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The author developed three instruments which operationally defined certain probability concepts and combinatorial abilities and subsequently used the instruments to classify the 90 subjects of the study according to their conceptual strategies or ability levels. The subjects were high school biology students from one school; all were taught by the same teacher. Two treatment groups were formed using three limitations on randomization--age, ability, and previous participation in the ISCS program. One group took the test sequence before commencing genetics instruction; the other group took the same test sequence after completing the unit. Results of the Probability Reasoning Differential Test showed that over 90 percent of the subjects fell into one of three postulated categories of strategies with the most frequently used strategy being that of consistency in predictions regarding correct behavior. The Test on Forming Combinations indicated that some students already possessed the competencies for forming combinations needed in solving genetic cross problems. The Probability Reasoning Survey provided data on the ability to form subsets of a sample space and associate the appropriate probabilities with them. Comparisons of pre- and post-test results failed to demonstrate any effect of genetics instruction on the concepts and abilities studied. (Author/EB)

Many ecosystems can be described as dynamic, open, and connected systems that are intrinsically linked to the attributes and processes of neighboring ecosystems. Connectivity among these ecosystems is critically important across multiple levels of biological organization. The cross-ecosystem exchange of organic matter is a well-described example of high variability in ecosystem connectivity. Subsidies of material and energy from donor ecosystems can significantly influence the structure and dynamics of recipient communities and food webs. In systems with pulsed resource subsidies variability in subsidy type, amount, and frequency affects consumer populations, species interactions, and food web complexity causing them to differ from systems with consistent in situ production. The largest magnitude in observed cross-ecosystem fluxes are from marine to terrestrial systems. An exceptional example of this flux is the substantial subsidy of organic matter exported by highly productive nearshore kelp forests to sandy beaches.In the marine realm, canopy forming kelps are considered foundation species that structure the surrounding nearshore reef community. Kelp forests provide many ecosystem functions including food and habitat provisioning, nutrient cycling, increased biodiversity, and subsidies to other habitats. The high turnover of biomass in kelp forests leads to the export of the majority of their net primary production as detritus to adjacent marine ecosystems. A large fraction of that kelp detritus from the donor ecosystem washes ashore on nearby sandy beaches, the recipient ecosystem. Beach ecosystems are characterized by frequent disturbance, low in situ primary productivity, and a reliance on marine subsidies. The inputs of wrack to beaches are strongly linked to their community and food web structure and to ecological functioning. Wrack subsidies from kelp forests to beaches are highly variable across space and time. This variation can affect species and populations directly via changes in habitat and food, indirectly through changes in species interactions, and functionally through effects on ecological processes on the beach. In my first chapter, I examine the role of habitat partitioning in reducing potential negative interspecific interactions across space and time among four co-habiting species of wrack detritivores, talitrid amphipods (Megalorchestia spp.), and how this niche partitioning is mediated by tide phase using field studies and mesocosm experiments. In my second chapter, I evaluate the hypothesis that biodiversity promotes ecosystem function by testing the effects of consumer species diversity and identity on kelp wrack processing rates using laboratory experiments and field consumption assays. In my third chapter, I further explore ecosystem functioning on sandy beaches by comparing in situ fluxes of CO2 from intertidal sediment to wrack and detritivore abundance and in relation to laboratory measured respiration rates of detritivore species. In my final chapter, I explore how wrack subsidies structure the sandy beach macroinvertebrate community, enhance species richness and abundance across multiple trophic levels, and stimulate multiple ecological and biogeochemical ecosystem functions. While the important role of linkages between donor and recipient ecosystems is widely known, there is much to learn with respect to how these links function across spatial and temporal scales and across species. Overall, my findings suggest that the inputs of wrack from kelp forests to beaches is a critical driver of important biotic and abiotic processes that interact to shape the community and promote ecosystem functioning. Four congeners of talitrid species reduce possible negative biotic interactions by partitioning their habitat and surface-active periods and the magnitude of these separations is mediated by tide phase. For the key ecosystem function of kelp wrack processing, consumption rates were not dependent on species richness in the laboratory or field trials. Those rates are body-size dependent and pointed to species identity as a significant factor in this ecosystem function, especially the presence of the two large-bodied species. Expanding on sandy beach ecosystem functioning, I found that wrack abundance across a natural range in variability is a significant driver of sediment CO2 flux and that wrack consumer respiration rates, determined by laboratory measurements, are a notable component (10.5%) of the net CO2 flux. The role of wrack in the sandy beach ecosystem is multifaceted, as demonstrated by my field survey of sites across a large natural range of wrack inputs. Wrack abundance structures the detritivore, predator and full wrack-associated macroinvertebrate communities, increases redundancy within the invertebrate food web, enhances shorebird diversity and abundance, and increases ecosystem multifunctionality. My results demonstrate how the dynamic coupling between sandy beach ecosystems and nearshore giant kelp forests is reflected in the role of kelp wrack subsidies in mediating ecosystem diversity, community structure, and ecosystem functioning of beaches.

ObjectivesThe human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking are the most prominent genetic and environmental risk factors for rheumatoid arthritis (RA). However, at which pre-arthritis stage (asymptomatic/symptomatic) they exert their effect is unknown. We aimed to determine whether HLA-SE and smoking are involved in the onset of autoantibody positivity, symptoms (clinically suspect arthralgia (CSA)) and/or progression to clinical arthritis.MethodsWe performed meta-analyses on results from the literature on associations of HLA-SE and smoking with anti-citrullinated protein antibodies (ACPAs) in the asymptomatic population. Next, we studied associations of HLA-SE and smoking with autoantibody positivity at CSA onset and with progression to clinical inflammatory arthritis (IA) during follow-up. Associations in ACPA-positive patients with CSA were validated in meta-analyses with other arthralgia cohorts. Analyses were repeated for rheumatoid factor (RF), anti-carbamylated protein antibodies (anti-CarP) and anti-acetylated protein antibodies (AAPA).ResultsMeta-analyses showed that HLA-SE is not associated with ACPA positivity in the asymptomatic population (OR 1.06 (95% CI:0.69 to 1.64)), whereas smoking was associated (OR 1.37 (95% CI: 1.15 to 1.63)). At CSA onset, both HLA-SE and smoking associated with ACPA positivity (OR 2.08 (95% CI: 1.24 to 3.49), OR 2.41 (95% CI: 1.31 to 4.43)). During follow-up, HLA-SE associated with IA development (HR 1.86 (95% CI: 1.23 to 2.82)), in contrast to smoking. This was confirmed in meta-analyses in ACPA-positive arthralgia (HR 1.52 (95% CI: 1.08 to 2.15)). HLA-SE and smoking were not associated with RF, anti-CarP or AAPA-positivity at CSA onset. Longitudinally, AAPA associated with IA development independent from ACPA and RF (HR 1.79 (95% CI: 1.02 to 3.16)), anti-CarP did not.ConclusionsHLA-SE and smoking act at different stages: smoking confers risk for ACPA and symptom development, whereas HLA-SE mediates symptom and IA development. These data enhance the understanding of the timing of the key risk factors in the development of RA.

Purpose SARS-CoV-2 RNA has been detected in ocular tissues, but their susceptibility to SARS-CoV-2 infection is unclear. Here, we tested whether SARS-CoV-2 can infect human conjunctival epithelial cells (hCECs) and induce innate immune response. Methods Conjunctival tissue from COVID-19 donors was used to detect SARS-CoV-2 spike and envelope proteins. Primary hCECs isolated from cadaver eyes were infected with the parental SARS-CoV-2 and its beta variant of concern (VOC). Viral genome copy number, and expression of viral entry receptors, TLRs, interferons, and innate immune response genes were determined by qPCR. Viral entry receptors were examined in hCECs and tissue sections by immunostaining. Spike protein was detected in the cell culture supernatant by dot blot. Results Spike and envelope proteins were found in conjunctiva from COVID-19 patients. SARS-CoV-2 infected hCECs showed high viral copy numbers at 24–72h post-infection; spike protein levels were the highest at 24hpi. Viral entry receptors ACE2, TMPRSS2, CD147, Axl, and NRP1 were detected in conjunctival tissue and hCECs. SARS-CoV-2 infection-induced receptor gene expression peaked at early time points post-infection, but gene expression of most TLRs peaked at 48 or 72hpi. SARS-CoV-2 infected hCECs showed higher expression of genes regulating antiviral response, RIG-I, interferons (α, β, & λ), ISG15 & OAS2, cytokines (IL6, IL1β, TNFα), and chemokines (CXCL10, CCL5). Compared to the parental strain, beta VOC induced increased viral copy number and innate response in hCECs. Conclusions Conjunctival epithelial cells are susceptible to SARS-CoV-2 infection. Beta VOC is more infectious than the parental strain and evokes a higher antiviral and inflammatory response.

Background Vaccines that are shelf stable and easy to administer are crucial to improve vaccine access and reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission around the world. Methods In this study, we demonstrate that an oral, adenovirus-based vaccine candidate protects against SARS-CoV-2 in a Syrian hamster challenge model. Results Hamsters administered 2 doses of VXA-CoV2-1 showed a reduction in weight loss and lung pathology and had completely eliminated infectious virus 5 days postchallenge. Oral immunization induced antispike immunoglobulin G, and neutralizing antibodies were induced upon oral immunization with the sera, demonstrating neutralizing activity. Conclusions Overall, these data demonstrate the ability of oral vaccine candidate VXA-CoV2-1 to provide protection against SARS-CoV-2 disease., Vaccinating Syrian hamsters with an oral vaccine against SARS-CoV-2 protects against disease-associated symptoms of weight loss and lung pathology. Spike-specific IgG was detected in the sera and nasal wash postvaccination, and viral loads were decreased upon challenge with SARS-CoV-2.

Mitochondria are dynamic, interactive organelles that connect cellular signaling and whole-cell homeostasis. This "mitochatting" allows the cell to receive information about the mitochondria's condition before accommodating energy demands. Mitofusin 2 (Mfn2), an outer mitochondrial membrane fusion protein specializes in mediating mitochondrial homeostasis. Early studies defined the biological significance of Mfn2, while latter studies highlighted its role in substrate metabolism. However, determining Mfn2 potential to contribute to energy homeostasis needs study. This review summarizes current literature on mitochondrial metabolic processes, dynamics, and evidence of interactions among Mfn2 and regulatory processes that may link Mfn2's role in maintaining mitochondrial function and substrate metabolism.

SARS-CoV-2 infection results in a spectrum of outcomes from no symptoms to widely varying degrees of illness to death. A better understanding of the immune response to SARS-CoV-2 infection and subsequent, often excessive, inflammation may inform treatment decisions and reveal opportunities for therapy. We studied immune cell subpopulations and their associations with clinical parameters in a cohort of 26 patients with COVID-19. Following informed consent, we collected blood samples from hospitalized patients with COVID-19 within 72 h of admission. Flow cytometry was used to analyze white blood cell subpopulations. Plasma levels of cytokines and chemokines were measured using ELISA. Neutrophils undergoing neutrophil extracellular traps (NET) formation were evaluated in blood smears. We examined the immunophenotype of patients with COVID-19 in comparison to that of SARS-CoV-2 negative controls. A novel subset of pro-inflammatory neutrophils expressing a high level of dual endothelin-1 and VEGF signal peptide-activated receptor (DEspR) at the cell surface was found to be associated with elevated circulating CCL23, increased NETosis, and critical-severity COVID-19 illness. The potential to target this subpopulation of neutrophils to reduce secondary tissue damage caused by SARS-CoV-2 infection warrants further investigation.

Individuals engaged in cooperative or mutualistic interactions can benefit from adjusting their strategic behaviour according to the bargaining positions of their partners. In interaction networks in which individuals cooperate with a variety of partners, assessing partners' options and expected interaction payoffs might be crucial for such behavioural optimization to occur. However, the cognitive challenges involved in this assessment and the constraints they may pose for strategic behaviour are currently poorly understood. Here, we investigated whether cleaner fish, Labroides dimidiatus, can learn to optimally prioritize partners, when faced with an ecologically relevant challenge that requires them to distinguish between interaction contexts. We expanded on a relatively well-studied problem that cleaners face in nature: choosing between client fish that simultaneously seek service. In such situations, cleaners should give priority to visitor client species as these tend to leave if not serviced immediately, while resident species will readily await inspection. In laboratory ‘biological market’ experiments simulating this choice using ephemeral and permanent food-offering plates, cleaners could learn to prefer visitors. However, the actual challenge cleaners face is more complex: interactions in the reef form a ‘complex market’ whereby clients appear in all possible configurations. In homogeneous configurations (visitor–visitor and resident–resident), the payoffs received from tending residents are higher which may slow down or even prevent learning to prioritize visitors. Adjusting the plate paradigm to this more complex scenario, we found that exposure to homogeneous configurations indeed posed a challenge to cleaners' strategic prioritization, diverting their preferences towards residents. Our results also revealed individual differences in performance, as some cleaners still learned to prefer visitors. This suggests that cleaners may not only learn about each client type separately, and that learning about cue configurations, a precursor of learning complex structures, may play a role in shaping their strategic behaviour in cooperative interactions.

In the Democratic Republic of Congo, banana is the third largest source of starch after cassava and maize and is the primary fruit crop. However, its cultivation remains secondary due to its high mineral demand, the infertility of tropical soils (ferralsol) and the low income of farmers. The dynamics of mineral elements and the understanding of its influence on crop growth are a necessity for a better use of fertilizers and an optimal crop production. The objective of this study was to evaluate the influence of organo-mineral amendment inputs on banana growth and improvement of soil chemical properties. The experiment was set up in a randomised complete block design with 9 treatments spotted 6 times. The treatments consisted of T0 (unfertilized control) ; T1 : 250g NPK (10-20-10) +187.5g urea (46%) ; T2 : 500g NPK (10-20-10) + 375g urea (46%); T3 : 750g NPK (10-20-10) +562.5g urea (46%) ; T4 : 1000g NPK compound fertilizer (10-20-10) +750g urea (46%), T5 : 12 kg (chicken droppings) ; T6 : 18 kg (chicken droppings) ; T7 : 24 kg (chicken droppings) and T8 : 30 kg (chicken droppings) per plant. The vitroplant banana of the great dwarf cultivar were planted and vegetative parameters were observed. Before and during the trial, soil samples were taken from the surface layer for chemical analysis. The results show that the different doses applied to the soil significantly decreased the sodium content (p=0.008) after the trial. On the other hand, similar effects were observed on the other chemical parameters (K+, Mg2+, Ca2+, Total C and Organic N) before and during the experiment. On the crop, the results show positive effects of organic amendments and mineral fertilization on the development of banana vitroplants. The mineral fertilizer treatments showed a high performance on the crop followed by the manure treatments compared to the control treatment which showed a low performance. However, positive relationships were obtained on soil mineral elements with some crop parameters. Based on these results, it should be noted that the use of organo-mineral amendments contributes to the growth of banana and the reduction of sodium in the soil through the presence of organic matter.

Despite the unequivocal success of hematopoietic stem and progenitor cell gene therapy, limitations still exist including genotoxicity and variegation/silencing of transgene expression. A class of DNA regulatory elements known as chromatin insulators (CIs) can mitigate both vector transcriptional silencing (barrier CIs) and vector-induced genotoxicity (enhancer-blocking CIs) and have been proposed as genetic modulators to minimize unwanted vector/genome interactions. Recently, a number of human, small-sized, and compact CIs bearing strong enhancer-blocking activity were identified. To ultimately uncover an ideal CI with a dual, enhancer-blocking and barrier activity, we interrogated these elements in vitro and in vivo. After initial screening of a series of these enhancer-blocking insulators for potential barrier activity, we identified three distinct categories with no, partial, or full protection against transgene silencing. Subsequently, the two CIs with full barrier activity (B4 and C1) were tested for their ability to protect against position effects in primary cells, after incorporation into lentiviral vectors (LVs) and transduction of human CD34(+) cells. B4 and C1 did not adversely affect vector titers due to their small size, while they performed as strong barrier insulators in CD34(+) cells, both in vitro and in vivo, shielding transgene's long-term expression, more robustly when placed in the forward orientation. Overall, the incorporation of these dual-functioning elements into therapeutic viral vectors will potentially provide a new generation of safer and more efficient LVs for all hematopoietic stem cell gene therapy applications.

There is a clear demand for quantitative literacy in the life sciences, necessitating competent instructors in higher education. However, not all instructors are versed in data science skills or research-based teaching practices. We surveyed biological and environmental science instructors (n = 106) about the teaching of data science in higher education, identifying instructor needs and illuminating barriers to instruction. Our results indicate that instructors use, teach, and view data management, analysis, and visualization as important data science skills. Coding, modeling, and reproducibility were less valued by the instructors, although this differed according to institution type and career stage. The greatest barriers were instructor and student background and space in the curriculum. The instructors were most interested in training on how to teach coding and data analysis. Our study provides an important window into how data science is taught in higher education biology programs and how we can best move forward to empower instructors across disciplines.

The role of the sports cardiologist has evolved into an essential component of the medical care of athletes. In addition to the improvement in health outcomes caused by reductions in cardiovascular risk, exercise results in adaptations in cardiovascular structure and function, termed exercise-induced cardiac remodeling. As diagnostic modalities have evolved over the last century, we have learned much about the healthy athletic adaptation that occurs with exercise. Sports cardiologists care for those with known or previously unknown cardiovascular conditions, distinguish findings on testing as physiological adaptation or pathological changes, and provide evidence-based and "best judgment" assessment of the risks of sports participation. We review the effects of exercise on the heart, the approach to common clinical scenarios in sports cardiology, and the importance of a patient/athlete-centered, shared decision-making approach in the care provided to athletes.

Schistosomiasis is a water-based neglected tropical disease that is prevalent in over 78 countries. It affects communities that are reliant on freshwater bodies contaminated with Schistosome cercariae for their daily water activities. Whilst treatment with the drug praziquantel is relatively effective, it does not prevent reinfection. One option for reducing schistosomiasis infection is providing at-risk communities with treated water, thereby reducing contact with cercaria-infested water for activities such as bathing or doing laundry. This study aims to establish design guidance for sand filtration to remove schistosome cercariae from water. Four sand filters were tested, varying from 300 to 2000 μm in sand grain size. Each filter was tested with a sand depth of 20 cm, which was increased until no cercariae were detected in the effluent. The required filter depth to remove 100% of cercariae ranged between 40 and 70 cm depending on sand grain size. Cercaria removal was more effective in filters with smaller sand grain size and larger filter depth. These results are valid for intermittent flow, for up to six cycle flushes. While more rigorous testing is needed, these initial results suggest that sand filters can be an effective way to treat cercaria-contaminated water in low-income settings.

Nutritional stability – a food system’s capacity to provide sufficient nutrients despite disturbance – is an important, yet challenging to measure outcome of diversified agriculture. Using 55 years of data across 184 countries, we assemble 22,000 bipartite crop-nutrient networks to quantify nutritional stability by simulating crop and nutrient loss in a country, and assess its relationship to crop diversity across regions, over time and between imports versus in country production. We find a positive, saturating relationship between crop diversity and nutritional stability across countries, but also show that over time nutritional stability remained stagnant or decreased in all regions except Asia. These results are attributable to diminishing returns on crop diversity, with recent gains in crop diversity among crops with fewer nutrients, or with nutrients already in a country’s food system. Finally, imports are positively associated with crop diversity and nutritional stability, indicating that many countries’ nutritional stability is market exposed., Crop diversification could be important for food security. Here, using methods from network science, the authors find that a positive relationship between crop diversity and nutritional stability globally does not necessarily equate to improving nutritional stability in a given country.

Oxidative stress is a physiological condition in which reactive oxygen species created through cellular respiration can potentially damage DNA and tissue. Oxidative stress may partially mediate trade-offs between reproductive effort and survival efforts. On the one hand, traits associated with reproductive effort, particularly costly male-male competition, are expected to raise oxidative stress. On the other hand, behavioral strategies may be a critical mediating mechanism, such that those who can better resist the physiological costs of oxidative damage exhibit increased mating effort. In a sample of 248 college students (173 men), we examined the associations between traits linked to mating effort—including personality features, athleticism, and history of illness—with levels of 8-OHdG, a biomarker of oxidative stress. 8-OHdG was measured twice, one week apart, once during active hours and once at awakening. In men, surgency, social dominance, and athleticism were all negatively associated with 8-OHdG levels in awakening, but not lab samples collected during active hours. In women, these same traits were positively associated with 8-OHdG levels, particularly in morning samples. Differences in associations based on sex and time of collection introduce additional complexities to understanding links between oxidative stress and mating effort.

Bempedoic acid is an inhibitor of adenosine triphosphate–citrate lyase approved for use in adults with hypercholesterolemia. Nonclinical studies assessed binding to the human ether‐a‐go‐go–related gene (hERG) potassium channel in vitro and the effect of bempedoic acid on QT/QTc in cynomolgus monkeys. A randomized, double‐blind, parallel‐design clinical study assessed the effects of steady‐state bempedoic acid at a supratherapeutic dose (240 mg/day, 33.3% higher the180 mg/day therapeutic dose), placebo, and moxifloxacin (400 mg) in healthy subjects. In vitro binding potency for bempedoic acid to the hERG potassium channel was weak, with half‐maximal inhibition (IC50) estimated at greater than 1000 μM (>1670‐fold the bempedoic acid 180 mg/day steady‐state unbound maximum concentration). In monkeys, individual rate‐corrected QT intervals showed no time‐ or dose‐dependent changes up to 100 mg/kg of bempedoic acid. In human subjects, the upper 90% confidence interval (CI) for the difference in QTc interval, corrected using Fridericia’s formula (QTcF), between bempedoic acid and placebo was less than 5 msec at all time points. Concentration‐QTcF analysis showed that maximum bempedoic acid concentration at steady‐state was attained at a median 2.1 h postdose, and the predicted mean change (90% CI) in QTcF at the observed mean bempedoic acid concentration 2 h postdose was −0.5 (−5.0, 4.0) msec. The lower bound of the moxifloxacin 90% CI exceeded 5 msec at prespecified time points, establishing study sensitivity. Steady‐state bempedoic acid at a supratherapeutic dose of 240 mg was generally well‐tolerated and not associated with QTc prolongation in healthy subjects.

Trichoderma is an important genus of symbiotic fungi, commonly used around the world as biocontrol agents and as biofertilizer. Although their beneficial effects are well known and are successfully exploited in sustainable agriculture practices, the biochemical mechanisms of plant growth-promoting actions of Trichoderma and their anti-pathogen characteristics are not well understood. This study biochemically surveyed 22 strains of Trichoderma and shows that Trichoderma produces cytokinins (CKs), which has not been reported to date. The phytohormone profiles ranged from 5.34 to 379.99 pmol CKs released to 10 mL of the growth medium and comprised riboside and nucleotide derivatives of cis-zeatin (cZ) and isopentenyladenine (iP), suggesting that fungal CKs originate from a tRNA degradation pathway. We reveal a connection between the levels of free base cZ produced by Trichoderma and the inhibition rate against the pathogen Fusarium graminearum among the tested strains. Furthermore, we analyzed CK profiles of Arabidopsis plants cultured in vitro in the presence of Trichoderma strains. The inoculated plants showed increased levels of cZ-type (cZR, cZROG) and iP-type (iP, iPR) CKs—the forms which dominated CK profiles of all the fungal in vitro cultures tested in this study. The increase in the levels of cZ derivatives was accompanied by a significant reduction in plant trans-zeatin (tZ)-type CKs (tZR, tZNT, tZOG, tZ7G, tZ9G) in Arabidopsis when co-cultured with the fungus. Our work suggests that CKs produced by plant symbiotic Trichoderma strains can be used for plant growth stimulation, may impact the colonization strategy of symbiotic fungi, and include alterations to the host plant phytohormones for enhanced plant resistance against pathogens.

Benzimidazole-2-carbamate (BZ) compounds, including Albendazole (Alb), are one of just two drug classes approved to treat the gastrointestinal protist Giardia duodenalis. Benzimidazoles bind to the tubulin dimer interface overlapping the colchicine binding site (CBS) of β-tubulin, thereby inhibiting microtubule polymerisation and disrupting microtubule networks. These BZ compounds are widely used as anthelmintic, anti-fungal and anti-giardial drugs. However, in helminths and fungi, BZ-resistance is widespread and caused by specific point mutations primarily occurring at F167, E198 and F200 in β-tubulin isoform 1. BZ-resistance in Giardia is reported clinically and readily generated in vitro, with significant implications for Giardia control. In Giardia, BZ mode of action (MOA) and resistance mechanisms are presumed but not proven, and no mutations in β-tubulin have been reported in association with Alb resistance (AlbR). Herein, we undertook detailed in vitro drug-susceptibility screens of 13 BZ compounds and 7 Alb structural analogues in isogenic G. duodenalis isolates selected for AlbR and podophyllotoxin, another β-tubulin inhibitor, as well as explored cross-resistance to structurally unrelated, metronidazole (Mtz). AlbR lines exhibited co-resistance to many structural variants in the BZ-pharmacophore, and cross-resistance to podophyllotoxin. AlbR lines were not cross-resistant to Mtz, but MtzR lines had enhanced survival in Alb. Lastly, Alb analogues with longer thioether substituents had decreased potency against our AlbR lines. In silico modelling indicated the Alb-β-tubulin interaction in Giardia partially overlaps the CBS and corresponds to residues associated with BZ-resistance in helminths and fungi (F167, E198, F200). Sequencing of Giardia β-tubulin identified a single nucleotide polymorphism resulting in a mutation from glutamic acid to lysine at amino acid 198 (E198K). To our knowledge, this is the first β-tubulin mutation reported for protistan BZ-resistance. This study provides insight into BZ mode of action and resistance in Giardia, and presents a potential avenue for a genetic test for clinically resistance isolates., Graphical abstract Image 1, Highlights • In vitro albendazole-resistant Giardia were broadly resistant to benzimidazole-2-carbamates. • Cross-resistance to structurally unrelated microtubule inhibitors was observed. • The predicted Giardia benzimidazole binding overlaps the colchicine binding site. • The E198K β-tubulin mutation was identified in the albendazole-resistant line. • Metronidazole-resistance may enhance acquisition of albendazole-resistance.

Background Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in aggressive timelines. Methods In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the severe acute respiratory syndrome coronavirus-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity. Results The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. Conclusions By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.

Context Causes of anterior knee pain (AKP) in jumping athletes include patellofemoral pain and patellar tendinopathy. The differential diagnosis of AKP is challenging, with variations in clinical presentations. No previous research has used pain location to describe AKP in basketball players. Objective To (1) describe the prevalence and pain distribution of AKP in collegiate basketball players and (2) report the prevalence of focal inferior pole pain using 2 outcome measures. Design Cross-sectional study. Setting University and collegiate basketball facilities in Alberta, Canada. Patients or Other Participants A total of 242 collegiate basketball athletes (138 women, 104 men). Main Outcome Measure(s) The single-legged decline squat test (SLDS) was used to capture pain location via pain mapping (dichotomized as focal or diffuse) and pain severity (numeric rating scale). The Oslo Sports Trauma Research Centre Knee questionnaire (OSTRC-Knee) and adapted version for patellar tendinopathy (OSTRC-Patellar Tendinopathy Questionnaire [OSTRC-P]) were used to report the prevalence of AKP and patellar tendinopathy, respectively. Focal inferior pole pain during the SLDS was used to classify patellar tendinopathy. Results Of the 242 players, 146 (60%) reported pain with the SLDS (unilateral = 64 [26%]; bilateral = 82 [34%]). A total of 101 (43%) described knee pain using the OSTRC-Knee. Pain mapping captured the variability in pain locations. Diffuse pain was more prevalent (left, 70%; right, 72%) than focal pain (left, 30%; right, 28%). Low prevalence of patellar tendinopathy was noted using the OSTRC-P (n = 21, 8.7%) and inferior pole pain during the SLDS (n = 25, 10.3%). Conclusions Diffuse AKP was common in Canadian basketball players; however, pain mapped to the inferior pole of the patella was not common. Few players reported tendinopathy using the OSTRC-P, suggesting that patellar tendinopathy was not a primary knee pain presentation in this jumping cohort. Pain location, rather than the presence or severity of pain alone, may better describe the clinical presentation of AKP in jumping athletes.

This study was supported by Grants from the US National Science Foundation including OCE 1458845 and the Santa Barbara Coastal Long-Term Ecological Research project (OCE 1232779, OCE 1831937). Declines in species diversity carry profound implications for ecosystem functioning. Communities of primary producers and consumers interact on evolutionary as well as ecological time scales, shaping complex relationships between biodiversity and ecosystem functioning. In subsidized ecosystems, resource inputs are independent of consumer actions, offering a simplified view of the relationship between species diversity and function for higher trophic levels. With food webs supported by substantial but variable inputs of detritus from adjacent marine ecosystems, sandy beaches are classic examples of subsidized ecosystems. We investigated effects of consumer species diversity and identity on a key ecological function, consumption of kelp wrack from nearshore giant kelp (Macrocystis pyrifera) forests. We assessed effects of species richness on kelp consumption by experimentally manipulating richness of six common species of invertebrate detritivores in laboratory mesocosms and conducting field assays of kelp consumption on beaches. Consumer richness had no effect on kelp consumption in the field and a slight negative effect in laboratory experiments. Kelp consumption was most strongly affected by the species composition of the detritivore community. Species identity and body size of intertidal detritivores drove variation in kelp consumption rates in both experiments and field assays. Our results provide further evidence that species traits, rather than richness per se, influence ecosystem function most, particularly in detrital-based food webs with high functional redundancy across species. On sandy beaches, where biodiversity is threatened by rising sea levels and expanding development, our findings suggest that loss of large-bodied consumer species could disproportionally impact ecosystem function. Postprint Postprint

Objectives We measured the production of cytokines, chemokines and antibodies involved in allergic responses and sCD23 levels during Schistosoma mansoni infection. Methods Individuals (n = 164) were selected using the ISAAC questionnaire and parasitological exams. The subjects were divided as follows: those infected individuals with allergy-related symptoms (A-I), those with allergy-related symptoms only (A-NI); those only infected (NA-I); and those non-infected individuals without allergy-related symptoms (NA-NI). We used supernatants from cell culture (mitogenic stimulation) to measure cytokine and chemokine levels using cytometric bead arrays. Serum levels of anti-Ascaris lumbricoides (Asc) and anti-Blomia tropicalis IgE were measured using ImmunoCAP, and sCD23 was measured using ELISA. Results Schistosoma mansoni infection was associated with a lower risk of allergy-related symptoms. In A-I, there were higher levels of TNF-α, IL-10, IL-6, IFN-γ and CXCL8 than in NA-NI group, with TNF-α and IL-6 also at higher levels compared to A-NI group. Levels of IL-6, CXCL8, total and anti-Asc IgE, as well as the numbers of eosinophils, were higher in NA-I than in NA-NI, and the antibodies were also lower in A-NI than in NA-I group. In AI and NA-I, there was less production of CCL2 than in NA-NI. There were no differences in the levels of IL-2, IL-4, IL-17, CCL5, sCD23 and anti-Blomia IgE. Conclusions Patients with allergy-related symptoms and infected (simultaneously) had higher levels of IL-10; due to the infection, there was increased production of IL-6 and CXCL8 and less CCL2. These data may characterize deviation to Th1 or attenuation of the Th2 response in allergy sufferers in areas endemic for schistosomiasis.

Background: Gut microbial communities are increasingly being linked to diseases in animals and humans. Obesity and its associated diseases are a concern for horse owners and veterinarians, and there is a growing interest in the link between diet, the intestinal microbiota and metabolic disease. Objectives: Assess the influence of long-term hay or haylage feeding on the microbiota and metabolomes of 20 Welsh mountain ponies. Study design: Longitudinal study. Methods: Urine, faeces and blood were collected from 20 ponies on a monthly basis over a 13-month period. Urine and faeces were analysed using proton magnetic resonance (1 H NMR) spectroscopy and faecal bacterial DNA underwent 16S rRNA gene sequencing. Results: Faecal bacterial community profiles were observed to be different for the two groups, with discriminant analysis identifying 102 bacterial groups (or operational taxonomic units, OTUs) that differed in relative abundance in accordance with forage type. Urinary metabolic profiles of the hay and haylage fed ponies were significantly different during 12 of the 13 months of the study. Notably, the urinary excretion of hippurate was greater in the hay fed ponies for the duration of the study, while ethyl-glucoside excretion was higher in the haylage fed ponies. Main limitations: The study was undertaken over a 13-month period and both groups of ponies had access to pasture during the summer months. Conclusions: The data generated from this study, suggest that the choice of forage may have implications for the intestinal microbiota and metabolism of ponies and therefore, potentially their health status. Understanding the potential implication of feeding a particular type of forage will enable horse owners to make more informed choices with regard to feed, especially if their horse or pony is prone to weight gain.

Previous work in several laboratories has demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of DNA and RNA viruses, yet the underlying mechanisms responsible for this positive effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how m6A readers regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is in large part mediated by the the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs while YTHDF2 binding correlates with increased HIV-1 transcript stability.Author SummaryThis manuscript reports that the expression of mRNAs encoded by the pathogenic human retrovirus HIV-1 is regulated by the methylation of a small number of specific adenosine residues. These in turn recruit a nuclear RNA binding protein, called YTHDC1, which modulates the alternative splicing of HIV-1 transcripts, as well as a cytoplasmic RNA binding protein, called YTHDF2, which stabilizes viral mRNAs. The regulation of HIV-1 gene expression by adenosine methylation is therefore critical for the effective and ordered expression of HIV-1 mRNAs and could represent a novel target for antiviral development.

Is it possible to slow the rate of ageing, or do biological constraints limit its plasticity? We test the ‘invariant rate of ageing’ hypothesis, which posits that the rate of ageing is relatively fixed within species, with a collection of 39 human and nonhuman primate datasets across seven genera. We first recapitulate, in nonhuman primates, the highly regular relationship between life expectancy and lifespan equality seen in humans. We next demonstrate that variation in the rate of ageing within genera is orders of magnitude smaller than variation in pre-adult and age-independent mortality. Finally, we demonstrate that changes in the rate of ageing, but not other mortality parameters, produce striking, species-atypical changes in mortality patterns. Our results support the invariant rate of ageing hypothesis, implying biological constraints on how much the human rate of ageing can be slowed., The ‘invariant rate of ageing’ hypothesis suggests that the rate of ageing tends to be constant within species. Here, Colchero et al. find support for the hypothesis across primates, including humans, suggesting biological constraints on the rate of ageing.

Cellular differentiation requires vast remodeling of transcriptomes, and therefore machinery mediating remodeling controls differentiation. Relative to transcriptional mechanisms governing differentiation, post-transcriptional processes are less well understood. As an important post-transcriptional determinant of transcriptomes, the RNA exosome complex (EC) mediates processing and/or degradation of select RNAs. During erythropoiesis, the erythroid transcription factor GATA1 represses EC subunit genes. Depleting EC structural subunits prior to GATA1-mediated repression is deleterious to erythroid progenitor cells. To assess the importance of the EC catalytic subunits Dis3 and Exosc10 in this dynamic process, we asked if these subunits function non-redundantly to control erythropoiesis. Dis3 or Exosc10 depletion in primary murine hematopoietic progenitor cells reduced erythroid progenitors and their progeny, while sparing myeloid cells. Dis3 loss severely compromised erythroid progenitor and erythroblast survival, rendered erythroblasts hypersensitive to apoptosis-inducing stimuli and induced γ-H2AX, indicative of DNA double-stranded breaks. Dis3 loss-of-function phenotypes were more severe than those caused by Exosc10 depletion. We innovated a genetic rescue system to compare human Dis3 with multiple myeloma-associated Dis3 mutants S447R and R750K, and only wild type Dis3 was competent to rescue progenitors. Thus, Dis3 establishes a disease mutation-sensitive, cell type-specific survival mechanism to enable a differentiation program.

The number of organisms that are known to produce cytokinins (CKs) continues to increase. In fact, species from all life kingdoms have now been shown to either produce CKs or at least have the genetic components to make it possible. In vitro growth of microorganisms, plant/animal cells, and tissue cultures often requires nutrient-rich media composed of ingredients with organic origins including: yeast extract, peptone, tryptone, or various plant or animal extracts. These compounds, derived from microbial, plant and animal materials, can be the source of significant levels of exogenous CKs in the culture medium. As CK investigative work continues to expand rapidly, it is of critical importance to draw attention to this complexity; the presence of CKs in growth medium affects CK metabolism of the cultured organism and interferes with the readings of analytical instrumentation used to profile CKs in tested microorganisms or cell cultures.

Context How specific coping strategies are associated with short-term outcomes among athletes after knee surgery is unknown. Objective To determine whether (1) specific coping strategies were associated with return to sport, satisfaction, self-reported knee function, or kinesiophobia after sport-related knee surgery and (2) these associations varied by age, sex, or surgical procedure. Study Design Case series. Methods Athletes (N = 184; men: n = 104, women: n = 80; aged 40 years: n = 39) who underwent outpatient knee surgery at a single center were enrolled. Use of specific coping strategies (self-distraction, venting, acceptance, positive reframing, emotional support, and instrumental support) was assessed preoperatively with the Brief-Coping Orientation to Problems Experienced inventory. Relationships among coping strategies and postoperative satisfaction, return to sport, International Knee Documentation Committee–Subjective and Tampa Scale for Kinesiophobia scores at a median 10.7 months follow-up were determined with consideration for age, sex, and surgical procedure. Results Return to the prior level of sport was 72%, and satisfaction was 86%. Most coping strategies had age-specific utilization rates; positive reframing was used least frequently in individuals aged 40 years. No other coping strategies were associated with outcomes. The surgical procedure was not related to an association between coping strategies and outcomes. Conclusions Coping strategies had age-specific associations with outcomes after knee surgery in athletes. Positive reframing was infrequently used in younger athletes. Greater use of positive reframing in this group may improve return to sport and satisfaction and reduce fear of reinjury.

A young French Bulldog was presented with clinical signs of chronic gastrointestinal disease, unresponsive to medical therapies. Parasite screenings and abdominal ultrasound failed to identify the etiology of the clinical signs. Cytologic evaluation of a rectal scraping sample diagnosed presumptive granulomatous colitis (GC) based on the presence of numerous macrophages with characteristic abundant, pink, granular cytoplasm, which showed an intense pink color when stained with periodic acid-Schiff. Tissue biopsy samples and Escherichia coli fluorescence in situ hybridization analysis confirmed the cytologic diagnosis. The cytologic, histopathologic, and clinical features and staining properties of GC in a French Bulldog are reported. Rectal scraping should be considered a part of the diagnostic evaluation in patients with suspected GC.

The interactive effect of seasonal temperatures and water potential (Ψ) were examined on the germination of four co-occurring Eucalyptus species (Eucalyptus melanophloia F.Muell., E. pilligaensis Maiden, E. populnea subsp. bimbil L.A.S.Johnson & K.D.Hill and E. viridis R.T.Baker) from the Brigalow Belt South bioregion of New South Wales. These eucalypts are important canopy species, and are a key inclusion for restoring native vegetation to areas that have had significant landscape modification. Final germination, time to first germination, and time to 50% germination were recorded at three alternating temperature regimes for winter (day/night, 19/5°C), spring–autumn (27/12°C) and summer (34/20°C), and eight different Ψ treatments between saturation point (0 MPa) and permanent wilting point (–1.5 MPa) under controlled laboratory conditions. Final germination was highest in all species when seeds were incubated at winter conditions, but spring–autumn and summer temperatures had mixed effects on final germination, depending on the species. Final germination consistently declined with lower Ψ across all temperatures for E. pilliagaensis, E. populnea subsp. bimbil and E. viridis, whereas E. melanophloia displayed a mixed response to temperature and Ψ. No germination occurred when Ψ was lower than –0.25 MPa. Time to first germination and time to reach 50% germination (t50) were lowest at summer temperatures for all species, and all species except E. pilligaensis experienced higher time to first germination and t50 (i.e. slower germination rates) as Ψ decreased from 0 to –0.25 MPa. Our results indicate that all four eucalypts should be treated as drought-sensitive species with a higher probability of field recruitment during cooler and wet conditions.

Plant lignin content and composition, which limit cell wall digestibility and efficiency of cellulose conversion to bioethanol, can be influenced by belowground biotic and abiotic factors. Switchgrass (Panicum virgatum L.) is a leading lignocellulosic biofuel crop and forms strong belowground associations with arbuscular mycorrhizal fungi (AMF), is susceptible to belowground plant-parasitic nematodes (PPN), and when grown in monoculture generally requires nitrogen (N) fertilization. The main objectives of the study were to investigate the effects of N fertilizer and belowground organisms on lignin content and composition of switchgrass. Leaf, stem, and root tissues were evaluated separately to test whether these factors had varying belowground (local) or aboveground (systemic) effects on plants. These factors were manipulated in a field study in 2017 using biocide applications to reduce soil fungi and nematodes. Combined biocide application reduced p-hydroxyphenyl (H) unit abundance in the leaves by 14% and increased the syringyl:guaiacyl (S:G) ratio in stems by 2%. Application of fungicide alone increased stem syringyl (S) unit by 12.4% as compared with control plots, and 11.1% as compared with nematicide plots. Overall, fertilizer increased total stem lignin by 3%, stem S unit by 6.7%, and stem S:G ratio by 10%, whereas it reduced the amount of H-unit in the roots by 11%. While the effects of N fertilizer were more pronounced in this study, changes to soil organisms had similar magnitudes of effect for some measures of lignin, indicating that these belowground interactions may be important for growers to consider in the future.

Background:Return to sports (RTS) is frequently considered an indicator of successful recovery after anterior cruciate ligament reconstruction (ACLR). However, despite the well-recognized health benefits of physical activity (PA), little is known about objectively measured PA in the 1 to 2 years after ACLR. Given that young female athletes have a high prevalence of ACLR and lower RTS rates as compared with their male counterparts, an in-depth examination of PA in this subgroup is warranted.Hypothesis:We hypothesized that female youth and young adults who have had ACLR in the previous 1 to 2 years would have less moderate or vigorous PA (MVPA) compared with healthy matched controls. We also hypothesized that the ACLR group would report lower levels of sports participation, patient-reported health outcomes, and physical function.Study Design:Cohort study; Level of evidence, 3.Methods:Participants included 51 female athletes with primary unilateral ACLR for a sports-related injury in the previous 1 to 2 years and 51 age- and sports-matched controls. Outcomes included objectively measured PA (GT3X accelerometers), previous and current sports participation and RTS, body mass index, Knee injury and Osteoarthritis Outcome Score (KOOS), triple single-leg hop, and one-leg rise. Mean within-pair differences with 95% CIs were used to assess differences between groups across all outcomes. Multivariable linear regression (clustered by pair) was used to examine whether the ACLR group had less MVPA than did the age- and sports-matched control group, adjusting for total wear time, age, time since injury, and body mass index.Results:Median age was 17.8 years (range, 14.6-22.6 years). There was no significant difference between groups in MVPA. However, the injury group had fewer mean minutes per day of vigorous PA (–1.22; 95% CI, −2.40 to −0.04), poorer KOOS values on all subscales, and shorter triple single-leg hop distance. In the injury group, 28 (55%) returned to sports, including 14 (27.5%) who returned at preinjury performance level. Across both groups, over one-third changed their most important sport, shifting toward an individual-based sport.Conclusion:At 1 to 2 years after ACLR, female athletes demonstrated no differences in combined MVPA and only a very small reduction in vigorous PA, yet they had higher levels of self-reported knee pain and symptoms, reduced knee function in sports, lower quality of life, and poorer objective knee function compared with matched controls.

Trichomonas vaginalis is a neglected urogenital parasitic protist that causes 170 million cases of trichomoniasis annually, making it the most prevalent non-viral, sexually transmitted disease. Trichomoniasis treatment relies on nitroheterocyclics, such as metronidazole. However, with increasing drug-resistance, there is an urgent need for novel anti-trichomonals. Little progress has been made to translate anti-trichomonal research into commercialised therapeutics, and the absence of a standardised compound-screening platform is the immediate stumbling block for drug-discovery. Herein, we describe a simple, cost-effective growth assay for T. vaginalis and the related Tritrichomonas foetus. Tracking changes in pH were a valid indicator of trichomonad growth (T. vaginalis and T. foetus), allowing development of a miniaturised, chromogenic growth assay based on the phenol red indicator in 96- and 384-well microtiter plate formats. The outputs of this assay can be quantitatively and qualitatively assessed, with consistent dynamic ranges based on Z′ values of 0.741 and 0.870 across medium- and high-throughput formats, respectively. We applied this high-throughput format within the largest pure-compound microbial metabolite screen (812 compounds) for T. vaginalis and identified 43 hit compounds. We compared these identified compounds to mammalian cell lines, and highlighted extensive overlaps between anti-trichomonal and anti-tumour activity. Lastly, observing nanomolar inhibition of T. vaginalis by fumagillin, and noting this compound has reported activity in other protists, we performed in silico analyses of the interaction of fumagillin with its molecular target methionine aminopeptidase 2 for T. vaginalis, Giardia lamblia and Entamoeba histolytica, highlighting potential for fumagillin as a broad-spectrum anti-protistal against microaerophilic protists. Together, this new platform will accelerate drug-discovery efforts, underpin drug-resistance screening in trichomonads, and contributing to a growing body of evidence highlighting the potential of microbial natural products as novel anti-protistals., Graphical abstract Image 1

Supplemental Digital Content is available in the text., Background: Cardiac involvement among hospitalized patients with severe coronavirus disease 2019 (COVID-19) is common and associated with adverse outcomes. This study aimed to determine the prevalence and clinical implications of COVID-19 cardiac involvement in young competitive athletes. Methods: In this prospective, multicenter, observational cohort study with data from 42 colleges and universities, we assessed the prevalence, clinical characteristics, and outcomes of COVID-19 cardiac involvement among collegiate athletes in the United States. Data were collected from September 1, 2020, to December 31, 2020. The primary outcome was the prevalence of definite, probable, or possible COVID-19 cardiac involvement based on imaging definitions adapted from the Updated Lake Louise Imaging Criteria. Secondary outcomes included the diagnostic yield of cardiac testing, predictors for cardiac involvement, and adverse cardiovascular events or hospitalizations. Results: Among 19 378 athletes tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, 3018 (mean age, 20 years [SD, 1 year]; 32% female) tested positive and underwent cardiac evaluation. A total of 2820 athletes underwent at least 1 element of cardiac triad testing (12-lead ECG, troponin, transthoracic echocardiography) followed by cardiac magnetic resonance imaging (CMR) if clinically indicated. In contrast, primary screening CMR was performed in 198 athletes. Abnormal findings suggestive of SARS-CoV-2 cardiac involvement were detected by ECG (21 of 2999 [0.7%]), cardiac troponin (24 of 2719 [0.9%]), and transthoracic echocardiography (24 of 2556 [0.9%]). Definite, probable, or possible SARS-CoV-2 cardiac involvement was identified in 21 of 3018 (0.7%) athletes, including 15 of 2820 (0.5%) who underwent clinically indicated CMR (n=119) and 6 of 198 (3.0%) who underwent primary screening CMR. Accordingly, the diagnostic yield of CMR for SARS-CoV-2 cardiac involvement was 4.2 times higher for a clinically indicated CMR (15 of 119 [12.6%]) versus a primary screening CMR (6 of 198 [3.0%]). After adjustment for race and sex, predictors of SARS-CoV-2 cardiac involvement included cardiopulmonary symptoms (odds ratio, 3.1 [95% CI, 1.2, 7.7]) or at least 1 abnormal triad test result (odds ratio, 37.4 [95% CI, 13.3, 105.3]). Five (0.2%) athletes required hospitalization for noncardiac complications of COVID-19. During clinical surveillance (median follow-up, 113 days [interquartile range=90 146]), there was 1 (0.03%) adverse cardiac event, likely unrelated to SARS-CoV-2 infection. Conclusions: SARS-CoV-2 infection among young competitive athletes is associated with a low prevalence of cardiac involvement and a low risk of clinical events in short-term follow-up.

Cyanobacteria are key primary producers in the environment, models for photosynthesis and the circadian clock in the lab, and emerging biological production platforms for industry. Despite their scope of importance some of the most fundamental components of their biology are poorly understood. We know little about the functions encoded by most of their genes, and research traditionally focused on constant light has left the organisms’ physiology in day-night conditions obscure. In the studies comprising my thesis, we worked to illuminate both of these knowledge gaps in the model cyanobacterium Synechococcus elongatus PCC 7942. We employed traditional molecular biology and -omic techniques, and developed and applied a high-throughput approach for whole genome mutant screens in S. elongatus (RB-TnSeq). Chapter one introduces what is known about cyanobacterial physiology in Light-Dark Cycles (LDCs). It also describes RB-TnSeq, which is used here to elucidate gene function both generally and specifically to LDCs. Chapter two reports the characterization of cellular activities upon light transitions that facilitate survival in LDCs. Chapter three presents the development of RB-TnSeq in S. elongatus and its use for the assignment of gene importance, the development of an improved metabolic model for S. elongatus, and the implementation of screens to further our understanding of these genes'. Chapter four reports the application of RB-TnSeq to understand survival in LDCs. In it, we identified the set of genes specifically important for survival of LDCs and followed up on them. This work resulted in improved understanding for the roles of the circadian clock and nucleotide signaling in LDC survival. Chapter five concludes by synthesizing the core achievements of the dissertation and suggesting future directions. Together, these chapters explain the development of a powerful genomic approach, RB-TnSeq, and its use to illuminate the genetic unknowns in Cyanobacteria as well as the organisms’ LDC physiology. These findings will be applicable to the basic understanding of this important phylum, its industrial use, and photosynthetic organisms more generally, for which Cyanobacteria are the most tractable models.

Introduction and objective: Sickle cell disease is an inherited pathology to an abnormality of hemoglobin precisely hemoglobin S for which there is no curative therapy. It mainly affects sub-Saharan African and Caribbean populations. Thus, this study aims to make the phytochemical screening ofCremaspora trifloraandMacaranga schweinfurthiias well as to evaluate the antisickling activity of their purified alkaloids. Methodology: Chemical screening was performed using color and precipitation tests as well as the foam index method. The extraction of the alkaloids was carried out with organic solvents in a basic medium while the purification by open column chromatography. The evaluation of the antisickling activity was carried out by Emmel’s test. Results: The chemical screening highlighted alkaloids, steroids, saponins, tannins, and terpenoids in the species studied. Flavonoids and anthocyanins were present in organs ofMacaranga schweinfurthii, but absent inCremaspora triflora. The extraction showed thatMacaranga schweinfurthiileaves contained 0.59% alkaloids and 0.73% alkaloids inCremaspora trifloraleaves. The alkaloids purification allowed to obtain an alkaloidal fraction MS1 (1.24 g, 70.05%) fromMacaranga schweinfurthiiand two fractions [CT2 (0.934 g, 63.97%) and CT3 (0.006 g, 0.41 %)] fromCremaspora triflorawhich tested positive with Dragendorff and Wagner reagents. The antisickling activity evaluation showed that the SIR varied between 36.00% (0.25 mg/ml) and 90.66% (1 mg/ml) for the alkaloid solutions ofCremaspora triflora(IC50of 0.51 mg/ml) as well as between 4.00% (0.25 mg/ml) and 33.33% (1 mg/ml) for the alkaloid solutions ofMacaranga schweinfurthii(IC50of 1.40 mg/ml). Conclusion: This study showed that the purified alkaloids of the studied plant species have an inhibitory power on sickling.&nbsp

BackgroundForkhead transcription factors control cell growth in multiple cancer types.Foxd1is essential for kidney development and mitochondrial metabolism, but its significance in renal cell carcinoma (ccRCC) has not been reported.MethodsTranscriptome data from the TCGA database was used to correlateFOXD1expression with patient survival.FOXD1was knocked out in the 786-O cell line and known targets were analyzed. Reduced cell growth was observed and investigated in vitro using growth rate and Seahorse XF metabolic assays and in vivo using a xenograft model. Cell cycle characteristics were determined by flow cytometry and immunoblotting. Immunostaining for TUNEL and γH2AX was used to measure DNA damage. Association of theFOXD1pathway with cell cycle progression was investigated through correlation analysis using the TCGA database.ResultsFOXD1expression level in ccRCC correlated inversely with patient survival. Knockout ofFOXD1in 786-O cells altered expression of FOXD1 targets, particularly genes involved in metabolism (MICU1) and cell cycle progression. Investigation of metabolic state revealed significant alterations in mitochondrial metabolism and glycolysis, but no net change in energy production. In vitro growth rate assays showed a significant reduction in growth of 786-OFOXD1null. In vivo, xenografted 786-OFOXD1nullshowed reduced capacity for tumor formation and reduced tumor size. Cell cycle analysis showed that 786-OFOXD1nullhad an extended G2/M phase. Investigation of mitosis revealed a deficiency in phosphorylation of histone H3 in 786-OFOXD1null, and increased DNA damage. Genes correlate withFOXD1in the TCGA dataset associate with several aspects of mitosis, including histone H3 phosphorylation.ConclusionsWe show that FOXD1 regulates the cell cycle in ccRCC cells by control of histone H3 phosphorylation, and that FOXD1 expression governs tumor formation and tumor growth. Transcriptome analysis supports this role for FOXD1 in ccRCC patient tumors and provides an explanation for the inverse correlation between tumor expression ofFOXD1and patient survival. Our findings reveal an important role for FOXD1 in maintaining chromatin stability and promoting cell cycle progression and provide a new tool with which to study the biology of FOXD1 in ccRCC.

The Trans-Omics for Precision Medicine (TOPMed) programme seeks to elucidate the genetic architecture and biology of heart, lung, blood and sleep disorders, with the ultimate goal of improving diagnosis, treatment and prevention of these diseases. The initial phases of the programme focused on whole-genome sequencing of individuals with rich phenotypic data and diverse backgrounds. Here we describe the TOPMed goals and design as well as the available resources and early insights obtained from the sequence data. The resources include a variant browser, a genotype imputation server, and genomic and phenotypic data that are available through dbGaP (Database of Genotypes and Phenotypes)1. In the first 53,831 TOPMed samples, we detected more than 400 million single-nucleotide and insertion or deletion variants after alignment with the reference genome. Additional previously undescribed variants were detected through assembly of unmapped reads and customized analysis in highly variable loci. Among the more than 400 million detected variants, 97% have frequencies of less than 1% and 46% are singletons that are present in only one individual (53% among unrelated individuals). These rare variants provide insights into mutational processes and recent human evolutionary history. The extensive catalogue of genetic variation in TOPMed studies provides unique opportunities for exploring the contributions of rare and noncoding sequence variants to phenotypic variation. Furthermore, combining TOPMed haplotypes with modern imputation methods improves the power and reach of genome-wide association studies to include variants down to a frequency of approximately 0.01%., The goals, resources and design of the NHLBI Trans-Omics for Precision Medicine (TOPMed) programme are described, and analyses of rare variants detected in the first 53,831 samples provide insights into mutational processes and recent human evolutionary history.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z′ values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

We evaluated interleukin-6 (IL-6) receptor-α subunit (IL-6Rα) signaling inhibition with sarilumab and tocilizumab, the association between IL-6Rα receptor occupancy (RO) and C-reactive protein (CRP), and the potential clinical relevance of any differences. For this, we measured IL-6Rα binding and signaling inhibition with sarilumab and tocilizumab in vitro, simulated soluble IL-6Rα RO over time for approved sarilumab subcutaneous (SC) and tocilizumab intravenous (IV) and SC doses, and assessed associations between calculated RO and CRP reduction, 28-joint Disease Activity Score based on CRP, and 20%/50%/70% improvement in American College of Rheumatology responses from clinical data. Sarilumab binds IL-6Rα in vitro with 15- to 22-fold higher affinity than tocilizumab, and inhibits IL-6–mediated classical and trans signaling via membrane-bound and soluble IL-6Rα. Sarilumab 200 and 150 mg SC every 2 weeks achieved >90% RO after first and second doses, respectively, maintained throughout the treatment period. At steady-state trough, RO was greater with sarilumab 200 mg (98%) and 150 mg SC every 2 weeks (94%), and tocilizumab 162 mg SC weekly (>99%) and 8 mg/kg IV every 4 weeks (99%), vs tocilizumab 162 mg SC every 2 weeks (84%) and 4 mg/kg IV every 4 weeks (60%). Higher RO was associated with greater CRP reduction and 28-joint Disease Activity Score based on CRP reduction, and more sarilumab patients achieving 20%/50%/70% improvement in American College of Rheumatology responses. The greatest reduction in CRP levels was observed with sarilumab (both doses) and tocilizumab 8 mg/kg IV every 4 weeks (reductions proportionally smaller with 4 mg/kg IV every 4 weeks). Higher IL-6Rα binding affinity translated into higher RO with sarilumab vs tocilizumab 4 mg/kg every 4 weeks or 162 mg every 2 weeks; tocilizumab required the higher dose or increased frequency to maintain the same degree of RO and CRP reduction. Higher RO was associated with clinical parameter improvements.

It is generally agreed that variation in social and/or environmental complexity yields variation in selective pressures on brain anatomy, where more complex brains should yield increased intelligence. While these insights are based on many evolutionary studies, it remains unclear how ecology impacts brain plasticity and subsequently cognitive performance within a species. Here, we show that in wild cleaner fish (Labroides dimidiatus), forebrain size of high-performing individuals tested in an ephemeral reward task covaried positively with cleaner density, while cerebellum size covaried negatively with cleaner density. This unexpected relationship may be explained if we consider that performance in this task reflects the decision rules that individuals use in nature rather than learning abilities: cleaners with relatively larger forebrains used decision-rules that appeared to be locally optimal. Thus, social competence seems to be a suitable proxy of intelligence to understand individual differences under natural conditions., The causes and consequences of social intelligence are challenging to establish. A study on wild cleaner fish reports that large forebrains enable individuals to score higher in a social competence test, suggesting forebrain size is important for complex social decision-making.

Although conformational dynamics of RNA molecules are potentially important in microRNA (miRNA) processing, the role of the protein binding partners in facilitating the requisite structural changes is not well understood. In previous work, we and others have demonstrated that nonduplex structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing. However, recent studies showed that the protein DGCR8 binds primary miRNA and duplex RNA with similar affinities. Here, we study RNA binding by a small recombinant construct of the DGCR8 protein and the RNA conformation changes that result. This construct, the DGCR8 core, contains two double-stranded RNA-binding domains (dsRBDs) and a C-terminal tail. To assess conformational changes resulting from binding, we applied small-angle x-ray scattering with contrast variation to detect conformational changes of primary-miR-16-1 in complex with the DGCR8 core. This method reports only on the RNA conformation within the complex and suggests that the protein bends the RNA upon binding. Supporting work using smFRET to study the conformation of RNA duplexes bound to the core also shows bending. Together, these studies elucidate the role of DGCR8 in interacting with RNA during the early stages of miRNA processing.

Background Angiotensin II type 1 receptor antibody (AT1R-Ab) is a non-HLA antibody that has been reported to cause antibody-mediated rejection and graft loss in kidney transplantation. The prevalence of positive AT1R-Ab varies between 8% and 18% in different regions. Thus, this study aims to determine the prevalence of AT1R-Ab among the Malaysian population. Methodology All sera for AT1R-Ab were collected at the University Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. The sera were centrifuged and kept refrigerated at −80 °C before being transported to the South Australian Transplantation and Immunogenetics Laboratory (SATIS). Enzyme-linked immunosorbent assay kit (One Lambda) was used for the detection of AT1R-Ab, and it was performed according to the manufacturer's instructions. The level of >17.1 U/mL was considered to be AT1R-Ab positive; 10.0–17.1 U/mL at risk, and Results A total of 115 samples were collected from 99 patients pre and post-kidney transplant recipients. From the pre-transplant sera (n = 68) 17.7% were positive, 35.3% were at risk and 47.0% were negative. The positive AT1R-Ab cohort were relatively younger, with a mean age of 34.7 ± 8.3 years old and statistically significant, with a p-value of 0.028. Among the sera that were tested positive, 19.0% were from the Chinese ethnicity, 6.7% from Malay and 16.7% from Indian. There was no difference in the rejection episodes, persistent or de novo HLA-DSA, and graft function between the group (AT1R-Ab negative vs AT1R-Ab at risk and positive) and the results were consistent in a model adjusted for all potential confounders. Conclusion The prevalence of positive (>17.1 U/mL) pre-transplant AT1R-Ab was 17.7% and 35.3% were at risk (10.0–17.1 U/mL) in our pre-transplant cohort.

This study was implemented to evaluate the performance of biofortified bean under different integrated soil fertility management (ISFM) options and agro-ecological conditions in Democratic Republic of Congo (DRC). A Split-plot design with eight genotypes as main factors and four ISFM options as secondary factors was carried out in eight production zones distributed across South-Kivu, North-Kivu, and Katanga provinces. The application of lime + manure + NPK increased the bean yield by 173% in Lohutu. Compared to local variety in Lohutu, the CODMLB001 variety under the same option increased the yield by 252%. The same ISFM option allowed best response in terms of micronutrient content of bean in Rutshuru, inducing up to 80.3 mg.kg-1 Fe, representing increase 41%. For Zn, the best response was obtained with lime + NPK applied in Kipopo that induced up to 32.2 mg.kg-1 Zn. Lime + manure + NPK fertilizer option reduced root rot severity by 17.8% compared to the control. This study confirmed the potential of increasing bean productivity, micronutrient and reducing the severity of major diseases through application of soil fertility management options, which will vary with the bean genotype and the environment under which bean is cultivated. Key words: Integrated soil fertility management, bean yield, micronutrient content, disease control, Phaseolus vulgaris L.

The yellow mealworm, the larval stage of the darkling beetle Tenebrio molitor, shows great promise as an alternative source of animal protein. Herein we present the 312 Mb draft genome assembled using 10x Genomics linked-read technology to inform research efforts and to provide resources to optimise yellow mealworm for mass production and consumption. The genome with a contig N50 of 39,478 bp contains 89% of conserved arthropod genes among the > 20,000 genes assembled (complete and partial genes). This draft assembly represents a valuable resource to understanding T. molitor biology as a means of producing alternative, sustainable protein for the growing population and in the face of changing climates.

The blue-tailed skink (Cryptoblepharus egeriae) is endemic to Christmas Island but underwent rapid population declines in the 1990s and 2000s and was listed as Extinct in the Wild in 2017. As invasive giant centipedes (Scolependra subspinipes) were implicated as a cause of a failed reintroduction of captive bred skinks into a fenced enclosure, we undertook a mesocosm experiment to investigate if skink survival and body condition was negatively affected by the presence and density of S. subspinipes. In addition, we used DNA barcoding to determine if wild centipedes consume other reptile species on Christmas Island. In the mesocosm experiments, survival of skinks was reduced by 30% and 44% at low and high centipede densities respectively over 12 weeks, and skink body condition also declined significantly over this period. DNA barcoding confirmed that skinks that were lost during the mesocosm experiment had been consumed by centipedes. Further, we detected DNA of two invasive reptiles (the common wolf snake Lycodon capucinus and the Asian House gecko Hemidactylus frenatus) in the stomachs of wild-caught centipedes, suggesting that centipedes are a generalist predator of reptiles in this island ecosystem. Based on these results, we recommend that attempts to reintroduce C. egeriae to Christmas Island should include the control of centipedes to increase the likelihood of success.

Elevated postprandial triacylglycerols (TAG) are an important risk factor for cardiovascular disease. Men have higher plasma TAG and impaired TAG clearance compared to women, which may contribute to sex differences in risk of cardiovascular disease. Understanding mechanisms of sex differences in TAG metabolism may yield novel therapeutic targets to prevent cardiovascular disease. Cholesteryl ester transfer protein (CETP) is a lipid shuttling protein known for its effects on high‐density lipoprotein (HDL) cholesterol levels. Although mice lack CETP, we previously demonstrated that transgenic CETP expression in female mice alters TAG metabolism. The impact of CETP on TAG metabolism in males, however, is not well understood. Here, we demonstrate that CETP expression increases plasma TAG in males, especially in very‐low density lipoprotein (VLDL), by impairing postprandial plasma TAG clearance compared to wild‐type (WT) males. Gonadal hormones were required for CETP to impair TAG clearance, suggesting a role for sex hormones for this effect. Testosterone replacement in the setting of gonadectomy was sufficient to restore the effect of CETP on TAG. Lastly, liver androgen receptor (AR) was required for CETP to increase plasma TAG. Thus, expression of CETP in males raises plasma TAG by impairing TAG clearance via testosterone signaling to AR. Further understanding of how CETP and androgen signaling impair TAG clearance may lead to novel approaches to reduce TAG and mitigate risk of cardiovascular disease.