SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells.

Scaffold attachment factor B (SAFB) is a conserved RNA-binding protein that is essential for early mammalian development. However, the functions of SAFB in mouse embryonic stem cells (ESCs) have not been characterized. Using RNA immunoprecipitation followed by RNA-seq (RIP-seq), we examined the RNAs associated with SAFB in wild-type and SAFB/SAFB2 double-knockout ESCs. SAFB predominantly associated with introns of protein-coding genes through purine-rich motifs. The transcript most enriched in SAFB association was the lncRNA Malat1, which also contains a purine-rich region in its 5' end. Knockout of SAFB/SAFB2 led to differential expression of approximately 1000 genes associated with multiple biological processes, including apoptosis, cell division, and cell migration. Knockout of SAFB/SAFB2 also led to splicing changes in a set of genes that were largely distinct from those that exhibited changes in expression level. The spliced and nascent transcripts of many genes whose expression levels were positively regulated by SAFB also associated with high levels of SAFB, implying that SAFB binding promotes their expression. Reintroduction of SAFB into double-knockout cells restored gene expression toward wild-type levels, an effect again observable at the level of spliced and nascent transcripts. Proteomics analysis revealed a significant enrichment of nuclear speckle-associated and RS domain-containing proteins among SAFB interactors. Neither Xist nor Polycomb functions were dramatically altered in SAFB/2 knockout ESCs. Our findings suggest that among other potential functions in ESCs, SAFB promotes the expression of certain genes through its ability to bind nascent RNA.

Social factors associated with changes in nutrition risk scores measured using SCREEN-8: data from the Canadian Longitudinal Study on Aging.

Purpose: To examine the social network factors associated with changes in nutrition risk scores, measured by SCREEN-8, over three years, in community-dwelling Canadians aged 45 years and older, using data from the Canadian Longitudinal Study on Aging (CLSA).Methods: Change in SCREEN-8 scores between the baseline and first follow-up waves of the CLSA was calculated by subtracting SCREEN-8 scores at follow-up from baseline scores. Multivariable linear regression was used to examine the factors associated with change in SCREEN-8 score.Results: The mean SCREEN-8 score at baseline was 38.7 (SD = 6.4), and the mean SCREEN-8 score at follow-up was 37.9 (SD = 6.6). The mean change in SCREEN-8 score was -0.90 (SD = 5.99). Higher levels of social participation (participation in community activities) were associated with increases in SCREEN-8 scores between baseline and follow-up, three years later.Conclusions: Dietitians should be aware that individuals with low levels of social participation may be at risk for having their nutritional status decrease over time and consideration should be given to screening them proactively for nutrition risk. Dietitians can develop and support programs aimed at combining food with social participation.

The synaptosome-associated protein 23 (SNAP23) is necessary for proper myogenesis.

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.

The Evidence for Screening Older Adults for Nutrition Risk in Primary Care: An Umbrella Review.

It is not known if nutrition risk screening of older adults should be a standard practice in primary care. The evidence in support of nutrition risk screening of older adults in primary care was examined and critically analyzed using an umbrella review. The peer reviewed and grey literature were searched for clinical practice guidelines (CPGs) and systematic reviews (SRs). Titles and abstracts were independently screened by the two authors. Resources were excluded if they did not apply to older adults, did not discuss nutrition/malnutrition risk screening, or were in settings other than primary care. Full texts were independently screened by both authors, resulting in the identification of six CPGs and three SRs that met the review criteria. Guidelines were appraised with the AGREE II tool and SRs with the AMSTAR 2 tool. The quality of the CPGs was high, while the quality of the SRs was low. The CPGs and SRs acknowledged a lack of high-quality research on the benefits of regular nutrition risk screening for older adults in primary care; however, CPGs recommended annual screening for older adults in primary care practices or other community settings. High-quality research investigating nutrition risk screening of older adults in primary care is needed.

Oasis Senior Supportive Living: Description of a Novel Aging-in-Place Model in Ontario.

Older adults are the fastest-growing demographic group in Canada, and the majority of older adults want to age-in-place within their communities. Many older adults live in naturally occurring retirement communities (NORCs), unplanned communities with a high proportion of older residents. NORC supportive services programs can help older adults successfully age-in-place. One such program is Oasis Senior Supportive Living, a partnership between older adults, building owners and managers, community partners, funders, and researchers. Using a qualitative approach, interviews were conducted with Oasis participants to understand their experiences of Oasis. This article will describe the three pillars upon which Oasis programming is based and provide insights from Oasis participants. It will discuss nutrition programming implemented in these NORCs and suggest how dietitians can support NORC residents.

Interprofessional primary care during COVID-19: a survey of the provider perspective.

BACKGROUND: Interprofessional primary care (IPC) teams provide comprehensive and coordinated care and are ideally equipped to support those populations most at risk of adverse health outcomes during the COVID-19 pandemic, including older adults, and patients with chronic physical and mental health conditions. There has been little focus on the experiences of healthcare teams and no studies have examined IPC practice during the early phase of the COVID-19 pandemic. The objective of the study was to describe the state of interprofessional health provider practice within IPC teams during the COVID-19 pandemic. METHODS: Observational cross-sectional design. A web-based survey was deployed to IPC providers working in team-based primary care clinics in the province of Ontario, Canada. The survey included 26 close-ended and six open-ended questions. Close-ended questions were analyzed using descriptive statistics. Content analysis was used to analyze the open-ended questions. RESULTS: 445 surveys were included in the final analysis. Service delivery shifted from in-person care (77% pre-COVID-19) to telephone (76.5% during the COVID-19 pandemic). Less than half of the respondents (40%) reported receiving any training for virtual delivery. Wait times to access team members were reported to have decreased. There has also been a shift in what IPC providers report as the most commonly seen conditions, with increases in visits related to mental health concerns, acute infections (including COVID-19), social isolation, and resource navigation. Respondents also reported a reduction in healthcare provision for multiple chronic conditions including diabetes, cardiovascular disease, and chronic pain. CONCLUSIONS: IPC teams are rapidly shifting their practice to supporting their patients during the pandemic. A surge in mental health issues has been seen and is expected to continue to increase in response to COVID-19. Understanding early experiences can help plan for future pandemic waves.

Food Insecurity in Older Adults in Canada and the United States: A Concept Analysis.

A concept analysis using the method of Walker and Avant was undertaken to clarify the concept of food insecurity in older adults in Canada and the United States. A literature review was undertaken to conduct a concept analysis of food insecurity in older people. Food insecurity is associated with multiple negative health outcomes and may be experienced differently by older adults as compared to younger adults. It is therefore important to understand the concept of food insecurity as is relates to older adults. Four defining attributes of food insecurity in older adults in Canada and the United States were identified: (i) inability to acquire or prepare enough food, (ii) compromising on food quality or preference, (iii) uncertainty or anxiety around the ability to acquire or prepare food, and (iv) socially unacceptable or non-normative practices. These attributes may allow for improved policies and programs aimed at addressing food insecurity in older adults by better meeting the needs of older individuals. Additional research into food insecurity as experienced by Canadian and American older adults could help to further clarify the concept.

Nucleolar and spindle-associated protein 1 (NUSAP1) interacts with a SUMO E3 ligase complex during chromosome segregation.

The mitotic spindle is composed of dynamic microtubules and associated proteins that together direct chromosome movement during mitosis. The spindle plays a vital role in accurate chromosome segregation fidelity and is a therapeutic target in cancer. Nevertheless, the molecular mechanisms by which many spindle-associated proteins function remains unknown. The nucleolar and spindle-associated protein NUSAP1 is a microtubule-binding protein implicated in spindle stability and chromosome segregation. We show here that NUSAP1 localizes to dynamic spindle microtubules in a unique chromosome-centric pattern, in the vicinity of overlapping microtubules, during metaphase and anaphase of mitosis. Mass spectrometry-based analysis of endogenous NUSAP1 interacting proteins uncovered a cell cycle-regulated interaction between the RanBP2-RanGAP1-UBC9 SUMO E3 ligase complex and NUSAP1. Like NUSAP1 depletion, RanBP2 depletion impaired the response of cells to the microtubule poison Taxol. NUSAP1 contains a conserved SAP domain (SAF-A/B, Acinus, and PIAS). SAP domains are common among many other SUMO E3s, and are implicated in substrate recognition and ligase activity. We speculate that NUSAP1 contributes to accurate chromosome segregation by acting as a co-factor for RanBP2-RanGAP1-UBC9 during cell division.

Undergraduate, Female, Nutrition Students' Perceptions of Curricular Influence on Attitudes toward Individuals with Obesity.

PURPOSE: This qualitative study, guided by a phenomenological approach, explored senior-level undergraduate, nutrition students' perceptions of how obesity and weight bias were addressed in the undergraduate curricula and how the curricula influenced their attitudes toward individuals with obesity. METHODS: Twenty senior-level undergraduate, nutrition students from the University of Guelph participated in interviews. Interviews were audio-recorded and transcribed verbatim. Thematic analysis entailed open, axial, and selective coding. RESULTS: Participants' sources of information about obesity in the curricula included nutrition courses, case studies, and non-nutrition courses. Regarding sources of information about weight bias in the curricula, they discussed nutrition courses, non-nutrition courses, and limited coverage of weight bias. Themes for curricular influence on attitudes toward people with obesity were increased knowledge of obesity, understanding the complexity of obesity, increased empathy toward individuals with obesity, and better ability to avoid stereotypes toward people with obesity. CONCLUSIONS: The perceptions among nutrition students varied regarding the amount and type of obesity and weight-bias information in the curricula, as well as the influence of the curricula on attitudes toward individuals with obesity, suggesting that obesity and weight bias warrant more coordinated coverage in the nutrition curricula.

FORTIETH ANNIVERSARY ISSUE OF THE INTERNATIONAL JOURNAL OF OROFACIAL MYOLOGY (IJOM): THE FACE OF THE IAOM.

This article presents an historical account of the International Journal of Orofacial Myology from its inception to the present. Highlights from individuals involved and perspectives are included.

Factors Associated with the Development of High Nutrition Risk: Data from the Canadian Longitudinal Study on Aging.

This study aimed to determine which social network, demographic, and health-indicator variables were able to predict the development of high nutrition risk in Canadian adults at midlife and beyond, using data from the Canadian Longitudinal Study on Aging. Multivariable binomial logistic regression was used to examine the predictors of the development of high nutrition risk at follow-up, 3 years after baseline. At baseline, 35.0 per cent of participants were at high nutrition risk and 42.2 per cent were at high risk at follow-up. Lower levels of social support, lower social participation, depression, and poor self-rated healthy aging were associated with the development of high nutrition risk at follow-up. Individuals showing these factors should be screened proactively for nutrition risk.

Social Factors Associated With Nutrition Risk in Community-Dwelling Older Adults in High-Income Countries: Protocol for a Scoping Review.

BACKGROUND: In high-income countries (HICs), between 65% and 70% of community-dwelling adults aged 65 and older are at high nutrition risk. Nutrition risk is the risk of poor dietary intake and nutritional status. Consequences of high nutrition risk include frailty, hospitalization, death, and reduced quality of life. Social factors (such as social support and commensality) are known to influence eating behavior in later life; however, to the authors' knowledge, no reviews have been conducted examining how these social factors are associated with nutrition risk specifically. OBJECTIVE: The objective of this scoping review is to understand the extent and type of evidence concerning the relationship between social factors and nutrition risk among community-dwelling older adults in HICs and to identify social interventions that address nutrition risk in community-dwelling older adults in HICs. METHODS: This review will follow the scoping review methodology as outlined by the JBI Manual for Evidence Synthesis and the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews) guidelines. The search will include MEDLINE (Ovid), CINAHL, PsycINFO, and Web of Science. There will be no date limits placed on the search. However, only resources available in English will be included. EndNote (Clarivate Analytics) and Covidence (Veritas Health Innovation Ltd) will be used for reference management and removal of duplicate studies. Articles will be screened, and data will be extracted by at least 2 independent reviewers using Covidence. Data to be extracted will include study characteristics (country, methods, aims, design, and dates), participant characteristics (population description, inclusion and exclusion criteria, recruitment method, total number of participants, and demographics), how nutrition risk was measured (including the tool used to measure nutrition risk), social factors or interventions examined (including how these were measured or determined), the relationship between nutrition risk and the social factors examined, and the details of social interventions designed to address nutrition risk. RESULTS: The scoping review was started in October 2023 and will be finalized by August 2024. The findings will describe the social factors commonly examined in the nutrition risk literature, the relationship between these social factors and nutrition risk, the social factors that have an impact on nutrition risk, and social interventions designed to address nutrition risk. The results of the extracted data will be presented in the form of a narrative summary with accompanying tables. CONCLUSIONS: Given the high prevalence of nutrition risk in community-dwelling older adults in HICs and the negative consequences of nutrition risk, it is essential to understand the social factors associated with nutrition risk. The results of the review are anticipated to aid in identifying individuals who should be screened proactively for nutrition risk and inform programs, policies, and interventions designed to reduce the prevalence of nutrition risk. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/56714.

Discovery of a cell-active chikungunya virus nsP2 protease inhibitor using a covalent fragment-based screening approach.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has been responsible for numerous large-scale outbreaks in the last twenty years. Currently, there are no FDA-approved therapeutics for any alphavirus infection. CHIKV non-structural protein 2 (nsP2), which contains a cysteine protease domain, is essential for viral replication, making it an attractive target for a drug discovery campaign. Here, we optimized a CHIKV nsP2 protease (nsP2pro) biochemical assay for the screening of a 6,120-compound cysteine-directed covalent fragment library. Using a 50% inhibition threshold, we identified 153 hits (2.5% hit rate). In dose-response follow up, RA-0002034, a covalent fragment that contains a vinyl sulfone warhead, inhibited CHIKV nsP2pro with an IC 50 of 58 ± 17 nM, and further analysis with time-dependent inhibition studies yielded a k inact /K I of 6.4 x 10 3 M -1 s -1 . LC-MS/MS analysis determined that RA-0002034 covalently modified the catalytic cysteine in a site-specific manner. Additionally, RA-0002034 showed no significant off-target reactivity against a panel of cysteine proteases. In addition to the potent biochemical inhibition of CHIKV nsP2pro activity and exceptional selectivity, RA-0002034 was tested in cellular models of alphavirus infection and effectively inhibited viral replication of both CHIKV and related alphaviruses. This study highlights the discovery and characterization of the chemical probe RA-0002034 as a promising hit compound from covalent fragment-based screening for development toward a CHIKV or pan-alphavirus therapeutic. Significance Statement: Chikungunya virus is one of the most prominent and widespread alphaviruses and has caused explosive outbreaks of arthritic disease. Currently, there are no FDA-approved drugs to treat disease caused by chikungunya virus or any other alphavirus-caused infection. Here, we report the discovery of a covalent small molecule inhibitor of chikungunya virus nsP2 protease activity and viral replication of four diverse alphaviruses. This finding highlights the utility of covalent fragment screening for inhibitor discovery and represents a starting point towards the development of alphavirus therapeutics targeting nsP2 protease.

Nutrition risk varies according to social network type: data from the Canadian Longitudinal Study on Aging.

OBJECTIVE: There were two primary objectives, namely: (1) to determine the social network types that Canadian adults aged 45 and older belong to and (2) to discover if social network type is associated with nutrition risk scores and the prevalence of high nutrition risk. DESIGN: A retrospective cross-sectional study. SETTING: Data from the Canadian Longitudinal Study on Aging (CLSA). PARTICIPANTS: 17 051 Canadians aged 45 years and older with data from baseline and first follow-up of the CLSA. RESULTS: CLSA participants could be classified into one of seven different social network types that varied from restricted to diverse. We found a statistically significant association between social network type and nutrition risk scores and percentage of individuals at high nutrition risk at both time points. Individuals with restricted social networks had lower nutrition risk scores and are more likely to be at nutrition risk, whereas individuals with diverse social networks had higher nutrition risk scores and are less likely to be at nutrition risk. CONCLUSIONS: Social network type was associated with nutrition risk in this representative sample of Canadian middle-aged and older adults. Providing adults with opportunities to deepen and diversify their social networks may decrease the prevalence of nutrition risk. Individuals with more restricted networks should be proactively screened for nutrition risk.

Barriers and facilitators for accessing and prescribing hormone therapy in primary care for transgender adults: a scoping review protocol.

OBJECTIVE: The objective of this scoping review is to understand the extent and type of evidence in relation to barriers and facilitators experienced by transgender adults in accessing hormone therapy. It will also explore the experiences of primary care practitioners in prescribing hormone therapy in primary care. INTRODUCTION: Providing care to transgender patients is a rapidly growing area of primary care. Despite the existence of clinical practice guidelines that support the prescription of gender-affirming hormone therapy in primary care, only a small number of primary care providers are offering this care. This review will seek to advance research on this topic by examining the barriers and facilitators of hormone prescription for transgender adults in primary care. INCLUSION CRITERIA: This review will consider research on primary care practitioners who prescribe hormone therapy to transgender adults. It will also focus on transgender adults who seek hormone therapy in primary care. Only studies that examine barriers and facilitators in primary care will be included. The review will include qualitative, quantitative, and mixed methods studies, in addition to systematic reviews and meta-analyses. METHODS: The search will include MEDLINE, CINAHL, EmCare, and Nursing and Allied Health Premium. No date limits will be applied to the search. Only articles written in English will be eligible for inclusion. Articles will be reviewed and data extracted by 2 independent reviewers. The results of the extracted data will be presented in a narrative summary with accompanying tables.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Immune response after rabies pre-exposure prophylaxis and a booster dose in Australian bat carers.

Periodic vaccination against rabies is essential for individuals at continuing risk of rabies exposure. There is limited evidence on long-term immunogenicity after a 3-dose intramuscular (3IM) pre-exposure prophylaxis (PrEP) and single IM booster dose, thus current guideline recommendations differ in the interval for serology tests following PrEP and boosters. This study investigated post-PrEP and post-booster persistence of antibodies in Australian bat carers. Bat carers who received 3IM PrEP/booster doses and had post-PrEP/booster serology test results were included. The proportion of antibody-negative (<0.5 EU/mL) individuals after PrEP/booster dose were examined. Three hundred and five participants (65.6% females, median age at PrEP 43.1 years) were included. The proportion who were antibody-negative varied depending on the time between 3IM PrEP and the serology test: 8.0% <1 year, 29.8% 1-2 years, 21.2% 2-3 years and 7.7% >3 years. Ninety-one participants receiving booster doses were further assessed. Only one participant was antibody-negative at >3 years after receiving one IM booster dose. Our findings support that a serology test should be performed 1 year after 3IM PrEP, followed by first booster if required. Rabies antibodies persist for many years after receiving the booster doses. The interval between subsequent serology tests and the first booster dose should be no longer than 3 years. Future studies are required to provide more insight into the most appropriate timing of subsequent boosters.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Immunogenicity of a single fractional intradermal dose of Japanese encephalitis live attenuated chimeric vaccine.

BACKGROUND: Japanese encephalitis (JE) is endemic in Asia and the western Pacific. Vaccination is recommended for travellers to endemic regions, but the high cost of the vaccine is a major barrier to uptake. METHODS: A quasi-experimental, pre-post intervention clinical trial without a control group was conducted to assess the immunogenicity and safety of intradermal (ID) JE vaccine. Healthy adults (18-45 years) received one dose of 0.1 mL (20% of standard dose) ID Imojev® (JE live attenuated chimeric vaccine, Sanofi-Aventis). Adverse events following immunization (AEFIs) were recorded 10 days post-vaccination. Blood samples were collected at baseline, 4 and 8 weeks post-vaccination. Neutralizing antibodies were measured using 50% plaque reduction neutralization test (PRNT50). Seroconversion was defined as PRNT50 titre ≥10. An in vitro study was also conducted to quantify the rate of decay of vaccine potency after reconstitution. RESULTS: In total, 51 participants (72.6% females, median age 31 years), all non-reactive to JE virus at baseline were enrolled. Mild and moderate AEFIs were reported by 19.6% of participants; none required medical attention or interfered with normal daily activities. All participants seroconverted at 4 weeks (GMT 249.3; 95%CI:192.8-322.5) and remained seropositive at 8 weeks (GMT 135.5; 95%CI:104.5-175.6). Vaccine potency declined at a rate of 0.14 log plaque-forming units/0.5 mL per hour. CONCLUSIONS: In healthy adults, a single 0.1 mL ID dose of Imojev was safe and immunogenic, at least in the short term. Reconstituted vials of Imojev vaccine may not retain their potency after 6 hours. Fractional JE ID vaccination could be a cheaper yet effective alternative for short-term travellers. Further studies need to investigate the immune response in a wider age range of individuals and the long-term immunogenicity of fractional JE ID vaccines. CLINICAL TRIALS REGISTRATION: ACTRN12621000024842.

APC/C prevents non-canonical order of cyclin/CDK activity to maintain CDK4/6 inhibitor-induced arrest.

Regulated cell cycle progression ensures homeostasis and prevents cancer. In proliferating cells, premature S phase entry is avoided by the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), although the APC/C substrates whose degradation restrains G1-S progression are not fully known. The APC/C is also active in arrested cells that exited the cell cycle, but it is not clear if APC/C maintains all types of arrest. Here by expressing the APC/C inhibitor, EMI1, we show that APC/C activity is essential to prevent S phase entry in cells arrested by pharmacological CDK4/6 inhibition (Palbociclib). Thus, active protein degradation is required for arrest alongside repressed cell cycle gene expression. The mechanism of rapid and robust arrest bypass from inhibiting APC/C involves cyclin-dependent kinases acting in an atypical order to inactivate RB-mediated E2F repression. Inactivating APC/C first causes mitotic cyclin B accumulation which then promotes cyclin A expression. We propose that cyclin A is the key substrate for maintaining arrest because APC/C-resistant cyclin A, but not cyclin B, is sufficient to induce S phase entry. Cells bypassing arrest from CDK4/6 inhibition initiate DNA replication with severely reduced origin licensing. The simultaneous accumulation of S phase licensing inhibitors, such as cyclin A and geminin, with G1 licensing activators disrupts the normal order of G1-S progression. As a result, DNA synthesis and cell proliferation are profoundly impaired. Our findings predict that cancers with elevated EMI1 expression will tend to escape CDK4/6 inhibition into a premature, underlicensed S phase and suffer enhanced genome instability.