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ABSTRACT: To comply with antibiotic restriction policies in the European Union, internal teat sealants (TS) are increasingly used at dry off (DO) in selective dry cow treatment protocols to maintain udder health. Postcalving TS residue attachment to milking equipment and associated cleaning difficulties is a reason some farmers stay away from blanket TS use. Our objective was therefore to improve insight into TS excretion visibility and to compare quantity, pattern, and presence versus absence of TS excretion postcalving between the typical 2 cow categories at DO: high (H)- and low (L)-SCC cows, treated with antibiotic (AB) plus TS (H-ABTS) or TS only (L-TS), respectively. In herds in the Netherlands (n = 3), and Germany (n = 4), cows were enrolled at DO, and categorized as H-ABTS (n = 93), or L-TS (n = 99). Postcalving, quarter-level TS visibility, quantities, patterns, and percentage of TS infused and excreted postcalving were recorded from 50 mL of premilk of every quarter at each of the first 15 or 16 milkings. Udder quarter health status was determined by bacteriological culture and SCC of quarter milk samples taken at DO and at d 3 postcalving and by clinical mastitis incidence from DO until 30 DIM. Univariable and multivariable models were created to explore associations of TS excretion presence versus absence at the first 3 milkings. Irrespective of SCC category, both laboratory personnel and farmers saw TS residues at the first milking in an equal 72% of quarters. Compared with laboratory as the gold standard, farmer sensitivity to spotting TS in premilk was 74.5% at the first milking and decreased to a maximum of 8.3% at the last 3 milkings. At the first milking, TS excretion quantities showed a bimodal distribution pattern and the mean percentage of TS infused (3.83 g) that was excreted in premilk at the first milking, was higher in the L-TS cows (45.5%) compared with the H-ABTS cows (32%). At the second and third milking, mean-adjusted TS percentage excreted was higher in the H-ABTS cows (8.5% and 1.8%, respectively) compared with the L-TS cows (4.6% and 0.4%, respectively). The multivariable model of the first 3 milkings showed parity at both the first and second milking, and the study group at both the second and third milking was significantly associated with TS presence. The univariable model showed no association between TS presence at the first milking and udder health. In conclusion, in premilk of the first milking, TS residue excretion was bimodal, higher in L-TS cows, more likely to be present in multiparous cows, and not associated with udder health. At the second and third milking, excretion was higher in H-ABTS cows and TS presence was only more likely in multiparous cows at the second milking.

IntroductionThere are limited data on prevalence of dementia in centenarians and near-centenarians (C/NC), its determinants, and whether the risk of dementia continues to rise beyond 100.MethodsParticipant-level data were obtained from 18 community-based studies (N = 4427) in 11 countries that included individuals ≥95 years. A harmonization protocol was applied to cognitive and functional impairments, and a meta-analysis was performed.ResultsThe mean age was 98.3 years (SD = 2.67); 79% were women. After adjusting for age, sex, and education, dementia prevalence was 53.2% in women and 45.5% in men, with risk continuing to increase with age. Education (OR 0.95;0.92-0.98) was protective, as was hypertension (odds ratio [OR] 0.51;0.35-0.74) in five studies. Dementia was not associated with diabetes, vision and hearing impairments, smoking, and body mass index (BMI).DiscussionAmong the exceptional old, dementia prevalence remains higher in the older participants. Education was protective against dementia, but other factors for dementia-free survival in C/NC remain to be understood.

Abstract Pediatric classic Hodgkin lymphoma (cHL) patients have a high survival rate but suffer from severe long‐term side effects induced by chemo‐ and radiotherapy. cHL tumors are characterized by the low fraction (0.1%–10%) of malignant Hodgkin and Reed–Sternberg (HRS) cells in the tumor. The HRS cells depend on the surrounding immune cells for survival and growth. This dependence is leveraged by current treatments that target the PD‐1/PD‐L1 axis in cHL tumors. The development of more targeted therapies that are specific for the tumor and are therefore less toxic for healthy tissue compared with conventional chemotherapy could improve the quality of life of pediatric cHL survivors. Here, we applied single‐cell RNA sequencing (scRNA‐seq) on isolated HRS cells and the immune cells from the same cHL tumors. Besides TNFRSF8 (CD30), we identified other genes of cell surface proteins that are consistently overexpressed in HRS cells, such as NRXN3 and LRP8, which can potentially be used as alternative targets for antibody–drug conjugates or CAR T cells. Finally, we identified potential interactions by which HRS cells inhibit T cells, among which are the galectin‐1/CD69 and HLA‐II/LAG3 interactions. RNAscope was used to validate the enrichment of CD69 and LAG3 expression on T cells near HRS cells and indicated large variability of the interaction strength with the corresponding ligands between patients and between tumor tissue regions. In conclusion, this study identifies new potential therapeutic targets for cHL and highlights the importance of studying heterogeneity when identifying therapy targets, specifically those that target tumor‐immune cell interactions.

Alzheimer's disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals-16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-β precursor protein processing, amyloid-β aggregation, lipid metabolism and microglial function in AD.

Eén op de drie ouderen herstelt onvoldoende na opname in het ziekenhuis. Er liggen veel kansen om met innovatieve interventies de geriatrische revalidatie slimmer vorm te geven. Dit is noodzakelijk gezien de toenemende vergrijzing, nijpende personeelstekorten en de wens om ouderen langer zelfstandig thuis te laten wonen. Geriatrische revalidatie moet daarom slimmer worden vormgegeven, onder meer door innovatieve interventies. Tijdens de lectorale rede op 7 maart j.l. is een toelichting gegeven op de onderzoekslijn behorende bij het bijzonder lectoraat Geriatrische Revalidatie ingesteld door Omring bij Inholland. Met als titel Slimmer én samen op weg naar toekomstbestendige zorg.

Abstract Background Visuospatial neglect (VSN) is a cognitive disorder after stroke in which patients fail to consciously process and interact with contralesional stimuli. Visual Scanning Training (VST) is the recommended treatment in clinical guidelines. At the moment, several mixed reality versions of Visual Scanning Training (VST) are being developed. The aim of this study was to explore the opinions of end-users (i.e., therapists) on the use of Virtual Reality (VR) and Augmented Reality (AR) in VSN treatment. Methods Therapists played one VR and two AR Serious Games, and subsequently filled out a questionnaire on User Experience, Usability, and Implementation. Results Sixteen therapists (psychologists, occupational, speech, and physiotherapists) played the games, thirteen of them evaluated the games. Therapists saw great potential in all three games, yet there was room for improvement on the level of usability, especially for tailoring the games to the patient’s needs. Therapists’ opinions were comparable between VR and AR Serious Games. For implementation, therapists stressed the urgency of clear guidelines and instructions. Discussion Even though VR/AR technology is promising for VSN treatment, there is no one-size-fits-all applicability. It may thus be crucial to move towards a plethora of training environments rather than a single standardized mixed reality neglect treatment. Conclusion As therapists see the potential value of mixed reality, it remains important to investigate the efficacy of AR and VR training tools.

Objective: Persons with high-risk for severe COVID-19 illness require special attention when considering university operations during the novel coronavirus pandemic. The objective of this study was to determine the number of students who fall within a high-risk category according to Centers for Disease Control and Prevention (CDC) guidelines using linked databases. Participants: Students enrolled at a large public University and who accessed the student health center between 2016 and 2020. Methods: Clinical data were linked with both university student enrollment and disability services databases to provide a comprehensive, de-identified dataset of students at higher medical risk of severe COVID-19 illness. Results: 1902 unique students (14% of the student health center population) were identified as having one or more high-risk condition. Conclusions: Utilizing a large and longitudinally linked student database provides universities with valuable information to make critical administrative decisions about how best to accommodate high-risk students to reduce their medical risk when returning to in-person instruction.

Genome-wide association studies (GWAS) have been highly informative in discovering disease-associated loci but are not designed to capture all structural variations in the human genome. Using long-read sequencing data, we discovered widespread structural variation within SINE-VNTR-Alu (SVA) elements, a class of great ape-specific transposable elements with gene-regulatory roles, which represents a major source of structural variability in the human population. We highlight the presence of structurally variable SVAs (SV-SVAs) in neurological disease-associated loci, and we further associate SV-SVAs to disease-associated SNPs and differential gene expression using luciferase assays and expression quantitative trait loci data. Finally, we genetically deleted SV-SVAs in the BIN1 and CD2AP Alzheimer's disease-associated risk loci and in the BCKDK Parkinson's disease-associated risk locus and assessed multiple aspects of their gene-regulatory influence in a human neuronal context. Together, this study reveals a novel layer of genetic variation in transposable elements that may contribute to identification of the structural variants that are the actual drivers of disease associations of GWAS loci.

Abstract Background Recovery after surgery intersects physical, psychological, and social domains. In this study we aim to assess the feasibility and usability of a mobile health application called PositiveTrends to track recovery in these domains amongst participants undergoing hip, knee arthroplasty or spine surgery. Our secondary aim was to generate procedure-specific, recovery trajectories within the pain and medication, psycho-social and patient-reported outcomes domain. Methods Prospective, observational study in participants greater than eighteen years of age. Data was collected prior to and up to one hundred and eighty days after completion of surgery within the three domains using PositiveTrends. Feasibility was assessed using participant response rates from the PositiveTrends app. Usability was assessed quantitatively using the System Usability Scale. Heat maps and effect plots were used to visualize multi-domain recovery trajectories. Generalized linear mixed effects models were used to estimate the change in the outcomes over time. Results Forty-two participants were enrolled over a four-month recruitment period. Proportion of app responses was highest for participants who underwent spine surgery (median = 78, range = 36–100), followed by those who underwent knee arthroplasty (median = 72, range = 12–100), and hip arthroplasty (median = 62, range = 12–98). System Usability Scale mean score was 82 ± 16 at 180 days postoperatively. Function improved by 8 and 6.4 points per month after hip and knee arthroplasty, respectively. In spine participants, the Oswestry Disability Index decreased by 1.4 points per month. Mood improved in all three cohorts, however stress levels remained elevated in spine participants. Pain decreased by 0.16 (95% Confidence Interval: 0.13–0.20, p

Objectives: We sought to characterize the SSRIs exposures reported to the U.S. National Poison Data System (NPDS).Background: More than 20 million antidepressants were prescribed between October and December 2020, a significant increase compared to the same months in the prior year. In 2017, a selective serotonin reuptake inhibitors (SSRIs) was mentioned in 57,254 single-substance toxic exposures reported to United States poison centers (PCs).Methods: The NPDS was queried for all human exposures to SSRIs reported to the U.S. Poison Centers (PCs) between 2015 and 2020. We descriptively assessed the demographic and clinical characteristics. Calls from acute care hospitals and hospital based EDs (ACH) were studied as a subgroup. Trends in SSRI exposures were analyzed using Poisson regression with percent changes being reported.Results: There were 346,082 SSRI exposure calls made to the PCs from 2015 to 2020, with the number of calls increasing from 51,791 to 62,504 during the study period. Single substance exposures accounted for 45.5% of such SSRI exposures. Of the total SSRI calls, the proportion of calls from acute care hospitals and EDs decreased from 56.2% to 53.2% from 2015 to 2020. Multiple substance exposures accounted for 65.5% of the overall SSRI calls from acute care hospitals and EDs. Approximately 15% of the patients reporting SSRI exposures were admitted to the critical care unit (CCU), with 18.8% patients admitted to a psychiatric unit. Residence was the most common site of exposure (94.2%), and 63.9% of these cases were enroute to the hospital via EMS when the PC was notified. Among the patients, 66.7% were male, with individuals between ages 13 and 19 years (31%) predominantly reported SSRI exposures. Suspected suicides (58.5%) and therapeutic errors (18.6%) were commonly observed reasons for exposure, with the former accounting for 83% cases reported by ACH. Major effects were seen in 3.7% cases and the case fatality rate for SSRI was 0.3%. Sertraline was the most commonly observed SSRI (23.6%). The most frequently co-occurring substances associated with the cases were atypical antipsychotics (9.3%) and benzodiazepines (8%). Tachycardia (19.7%) and drowsiness/lethargy (15.6%) were commonly observed clinical effects. During the study period, the frequency of SSRI exposures increased by 19.9% (95% CI: 16.2%, 22.7%; p

Objectives: The objective of the study is to characterize the risk markers of oxycodone misuse using the nationally representative National Survey of Drug Use and Health (NSDUH) data.Background: Drug overdoses continues to be a public health crisis with 70,630 fatalities in 2019. Approximately two-thirds of these deaths (66%) involved a prescription or illicit opioid. Synthetic opioids accounted for 72.9% of opioid-involved overdose deaths in 2019.Methods: The 2019 NSDUH public use cross-sectional data were analyzed. The respondents were classified into two groups, past year oxycodone misusers and non-misusers, based on the screening questions assessing past year misuse of oxycodone products. The prevalence of selected demographic, clinical factors and substance use and abuse, including prescription medications, was assessed descriptively for the two population groups using cross tabulated frequencies and chi-square tests. Logistic regression models using a backward selection process were used to identify predictors of oxycodone misuse adjusting for covariates. Adjusted odds ratios (OR) and corresponding 95% Confidence Intervals (CI) were calculated.Results: Overall, the 2019 NSDUH survey comprised of 56,136 respondents, of which 4,359 respondents (7.7%) reported using oxycodone products over the last year. Furthermore, 770 respondents reported misuse, accounting for 17.6% of the total oxycodone users or 1.4% of the survey sample. The proportion of past year oxycodone misusers was higher in males (54.1% vs 44.6%, p

Abstract Background Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. Methods We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and “fuses” evidence from RNA-seq and whole genome sequencing (WGS) using intron–exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. Results In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. Conclusions Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making.

Abstract Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Abstract Background The rs72824905 single-nucleotide polymorphism in the PLCG2 gene, encoding the p.P522R residue change in Phospholipase C gamma 2 (PLCγ2), associates with protection against several dementia subtypes and with increased likelihood of longevity. Cell lines and animal models indicated that p.P522R is a functional hypermorph. We aimed to confirm this in human circulating peripheral immune cells. Methods We compared effects of p.P522R on immune system function between carriers and non-carriers (aged 59-103y), using in-depth immunophenotyping, functional B-cell and myeloid cell assays, and in vivo SARS-CoV-2 vaccination. Results In line with expectations, p.P522R impacts immune cell function only slightly, but it does so across a wide array of immune cell types. Upon B-cell stimulation, we observed increased PLCγ2 phosphorylation and calcium release, suggesting increased B-cell sensitivity upon antigen recognition. Further, p.P522R-carriers had higher numbers of CD20++CD21-CD24+ naive B cells and IgG1+ memory B cells. In myeloid cells, normalized ROS production was higher upon PLCγ2-dependent stimulation. On classical monocytes, CD33 levels were elevated. Furthermore, carriers expressed lower levels of allergy-related FcεRI on several immune cell subsets. Nevertheless, carriers and non-carriers had similar serological responses to SARS-CoV-2 vaccination. Conclusion The immune system from p.P522R-carriers is slightly more responsive to stimulation than in non-carriers.

Aims: This study compares the incidence and risk-markers of student alcohol intoxication-related emergency department (ED) visits and alcohol-related incidents reported to student affairs. Methods: Enrollment data were linked to ED visits with alcohol intoxication and to alcohol-related incidents reported to campus authorities within one year following the first (index) annual enrollment. Incidence, annual trends and associated risk markers were analyzed. Results: The cohort consisted of 204,423 students aged 16-49. Incidence rates of first ED visit with alcohol intoxication and alcohol-related incidents were 59/10,000 and 311/10,000 person-years, respectively. Both outcomes shared common risk-markers including age, gender, race/ethnicity, fraternity and sorority (FSL) membership, an existing diagnosis of depression, etc. Being an athlete was associated with a lower risk of alcohol-related ED visits, and transfer students were at lower risk for alcohol-related incidents. Conclusion: Linking enrollment data with hazardous drinking events can help in better monitoring of student hazardous drinking and targeting interventions.

Mutations in PPARγ lead to lipodystrophy, but the mechanisms by which the mutations affect the activity in chromatin is unknown. Here, Madsen, Broekema et al. showed that mutations affecting two intermolecular interactions compromise chromatin remodeling.

Male subjects in animal and human studies are disproportionately used for toxicological testing. This discrepancy is evidenced in clinical medicine where females are more likely than males to experience liver-related adverse events in response to xenobiotics. While previous work has shown gene expression differences between the sexes, there is a lack of systems-level approaches to understand the direct clinical impact of these differences. Here, we integrate gene expression data with metabolic network models to characterize the impact of transcriptional changes of metabolic genes in the context of sex differences and drug treatment. We used Tasks Inferred from Differential Expression (TIDEs), a reaction-centric approach to analyzing differences in gene expression, to discover that several metabolic pathways exhibit sex differences including glycolysis, fatty acid metabolism, nucleotide metabolism, and xenobiotics metabolism. When TIDEs is used to compare expression differences in treated and untreated hepatocytes, we find several subsystems with differential expression overlap with the sex-altered pathways such as fatty acid metabolism, purine and pyrimidine metabolism, and xenobiotics metabolism. Finally, using sex-specific transcriptomic data, we create individual and averaged male and female liver models and find differences in the pentose phosphate pathway and other metabolic pathways. These results suggest potential sex differences in the contribution of the pentose phosphate pathway to oxidative stress, and we recommend further research into how these reactions respond to hepatotoxic pharmaceuticals.

IntroductionMetastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. MethodsHere, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and in a metastatic xenograft mouse model.ResultsOur results show that NK-92/5.28.z cells effectively kill RMS cells in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors.DiscussionThese findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS.

Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of fcp1 mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in fcp1 and cdk8Δ mutants and was required for the suppression of fcp1 mutant growth defects by loss of CDK8 under oxidative stress conditions. Targeted analysis revealed that mutating FCP1 decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes (TRX2 and TSA1) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of TRX2 and TSA1 observed in the fcp1 mutant. Interestingly, loss of CDK8 robustly normalized the mRNA levels of Skn7-dependent genes in the fcp1 mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of CDK8 could overcome transcriptional and/or posttranscriptional alterations in the fcp1 mutant through its regulatory effect on Skn7. Furthermore, our work also implicated FCP1 and CDK8 in the broader response to environmental stressors in yeast.

Human longevity is heritable, but genome-wide association (GWA) studies have had limited success. Here, we perform two meta-analyses of GWA studies of a rigorous longevity phenotype definition including 11,262/3484 cases surviving at or beyond the age corresponding to the 90th/99th survival percentile, respectively, and 25,483 controls whose age at death or at last contact was at or below the age corresponding to the 60th survival percentile. Consistent with previous reports, rs429358 (apolipoprotein E (ApoE) ε4) is associated with lower odds of surviving to the 90th and 99th percentile age, while rs7412 (ApoE ε2) shows the opposite. Moreover, rs7676745, located near GPR78, associates with lower odds of surviving to the 90th percentile age. Gene-level association analysis reveals a role for tissue-specific expression of multiple genes in longevity. Finally, genetic correlation of the longevity GWA results with that of several disease-related phenotypes points to a shared genetic architecture between health and longevity.

Abstract Rhabdomyosarcomas (RMS) are mesenchyme‐derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high‐risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4–8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.