoms. Secondary symptoms of fecal incontinence, infection, and pain were evaluated.ResultsEighteen patients (10 males, mean age 41 ± 13 years) were analyzed. Transphincteric fistula was the most common type (67%, N = 12). The mean number of previous fistula procedures was 1.33 ± 1.64. There was a 22% (N = 4) success rate at an average postoperative follow-up period of 29 ± 8 months (range 18-46 months). Of those who failed, 64% (N = 9) had a subsequent fistula procedure. There were no cases of fecal incontinence, but 3 cases (17%) of postoperative infection were reported and 8 patients (44%) had a subjective increase in pain at first follow-up appointment.ConclusionsOur data showed a much higher failure rate of laser fistulectomy compared to those reported in the literature. However, the small sample size, a large amount of heterogeneity in our patient population with a mixture of fistula types present, and various laser techniques applied decreased the power of this study.

Mounting evidence indicates that SARS-CoV-2 can infect multiple systemic tissues, but few studies have evaluated SARS-CoV-2 RNA dynamics in multiple specimen types due to their reduced accessibility and diminished performance of RT-qPCR with non-respiratory specimens. Here, we employed an ultrasensitive CRISPR-RT–PCR assay to analyze longitudinal mucosal (nasal, buccal, pharyngeal, and rectal), plasma, and breath samples from SARS-CoV-2-infected non-human primates (NHPs) to detect dynamic changes in SARS-CoV-2 RNA level and distribution among these specimens. We observed that CRISPR-RT–PCR results consistently detected SARS-CoV-2 RNA in all sample types at most time points post-infection, and that SARS-CoV-2 infection dose and administration route did not markedly affect the CRISPR-RT–PCR signal detected in most specimen types. However, consistent RT-qPCR positive results were restricted to nasal, pharyngeal, and rectal swab samples, and tended to decrease earlier than CRISPR-RT–PCR results, reflecting lower assay sensitivity. SARS-CoV-2 RNA was detectable in both pulmonary and extrapulmonary specimens from early to late infection by CRISPR-RT–PCR, albeit with different abundance and kinetics, with SARS-CoV-2 RNA increases detected in plasma and rectal samples trailing those detected in upper respiratory tract samples. CRISPR-RT–PCR assays for SARS-CoV-2 RNA in non-respiratory specimens may thus permit direct diagnosis of suspected COVID-19 cases missed by RT–PCR, while tracking SARS-CoV-2 RNA in minimally invasive alternate specimens may better evaluate the progression and resolution of SARS-CoV-2 infections.

This investigation (1) elucidates genuine interest in the context of learning from Dewey's perspective, (2) assesses construct validity evidence for a genuine interest conceptual model using data from the Factors Effecting Ethics Learning survey, and (3) assesses measurement invariance of the genuine interest constructs between male (n = 352) and female (n = 188) Taiwan high school students. Results provide support for genuine interest as a two-dimensional construct. Second-order factor analysis shows that latent variables of genuine interest in learning socio-scientific issues and interpersonal communication ethics-related issues are represented by sub-latent constructs of learning interest and learning enjoyment. Assessment of gender invariance in the conceptual model provides evidence of measurement equivalence.

OBJECTIVE: Adiposity has been hypothesized to interfere with the activity of bevacizumab (BEV), an anti-angiogenic agent. Measurements of adiposity, BMI, surface fat area (SFA), and visceral fat area (VFA) were investigated as prognostic of oncologic outcomes among patients treated with chemotherapy, with or without BEV, on GOG 218, a prospective phase III trial. METHOD: Pretreatment computed tomography (CT) for 1538 GOG 218 participants were analyzed. Proportional hazards models assessed association between adiposity and overall survival (OS) adjusted for other prognostic factors. The predictive value of adiposity as a function of BEV treatment was assessed in 1019 patients randomized to either chemotherapy (CT) + placebo (P) → P or CT + BEV → BEV. RESULTS: After adjusting for prognostic factors, SFA was not associated with the overall hazard of death (p = 0.981). There was a non-significant 0.1% (p = 0.062) increase in hazard of death associated with a unit increase in VFA. When comparing the treatment HRs for patients who did and did not receive BEV, there was no association with SFA (p = 0.890) or VFA (p = 0.106). A non-significant 0.8% increase in the hazard of death with unit increase in BMI (p = 0.086) was observed. BMI values were not predictive of a longer survival for patients with BEV vs placebo (p = 0.606). CONCLUSION: Measures of adiposity strongly correlated to one another but were not predictive of efficacy for BEV. VFA is a weak prognostic factor.

First trimester placental development comprises some of the most critical yet understudied events that impact fetal development. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence offspring throughout their lives. Thus, a paradigm to study early placental development is necessary, and this has spurred on the pursuit of new in vitro model systems that recapitulate specific aspects of placentation. One of the most complex and translationally valid models to arise are organoids, three-dimensional structures comprising multiple differentiated cell types that originate from a common progenitor population. Trophoblasts are the progenitor cells of the placenta, serving as the proliferative base for placental development. Recent advances have enabled the derivation of organoids from primary tissue, yet access to first trimester human samples is ethically constrained; derivation from established trophoblast stem cell lines is an alternative source. Organoids have already proven useful in generating insights into molecular events that underlie trophoblast differentiation, with the identification of new cell subtypes that are primed to differentiate down different paths. In this review, (1) we recap early pregnancy development events, (2) provide an overview of the cellular complexity of the placenta, (3) discuss the generation of organoids from tissue versus cellular sources, (4) highlight the value of translational animal models, and (5) focus on the complexities of the molecular regulation of trophoblast organoid development, differentiation, and function. [ABSTRACT FROM AUTHOR]

Animal health and feed intake are closely interrelated, with the latter being an important indicator of an animal’s health status. Automated sensors for dairy cattle have been developed to detect changes in indicators of health, such as decreased rumination or activity. Previous studies have identified associations between sensor measurements and feed intake. Thus, the objective of this study was to determine if health disorders impact the associations identified between sensors and dry matter intake (DMI), and to measure the impact of health disorders on DMI. A total of 934 cows with health disorders (lameness, mastitis, and other), of which 57, 94, and 333 cows had observations for a rumen bolus and one of two ear tags, were analyzed to determine how health disorders impact the association of sensors with DMI. Eleven sensor measurements were collected across the three sensors, including total and point-in-time activity, rumination time, inner-ear temperature, rumen pH and rumen temperature. Associations of health disorders and sensor measures with DMI were evaluated when accounting for systematic effects (i.e., contemporary group, parity, and days in milk) and energy sinks accounted for in determination of feed efficiency (e.g., milk production, body weight and composition). In order to determine if inclusion of health disorders or sensor measures improved model fit, model AICs were assessed. Health disorders were significantly associated with all sensor measurements (P< 0.0001), with the direction of association dependent on sensor measure and health disorder. Moreover, DMI decreased with all health disorders, with larger impacts observed in animals in third and higher lactations. Numerous sensor measurements were associated with DMI, including when DMI was adjusted for energy sink variables and health. Inclusion of rumen bolus temperature, rumination or activity with health data reduced model AIC when evaluating DMI as the dependent variable. Some sensor measures, including measurements of activity, temperature and rumination, accounted for additional variation in feed intake when adjusted for health disorders. Results from the study indicate that feed intake and sensor measures are impacted by health disorders. These findings may have implications for use of sensors in genetic evaluations and precision feeding of dairy cattle.

The polycomb repressive complex 2 (PRC2) is a histone methyltransferase regulating cell differentiation and identity. Here, the authors show that the vertebrate-specific PRC2 accessory subunit PALI1 facilitates substrate binding by the complex and elucidate the allosteric mechanism of PALI1- mediated PRC2 activation.

No clear consensus has emerged from the literature on the gene expression changes that occur in human whole blood with age. In this study we compared whole blood ageing genes from the published literature with data on gene specificity for leukocyte subtypes. Surprisingly we found that highly ranked ageing genes were predominantly expressed by naïve T cells, with limited expression from more common cell types. Highly ranked ageing genes were also more likely to have decreased expression with age. Taken together, it is plausible that much of the observed gene expression changes in whole blood is reflecting the decline in abundance of naïve T cells known to occur with age, rather than changes in transcription rates in common cell types. Correct attribution of the gene expression changes that occur with age is essential for understanding the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Feed efficiency is an important trait to dairy production because of its impact on sustainability and profitability. Measuring individual cow feed intake on commercial farms would be unfeasibly costly at present. Thus, developing cheap and portable indicators of feed intake would be highly beneficial for genetic selection and precision feeding management tools. Given the growing use of automated sensors on dairy farms, the objective of this study was to determine the relationship between measurements recorded from multiple wearable sensors and feed intake. A total of three different wearable sensors were evaluated for their association with dry mater intake (DMI). The sensors measured activity (sensors = 3), rumination (sensors = 1), ear temperature (sensors = 1), rumen pH (sensors = 1) and rumen temperature (sensors = 1). A range of 56–340 cows with assorted sensors from 24 to 313 days in milk (DIM) were modeled to evaluate associations with DIM, parity, and contemporary group (CG; comprised of pen and study cohort). Models extending upon these variables included known energy sinks (i.e., milk production, milk fat/protein and metabolic body weight), to characterize the association of sensors measures and DMI. Statistically significant (i.e., P < 0.05) regression coefficients for individual sensor measures with DMI ranged from 9.01E-07 to −3.45 kg DMI/day. When integrating all measures from a single sensor in a model, estimated regression coefficients ranged 8.83E-07 to −3.48 kg DMI/day. Significant associations were also identified for milk production traits, parity, DIM and CG. Associations tended to be highest for timepoints around the time of feeding and when multiple measurements within a sensor were integrated in a single model. The findings of this study indicate sensor measures are associated with feed intake and other energy sink traits and variables impacting feed efficiency. This information would be helpful to improve feed and feeding efficiency on commercial farms as proxy measurements for feed intake.

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host’s ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2’-5’ oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored.

Ducted fuel injection (DFI) has been shown to be effective at attenuating soot emissions from mixing-controlled compression-ignition engines. Furthermore, simultaneously low soot and nitrogen oxide (NOx) emissions have been achieved when DFI is combined with simulated exhaust-gas recirculation. Whereas previous studies investigating the effects of duct length and diameter have been conducted in combustion vessels, this is the first such study conducted in an engine. The work presented here quantifies the effects of duct length and diameter using 2- and 3-mm ducts that are 8, 12, and 16 mm long with a four-orifice fuel injector. This study also employs a larger fuel-injector-orifice diameter than has been studied previously. A comparison of 0.110-mm and 0.175-mm fuel-injector-orifice diameters with a single duct geometry is also presented. This study finds that the larger fuel-injector-orifice diameter provides better soot attenuation when it is used with a larger-diameter duct. The purpose of the larger orifice was to achieve a higher load than has been reported in previous DFI studies, namely 13.4 bar gross indicated mean effective pressure.