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BackgroundEvery bout of exercise mobilizes and redistributes large numbers of effector lymphocytes with a cytotoxic and tissue migration phenotype. The frequent redistribution of these cells is purported to increase immune surveillance and play a mechanistic role in reducing cancer risk and slowing tumor progression in physically active cancer survivors. Our aim was to provide the first detailed single cell transcriptomic analysis of exercise-mobilized lymphocytes and test their effectiveness as a donor lymphocyte infusion (DLI) in xenogeneic mice engrafted with human leukemia.MethodsPeripheral blood mononuclear cells (PBMCs) were collected from healthy volunteers at rest and at the end of an acute bout of cycling exercise. Flow cytometry and single-cell RNA sequencing was performed to identify phenotypic and transcriptomic differences between resting and exercise-mobilized cells using a targeted gene expression panel curated for human immunology. PBMCs were injected into the tail vein of xenogeneic NSG-IL-15 mice and subsequently challenged with a luciferase tagged chronic myelogenous leukemia cell line (K562). Tumor growth (bioluminescence) and xenogeneic graft-versus-host disease (GvHD) were monitored bi-weekly for 40-days.ResultsExercise preferentially mobilized NK-cell, CD8+ T-cell and monocyte subtypes with a differentiated and effector phenotype, without significantly mobilizing CD4+ regulatory T-cells. Mobilized effector lymphocytes, particularly effector-memory CD8+ T-cells and NK-cells, displayed differentially expressed genes and enriched gene sets associated with anti-tumor activity, including cytotoxicity, migration/chemotaxis, antigen binding, cytokine responsiveness and alloreactivity (e.g. graft-versus-host/leukemia). Mice receiving exercise-mobilized PBMCs had lower tumor burden and higher overall survival (4.14E+08 photons/s and 47%, respectively) at day 40 compared to mice receiving resting PBMCs (12.1E+08 photons/s and 22%, respectively) from the same donors (pConclusionExercise in humans mobilizes effector lymphocytes with an anti-tumor transcriptomic profile and their use as DLI extends survival and enhances the graft-versus-leukemia (GvL) effect without exacerbating GvHD in human leukemia bearing xenogeneic mice. Exercise may serve as an effective and economical adjuvant to increase the GvL effects of allogeneic cell therapies without intensifying GvHD.

Supplementary Methods and Materials from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

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Background The frass of several herbivorous insect species has been utilised as natural medicines in Asia; however, the metabolite makeup and pharmaceutical activities of insect frass have yet to be investigated. Oligophagous Papilionidae insects utilise specific kinds of plants, and it has been suggested that the biochemicals from the plants may be metabolised by cytochrome P450 (CYP) in Papilionidae insects. In this study, we extracted the components of the frass of Papilio machaon larvae reared on Angelica keiskei, Oenanthe javanica or Foeniculum vulgare and examined the biological activity of each component. Then, we explored the expression of CYP genes in the midgut of P. machaon larvae and predicted the characteristics of their metabolic system. Results The components that were extracted using hexane, chloroform or methanol were biochemically different between larval frass and the host plants on which the larvae had fed. Furthermore, a fraction obtained from the chloroform extract from frass of A. keiskei-fed larvae specifically inhibited the cell proliferation of the human colon cancer cell line HCT116, whereas fractions obtained from the chloroform extracts of O. javanica- or F. vulgare-fed larval frass did not affect HCT116 cell viability. The metabolites from the chloroform extract from frass of A. keiskei-fed larvae prevented cell proliferation and induced apoptosis in HCT116 cells. Next, we examined the metabolic system in A. keiskei-fed larvae by RNA-seq analysis and found that the A. keiskei-fed larval midgut had different characteristics from the O. javanica- or F. vulgare-fed larval metabolic systems. We found that the CYP6B2 transcript was highly expressed in the A. keiskei-fed larval midgut. Conclusions These findings indicate that P. machaon metabolites might be useful as pharmaceutical agents against human colon cancer subtypes. Importantly, our findings show that it might be possible to use insect metabolic enzymes for the chemical structural conversion of plant-derived compounds with complex structures.

IntroductionThe use of allogeneic hematopoietic cell transplantation (allo-HCT) for treating hematological disorders is increasing, but the development of graft-versus-host disease (GvHD) remains a major cause of morbidity and mortality. The use of post-transplant cyclophosphamide (CY) has significantly improved outcomes following allo-HCT, but complications of viral reactivation due to delayed immune reconstitution or relapse remain. Other laboratories are evaluating the potential benefit of lowering the dose of CY given post-transplant, whereas our laboratory has been focusing on whether partially replacing CY with another DNA alkylating agent, bendamustine (BEN) may be advantageous in improving outcomes with allo-HCT.MethodsHere, we utilized a xenogeneic GvHD (xGvHD) model in which immunodeficient NSG mice are infused with human peripheral blood mononuclear cells (PBMCs).ResultsWe show that a lower dose of CY (25 mg/kg) given on days +3 and +4 or CY (75 mg/kg) given on only day +3 post-PBMC infusion is not sufficient for improving survival from xGvHD, but can be improved with the addition of BEN (15 mg/kg) on day +4 to day +3 CY (75 mg/kg). CY/BEN treated mice when combined with cyclosporine A (CSA) (10mg/kg daily from days +5 to +18 and thrice weekly thereafter), had improved outcomes over CY/CY +CSA treated mice. Infiltration of GvHD target organs was reduced in both CY/CY and CY/BEN treatment groups versus those receiving no treatment. CY/CY +CSA mice exhibited more severe xGvHD at day 10, marked by decreased serum albumin and increased intestinal permeability. CY/BEN treated mice had reductions in naïve, effector memory and Th17 polarized T cells. RNAseq analysis of splenocytes isolated from CY/CY and CY/BEN treated animals revealed increased gene set enrichment in multiple KEGG pathways related to cell migration, proliferation/differentiation, and inflammatory pathways, among others with CY/BEN treatment.ConclusionTogether, we illustrate that the use of CY/BEN is safe and shows similar control of xGvHD to CY/CY, but when combined with CSA, survival with CY/BEN is significantly prolonged compared to CY/CY.

Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recent studies have highlighted EVs carry preassembled complex biological information that elicit pleiotropic responses in target cells. It is well recognized that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation (DC): the larger size class (referred to as “microvesicles” or “shed microvesicles,” sMVs) is heterogeneous (100–1500 nm), while the smaller size class (referred to as “exosomes”) is relatively homogeneous in size (50–150 nm). A key issue hindering progress in understanding underlying mechanisms of EV subtype biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV subpopulations suitable for molecular analysis. In this protocol we reveal a novel method for the isolation, purification, and characterization of distinct EV subtypes: exosomes and sMVs. This method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from conditioned medium from a human colon cancer cell model. For both EV subtypes, this protocol details extensive purification and characterization based on dynamic light scattering, cryoelectron microscopy, quantitation, immunoblotting, and comparative label-free proteome profiling. This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.

Context The use of dual-purpose crops (for grazing and grain) has increased in the high-rainfall zone in southern Australia. Aim A systems experiment examined the impact on livestock production and supplementary feeding when dual-purpose crops were incorporated into a production system based on Merino ewes producing yearling lambs for sale. Methods The experimental site near Canberra, ACT, was subdivided into nine experimental units (‘farmlets’) with three replicate farmlets for each of three production-system treatments. Each farmlet was managed as a self-contained unit with six Merino ewes and their progeny during 2013–16 (4 years). Ewes were joined in February, lambed in July and shorn in spring; the original cohort of ewes (born 2009) was replaced by a new cohort (born 2012) at the midpoint of the experiment. Six weaners were retained after weaning in each farmlet and sold as yearlings. Control farmlets were sown to pasture based on phalaris (Phalaris aquatica L.) and subterranean clover (Trifolium subterraneum L.) and comprised sub-paddocks to allow rotational grazing. Farmlets in treatments that included dual-purpose crops comprised six sub-paddocks (0.231 ha), with two sown to permanent pasture, and four supporting a rotation of pasture–pasture–dual-purpose canola (Brassica napus L.)–dual-purpose wheat (Triticum aestivum L.). In one of the crop–pasture production system treatments, crop-grazing was prioritised for ewes (ECG treatment); in the other, crop-grazing was prioritised for their progeny weaners (WCG treatment). Key results Greasy fleece weight from ECG (5.3 kg) and WCG (5.1 kg) ewes was higher (P < 0.001) than from control ewes (4.7 kg) averaged over the 4 years. The final sale weight of yearling weaners from the WCG system (44.3 kg) was higher (P < 0.001) than from the control (39.2 kg) or ECG (39.1 kg) systems when averaged over the 4 years. The benefit was predominantly due to greater weight gain during the period when weaners grazed the crop during late autumn and winter. Sale weight of lamb per hectare was higher (P = 0.003) in the WCG treatment (216 kg) compared with the ECG treatment (186 kg) when averaged over the 4 years of the experiment but did not differ (P > 0.05) to the control (201 kg). Meat production over the 4 years was higher (P < 0.001) in the WCG system (226 kg/ha) than other treatments when weight gain from wethers in 2014 was included. The impact of including dual-purpose crops on supplementary feeding was variable and depended on seasonal conditions. Conclusions Incorporation of dual-purpose crops into the high-rainfall production system can increase meat and wool production, with the highest meat production being obtained when crop grazing was prioritised for young carry-over livestock. Implications Prioritising dual-purpose crops for young growing livestock can increase meat production from the system while allowing other livestock classes (wethers or ewes) to graze the crops in better seasons when there was excess forage that would otherwise have been under-utilised.

Context Growing of dual-purpose crops for grazing by livestock has increased in popularity in the high-rainfall zone of southern Australia, a livestock production zone traditionally based on permanent perennial grass species. Aims A systems experiment examined the impact on pasture forage availability, sheep grazing days and crop yields when one-third of a farmlet was sown to dual-purpose wheat (Triticum aestivum L.) and canola (Brassica napus L.) crops. Methods The experiment comprised nine experimental units (farmlets) divided into three treatments with three replicate farmlets per treatment: control farmlets sown to phalaris (Phalaris aquatica L.)-based pastures; and two treatments with grazing of crops prioritised for either ewes or their progeny. Control farmlets comprised four sub-paddocks (0.231 ha each) in 2013 and six sub-paddocks in 2014–2016. Farmlets in treatments that included dual-purpose crops comprised six sub-paddocks (0.231 ha), with two sub-paddocks sown to permanent pasture and the other four sub-paddocks supporting a pasture–pasture–canola–wheat rotation. Key results Crops were sown in February or early March and grazing commenced by mid-May in all years. Canola was grazed first in the sequence in 3 of 4 years. Treatments had similar total sheep grazing days per year, except for the progeny-prioritised treatment in 2014 when agistment wethers were introduced to utilise excess crop forage. Grazing did not affect wheat yields (3.9 vs 3.7 t/ha, P > 0.05) but did reduce canola yields (3.6 vs 3.0 t/ha, P = 0.007). Pasture availability (dry matter per ha in the pasture paddock at entry by sheep) was higher in the control during late summer and autumn when the crops were being established; however, resting of pastures during late autumn and winter while crops were grazed resulted in no difference in pasture availability among treatments during spring. Conclusion and implications The key feed-gap is in late summer and autumn when dual-purpose crops are included in the system. Early and timely sowing of crops increases the grazing opportunity from dual-purpose crops before lock-up. Growing wheat plus canola provided some hedge against poor establishment and/or slow growth rates in one of the crops.

The Antarctic environment is characterized by many of the same extreme stressors as long-duration space flight (LDSE), thereby providing a useful earth-based analog for examining changes in and predictors of mental health over time. At coastal (n = 88) and inland (n = 22) Antarctic stations we tracked mental health symptoms across a nine-month period including winter-over using the Mental Health Checklist (MHCL; Bower et al., 2019). Our monthly assessment battery also examined changes in physical complaints, biomarkers of stress, and the use of different emotion regulation strategies. MHCL positive adaptation scores showed linear decreases whereas MHCL poor self-regulation scores and severity of physical symptoms increased across the study period. During-mission use of emotion regulation strategies and dehydroepiandrosterone (DHEA) levels predicted end-of-study MHCL scores, whereas trait-based psychological measures collected at the start of the mission showed little predictive utility. Results suggest that interventions and counter measures aimed at enhancing positive affect/emotion during prolonged exposure to extreme environments may be useful in reducing psychological risk.

Habitual endurance exercise results in increased erythropoiesis, which is primarily controlled by erythropoietin (EPO), yet studies demonstrating upregulation of EPO via a single bout of endurance exercise have been equivocal. This study compares the acute EPO response to 30 min of high versus 90 min of moderate-intensity endurance exercise and whether that response can be upregulated via selective adrenergic receptor blockade. Using a counterbalanced, cross-over design, fifteen participants (age 28 ± 8) completed two bouts of running (30-min, high intensity vs 90-min, moderate intensity) matched for overall training stress. A separate cohort of fourteen participants (age 31 ± 6) completed three bouts of 30-min high-intensity cycling after ingesting the preferential β1-adrenergic receptor (AR) antagonist bisoprolol, the non-preferential β1 + β2 antagonist nadolol or placebo. Venous blood was collected before, during, and after exercise, and serum EPO levels were determined by ELISA. No detectable EPO response was observed during or after high intensity running, however, in the moderate-intensity trial EPO was significantly elevated at both during-exercise timepoints (+ 6.8% ± 2.3% at 15 min and + 8.7% ± 2.2% at 60 min). No significant change in EPO was observed post-cycling or between the trials involving βAR blockade. Neither training mode (running or cycling), nor beta-blockade significantly influenced the EPO response to 30 min of high-intensity exercise, however, 90 min of moderate-intensity running elevated EPO during exercise, returning to baseline immediately post-exercise. Identifying the optimal mode, duration and intensity required to evoke an EPO response to exercise may help tailor exercise prescriptions designed to maximize EPO response for both performance and clinical applications.

Purpose Preclinical evidence suggests that natural killer cell (NK-cell) function and myokines facilitate the protective effects of exercise for breast cancer prevention. Since higher-intensity exercise acutely promotes greater mobilization and larger changes in NK-cell cytotoxicity than lower-intensity, high-intensity interval training (HIIT) might offer increased immune protection compared to moderate-intensity continuous-training (MICT). This study compared a 12-week HIIT program to a 12-week MICT program and usual care on changes in resting NK-cell function and circulating myokines among women at high risk for breast cancer. Methods Thirty-three women were randomized to HIIT, MICT, or usual care, for a supervised exercise intervention. Blood was collected at baseline and end-of-study. The cytotoxic activity of CD3−/CD56+ NK-cells against the K562 target cell line in vitro was determined by flow cytometry. Circulating myokines (IL-15, IL-6, irisin, OSM, osteonectin, IL-7) were assessed with luminex multiplex assays and ELISA. One-way ANOVA and paired sample t-tests assessed between- and within-group differences, respectively. Pearson correlation coefficients determined relationships between baseline fitness and change variables. Results Significant differences were not observed between groups for change in NK-cell function or circulating myokines (p > 0.05). Significant correlations were only observed for baseline peak aerobic capacity (ml/kg/min) and change in NK-cell-specific lysis (r = − 0.43, p = 0.02) and hemacytotoxicity for the total sample (r = − 0.46, p = 0.01). Conclusion Our findings suggest that exercise intensity may not significantly impact change in resting NK-cell function and circulating myokines among women at high risk for breast cancer. Structured exercise training may have a larger impact on NK-cell function in those with lower levels of cardiorespiratory fitness. Clinical trial registration: NCT02923401; Registered on October 4, 2016

Long-duration spaceflight impairs natural killer (NK) cell function, which could compromise immune surveillance in exploration class mission crew. To determine if microgravity can impair NK-cell function, we established a rotary cell culture system to expose human peripheral blood mononuclear cells to simulated microgravity (SMG) in vitro. We found that 12 h of SMG suppressed NK-cell cytotoxic activity (NKCA) by ~50% against K562, U266 and 721.221 tumor target cells when returned to the 1G environment. Mass cytometry was used to identify 37 individual markers associated with NK-cell activation, maturation and cytotoxicity, revealing that SMG causes reductions in NK-cell degranulation and effector cytokine production. Extended flow cytometry confirmed that SMG lowered NK cell perforin and granzyme b expression by 25% and 17% respectively, but did not affect the surface expression of various activating (NKG2D, NKp30) and inhibitory (NKG2A, KLRG1) receptors or the ability of NK-cells to conjugate with target cells. Flow cytometry further revealed that SMG impaired NK-cell degranulation (reduced CD107a+ expression) and suppressed TNFα and IFNγ secretion in response to stimulation with K562 target cells. These findings indicate that SMG ‘disarms’ human NK-cells of cytolytic granules and impairs NKCA against a range of tumor target cells in vitro. Exposure to microgravity could be a factor that contributes to impaired NK-cell function during long duration space travel.

Phosphorus (P) is usually stratified in the topsoil layer under pasture, due to the broadcast application of fertiliser, excreta and leaf-litter deposition on the soil surface, and minimal soil disturbance. The objective of this study was to investigate root proliferation and P acquisition in response to P stratification by comparing two Trifolium subterraneum cultivars with contrasting root morphologies. Clover micro-swards were grown with deficient, constrained and sufficient P supplied in a topsoil layer overlying a P-deficient subsoil that mimicked the stratification of P that occurs under pasture. Phosphorus labelled with 33P- and 32P-radioisotope tracer was mixed throughout the topsoil and subsoil layers, respectively. The shoot yield and total plant P uptake of the cultivars increased in response to increased topsoil P supply. The length of roots produced by the cultivars was equivalent in each of the P treatments, although the specific root length achieved by the cultivars was substantially different. In the P-constrained and P-sufficient treatments, ~91% and ~ 99% of total plant P was acquired by topsoil roots, respectively. In contrast, subsoil roots acquired 60–74% of total plant P in the P-deficient treatment. Topsoil roots were most important for P acquisition when P was highly stratified, whereas subsoil roots contributed to P acquisition when P was uniformly distributed throughout the P-deficient soil profile. Selection for prolific nutrient-foraging roots, in conjunction with plasticity for subsoil exploration, may improve the P-acquisition efficiency of T. subterraneum genotypes and confer adaptability across a range of soil-P environments.

The experiment was conducted to understand how root morphological traits contributed to the contrasting P-acquisition efficiencies of two Trifolium subterraneum cultivars. Phosphorus acquisition, root length proliferation and root acclimation to P deficiency were investigated by varying the P supply (20–300 mg P kg−1) and planting density (1–12 plants pot−1) of clover micro-swards. The shoot yield of both cultivars increased in response to increased P supply and increased planting density. Higher planting densities increased shoot yield by reducing the time to leaf canopy closure. In P-deficient soil, the ‘less P-efficient’ cultivar acquired less P and yielded relatively poorly compared with the ‘more P-efficient’ cultivar when planting density was low (

CD3+/CD56+Natural killer (NK) cell-like T-cells (NKT-like cells) represent ex vivointo cytokine-induced killer (CIK) cells, however this therapeutic cell product has had mixed results against hematological malignancies in clinical trials. The aim of this study was to determine if NKT-like cells mobilized during acute cycling exercise could be used to generate more potent anti-tumor CIK cells from healthy donors. An acute exercise bout increased NKT-like cell numbers in blood 2-fold. Single cell RNA sequencing revealed that exercise mobilized NKT-like cells have an upregulation of genes and transcriptomic programs associated with enhanced anti-tumor activity, including cytotoxicity, cytokine responsiveness, and migration. Exercise, however, did not augment theex vivoexpansion of CIK cells or alter their surface phenotypes after 21-days of culture. CIK cells expanded at rest, during exercise (at 60% and 80% VO2max) or after (1h post) were equally capable of killing leukemia, lymphoma, and multiple myeloma target cells with and without cytokine (IL-2) and antibody (OKT3) primingin vitro. We conclude that acute exercise in healthy donors mobilizes NKT-like cells with an upregulation of transcriptomic programs involved in anti-tumor activity, but does not augment theex vivoexpansion of CIK cells.

Background Immunosenescence is described as age-associated changes within the immune system that are responsible for decreased immunity and increased cancer risk. Physically active individuals have fewer ‘senescent’ and more naïve T-cells compared to their sedentary counterparts, but it is not known if exercise training can rejuvenate ‘older looking’ T-cell profiles. We determined the effects of 12-weeks supervised exercise training on the frequency of T-cell subtypes in peripheral blood and their relationships with circulating levels of the muscle-derived cytokines (i.e. ‘myokines’) IL-6, IL-7, IL-15 and osteonectin in older women at high risk of breast cancer. The intervention involved 3 sessions/week of either high intensity interval exercise (HIIT) or moderate intensity continuous exercise (MICT) and were compared to an untrained control (UC) group. Results HIIT decreased total granulocytes, CD4+ T-cells, CD4+ naïve T-cells, CD4+ recent thymic emigrants (RTE) and the CD4:CD8 ratio after training, whereas MICT increased total lymphocytes and CD8 effector memory (EM) T-cells. The change in total T-cells, CD4+ naïve T-cells, CD4+ central memory (CM) T-cells and CD4+ RTE was elevated after MICT compared to HIIT. Changes in $$ \dot{\mathrm{V}}{\mathrm{O}}_{2\max } $$ V ̇ O 2 max after training, regardless of exercise prescription, was inversely related to the change in highly differentiated CD8+ EMRA T-cells and positively related to changes in β2-adrenergic receptor (β2-AR) expression on CM CD4+ and CM CD8+ T-cells. Plasma myokine levels did not change significantly among the groups after training, but individual changes in IL-7 were positively related to changes in the number of β2-AR expressing CD4 naïve T cells in both exercise groups but not controls. Further, CD4 T-cells and CD4 naive T-cells were negatively related to changes in IL-6 and osteonectin after HIIT but not MICT, whereas CD8 EMRA T-cells were inversely related to changes in IL-15 after MICT but not HIIT. Conclusions Aerobic exercise training alters the frequency of peripheral T-cells associated with immunosenescence in middle aged/older women at high risk of breast cancer, with HIIT (pro-senescent) and MICT (anti-senescent) evoking divergent effects. Identifying the underlying mechanisms and establishing whether exercise-induced changes in peripheral T-cell numbers can alter the risk of developing breast cancer warrants investigation.

Chtop (chromatin target of Prmt1) regulates various aspects of gene expression including transcription and mRNA export. Despite these important functions, the regulatory mechanism underlying Chtop expression remains undetermined. Using Chtop-expressing human cell lines, we demonstrate that Chtop expression is controlled via an autoregulatory negative feedback loop whereby Chtop binds its own mRNA to retain intron 2 during splicing; a premature termination codon present at the 5' end of intron 2 leads to nonsense-mediated decay of the mRNA. We also show that Chtop interacts with exon 2 of Chtop mRNA via its arginine-glycine-rich (RG) domain, and with intron 2 via its N-terminal (N1) domain; both are required for retention of intron 2. In addition, we show that hnRNP H accelerates intron 2 splicing of Chtop mRNA in a manner dependent on Chtop expression level, suggesting that Chtop and hnRNP H regulate intron 2 retention of Chtop mRNA antagonistically. Thus, the present study provides a novel molecular mechanism by which mRNA and protein levels are constitutively regulated by intron retention.

The use of immunotherapies has shown promise against selective human cancers. Identifying novel combinations of innate and adaptive immune cell-activating agents that can work synergistically to suppress tumor growth and provide additional protection against resistance or recurrence is critical. The A20 murine lymphoma model was used to evaluate the effect of various combination immunotherapies administered intratumorally. We show that single-modality treatment with Poly(I:C) or GM-CSF-secreting allogeneic cells only modestly controls tumor growth, whereas when given together there is an improved benefit, with 50% of animals clearing tumors and surviving long-term. Neither heat nor irradiation of GM-CSF-secreting cells enhanced the response over use of live cells. The use of a TIM-3 inhibitory antibody and an OX40 agonist in combination with Poly(I:C) allowed for improved tumor control, with 90% of animals clearing tumors with or without a combination of GM-CSF-secreting cells. Across all treatment groups, mice rejecting their primary A20 tumors were immune to subsequent challenge with A20, and this longstanding immunity was T-cell dependent. The results herein support the use of combinations of innate and adaptive immune activating agents for immunotherapy against lymphoma and should be investigated in other cancer types.

Athletes are advised to receive the COVID-19 vaccination to protect them from SARS CoV-2 infection during major competitions. Despite this, many athletes are reluctant to get the COVID-19 vaccine due to concerns that symptoms of vaccinosis may impair athletic performance. OBJECTIVE: To determine the effects of COVID-19 vaccination on the physiological responses to graded exercise. METHODS: Healthy physically active participants completed a 20-minute bout of graded cycling exercise at intensities corresponding to 50, 60, 70 and 80% of the pre-determined VO2max before and ~21 days after receiving the COVID-19 vaccine (2 dose Pfizer mRNA or 1 dose Johnson&Johnson). RESULTS: Vaccination had no effect on a large number of physiological responses to exercise measured in blood (e.g. lactate, epinephrine, cortisol) and by respiratory gas exchange (e.g. oxygen uptake, CO2 production, ventilation, respiratory exchange ratio, predicted VO2max, ventilatory threshold) (p>0.05). We did, however, find significant elevations in heart rate (~5 bpm) and norepinephrine (p = 0.006 and 0.04, respectively) in response to vigorous (e.g. 70-80% VO2max) intensity exercise after vaccination, particularly in those that received the two shot Pfizer mRNA vaccine regimen. These findings held true when compared to demographically matched controls who completed identical bouts of exercise several weeks apart without receiving a vaccine; delta values for heart rate (p=0.03) and norepinephrine (p=0.01) were elevated in the second trial for those that received the Pfizer mRNA vaccine compared to the controls at the 70% and 80% VO2max stages, respectively. CONCLUSION: Recent COVID-19 vaccination has minimal effects on the physiological responses to graded exercise in physically active healthy people. The small elevations in cardiovascular and neuroendocrine responses to exercise after the Pfizer mRNA vaccine regimen could have implications for athletes at the elite level and warrants investigation.

Aim: To report the first study of temelimab, a monoclonal antibody neutralizing the pathogenic human endogenous retrovirus type W envelope, in patients with type 1 diabetes (T1D). Materials and Methods: This double-blind, placebo-controlled, randomized clinical trial recruited adult patients with T1D within 4 years postdiagnosis and remaining C-peptide secretion. Sixty-four patients were randomized (2:1) to monthly temelimab 6 mg/kg or placebo during 24 weeks, followed by a 24-week, open-label extension, during which all patients received temelimab. The primary objective was the safety and tolerability of temelimab. The secondary objective was to assess the pharmacodynamics response such as C-peptide levels, insulin use, HbA1c, hypoglycaemia and autoantibodies. Results: Temelimab was well tolerated without any group difference in the frequency or severity of adverse events. Concerning exploratory endpoints, there was no difference in the levels of C-peptide, insulin use or HbA1c between treatment groups at weeks 24 and 48. The frequency of hypoglycaemia events was reduced with temelimab (P = 0.0004) at week 24 and the level of anti-insulin antibodies was lower with temelimab (P < 0.01); the other autoantibodies did not differ between groups. Conclusions: Temelimab appeared safe in patients with T1D. Pharmacodynamics signals (hypoglycaemia and anti-insulin antibodies) under temelimab were observed. Markers of β-cell functions were not modified by treatment. These results need to be further explored in younger patients with T1D with earlier disease onset.

Methamphetamine (Meth) abuse has reached epidemic proportions in many countries and can induce psychotic episodes mimicking the clinical profile of schizophrenia. Brain-derived neurotrophic factor (BDNF) is implicated in both Meth effects and schizophrenia. We therefore studied the long-term effects of chronic Meth exposure in transgenic mice engineered to harbor the human BDNFVal66Met polymorphism expressed via endogenous mouse promoters. These mice were chronically treated with an escalating Meth regime during late adolescence. At least 4 weeks later, all hBDNFVal66Met Meth-treated mice exhibited sensitization confirming persistent behavioral effects of Meth. We used high-resolution quantitative mass spectrometry-based proteomics to biochemically map the long-term effects of Meth within the brain, resulting in the unbiased detection of 4808 proteins across the mesocorticolimbic circuitry. Meth differentially altered dopamine signaling markers (e.g., Dat, Comt, and Th) between hBDNFVal/Val and hBDNFMet/Met mice, implicating involvement of BDNF in Meth-induced reprogramming of the mesolimbic proteome. Targeted analysis of 336 schizophrenia-risk genes, as well as 82 growth factor cascade markers, similarly revealed that hBDNFVal66Met genotype gated the recruitment of these factors by Meth in a region-specific manner. Cumulatively, these data represent the first comprehensive analysis of the long-term effects of chronic Meth exposure within the mesocorticolimbic circuitry. In addition, these data reveal that long-term Meth-induced brain changes are strongly dependent upon BDNF genetic variation, illustrating how drug-induced psychosis may be modulated at the molecular level by a single genetic locus.

Grasslands are a globally important use of land for food and fibre production, which often require the addition of phosphorus (P) fertiliser to maximise plant production. However, a large proportion of the added P can accumulate in pools of poorly available inorganic and organic P in the surface soil layer under grasslands. The aim of this study was to identify the chemical nature of the organic P in soils from a long-term fertiliser by grazing permanent pasture experiment that have received varying additions of phosphatic fertiliser (cumulative P input of 27, 169, 311, 513, 745 and 1035 kg P ha−1) over a period of 37 years. The design of the experiment uniquely provides insight into the response of soil organic P to the addition of fertiliser P on the decadal scale. On average, 46% of the added fertiliser P was recovered as total P in the 0–100 mm soil layer after 37 years of phosphate addition. The content of both inorganic and organic forms of soil P increased with the addition of fertiliser P. The accumulation of organic P increased linearly up to a cumulative P input of 745 kg P ha−1 and plateaued thereafter. The majority of organic P in all treatments was detected as a broad signal in the phosphomonoester region of solution 31P nuclear magnetic resonance (NMR) spectra; this also accounted for 79% of the accumulated organic P in fertilised soil. Our results indicate that accumulation of P in the organic portion as complex forms eventually reaches a new equilibrium where no net accumulation would be expected with further addition of phosphate.

The file attached to this record is the author's final peer reviewed version. Although human psychological risks gravely threaten the safety and success of future Mars missions, current knowledge of the mental health problems most likely to manifest during long duration space exploration (LDSE) is surprisingly inadequate. Previous research conducted during spaceflight and in analog settings has produced discrepant, sometimes contradictory findings and relied on measures that have not been validated for use in extreme environments, where the number, intensity, and duration of stressors exceed typical human experience. We therefore developed the Mental Health Checklist (MHCL) based on subject matter interviews and comprehensive literature reviews. In study one, exploratory and confirmatory factor analyses resulted in 3 reliable subscales (positive adaptation, poor self-regulation, and anxious apprehension) explaining 53% of the total variance. In study two, we examined the reliability and convergent validity of the MHCL in large sample of participants stationed in Antarctica. Findings suggest the MHCL to have acceptable psychometric properties for use in extreme settings. We encourage other researchers to incorporate the MHCL in future studies, including spaceflight research, and to examine its sensitivity for capturing intra-individual symptom changes over time.

Background Nurse staffing shortages are often managed by using temporary staff. This review discovers what is known about nurses who choose to work in this way. Procedure A literature search found eight international studies investigating the lived experience of agency nurses. Results Nurses actively choose agency nursing to work flexibly and avoid office politics or as a career stop-gap. However, respondents describe feeling isolated, working difficulties with permanent staff and fewer opportunities for training. Conclusions Though participants described positive reasons for choosing agency nursing, there were significant downsides. The findings must be viewed in the context of the continued feminized nature of the nursing profession as care responsibilities were cited as a reason for choosing this study pattern. However, the lack of security and provision of adequate pensions and career advancement are considerable issues. The costs of using agency staff are high and consideration must be given to encouraging these nurses into substantive contracts. These individuals show considerable resilience, flexibility, and varied expertise. Such qualities are vital for health care in the 21st century and understanding why they opt out of permanent nursing will enable employers to adapt practices to harness this. Suggestions for practice and further research are presented.

Remodelling of the immune system with age — immunosenescence — is a substantial contributor to poor health in older adults, with increasing risk of infections, cancer and chronic inflammatory disease contributing to age-related multi-morbidity. What is seldom considered when examining the immune response of an aged individual is that the immune system is profoundly influenced by physical activity. Habitual physical activity levels decline with age, with significant consequences for muscle mass and function. Skeletal muscle is a major immune regulatory organ and generates a range of proteins, termed myokines, which have anti-inflammatory and immunoprotective effects. Several studies indicate that maintaining physical activity has immune benefits in older adults, for example, it reduces the systemic inflammation associated with chronic age-related diseases. Here, we discuss how physical activity can prevent or ameliorate age-related multi-morbidity by boosting immune function, and we consider whether physical activity could improve immunotherapy outcomes in age-related conditions such as cancer.

Athletes are advised to receive the COVID-19 vaccination to protect them from SARS CoV-2 infection during major competitions. Despite this, many athletes are reluctant to get the COVID-19 vaccine due to concerns that symptoms of vaccinosis may impair athletic performance.OBJECTIVETo determine the effects of COVID-19 vaccination on the physiological responses to graded exercise.METHODSHealthy participants completed a 20-minute bout of graded cycling exercise before and ~21 days after COVID-19 vaccination (2 dose Pfizer mRNA or 1 dose Johnson & Johnson).RESULTSOxygen uptake, CO2 production, respiratory exchange ratio, ventilation, heart rate, serum noradrenaline, and rating of perceived exertion were significantly elevated in the post vaccine trial. However, vaccination did not affect serum lactate, adrenaline, cortisol, predicted , and ventilatory threshold. Post-vaccine effects on heart rate and noradrenaline remained significant in non-infected participants that received the Pfizer vaccine. No significant effects in respiratory gas exchange parameters were found after vaccination in those previously exposed to SARS-CoV-2, but exercise adrenaline levels were significantly lower and serum lactate levels trending (p= 0.10) lower after vaccination. No changes in any physiological responses to exercise were found in control participants who completed two bouts of exercise separated by ~5 weeks without vaccination.CONCLUSIONRecent COVID-19 vaccination is associated with modest increases in the physiological demands to graded exercise in non-infected healthy people but may actually improve metabolic responses to exercise in those previously infected with SARS-CoV-2. Whether or not these small effects could impact athletic performance at the elite level warrants investigation.

Immunosenescence is described as age-associated changes within the immune system that are responsible for decreased immunity and increased cancer risk. Physically active individuals have fewer 'senescent' and more naïve T-cells compared to their sedentary counterparts, but it is not known if exercise training can rejuvenate 'older looking' T-cell profiles. We determined the effects of 12-weeks supervised exercise training on the frequency of T-cell subtypes in peripheral blood and their relationships with circulating levels of the muscle-derived cytokines (i.e. 'myokines') IL-6, IL-7, IL-15 and osteonectin in older women at high risk of breast cancer. The intervention involved 3 sessions/week of either high intensity interval exercise (HIIT) or moderate intensity continuous exercise (MICT) and were compared to an untrained control (UC) group.HIIT decreased total granulocytes, CD4+ T-cells, CD4+ naïve T-cells, CD4+ recent thymic emigrants (RTE) and the CD4:CD8 ratio after training, whereas MICT increased total lymphocytes and CD8 effector memory (EM) T-cells. The change in total T-cells, CD4+ naïve T-cells, CD4+ central memory (CM) T-cells and CD4+ RTE was elevated after MICT compared to HIIT. Changes in [Formula: see text] after training, regardless of exercise prescription, was inversely related to the change in highly differentiated CD8+ EMRA T-cells and positively related to changes in β2-adrenergic receptor (β2-AR) expression on CM CD4+ and CM CD8+ T-cells. Plasma myokine levels did not change significantly among the groups after training, but individual changes in IL-7 were positively related to changes in the number of β2-AR expressing CD4 naïve T cells in both exercise groups but not controls. Further, CD4 T-cells and CD4 naive T-cells were negatively related to changes in IL-6 and osteonectin after HIIT but not MICT, whereas CD8 EMRA T-cells were inversely related to changes in IL-15 after MICT but not HIIT.Aerobic exercise training alters the frequency of peripheral T-cells associated with immunosenescence in middle aged/older women at high risk of breast cancer, with HIIT (pro-senescent) and MICT (anti-senescent) evoking divergent effects. Identifying the underlying mechanisms and establishing whether exercise-induced changes in peripheral T-cell numbers can alter the risk of developing breast cancer warrants investigation.

International audience; Differences in root morphology and acclimation to low-phosphorus (P) soil were examined among eight legume species from the Trifolium Section Tricocephalum to understand how these root attributes determine P acquisition. Ornithopus sativus was included as a highly P-efficient benchmark species. Plants were grown as microswards in pots with five rates of P supplied in a topsoil layer to mimic uneven P distribution within a field soil profile. Topsoil and subsoil roots were harvested separately to enable measurement of the nutrient-foraging responses. Critical P requirement (lowest P supply for maximum yield) varied over a threefold range, reflecting differences in root morphology and acclimation of nutrient-foraging roots to P stress. Among the species, there was a 3.2-fold range in root length density, a 1.7-fold range in specific root length, and a 2.1-fold range in root hair length. O. sativus had the lowest critical P requirement, displayed a high root length density, the highest specific root length, and the longest root hairs. Acquisition of P from P-deficient soil was facilitated by development of a large root hair cylinder (i.e. a large root–soil interface). This, in turn, was determined by the intrinsic root morphology attributes of each genotype, and the plasticity of its root morphology response to internal P stress. Root acclimation in low-P soil by all species was mostly associated with preferential allocation of mass to nutrient-foraging roots. Only O. sativus and four of the Trifolium species adjusted specific root length beneficially, and only O. sativus increased its root hair length in low-P soil.

The remarkable success of cancer immunotherapies has provided new hope to cancer patients. Unfortunately, a significant proportion of patients remain unable to respond to immunotherapy or maintain durable clinical responses. The lack of objective responses likely results from profound immune dysfunction often observed in patients with cancer. There is substantial evidence that exercise and physical activity can reduce incidence and improve outcomes in cancer patients. As the immune system is highly responsive to exercise, one potential avenue to improve immune function is through exercise and physical activity. A single event of dynamic exercise results in the substantial mobilization of leukocytes with increased functional capacities into the circulation. Chronic, or long-term, exercise leads to higher physical fitness in terms of greater cardiorespiratory function and/or muscle strength and endurance. High aerobic capacity, as measured by maximal oxygen uptake, has been associated with the reduction of dysfunctional T cells and improvements in the abundance of some T cell populations. To be sure, however, the mechanisms of exercise-mediated immune changes are both extensive and diverse. Here, we examine the evidence and theorize how acute and chronic exercise could be used to improve responses to cancer immunotherapies including immune checkpoint inhibitors, dendritic cell vaccines, natural killer cell therapies, and adoptive T cell therapies such as chimeric antigen receptor (CAR) T cells. Although the parameters of optimal exercise to yield defined outcomes remain to be determined, the available current data provide a compelling justification for additional human studies and clinical trials investigating the adjuvant use of exercise in immuno-oncology.

Objective Insects are the most evolutionarily successful groups of organisms, and this success is largely due to their flight ability. Interestingly, some stick insects have lost their flight ability despite having wings. To elucidate the shift from wingless to flying forms during insect evolution, we compared the nutritional metabolism system among flight-winged, flightless-winged, and flightless-wingless stick insect groups. Results Here, we report RNA sequencing of midgut transcriptome of Entoria okinawaensis, a prominent Japanese flightless-wingless stick insect, and the comparative analysis of its transcriptome in publicly available midgut transcriptomes obtained from seven stick insect species. A gene enrichment analysis for differentially expressed genes, including those obtained from winged vs wingless and flight vs flightless genes comparisons, revealed that carbohydrate metabolic process-related genes were highly expressed in the winged stick insect group. We also found that the expression of the mitochondrial enolase superfamily member 1 transcript was significantly higher in the winged stick insect group than in the wingless stick insect group. Our findings could indicate that carbohydrate metabolic processes are related to the evolutionary process through which stick insects gain the ability of flight.

BACKGROUND AND PURPOSE: The relationship between carotid intraplaque hemorrhage and luminal stenosis severity is not well-established. We sought to determine whether intraplaque hemorrhage is related to carotid stenosis and at what degree of stenosis intraplaque hemorrhage most likely contributes to ischemic symptoms. MATERIALS AND METHODS: Consecutive patients who underwent MR carotid plaque imaging with MPRAGE sequences to identify intraplaque hemorrhage were retrospectively reviewed. Degrees of stenoses were categorized as minimal (70%). Arteries were categorized into 2 groups: symptomatic (ipsilateral to a cerebral ischemic event) and asymptomatic (from a patient without an ischemic event). Multiple regression analyses were used to determine independent associations between the degree of stenosis and intraplaque hemorrhage and the presence of intraplaque hemorrhage with symptoms among categories of stenosis. RESULTS: We included 449 patients with 449 carotid arteries: Two hundred twenty-five (50.1%) were symptomatic, and 224 (49.9%) were asymptomatic. An increasing degree of stenosis was independently associated with the presence of intraplaque hemorrhage (OR = 1.02; 95% confidence interval, 1.01–1.03). Intraplaque hemorrhage was independently associated with ischemic events in arteries with 30% stenosis. Of symptomatic arteries with minimal stenosis, 8.7% had intraplaque hemorrhage versus 1.7% of asymptomatic arteries (P = .02). No differences in intraplaque hemorrhage prevalence were found between symptomatic and asymptomatic groups with moderate (P = .18) and severe stenoses (P = .99). CONCLUSIONS: The presence of intraplaque hemorrhage on high-resolution plaque imaging is likely most useful in identifying symptomatic plaques in cases of minimal stenosis.

summary The occurrence and type of storage carbohydrate (fructan, starch or sucrose) was determined in stems of reproductive tillers of a number of species of the family Gramineae representing major taxonomic groups. Plants were collected from two grassland sites in Victoria (Australia). Fructans were detected in all species examined, but particularly high concentrations were found in species of the subfamily Pooideae where fructans accounted for 75–95% of the storage carbohydrates. In most other subfamilies, fructans varied between 5 and 30% of the storage carbohydrates. However, fructans accounted for 50% of the storage carbohydrates in species of the subfamily Arundinoideae. Separation of fructans (to DP 14) by thin-layer chromatography demonstrated that different isomers of trisaccharides and different proportions of oligosaccharides were present, depending on the species. In some species there was evidence of continuous series of oligosaccharides between sucrose and high molecular weight polymers. However, these series differed markedly from the series of oligosaccharides present in Helianthus tuberosus. The polymerization of fructans and trisaccharide forms present in the grasses examined were similar on generic and, in a number of cases, subtribal and tribal levels and reflected phylogenetic relationships between taxa. The possible presence of fructans in all subfamilies may indicate that fructan synthesis is not necessarily exclusive to the subfamily Pooideae.

SUMMARY Fructan- and sucrose-hydrolyzing activity was extracted from plants of Lolium rigidum Gaudin. Sucrose-hydrolyzing activity varied 30-fold between different plant parts and parts of different ages. In contrast, fructan-hydrolyzing activity only varied two-fold. At least four β-fructofuranosidase activities were partially purified from mature vegetative tissues of Lolium rigidum. The β-fructofuranosidases were characterized by their different elution patterns on various chromatographic media and their relative rates of hydrolysis of sucrose and fructan extracted from different sources. Fructan-hydrolyzing activities were partially purified that hydrolyzed fructan containing predominantly 2–1 glycosidic linkages relatively more rapidly than fructan containing predominantly 2–6 glycosidic linkages. β-fructofuranosidases that were relatively more active against fructan than sucrose when compared with activity present in the crude extract, were inhibited by sucrose. It is concluded that it is necessary to use appropriate fructan substrates and to consider the possible involvement of all β-fructofuranosidase activities in hydrolysis of fructans in grasses.