Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

A pals-25 gain-of-function allele triggers systemic resistance against natural pathogens of C. elegans.

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, with upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that PALS-22/25-regulated epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans.

The interactive effect of sustained sleep restriction and resistance exercise on skeletal muscle transcriptomics in young females.

Both sleep loss and exercise regulate gene expression in skeletal muscle, yet little is known about how the interaction of these stressors affects the transcriptome. The aim of this study was to investigate the effect of nine nights of sleep restriction (SR), with repeated resistance exercise (REx) sessions, on the skeletal muscle transcriptome of young, trained females. Ten healthy females aged 18-35 yr old undertook a randomized cross-over study of nine nights of SR (5 h time in bed) and normal sleep (NS; ≥7 h time in bed) with a minimum 6-wk washout. Participants completed four REx sessions per condition (days 3, 5, 7, and 9). Muscle biopsies were collected both pre- and post-REx on days 3 and 9. Gene and protein expression were assessed by RNA sequencing and Western blot, respectively. Three or nine nights of SR had no effect on the muscle transcriptome independently of exercise. However, close to 3,000 transcripts were differentially regulated (false discovery rate < 0.05) 48 h after the completion of three resistance exercise sessions in both NS and SR conditions. Only 39% of downregulated genes and 18% of upregulated genes were common between both conditions, indicating a moderating effect of SR on the response to exercise. SR and REx interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.NEW & NOTEWORTHY This study investigated the effect of nine nights of sleep restriction, with repeated resistance exercise sessions, on the skeletal muscle transcriptome of young, trained females. Sleep restriction and resistance exercise interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.

Feasibility of same-day discharge of robotic-assisted laparoscopic radical prostatectomy with pelvic lymph node dissection.

PURPOSE: Prostate cancer is one of the most common oncologic diseases. Outpatient robotic-assisted laparoscopic radical prostatectomy (RALP) has gained popularity due to its ability to minimize patient costs while maintaining low complication rates. Few studies have analyzed the possibility of performing outpatient RALP specifically in patients undergoing concurrent pelvic lymph node dissections (PLND). METHODS: Using the National Surgical Quality Improvement Program Database (NSQIP), we identified total number of RALP, stratified into inpatient and outpatient groups including those with and without PLND from 2016 to 2021. Baseline characteristics, intraoperative and postoperative complications, and unplanned readmission rates were summarized. Proportions of outpatient procedures were calculated to assess adoption of outpatient protocol. RESULTS: Between 2016 and 2021, a total of 58,527 RALP were performed, 3.7% (2142) outpatient and 96.3% inpatient. Altogether, patients undergoing outpatient RALP without PLND were more likely to have hypertension (52.6% vs. 46.3%, p < 0.01). Patients undergoing outpatient RALP without PLND were more likely to have sepsis or urinary tract infections (3.4% vs. 1.9%, p = 0.04) when compared to outpatient RALP with PLND. Cardiopulmonary, renal, thromboembolic complications, and 30-day events such as unplanned readmission, reoperation rates, and mortality were similar in both groups. However, among multivariate analysis regarding 30-day readmission and complications, there were no significant differences between outpatient RALP with or without PLND. CONCLUSION: Patients undergoing outpatient RALP without PLND were more likely to have baseline hypertension and higher rates of postoperative infection, when compared to outpatient RALP with PLND. No significant differences were seen regarding 30-day readmission or complications on multivariate analysis.

Microsporidia: Pervasive natural pathogens of Caenorhabditis elegans and related nematodes.

The nematode Caenorhabditis elegans is an invaluable host model for studying infections caused by various pathogens, including microsporidia. Microsporidia represent the first natural pathogens identified in C. elegans, revealing the previously unknown Nematocida genus of microsporidia. Following this discovery, the utilization of nematodes as a model host has rapidly expanded our understanding of microsporidia biology and has provided key insights into the cell and molecular mechanisms of antimicrosporidia defenses. Here, we first review the isolation history, morphological characteristics, life cycles, tissue tropism, genetics, and host immune responses for the four most well-characterized Nematocida species that infect C. elegans. We then highlight additional examples of microsporidia that infect related terrestrial and aquatic nematodes, including parasitic nematodes. To conclude, we assess exciting potential applications of the nematode-microsporidia system while addressing the technical advances necessary to facilitate future growth in this field.

Characterization and RNA interference-mediated silencing of tryptophan 2,3-dioxygenase gene in Carpophilus hemipterus (L.) (Coleoptera: Nitidulidae).

Dried fruit beetle, Carpophilus hemipterus (Linnaeus, 1758) (Coleoptera: Nitidulidae), is a serious pest of ripened fresh fruit in the orchard and dried fruit in postprocessing storage. Despite the economic impact and widespread distribution of C. hemipterus, there is a lack of functional genomics research seeking to elucidate features of molecular physiology for improved pest management. Here, we report the characterization of the gene named Vermilion in C. hemipterus (ChVer) that encodes for tryptophan 2,3-dioxygenase. The Vermilion is frequently used as a visual marker for genomics approaches as tryptophan 2,3-dioxygenase is involved in the biosynthesis of eye coloration pigments in insects. We identified 1628 bp long full-length transcript of ChVer from transcriptomic database of C. hemipterus. The expression analysis among adult body parts revealed peak ChVer expression in head compared to thorax and abdomen, which is consistent with its role. Among the C. hemipterus developmental stages, peak ChVer expression was observed in first instar larva, second instar larva, and adult male stages, whereas the lowest levels of expression were seen in third instar larva, prepupa, and pupa. The nanoinjection of ChVer double-stranded RNA in larval C. hemipterus resulted in a significant reduction in ChVer transcript levels as well as caused a loss of eye color, that is, the white-eyed phenotype in adults. Characterization of visually traceable marker gene and robust RNA interference response seen in this study will enable genomics research is this important pest.

The power of effective study design in animal experimentation: Exploring the statistical and ethical implications of asking multiple questions of a data set.

One of the chief advantages of using highly standardised biological models including model organisms is that multiple variables can be precisely controlled so that the variable of interest is more easily studied. However, such an approach often obscures effects in sub-populations resulting from natural population heterogeneity. Efforts to expand our fundamental understanding of multiple sub-populations are in progress. However, such stratified or personalised approaches require fundamental modifications of our usual study designs that should be implemented in Brain, Behavior and Immunity (BBI) research going forward. Here we explore the statistical feasibility of asking multiple questions (including incorporating sex) within the same experimental cohort using statistical simulations of real data. We illustrate and discuss the large explosion in sample numbers necessary to detect effects with appropriate power for every additional question posed using the same data set. This exploration highlights the strong likelihood of type II errors (false negatives) for standard data and type I errors when dealing with complex genomic data, where studies are too under-powered to appropriately test these interactions. We show this power may differ for males and females in high throughput data sets such as RNA sequencing. We offer a rationale for the use of alternative experimental and statistical strategies based on interdisciplinary insights and discuss the real-world implications of increasing the complexities of our experimental designs, and the implications of not attempting to alter our experimental designs going forward.

Evaluating Scalable Supervised Learning for Synthesize-on-Demand Chemical Libraries.

Traditional small-molecule drug discovery is a time-consuming and costly endeavor. High-throughput chemical screening can only assess a tiny fraction of drug-like chemical space. The strong predictive power of modern machine-learning methods for virtual chemical screening enables training models on known active and inactive compounds and extrapolating to much larger chemical libraries. However, there has been limited experimental validation of these methods in practical applications on large commercially available or synthesize-on-demand chemical libraries. Through a prospective evaluation with the bacterial protein-protein interaction PriA-SSB, we demonstrate that ligand-based virtual screening can identify many active compounds in large commercial libraries. We use cross-validation to compare different types of supervised learning models and select a random forest (RF) classifier as the best model for this target. When predicting the activity of more than 8 million compounds from Aldrich Market Select, the RF substantially outperforms a naïve baseline based on chemical structure similarity. 48% of the RF's 701 selected compounds are active. The RF model easily scales to score one billion compounds from the synthesize-on-demand Enamine REAL database. We tested 68 chemically diverse top predictions from Enamine REAL and observed 31 hits (46%), including one with an IC50 value of 1.3 µM.

Chemosensory mechanisms of host seeking and infectivity in skin-penetrating nematodes.

Approximately 800 million people worldwide are infected with one or more species of skin-penetrating nematodes. These parasites persist in the environment as developmentally arrested third-stage infective larvae (iL3s) that navigate toward host-emitted cues, contact host skin, and penetrate the skin. iL3s then reinitiate development inside the host in response to sensory cues, a process called activation. Here, we investigate how chemosensation drives host seeking and activation in skin-penetrating nematodes. We show that the olfactory preferences of iL3s are categorically different from those of free-living adults, which may restrict host seeking to iL3s. The human-parasitic threadworm Strongyloides stercoralis and hookworm Ancylostoma ceylanicum have highly dissimilar olfactory preferences, suggesting that these two species may use distinct strategies to target humans. CRISPR/Cas9-mediated mutagenesis of the S. stercoralis tax-4 gene abolishes iL3 attraction to a host-emitted odorant and prevents activation. Our results suggest an important role for chemosensation in iL3 host seeking and infectivity and provide insight into the molecular mechanisms that underlie these processes.

A cullin-RING ubiquitin ligase promotes thermotolerance as part of the intracellular pathogen response in Caenorhabditis elegans.

Intracellular pathogen infection leads to proteotoxic stress in host organisms. Previously we described a physiological program in the nematode Caenorhabditis elegans called the intracellular pathogen response (IPR), which promotes resistance to proteotoxic stress and appears to be distinct from canonical proteostasis pathways. The IPR is controlled by PALS-22 and PALS-25, proteins of unknown biochemical function, which regulate expression of genes induced by natural intracellular pathogens. We previously showed that PALS-22 and PALS-25 regulate the mRNA expression of the predicted ubiquitin ligase component cullin cul-6, which promotes thermotolerance in pals-22 mutants. However, it was unclear whether CUL-6 acted alone, or together with other cullin-ring ubiquitin ligase components, which comprise a greatly expanded gene family in C. elegans Here we use coimmunoprecipitation studies paired with genetic analysis to define the cullin-RING ligase components that act together with CUL-6 to promote thermotolerance. First, we identify a previously uncharacterized RING domain protein in the TRIM family we named RCS-1, which acts as a core component with CUL-6 to promote thermotolerance. Next, we show that the Skp-related proteins SKR-3, SKR-4, and SKR-5 act redundantly to promote thermotolerance with CUL-6. Finally, we screened F-box proteins that coimmunoprecipitate with CUL-6 and find that FBXA-158 and FBXA-75 promote thermotolerance. In summary, we have defined the three core components and two F-box adaptors of a cullin-RING ligase complex that promotes thermotolerance as part of the IPR in C. elegans, which adds to our understanding of how organisms cope with proteotoxic stress.

Increased risk of lower limb osteoarthritis among former professional soccer (football) players.

BACKGROUND: Soccer is a high-speed contact sport with risk of injury. Despite long-standing concern, evidence to date remains inconsistent as to the association between playing professional-level soccer and lifelong musculoskeletal consequences. AIMS: The objectives were to assess risk of osteoarthritis in former professional soccer players compared to matched general population controls, and subsequently assess associated musculoskeletal disorders which may contribute to, or result from, osteoarthritis-specifically meniscal injury and joint replacement. METHODS: We conducted a retrospective cohort study using national electronic health records (EHRs) on a cohort of 7676 former professional soccer players aged 40 or over at recruitment, matched on year of birth, sex (all male) and socio-economic status with 23 028 general population controls. Outcomes of interest were obtained by utilizing individual-level record linkage to EHRs from general hospital inpatient and day-case admissions. RESULTS: Compared to controls, former soccer players showed a greater risk of hospital admission for osteoarthritis (hazard ratio [HR] 3.01; 95% confidence interval [CI] 2.80-3.25; P < 0.001). This increased risk appeared age dependant, normalizing over age 80 years and reflective of increased risk of lower limb osteoarthritis. Further, risk of hospital admissions for meniscal injury (HR 2.73; 95% CI 2.42-3.08; P < 0.001) and joint replacement (HR 2.82; 95% CI 2.23-3.57; P < 0.001) were greater among former soccer players. CONCLUSIONS: We report an increased risk of lower limb osteoarthritis in former soccer players when compared with matched population controls. The results of this research add data in support of lower limb osteoarthritis among former soccer players representing a potential industrial injury.

Requirement for scleraxis in the recruitment of mesenchymal progenitors during embryonic tendon elongation.

The transcription factor scleraxis (Scx) is required for tendon development; however, the function of Scx is not fully understood. Although Scx is expressed by all tendon progenitors and cells, only long tendons are disrupted in the Scx-/- mutant; short tendons appear normal and the ability of muscle to attach to skeleton is not affected. We recently demonstrated that long tendons are formed in two stages: first, by muscle anchoring to skeleton via a short tendon anlage; and second, by rapid elongation of the tendon in parallel with skeletal growth. Through lineage tracing, we extend these observations to all long tendons and show that tendon elongation is fueled by recruitment of new mesenchymal progenitors. Conditional loss of Scx in mesenchymal progenitors did not affect the first stage of anchoring; however, new cells were not recruited during elongation and long tendon formation was impaired. Interestingly, for tenocyte recruitment, Scx expression was required only in the recruited cells and not in the recruiting tendon. The phenotype of Scx mutants can thus be understood as a failure of tendon cell recruitment during tendon elongation.

A sorghum genome-wide association study (GWAS) identifies a WRKY transcription factor as a candidate gene underlying sugarcane aphid (Melanaphis sacchari) resistance.

MAIN CONCLUSION: A WRKY transcription factor identified through forward genetics is associated with sorghum resistance to the sugarcane aphid and through heterologous expression reduces aphid populations in multiple plant species. Crop plant resistance to insect pests is based on genetically encoded traits which often display variability across diverse germplasm. In a comparatively recent event, a predominant sugarcane aphid (SCA: Melanaphis sacchari) biotype has become a significant agronomic pest of grain sorghum (Sorghum bicolor). To uncover candidate genes underlying SCA resistance, we used a forward genetics approach combining the genetic diversity present in the Sorghum Association Panel (SAP) and the Bioenergy Association Panel (BAP) for a genome-wide association study, employing an established SCA damage rating. One major association was found on Chromosome 9 within the WRKY transcription factor 86 (SbWRKY86). Transcripts encoding SbWRKY86 were previously identified as upregulated in SCA-resistant germplasm and the syntenic ortholog in maize accumulates following Rhopalosiphum maidis infestation. Analyses of SbWRKY86 transcripts displayed patterns of increased SCA-elicited accumulation in additional SCA-resistant sorghum lines. Heterologous expression of SbWRKY86 in both tobacco (Nicotiana benthamiana) and Arabidopsis resulted in reduced population growth of green peach aphid (Myzus persicae). Comparative RNA-Seq analyses of Arabidopsis lines expressing 35S:SbWRKY86-YFP identified changes in expression for a small network of genes associated with carbon-nitrogen metabolism and callose deposition, both contributing factors to defense against aphids. As a test of altered plant responses, 35S:SbWRKY86-YFP Arabidopsis lines were activated using the flagellin epitope elicitor, flg22, and displayed significant increases in callose deposition. Our findings indicate that both heterologous and increased native expression of the transcription factor SbWRKY86 contributes to reduced aphid levels in diverse plant models.

Cotesting for Human Immunodeficiency Virus and Sexually Transmitted Infections in the Emergency Department.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) and US Preventive Services Task Force (USPSTF) guidelines recommend screening for human immunodeficiency virus (HIV) in patients aged 15 to 65 years, as well as those at increased risk. Patients screened in the emergency department (ED) for gonorrhea (GC) and/or chlamydia represent an increased-risk population. Our aim was to assess compliance with CDC and USPSTF guidelines for HIV testing in a national sample of EDs. METHODS: We examined data from the 2010 to 2018 Nationwide Emergency Department Sample, which can be used to create national estimates of ED care to query tests for GC, chlamydia, HIV, and syphilis testing. Weighted proportions and 95% confidence intervals (CIs) were reported, and Rao-Scott χ 2 tests were used. RESULTS: We identified 13,443,831 (weighted n = 3,094,214) high-risk encounters in which GC/chlamydia testing was performed. HIV screening was performed in 3.9% (95% CI, 3.4-4.3) of such visits, and syphilis testing was performed in 2.9% (95% CI, 2.7-3.2). Only 1.5% of patients with increased risk encounters received both HIV and syphilis cotesting. CONCLUSIONS: Despite CDC and USPSTF recommendations for HIV and syphilis screening in patients undergoing STI evaluation, only a small proportion of patients are being tested. Further studies exploring the barriers to HIV screening in patients undergoing STI assessment in the ED may help inform future projects aimed at increasing guidance compliance.

Defining the micro and macro roles of a hemophilia treatment center social worker in the United States from an interdisciplinary team perspective.

INTRODUCTION: The social worker (SW) role in the Hemophilia Treatment Center (HTC) is complex and broad, providing direct support, spanning across micro, mezzo and macro levels of care. AIM: Research demonstrates discrepancy between actual and ideal SW roles among the HTC SW community. Soliciting perceptions from HTC staff about the SW role can provide a deeper understanding of this discrepancy and improve collaboration amongst care team members in meeting the psychosocial needs of HTC patients. METHODS: Funded by the National Hemophilia Foundation (NHF), a national online survey was conducted in 2020 to determine the views and attitudes of what the SW role is by HTC staff. Separate surveys were emailed to active HTC SWs and staff to collect anonymous data. Demographics of SWs gathered included age, education, years of practice, full time equivalent (FTE) status, and caseload. All disciplines were asked questions about perceptions, barriers, and potential ways to enhance and strengthen the SW role within HTCs. RESULTS: Results demonstrated that subcategory-oriented questions (40 in total) and qualitative responses highlighted diverse viewpoints and offered clarity about these differences. CONCLUSION: Findings indicated most HTC staff value the multi-faceted role of SW at their centres, and both groups identified time, limited resources, and role confusion as barriers to utilizing SW services. Outcomes will inform the development of a "standards of practice" tool that will provide education for HTC staff, patients, and families, and serve as an empowerment tool for SW to highlight their skillset and define their role.

Do changes in primary care service use over time differ by neighbourhood income? Population-based longitudinal study in British Columbia, Canada.

BACKGROUND: Strong primary care systems have been associated with improved health equity. Primary care system reforms in Canada may have had equity implications, but these have not been evaluated. We sought to determine if changes in primary care service use between 1999/2000 and 2017/2018 differ by neighbourhood income in British Columbia. METHODS: We used linked administrative databases to track annual primary care visits, continuity of care, emergency department (ED) visits, specialist referrals, and prescriptions dispensed over time. We use generalized estimating equations to examine differences in the magnitude of change by neighbourhood income quintile, adjusting for age, sex/gender, and comorbidity, and stratified by urban/rural location of residence. We also compared the characteristics of physicians providing care to people living in low- and high-income neighbourhoods at two points in time. RESULTS: Between 1999/2000 and 2017/8 the average number of primary care visits per person, specialist referrals, and continuity of care fell in both urban and rural settings, while ED visits and prescriptions dispensed increased. Over this period in urban settings, primary care visits, continuity, and specialist referrals fell more rapidly in low vs. high income neighbourhoods (relative change in primary care visits: Incidence Rate Ratio (IRR) 0.881, 95% CI: 0.872, 0.890; continuity: partial regression coefficient -0.92, 95% CI: -1.18, -0.66; specialist referrals: IRR 0.711, 95%CI: 0.696, 0.726), while ED visits increased more rapidly (IRR 1.06, 95% CI: 1.03, 1.09). The percentage of physicians who provide the majority of visits to patients in neighbourhoods in the lower two income quintiles declined from 30.6% to 26.3%. CONCLUSION: Results raise concerns that equity in access to primary care has deteriorated in BC. Reforms to primary care that fail to attend to the multidimensional needs of low-income communities may entrench existing inequities. Policies that tailor patterns of funding and allocation of resources in accordance with population needs, and that align accountability measures with equity objectives are needed as part of further reform efforts.

The Role of Cardiac Computed Tomography in Heart Failure.

PURPOSE OF REVIEW: Cardiac computed tomography (CT) is becoming a more widely applied tool in the diagnosis and management of a variety of cardiovascular conditions, including heart failure. The aim of this narrative review is to examine the role of cardiac CT in patients with heart failure. RECENT FINDINGS: Coronary computed tomographic angiography has robust diagnostic accuracy for ruling out coronary artery disease. These data are reflected in updated guidelines from major cardiology organizations. New roles for cardiac CT in myocardial imaging, perfusion scanning, and periprocedural planning, execution, and monitoring are being implemented. Cardiac CT is useful in ruling out coronary artery disease its diagnostic accuracy, accessibility, and safety. It is also intricately linked to invasive cardiac procedures that patients with heart failure routinely undergo.

Sex differences among endurance athletes in the pre-race relationships between sleep, and perceived stress and recovery.

This study examined sex differences among endurance athletes in pre-race relationships between sleep, and perceived stress and recovery. Thirty-six athletes completed the Short Recovery and Stress Scale, and had sleep monitored via actigraphy, over four consecutive days prior to an ultra-marathon. Overall, compared with males, females had shorter wake after sleep onset (mean ± SD, 50 ± 23 vs 65 ± 23 min, p = .04) and lower emotional balance (3.9 ± 1.1 vs 4.8 ± 1.1 arbitrary units, p = .001). The day before the race, females scored higher for all stress-related items (p < 0.05). Among females, higher scores for emotional balance (ß = -31 min, p = .01) and negative emotional state (ß = -21 min, p < .001) were associated with reduced sleep duration. Among males, higher scores for overall stress were associated with increased sleep duration (ß = 22 min, p = .01). Across all athletes, longer sleep duration was associated with improved overall recovery (ß = 0.003 arbitrary units, p = .02). Females experienced greater pre-race stress than males, and their sleep duration was associated with emotional factors. The SRSS may help identify female athletes at risk of sleep difficulties prior to competition.

Monitoring Effects of Sleep Extension and Restriction on Endurance Performance Using Heart Rate Indices.

ABSTRACT: Roberts, SSH, Aisbett, B, Teo, W-P, and Warmington, S. Monitoring effects of sleep extension and restriction on endurance performance using heart rate indices. J Strength Cond Res 36(12): 3381-3389, 2022-Heart rate (HR) indices are useful for monitoring athlete fatigue or "readiness to perform." This study examined whether HR indices are sensitive to changes in readiness following sleep restriction (SR) and sleep extension (SE). Nine athletes completed a crossover study with 3 conditions: SR, normal sleep (NS), and SE. Each condition required completion of an endurance time trial (TT) on 4 consecutive days (D1-D4). Athletes slept habitually before D1; however, time in bed was reduced by 30% (SR), remained normal (NS), or extended by 30% (SE), on subsequent nights (D1-D3). Daily resting HR and HR variability were recorded. The maximal rate of HR increase and HR recovery was determined from a constant-load test before TTs. Exercise intensity ratios incorporating mean HR, mean power (W), and perceived exertion (RPE) were recorded at steady state during constant-load tests (W:HR SS ) and during TTs (W:HR TT , RPE:HR TT ). Compared with D4 of NS, RPE:HR TT was lower on D4 of SE ( p = 0.008)-when TT performances were faster. Compared with D1 of SR, RPE:HR TT was higher on D3 and D4 of SR ( p < 0.02). Moderate correlations were found between percentage changes in W:HR TT and changes in TT finishing time in SR ( r = -0.67, p = 0.049) and SE ( r = -0.69, p = 0.038) conditions. Intensity ratios incorporating mean HR seem sensitive to effects of sleep duration on athlete readiness to perform. When interpreting intensity ratios, practitioners should consider potential effects of prior sleep duration to determine whether sleep-promoting interventions are required (e.g., SE).

Sensitivity to targeted therapy differs between HER2-amplified breast cancer cells harboring kinase and helical domain mutations in PIK3CA.

BACKGROUND: HER2-amplified breast cancer is a clinically defined subtype of breast cancer for which there are multiple viable targeted therapies. Resistance to these targeted therapies is a common problem, but the mechanisms by which resistance occurs remain incompletely defined. One mechanism that has been proposed is through mutation of genes in the PI3-kinase pathway. Intracellular signaling from the HER2 pathway can occur through PI3-kinase, and mutations of the encoding gene PIK3CA are known to be oncogenic. Mutations in PIK3CA co-occur with HER2-amplification in ~ 20% of cases within the HER2-amplified subtype. METHODS: We generated isogenic knockin mutants of each PIK3CA hotspot mutation in HER2-amplified breast cancer cells using adeno-associated virus-mediated gene targeting. Isogenic clones were analyzed using a combinatorial drug screen to determine differential responses to HER2-targeted therapy. Western blot analysis and immunofluorescence uncovered unique intracellular signaling dynamics in cells resistant to HER2-targeted therapy. Subsequent combinatorial drug screens were used to explore neuregulin-1-mediated resistance to HER2-targeted therapy. Finally, results from in vitro experiments were extrapolated to publicly available datasets. RESULTS: Treatment with HER2-targeted therapy reveals that mutations in the kinase domain (H1047R) but not the helical domain (E545K) increase resistance to lapatinib. Mechanistically, sustained AKT signaling drives lapatinib resistance in cells with the kinase domain mutation, as demonstrated by staining for the intracellular product of PI3-kinase, PIP3. This resistance can be overcome by co-treatment with an inhibitor to the downstream kinase AKT. Additionally, knockout of the PIP3 phosphatase, PTEN, phenocopies this result. We also show that neuregulin-1, a ligand for HER-family receptors, confers resistance to cells harboring either hotspot mutation and modulates response to combinatorial therapy. Finally, we show clinical evidence that the hotspot mutations have distinct expression profiles related to therapeutic resistance through analysis of TCGA and METABRIC data cohorts. CONCLUSION: Our results demonstrate unique intracellular signaling differences depending on which mutation in PIK3CA the cell harbors. Only mutations in the kinase domain fully activate the PI3-kinase signaling pathway and maintain downstream signaling in the presence of HER2 inhibition. Moreover, we show there is potentially clinical importance in understanding both the PIK3CA mutational status and levels of neuregulin-1 expression in patients with HER2-amplified breast cancer treated with targeted therapy and that these problems warrant further pre-clinical and clinical testing.