Mind the GAP: RASA2 and RASA3 GTPase-activating proteins as gatekeepers of T cell activation and adhesion.

Following stimulation, the T cell receptor (TCR) and its coreceptors integrate multiple intracellular signals to initiate T cell proliferation, migration, gene expression, and metabolism. Among these signaling molecules are the small GTPases RAS and RAP1, which induce MAPK pathways and cellular adhesion to activate downstream effector functions. Although many studies have helped to elucidate the signaling intermediates that mediate T cell activation, the molecules and pathways that keep naive T cells in check are less understood. Several recent studies provide evidence that RASA2 and RASA3, which are GAP1-family GTPase-activating proteins (GAPs) that inactivate RAS and RAP1, respectively, are crucial molecules that limit T cell activation and adhesion. In this review we describe recent data on the roles of RASA2 and RASA3 as gatekeepers of T cell activation and migration.

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

Occupational physical activity and risk of mortality in women and men: the Tromsø Study 1986-2021.

OBJECTIVE: Associations between occupational physical activity (OPA) and mortality risks are inconclusive. We aimed to examine associations between (1) OPA separately and (2) jointly with leisure time physical activity (LTPA), and risk of all-cause, cardiovascular disease (CVD) and cancer mortality, over four decades with updated exposure and covariates every 6-8 years. METHODS: Adults aged 20-65 years from the Tromsø Study surveys Tromsø3-Tromsø7 (1986-2016) were included. We categorised OPA as low (sedentary), moderate (walking work), high (walking+lifting work) or very high (heavy manual labour) and LTPA as inactive, moderate and vigorous. We used Cox/Fine and Gray regressions to examine associations, adjusted for age, body mass index, smoking, education, diet, alcohol and LTPA (aim 1 only). RESULTS: Of 29 605 participants with 44 140 total observations, 4131 (14.0%) died, 1057 (25.6%) from CVD and 1660 (40.4%) from cancer, during follow-up (median: 29.1 years, 25th-75th: 16.5.1-35.3). In men, compared with low OPA, high OPA was associated with lower all-cause (HR 0.83, 95% CI 0.74 to 0.92) and CVD (subdistributed HR (SHR) 0.68, 95% CI 0.54 to 0.84) but not cancer mortality (SHR 0.99, 95% CI 0.84 to 1.19), while no association was observed for moderate or very high OPA. In joint analyses using inactive LTPA and low OPA as reference, vigorous LTPA was associated with lower all-cause mortality combined with low (HR 0.75, 95% CI 0.64 to 0.89), high (HR 0.67, 95% CI 0.54 to 0.82) and very high OPA (HR 0.74, 95% CI 0.58 to 0.94), but not with moderate OPA. In women, there were no associations between OPA, or combined OPA and LTPA, with mortality. CONCLUSION: High OPA, but not moderate and very high OPA, was associated with lower all-cause and CVD mortality risk in men but not in women. Vigorous LTPA was associated with lower mortality risk in men with low, high and very high OPA, but not moderate OPA.

Ten-year mortality among older male recreational endurance athletes in the Birkebeiner Aging Study in comparison with older men from the Tromsø Study.

BACKGROUND: Physical activity (PA) is associated with reduced mortality. However, whether there is an added benefit of long-term endurance training is unclear. Thus, we aimed to examine 10-year mortality in older male endurance athletes compared with an older male general population. METHOD: Male athletes (n = 503) participating in an annual long-distance ski race (median years of participation: 14, range: 1-53) from the Norwegian Birkebeiner Aging study (BiAS), and non-athletic men (n = 1867) attending the sixth Tromsø Study (Tromsø6) aged ≥65 years were included. Associations with endurance sport practice and joint exposures of endurance sport practice and self-reported leisure-time PA with all-cause mortality were examined. We analyzed the data with Cox proportional hazard models and regression standardization. RESULTS: After 10 years (median: 10.4, range: 0.5-11.1) the mortality rate was lower in athletes (hazard ratio (HR) 0.34, 95% confidence interval (CI): 0.24-0.49) compared with non-athletes, corresponding to a 15% (95% CI: 12-19%) absolute risk reduction associated with endurance sport practice. In joint analyses categorized according to PA and endurance sport practice, we observed an inverse dose-response relationship with mortality (p < 0.001). Compared to inactive non-athletes, PA was associated with lower mortality in both active non-athletes and athletes. However, the observed benefit among participants reporting moderate-to-vigorous PA was larger in athletes (HR: 0.21, 95% CI: 0.14-0.32) than non-athletes (HR: 0.43, 95% CI: 0.31-0.59) (p < 0.01). CONCLUSION: Endurance sport practice was associated with reduced 10-year mortality, beyond the effect of PA in older men. This study suggests that long-term endurance sport practice maintained into older adulthood promotes longevity.

Rationale and design of the Birkebeiner Ageing Study - a prospective cohort study of older endurance athletes.

BACKGROUND: While regular physical activity is associated with reduced mortality and morbidity in general populations, health outcomes and functional capacity related to upholding strenuous endurance exercise beyond the age of 65 years are only sparsely studied. The aim of this study is to assess associations of prolonged strenuous endurance sport practice with ageing, functional decline, morbidity and longevity among older recreational endurance athletes, during long-term follow-up. METHODS: Prospective cohort study of older recreational endurance athletes in Norway. All skiers aged 65 years and older who participated in a long-distance endurance competition, the annual 54-km Birkebeiner cross-country ski race in 2009 or 2010, were invited. The participants answered an extensive baseline questionnaire about lifestyle habits, including leisure-time physical activity and endurance sport participation, diseases, medication use and physical and mental health, with follow-up questionnaires planned every fifth year until 2029. New participants may be invited with the aim to increase the study size. Endpoints such as all-cause and disease-specific mortality, incidence and cumulative prevalence of diseases, use of medication, physical and mental health and functional decline will be assessed subsequently. Out of 658 invited skiers (51 women), 551(84%) completed the baseline questionnaire and were included in the study. The mean age was 68.8 years (median 68, range 65- 90). At baseline, the participants had completed the Birkebeiner race for an average of 16.6 years and reported an average of 33.4 years of regular endurance exercise, with one out of five reporting at least 50 years of exercise. In all, 479 (90%) reported that they were still practicing leisure-time physical activity of moderate or vigorous intensity at least twice weekly. The prevalence of cardiovascular risk factors and diseases was low. DISCUSSION: This prospective study of a cohort of recreational athletes exposed to prolonged and strenuous endurance exercise, could complement population-based studies by providing data on associations between life-long endurance sport participation, aging, functional decline and health outcomes during long-term follow-up.

Prenatal exposure to paracetamol/acetaminophen and precursor aniline impairs masculinisation of male brain and behaviour.

Paracetamol/acetaminophen (N-Acetyl-p-Aminophenol; APAP) is the preferred analgesic for pain relief and fever during pregnancy. It has therefore caused concern that several studies have reported that prenatal exposure to APAP results in developmental alterations in both the reproductive tract and the brain. Genitals and nervous system of male mammals are actively masculinised during foetal development and early postnatal life by the combined actions of prostaglandins and androgens, resulting in the male-typical reproductive behaviour seen in adulthood. Both androgens and prostaglandins are known to be inhibited by APAP. Through intrauterine exposure experiments in C57BL/6 mice, we found that exposure to APAP decreased neuronal number in the sexually dimorphic nucleus (SDN) of the preoptic area (POA) in the anterior hypothalamus of male adult offspring. Likewise, exposure to the environmental pollutant and precursor of APAP, aniline, resulted in a similar reduction. Decrease in neuronal number in the SDN-POA is associated with reductions in male sexual behaviour. Consistent with the changes, male mice exposed in uteri to APAP exhibited changes in urinary marking behaviour as adults and had a less aggressive territorial display towards intruders of the same gender. Additionally, exposed males had reduced intromissions and ejaculations during mating with females in oestrus. Together, these data suggest that prenatal exposure to APAP may impair male sexual behaviour in adulthood by disrupting the sexual neurobehavioral programming. These findings add to the growing body of evidence suggesting the need to limit the widespread exposure and use of APAP by pregnant women.

Covert cavitation: Spectral peak suppression in the acoustic emissions from spatially configured nucleations.

Dual laser-nucleation is used to precisely configure two cavitation bubbles within a focused ultrasound field of f0 = 692 kHz, in proximity to the tip of a needle hydrophone. With both bubbles responding in the f0/2 sub-harmonic regime, confirmed via ultra-high speed shadowgraphic imaging, an emission spectrum with no sub-harmonic content is demonstrated, for an inter-bubble spacing ≈λ0. A spectral model for periodic shock waves from multiple nucleations demonstrates peak suppressions at nf0/2 when applied to the experiment, via a windowing effect in the frequency domain. Implications for single-element passive detection of cavitation are discussed.

An analysis of the acoustic cavitation noise spectrum: The role of periodic shock waves.

Research on applications of acoustic cavitation is often reported in terms of the features within the spectrum of the emissions gathered during cavitation occurrence. There is, however, limited understanding as to the contribution of specific bubble activity to spectral features, beyond a binary interpretation of stable versus inertial cavitation. In this work, laser-nucleation is used to initiate cavitation within a few millimeters of the tip of a needle hydrophone, calibrated for magnitude and phase from 125 kHz to 20 MHz. The bubble activity, acoustically driven at f0 = 692 kHz, is resolved with high-speed shadowgraphic imaging at 5 × 106 frames per second. A synthetic spectrum is constructed from component signals based on the hydrophone data, deconvolved within the calibration bandwidth, in the time domain. Cross correlation coefficients between the experimental and synthetic spectra of 0.97 for the f0/2 and f0/3 regimes indicate that periodic shock waves and scattered driving field predominantly account for all spectral features, including the sub-harmonics and their over-harmonics, and harmonics of f0.

Comparison of resistance training using barbell half squats and trap bar deadlifts on maximal strength, power performance, and lean mass in recreationally active females: an eight-week randomised trial.

BACKGROUND: The aim of this study was to investigate the effect of high load resistance training using barbell half squats compared with trap bar deadlifts on maximal strength, power performance, and lean mass in recreationally active females. METHODS: Twenty-two recreationally active female participants (age: 26.9 ± 7.7 yrs.; height: 166.0 ± 5.1 cm; weight: 68.6 ± 9.9 kg) were randomly assigned to either a barbell half squat group (SG: n = 10) or trap bar deadlift group (DG: n = 12). Training consisted of twice-weekly sessions for eight weeks. Both groups completed one-repetition maximum (1RM) testing for both barbell half squat and trap bar deadlift groups. Countermovement jump (CMJ) and sprint performance were also assessed. Total body (TBLM) and leg lean mass (LLM) were measured with dual-energy x-ray absorptiometry. Between-group differences were analysed using analysis of covariance. RESULTS: SG tended to improve 1RM half squat (21.0 ± 11.5 kg vs. 13.1 ± 7.5 kg) more than DG (mean difference (MD): 8.0 kg, 95% CI: -0.36 - 16.3 kg). A similar pattern in favour of DG (18.4 ± 11.2 vs. 11.7 ± 8.1 kg) compared to SG was observed (MD: 6.5 kg, 95% CI: -2.5 - 15.6 kg). No between-group differences for sprint, jump or lean body mass changes was observed. For groups combined, the following changes in CMJ (2.0 ± 2.4 cm), 5-m sprint (-0.020 ± 0.039 s), 15-m sprint (-0.055 ± 0.230 s), TBLM (0.84 ± 1.12 kg), and LLM (0.27 ± 0.59 kg) was observed. CONCLUSIONS: An exercise intervention consisting of half squats or trap bar deadlift were associated with improved muscle strength, power, and lean mass. Our findings suggests that in recreationally active females, exercise selection is less of a concern provided that heavy loads are applied, and relevant muscle groups are targeted.

Females Display Lower Risk of Myocardial Infarction From Higher Estimated Cardiorespiratory Fitness Than Males: The Tromsø Study 1994-2014.

Objective: To examine the dose-response association between estimated cardiorespiratory fitness (eCRF) and risk of myocardial infarction (MI). Patients and Methods: Adults who attended Tromsø Study surveys 4-6 (Janurary 1,1994-December 20, 2008) with no previous cardiovascular disease were followed up through December 31, 2014 for incident MI. Associations were examined using restricted cubic splines Fine and Gray regressions, adjusted for education, smoking, alcohol, diet, sex, adiposity, physical activity, study survey, and age (timescale) in the total cohort and subsamples with hyperlipidemia (n=2956), hypertension (n=8290), obesity (n=5784), metabolic syndrome (n=1410), smokers (n=3823), and poor diet (n=3463) and in those who were physically inactive (n=6255). Results: Of 14,285 participants (mean age ± SD, 53.7±11.4 years), 979 (6.9%) experienced MI during follow-up (median, 7.2 years; 25th-75th, 5.3-14.6 years). Females with median eCRF (32 mL/kg/min) had 43% lower MI risk (subdistributed hazard ratio [SHR], 0.57; 95% CI, 0.48-0.68) than those at the 10th percentile (25 mL/kg/min) as reference. The lowest MI risk was observed at 47 mL/kg/min (SHR, 0.02; 95% CI, 0.01-0.11). Males had 26% lower MI risk at median eCRF (40 mL/kg/min; SHR, 0.74; 95% CI, 0.63-0.86) than those at the 10th percentile (32 mL/kg/min), and the lowest risk was 69% (SHR, 0.31; 95% CI, 0.14-0.71) at 60 mL/kg/min. The associations were similar in subsamples with cardiovascular disease risk factors. Conclusion: Higher eCRF associated with lower MI risk in females and males, but associations were more pronounced among females than those in males. This suggest eCRF as a vital estimate to implement in medical care to identify individuals at high risk of future MI, especially for females.

How CRISPR/Cas9 Gene Editing Is Revolutionizing T Cell Research.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows for precise gene targeting in mammalian cells, including T cells, allowing scientists to disrupt or edit specific genes of interest. This has enabled immunologists to investigate T cell functions as well as opened the path for novel therapeutics involving gene editing of T cells ex vivo before transferring these back to patients to increase T cell efficacy. This review outlines how CRISPR/Cas9 has transformed T cell research allowing immunologists to rapidly probe the roles of genes in T cells thus paving the way for novel therapeutics. Furthermore, this review describes how these tools reduce the requirement for genetic mouse models, while increasing the translational potential of T cell research.

Risk of atrial fibrillation and stroke among older men exposed to prolonged endurance sport practice: a 10-year follow-up. The Birkebeiner Ageing Study and the Tromsø Study.

AIMS: Endurance sport practice is associated with a high prevalence of atrial fibrillation (AF), which increases the risk of stroke in the general population. However, stroke risk in endurance athletes with AF is sparsely investigated. Most studies have been limited by design and are largely restricted to younger and middle-aged populations. Thus, we aimed to investigate AF and stroke risk in older athletes exposed to prolonged endurance training. METHOD: During a 10-year period, 505 male athletes aged ≥65 years frequently participating in a long-distance ski race were compared with 1867 men of the same age from the general population. The main exposure was endurance sport practice with self-reported AF and stroke as outcomes. Stroke risk was further examined by joint modelling of AF and endurance practice. Statistical analysis was conducted with a modified Poisson model. RESULTS: Athletes (median age: 68, range: 65-90) participated in a long-distance ski race over a median of 14 years (range: 1-53). Prevalence (28.5% vs 17.8%) and adjusted risk of AF (risk ratio (RR): 1.88, 95% CI: 1.49 to 2.37) were higher in athletes compared with non-athletes, whereas the prevalence (5.4% vs 9.7%) and risk of stroke were lower (RR: 0.60, 95% CI: 0.37 to 0.95). Compared with athletes without AF, risk of stroke was twofold in athletes (RR: 2.38, 95% CI: 1.08 to 5.24) and nearly fourfold in non-athletes (RR: 3.87, 95% CI: 1.98 to 7.57) with AF. CONCLUSION: Although older male endurance athletes experienced an increased risk of AF, the long-term risk of stroke was substantially reduced compared with non-athletes.

A CRISPR screen targeting PI3K effectors identifies RASA3 as a negative regulator of LFA-1-mediated adhesion in T cells.

The integrin lymphocyte function-associated antigen 1 (LFA-1) helps to coordinate the migration, adhesion, and activation of T cells through interactions with intercellular adhesion molecule 1 (ICAM-1) and ICAM-2. LFA-1 is activated during the engagement of chemokine receptors and the T cell receptor (TCR) through inside-out signaling, a process that is partially mediated by phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol 3,4,5-trisphosphate (PIP3). To evaluate potential roles of PI3K in LFA-1 activation, we designed a library of CRISPR/single guide RNAs targeting known and potential PIP3-binding proteins and screened for effects on the ability of primary mouse T cells to bind to ICAM-1. We identified multiple proteins that regulated the binding of LFA-1 to ICAM-1, including the Rap1 and Ras GTPase-activating protein RASA3. We found that RASA3 suppressed LFA-1 activation in T cells, that its expression was rapidly reduced upon T cell activation, and that its activity was inhibited by PI3K. Loss of RASA3 in T cells led to increased Rap1 activation, defective lymph node entry and egress, and impaired responses to T-dependent immunization in mice. Our results reveal a critical role for RASA3 in T cell migration, homeostasis, and function.

In vivo CRISPR screens reveal a HIF-1α-mTOR-network regulates T follicular helper versus Th1 cells.

T follicular helper (Tfh) cells provide signals to initiate and maintain the germinal center (GC) reaction and are crucial for the generation of robust, long-lived antibody responses, but how the GC microenvironment affects Tfh cells is not well understood. Here we develop an in vivo T cell-intrinsic CRISPR-knockout screen to evaluate Tfh and Th1 cells in an acute viral infection model to identify regulators of Tfh cells in their physiological setting. Using a screen of druggable-targets, alongside genetic, transcriptomic and cellular analyses, we identify a function of HIF-1α in suppressing mTORC1-mediated and Myc-related pathways, and provide evidence that VHL-mediated degradation of HIF-1α is required for Tfh development; an expanded in vivo CRISPR screen reveals multiple components of these pathways that regulate Tfh versus Th1 cells, including signaling molecules, cell-cycle regulators, nutrient transporters, metabolic enzymes and autophagy mediators. Collectively, our data serve as a resource for studying Tfh versus Th1 decisions, and implicate the VHL-HIF-1α axis in fine-tuning Tfh generation.

Tetherin antagonism by SARS-CoV-2 enhances virus release: multiple mechanisms including ORF3a-mediated defective retrograde traffic.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrated that SARS-CoV-2 infection causes tetherin downregulation, and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigated the potential viral proteins involved in abrogating tetherin function and found that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles via reduced retrograde recycling and increases tetherin localisation to late endocytic organelles. By removing tetherin from the Coronavirus budding compartments, ORF3a enhances virus release. We also found expression of Spike protein caused a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

PI3K in T Cell Adhesion and Trafficking.

PI3K signalling is required for activation, differentiation, and trafficking of T cells. PI3Kδ, the dominant PI3K isoform in T cells, has been extensively characterised using PI3Kδ mutant mouse models and PI3K inhibitors. Furthermore, characterisation of patients with Activated PI3K Delta Syndrome (APDS) and mouse models with hyperactive PI3Kδ have shed light on how increased PI3Kδ activity affects T cell functions. An important function of PI3Kδ is that it acts downstream of TCR stimulation to activate the major T cell integrin, LFA-1, which controls transendothelial migration of T cells as well as their interaction with antigen-presenting cells. PI3Kδ also suppresses the cell surface expression of CD62L and CCR7 which controls the migration of T cells across high endothelial venules in the lymph nodes and S1PR1 which controls lymph node egress. Therefore, PI3Kδ can control both entry and exit of T cells from lymph nodes as well as the recruitment to and retention of T cells within inflamed tissues. This review will focus on the regulation of adhesion receptors by PI3Kδ and how this contributes to T cell trafficking and localisation. These findings are relevant for our understanding of how PI3Kδ inhibitors may affect T cell redistribution and function.

Efficient CRISPR/Cas9-Mediated Mutagenesis in Primary Murine T Lymphocytes.

The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling, and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene-silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9-based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation of many cell types with greater precision and ease. This article outlines a protocol for CRISPR/Cas9-mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs. © 2018 by John Wiley & Sons, Inc.

Performance characterisation of a passive cavitation detector optimised for subharmonic periodic shock waves from acoustic cavitation in MHz and sub-MHz ultrasound.

We describe the design, construction and characterisation of a broadband passive cavitation detector, with the specific aim of detecting low frequency components of periodic shock waves, with high sensitivity. A finite element model is used to guide selection of matching and backing layers for the shock wave passive cavitation detector (swPCD), and the performance is evaluated against a commercially available device. Validation of the model, and characterisation of the swPCD is achieved through experimental detection of laser-plasma bubble collapse shock waves. The final swPCD design is 20 dB more sensitive to the subharmonic component, from acoustic cavitation driven at 220 kHz, than the comparable commercial device. This work may be significant for monitoring cavitation in medical applications, where sensitive detection is critical, and higher frequencies are more readily absorbed by tissue.

Pros and cons of different therapeutic antibody formats for recombinant antivenom development.

Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different molecular formats each have their own therapeutic characteristics. In this review, we provide an overview of the advances made in the development of toxin-targeting antibodies, and discuss the benefits and drawbacks of different antibody formats in relation to their ability to neutralize toxins, pharmacokinetic features, propensity to cause adverse reactions, formulation, and expression for research and development (R&D) purposes and large-scale manufacturing. A research trend seems to be emerging towards the use of human antibody formats as well as camelid heavy-domain antibody fragments due to their compatibility with the human immune system, beneficial therapeutic properties, and the ability to manufacture these molecules cost-effectively.