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Intracellular events in the synthesis, glycosylation, and transport of the lymphocytic choriomeningitis virus (LCMV) glycoproteins have been examined. We have shown by N-glycanase digestion that LCMV strain Arm-4 bears five oligosaccharides on GP-1 and two on GP-2. By pulse-chase labeling experiments in the presence of drugs which inhibit N-linked oligosaccharide addition and processing we demonstrate that addition of high mannose precursor oligosaccharides is necessary for transport and cleavage of the viral GP-C glycoprotein. Moreover, in the presence of tunicamycin which inhibits en bloc addition of these mannose-rich side chains, virus budding was substantially decreased and infectious virions were reduced by more than 1000-fold in the supernatant medium. Incubation in the presence of castantospermine, which permits addition of oligomannosyl-rich chains but blocks further processing, restored transport and cleavage of GP-C and maturation of virions. Finally, by temperature block experiments we have determined that maturation of GP-C oligosaccharides to an endoglycosidase H resistant form precedes cleavage to GP-1 and GP-2. The latter process is most likely to occur in the Golgi or post-Golgi compartment.

Doping control and drug testing describe the process of assessing an athlete's status regarding medications and other compounds on the banned substance lists. Medications can be over the counter, prescription, or illicit. Compounds can include supplements, vitamins, and herbal mixtures. At the collegiate level, specific groups have been identified as reliable sources to conduct and evaluate drug tests. The adjudication process has been outlined, as well as the sanctions for testing positive. In this paper, the substances that are prohibited will be identified and the drug testing process will be explained. [ABSTRACT FROM AUTHOR]

Abstract Background Since females often pay a higher cost for heterospecific matings, mate discrimination and species recognition are driven primarily by female choice. In contrast, frequent indiscriminate matings are hypothesized to maximize male fitness. However, recent studies show that previously indiscriminate males (e.g., Drosophila melanogaster and Poecilia reticulata) can learn to avoid heterospecific courtship. This ability of males to discriminate against heterospecific courtship may be advantageous in populations where two species co-occur if courtship or mating is costly. Results Here, we tested whether Drosophila pseudoobscura males learn to discriminate against heterospecific females after being exposed to and rejected by D. persimilis females. In most of our assays, we failed to observe differences in D. pseudoobscura courtship intensity of heterospecific females by males that had previously courted heterospecific females vs. males that had been maintained in isolation. Conclusion We conclude that learning to avoid heterospecific courtship may not be universal, even within the genus Drosophila, and may possibly be dependent on the natural history of the species.

Objective: Increased attention has been directed toward assessing and improving academic quality in athletic training education. The educational process has been assessed from a global level, but little is known about how athletic training students learn. The purpose of this investigation was to assess the learning styles of undergraduate athletic training students. Design and Setting: Undergraduate students enrolled in a Committee on Accreditation of Allied Health Education Programs (CAAHEP)-accredited athletic training education program completed a learning styles inventory during a regularly scheduled athletic training class at the start of the spring semester. Subjects: Twenty-seven student athletic trainers (age range, 19-30 yrs, mean age = 20.5 yrs) served as subjects. Sixteen subjects (7 male, 9 female) were in the first year of this 3-year program. Eleven subjects (7 male, 4 female) were second-year students. Measurements: Learning style was assessed using the Productivity Environmental Preference Survey. Results: Parametric and nonparametric one-way analyses of variance for each learning subscale by sex and by year in program revealed significant differences (P < .05) in light preferences for male and female students. There were also significant differences (P < .05) between first- and second-year students in preferences for afternoon learning activities. Conclusions: These findings suggest that undergraduate athletic training students function best as learners in a well-lit learning environment. The significance of afternoon as the preferred time for learning reinforces the importance of the clinical setting in the introduction and mastery of skills. Athletic training educators and clinical instructors can use these results as they examine their teaching strategies and educational environments. [ABSTRACT FROM AUTHOR]

The purpose of this review was to summarize the evidence with regard to behavioral and psychosocial assessment of the periodontitis patient, the candidate for implant therapy, and the peri-implantitis patient. Periodontitis has an adverse effect on quality of life and its treatment can lead to significant improvements experienced by the patient. The latter is true for rehabilitation with dental implants, although patients harbor diverse expectations and perceptions of implant therapy, which can often interfere with satisfaction and/or influence long-term success. A thorough behavioral assessment of the candidate for implant therapy is essential, which should include, perceptions, expectations, as well as risk for behavioral disorders. Remedial action is essential to correct misperceptions and any identified risks. Finally, patients have limited awareness of limited ability to identify signs of peri-implantitis. The diagnosis of peri-implantitis can be a cause of significant distress, resentment, and loss of trust to the treatment and the caregivers. Despite documented value in clinical research, currently available instruments assessing patient-reported outcomes have little application in day-to-day clinical practice. Face-to-face patient to doctor open-ended communication remains the most effective way to comprehensively establish the long-term "therapeutic alliance" essential for the long journey for the periodontitis patient., (© 2024 The Author(s). Periodontology 2000 published by John Wiley & Sons Ltd.)

Introduction: The growing ability to provide online services has enabled the proliferation of exercise-based telehealth interventions; however, adoption in older adults may be impacted by low digital literacy and "technophobia.", Objectives: The aim of this study was to explore the experience of community and aged-care dwelling older adults following exercise-based telehealth services to provide insights that could guide future telehealth exercise delivery., Design: Semi-structured interviews for qualitative analysis., Methods: Participants (age ≥60) who had completed at least one online exercise session from a registered health professional were recruited through a combination of purposeful and snowball sampling methods via their exercise facility or provider. A semi-structured interview guide was used by 2 interviewers to investigate participants' experiences and a "critical friends" approach used to identify common themes., Results: Thirteen interviews with 21 participants were conducted from 2 different facilities. Analysis identified meaning units within 3 themes and subthemes. Technology subthemes related to digital confidence prior to telehealth and changes during interventions, as well as the usability of technology for telehealth. Clinical practice subthemes described the different motivations to exercise, perceived benefits of telehealth, important implications for practitioners, and perceptions of safety. The social connection theme related to the social benefits of telehealth., Conclusions: Older adults in our sample were technologically confident and capable of performing exercise sessions delivered via telehealth. They notice benefits from this form of exercise delivery however, prefer face-to-face exercise delivery.

People with osteoarthritis often experience pain and depression. These meta-analyses examined and compared nonpharmacological randomized controlled trials (RCTs) for pain and symptoms of depression in people living with osteoarthritis. RCTs published up until April 2022 were sourced by searching electronic databases EMBASE, PUBMED & MEDLINE, Web of Science, CINAHL and PEDro. Random-effects meta-analyses were performed to calculate pooled effect sizes (ES) and 95% confidence intervals (CI) for pain and depression. Subgroup analyses examined intervention subtypes. For pain, 29 interventions (n = 4382; 65 ± 6.9 years; 70% female), revealed a significant effect on reducing pain (ES = 0.43, 95% CI [0.25, 0.61], p < 0.001). Effect sizes were significant (p < 0.001) for movement meditation (ES = 0.52; 95% CI [0.35, 0.69]), multimodal approaches (ES = 0.37; 95% CI [0.22, 0.51]), and psychological therapy (ES = 0.21; 95% CI [0.11, 0.31]), and significant (p = 0.046) for resistance exercise (ES = 0.43, 95% CI [- 0.07, 0.94]. Aerobic exercise alone did not improve pain. For depression, 28 interventions (n = 3377; 63 ± 7.0 years; 69% female), revealed a significant effect on reducing depressive symptoms (ES = 0.29, 95% CI [0.08, 0.49], p < 0.001). Effect sizes were significant for movement meditation (ES = 0.30; 95% CI [0.06, 0.55], p = 0.008) and multimodal interventions (ES = 0.12; 95% CI [0.07, 0.18], p < 0.001). Resistance/aerobic exercise or therapy alone did not improve depressive symptoms. Mind-body approaches were more effective than aerobic/resistance exercise or therapy alone for reducing pain and depression in people with osteoarthritis.Systematic review registration: PROSPERO CRD42022338051., (© 2023. Springer Nature Limited.)

Purpose: Examine the reliability of field-based fitness assessments in school-aged children with emotional or behavioral difficulties (EBD). Understanding the impact of fitness on physical activity participation for children with EBD is limited by our ability to reliably measure it., Methods: Fifteen children aged 7-12 years with EBD completed 7 assessments-standing broad jump, overhead throw, grip strength, isometric plank hold, isometric wall squat, unilateral heel raise, and modified 6-minute walk test-in a random order on 2 separate occasions. Intraclass correlation coefficients (ICCs) were computed to evaluate reliability., Results: ICCs ranged from .65 to .99 representing moderate to excellent reliability for all assessments. Shorter assessments requiring less attention and behavior regulation tended to demonstrate higher ICC values while assessments with greater attention or behavioral regulation demands tended to have lower ICC values., Conclusion: Results demonstrate varied reliability for fitness tests in children with EBD. Practitioners can use grip strength and standing broad jump assessments with confidence. Other assessments have good reliability but greater variability indicating they may be a challenge for some children with EBD.

While blood antibodies mediate protective immunity in most organs, whether they protect nasal surfaces in the upper airway is unclear. Using multiple viral infection models in mice, we found that blood-borne antibodies could not defend the olfactory epithelium. Despite high serum antibody titers, pathogens infected nasal turbinates, and neurotropic microbes invaded the brain. Using passive antibody transfers and parabiosis, we identified a restrictive blood-endothelial barrier that excluded circulating antibodies from the olfactory mucosa. Plasma cell depletions demonstrated that plasma cells must reside within olfactory tissue to achieve sterilizing immunity. Antibody blockade and genetically deficient models revealed that this local immunity required CD4 + T cells and CXCR3. Many vaccine adjuvants failed to generate olfactory plasma cells, but mucosal immunizations established humoral protection of the olfactory surface. Our identification of a blood-olfactory barrier and the requirement for tissue-derived antibody has implications for vaccinology, respiratory and CNS pathogen transmission, and B cell fate decisions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Receptor tyrosine kinase inhibitors improve cancer survival but their cardiotoxicity requires investigation. We investigated these inhibitors' effects on human cardiac progenitor cells in vitro and rat heart in vivo. We applied imatinib, sunitinib or sorafenib to human cardiac progenitor cells, assessing cell viability, proliferation, stemness, differentiation, growth factor production and second messengers. Alongside, sunitinib effects were assessed in vivo. Inhibitors decreased (p < 0.05) cell viability, at levels equivalent to 'peak' (24 h; imatinib: 91.5 ± 0.9%; sunitinib: 83.9 ± 1.8%; sorafenib: 75.0 ± 1.6%) and 'trough' (7 days; imatinib: 62.3 ± 6.2%; sunitinib: 86.2 ± 3.5%) clinical plasma levels, compared to control (100% viability). Reduced (p < 0.05) cell cycle activity was seen with imatinib (29.3 ± 4.3% cells in S/G2/M-phases; 50.3 ± 5.1% in control). Expression of PECAM-1, Nkx2.5, Wnt2, linked with cell differentiation, were decreased (p < 0.05) 2, 2 and 6-fold, respectively. Expression of HGF, p38 and Akt1 in cells was reduced (p < 0.05) by sunitinib. Second messenger (p38 and Akt1) blockade affected progenitor cell phenotype, reducing c-kit and growth factor (HGF, EGF) expression. Sunitinib for 9 days (40 mg/kg, i.p.) in adult rats reduced (p < 0.05) cardiac ejection fraction (68 ± 2% vs. baseline (83 ± 1%) and control (84 ± 4%)) and reduced progenitor cell numbers. Receptor tyrosine kinase inhibitors reduce cardiac progenitor cell survival, proliferation, differentiation and reparative growth factor expression., (© 2022. The Author(s).)

The purpose of this study was to explore the impact of cultural food insecurity on identity and well-being in second-generation American and international university students. Thirty-one semi-structured interviews were conducted from January-April 2020. Audio transcripts were analyzed using continuous and abductive thematic analysis. Students indicated that cultural foodways enhanced their well-being by facilitating their cultural/ethnic identity maintenance, connection, and expression. Conversely, cultural food insecurity diminished student well-being due to reduced cultural anchors, highlighting the importance of cultural food in this population. Universities that reduce cultural foodways barriers may mitigate cultural food insecurity for second-generation American and international university students. (100/100).

We sought to examine the strategies promoting and countering state preemption of local sugar-sweetened beverage (SSB) taxes in the United States. Using Crosbie and Schmidt's tobacco preemption framework, we analyzed key tactics used by the SSB industry to achieve state preemption of local taxes identified in news sources, industry Web sites, government reports, and public documents.Starting in 2017, 4 states rejected and 4 passed laws preempting local SSB taxes. The beverage industry attempted to secure state preemption through front groups and trade associations, lobbying key policymakers, inserting preemptive language into other legislation, and issuing legal threats and challenges. The public health community's response is in the early stages of engaging in media advocacy, educating policymakers, mobilizing national collaboration, and expanding legal networks.State preemption of local SSB taxes is in the early stages but will likely scale up as local tax proposals increase. The public health community has a substantial role in proactively working to prevent preemption concurrent with health policy activity and using additional strategies successfully used in tobacco control to stop preemption diffusion.

Objective: Despite support for the role of exercise in improving physical and mental health for various psychiatric disorders, few service implementation evaluations within diverse hospital environments exist. This study presents the feasibility and implementation of a clinical exercise physiology service within a hospital mental health service., Method: Feasibility and service data were collected from databases and self-report (consumer and staff) for 6 months from the commencement of new exercise services (gym and group sessions) for community and inpatients (one secure and three open wards)., Results: One hundred and twenty consumers engaged with exercise services with 70 direct referrals over the 6-month audit period (mean age 40 ± 13 years (19-69); 41% male). The overwhelming reason for referral was related to weight loss/management (65.7%), with the majority of patients (51%) presenting with schizophrenia spectrum and other psychotic disorders. Further, 549 exercise service interactions were delivered and 78% gym attendees and 69% group session attendees rated the exercise sessions as 'Good' or 'Great', and intention to return ranged from 78% for inpatient gym sessions to 91% for community gym sessions., Conclusions: Embedding exercise physiology into a mental health service is feasible and well accepted and the evaluation of long-term consumer outcomes in 'real-world' will serve as a crucial step.

Purpose: There are multidien patterns of seizure occurrence. Predicting seizure risk may be easier with biomarker correlates to multidien patterns. We hypothesize multiday hyper or hypoglycemia contributes to seizure risk., Methods: In a type I diabetic (T1D) with focal onset epilepsy with continuous glucose monitoring (CGM) and responsive neurostimulation (RNS) devices, we studied multiday interictal activities (IEA), seizures, and glucose. Hourly CGM data was matched to hourly RNS captures of interictal and ictal activities over 33 months. RNS detection settings were unchanged. Multidien cycles were analyzed, active blocks of IEA and ictal episodes defined, and tissue glucose averages studied., Results: Average glucose was 161 mg/dl. A 40-day cycle of interictal and ictal activities occurred, though no similar glucose cycle was evident. Glucose elevations relative to patient average were associated with increases in IEA but not seizure. Frequent seizures were not associated with obvious elevations or decreases of glucose from baseline, most seizures occurred at +/- 10 mg/dl of average daily glucose (i.e. 150-170 mg/dl)., Conclusion: Tissue glucose may influence IEA but may not influence multiday seizure activity or very frequent seizures. In an ambulatory T1D patient multiday hypo or hyperglycemic extremes do not appear to provoke seizure activities., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Food contributes to an individual's physical and mental well-being and expresses one's cultural identity through preparation, sharing, and consumption (i.e., foodways). Inadequate access to cultural foods can create cultural stress and affect one's identity and well-being. In particular, second-generation U.S. American student populations may have a higher risk for cultural stress due to being away from family, academic stress, environmental changes, and diminished financial stability to purchase cultural foods. Thus, an exploratory qualitative methodology was used to elicit information about second-generation U.S. Americans' food experiences to identify how cultural foods play a role in individual identity and how individual well-being is influenced by the presence or lack of cultural foods. Sixteen semi-structured interviews were conducted with second-generation American students at the University of Nevada, Reno, who self-identified as a cultural or ethnic minority. A standard thematic analysis was conducted. The authors identified that cultural food security influenced the ability to practice foodways, which tied Second-generation American students to their cultural identities. The absence of foodways led to anxiety and depression among students, amplifying the feelings of identity degradation. Second-generation American students discussed that the ability to practice their foodways improved multiple well-being components and led to feelings of happiness, decreased stress, warmth, better digestion, and a sense of belonging, comfort, and safety. College populations continue to grow and become more diverse, and with the increasing Second-generation American students, it is essential to improve the access and availability of cultural foods to improve their overall well-being. (245/250 words)., Supplementary Information: The online version contains supplementary material available at 10.1007/s12571-020-01140-w., Competing Interests: Conflicts of interest/competing interestsThe authors declare that they have no conflict of interest., (© International Society for Plant Pathology and Springer Nature B.V. 2021.)

Objectives: To evaluate if neuromuscular performance and hypermobility are factors associated with children's motor competence., Design: Cross-sectional observation study., Methods: Data was collected on 60 children aged 6-12 years; motor competence was determined using the Movement Assessment Battery for Children-2 test, with children classified into 3 groups (Typically Developing n=30; 'At Risk' of low motor competence (LMC) n=9; LMC n=21). Neuromuscular performance was determined utilising the Resistance Training Skills Battery for Children (RTSBc), 5-repetition maximum (5RM) leg press and Biodex dynamometry to assess isometric and isokinetic peak torque of the knee flexors and extensors. Hypermobility was measured using the Beighton and Lower Limb Assessment Score., Results: Between-groups MANCOVA revealed typically developing children scored significantly higher on the RTSBc than those 'at risk' of LMC (p=0.021) and those in the LMC group (p<0.001). 5RM scores also differed between groups, with typically developing children achieving significantly higher scores than the LMC group. No differences were found between groups for isometric or isokinetic measures of strength. Sequential regression analysis revealed neuromuscular performance variables explained 44.7% of the variance in motor competence, with RTSBc (p<0.001) and 5RM (p=0.019) emerging as positive significant predictors. Hypermobility failed to explain significant variance in motor competence beyond that explained by neuromuscular performance., Conclusions: Neuromuscular performance of children varies according to levels of motor competence, with those with LMC performing poorly on tasks requiring multi-joint movement. Furthermore, neuromuscular performance predicted almost half the variance observed in motor competence and highlights a novel intervention strategy., (Copyright © 2020 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it-and the other parasite proteins it interacts with-promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet., (Copyright © 2020 Campeotto et al.)

Objectives: In a sample of children with, or at risk of, movement difficulties, (1) To test the direct effects of children's perceptions of parents' logistic support for physical activity on children's physical activity-related self-perceptions and on children's physical activity levels, and (2) To explore the indirect relationship between children's perceptions of parents' logistic support for physical activity and children's physical activity levels through children's physical activity-related self-perceptions., Design: Cross-sectional observation study., Methods: Data were collected from 120 children aged 6 to 12 years; movement proficiency levels were determined using the movement assessment battery for children-2 test. Children's perspectives of parental support for physical activity were captured using the Activity Support Scale for Multiple Groups child report. Children's self-perceptions towards physical activity were reported with the Children's Self- perceptions of Adequacy in and Predilections for physical activity, and time spent in physical activity were measured using accelerometers., Results: There was no significant direct effect between perceived levels of parents' logistic support for physical activity and children's physical activity. A significant indirect relationship between these variables was discovered, with higher perceived levels of parent logistical support for physical activity predicting stronger perceptions of adequacy (i.e., confidence) toward physical activity participation among children, which in turn was associated with increased physical activity minutes., Conclusions: The results demonstrate that irrespective of a child's movement ability, children's perceptions of parents' logistic support for physical activity indirectly and positively predicts children's physical activity levels, via children's confidence for physical activity., (Copyright © 2019. Published by Elsevier Ltd.)

Despite significant progress in controlling malaria, the disease remains a global health burden. The intricate interactions the parasite Plasmodium falciparum has with its host allows it to grow and multiply in human erythrocytes. The mechanism by which P. falciparum merozoites invade human erythrocytes is complex, involving merozoite proteins as well as erythrocyte surface proteins. Members of the P. falciparum reticulocyte binding-like protein homolog (PfRh) family of proteins play a pivotal role in merozoite invasion and hence are important targets of immune responses. Domains within the PfRh2b protein have been implicated in its ability to stimulate natural protective antibodies in patients. More specifically, a 0.58 kbp deletion, at the C-terminus has been reported in high frequencies in Senegalese and Southeast Asian parasite populations, suggesting a possible role in immune evasion. We analysed 1218 P. falciparum clinical isolates, and the results show that this deletion is present in Ghanaian parasite populations (48.5% of all isolates), with Kintampo (hyper-endemic, 53.2%), followed by Accra (Hypo-endemic, 50.3%), Cape Coast (meso-endemic, 47.9%) and Sogakope (meso-endemic, 43.15%). Further analysis of parasite genomes stored in the MalariaGEN database revealed that the deletion variant was common across transmission areas globally, with an overall frequency of about 27.1%. Interestingly, some parasite isolates possessed mixed PfRh2b deletion and full-length alleles. We further showed that levels of antibodies to the domain of PfRh2 protein were similar to antibody levels of PfRh5, indicating it is less recognized by the immune system.

Many human pathogens use host cell-surface receptors to attach and invade cells. Often, the host-pathogen interaction affinity is low, presenting opportunities to block invasion using a soluble, high-affinity mimic of the host protein. The Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) provides an exciting candidate for mimicry: it is highly conserved and its moderate affinity binding to the human receptor basigin (K D  ≥1 μM) is an essential step in erythrocyte invasion by this malaria parasite. We used deep mutational scanning of a soluble fragment of human basigin to systematically characterize point mutations that enhance basigin affinity for RH5 and then used Rosetta to design a variant within the sequence space of affinity-enhancing mutations. The resulting seven-mutation design exhibited 1900-fold higher affinity (K D approximately 1 nM) for RH5 with a very slow binding off rate (0.23 h -1 ) and reduced the effective Plasmodium growth-inhibitory concentration by at least 10-fold compared to human basigin. The design provides a favorable starting point for engineering on-rate improvements that are likely to be essential to reach therapeutically effective growth inhibition., (© 2019 Wiley Periodicals, Inc.)

Detection of protein biomarkers is an important tool for medical diagnostics, typically exploiting concentration of particular biomarkers or biomarker release from tissues. We sought to establish whether proteins not normally released by living cells can be extracted without harming cells, with a view to extending this into biomarker harvest for medical diagnosis and other applications. Styrene maleic acid (SMA) is a polymer that extracts nanodiscs of biological membranes (containing membrane proteins) from cells. Hitherto it has been used to harvest SMA-lipid-membrane protein particles (SMALP) for biochemical study, by destroying the living cellular specimen. In this study, we applied SMA at low concentration to human primary cardiovascular cells and rat vascular tissue, to 'biopsy' cell proteins while avoiding significant reductions in cell viability. SMA at 6.25 parts per million harvested proteins from cells and tissues without causing significant release of cytosolic dye (calcein) or reduction in cell viability at 24 and 72 hours post-SMA (MTT assay). A wide range of proteins were recovered (20-200 kDa) and a number identified by mass spectrometry: this confirmed protein recovery from plasma membrane, intracellular membranes and cell cytosol without associated cell death. These data demonstrate the feasibility of non-lethally sampling proteins from cells, greatly extending our sampling capability, which could yield new physiological and/or pathological biomarkers.

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion.

Malaria is caused by Plasmodium parasites, which invade and replicate in erythrocytes. For Plasmodium falciparum, the major cause of severe malaria in humans, a heterotrimeric complex comprised of the secreted parasite proteins, PfCyRPA, PfRIPR and PfRH5 is essential for erythrocyte invasion, mediated by the interaction between PfRH5 and erythrocyte receptor basigin (BSG). However, whilst CyRPA and RIPR are present in most Plasmodium species, RH5 is found only in the small Laverania subgenus. Existence of a complex analogous to PfRH5-PfCyRPA-PfRIPR targeting BSG, and involvement of CyRPA and RIPR in invasion, however, has not been addressed in non-Laverania parasites. Here, we establish that unlike P. falciparum, P. knowlesi and P. vivax do not universally require BSG as a host cell invasion receptor. Although we show that both PkCyRPA and PkRIPR are essential for successful invasion of erythrocytes by P. knowlesi parasites in vitro, neither protein forms a complex with each other or with an RH5-like molecule. Instead, PkRIPR is part of a different trimeric protein complex whereas PkCyRPA appears to function without other parasite binding partners. It therefore appears that in the absence of RH5, outside of the Laverania subgenus, RIPR and CyRPA have different, independent functions crucial for parasite survival., Competing Interests: The authors have declared that no competing interests exist.

Children with low movement proficiency have been identified as having poorer physiological and psychosocial outcomes; however, the varied measurement approaches used to assess these outcomes have varied resulting in conflicting evidence regarding the presence and magnitude of differences compared to Typically Developing (TD) children. Additionally, there has been limited research into the role of parental support for physical activity (PA) in this group. We compared children with varying levels of movement proficiency on physiological characteristics and self-perceptions regarding PA. In addition, these children's parents were compared on physiological characteristics and support of their children's PA. Children (N = 117) aged 6 to 12 years, along with their parent/guardian, participated in this study. Children were classified according to the Movement Assessment Battery for Children-2 test (Typically Developing (TD) = 60; At Risk = 19; Developmental Coordination Disorder (DCD) = 38). Children's PA, muscle strength, cardio-respiratory fitness (CRF), body composition, and self-perceptions regarding PA were assessed, with parents assessed on CRF, body composition, and PA support. Compared to TD children, children with DCD had lower PA (p = 0.036), predilection (p ≤0.001) and adequacy (p ≤0.001) regarding PA, higher body fat percentage (p = 0.019), and received less logistic support (i.e., transportation) from their parents (p = 0.012). TD children had increased muscle strength compared to the DCD (p ≤ 0.001) and At Risk (p ≤ 0.001) groups. Results indicated that, relative to TD children, children with DCD have multiple physiological deficits, receive less parental logistic support for PA involvement, and report lower scores on psychological constructs that are predictive of PA involvement., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objectives: Resistance training (RT) is emerging as a training modality to improve motor function and facilitate physical activity participation in children across the motor proficiency spectrum. Although RT competency assessments have been established and validated among adolescent cohorts, the extent to which these methods are suitable for assessing children's RT skills is unknown. This project aimed to assess the psychometric properties of the adapted Resistance Training Skills Battery for Children (RTSBc), in children with varying motor proficiency., Design: Repeated measures design with 40 participants (M age=8.2±1.7years) displaying varying levels of motor proficiency., Methods: Participants performed the adapted RTSBc on two occasions, receiving a score for their execution of each component, in addition to an overall RT skill quotient child (RTSQc). Cronbach's alpha, intra-class correlation (ICC), Bland-Altman analysis, and typical error were used to assess test-retest reliability. To examine construct validity, exploratory factor analysis was performed alongside computing correlations between participants' muscle strength, motor proficiency, age, lean muscle mass, and RTSQc., Results: The RTSBc displayed an acceptable level of internal consistency (alpha=0.86) and test-retest reliability (ICC range=0.86-0.99). Exploratory factor analysis supported internal test structure, with all six RT skills loading strongly on a single factor (range 0.56-0.89). Analyses of structural validity revealed positive correlations for RTSQc in relation to motor proficiency (r=0.52, p<0.001) and strength scores (r=0.61, p<0.001)., Conclusions: Analyses revealed support for the construct validity and test-retest reliability of the RTSBc, providing preliminary evidence that the RTSBc is appropriate for use in the assessment of children's RT competency., (Copyright © 2018 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.)

Plasmodium knowlesi, a zoonotic parasite causing severe-to-lethal malaria disease in humans, has only recently been adapted to continuous culture with human red blood cells (RBCs). In comparison with the most virulent human malaria, Plasmodium falciparum, there are, however, few cellular tools available to study its biology, in particular direct investigation of RBC invasion by blood-stage P. knowlesi merozoites. This leaves our current understanding of biological differences across pathogenic Plasmodium spp. incomplete. Here, we report a robust method for isolating viable and invasive P. knowlesi merozoites to high purity and yield. Using this approach, we present detailed comparative dissection of merozoite invasion (using a variety of microscopy platforms) and direct assessment of kinetic differences between knowlesi and falciparum merozoites. We go on to assess the inhibitory potential of molecules targeting discrete steps of invasion in either species via a quantitative invasion inhibition assay, identifying a class of polysulfonate polymer able to efficiently inhibit invasion in both, providing a foundation for pan-Plasmodium merozoite inhibitor development. Given the close evolutionary relationship between P. knowlesi and P. vivax, the second leading cause of malaria-related morbidity, this study paves the way for inter-specific dissection of invasion by all three major pathogenic malaria species.

Mucormycosis is responsible for an increasing proportion of deaths after allogeneic bone marrow transplantation. Because this disease is associated with severe immunodeficiency and has shown resistance to even the newest antifungal agents, we determined the feasibility of reactivating and expanding Rhizopus oryzae -specific T cells for use as adoptive immunotherapy in transplant recipients. R. oryzae extract - pulsed monocytes were used to stimulate peripheral blood mononuclear cells from healthy donors, in the presence of different cytokine combinations. The generated R. oryzae -specific T cell products were phenotyped after the third stimulation and further characterized by the use of antibodies that block class I/II molecules, as well as pattern recognition receptors. Despite the very low frequency of R. oryzae -specific T cells of healthy donors, we found that stimulation with interleukin-2 (IL-2)/IL-7 cytokine combination could expand these rare cells. The expanded populations included 17%-83% CD4 + T cells that were specific for R. oryzae antigens. Besides interferon-γ (IFN-γ), these cells secreted IL-5, IL-10, IL-13, and tumor necrosis factor alpha (TNF-α), and recognized fungal antigens presented by HLA-II molecules rather than through nonspecific signaling. The method described herein is robust and reproducible, and could be used to generate adequate quantities of activated R. oryzae -specific T cells for clinical testing of safety and antifungal efficacy in patients with mucormycosis.

Human papilloma virus (HPV) is a known cause of cervical cancer, squamous cell carcinoma and laryngeal cancer. Although treatments exist for HPV-associated malignancies, patients unresponsive to these therapies have a poor prognosis. Recent findings from vaccine studies suggest that T-cell immunity is essential for disease control. Because Epstein-Barr Virus (EBV)-specific T cells have been highly successful in treating or preventing EBV-associated tumors, we hypothesized that the development of a manufacturing platform for HPV-specific T cells from healthy donors could be used in a third-party setting to treat patients with high-risk/relapsed HPV-associated cancers. Most protocols for generating virus-specific T cells require prior exposure of the donor to the targeted virus and, because the seroprevalence of high-risk HPV types varies greatly by age and ethnicity, manufacturing of donor-derived HPV-specific T cells has proven challenging. We, therefore, made systematic changes to our current Good Manufacturing Practice (GMP)-compliant protocols to improve antigen presentation, priming and expansion for the manufacture of high-efficacy HPV-specific T cells. Like others, we found that current methodologies fail to expand HPV-specific T cells from most healthy donors. By optimizing dendritic cell maturation and function with lipopolysaccharide (LPS) and interferon (IFN)γ, adding interleukin (IL)-21 during priming and depleting memory T cells, we achieved reliable expansion of T cells specific for oncoproteins E6 and E7 to clinically relevant amounts (mean, 578-fold expansion; n = 10), which were polyfunctional based on cytokine multiplex analysis. In the third-party setting, such HPV-specific T-cell products might serve as a potent salvage therapy for patients with HPV-associated diseases., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Peritoneal carcinomatosis results when tumour cells implant and grow within the peritoneal cavity. Treatment and prognosis vary based on the primary cancer. Although therapy with intention-to-cure is offered to selective patients using cytoreductive surgery with chemotherapy, the prognosis remains poor for most of the patients. Photodynamic therapy (PDT) is a cancer-therapeutic modality where a photosensitiser is administered to patients and exerts a cytotoxic effect on cancer cells when excited by light of a specific wavelength. It has potential application in the treatment of peritoneal carcinomatosis., Methods: We systematically reviewed the evidence of using PDT to treat peritoneal carcinomatosis in both animals and humans (Medline/EMBASE searched in June 2017)., Results: Three human and 25 animal studies were included. Phase I and II human trials using first-generation photosensitisers showed that applying PDT after surgical debulking in patients with peritoneal carcinomatosis is feasible with some clinical benefits. The low tumour-selectivity of the photosensitisers led to significant toxicities mainly capillary leak syndrome and bowel perforation. In animal studies, PDT improved survival by 15-300%, compared to control groups. PDT led to higher tumour necrosis values (categorical values 0-4 [4=highest]: PDT 3.4±1.0 vs. control 0.4±0.6, p<0.05) and reduced tumour size (residual tumour size is 10% of untreated controls, p<0.001)., Conclusion: PDT has potential in treating peritoneal carcinomatosis, but is limited by its narrow therapeutic window and possible serious side effects. Recent improvement in tumour-selectivity and light delivery systems is promising, but further development is needed before PDT can be routinely applied for peritoneal carcinomatosis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Family cars represent ∼74% of the yearly global output of motorized vehicles. With a life expectancy of ∼8 decades in many countries, the average person spends >100 min daily inside the confined and often shared space of the car, with exposure to a mix of potentially harmful microbes. Can commercial in-car microbial air decontamination devices mitigate the risk? Three such devices (designated devices 1 to 3) with HEPA filters were tested in the modified passenger cabin (3.25 m 3 ) of a four-door sedan housed within a biosafety level 3 containment facility. Staphylococcus aureus (ATCC 6538) was suspended in a soil load to simulate the presence of body fluids and aerosolized into the car's cabin with a 6-jet Collison nebulizer. A muffin fan (80 mm by 80 mm, with an output of 0.17 m 3 /min) circulated the air inside. Plates (150 mm diameter) of Trypticase soy agar (TSA), placed inside a programmable slit-to-agar sampler, were held at 36 ± 1°C for 18 to 24 h and examined for CFU. The input dose of the test bacterium, its rate of biological decay, and the log 10 reductions by the test devices were analyzed. The arbitrarily set performance criterion was the time in hours a device took for a 3-log 10 reduction in the level of airborne challenge bacterium. On average, the level of S. aureus challenge in the air varied between 4.2 log 10 CFU/m 3 and 5.5 log 10 CFU/m 3 , and its rate of biological decay was -0.0213 ± 0.0021 log 10 CFU/m 3 /min. Devices 1 to 3 took 2.3, 1.5, and 9.7 h, respectively, to meet the performance criterion. While the experimental setup was tested using S. aureus as an archetypical airborne pathogen, it can be readily adapted to test other types of pathogens and technologies. IMPORTANCE This study was designed to test the survival of airborne pathogens in the confined and shared space of a family automobile as well as to assess claims of devices marketed for in-car air decontamination. The basic experimental setup and the test protocols reported are versatile enough for work with all major types of airborne human pathogens and for testing a wide variety of air decontamination technologies. This study could also lay the foundation for a standardized test protocol for use by device makers as well as regulators for the registration of such devices., (Copyright © 2017 American Society for Microbiology.)

Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry., Competing Interests: Conflict of interest statement: O.O. is a cofounder of a company commercializing real-time deformability cytometry.

Many promising vaccine candidates from pathogenic viruses, bacteria, and parasites are unstable and cannot be produced cheaply for clinical use. For instance, Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is essential for erythrocyte invasion, is highly conserved among field isolates, and elicits antibodies that neutralize in vitro and protect in an animal model, making it a leading malaria vaccine candidate. However, functional RH5 is only expressible in eukaryotic systems and exhibits moderate temperature tolerance, limiting its usefulness in hot and low-income countries where malaria prevails. Current approaches to immunogen stabilization involve iterative application of rational or semirational design, random mutagenesis, and biochemical characterization. Typically, each round of optimization yields minor improvement in stability, and multiple rounds are required. In contrast, we developed a one-step design strategy using phylogenetic analysis and Rosetta atomistic calculations to design PfRH5 variants with improved packing and surface polarity. To demonstrate the robustness of this approach, we tested three PfRH5 designs, all of which showed improved stability relative to wild type. The best, bearing 18 mutations relative to PfRH5, expressed in a folded form in bacteria at >1 mg of protein per L of culture, and had 10-15 °C higher thermal tolerance than wild type, while also retaining ligand binding and immunogenic properties indistinguishable from wild type, proving its value as an immunogen for a future generation of vaccines against the malaria blood stage. We envision that this efficient computational stability design methodology will also be used to enhance the biophysical properties of other recalcitrant vaccine candidates from emerging pathogens., Competing Interests: The authors declare no conflict of interest.

Temperature sensitivity is a phenotype often associated with attenuation of viruses. Previously, we purified several mumps variants from an incompletely attenuated Urabe strain live attenuated vaccine. Here we characterize one isolate that is sensitive to growth at high temperature. This virus was attenuated in a small animal model of mumps virulence, and we identified unique coding substitutions in the hemagglutinin-neuraminidase (HN), the viral polymerase (L) gene, and a non-coding substitution close to the anti-genome promoter sequences. At the non-permissive temperature, transcription of viral mRNAs and production of the replication intermediate were reduced compared to events at the permissive temperature and to a non-ts virulent Urabe virus. As well, synthesis of viral proteins was also reduced at the higher temperature. While the actual sequence substitutions in the ts virus were unique, the pattern of substitutions in HN, L and genome end sequences is similar to another attenuated Urabe virus previously described by us., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background Aims: Human parainfluenza virus-3 (HPIV) is a common cause of respiratory infection in immunocompromised patients and currently has no effective therapies. Virus-specific T-cell therapy has been successful for the treatment or prevention of viral infections in immunocompromised patients but requires determination of T-cell antigens on targeted viruses., Methods: HPIV3-specific T cells were expanded from peripheral blood of healthy donors using a rapid generation protocol targeting four HPIV3 proteins. Immunophenotyping was performed by flow cytometry. Viral specificity was determined by interferon (IFN)-γ ELISpot, intracellular cytokine staining and cytokine measurements from culture supernatants by Luminex assay. Cytotoxic activity was tested by 51 Cr release and CD107a mobilization assays. Virus-specific T cells targeting six viruses were then produced by rapid protocol, and the phenotype of HPIV3-specific T cells was determined by immunomagnetic sorting for IFN-γ-producing cells., Results: HPIV3-specific T cells were expanded from 13 healthy donors. HPIV3-specific T cells showed a CD4 + predominance (mean CD4:CD8 ratio 2.89) and demonstrated specificity for multiple HPIV3 antigens. The expanded T cells were polyfunctional based on cytokine production but only had a minor cytotoxic component. T cells targeting six viruses in a single product similarly showed HPIV3 specificity, with a predominant effector memory phenotype (CD3 + /CD45RA - /CCR7 - ) in responder cells., Discussion: HPIV3-specific T cells can be produced using a rapid ex vivo protocol from healthy donors and are predominantly CD4 + T cells with Th1 activity. HPIV3 epitopes can also be successfully targeted alongside multiple other viral epitopes in production of six-virus T cells, without loss of HPIV3 specificity. These products may be clinically beneficial to combat HPIV3 infections by adoptive T-cell therapy in immune-compromised patients., Competing Interests: COI Statement: The authors have no relevant conflicts of interest., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Although indoor air can spread many pathogens, information on the airborne survival and inactivation of such pathogens remains sparse., Methods: Staphylococcus aureus and Klebsiella pneumoniae were nebulized separately into an aerobiology chamber (24.0 m 3 ). The chamber's relative humidity and air temperature were at 50% ± 5% and 20°C ± 2°C, respectively. The air was sampled with a slit-to-agar sampler. Between tests, filtered air purged the chamber of any residual airborne microbes., Results: The challenge in the air varied between 4.2 log 10 colony forming units (CFU)/m 3 and 5.0 log 10 CFU/m 3 , sufficient to show a ≥3 log 10 (≥99.9%) reduction in microbial viability in air over a given contact time by the technologies tested. The rates of biologic decay of S aureus and K pneumoniae were 0.0064 ± 0.00015 and 0.0244 ± 0.009 log 10 CFU/m 3 /min, respectively. Three commercial devices, with ultraviolet light and HEPA (high-efficiency particulate air) filtration, met the product efficacy criterion in 45-210 minutes; these rates were statistically significant compared with the corresponding rates of biologic decay of the bacteria. One device was also tested with repeated challenges with aerosolized S aureus to simulate ongoing fluctuations in indoor air quality; it could reduce each such recontamination to an undetectable level in approximately 40 minutes., Conclusions: The setup described is suitable for work with all major classes of pathogens and also complies with the U.S. Environmental Protection Agency's guidelines (2012) for testing air decontamination technologies., (Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Computer-aided design and draft, along with computer-aided engineering software, are used widely in different fields to create, modify, analyze, and optimize designs., Methods: We used computer-aided design and draft software to create a 3-dimensional model of an aerobiology chamber built in accordance with the specifications of the 2012 guideline from the Environmental Protection Agency for studies on survival and inactivation of microbial pathogens in indoor air. The model was used to optimize the chamber's airflow design and the distribution of aerosolized bacteria inside it., Results: The findings led to the identification of an appropriate fan and its location inside the chamber for uniform distribution of microbes introduced into the air, suitability of air sample collection from the center of the chamber alone as representative of its bacterial content, and determination of the influence of room furnishings on airflow patterns inside the chamber., Conclusions: The incorporation of this modeling study's findings could further improve the design of the chamber and the predictive value of the experimental data using it. Further, it could make data generation faster and more economical by eliminating the need for collecting air samples from multiple sites in the chamber., (Copyright © 2016 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.)

Indoor air can be an important vehicle for a variety of human pathogens. This review provides examples of airborne transmission of infectious agents from experimental and field studies and discusses how airborne pathogens can contaminate other parts of the environment to give rise to secondary vehicles leading air-surface-air nexus with possible transmission to susceptible hosts. The following groups of human pathogens are covered because of their known or potential airborne spread: vegetative bacteria (staphylococci and legionellae), fungi (Aspergillus, Penicillium, and Cladosporium spp and Stachybotrys chartarum), enteric viruses (noro- and rotaviruses), respiratory viruses (influenza and coronaviruses), mycobacteria (tuberculous and nontuberculous), and bacterial spore formers (Clostridium difficile and Bacillus anthracis). An overview of methods for experimentally generating and recovering airborne human pathogens is included, along with a discussion of factors that influence microbial survival in indoor air. Available guidelines from the U.S. Environmental Protection Agency and other global regulatory bodies for the study of airborne pathogens are critically reviewed with particular reference to microbial surrogates that are recommended. Recent developments in experimental facilities to contaminate indoor air with microbial aerosols are presented, along with emerging technologies to decontaminate indoor air under field-relevant conditions. Furthermore, the role that air decontamination may play in reducing the contamination of environmental surfaces and its combined impact on interrupting the risk of pathogen spread in both domestic and institutional settings is discussed., (Copyright © 2016. Published by Elsevier Inc.)