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Introduction: In the setting of pediatric and adolescent young adult cancer, increased access to genomic profiling has enhanced the detection of genetic variation associated with cancer predisposition, including germline syndromic conditions. Noonan syndrome (NS) is associated with the germline RAS pathway activating alterations and increased risk of cancer. Herein, we describe our comprehensive molecular profiling approach, the association of NS with glioma and glioneuronal tumors, and the clinical and histopathologic characteristics associated with the disease., Methods: Within an institutional pediatric cancer cohort (n = 314), molecular profiling comprised of paired somatic disease-germline comparator exome analysis, RNA sequencing, and tumor classification by DNA methylation analysis was performed., Results: Through the implementation of paired analysis, this study identified 4 of 314 (1.3%) individuals who harbored a germline PTPN11 variant associated with NS, of which 3 individuals were diagnosed with a glioma or glioneuronal tumor. Furthermore, we extend this study through collaboration with a peer institution to identify two additional individuals with NS and a glioma or glioneuronal tumor. Notably, in three of five (60%) individuals, paired genomic profiling led to a previously unrecognized diagnosis of Noonan syndrome despite an average age of cancer diagnosis of 16.8 years. The study of the disease-involved tissue identified signaling pathway dysregulation through somatic alteration of genes involved in cellular proliferation, survival, and differentiation., Discussion: Comparative pathologic findings are presented to enable an in-depth examination of disease characteristics. This comprehensive analysis highlights the association of gliomas and glioneuronal tumors with RASopathies and the potential therapeutic challenges and importantly demonstrates the utility of genomic profiling for the identification of germline cancer predisposition., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shatara, Schieffer, Melas, Varga, Thomas, Bucknor, Costello, Wheeler, Kelly, Miller, Rodriguez, Mathew, Lee, Crotty, Leary, Paulson, Cole, Abdelbaki, Finlay, Lazow, Salloum, Fouladi, Boué, Mardis and Cottrell.)

Objectives: Oral health is an important part of general health and well-being and shares risk factors, such as poor diet, with obesity. The published literature assessing the association between obesity and oral health in early childhood is sparse and inconsistent. The objective of this study was to investigate associations between overweight/obesity (measured by body mass index) and dental outcomes (caries, plaque index and gingival index) both cross-sectionally and longitudinally, taking account of potential confounding factors, based on data collected at age 2 and age 5 within the Australian Study of Mothers' and Infants' Life Events Affecting Oral Health (SMILE) birth cohort study., Methods: This study used data from 1174 SMILE participants. Associations between overweight/obesity and dental outcomes were assessed using generalized linear regression models for the modified Poisson family with log link to estimate prevalence ratios. Cross-sectional and longitudinal models were fitted, after minimal and full adjustment for potential confounders., Results: Approximately 12% of the participants were overweight/obese at 2 years and 9% at 5 years. Between 2 and 5 years, the prevalence of caries increased from approximately 4% to 24%, at least mild plaque accumulation increased from 37% to 90% and at least mild inflammation from 27% to 68%. There were no associations between overweight/obesity and the prevalence of dental caries; prevalence ratios (PR) [95% confidence interval (CI)] after adjustment for age and sex were 0.9 (0.3, 2.4) cross-sectionally at 2 years, 1.0 (0.6, 1.5) cross-sectionally at 5 years, and 1.0 (0.6, 1.5) for overweight/obesity at 2 years and caries at 5 years. Prevalence ratios were all around the value of 1 for the other dental outcomes and also after adjustment for additional confounders., Conclusions: There were no associations between overweight/obesity and dental caries, plaque index or gingival index in this cohort of preschool children. However, associations may emerge as the children become older, and it will be possible to extend analyses to include data collected at age 7 in the near future., (© 2024 The Author(s). Community Dentistry and Oral Epidemiology published by John Wiley & Sons Ltd.)

Later eating rhythm (LER) refers to later timing, greater energy intake (EI), and higher frequency of eating occasions (meal/snack) in the evening. The significance of LER in child health is becoming increasingly recognised. However, the lack of consensus regarding definitions of LER make it challenging to fully comprehend its role. This data note describes LER variables derived in the Avon Longitudinal Study of Parents and Children (ALSPAC), an ongoing birth cohort which enrolled 14,541 pregnant women living in Avon, UK, with an expected date of delivery between April 1991 - December 1992. When children were 7 years, parents completed a structured 3-day food diary, recording all foods/drinks consumed over 3 days (preferably 1 weekend day and 2 weekdays). Data was available for 7,285 children (50.1% response rate). A subsample of 4,869 children had exact time of eating occasions added to the existing database, which only included broad indications of eating timing based on 2-7 hour long meal slots. 13 LER variables were derived for the entire week and weekdays/weekend days separately. These comprise: 1) eating around individual bedtime (number days); 2) eating around average bedtime (number days); 3) time of evening main meal (hrs:mins); 4) time of last eating occasion (hrs:mins); 5) EI in the evening (percentage of total daily energy intake, %TDEI); 6) EI within 2hrs before bedtime (%TDEI); 7) EI for evening main meal (%TDEI); 8) EI for evening snacks (%TDEI); 9) Night eating1 (NE1): eating over 30% of total daily energy intake after 18:00 (number days); 10) NE2: eating over 25% of total daily energy intake within 2hrs before bedtime (number days); 11) eating frequency after 17:00 (number of eating occasions); 12) regularity of dinner (number of days); 13) frequency of evening snacks (number days). We describe the derivation, prevalence and inter-corelations between LER variables., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Zou M et al.)

Introduction: Syphilis, a bacterial infection caused by Treponema pallidum, can affect a wide variety of organ systems of its host. We aim to present the cases of two patients who presented to an urban, tertiary care, academic Emergency Department with ocular symptoms who underwent imaging, laboratory, and specialist evaluation and were ultimately diagnosed with ocular syphilis., Case Report: The first patient is a 46-year-old female who presented to the Emergency Department with painless complete vision loss in a unilateral eye. Her exam was significant for bilateral papilledema, and further workup revealed a retinal detachment in the affected eye. Rapid plasma reagin (RPR) titers resulted positive with 1:128 and a reactive fluorescent treponemal antibody (FTA). Further workup including lumbar puncture and magnetic resonance imaging were unable to be obtained due to patient refusal and multiple discharges against medical advice. The second patient is a 38-year-old female with a history of intravenous drug use who presented for bilateral circumferential peripheral vision loss with central sparing. The examination showed bilateral papilledema. Lumbar puncture was performed with normal intracranial pressure. RPR titers resulted positive with 1:128. MRI and ophthalmology evaluation did not reveal any other etiology or explanation for papilledema., Conclusion: This case series highlights the importance of considering syphilis as a possible cause of ocular symptoms in high risk patient populations and the need for prompt and appropriate treatment given the increasing prevalence of syphilis worldwide., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Importance: The COVID-19 pandemic has led to 775 million documented cases and over 7 million deaths worldwide as of March 2024 and is an ongoing health crisis. To limit viral spread within households and in the community, public health officials have recommended self-isolation, self-quarantine of exposed household contacts, and mask use. Yet, risk of household transmission (HHT) may be underestimated due to low frequency of sampling, and risk factors for HHT are not well understood., Objectives: To estimate the secondary attack rate of SARS-CoV-2 within households and to define the risk factors for new infections in household members who are in close contact with the index case., Design, Setting, and Participants: In this prospective cohort study, from March 2020-December 2021 we enrolled 60 households with index cases who tested positive for SARS-CoV-2. All household contacts and index cases were tested daily for SARS-CoV-2 via reverse transcription polymerase chain reaction (RT-PCR) using self-collected anterior nares specimens. Households were followed until all study participants in the household tested negative for SARS-CoV-2 for seven consecutive days. We collected sex, age, race/ethnicity, comorbidities, and relationship to index case for secondary contacts, household level characteristics including primary income, household density, and square feet per person on property. We compared the sociodemographic variables between COVID-19 positive and negative household members and between households where secondary transmission did and did not occur., Main Outcomes and Measures: Daily anterior nares swabs were tested for SARS-CoV-2 using RT-PCR, in order to assess duration of nasal shedding of SARS-CoV-2, as well as risk of transmission to secondary household contacts., Results: Of the 163 participants in this study, 84 (51.5%) were women; median age (IQR) was 36.0 (17.0-54.0) years of age; 78 (47.8%) were white and 48 (29.5%) were Hispanic/LatinX. Of the fifty households with household contacts, at least one secondary case occurred in twenty-six households (52.0%) and forty-five household contacts (43.7%) were infected. Secondary attack rate was lowest among children of index cases (6/23, 26.1%). Modified Poisson regression identified that the risk of transmission to household contacts increases significantly with age (Risk ratio for each increase in years of age = 1.01, 95% CI = 1.00-1.02). Mixed effects regression models identified that participants with chronic diseases, such as asthma, diabetes, cancer, or cardiac disease, had higher Cts at baseline when compared to participants without chronic diseases (6.62, 95% CI: 1.46-11.77, p = 0.02) and show a slower rate of increase in Ct over time (-0.43, 95% CI: -0.77 to -0.09, p = 0.02)., Conclusions and Relevance: This study suggests that HHT represents a key source of community-based infection of SARS-CoV-2. Allocation of resources for contact investigations and prevention interventions should focus on the individuals at highest risk of infection in households, especially those with higher density homes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Altamirano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Encephalocraniocutaneous lipomatosis (ECCL) is a rare genetic condition with well-described skin, ocular, and central nervous system findings. Several case reports have been documented demonstrating the presence of low-grade gliomas in patients with ECCL and the association with certain FGFR1 mutations. We report on a case of diffuse low-grade glioma, mitogen activated protein kinase pathway altered in a patient with ECCL, who was found to have a distinct FGFR1 mutation., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objectives: Investigate associations between cleft laterality in patients with non-syndromic unilateral cleft lip and palate (UCLP) and oral-health, dental-arch, speech, audiological, psychological and nasolabial-aesthetic outcomes., Methods: Secondary data analysis of the outcomes of 5-year-old children with non-syndromic complete UCLP identified from three studies: Cleft Collective (n = 155), Cleft Care UK (CCUK) (n = 266) and Clinical Standards Advisory Group (CSAG) study (n = 238). Outcome measures included occlusal assessment using the 5-year-old's index score, speech intelligibility rating using the CAPS-A Audit tool, audiological assessment using pure tone audiometry, nasolabial aesthetic assessment using the Asher-McDade tool, oral-health assessment using decayed, missing, filled teeth scores and parent-reported outcomes. Logistic regression with adjustment for age, sex and index-of-multiple-deprivation scores were performed., Results: No differences were found in patient-reported outcomes between the left and right clefts in the Cleft Collective study. From the CCUK study, right clefts had poorer speech (n = 236; 95% CI 1.09, 3.42; and P = .03) and hearing outcomes (n = 211; 95% CI 1.03, 3.43; P = .04). In the CSAG study, patients with left clefts were more likely to be teased (n = 213; 95% CI 0.26, 0.85; and P = .01)., Conclusion: Weak associations between cleft laterality, speech, hearing and psychological outcomes were found, however the findings were inconsistent across the studies. This study contributes to evidence of associations between laterality and outcomes in children born with UCLP., (© 2024 The Authors. Orthodontics & Craniofacial Research published by John Wiley & Sons Ltd.)

Purpose: To determine whether tranexamic acid (TXA) is safe to administer preoperatively in patients undergoing hip arthroscopy by comparing the venous thromboembolic rate and complication rate between patients who did and did not receive TXA preoperatively., Methods: This was a multicenter consecutive-cohort series of patients who underwent arthroscopic hip surgery between 2014 and 2021. The 2 cohorts comprised patients who did and did not receive TXA preoperatively (single dose of 1-2 g), after a practice change. Data were collected via chart review. Surgical outcomes included days until follow-up, visual analog scale pain score at first follow-up, total operating room (OR) time, number of arthroscopic fluid bags (3 L/bag), and complications and revision operations up to 1 year after surgery. The Mann-Whitney U test was performed for continuous variables, and the χ 2 test, for categorical variables., Results: A total of 862 patients were identified: 449 (52%) received TXA and 413 (48%) did not. Patient demographic characteristics including age, sex, height, weight, body mass index, smoking status, and procedures performed, as well as number of anchors used (3.5 anchors for no TXA vs 3.7 anchors for TXA) and traction time (38 minutes for no TXA vs 40 minutes for TXA), did not significantly differ between groups. Significantly more patients underwent prior hip arthroscopy in the TXA group (n = 45; primary, n = 404) than in the group that did not receive TXA (n = 25; primary, n = 388) (P = .03). Visual analog scale pain scores at the first follow-up visit (2.61 for no TXA vs 2.62 for TXA, P = .62) and the need for subsequent revision surgery (24 patients with no TXA vs 18 patients with TXA, P = .68) were not significantly different. TXA use was associated with less arthroscopic fluid utilization (5.9 bags of 3 L of fluid for no TXA vs 5.3 bags of 3 L of fluid for TXA, P < .01) and less total OR time (99.5 minutes for no TXA vs 90.0 minutes for TXA, P < .01). There was a higher overall complication rate in the group that did not receive TXA (n = 27) than in the group that did (n = 10) (P = .01). However, if lateral femoral cutaneous nerve neurapraxia was excluded, then no difference in complication rate was observed (P = .24)., Conclusions: There was no difference in the incidence of venous thromboembolic complications between patients who did and did not receive TXA preoperatively. We observed a lower overall complication rate in patients who received TXA preoperatively; however, this normalized between the 2 groups when lateral femoral cutaneous nerve neuritis was excluded. No difference in early pain control or revision surgery rate was observed between groups. Although there was statistically less arthroscopic fluid utilization and less total OR time in the group that received TXA, further studies are needed to clarify whether this is clinically meaningful. Preoperatively administered TXA is a safe adjunct medication in patients undergoing arthroscopic hip surgery., Level of Evidence: Level III, retrospective multicenter consecutive series., Competing Interests: Disclosures The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R.W. reports board membership and a consulting or advisory relationship with University of Iowa. M.B. reports board membership and a consulting or advisory relationship with Cedars-Sinai Kerlan-Jobe Institute. All other authors (S.A.S., S.C.M., S.L., K.G., A.H.) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Time-limited eating is a dietary intervention whereby eating is limited to a specific window of time during the day. The usual eating windows of adults, and how these can be manipulated for dietary interventions, is well documented. However, there is a paucity of data on eating windows of young people, the manipulation of which may be a useful intervention for reducing obesity. This paper reviewed the existing literature on the eating windows of children and adolescents, aged 5-18 years, plus clock times of first and last intakes and variations by subgroup. Two databases (Medline and Embase) were searched for eligible papers published between February 2013 and February 2023, with forward searching of the citation network of included studies on Web of Science. Articles were screened, and data extracted, in duplicate by two independent reviewers. Ten studies were included, with both observational and experimental designs. Narrative synthesis showed large variations in eating windows with average values ranging from 9.7 h to 16.4 h. Meta-analysis, of five studies, showed a pooled mean daily eating window of 11.3 h (95% CI 11.0, 11.7). Large variations in eating windows exist across different study populations; however, the pooled data suggest that it may be possible to design time-limited eating interventions in paediatric populations aimed at reducing eating windows. Further high-quality research, investigating eating windows and subsequent associations with health outcomes, is needed.

Background: Wound healing is particularly important for sarcoma patients who undergo neoadjuvant radiation therapy. Previous studies have demonstrated wound complications in this population approaching 35%. With this high rate of wound healing issues, identifying treatment modalities to minimize these complications is of paramount importance., Methods: All patients with high grade bone and soft tissue sarcoma received 15 days of twice daily amino acid supplementation starting in the immediate post-operative period. We documented the healing status of the surgical wound, the primary outcome, at all follow up appointments until six months after surgery. Non-healing wounds were defined as any wound requiring 1) a return visit to the OR for debridement, 2) IV antibiotics (ABX), and 3) unhealed wounds at 6 months post-operatively. 1 For each patient, we collected biometrics with lean body mass analysis at preoperative appointment, and two and six weeks postoperatively. The proportion with non-healing wounds was compared with a historical patient cohort using the chi-square test. In a subgroup of participants with body composition measurements, we also compared changes in mean fat mass, lean mass, and psoas index from pre-operative baseline to 6 months post-operative using generalized linear models., Results: A total of 33 consecutive patients were supplemented with a branched chain amino acid (BCAA) formulation. The historical cohort included 146 participants from the previous 7 years (2010-2017). 26% of patients in the historical cohort experienced wound complications compared to 30% in the supplemented group. (p=0.72) When focusing specifically on lower extremity sarcomas treated with neoadjuvant radiation therapy, 46% of patients in the supplemented group experienced wound healing complications compared to 39% in the non-supplemented group (p=0.68). BCAA supplementation was found to be protective with regards to decreasing muscle wasting with no difference in psoas index measurements throughout the study period compared to a 20% muscle loss in the historical cohort (p=0.02)., Conclusion: In our limited sample size, there was no difference in wound healing complications between sarcoma patients who received postoperative BCAA supplementation compared to a historical cohort who were not supplemented. Patients who did not receive supplementation had a significant decline in post-operative psoas index following operative sarcoma removal. Level of Evidence: III ., Competing Interests: Disclosures: Funding was made possible by philanthropic donations to the University of Iowa Sarcoma Program., (Copyright © The Iowa Orthopaedic Journal 2023.)

Background: Later-eating rhythm (LER) refers to a later timing, greater energy intake, and higher meal frequency in the evening. The role of childhood LER in obesity development is emerging, but most evidence is cross-sectional. Cross-context comparison allows the improvement of causal inference in observational studies by comparing cohorts with different confounding structures. This method is applied to assess the causal effects of LER on adiposity, by exploring the likelihood of residual confounding due to socioeconomic status., Methods: In this cross-cohort analysis, we used ongoing birth cohort data from the UK Avon Longitudinal Study of Parents and Children (ALSPAC) established in 1991, and the nationally representative China Health and Nutrition Survey (CHNS) collected in 1989-2011. Children with available data at age 7 years were eligible. We applied indices of inequality for assessing confounding structure by regressing LER/adiposity on the standardised score of socioeconomic status (SES) in each cohort. We used multivariable linear and binary logistic regressions to model cross-sectional and prospective associations between LER at 7 years of age and body-mass index (BMI) at ages 7 and 9 years in both cohorts. Analyses were adjusted by age, sex, ethnicity, residency, and socioeconomic status. We used a p value for the Cochrane Q-test obtained from meta-analysis to test for heterogeneity between cohorts., Findings: We analysed data from 4019 children (2170 [54·0%] female; 1849 [46.0%] male) in ALSPAC and 1749 (788 [45·1%] female; 961 [54.9%] male) in CHNS. The associations between SES and LER or adiposity differed between ALSPAC and CHNS (SES and energy intake for evening main meal: b=1·81 [95% CI 0·81 to 2·81] vs -3·02 [-4·76 to -1·27]; SES and frequency of evening snacks: odds ratio [OR]=0·51 [95% CI 0·41-0·63] vs 5·71 [3·54-9·22]; SES and BMI: b=-0·42 [-0·65 to -0·18] vs 1·29 [0·75 to 1·84]). Positive associations between frequency of evening snacks and BMI were seen in both cross-sectional and longitudinal analyses in both cohorts (mean change of BMI with 1 day increase of consuming evening snacks b=0·09 [0·02 to 0·15]; 0·13 [0·03 to 0·22] kg/m 2 per day in ALSPAC, and b=0·11 [-0·07 to 0·28]; 0·30 [0·07 to 0·52] kg/m 2 per day in CHNS). No associations were found for energy intake. p values for heterogeneity ranged from 0·107 to 0·932., Interpretation: Both cohorts showed consistent results despite varied dietary cultures and SES patterning of LER or adiposity. Energy intake in the evening or night was not associated with adiposity, whereas evening snacking was. More recent, high-quality cohorts are warranted to enhance the strength of the conclusions., Funding: None., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Physical activity (PA) is a key component in the management of type 2 diabetes. However, this population have low rates of PA engagement. Electrically assisted cycling has been identified as a means through which to increase PA by incorporating activity into daily life, while overcoming some of the barriers to conventional cycling. The determinants of e-cycling among people living with chronic disease are largely unknown. The aim of this research was to explore the determinants of e-cycling among individuals with type 2 diabetes using the Theoretical Domains Framework (TDF) and the Capability, Opportunity and Motivation for Behaviour change model (COM-B). This information is important for determining the suitability of future e-cycling initiatives and, if appropriate, inform future e-cycling interventions., Method: One-to-one semi structured interviews were conducted with 16 participants from the e-cycling arm of a pilot randomised controlled trial between September 2019 and April 2020. The TDF was used to develop the interview guide. The framework method of analysis was used, incorporating both deductive and inductive reasoning. A conceptual model of the factors that influence e-cycling in this population was created., Results: The most commonly reported TDF domains were skills, knowledge, belief about capabilities, belief about consequences and environmental context and resources. Specifically, e-bike training facilitated e-cycling engagement by providing participants with the skills, knowledge, and confidence needed to ride the e-bike and ride on the road. In addition, the enjoyment of e-cycling was a key facilitator to engagement. Participants engaged in e-cycling to improve their health rather than for environmental reasons. Most barriers to e-cycling related to the natural or physical environment., Conclusion: This study provides insight into the personal, social, and environmental factors associated with e-cycling in this population. The findings of this study can be used to develop a more refined e-cycling intervention targeting the factors identified as influencing e-cycling engagement. In addition, this information will help in the selection of mechanistic outcome measures for evaluation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Bourne, Leary, England and Searle.)

Background: People with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) experience core symptoms of post-exertional malaise, unrefreshing sleep, and cognitive impairment. Despite numbering 0.2-0.4% of the population, no laboratory test is available for their diagnosis, no effective therapy exists for their treatment, and no scientific breakthrough regarding pathogenesis has been made. It remains unknown, despite decades of small-scale studies, whether individuals experience different types of ME/CFS separated by onset-type, sex or age., Methods: DecodeME is a large population-based study of ME/CFS that recruited 17,074 participants in the first 3 months following full launch. Detailed questionnaire responses from UK-based participants who all reported being diagnosed with ME/CFS by a health professional provided an unparalleled opportunity to investigate, using logistic regression, whether ME/CFS severity or onset type is significantly associated with sex, age, illness duration, comorbid conditions or symptoms., Results: The well-established sex-bias among ME/CFS patients is evident in the initial DecodeME cohort: 83.5% of participants were females. What was not known previously was that females tend to have more comorbidities than males. Moreover, being female, being older and being over 10 years from ME/CFS onset are significantly associated with greater severity. Five different ME/CFS onset types were examined in the self-reported data: those with ME/CFS onset (i) after glandular fever (infectious mononucleosis); (ii) after COVID-19 infection; (iii) after other infections; (iv) without an infection at onset; and, (v) where the occurrence of an infection at or preceding onset is not known. Among other findings, ME/CFS onset with unknown infection status was significantly associated with active fibromyalgia., Conclusions: DecodeME participants differ in symptoms, comorbid conditions and/or illness severity when stratified by their sex-at-birth and/or infection around the time of ME/CFS onset., Competing Interests: Competing interests: ADC is a committee member of the Science for ME online support discussion forum. CPP is Deputy Chair of the ME/CFS Research Collaborative and has had PhD studentships funded by Action for ME & the Chief Scientist Office (Scotland), or by ME Research UK. JW was on the ME/CFS Research Collaborative Patient Advisory Group and received travel expenses for participation in Board Meetings. SC is CEO of Action for ME; Co-Chair, World ME Alliance; and, Non-Executive Director, Curo Group. SL is Head of Advocacy and Communications of the World ME Alliance., (Copyright: © 2023 Bretherick AD et al.)

Objective: Combination therapy with infliximab and a thiopurine has been shown to be more effective than monotherapy in patients with inflammatory bowel disease (IBD). The therapeutic efficacy of thiopurines is correlated with 6-thioguanine (6-TGN) levels between 235 and 450 pmol/8×10 8 erythrocytes. The primary aim of the study was to investigate the association between 6-TGN levels and inhibition prevention of the production of antibodies to infliximab (ATI)., Design: We performed a retrospective review of the medical records of patients being treated with infliximab for IBD at University Hospitals Bristol NHS Foundation Trust. Demographic and biochemical data were extracted, alongside thiopurine metabolite levels, trough levels of infliximab and the presence of ATI. χ 2 tests were used to investigate the association between 6-TGN levels and prevention of ATI. Logistic regression was used to compare the odds of prevented ATI between those with a 6-TGN level between 235 and 450 pmol/8×10 8 erythrocytes, those with a 6-TGN level outside of this range, and the baseline group who were on infliximab monotherapy., Results: Data were extracted for 100 patients. Six of 32 patients with a 6-TGN level between 235 and 450 pmol/8×10 8 erythrocytes developed ATI (18.8%) compared with 14 out of 22 (63.6%) patients with a 6-TGN outside of this range and 32 out of 46 (69.6%) patients on monotherapy (p=0.001). The OR (95% CI) for prevented ATI in those with a 6-TGN between 235 and 450 pmol/8×10 8 erythrocytes compared with a 6-TGN outside of this range was 7.6 (2.2, 26.3) (p=0.001) and compared with monotherapy was 9.9 (3.3, 29.4) (p=0.001)., Conclusion: 6-TGN levels between 235 and 450 pmol/8×10 8 erythrocytes prevented production of ATI. This supports therapeutic drug monitoring to help guide treatment and maximise the beneficial effects of combination therapy for patients with IBD., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Female breast cancer is the most frequently diagnosed cancer. The long-term survival rates for this disease have increased; however, the unique demand for high-quality healthcare to improve breast-cancer survivorship are commonly unmet. The Mediterranean diet (MD) is associated with reduced breast-cancer risk and various health-related benefits in the general population, but its effect on breast-cancer survivors remains uncertain. The objective of this systematic review and meta-analysis was to assess current evidence from randomised controlled trials (RCTs) and observational studies (cohort, cross-sectional and case-control) regarding the effect of the MD on survival, quality of life (QoL) and health-related outcomes in female breast-cancer survivors. MEDLINE, EMBASE, Web of Science and the Cochrane library were searched for studies published before and including April 2022. Two reviewers independently screened the literature and completed the data extraction and risk-of-bias assessment. Eleven studies (fifteen reports) were included, including two RCTs, four cohort and five cross-sectional studies. The meta-analysis of the cohort studies showed strong evidence of an inverse association between high adherence to the MD and all-cause mortality (hazard ratio (HR) 0.78, 95% confidence interval (CI) 0.66-0.93, I 2 : 0%, Grading of Recommendations Assessment, Development and Evaluation (GRADE) = low certainty of evidence) and non-breast-cancer mortality (HR 0.67, 95% CI 0.50-0.90, I 2 : 0%, GRADE = very low certainty of evidence). The associations between high adherence to the MD and QoL and health-related parameters were not consistent. These findings highlight the potential of adherence to the MD to reduce the risk of mortality. Future research with better study designs, as well as more consistent measurements of QoL and MD adherence, taking into account changes in MD adherence over time and population subgroups, is needed to provide more robust evidence on the survival, QoL and health-related outcomes in BC survivors.

Background: Type 2 diabetes mellitus (T2DM) and its associated complications puts considerable strain on healthcare systems. With the global incidence of T2DM increasing, effective disease management is essential. Physical activity (PA) is a key component of T2DM management; however, rates of PA engagement are low in this population. Developing effective and sustainable interventions that encourage PA is a high priority. Electrically assisted bicycles are becoming increasingly popular and may increase PA in healthy adults. This study aimed to provide evidence of the feasibility of conducting a randomized controlled trial to evaluate the efficacy of an e-cycling intervention to increase PA and improve health in individuals with T2DM., Methods: A parallel-group two-arm randomized, waitlist-controlled pilot study was conducted. Individuals were randomized to either an e-bike intervention or standard care. The intervention incorporated two one-to-one e-bike skills training and behavioural counselling sessions delivered by a community-based cycling charity, followed by a 12-week e-bike loan with two further sessions with the instructors. Feasibility was assessed via measures related to recruitment, retention and intervention implementation. Post-intervention interviews with instructors and participants explored the acceptability of the study procedures and intervention. Clinical, physiological and behavioural outcomes were collected at baseline and post-intervention to evaluate the intervention's potential., Results: Forty participants (M age  = 57) were randomized, of which 34 were recruited from primary care practices. Thirty-five participants were retained in the trial. The intervention was conducted with high fidelity (> 80% content delivered). E-bike training provided participants with the skills, knowledge and confidence needed to e-bike independently. Instructors reported being more confident delivering the skills training than behavioural counselling, despite acknowledging its importance. The study procedures were found to be acceptable to participants. Between-group differences in change during the intervention were indicative of the interventions potential for improving glucose control, health-related quality of life and cardiorespiratory fitness. Increases in overall device measured moderate-to-vigorous PA behaviour following the intervention were found, and there was evidence that this population self-selected to e-cycle at a moderate intensity., Conclusions: The study's recruitment, retention, acceptability and potential efficacy support the development of a definitive trial subject to identified refinements., Trial Registration: ISRCTN, ISRCTN67421464 . Registered 17/12/2018., (© 2023. The Author(s).)

Introduction: A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear., Methods: In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in individuals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR diversity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing., Results and Discussion: The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p < 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Currenti, Simmons, Oakes, Gaudieri, Warren, Gangula, Alves, Ram, Leary, Armitage, Smith, Chopra, Halasa, Pilkinton and Kalams.)

Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Society for Histocompatibility and Immunogenetics. All rights reserved.)

Background: The limited variation observed among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consensus sequences makes it difficult to reconstruct transmission linkages in outbreak settings. Previous studies have recovered variation within individual SARS-CoV-2 infections but have not yet measured the informativeness of within-host variation for transmission inference., Methods: We performed tiled amplicon sequencing on 307 SARS-CoV-2 samples, including 130 samples from 32 individuals in 14 households and 47 longitudinally sampled individuals, from 4 prospective studies with household membership data, a proxy for transmission linkage., Results: Consensus sequences from households had limited diversity (mean pairwise distance, 3.06 single-nucleotide polymorphisms [SNPs]; range, 0-40). Most (83.1%, 255 of 307) samples harbored at least 1 intrahost single-nucleotide variant ([iSNV] median, 117; interquartile range [IQR], 17-208), above a minor allele frequency threshold of 0.2%. Pairs in the same household shared significantly more iSNVs (mean, 1.20 iSNVs; 95% confidence interval [CI], 1.02-1.39) than did pairs in different households infected with the same viral clade (mean, 0.31 iSNVs; 95% CI, .28-.34), a signal that decreases with increasingly stringent minor allele frequency thresholds. The number of shared iSNVs was significantly associated with an increased odds of household membership (adjusted odds ratio, 1.35; 95% CI, 1.23-1.49). However, the poor concordance of iSNVs detected across sequencing replicates (24.8% and 35.0% above a 0.2% and 1% threshold) confirms technical concerns that current sequencing and bioinformatic workflows do not consistently recover low-frequency within-host variants., Conclusions: Shared within-host variation may augment the information in consensus sequences for predicting transmission linkages. Improving sensitivity and specificity of within-host variant identification will improve the informativeness of within-host variation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Children's Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community. The CBTN's 32 member institutions utilize a shared regulatory governance architecture at the Children's Hospital of Philadelphia to accelerate and maximize the use of biospecimens and data. As of August 2022, CBTN has enrolled over 4700 subjects, over 1500 parents, and collected over 65,000 biospecimen aliquots for research. Additionally, over 80 preclinical models have been developed from collected tumors. Multi-omic data for over 1000 tumors and germline material are currently available with data generation for > 5000 samples underway. To our knowledge, CBTN provides the largest open-access pediatric brain tumor multi-omic dataset annotated with longitudinal clinical and outcome data, imaging, associated biospecimens, child-parent genomic pedigrees, and in vivo and in vitro preclinical models. Empowered by NIH-supported platforms such as the Kids First Data Resource and the Childhood Cancer Data Initiative, the CBTN continues to expand the resources needed for scientists to accelerate translational impact for improved outcomes and quality of life for children with brain and spinal cord tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. David S. Ziegler is a consultant, or on the advisory board, of Bayer, AstraZeneca, Accendatech, Novartis, Day One, FivePhusion, Amgen, Alexion, and Norgine. Angela J. Waanders is on the advisory board of Alexion and Day One., (Copyright © 2022. Published by Elsevier Inc.)

Medulloblastoma (MB) is the most common malignant central nervous system tumor of childhood, comprising a heterogenous group of tumors each with distinct biology, clinical behavior, and prognosis. Long-term survival remains unacceptable, and those who do survive face high late mortality risk, new chronic treatment-related medical conditions, neurocognitive impairments, and poor health-related quality of life. Up-front treatment strategies now integrate molecular subgrouping with standard clinico-radiological factors to more actually risk stratify newly-diagnosed patients. To what extent this new stratification will lead to improvements in treatment outcome will be determined in the coming years. In parallel, discovery and appreciation for medulloblastoma's inter- and intra-tumoral heterogeneity continues growing. Clinical trials treating relapsed disease now encompass precision medicine, epigenetic modification, and immune therapy approaches. The Pacific Pediatric Neuro-Oncology (PNOC) Medulloblastoma Working Group is committed to developing clinical trials based on these evolving therapeutic strategies and supports translational efforts by PNOC researchers and the multi-stakeholder medulloblastoma community at large., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Background: Wrestling is known to be a sport of relatively high injury incidence, and knee injuries account for a large percentage of those injuries. Treatment of these injuries varies considerably depending on injury and wrestler characteristics, leading to variability in complete recovery and return to sport (RTS). The purpose of this study was to evaluate injury trends, treatment strategies, and RTS characteristics after knee injuries in competitive collegiate wrestling., Methods: NCAA Division I collegiate wrestlers who sustained knee injuries between January 2010 and May 2020 were identified using an institutional Sports Injury Management System (SIMS). Wrestling-related knee, meniscus, and patella injuries were identified, and treatment strategies were documented to investigate potential recurrent injury trends. Descriptive statistics were used to quantify the number of days, practices, and competitions missed, return to sport times, and recurrent injuries among wrestlers., Results: Overall, 184 knee injuries were identified. After excluding non-wrestling injuries (n=11), 173 injuries remained (77 wrestlers). The mean age at time of injury was 20.8 ± 1.4 years, and the mean BMI was 25.9 ± 3.8 kg/m2. There were 135 primary injuries (74 wrestlers), which consisted of 72 (53%) ligamentous injuries, 30 (22%) meniscus injuries, 14 patellar injuries (10%), and 19 other injuries (14%). The majority of ligamentous injuries (93%) and patellar injuries (79%) were treated non-operatively, while the majority of meniscus tears (60%) underwent surgery. Twenty-three wrestlers (22%) sustained recurrent knee injuries, of which 76% were treated non-operatively after their initial injury. Recurrent injuries consisted of 12 (32%) ligamentous injuries, 14 (37%) meniscus injuries, eight (21%) patellar injuries, and four (11%) other injuries. Fifty percent of recurrent injuries were treated operatively. When comparing recurrent injuries to primary injuries, recurrent injuries had a significantly longer return to sport time (Recurrent 68.3 ± 96.0 days vs. Primary 26.0 ± 56.4 days, p=0.01)., Conclusion: The majority of NCAA Division I collegiate wrestlers who sustained knee injuries were initially treated non-operatively, and approximately one in five wrestlers sustained recurrent injuries. Return to sport time was significantly increased after a recurrent injury. Level of Evidence: IV ., (Copyright © The Iowa Orthopaedic Journal 2023.)

It is important to both protect the healthy development and maintain the oral health of the child population. The study examined the effect of early childhood exposures to water fluoridation on measures of school-age executive functioning and emotional and behavioral development in a population-based sample. This longitudinal follow-up study used information from Australia's National Child Oral Health Study 2012-14. Children aged 5 to 10 y at baseline were contacted again after 7 to 8 y, before they had turned 18 y of age. Percent lifetime exposed to fluoridated water (%LEFW) from birth to the age 5 y was estimated from residential history and postcode-level fluoride levels in public tap water. Measures of children's emotional and behavioral development were assessed by the Strength and Difficulties Questionnaire (SDQ), and executive functioning was measured by the Behavior Rating Inventory of Executive Function (BRIEF). Multivariable regression models were generated to compare the associations between the exposure and the primary outcomes and controlled for covariates. An equivalence test was also conducted to compare the primary outcomes of those who had 100% LEFW against those with 0% LEFW. Sensitivity analysis was also conducted. A total of 2,682 children completed the SDQ and BRIEF, with mean scores of 7.0 (95% confidence interval, 6.6-7.4) and 45.3 (44.7-45.8), respectively. Those with lower %LEFW tended to have poorer scores of the SDQ and BRIEF. Multivariable regression models reported no association between exposure to fluoridated water and the SDQ and BRIEF scores. Low household income, identifying as Indigenous, and having a neurodevelopmental diagnosis were associated with poorer SDQ/BRIEF scores. An equivalence test confirmed that the SDQ/BRIEF scores among those with 100% LEFW were equivalent to that of those who had 0% LEFW. Exposure to fluoridated water during the first 5 y of life was not associated with altered measures of child emotional and behavioral development and executive functioning.

Introduction: Subependymal giant cell astrocytoma (SEGA) is the most common CNS tumor in patients with tuberous sclerosis complex (TSC). Although these are benign, their proximity to the foramen of Monroe frequently causes obstructive hydrocephalus, a potentially fatal complication. Open surgical resection has been the mainstay of treatment; however, this can cause significant morbidity. The development of mTOR inhibitors has changed the treatment landscape, but there are limitations to their use. Laser interstitial thermal therapy (LITT) is an emerging treatment modality that has shown promise in treatment of a variety of intracranial lesions, including SEGAs. We present a single institution, retrospective study of patients treated for SEGAs with LITT, open resection, mTOR inhibitors, or a combination of these modalities. The primary study outcome was tumor volume at most recent follow-up compared with volume at treatment initiation. The secondary outcome was clinical complications associated with treatment modality., Methods: Retrospective chart review was performed to identify patients with SEGAs treated at our institution from 2010 to 2021. Demographics, treatment information, and complications were collected from the medical record. Tumor volumes were calculated from imaging obtained at initiation of treatment and at most recent follow-up. Kruskal-Wallis nonparametric testing was used to assess differences in tumor volume and follow-up duration between groups., Results: Four patients underwent LITT (3 with LITT only), three underwent open surgical resection, and four were treated with mTOR inhibitors only. Mean percent tumor volume reduction for each group was 48.6 ± 13.8, 90.7 ± 39.8, and 67.1 ± 17.2%, respectively. No statistically significant difference was identified comparing percent tumor volume reduction between the three groups (p = 0.0513). Additionally, there was no statistically significant difference in follow-up duration between groups (p = 0.223). Only 1 patient in our series required permanent CSF diversion and 4 discontinued or decreased the dose of mTOR inhibitor due to either cost or side effects., Conclusions: Our study suggests that LITT could be considered as a treatment option for SEGAs as it was effective in reducing tumor volume with very few complications. This modality is less invasive than open resection and may be an alternative for patients who are not candidates for mTOR inhibitors. We recommend an updated paradigm for SEGA treatment which includes LITT in select cases after consideration of patient-specific factors., (The Author(s). Published by S. Karger AG, Basel.)

Adaptation to human leukocyte antigen (HLA)-associated immune pressure represents a major driver of human immunodeficiency virus (HIV) evolution at both the individual and population level. To date, there has been limited exploration of the impact of the initial cellular immune response in driving viral adaptation, the dynamics of these changes during infection and their effect on circulating transmitting viruses at the population level. Capturing detailed virological and immunological data from acute and early HIV infection is challenging as this commonly precedes the diagnosis of HIV infection, potentially by many years. In addition, rapid initiation of antiretroviral treatment following a diagnosis is the standard of care, and central to global efforts towards HIV elimination. Yet, acute untreated infection is the critical period in which the diversity of proviral reservoirs is first established within individuals, and associated with greater risk of onward transmissions in a population. Characterizing the viral adaptations evident in the earliest phases of infection, coinciding with the initial cellular immune responses is therefore relevant to understanding which changes are of greatest impact to HIV evolution at the population level. In this study, we utilized three separate cohorts to examine the initial CD8+ T cell immune response to HIV (cross-sectional acute infection cohort), track HIV evolution in response to CD8+ T cell-mediated immunity over time (longitudinal chronic infection cohort) and translate the impact of HLA-driven HIV evolution to the population level (cross-sectional HIV sequence data spanning 30 years). Using next generation viral sequencing and enzyme-linked immunospot interferon-gamma recall responses to peptides representing HLA class I-specific HIV T cell targets, we observed that CD8+ T cell responses can select viral adaptations prior to full antibody seroconversion. Using the longitudinal cohort, we uncover that viral adaptations have the propensity to be retained over time in a non-selective immune environment, which reflects the increasing proportion of pre-adapted HIV strains within the Western Australian population over an approximate 30-year period., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Objective: To describe breastfeeding prevalence and maternal experience in infants with trisomy 21., Design: Longitudinal cohort study., Setting: Participants from UK recruited through websites, social media and local collaborators: neonatologists, community paediatricians and research nurses., Subjects: Infants under the age of 8 months with Down syndrome (DS) recruited to the Feeding and Autoimmunity in Down Syndrome Evaluation Study between 1 September 2014 and 31 August 2017. Seventy participants: median age 20 weeks (IQR 13-29 weeks) at initial questionnaire., Main Outcome Measure: Breastfeeding prevalence at 6 weeks and 6 months among infants with DS., Results: The prevalence of exclusive breast feeding among study participants was similar to the general population (13/61, 21% vs 23% at 6 weeks, 2/54, 4% vs 1% at 6 months). However, the prevalence of breast feeding (exclusive or combination feeding) among the study participants was higher than the general population (39/61 64% vs 55% at 6 weeks, 32/59 54% vs 34% at 6 months)., Conclusion: Although there may be challenges in establishing breast feeding in infants with DS, our data suggest that exclusive breast feeding is possible for some, and the prevalence of breast feeding is comparable to the prevalence in the general population., Trial Registration Number: ISRCTN12415856., Competing Interests: Competing interests: None delclared, (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Around 80% of adults worldwide carry human cytomegaloviris (HCMV). The HCMV gene UL18 is a homolog of HLA class I genes and encodes a protein with high affinity for the NK and T-cell cytotoxicity inhibitor LIR-1. UL18 was deep sequenced from blood, saliva or urine from Indonesian people with HIV (PWH) ( n = 28), Australian renal transplant recipients (RTR) ( n = 21), healthy adults ( n = 7) and neonates ( n = 4). 95% of samples contained more than one variant of HCMV UL18 , as defined by carriage of nonsynonymous variations. When aligned with immunological markers of the host's burden of HCMV, the S318N variation associated with high levels of antibody reactive with HCMV lysate in PWH over 12 months on antiretroviral therapy. The A107T variation associated with HCMV antibody levels and inflammatory biomarkers in PWH at early timepoints. Variants D32G, D248N, V250A and E252D aligned with elevated HCMV antibody levels in RTR, while M191K, E196Q and F165L were associated with HCMV-reactive T-cells and proportions of Vδ2 - γδ T-cells-populations linked with high burdens of HCMV. We conclude that UL18 is a highly variable gene, where variation may alter the persistent burden of HCMV and/or the host response to that burden.

Background: Cross-sectional tumor measures are traditional clinical trial endpoints; however volumetric measures may better assess tumor growth. We determined the correlation and compared the prognostic impact of cross-sectional and volumetric measures of progressive disease (PD) among patients with DIPG., Methods: Imaging and clinical data were abstracted from the International DIPG Registry. Tumor volume and cross-sectional product (CP) were measured with mint Lesion™ software using manual contouring. Correlation between CP and volume (segmented and mathematical [ellipsoid] model) thresholds of PD were assessed by linear regression. Landmark analyses determined differences in survival (via log-rank) between patients classified as PD versus non-PD by CP and volumetric measurements at 1, 3, 5, 7, and 9 months postradiotherapy (RT). Hazard ratios (HR) for survival after these time points were calculated by Cox regression., Results: A total of 312 MRIs (46 patients) were analyzed. Comparing change from the previous smallest measure, CP increase of 25% (PD) correlated with a segmented volume increase of 30% (R2 = 0.710), rather than 40% (spherical model extrapolation). CP-determined PD predicted survival at 1 month post-RT (HR = 2.77), but not other time points. Segmented volumetric-determined PD (40% threshold) predicted survival at all imaging timepoints (HRs = 2.57, 2.62, 3.35, 2.71, 16.29), and 30% volumetric PD threshold predicted survival at 1, 3, 5, and 9 month timepoints (HRs = 2.57, 2.62, 4.65, 5.54). Compared to ellipsoid volume, segmented volume demonstrated superior survival associations., Conclusions: Segmented volumetric assessments of PD correlated better with survival than CP or ellipsoid volume at most time points. Semiautomated tumor volume likely represents a more accurate, prognostically-relevant measure of disease burden in DIPG., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) is a common, long-term condition characterised by post-exertional malaise, often with fatigue that is not significantly relieved by rest. ME/CFS has no confirmed diagnostic test or effective treatment and we lack knowledge of its causes. Identification of genes and cellular processes whose disruption adds to ME/CFS risk is a necessary first step towards development of effective therapy., Methods: Here we describe DecodeME, an ongoing study co-produced by people with lived experience of ME/CFS and scientists. Together we designed the study and obtained funding and are now recruiting up to 25,000 people in the UK with a clinical diagnosis of ME/CFS. Those eligible for the study are at least 16 years old, pass international study criteria, and lack any alternative diagnoses that can result in chronic fatigue. These will include 5,000 people whose ME/CFS diagnosis was a consequence of SARS-CoV-2 infection. Questionnaires are completed online or on paper. Participants' saliva DNA samples are acquired by post, which improves participation by more severely-affected individuals. Digital marketing and social media approaches resulted in 29,000 people with ME/CFS in the UK pre-registering their interest in participating. We will perform a genome-wide association study, comparing participants' genotypes with those from UK Biobank as controls. This should generate hypotheses regarding the genes, mechanisms and cell types contributing to ME/CFS disease aetiology., Discussion: The DecodeME study has been reviewed and given a favourable opinion by the North West - Liverpool Central Research Ethics Committee (21/NW/0169). Relevant documents will be available online ( www.decodeme.org.uk ). Genetic data will be disseminated as associated variants and genomic intervals, and as summary statistics. Results will be reported on the DecodeME website and via open access publications., (© 2022. The Author(s).)

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern., (© 2022. The Author(s).)

Background: The study aimed to evaluate whether simplified chemotherapy followed by dose-reduced irradiation was effective for treating patients (ages 3-21 years) with localized germinoma. The primary endpoint was 3-year progression-free survival (PFS) rate., Methods: Patients with a complete response to chemotherapy with carboplatin and etoposide received 18 Gy WVI + 12 Gy boost to the tumor bed. Patients with partial response proceeded to 24 Gy WVI + 12 Gy. Longitudinal cognitive functioning was evaluated prospectively on ALTE07C1 and was a primary study aim., Results: One hundred and fifty-one patients were enrolled; 137 were eligible. Among 90 evaluable patients, 74 were treated with 18 Gy and 16 with 24 Gy WVI. The study failed to demonstrate noninferiority of the 18 Gy WVI regimen compared to the design threshold of 95% 3-year PFS rate, where, per design, patients who could not be assessed for progression at 3 years were counted as failures. The Kaplan-Meier (KM)-based 3-year PFS estimates were 94.5 ± 2.7% and 93.75 ± 6.1% for the 18 Gy and 24 Gy WVI cohorts, respectively. Collectively, estimated mean IQ and attention/concentration were within normal range. A lower mean attention score was observed at 9 months for patients treated with 24 Gy. Acute effects in processing speed were observed in the 18 Gy cohort at 9 months which improved at 30-month assessment., Conclusions: While a failure according to the prospective statistical noninferiority design, this study demonstrated high rates of chemotherapy responses, favorable KM-based PFS and OS estimates in the context of reduced irradiation doses and holds promise for lower long-term morbidities for patients with germinoma., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Childhood adiposity, an important predictor of adult chronic disease, has been rising dramatically. Later eating rhythm, termed night eating, is increasing in adults but rarely studied in younger ages., Objective: The objective of this study was to review the association between later eating rhythm and adiposity in children and adolescents. The aspects of later eating being considered included: energy intake (for evening main meal, evening snack, whole evening period, and around bedtime); timing (any food eaten at later timing); and meal frequency in the evening/night (evening main meal skipping, evening snack consumption)., Data Sources: Five databases (the Cochrane Library, CINAHL, Embase, MEDLINE (via OVID), and Web of Science) were searched for eligible articles published prior to and including August 2020., Data Extraction: Data extraction and quality assessment were conducted by 2 reviewers independently., Data Analysis: Forty-seven studies were included, all of which were observational. Meta-analysis showed positive associations between both higher energy intake around bedtime (odds ratio [OR] 1.19, 95% CI 1.06, 1.33) and evening main meal skipping (OR 1.30, 95% CI 1.14, 1.48), and adiposity. There was evidence to suggest that consuming evening snacks reduced adiposity, but it was very weak (OR 0.80, 95% CI 0.62, 1.05). No association was seen between eating later and adiposity (OR 1.04, 95% CI 0.68, 1.61). In the narrative analysis, approximately half of the studies suggested that there was no association between later eating rhythm and adiposity, either as a whole or within exposure subsets., Conclusion: The magnitude of the relationship between later eating rhythm and adiposity is very small, and may vary depending on which aspects of later eating rhythm are under consideration; however, the evidence for this conclusion is of very low certainty . Further research with a more consistent definition of "later timing", and longitudinal studies in different populations, may lead to different conclusions., Systematic Review Registration: PROSPERO registration no. CRD42019134187., (© The Author(s) 2021. Published by Oxford University Press on behalf of the International Life Sciences Institute.)

Background: Postoperative complications and substantial loss of physical function are common after musculoskeletal trauma. We conducted a prospective randomized controlled trial to assess the impact of conditionally essential amino acid (CEAA) supplementation on complications and skeletal muscle mass in adults after operative fixation of acute fractures., Methods: Adults who sustained pelvic and extremity fractures that were indicated for operative fixation at a level-I trauma center were enrolled. The subjects were stratified based on injury characteristics (open fractures and/or polytrauma, fragility fractures, isolated injuries) and randomized to standard nutrition (control group) or oral CEAA supplementation twice daily for 2 weeks. Body composition (fat-free mass [FFM]) was measured at baseline and at 6 and 12 weeks postoperatively. Complications were prospectively collected. An intention-to-treat analysis was performed. The relative risk (RR) of complications for the control group relative to the CEAA group was determined, and linear mixed-effects models were used to model the relationship between CEAA supplementation and changes in FFM., Results: Four hundred subjects (control group: 200; CEAA group: 200) were enrolled. The CEAA group had significantly lower overall complications than the control group (30.5% vs. 43.8%; adjusted RR = 0.71; 95% confidence interval [CI] = 0.55 to 0.92; p = 0.008). The FFM decreased significantly at 6 weeks in the control subjects (-0.9 kg, p = 0.0205), whereas the FFM was maintained at 6 weeks in the CEAA subjects (-0.33 kg, p = 0.3606). This difference in FFM was not seen at subsequent time points., Conclusions: Our results indicate that CEAA supplementation has a protective effect against common complications and early skeletal muscle wasting after operative fixation of extremity and pelvic fractures. Given the potential benefits of this inexpensive, low-risk intervention, multicenter prospective studies in focused trauma populations are warranted., Level of Evidence: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G920)., (Copyright © 2022 by The Journal of Bone and Joint Surgery, Incorporated.)

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ~80% of adults worldwide. Acute infections are often asymptomatic in healthy individuals but generate diverse syndromes in neonates, renal transplant recipients (RTR), and people with HIV (PWH). The HCMV gene UL111a encodes a homolog of human interleukin-10 (IL-10) that interacts with the human IL-10 receptor. Deep sequencing technologies were used to sequence UL111a directly from 59 clinical samples from Indonesian PWH and Australian RTR, healthy adults, and neonates. Overall, 93% of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous variation. Carriage of these variants differed between neonates and adults, Australians and Indonesians, and between saliva and blood leukocytes. The variant alleles of N41D and S71Y occurred together in Australian RTR and were associated with higher T-cell responses to HCMV pp65. The variant P122S was associated with lower levels of antibodies reactive with a lysate of HCMV-infected fibroblasts. L174F was associated with increased levels of antibodies reactive with HCMV lysate, immediate-early 1 (IE-1), and glycoprotein B (gB) in Australian RTR and Indonesians PWH, suggesting a higher viral burden. We conclude that variants of UL111a are common in all populations and may influence systemic responses to HCMV.

Background: Pineoblastoma is a rare brain tumor usually diagnosed in children. Given its rarity, no pineoblastoma-specific trials have been conducted. Studies have included pineoblastoma accruing for other embryonal tumors over the past 30 years. These included only occasional children with pineoblastoma, making clinical features difficult to interpret and determinants of outcome difficult to ascertain., Patients and Methods: Centrally or independently reviewed series with treatment and survival data from North American and Australian cases were pooled. To investigate associations between variables, Fisher's exact tests, Wilcoxon-Mann-Whitney tests, and Spearman correlations were used. Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models were used in survival analyses., Results: We describe a pooled cohort of 178 pineoblastoma cases from Children's Oncology Group (n = 82) and institutional series (n = 96) over 30 years. Children <3 years of age have significantly worse survival compared to older children, with 5-year progression-free survival (PFS) and overall survival (OS) estimates of 13.5 ± 5.1% and 16.2 ± 5.3%, respectively, compared with 60.8 ± 5.6% and 67.3 ± 5.0% for ≥3 years old (both P < .0001). Multivariable analysis showed male sex was associated with worse PFS in children <3 years of age (hazard ratio [HR] 3.93, 95% CI 1.80-8.55; P = .0006), suggestive of sex-specific risks needing future validation. For children ≥3 years of age, disseminated disease at diagnosis was significantly associated with an inferior 5-year PFS of 39.2 ± 9.7% (HR 2.88, 95% CI 1.52-5.45; P = .0012) and 5-year OS of 49.8 ± 9.1% (HR 2.87, 95% CI 1.49-5.53; P = .0016)., Conclusion: Given the rarity of this tumor, prospective, collaborative international studies will be vital to improving the long-term survival of these patients., (© The Author(s) 2022. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Glioblastoma (GBM) displays diffusive invasion throughout the brain microenvironment, which is partially responsible for its short median survival rate (<15 months). Stem-like subpopulations (GBM stem-like cells, GSCs) are believed to play a central role in therapeutic resistance and poor patient prognosis. Given the extensive tissue remodeling and processes such as vessel co-option and regression that occur in the tumor microenvironment, it is essential to understand the role of metabolic constraint such as hypoxia on GBM cell populations. This work describes the use of a multidimensional gelatin hydrogel to culture patient-derived GBM cells, to evaluate the influence of hypoxia and the inclusion brain-mimetic hyaluronic acid on the relative activity of GSCs versus overall GBM cells. Notably, CD133 + GBM cell fraction is crucial for robust formation of tumor spheroids in multidimensional cultures. In addition, while the relative size of the CD133 + GBM subpopulation increased in response to both hypoxia and matrix-bound hyaluronan, we did not observe cell subtype-specific changes in invasion signaling pathway activation. Taken together, this study highlights the potential of biomimetic culture systems for resolving changes in the population dynamics and behavior of subsets of GBM specimens for the future development of precision medicine applications. Impact Statement This study describes a gelatin hydrogel platform to investigate the role of extracellular hyaluronic acid and hypoxia on the behavior of a CD133 + subset of cells within patient-derived glioblastoma (GBM) specimens. We report that the relative expansion of the CD133 + GBM stem cell-like population is strongly responsive to extracellular cues, highlighting the significance of biomimetic hydrogel models of the tumor microenvironment to investigate invasion and therapeutic response.

Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions., (© 2022. The Author(s).)

Background and Aims: Patients with differentiated thyroid cancer are often advised to follow a low iodine diet (LID) one to two weeks before radioiodine remnant ablation (RRA). We describe treatment practices and ablation success rates in centres (C1, C2, C3) in the UK with different approaches to LID advice., Methods: Historic cohort of patients with differentiated thyroid cancer treated with RRA in 2015/16 in C1 (n = 50, 1-week LID), C2 (n = 59, 2-week LID) and C3 (n = 108, no LID advice). Response to RRA was stratified as excellent, indeterminate, or incomplete by the adapted American Thyroid Association Dynamic Risk Stratification Score., Results: There was little difference in age, sex and staging between centres, but the percentage receiving 1.1 GBq vs higher administered activities differed (C1:22%, C2:44%, C3:15%, p < 0.001). Excellent response was recorded for: C1:48%, C2:36%, C3:49% (p = 0.61). Differences in RRA preparation and outcome assessment at C3 precluded comparison across all centres. Adjusted odds ratio for excellent response at C2 vs C1 was 0.57 (95%CI: 0.25,1.32), p = 0.19., Conclusions: There was no evidence that advising a LID for 2-weeks before RRA improves outcomes compared to 1-week. For definitive recommendations on LIDs prior to RRA, a prospective multi-centre study with a more homogenous approach to patient management or, randomised controlled trial, is needed., Competing Interests: Declaration of competing interest IHS is the lead and LM is a member of the UK Low Iodine Diet Working Group. The remaining authors have nothing to declare., (Copyright © 2021 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Background: Diffuse intrinsic pontine gliomas (DIPG) generally occur in young school-age children, although can occur in adolescents and young adults. The purpose of this study was to describe clinical, radiological, pathologic, and molecular characteristics in patients ≥10 years of age with DIPG enrolled in the International DIPG Registry (IDIPGR)., Methods: Patients ≥10 years of age at diagnosis enrolled in the IDIPGR with imaging confirmed DIPG diagnosis were included. The primary outcome was overall survival (OS) categorized as long-term survivors (LTS) (≥24 months) or short-term survivors (STS) (<24 months)., Results: Among 1010 patients, 208 (21%) were ≥10 years of age at diagnosis; 152 were eligible with a median age of 12 years (range 10-26.8). Median OS was 13 (2-82) months. The 1-, 3-, and 5-year OS was 59.2%, 5.3%, and 3.3%, respectively. The 18/152 (11.8%) LTS were more likely to be older (P < .01) and present with longer symptom duration (P < .01). Biopsy and/or autopsy were performed in 50 (33%) patients; 77%, 61%, 33%, and 6% of patients tested had H3K27M (H3F3A or HIST1H3B), TP53, ATRX, and ACVR1 mutations/genome alterations, respectively. Two of 18 patients with IDH1 testing were IDH1-mutant and 1 was a LTS. The presence or absence of H3 alterations did not affect survival., Conclusion: Patients ≥10 years old with DIPG have a median survival of 13 months. LTS present with longer symptom duration and are likely to be older at presentation compared to STS. ATRX mutation rates were higher in this population than the general DIPG population., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: This study examines Irish undergraduate students' behaviours and motives regarding alcohol consumption. The study explores both levels and patterns of consumption., Method: A cross-sectional design using a convenience sample of (n = 213) students from a selection of different courses in Health Sciences at Trinity College Dublin was used to obtain this data. The study used a peer-led approach to design and data collection. Peer-led research is emerging as a robust methodology. Evidence supports it as an effective approach, particularly with sensitive questions, which may be shared with more ease between persons with common interests and experiences., Results: In terms of alcohol consumption levels and patterns, of those who drank almost three quarters (149/71%) met the threshold for binge drinking (i.e. six of more consecutive drinks in one session). Males (n = 36/73.4%) were more likely than females (n = 113/69.7%) to binge drink. Moreover, one in 5 males (n = 10/20.4%) said that they drank ten or more drinks in one session. Males were more likely to drink for conformity reasons. Despite this, a significant proportion (69.2%) of participants reported alcohol-related problems. The Drinking Motives Questionnaire-Revised (DMQR) results showed that overall students were more likely to drink for social and enhancement reasons rather than coping or conformity reasons, consistent with other studies. Nonetheless, males in the current study were more likely to drink for conformity reasons., Conclusion: Given the high rates of hazardous drinking, the development of an alcohol intervention may be justified, given the high response rates to peer-screening, a peer-led intervention for alcohol-related harms may yield positive results., (© 2021. Royal Academy of Medicine in Ireland.)

Medulloblastoma, a common pediatric malignant central nervous system tumour, represent a small proportion of brain tumours in adults. Previously it has been shown that in adults, Sonic Hedgehog (SHH)-activated tumours predominate, with Wingless-type (WNT) and Group 4 being less common, but molecular risk stratification remains a challenge. We performed an integrated analysis consisting of genome-wide methylation profiling, copy number profiling, somatic nucleotide variants and correlation of clinical variables across a cohort of 191 adult medulloblastoma cases identified through the Medulloblastoma Advanced Genomics International Consortium. We identified 30 WNT, 112 SHH, 6 Group 3, and 41 Group 4 tumours. Patients with SHH tumours were significantly older at diagnosis compared to other subgroups (p < 0.0001). Five-year progression-free survival (PFS) for WNT, SHH, Group 3, and Group 4 tumours was 64.4 (48.0-86.5), 61.9% (51.6-74.2), 80.0% (95% CI 51.6-100.0), and 44.9% (95% CI 28.6-70.7), respectively (p = 0.06). None of the clinical variables (age, sex, metastatic status, extent of resection, chemotherapy, radiotherapy) were associated with subgroup-specific PFS. Survival among patients with SHH tumours was significantly worse for cases with chromosome 3p loss (HR 2.9, 95% CI 1.1-7.6; p = 0.02), chromosome 10q loss (HR 4.6, 95% CI 2.3-9.4; p < 0.0001), chromosome 17p loss (HR 2.3, 95% CI 1.1-4.8; p = 0.02), and PTCH1 mutations (HR 2.6, 95% CI 1.1-6.2; p = 0.04). The prognostic significance of 3p loss and 10q loss persisted in multivariable regression models. For Group 4 tumours, chromosome 8 loss was strongly associated with improved survival, which was validated in a non-overlapping cohort (combined cohort HR 0.2, 95% CI 0.1-0.7; p = 0.007). Unlike in pediatric medulloblastoma, whole chromosome 11 loss in Group 4 and chromosome 14q loss in SHH was not associated with improved survival, where MYCN, GLI2 and MYC amplification were rare. In sum, we report unique subgroup-specific cytogenetic features of adult medulloblastoma, which are distinct from those in younger patients, and correlate with survival disparities. Our findings suggest that clinical trials that incorporate new strategies tailored to high-risk adult medulloblastoma patients are urgently needed., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ∼80% of the world's population. Acute infections are asymptomatic in healthy individuals but generate diverse syndromes in neonates, solid organ transplant recipients, and HIV-infected individuals. The HCMV gene US28 encodes a homolog of a human chemokine receptor that is able to bind several chemokines and HIV gp120. Deep sequencing technologies were used to sequence US28 directly from 60 clinical samples from Indonesian HIV patients and Australian renal transplant recipients, healthy adults, and neonates. Molecular modeling approaches were used to predict whether nine nonsynonymous mutations in US28 may alter protein binding to a panel of six chemokines and two variants of HIV gp120. Ninety-two percent of samples contained more than one variant of HCMV, as defined by at least one nonsynonymous mutation. Carriage of these variants differed between neonates and adults, Australian and Indonesian samples, and saliva samples and blood leukocytes. Two nonsynonymous mutations (N170D and R267K) were associated with increased levels of immediate early protein 1 (IE-1) and glycoprotein B (gB) HCMV-reactive antibodies, suggesting a higher viral burden. Seven of the nine mutations were predicted to alter binding of at least one ligand. Overall, HCMV variants are common in all populations and have the potential to affect US28 interactions with human chemokines and/or gp120 and alter responses to the virus. The findings relied on deep sequencing technologies applied directly to clinical samples, so the variants exist in vivo . IMPORTANCE Human cytomegalovirus (HCMV) is a common viral pathogen of solid organ transplant recipients, neonates, and HIV-infected individuals. HCMV encodes homologs of several host genes with the potential to influence viral persistence and/or pathogenesis. Here, we present deep sequencing of an HCMV chemokine receptor homolog, US28, acquired directly from clinical specimens. Carriage of these variants differed between patient groups and was associated with different levels of circulating HCMV-reactive antibodies. These features are consistent with a role for US28 in HCMV persistence and pathogenesis. This was supported by in silico analyses of the variant sequences demonstrating altered ligand-binding profiles. The data delineate a novel approach to understanding the pathogenesis of HCMV and may impact the development of an effective vaccine.

Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8 + T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8 + T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8 + T cells were predominantly dual tetramer + , confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8 + T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8 + T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an 'effective' immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNɣ, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8 + T cells and potentially selecting for less effective T cell clones from the acute to chronic phase., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Currenti, Law, Qin, John, Pilkinton, Bansal, Leary, Ram, Chopra, Gangula, Yue, Warren, Barnett, Alves, McDonnell, Sooda, Heath, Mallal, Goepfert, Kalams and Gaudieri.)

Background: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host's anti-viral immune response, in turn affecting the frequency of variants over time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally., Methods: Deep-sequencing data of SARS-CoV-2 from public databases and from clinical samples were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Results: Sequence analysis suggests that the 3 adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep-sequencing data from 981 clinical samples confirmed the presence of the novel TRS-CS-dimerization domain RNA in individuals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames., Conclusions: The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans, resulting in both coding changes and novel sub-genomic RNA transcripts, suggests this as a mechanism for diversification and adaptation within its new host., Competing Interests: The authors declare that they have no conflicts of interests., (© Pathogens and Immunity 2021.)

Type B adverse drug reactions (ADRs) are iatrogenic immune-mediated syndromes with mechanistic etiologies that remain incompletely understood. Some of the most severe ADRs, including delayed drug hypersensitivity reactions, are T-cell mediated, restricted by specific human leukocyte antigen risk alleles and sometimes by public or oligoclonal T-cell receptors (TCRs), central to the immunopathogenesis of tissue-damaging response. However, the specific cellular signatures of effector, regulatory, and accessory immune populations that mediate disease, define reaction phenotype, and determine severity have not been defined. Recent development of single-cell platforms bringing together advances in genomics and immunology provides the tools to simultaneously examine the full transcriptome, TCRs, and surface protein markers of highly heterogeneous immune cell populations at the site of the pathological response at a single-cell level. However, the requirement for advanced bioinformatics expertise and computational hardware and software has often limited the ability of investigators with the understanding of diseases and biological models to exploit these new approaches. Here we describe the features and use of a state-of-the-art, fully integrated application for analysis and visualization of multiomic single-cell data called Visual Genomics Analysis Studio (VGAS). This unique user-friendly, Windows-based graphical user interface is specifically designed to enable investigators to interrogate their own data. While VGAS also includes tools for sequence alignment and identification of associations with host or organism genetic polymorphisms, in this review we focus on its application for analysis of single-cell TCR-RNA-Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-seq, enabling holistic cellular characterization by unbiased transcriptome and select surface proteome. Critically, VGAS does not require user-directed coding or access to high-performance computers, instead incorporating performance-optimized hidden code to provide application-based fast and intuitive tools for data analyses and production of high-resolution publication-ready graphics on standard specification laptops. Specifically, it allows analyses of comprehensive single-cell TCR sequencing (scTCR-seq) data, detailing (i) functional pairings of α-β heterodimer TCRs, (ii) one-click histograms to display entropy and gene rearrangements, and (iii) Circos and Sankey plots to visualize clonality and dominance. For unbiased single-cell RNA sequencing (scRNA-seq) analyses, users extract cell transcriptome signatures according to global structure via principal component analysis, t-distributed stochastic neighborhood embedding, or uniform manifold approximation and projection plots, with overlay of scTCR-seq enabling identification and selection of the immunodominant TCR-expressing populations. Further integration with similar sequence-based detection of surface protein markers using oligo-labeled antibodies (CITE-seq) provides comparative understanding of surface protein expression, with differential gene or protein analyses visualized using volcano plot or heatmap functions. These data can be compared to reference cell atlases or suitable controls to reveal discrete disease-specific subsets, from epithelial to tissue-resident memory T-cells, and activation status, from senescence through exhaustion, with more finite transcript expression displayed as violin and box plots. Importantly, guided tutorial videos are available, as are regular application updates based on the latest advances in bioinformatics and user feedback., Competing Interests: VGAS is proprietary software owned by Murdoch University who employ many of the lead authors. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hertzman, Deshpande, Leary, Li, Ram, Chopra, Cooper, Watson, Palubinsky, Mallal, Gibson and Phillips.)

We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed "subgenomic RNAs." sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5' UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5' end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2 +/- cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 samples worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA., (© 2021 Parker et al.; Published by Cold Spring Harbor Laboratory Press.)