Male MS-NASH mice were maintained on a high-fat diet for 16 weeks with and without red algae-derived minerals. Obeticholic acid (OCA) was used as a comparator in the same strain and diet. C57BL/6 mice maintained on a standard (low-fat) rodent chow diet were used as a control. At the end of the in-life portion of the study, body weight, liver weight, liver enzyme levels and liver histology were assessed. Samples obtained from individual livers were subjected to Tandem Mass Tag labeling / mass spectroscopy for protein profile determination. As compared to mice maintained on the low-fat diet, all high-fat-fed mice had increased whole-body and liver weight, increased liver enzyme (aminotransferases) levels and widespread steatosis / ballooning hepatocyte degeneration. Histological evidence for liver inflammation and collagen deposition was also present, but changes were to a lesser extent. A moderate reduction in ballooning degeneration and collagen deposition was observed with mineral supplementation. Control mice on the high-fat diet alone demonstrated multiple protein changes associated with dysregulated fat and carbohydrate metabolism, lipotoxicity and oxidative stress. Cholesterol metabolism and bile acid formation were especially sensitive to diet. In mice receiving multi-mineral supplementation along with the high-fat diet, there was reduced liver toxicity as evidenced by a decrease in levels of several cytochrome P450 enzymes and other oxidant-generating moieties. Additionally, elevated expression of several keratins was also detected in mineral-supplemented mice. The protein changes observed with mineral supplementation were not seen with OCA. Our previous studies have shown that mice maintained on a high-fat diet for up to 18 months develop end-stage liver injury including hepatocellular carcinoma. Mineral-supplemented mice were substantially protected against tumor formation and other end-state consequences of high-fat feeding. The present study identifies early (16-week) protein changes occurring in the livers of the high-fat diet-fed mice, and how the expression of these proteins is influenced by mineral supplementation. These findings help elucidate early protein changes that contribute to end-stage liver injury and potential mechanisms by which dietary minerals may mitigate such damage. Non-alcoholic fatty liver disease (NAFLD) is rapidly becoming the most common cause of liver injury in Western society and throughout the world –3. It isThe high content of saturated fat and sugar in the typical Western diet is thought to underlie fat accumulation –8. ExperIn our own previous studies , 21, C57While an extended feeding period is essential to see the full spectrum of liver disease develop in rodents, long-term studies are not optimal for identifying the early events that lead to steatosis and disease progression in high-fat-fed mice. Likewise, long-term studies are not ideal for understanding how dietary minerals (or other interventions) might mitigate disease progression. As a way to help understand the early events that drive liver disease progression and its prevention, cohorts of mice (MS-NASH strain) were placed on a high-fat diet with fructose added to the drinking water (high-fat diet) with and without mineral supplementation (Aquamin®) for a period of 16 weeks. An additional cohort of high-fat diet mice was treated with a comparator, obeticholic acid (OCA), a drug that acts as a Farnesoid X Receptor (FXR) agonist, that has been shown to reduce liver damage in both humans –25 and mDiet D12079B  with 5% fructose added to the drinking water was used as the high-fat Western diet for this study. This diet was designed to provide 500 gm/kg of carbohydrates, 198 gm/kg of proteins and 210 gm/kg of fat. The percentage of calories from carbohydrates, proteins and fat in this diet was 43, 17, and 40%, respectively. This diet also contained 39 gm/kg of a standard mineral mix (S10001A) containing calcium carbonate and calcium phosphate and other essential trace elements. Diet 5,008 (Purina) was used as the low-fat control diet. The caloric distribution from this low-fat diet was approximately 57, 27, and 16% from carbohydrates, proteins and fat, respectively. Diet 5,008 was supplemented with standard minerals (2.5%) as part of the diet formula. High-fat diet alone was considered as the control for comparison with the low-fat diet or with Aquamin® and OCA as interventions in the high-fat diet.(ICP-OES). Aquamin® is a product rich in calcium, magnesium, and trace elements, obtained from the skeletal remains of red marine algae  Marigot–30], andObeticholic acid (OCA) was prepared as a 3 mg/mL suspension in 0.5% methylcellulose. Animals received 0.25 mL OCA (approximately 30 mg/kg) by oral gavage five times per week beginning on week-8 of high-fat feeding.ad libitum. The animal environment was maintained at 70–74°F with a 12-h light-dark cycle. During the in-life portion of the study, health-checks were done daily. At termination (after 16 weeks on diet), animals were euthanized by carbon dioxide asphyxiation. The entire in-life portion of the study and termination (euthanasia and tissue harvesting) was carried out by Crown Bio, Inc.  under the approved Standard Operating Procedures at the testing site. All procedures involving live animals were approved by the Institutional Animal Care and Use Committee (IACUC). Crown Bio, Inc., is an AAALAC-accredited institution.The study was conducted in a mouse model - MS-NASH mouse  by) by34)])At euthanasia, blood was obtained by cardiac puncture and used to assess liver enzyme  levels as a measure of injury and triglyceride (TG) levels as an indicator of metabolic function. Livers were removed and weighed. After gross examination, a piece of tissue from the left lobe was immediately frozen in liquid nitrogen and used for proteomic analysis. Another tissue piece from the left lobe was fixed in 10% buffered formalin and used for histology.Serum levels of AST, ALT and triglycerides were assessed using a Beckman chemical analyzer. All blood chemistry measurements were made following a standard operating procedure at Crown Bioscience.Formalin-fixed liver tissue samples were processed for histology and 3–4 μm-thick sections were stained with hematoxylin and eosin (H&E) and picrosirius red (PSR), independently. H&E-stained tissue sections were evaluated under light microscopy for evidence of steatosis, ballooning hepatocyte degeneration and inflammation using the Kleiner scoring system . KleinerAfter microscopic evaluation, slides were digitized using the Aperio AT2 whole slide scanner (Leica Biosystems) at 40x with a resolution of 0.5 μm per pixel with a 20x objective. The scanned images were housed on a server and accessed using Leica Aperio eSlide Manager (Version 12.3.2.5030), a digital pathology management software. These digitized histological sections were viewed and analyzed using Aperio ImageScope (Version 12.3.3.5048), a slide viewing software by Leica.UniProt.org, and Reactome V78 (reactome.org) was used for pathway enrichment analyses (Mus musculus. STRING database-v11 (string-db.org) was used to detect protein-protein interactions and additional enrichment analyses provide information related to cellular components, molecular functions, and biological processes by Gene Ontology (GO) annotation. It also offered WikiPathways and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases to curate pathways. Only proteins with a ≤ 2% FDR confidence of detection were included in the analyses. The individual differential protein expression was established by calculating the abundance ratio of normalized abundances of each intervention sample to the high-fat alone samples. For the group analysis, individual data points were merged by groups to obtain means and standard deviations. The initial analysis was an unbiased search of differentially-expressed proteins and significantly altered proteins. Subsequently, we searched the database, specifically, for changes in the expression of keratins and other differentiation-related proteins. Mass spectrometry-based proteomics data were placed in a data repository known as ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD030954.Proteomic experiments were carried out in the Proteomics Resource Facility (PRF), a core laboratory in the Department of Pathology at the University of Michigan. For protein mass spectrometry analysis, we employed liver tissue from five mice from each group and assessed each separately. A cryopreserved tissue piece from the left lobe (of each mouse liver) was weighed and homogenized in Radioimmuno-Precipitation Assay (RIPA) - lysis and extraction buffer  for protein isolation, as described in our previous publications –40. Fiftanalyses  for spect-test (two-tailed) for comparison using GraphPad Prism, version 9. Pathways enrichment data reflect Reactome-generated p-values based on the number of entities identified in a given pathway as compared to total proteins responsible for that pathway. The significance  is calculated by the overrepresentation analysis (hypergeometric distribution). For STRING enrichment analysis, the whole genome statistical background was assumed and FDR stringency was ≤ 1%. Data were considered significant at p < 0.05.Group means and standard deviations were obtained for discrete gross and histochemical features as well as for individual biochemical values  and individual protein values obtained in the proteomic analysis. Data generated in this way were analyzed by analysis of variance (ANOVA) followed by unpaired Animal weights and liver weights were assessed at euthanasia . As expeSections of formalin-fixed liver tissue were stained with hematoxylin and eosin and evaluated for steatosis, ballooning hepatocyte degeneration and inflammation. Additional sections were stained with PSR and assessed for collagen deposition. Data and histological representations of all four groups are shown in The number of upregulated and downregulated proteins in C57BL/6 mice on the low-fat diet (relative to high-fat control mice) and the number of up- and downregulated proteins with Aquamin® or OCA in comparison to the high-fat control mice is presented as Venn diagrams  show thep < 0.05 are shown in blue while downregulated proteins are shown in red. The bold colors (blue and red) indicate proteins that were statistically different and also at least 1.5-fold up- or downregulated.p < 0.05 and at least 1.5-fold increase) in response to Aquamin®. lipid metabolism and trafficking of lipids  including three  that were among the most highly downregulated  gene is considered a tumor suppressor and its loss can contribute to liver injury  with, presumably, different mechanisms of action were also separately evaluated.In comparison with low-fat-fed control mice, livers were substantially enlarged in high-fat mice. However, there was no gross evidence of more serious liver damage  at the 16-week time-point. At the histological level, steatosis was widespread in virtually every mouse on the high-fat diet (not observed in low-fat controls). Ballooning hepatocyte degeneration was also prominent, but inflammation and collagen deposition were minimal. These effects were accompanied by an increase in serum triglycerides and an increase in serum ALT and AST levels. Consistent with previous findings in both rat  and mousIn contrast to the substantial differences distinguishing low-fat and high-fat feeding, mineral supplementation - along with the high-fat diet - had no effect on whole-body weight, gross liver weight or steatosis. Likewise, there was no improvement in serum triglycerides and no decrease in AST and ALT values as compared to what was seen in high-fat control mice. This is consistent with findings reported earlier that mineral supplementation does not prevent steatosis from developing , 21 and The protein profile observed in response to intervention with Aquamin® supports this view. While a select few proteins involved directly in fat metabolism / lipid transport were modulated with Aquamin®, the most striking Aquamin®-induced protein changes relate to decreased amino acid metabolism as well as a reduction in purine and pyrimidine catabolism. Also prominent was the increased expression of moieties that are part of the epithelial cell differentiation process. Reactome pathway analysis identified keratinization, cornified envelope formation, hemidesmosome formation, junction formation and gap junction assembly as significantly altered pathways. Proteins involved in microtubule assembly were also upregulated in response to Aquamin® and several pathways related to protein trafficking within the cell or between interior and cell surface were identified.per se, was not addressed in these earlier studies, reduced oxidative stress and reduced tumor formation are, certainly, consistent.How the protein changes observed in response to Aquamin® in the current study and the reduction in liver tumor formation seen previously in our earlier long-term studies , 21 are Aquamin®-induced epithelial cell differentiation provides another potential mechanism to counteract cancer-promotion. A wide range of empirical information supports this view. Epidemiological studies have clearly demonstrated the inverse relationship between differentiation in epithelial cells and tumor incidence in many tissues including those of the gastrointestinal tract –66. AnimIn both Aquamin®-treated animals and low-fat-fed mice, the strong keratin upregulation reflected a response in a subset of animals. Why only some animals demonstrated this protein signature is not known, but the finding raises an interesting question: Does the variability in response from animal-to-animal help explain why virtually every mouse fed a high-fat diet rapidly develops steatosis but only a subset progresses to more serious disease? This question cannot be addressed with the presently available data but we have recently initiated a large linear study in which liver biopsies are obtained from mice on a high-fat diet after 16-weeks on diet (as in the present study) following which the animals are maintained for a total of 18-months in order to determine if there is an inverse correlation between keratin expression at the early time-point and tumor formation later.Although the focus of this study was on mineral supplementation, we also included OCA as a separate intervention for comparison. OCA has been shown to inhibit features of NAFLD / NASH in human patients –25 and iNAFLD is a rising public health challenge. Currently, public health measures focus on “lifestyle” changes, especially diet, to prevent the development of steatosis. Such an approach will not work with everyone. Perhaps efforts directed at reducing the downstream consequences of fatty liver infiltration would be of value. It should be noted in this regard that a majority of individuals in Western society do not meet USDA daily intake guidelines for calcium , 81. ManIn summary, our past studies have clearly demonstrated the importance of adequate mineral intake for preventing the downstream consequences of fatty liver accumulation in a murine model (as summarized in the graphic cartoon – All relevant data are within the manuscript and its Supporting Information files. The mass spectrometry proteomics data are available on ProteomeXchange Consortium (PRIDE partner repository) – dataset identifier PXD030954.The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). Crown Bio, Inc., is an American Association for Accreditation of Laboratory Animal Care (AAALAC)-Accredited Institution.All listed authors have contributed toward this project to attain authorship status according to ICMJE criteria. Their specific contributions are as follows: MA and JV: conceptualization, methodology, resources, writing—original draft preparation, supervision, and project administration. MA: software and visualization. MA, SM, RK, and JV: validation. MA, SM, RK, IH, DZ, and MJ-M: formal analysis and data curation. MA, SM, RK, IH, DZ, MJ-M, MA, and JV: investigation and writing—review and editing. MA and JV: funding acquisition. All authors have read and agreed to this version of the manuscript.This study was funded by NIH grant CA201782 including supplemental funding through the Office of Dietary Supplements to JV and funding from the American Society for Investigative Pathology (ASIP) Summer Research Opportunity Program in Pathology (SROPP) to MA.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


While it is known that poor sleep is a seizure precipitant, this association remains poorly quantified. This study investigated whether seizures are preceded by significant changes in sleep efficiency as measured by a wearable equipped with an electrocardiogram, respiratory bands, and an accelerometer.Nocturnal recordings from 47 people with epilepsy hospitalized at our epilepsy monitoring unit were analyzed (304 nights). Sleep metrics during nights followed by epileptic seizures (24 h post-awakening) were compared to those of nights which were not.p < 0.05). Each standard deviation decrease in sleep efficiency and increase in wake after sleep onset was respectively associated with a 1.25-fold  and 1.49-fold  increased odds of seizure occurrence the following day. Furthermore, nocturnal seizures were associated with significantly lower sleep efficiency and higher wake after sleep onset (p < 0.05), as well as increased odds of seizure occurrence following wake .Lower sleep efficiency (percentage of sleep during the night) was found in the nights preceding seizure days (Findings indicate lower sleep efficiency during nights preceding seizures, suggesting that wearable sensors could be promising tools for sleep-based seizure-day forecasting in people with epilepsy. Epilepsy is a chronic neurological condition which affects about 60 million people worldwide and is characterized by a predisposition to spontaneous and recurrent epileptic seizures . It has Monitoring sleep could possibly improve the management of seizures for people with epilepsy (PWE) , 5, 6. FCarré Technologies Inc.). The Hexoskin sleep analysis algorithm, which classifies signals into wake and sleep, was developed and validated on healthy young adults  which combines scalp-EEG, electro-oculography, electromyography, and respiration recordings for the detailed analysis of sleep macrostructure. PSG is currently the most accurate sleep staging technique and the gold standard for short-term sleep-disorder diagnosis in dedicated sleep clinics . Howeverg adults . This weIn this study, we used the Hexoskin smart shirt to monitor nocturnal sleep in a large cohort of PWE in a controlled clinical setting. We aimed to assess the feasibility of monitoring sleep quality in PWE using a novel smart shirt and to evaluate whether wearable-measured changes in nocturnal sleep quality are associated with the occurrence of seizures within the 24 h following wake.n = 9 days, range = ] out of 11.8 days at the EMU . The shirts were removed and replaced with clean ones when patients took a shower. Patients underwent simultaneous video-EEG monitoring with the Nihon Kohden system which allowed for all seizures to be accurately annotated by neurologists. A total of 393 days of continuous smart-shirt data from 53 patients, with a confirmed diagnosis of epilepsy, were recorded. Six patients who had seizures every day/night were excluded to avoid reporting overly optimistic results. Recordings from 47 patients  were analyzed. Both patients with focal (43/47) and generalized (4/47) epilepsy were recruited and included in the study. For patients with focal epilepsy, the suspected localization of the epileptic focus is provided in the Patients admitted to the University of Montreal Hospital Center Epilepsy Monitoring Unit (EMU) (April 2019 to March 2021) were recruited on the day of their admission to wear the smart shirt during their hospitalization. For most patients, the Hexoskin smart shirt was installed at the beginning of their hospitalization and removed prior to their discharge by research personnel (median starting day: day 2 following EMU admission). On average, patients wore the Hexoskin smart shirt for 9.2 days [mediaCarré Technologies Inc.), a garment embedded with a single-lead ECG (256 Hz) and two  inductive respiratory bands (128 Hz each). It also includes a detachable telemetry device which holds a three-axis accelerometer (64 Hz) and collects, preprocesses, and transmits all recordings to the Hexoskin Connected Health Platform. The smart shirt sensors as well as an example of raw acquired recordings are shown in Patients wore the Hexoskin smart shirt (lead ECG 56 Hz andSeizures were annotated by epileptologists based on video-EEG recordings (blinded to the Hexoskin data). Each night was then labeled as either “pre-seizure” if it was followed by at least one epileptic seizure in the 24-h post-awakening  or “non-pre-seizure” if it was not followed by any epileptic seizure in the 24-h post-awakening. The presence or absence of nocturnal seizures was also noted as a dichotomous variable. A detailed description of the data inclusion criteria is provided in For all patients, in accordance with standard sleep analysis common practices, the first night at the EMU was excluded to avoid contaminating the data with recordings during the adaptation period. The first night of sleep analysis is often accompanied by lower sleep efficiency due to a combination of factors such as a change in environment and stress . MoreoveThe Hexoskin sleep algorithm used was previously developed and validated in young healthy adults. The algorithm analyzes multimodal recordings to classify continuous, non-overlapping 20-second segments as either sleep or wake with 90.8% agreement with standard PSG recordings . The HexThe four sleep metrics which were analyzed in this study are the following: total sleep duration (hours), sleep latency (minutes), WASO (minutes), and sleep efficiency (%).t-tests or Wilcoxon rank-sum tests were then used according to the distribution of the data. Bonferroni correction was applied to account for multiple comparisons with an adjusted alpha level of 0.0125  are preceded by significantly lower quality of sleep: decreases in sleep efficiency or total sleep duration and increases in sleep latency or WASO. One tailed Welch f 0.0125 . LogistiThree sub-analyses were conducted between pre-seizure and non-pre-seizure nights. The first included all nights , corresponding to 80 pre-seizure nights and 224 non-pre-seizure nights. This analysis was conducted to evaluate the existence of an overall significant difference in sleep metrics during nocturnal sleep in the eve of a clinical seizure. To assess the possible effect of nocturnal seizures during sleep, the second analysis compared sleep metrics between pre-seizure nights and non-pre-seizure nights including only nights without a nocturnal seizure (261 nights), corresponding to 54 pre-seizure nights and 207 non-pre-seizure nights. Similarly, the third analysis included only nights with the presence of at least one nocturnal seizure (43 nights), corresponding to 26 pre-seizure nights and 17 non-pre-seizure nights.Due to the direct link between the occurrence of clinical seizures during sleep and sleep quality, a sub-analysis to study the effect of nocturnal seizures on sleep metrics was also performed. The four sleep metrics of all nights with at least one nocturnal seizure (43) were compared to nights without a nocturnal seizure (261) regardless of whether the night was pre-seizure or non-pre-seizure. The relationship between the presence of nocturnal seizures and the night being a pre-seizure night was also analyzed using a Chi-Square test of independence.In the EMU, patients' anti-seizure medications are often reduced or stopped. There were no medication changes prior to EMU admission. Reduction in anti-seizure medication is known to directly  , 19 and Of the 53 patients recruited, 47 were included for analysis. During their hospitalization, 13 patients solely had focal to bilateral tonic-clonic seizures, 12 only had focal impaired awareness seizures, 11 only had focal aware seizures, 7 experienced mixed types of focal seizures, and 4 had generalized tonic-clonic seizures. Prior to analysis, 59 nights were excluded due to sleep deprivation, the first night at the EMU, significant daytime sleep (>4 h of continuous sleep), missing video-EEG recordings, and the smart shirt removed by the patient. A total of 304 nights  were included, according to the criteria depicted in n = 80) were statistically compared to those of all of non-pre-seizure nights (n = 224). Total sleep duration was analyzed with the Welch t-test. The other variables were analyzed with the Wilcoxon rank-sum test. Significantly lower sleep efficiency was observed in pre-seizure nights compared to non-pre-seizure nights  at a Bonferroni-corrected alpha of 0.0125. Increased time in WASO during pre-seizure nights  was significant at 95% confidence only before correcting for multiple comparisons. Distributions of these sleep metrics are shown in p < 0.001).First, the four sleep metrics of all pre-seizure nights (p = 0.033). The reduced sample sizes resulted in low statistical power for these comparisons which may have caused any existing differences in other sleep metrics to not be captured. Average values of the metrics per group as well as p values are detailed in Pre-seizure and non-pre-seizure sleep metrics were then compared during nights with and without one or more nocturnal clinical seizure separately to evaluate if the presence of nocturnal seizures during sleep was a confounding factor. No significant difference in sleep metrics between pre-seizure and non-pre-seizure nights was found. However, before correcting for multiple comparisons, sleep efficiency was significantly lower for pre-seizure nights than non-pre-seizure nights without a nocturnal seizure at 95% confidence  were compared to all of the nights without a nocturnal seizure (n = 261), regardless of whether or not the nights were pre-seizure or non-pre-seizure, using the Wilcoxon rank-sum test. Significantly lower sleep efficiency was observed in nights with nocturnal seizures than nights without . Nights with nocturnal seizures also showed significantly greater WASO .Since the occurrence of nocturnal seizures was significantly greater in pre-seizure nights according to the Chi-Square test described in Section 3.2.1, the effect of nocturnal seizures on sleep metrics was assessed in a Distributions of significant metrics, illustrated in p = 0.02). Each standard deviation increase in time in WASO (19.96 min) was associated with a 1.49-fold increase in the odds of seizure occurrence in the 24 h following wake . Given the high occurrence of nocturnal seizures during pre-seizure nights, the odds of seizures occurring after a night disrupted by one or more nocturnal seizures were also assessed. Interestingly, there was a 5.86-fold increased odds of seizure occurrence in the 24 h following wake associated with a nocturnal seizure occurring during sleep .Each standard deviation decrease in sleep efficiency (5.70%) was associated with a 1.25-fold increase in the odds of seizure occurrence in the 24 h following wake according to a logistic regression analysis . In addition, a thorough investigation of the reduction/discontinuation of other anti-seizure medication was conducted. For each patient, specific medications and dose changes are provided in Nineteen patients (40.42%) received lorazepam at least once during their hospitalization for various reasons . The 151 nights of patients who took lorazepam had on average an increased sleep efficiency of 0.73% as compared to the 153 nights of the 27 patients who did not (μ = 92.64 > 91.91%). This difference was not statistically significant according to the Wilcoxon rank-sum test  in the EMU. We evaluated the existence of lower sleep quality during nights followed by a seizure in the 24 h post-awakening. To our knowledge, this is the first study to perform sleep monitoring using a multimodal smart shirt in PWE to quantitatively evaluate changes in sleep quality preceding seizure occurrence in a clinical setting.p values  may be driving seizure occurrence. However, low sleep efficiency in the EMU is one of many seizure-provoking factors.In addition, albeit being statistically significant, the differences measured in sleep efficiency and WASO between pre-seizure and non-pre-seizure nights were relatively small. We surmise that these differences could be larger at home where sleep is not as ‘controlled' as it is in the EMU. In the near future, we believe that such metrics could be combined with other personalized seizure risk markers (for example multi day cycles of seizures and/or daily levels of stress) to enhance seizure day forecasting performances. In this study, simple metrics of sleep quality measured solely by a wearable were studied. Although these metrics were based on sleep-wake classifications previously validated with polysomnography , more coPresented results provide new insights regarding the complex relationship between sleep and seizure occurrence; nonetheless, several avenues of improvement exist. In the study by Dell et al. , 10 patiOur findings reveal lower sleep efficiency, measured by the Hexoskin smart shirt, during nights preceding seizures in a large cohort of patients with epilepsy. Increased wake during nocturnal sleep and the occurrence of seizures during nocturnal sleep could be early precursors of increased seizure odds the following day. These findings will guide future work on the development of probabilistic seizure-day forecasting based on long-term wearable sleep monitoring. Further studies looking at different seizure types, patient-specific baseline sleep macrostructure, and the effect of age and sex in a real-life setting would greatly improve our understanding of the bidirectional relationship between sleep and seizures.elie.bou.assi.chum@ssss.gouv.qc.ca.The datasets presented in this article are not readily available because sharing of raw data is conditional to approval by our Institution's Research Ethics Board. Requests to access the datasets should be directed to EB, This study involving human participants was reviewed and approved by the Research Ethics Board of the Centre de Recherche du Centre Hospitalier de L'Université de Montréal. The patients/participants provided their written informed consent to participate in this study.DN and EB are equally contributing principal investigators. LG and TD wrote the main manuscript text and performed the statistical analysis. LG, TD, DT, LB, MR, and EB performed the study design and data retrieval. TD, LG, and EB prepared the figures. All authors contributed to the article and approved the submitted version.


Metabolic alterations, particularly disorders of lipoprotein metabolism in COVID-19, may affect the course and outcome of the disease. This study aims at evaluating the lipoprotein profile and redox status in SARS-CoV-2 infected patients with different pneumonia severity and their association with lethal outcomes.  The prospective cohort study was performed on 98 COVID-19 patients with mild, moderate, and severe pneumonia. Lipid and inflammatory parameters, lipoprotein subclasses, and redox status biomarkers were determined at the study entry and after one week.  Compared to patients with mild and moderate pneumonia, severely ill patients had higher oxidised low-density lipoprotein (oxLDL) and malondialdehyde levels and lower high-density lipoprotein cholesterol (HDL-C) concentrations and paraoxonase 1 activity. Reduction in the proportion of large HDL 2a subclasses with a concomitant increase in the proportion of smallest HDL 3c and small dense LDL (sdLDL) particles was observed in patients with severe disease during the time. However, these changes were reversed in the mild and moderate groups. The results showed a positive association between changes in oxLDL and total antioxidative status. However, prooxidants and antioxidants in plasma were lower in patients with lethal outcomes.  Increased levels of oxLDL and sdLDL particles may contribute to the severity of COVID-19. The role of oxidative stress should be clarified in further studies, mainly its association with lethal outcomes. COVID-19 pandemic caused by SARS-CoV-2 virus still represents a central public health concern . It was Moreover, some studies reported increased prooxidants and biomarkers of lipid peroxidation in the blood of SARS-CoV-2 infected patients . This exThe prospective cohort study was performed at the Clinical Hospital Center, Dr Dragisa Misovic-Dedinje, from July 2020 to October 2021. It encompassed 98 COVID-19 patients with positive results on the real-time fluorescent RT-polymerase chain reaction performed according to WHO guidelines . For pat2−) and malondialdehyde (MDA) were measured using methods developed by Auclair and Voisin [Blood was sampled (the first time point) the day after CT scoring. Since the usual time for recovery from the progressive disease stage is seven days , blood wd Voisin  and Girod Voisin , respectd Voisin .U test with Bonferroni correction for subgroup differences (for skewed data) were used for mild, moderate, and severe group data comparison. Student's t-test and Mann–Whitney U test were used to test data differences between deceased and recovered patients. Categorical variables were tested by the Chi-square test. A two-way repeated measured ANOVA test was utilised to evaluate the effect of the time (changes in two repeated observations—the first and the second point) and the effect of time-outcome interaction for normally distributed data. We used Wilcoxon signed-rank to test whether the changes of skewed data in two repeated observations in deceased and recovered patients were statistically significant. Data are shown as mean ± standard deviation for normally distributed variables. The median for independent data or the median of difference for dependent data with interquartile range were presented for nonnormally distributed variables. Relative or absolute frequencies are shown for categorical variables. Moreover, to analyse whether the changes of examined parameters between the two-time points are interrelated, univariate and multivariate linear fixed-effect panel model regression was used. Cox regression was employed to model time to in-hospital death. The data were presented as hazard ratios with a 95% confidence interval (CI). The statistical analyses were performed with PASW® Statistic v. 27  software. A two-tailed p value ≤0.05 was considered to indicate statistical significance.Data distribution was tested using the Kolmogorov-Smirnov test. ANOVA with Tukey's post hoc test  and the Kruskal-Wallis with Mann–Whitney 2) and received high-flow oxygen therapy. One patient with moderate pneumonia and one-third of patients with severe pneumonia required intubation. Severely ill patients received anticoagulant therapy, including aspirin (100%), fraxiparine (82%), and corticosteroids (97%). Besides anticoagulants, some patients with mild and moderate pneumonia also received antiviral medication. All patients were supplemented by vitamins C and D.2− and antioxidants TAS, SOD, and PON1 levels. Additional differences were found between severe and mild pneumonia patients; those with severe pneumonia had higher IL-6 and PAB levels and lower total SH-groups.We further analysed changes of examined parameters after one week. The results demonstrated that TC, HDL-C, and LDL-C concentrations increased in all three groups. At the same time, we observed a reduction of HDL 2 subclasses proportion in all groups. A significant interaction between disease severity and time was demonstrated for the relative proportion of HDL 3c and HDL 2a subclasses as well as the proportion of sdLDL particles. The proportions of HDL 3c subclasses and sdLDL particles decreased in mild and moderate pneumonia patients but increased in those with severe disease. In contrast, the proportion of HDL 2a particles increased in patients with mild and moderate illness and declined in patients with severe pneumonia .The median of the difference between the two-time points revealed that PAB activity, IL-6, and CRP concentrations significantly decreased in severely and moderately ill patients. A significant reduction of total SH-groups and oxLDL concentrations was demonstrated in severely ill patients with a concomitant increase in TOS. In the patients with mild pneumonia, only SOD activity was decreased, and MDA was increased over time .2 was lower in deceased patients. Both prooxidants and antioxidants in plasma were lower in patients with lethal outcomes.During the follow-up period, 18 patients (20%) died. p = 0.05).Cox regression analysis was employed to analyse the association between the examined laboratory parameter levels in the first point and the risk of fatal outcomes. The results showed that the association between higher oxLDL concentrations and increased mortality  was borderline significant  could be explained by a probable rise in reducing blood components  which are capable of changing and neutralising prooxidants chemically. Mentioned blood reductants rise is well known in severely ill patients, sepsis and COVID-19 [Evidently, our patients with COVID-19 are in a state of severe immune system activation, which in turn causes oxidative stress burden. Olwal et al.  elaboratCOVID-19 , 30. HowCOVID-19 . Althoug2O2) [On the other hand, increase in oxLDL could be explained by PON1 protective role exhaustion documented here as a significant fall in this enzyme activity but also as a consequence of fast LDL lipid-hydroperoxides decomposition to their end products such as MDA and hydroxy-nonennal and other toxic aldehydes . The pro2O2) . Conside2O2) , it is r2O2) . In SARS2O2) . Moreove2O2) . Our stuAdditionally, changes in oxLDL were strongly influenced by alterations in antioxidant activity. The positive association between oxLDL and TAS changes throughout time suggests that prooxidants can stimulate total antioxidant capacity. On the other hand, an increase in antioxidant enzymes may be insufficient for cell and tissue protection . Yet, weSome limitations of our study should be taken into account when comparing our data with the results of other studies—the most significant being differences in ethnicity, age, and prevalence of comorbid conditions among studied COVID-19 patients. Also, the sampling time and follow-up period should be considered when comparing prognostic information based on lipid and lipoprotein data across different studies. It should also be acknowledged that the vast majority of our patients with severe COVID-19 received corticosteroid therapy, which might be responsible for adverse modulation of LDL and HDL subclasses distribution observed in this group during follow up. However, corticosteroid treatment is commonly associated with increased TG levels . Still, In conclusion, our study results indicated rapid changes in the lipid profile during the hospitalisation of COVID-19 patients, especially levels of oxLDL and sdLDL particles, which increase may contribute to the severity of the disease. Further studies should clarify the role of oxidative stress in the disease severity and prognosis.


Protein tyrosine phosphatases function in dephosphorylating target proteins to regulate signaling pathways that control a broad spectrum of fundamental physiological and pathological processes. Detailed knowledge concerning the roles of classical PTPs in human cancer merits in-depth investigation. We comprehensively analyzed the regulatory mechanisms and clinical relevance of classical PTPs in more than 9000 tumor patients across 33 types of cancer. The independent datasets and functional experiments were employed to validate our findings. We exhibited the extensive dysregulation of classical PTPs and constructed the gene regulatory network in human cancer. Moreover, we characterized the correlation of classical PTPs with both drug-resistant and drug-sensitive responses to anti-cancer drugs. To evaluate the PTP activity in cancer prognosis, we generated a PTPscore based on the expression and hazard ratio of classical PTPs. Our study highlights the notable role of classical PTPs in cancer biology and provides novel intelligence to improve potential therapeutic strategies based on pTyr regulation. Protein tyrosine phosphorylation is a dynamic and reversible post-translational modification regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). It is a fundamental mechanism for regulating innumerable biological processes , 2. SincThis study comprehensively characterized the molecular regulatory mechanisms and clinical relevance of classical PTPs in 33 types of cancer. We discovered that copy number variations (CNVs) are the predominant factor leading to abnormal expression of classical PTPs across cancer types. We further displayed the architecture and features of gene regulatory networks (GRN) by transcription factors (TFs) and microRNAs (miRNAs). In addition, we assessed the clinical prognosis for classical PTPs and constructed a rigorous model to evaluate the PTP activity in cancer. Finally, we explored the interaction network of classical PTPs with FDA-approved drugs and identified that classical PTPs are promising drug targets for improving therapeutic strategies.All human samples used in this study were collected from patients subjected to clinical surgery in the First affiliated hospital of Bengbu medical college. Before RNA isolation and protein extraction, samples were stored at -80 ℃. Collections of human samples were obtained and approved by the ethics committee of Bengbu medical college. Written informed consent was obtained from individual or guardian participants.https://gdac.broadinstitute.org/). GISTIC2 was employed to define the significant gain or loss in genomic regions  Research Network. We systematically analyzed 38 classical PTPs in 33 TCGA cancer projects. Every project indicates a particular cancer type. RNA-seq data for the transcriptional expression of 33 types of cancer were downloaded using the TCGAbiolinks R package . The CNV regions . The cli regions  or downl regions , the Gen regions , and the regions  were useThe omics data of cell lines, including gene expression profiles, somatic mutations, and CNVs, were acquired from the Broad Institute Cancer Cell Line Encyclopedia (CCLE) and the Genomics of Drug Sensitivity in Cancer databases (GDSC) , 16. TheTo avoid instability between tumor and normal samples in differential analyses caused by batch effect, we downloaded the gene expression profiles from the TCGA project, and the Genotype-Tissue Expression (GTEx) project that is re-computed by the UCSC Xena project depended on a defined pipeline. Thirty-eight differentially classical PTPs were acquired using the limma R package . The BH-Gene Set Variation Analysis (GSVA) was performed to compute the activity of 50 cancer hallmark-related pathways across 33 types of cancer. After that, the spearman correlation between the expression to classical PTPs and pathway activity was estimated. BH-adjusted p-valued< 0.05 and |Rs| > 0.3 were defined as significant.To identify the interactions between miRNA and target genes , we collected miRNAs from four target prediction databases: miRDB, miRTarBase, PITA, and TargetScan –23. The Next, we downloaded the promoter region sequence (from -1000 to +200 bp around the transcription start site) of target genes from the UCSC Table Brower depending on the human wgEncodeGencodeBasic V37 database. TF-binding sites were identified using the TFBSTools R package combined with JASPAR2020 datasets , 25. P-vhttps://www.uth.edu/bioinfo/software.htm). The raw FFLs networks were further refined using TCGA expression profiles to improve the accuracy. The spearman correlation calculated TFs, miRNAs, and target genes for every FFLs in the raw network. For miRNAs-genes pairs, p-value< 0.05 and Rs< 0 was defined as significant. For TFs-genes pairs, p-value< 0.05 and Rs > 0 was defined as significant. The less significant FFLs networks were removed from raw networks to produce the final refined co-regulatory networks in each cancer type. In addition, the refined pan-cancer network was used to perform centrality measure and MCC analysis using the cytoHubba plugin  were extracted from the combination of miRNAs-genes and TFs-genes using MotifPredictor  were retrieved from prior research as FDA-approved therapeutic drugs or biomarker targets . The theWe obtained the drug sensitivity area under the dose-response curve (AUC) and gene expression profiles for cancer cell lines from GDSC to examine drug sensitivity in cancer cell lines . We compThe survival analysis of classical PTPs was analyzed using the Kaplan-Meier method with the log-rank test by the survival R package. The cut-off point in each set was estimated using the survminer R package. P-value< 0.05 was defined as significant. Moreover, the Cox proportional hazard regression model was applied to estimate the hazard ratio (HR) for every classical PTP.The log2 transformed expression profiles were first z-normalized for the classical PTPs with significant HR values across the analyzed samples . PTPscorn is the number of analyzed samples, and e is the z-normalized expression data of involved PTPs with a significant HR.where To identify classical PTPs essential to the proliferation and survival of cancer cells, we applied CERES across 342 cancer cell lines . CERES i2 chamber. Lipofectamine 3000 (Thermo Fisher) was applied to transiently transfection for plasmids and siRNAs following the manufacture`s instruction. After 48 h transfection, cells were lysed on the ice, and western blotting analysis was performed as previously described  supplemented with 10% fetal bovine serum  and 1% Penicillin/Streptomycin. U-87 cell line was cultured in Eagle’s Minimum Essential Medium  supplemented with 20% fetal bovine serum and 1% Penicillin/Streptomycin. All cell lines were maintained at 37℃ in a humidified 5% COescribed .Cells were co-transfected with siRNA or plasmid and luciferase reporter plasmid containing the target promoter for 48 h. The luciferase activity was detected by the Luciferase Assay System  and plate reader .Cell cycle and apoptosis were analyzed using propidium iodide (1 mg/ml) and ribonuclease-A (10 g/ml) , and Annexin V/PI assay by flow cytometry  respectively as we previously described .Cells were seeded in 96-well plates and evaluated with Cell Counting Kit-8  according to the manufacturer`s instruction at the indicated time points. A plate reader  was used at the endpoint to assess the results.Cells were seeded in six-well plates at a density of 1000 cells. After two weeks of growth, colonies were fixed with paraformaldehyde for 30 minutes and marked with 0.1% crystal violet solution for 15 min. Finally, an optical microscope was used to counter the number of colonies.6 cells in serum-free DMEM). Mice were anatomized after two weeks. Western blot was used to detect the protein level of the target gene. The animal study was reviewed and approved by the Animal Care and Use Committee of Northeastern University.Male mice  were acquired from Charles River  and fed in the house in the pathogen-free condition. All procedures were approved by the Institutional Committee on Animal Care of Northeastern University. U-257 cells stably expressing PTPN12 and empty vector were injected subcutaneously into nude mice’s right super lateral tissue . The student’s t-test and the Wilcoxon rank-sum test were utilized to compare normally distributed variables and non-normally distributed variables. The p-values were two-sided and adjusted according to the Benjamini–Hochberg (BH) approach to control the false discovery rate (FDR). A BH-adjusted p-value< 0.05 was considered statistically significant unless otherwise indicated.Statistical analysis and graphical visualization were performed in R, version 4.0.0 (-8), and PTPN6 was negatively correlated with PTPRG .In this study, we reviewed the literature and explored the roles of classical PTPs in human cancer (-32) and PTPN2 in UCS , and some abnormal down-regulations were associated with CNV loss such as PTPN23 in UCS  and PTPRS in OV   into three categories referenced in the published study , includiTo describe the regulatory mechanisms of GRNs in extenso, we constructed the co-regulatory network and integrated TCGA expression data to refine the FFL patterns  and linked to drug resistance to 33 anti-cancer drugs . Dysregulation of the EGFR signaling pathway is an established feature in multiple cancer types, and EGFR signaling can be regulated by classical PTPs  and RTK pathway inhibitor Axitinib , Lestaurtinib , Sorafenib  and PD-173074   as an example, we identified 20 PTPs that are highly associated with the sensitivity of 94 anti-cancer drugs and are targeted by 34 TFs and 42 miRNAs. The expression of these PTPs and paired TFs is negatively associated (drug-sensitive) with the response to anti-cancer drugs. In contrast, their paired miRNAs are positively associated (drug-resistant) with identical drugs , 44, andImmunotherapy has become an efficient clinical approach for cancer treatment. We, therefore, estimated the roles of classical PTPs in anti-PD-L1 treatment  is widely regarded as the gold standard in cancer research, having the most straightforward and clinically meaningful endpoint definition: dead or alive. Furthermore, disease-specific survival (DSS) was superior to OS in patient outcomes . To expl-10; LGG, logFC = 1.50, FDR< 3.13 × 10-10), protein alteration in three cancers , CNV alteration in two cancers  and methylation alteration in nine cancers , logFC = 0.48, FDR< 2.55× 10-11). Surprisingly, 98.3 percent (116/118) of CGAs were connected with at least one kind of PTPscore-associated molecular signature in at least one cancer type. Interestingly, PTPs has an effect on a number of immunotherapeutic targets. PDCD1 (PD-1) was shown to be strongly expressed in PTPscore-high LUSC samples , suggesting that PDCD1 inhibitors, such as nivolumab and pembrolizumab , which is in line with the Phase II multicenter trial in melanoma patients (-24) and Axitinib in LIHC , indicating a possible therapeutic advantage of combining cancer therapy with phosphatase-targeted therapy for individuals with THYM or LIHC. Strikingly, some high-PTPsocre tumors may develop sensitivity to other drugs, including Gefitinib in uveal melanoma (UVM)  and AZD6244 in TGCT , which shows that phosphatase-targeted treatment may not be beneficial for patients with these malignancies.We next assessed the PTPscore on drug response using the imputed drug data from the TCGA samples. We observed that the response to sorafenib was positively associated (drug-resistant) to PTPscore in BRCA , Key Research and Development Plan of Liaoning Province (2020JH2/10300080), National Natural Science Foundation of China , and the Fundamental Research Funds for the Central Universities of China .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


University students are a critical group for vaccination programmes against COVID-19, meningococcal disease (MenACWY) and measles, mumps and rubella (MMR). We aimed to evaluate risk factors for vaccine hesitancy and views about on-campus vaccine delivery among university students. Data were obtained through a cross-sectional anonymous online questionnaire study of undergraduate students in June 2021 and analysed by univariate and multivariate tests to detect associations. Complete data were obtained from 827 participants (7.6% response-rate). Self-reporting of COVID-19 vaccine status indicated uptake by two-thirds , willing for 23% (194/827), refusal by 5% (40/827) and uncertain results for 8% (66/827). Hesitancy for COVID-19 vaccines was 5% (40/761). COVID-19 vaccine hesitancy was associated with Black ethnicity  and concerns about vaccine side-effects . Uncertainty about vaccine status was frequently observed for MMR (11%) and MenACWY (26%) vaccines. Campus-associated COVID-19 vaccine campaigns were favoured by UK-based students  and UK-based international students . Limitations of this study were use of use of a cross-sectional approach, self-selection of the response cohort, slight biases in the demographics and a strict definition of vaccine hesitancy. Vaccine hesitancy and uncertainty about vaccine status are concerns for effective vaccine programmes. Extending capabilities of digital platforms for accessing vaccine information and sector-wide implementation of on-campus vaccine delivery are strategies for improving vaccine uptake among students. Future studies of vaccine hesitancy among students should aim to extend our observations to student populations in a wider range of university settings and with broader definitions of vaccine hesitancy. Young people are an important risk group for vaccination programmes due to their high mobility, inexperience of accessing medical systems and relatively high levels of vaccine hesitancy compared to older populations ,2. Within = 97) with lower socioeconomic status being the most impactful contributory factor in lower-middle income countries/regions and perceived vaccine safety in more affluent countries/regions [Multiple previous studies have been conducted to determine the reasons for COVID-19 vaccine hesitancy in the general population. Vaccine hesitancy or acceptance has been assessed by a range of measures including use of the SAGE guidelines, Likert-scaled acceptance questions, attitudinal measures and actual uptake or intention to uptake (as utilised herein) ,9,10,11./regions . Common /regions ,13,14. S/regions ,18,19,20/regions ,22,23. A/regions ,25.Prior to the COVID-19 pandemic, a major concern for student populations was the prevention of cases and outbreaks of meningococcal disease, measles and mumps. Rising levels of infections due to a MenW cc11-lineage strain led to introduction of the MenACWY vaccine into the UK school-age vaccination programme and new university entrants from August 2015 ,27. OutbStudies prior to the COVID-19 pandemic reported uptake rates of the MenACWY vaccines among students at 68–71% ,31. In gTo evaluate the barriers to uptake of vaccines among students and to inform university vaccination policies, we assessed the attitudes, knowledge, perceived vaccine status and willingness for uptake of COVID-19, MMR and MenACWY vaccines among university students during the roll-out of COVID-19 vaccines to 18-year-olds and above in the UK.All study participants gave their informed consent for inclusion before participation in the study. The study was conducted in accordance with the Helsinki Declaration and ethical approval was given by the University of Leicester (UoL) Medicine and Biological Sciences Research Ethics Committee (reference number 29522).https://www.england.nhs.uk/2021/06/21-and-22-year-olds-to-be-offered-covid-19-jab-from-today/, accessed on 9 August 2022 and https://www.england.nhs.uk/2021/06/nhs-invites-all-adults-to-get-a-covid-jab-in-final-push/, accessed on 9 August 2022). Prior to these dates, only healthcare workers and medical students as well as individuals in vulnerable categories were eligible for COVID-19 vaccines in the <30-year-old age bracket. At the time the questionnaire was designed, we assumed most students would be unvaccinated when they completed the questionnaire.The questionnaire was emailed to students on three occasions between the 1st and 21st of June 2021. Access to COVID-19 vaccines in the UK was extended to the 25–29, 23–24, 21–22 and 18–20 age brackets on the 7th, 15th, 16th and 17th June 2021, respectively . In order to determine if the demographics of our study participants were similar to those of other UK universities and for weighting of the multivariable analyses, we obtained demographic data from HESA . Similarly, we compared vaccination rates in our study with local, regional and national vaccination rates obtained from the UK Coronavirus Dashboard .Sample size was determined by the total number of surveys completed with all responses being considered in the analysis. De-identified survey responses were analysed using R version 4.0.3 with the tidyverse (data handling), jsonlite 1.7.1 (data extraction), ggplot2 3.32 , gtsummary 1.4.2 (tabulation), UpSetR 1.4.0 (graphing of sets) and likert 1.3.5 (graphing of Likert-style responses) packages ,38,39,40p-values for multiple testing. COVID-19 vaccine hesitancy and preference for on-campus vaccinations (COVID-19 and MMR) were dichotomised and used as dependent variables. Univariable analyses were performed on unweighted survey results using chi-squared, Fisher’s exact or Wilcoxon’s rank sum tests. False discovery rate (FDR) correction was used to adjust n = 6). Given the low rate of vaccine hesitancy and the inherent limitations on creating meaningful and representative training and test data subsets, no attempt was made to assess model performance . Multivariate models were used to identify significant factors influencing vaccine hesitancy rather than for the purpose of generating an effective predictive model of hesitancy.Multivariable analysis was performed using logistic regression  on both unweighted and weighted survey results, with vaccine hesitancy and preference for on-campus vaccinations (COVID-19 and MMR) as dependent variables. Predictors included gender, ethnic group, age group, course studied, year of study, experience of harassment, experience of COVID-19-related death, concern over side-effects from the Oxford/AstraZeneca vaccine, concern over hospitalisation from COVID-19, concern over spreading COVID-19, home area , residence while studying  and a psychometric score on self-determination/fatalism. Home area was derived using information on postcodes and international status with students being classified as local if they came from either Leicester or the wider county of Leicestershire, ‘national’ for students from the rest of the UK and ‘international’ for students ordinarily living overseas. Experiences of COVID-19-related deaths were classified into a Yes or No category according to responses to question 21  with theStatistical differences between the distributions of VAX scores for different groups were determined using pairwise Dunn tests with FDR correction.p-value (FDR correction) of <0.05 considered significant.All tests were two-sided with a corrected n = 86) international students. Response rates were higher among females (11% above the level for UK universities and 14% above the UoL level) and had an ethnicity profile intermediate between UoL and UK university undergraduate populations  undergraduate students were invited to participate in a study of the uptake and attitudes to COVID-19 vaccines. Complete answers were provided by 7.6%  of participants. Respondents were young (94% 18–25-year-olds), ethnically diverse  and included 10%  who indicated that they had refused or would refuse a COVID-19 vaccine.Ethnicity, concerns around side-effects (particularly the AstraZeneca vaccine), concerns around spreading COVID-19 to others, place of residence while studying and VAX score were all found to be significantly associated with hesitancy after correcting for multiple testing. There was a weak trend for an association of age with vaccine hesitancy; this could not, however, be explored further due to banded collection of age data and the narrow age range of this cohort.Analysis of the individual VAX scale questions see  showed tA surprising observation was that high proportions of both vaccine-willing  and vaccine-hesitant  students had experienced a COVID-19 death among relatives or other acquaintances . This oup = 0.021) compared to humanities, law and social sciences. For ethnicity, hesitancy among Black students had a high odds ratio  as compared to White students (p = 0.004) were less vaccine-hesitant than students living at home.The multivariable analysis identified associations between vaccine hesitancy and ethnicity, course of study, side-effects and place of term-time residence, as found in the univariate analysis, and additionally with experiences of death among contacts . For coustudents . Studentp < 0.001; data not shown). Concerns about side-effects were, however, lower for the Pfizer/BioNTech vaccine .Hesitancy was strongly associated with concerns over side-effects from the AstraZeneca  and Pfiz vaccine . Surpris vaccine . This asr = 0.63; Wilcoxon rank sum test p < 0.0001), as expected. The Asian ethnic group had significantly lower VAX scores than both White and other ethnic groups, while the Black ethnic group had significantly lower VAX scores than White ethnicity, indicating a higher level of vaccine hesitancy in Asian and Black groups . SimilarViews on MMR and MenACWY vaccines are shown in p = 0.021) or private accommodation  were in favour of this provision.The univariable analysis of on-campus MMR/MenACWY vaccine programmes identified a significant association with MMR vaccine status indicating that those who responded with either a ‘Yes’ or ‘Don’t Know’ response for their vaccine status were in favour of these programmes . HoweverUniversity students are a critical group for illness and spread of infectious diseases and hence are an important target for vaccination programmes. Our survey of University of Leicester students was unique in that we evaluated both attitudes to and mechanisms for uptake of the three major vaccines targeted to this population group in the UK. Our study indicates that ethnicity, concerns over side-effects and place of residence are key determinants of COVID-19 vaccine hesitancy. We also found high levels of uncertainty among students about their MenACWY and MMR vaccine status. As an approach to facilitating vaccine uptake, students were asked about on-campus provision of vaccines and reported being in favour of this approach. Based on our findings we elaborate key recommendations for improved vaccine delivery to this population sector. despite this age group only becoming eligible for COVID-19 vaccines during the data collection period. These uptake levels were significantly higher than the wider young adult population at that time Identifying vaccine-hesitant individuals is a key concern for vaccination programmes. In our study, uptake of the COVID-19 vaccine or intention to vaccinate was found to be relatively high concurring with findings from other studies conducted with student groups. For example, Di Giuseppe et al. ,44.Although vaccine hesitancy was low, we found that hesitancy was strongly linked to ethnicity and more specifically to Black ethnicity in our univariate and multivariate analyses, respectively. Furthermore, analysis of VAX scores for all individuals showed that Asian and Black ethnic groups had significantly lower VAX scores indicating a general trend towards hesitancy among the minority ethnic groups . Other sp = 0.024) as also observed by Szmyd et al. [A novel finding was of an association between vaccine hesitancy and students who lived at home and significantly lower average VAX scores for students living at home as compared to those living in private accommodation or other accommodation types . While td et al.  for a coOur study was performed a few months after concerns about side-effects of the AstraZeneca vaccine were widely publicised. Associations between these concerns and vaccine hesitancy were detected in both our univariate and multivariate models, indicating that this important factor could reduce vaccine uptake. Nevertheless, uptake and willingness levels were high, suggesting that other factors may override the perceived risks of side-effects.An important strategy for increasing MMR and MenACWY vaccine uptake among young adults is making them aware of their vaccination status . This isHarnessing new approaches developed during the UK COVID-19 vaccine roll-out is a potential positive legacy of the pandemic to build back better for future generations. Empowering digitally aware young people to take responsibility for their own health and to engage in community health policies is an achievable, cost-efficient outcome with far-reaching personal and population benefits. A key recommendation is for provision of vaccine status and access information for all vaccines on digital platforms  so that individuals can make informed decisions about taking up missed vaccinations. Specific delivery of vaccines to international students should be a gold standard for the university sector combined with wide adoption of on-campus vaccination programmes. The benefits of implementation of these recommendations would be protection of more individuals and improved population protection through reduced transmission.A strength of this study is that it is the first to simultaneously evaluate uptake, knowledge and attitudes to COVID-19, MMR and MenACWY vaccines among university students. A further strength is the high number and ethnic diversity of the participant population. The use of multivariable regression was a strength that allowed for adjustment for confounders and for identification of significant associations between variables and vaccination parameters with the potential to inform vaccination policies.Findings from this study may, however, be affected by the inherent limitations of cross-sectional studies. Our approach of utilising emails to send out invitations and an online survey form may have limited access for some potential participants. The self-selecting nature of the response cohort and a response rate of 8%, despite incentives, indicates that there may be bias due to demographics. The strengths and limitations arising from a range of demographics were considered above, but it is possible that biases from other unaccounted demographics  may confound generalisability of our data to the wider UK student population. We note that distribution in ethnic group and gender differ in our response cohort to both the University of Leicester and the wider UK student demographic and have attempted to mitigate these effects by using a weighted multivariate analysis. A further limitation is that a pilot study was not completed with the entire questionnaire. A significant potential limitation of our study, and inherent in many studies of vaccination, is enhanced participation by individuals with pro-vaccine attitudes and reduced participation by vaccine-hesitant individuals. Our study may also have been subject to social desirability bias due to the study survey being delivered through the University of Leicester email system and being promoted by the senior university team. Our fully anonymised survey system and online formats was designed to counter both of these biases while inducements to participate was designed to minimise the former bias. Our level of vaccine hesitancy as determined by vaccine uptake is similar to other studies. However, this strict determination of vaccine hesitancy may have missed the full range of hesitancy and excluded students who obtained the vaccine despite having a degree of vaccine hesitancy.The findings from this study indicate that there may be differences in uptake and access to the COVID-19, MenACWY and MMR vaccines among university students. Students of Black ethnicity and those residing at home were less likely to be vaccinated with COVID-19 vaccines. Further research on the reasons for hesitancy may be required in order to delivery more effective, ‘tailored’ vaccine information and to develop methods for enhancing trust and acceptance of vaccines in these groups. High levels of uncertainty about personal vaccine status and availability of the MMR and MenACWY vaccines were observed and are likely to impact vaccine uptake. On campus vaccination delivery was found to be widely favoured particularly by on-campus and international students. These knowledge gaps and delivery approaches should be considered in future student-focussed vaccination campaigns and explored through additional research. Our findings indicate that adopting ‘best practices’ of easy access and digital vaccine information within the university-sector may break down barriers and future-proof uptake of all required vaccines among students.


TBI diagnoses of mild TBI (mTBI) or moderate-to-severe TBI (MS-TBI) along with LOC status were identified using International Classification of Diseases, Ninth Revision (ICD-9) codes and the Defense and Veterans Brain Injury Center Standard Surveillance Case Definition for TBI. Self-reported psychiatric symptoms were evaluated for post-traumatic stress disorder (PTSD) with the PTSD Checklist, Civilian Version for PTSD, the Patient Health Questionnaire-9 for major depressive disorder (MDD), and the Patient Health Questionnaire-15 for somatic symptom disorder (SSD) in two time periods post-injury: Assessment Period 1  and Assessment Period 2 . mTBI, but not MS-TBI, was associated with increased neuropsychiatric symptoms: PTSD in AP1 and AP2; MDD in AP1; and SSD in AP2. A subgroup analysis of mTBI with and without LOC revealed that mTBI with LOC, but not mTBI without LOC, was associated with increased symptoms as compared to non-TBI: PTSD in AP1 and AP2; MDD in AP1; and SSD in AP1 and AP2. Moreover, mTBI with LOC was associated with increased MDD symptoms in AP2, and SSD symptoms in AP1 and AP2, compared to mTBI without LOC. These findings reinforce the need for the accurate characterization of TBI severity and a multi-disciplinary approach to address the devastating impacts of TBI in injured SMs.The impact of traumatic brain injury (TBI) severity and loss of consciousness (LOC) on the development of neuropsychiatric symptoms was studied in injured service members (SMs;  Previous studies have proposed that there is a “dose-response relationship” between TBI severity and neuropsychiatric outcomes, in which greater TBI severity (moderate or severe) is proportional to worse long-term neurocognitive and -behavioral symptoms.2 It has been suggested that LOC may be protective against post-traumatic stress disorder (PTSD) development attributable to amnesia and lack of memory of the traumatic event.37–39 However, recent studies, especially those considering data from the conflicts in Iraq and Afghanistan, have demonstrated that TBI, even those with LOC, is an important risk factor for PTSD and other psychiatric outcomes.41 Yet, given other evidence that mTBI carries a higher risk of PTSD than more severe TBI,42 it remains possible that extended LOC and amnesia present in severe TBI (as opposed to mTBI) could exert protective effects on neuropsychiatric sequelae. Whether this phenomenon is related to certain aspects of traumatic memory consolidation and mediated by LOC or other factors remains to be determined.Ambiguity regarding TBI severity raises the question of the potential role of loss of consciousness (LOC) on neuropsychiatric sequelae. According to the Veterans Affairs and DoD clinical practice guidelines for the management of concussion, TBI severity can be graded by duration of LOC, in which mTBI is defined as a concussion with LOC <30 min and MS-TBI with LOC >30 min.The purpose of the current study was to 1) determine the association of TBI severity and self-reported neuropsychiatric symptoms related to PTSD, major depressive disorder (MDD), and somatic symptom disorder (SSD) and 2) examine neuropsychiatric outcomes of TBI patients based on LOC status. We hypothesized that mTBI may lead to greater symptom severity compared to MS-TBI, and mTBI with LOC may contribute to increased self-reported symptoms.43 They were administered similar follow-up assessments in the subsequent months. Medical diagnoses were derived from International Classification of Diseases, Ninth Revision (ICD-9) codes as part of routine care. Permission to analyze data from these deidentified subjects was provided by the institutional review board at the Walter Reed National Military Medical Center, and the study was deemed exempt.The study sites included Walter Reed Army Medical Center (2003–2011) and Walter Reed National Military Medical Center (2011–2012). Study subjects were hospitalized service members (SMs) medically evacuated from Iraq and Afghanistan because of their need for continued medical or surgical inpatient treatment. They typically arrived within a few days post-injury and within 72 h of arrival or when medically stable received an initial behavioral health assessment that included self-reported psychiatric screening with standardized assessments.To determine longitudinal effects of TBI severity on neuropsychiatric symptoms, two post-injury time periods were pre-determined as Assessment Periods (APs) based on the distribution of when the behavioral health assessments had been completed. The first AP included the first 75 days post-injury, referred to as Assessment Period 1 , and the second AP occurred between post-injury days 90 and 365 . A total of 1278 wounded SMs with ICD-9 codes completed the behavioral health assessments in both AP1 and AP2. 44 TBI diagnoses were identified, and the severity of each TBI was based on the most severe injury charted and classified as mild or moderate-to-severe using the DVBIC 2015 criteria , Armed Forces Health Surveillance Branch, and the Centers for Disease Control and Prevention have collaborated to develop a standard TBI surveillance case definition using ICD-9 codes.criteria , similar47 we incorporated combat trauma exposure into the analysis. SMs completed a survey containing 11 potentially stressful combat experiences during the deployments. Total Combat Exposure Severity Score (CESS) for each subject was computed by totaling the tally of positive responses for all 11 questions , Patient Health Questionnaire-9 for MDD, and Patient Health Questionnaire-15 for SSD. All three screening tools were utilized at AP1 and AP2  for all uestions  with a r49 The ISS is a composite score derived from AIS values from six body regions and is considered the gold standard of injury severity in PTSD studies of civilian and military populations.51 For each SM, AIS values were determined by clinicians based on injury severity at onset and did not change over time. Severe injury was defined as ISS ≥16, like other studies.52–56Physical injuries were assessed with the Abbreviated Injury Scale (AIS) and Injury Severity Score (ISS), which utilize clinician-rated scales to grade the severity of physical injury in patients.All data were analyzed using the Statistical Package for the Social Sciences . Binary logistic regression models were used to assess relationships between the presence of self-reported PTSD, MDD, and SSD at AP1 and AP2 with TBI severity (mTBI or MS-TBI). In the first model, the odds of developing probable PTSD, MDD, and SSD after mTBI and MS-TBI were calculated relative to the no-TBI reference group. In a subgroup analysis within the same model, the odds of developing probable PTSD, MDD, and SSD after mTBI without and with LOC were calculated relative to the no-TBI reference group. In a second model, the odds of developing probable PTSD, MDD, and SSD after mTBI with LOC and MS-TBI were calculated relative to the mTBI without LOC reference group. All subjects' demographic variables were included in the multiple logistic regression models, unless otherwise noted. Subjects' characteristics are shown in n = 1278) of subjects, 39% were diagnosed with TBI (n = 493), whereas 12.3% and 26% were diagnosed with mTBI (n = 157) and MS-TBI (n = 336), respectively (Of the entire cohort (ectively . The majIn the early period post-injury (AP1), rates of PTSD (225%) and MDD (281%) were higher in the mTBI group whereas rates of PTSD (58%) and MDD (55%) were lower in the MS-TBI group, as compared to the no-TBI group. However, in the late period post-injury (AP2), rates of PTSD were higher in both the mTBI (183%) and MS-TBI groups (125%), as compared to the no-TBI group .Rates of probable PTSD, MDD, and SSD among TBI subjects with and without LOC are delineated in p < 0.05) and AP2 , MDD in AP1 , and SSD in AP2 . However, MS-TBI was not significantly associated with increased rates of PTSD, MDD, or SSD in either AP1 or AP2.Results of the logistic regression analyses for the mTBI and MS-TBI groups are summarized in p < 0.01) and AP2 . The mTBI with LOC group also had increased odds of MDD in AP1  and SSD in both AP1  and AP2 . However, mTBI without LOC was not significantly associated with any of the syndromes in AP1 or AP2.p < 0.05) and SSD in AP1  and AP2 .Using mTBI without LOC as a reference group, associations between mTBI with LOC and symptoms were analyzed as shown in 26 However, our findings support that those with mTBI tend to endorse significantly more symptoms, including those related to pain, self-perceived difficulties, and emotional distress, compared to those with MS-TBI.57–62 Additionally, our results are consistent with several studies demonstrating that PCL scores reported by mTBI subjects are higher than those reported by MS-TBI subjects, and that PTSD is more likely to occur in the context of mTBI.63–65Findings demonstrated that mTBI, not MS-TBI, is associated with increased risk for developing neuropsychiatric symptoms within the first year post-injury. Additionally, mTBI with LOC, not mTBI without LOC, increased the risk for PTSD, MDD, and SSD symptoms compared to non-TBI. Our findings dispute the notion that there is a positive association between TBI severity and neurocognitive outcomes.66 In other words, the strength with which traumatic memories are encoded may correlate with PTSD symptom development.67–70The precise reason why mTBI subjects present with increased symptoms in the early period post-injury is not fully understood. One interpretation is that, in contrast to MS-TBI which involves more prolonged LOC, mTBI may be associated with greater memory encoding of the traumatic event. This notion is consistent with a study that reported an inverse relationship between the duration of post-traumatic amnesia and severity of re-experiencing symptoms related to PTSD in patients with mTBI at the 3-month follow-up.71 Another study reported that SMs had fewer self-reported neurobehavioral and post-traumatic stress symptoms after MS-TBI compared to mTBI.72Similar to past studies, MS-TBI subjects had decreased odds of symptoms in AP1 compared to those without TBI, suggesting that MS-TBI may be protective against early-onset neuropsychiatric symptoms. For instance, a study found that MS-TBI patients, not mTBI patients, had fewer psychiatric diagnoses than their non-TBI counterparts.74 Importantly, such a lack of awareness can negatively impact treatment and recovery post-TBI because of patients' underestimating of impairments and overestimating of functional abilities, leading to missed diagnoses.75–77 Other explanations for why MS-TBI patients may present with fewer symptoms in the early period include variations in post-injury factors, such as length of hospitalization, during which significant support is provided to ameliorate such symptomology.79 MS-TBI patients are more likely to require hospital intervention and intensive rehabilitation. Thus, these patients may not realize the extent of their non-physical impairments, given that the controlled hospital environment may limit the cognitive and social demands of everyday life.There are several interpretations for such a phenomenon. First, some MS-TBI patients may lack awareness and have impaired insight or apathy of their condition, whereas mTBI subjects may generally be more self-aware of their distress, leading to greater symptom reporting.42 it is conceivable that the longer duration of LOC, commonly observed in MS-TBI, may be protective against PTSD in the early period post-injury. One study, evaluating 47 SMs with moderate TBI who had no recollection of the event, postulated that the lack of intrusive memories and re-experiencing was a direct result of LOC-related amnesia.38Compared to the mTBI without LOC group, MS-TBI exhibited decreased PTSD symptoms, whereas mTBI with LOC showed increased PTSD symptoms, although not statistically significant. These findings may have relevance to the complex role of LOC on traumatic memories post-TBI. Given the current and previous findings that mTBI carries a higher risk of PTSD than severe TBI,80 It is possible that mTBI patients with LOC are still able to develop emotional reactions to traumatic events despite the lack of overt memory and conscious recall. For instance, studies suggested that those with mTBI and LOC may develop ”pseudo-memories” or collections of memory, despite being amnesic for the traumatic event.81–83 Such pseudo-memories can trigger declarative memory of the traumatic event by secondhand information, such as newspaper coverage, subjectively generated images, or other indirect stimuli. Another consideration is that certain persons with LOC may have some memory of the trauma leading up to the TBI and thus not be fully amnesic. This may be more common in those with relatively shorter duration of LOC and mTBI and hence contribute to the current findings. Yet, despite these possible explanations, the precise relationship between LOC and memory of the traumatic event is unclear, and thus further investigation is needed to unravel the neurological mechanisms of LOC and their effects on memory formation and neuropsychiatric symptom development.84–86In contrast, the protective effects of LOC were less pronounced when comparing mTBI with or without LOC. mTBI subjects with LOC, not mTBI subjects without LOC, exhibited increased neuropsychiatric symptoms compared to subjects without TBI. This is consistent with past studies discussing LOC as an indicator of injury severity and that mTBI with LOC patients have worse neuropsychiatric outcomes compared to mTBI without LOC.88There are several limitations in the current study. First, although this study used a longitudinal study design to determine the association between TBI severity and development of symptoms, only two time periods in the first year post-injury were used for data analysis because of data availability. Second, there is an incomplete body of clinical information, such as clinician-determined psychiatric evaluations and pre-injury factors  that may influence the onset and trajectory of symptom development. Third, TBI diagnoses were based on the DoD's classification of TBI severity using ICD-9 codes without specific information regarding injury mechanisms. The possible inaccuracy of categorizing TBI severity using ICD-9 codes has been described in past studies.Further, the timing of TBI relative to other physical trauma cannot be definitively known, though it is likely that such injuries occurred concurrently considering that SMs were evacuated because of polytrauma, including TBI. Fourth, additional follow-up data were not available to determine whether there were group differences in response to therapy. Finally, self-reported symptoms were used to categorize participants into either syndrome-present or -absent group rather than using a clinician-determined diagnosis. This approach may have resulted in some patients being misclassified because of differences in individual interpretation of questions and rating scales, and personal biases.89–95 Ultimately, TBI-related neuropsychiatric disorders constitute an imperative health burden in the military population, and a concerted effort is much warranted to address the devastating impact of TBI on military SMs and their families.We recommend that anyone exposed to trauma that could cause TBI be immediately professionally screened for such with thorough documentation of LOC. Further, though MS-TBI patients may report fewer symptoms in the early period after brain injury, symptom onset may be delayed, and thus long-term follow-up is recommended. Finally, the complex relationship between TBI severity and development of neuropsychiatric symptoms in injured SMs reinforce the need for a multi-disciplinary approach. Holistic and collaborative care approaches to the evaluation and treatment of coexisting symptom-based disorders are important in designing intervention strategies.


Human papillomavirus (HPV) vaccination plays a key role in the prevention ofcervical cancer. Yet, disparities in HPV vaccination in Sweden havepersisted. Previous research on such disparities has typically focused onsingular sociodemographic variables and measures of average risk. Using amulti-categorical approach and drawing on intersectionality theory, thisstudy aimed to provide a more precise mapping of HPV non-vaccination amonggirls in different sociodemographic groups and geographical areas in Swedenduring 2013–2020.Using nationwide register data, we conducted a multi-categorical analysis ofindividual heterogeneity and discriminatory accuracy complemented by amultilevel geographical analysis. We mapped HPV non-vaccination prevalenceacross 54 strata defined by parental income, education and country of birth,and urban versus rural place of residence. We also disentangled municipaland regional influences on HPV non-vaccination.HPV non-vaccination was more common in groups with a low income, a loweducation and an immigration background, whereas among those with animmigration background, the association between income, education and HPVnon-vaccination was more complex. Geographical differences were foundbetween municipalities. However, the discriminatory accuracy of thesociodemographic and geographical groups was weak, and 50% of thenon-vaccination cases were observed in eight strata, of which some are amongthose with low risk.Our findings underscore the importance of universal yet tailoredapproaches, including providing adequate information about HPVvaccination in Swedish and other languages, and of health-careprofessionals displaying sensitivity to patients’ and parents’ questionsor needs. In order to prevent human papillomavirus (HPV) infections and cervical cancer , in 2010While HPV vaccine coverage varies between countries , in Swed5Still, studies of disparities in HPV vaccination uptake in Sweden are relativelysparse and mainly investigate the effects of singular socio-economic or geographicaldimensions . This maOur study draws on intersectionality theory , which iWe applied an analysis of individual heterogeneity and discriminatory accuracy(AIHDA), which is suitable for the multi-categorical study of health disparities, andcomUsing multi-categorical AIHDA, complemented by a multilevel geographicalanalysis, we aimed to provide an improved mapping of the sociodemographic andgeographic distribution of HPV vaccine uptake in Sweden.After approval by the Swedish Ethical Review Authority (no. 2020-05688), theNational Vaccination Register (NVR) administered by the Public Health Agency waslinked to the Register of the Total Swedish Population (TPR) and theLongitudinal Integration Database for Health Insurance and Labour Market Studies(LISA), which provides demographic and socio-economic information. The lattertwo are administered by Statistics Sweden, who performed the record linkage.N=315,652). Each agegroup was followed up during the period in which they were 10–12 years old . Weexcluded those who died (n=95) or emigrated(n=1134) during the follow-up period and those with missinginformation . The final studypopulation consisted of 311,656 girls .Our study population consisted of all girls between two and seven years of ageliving in Sweden on 31 December 2010 .We computed a cumulative measure of individualised equivalised disposable familyincome by using information on absolute income for the years 2000, 2005 and2010. For each of the three years, incomes were categorised into 25 groups byquantiles using the complete Swedish population. These groups were summed up,assigning to each individual a value between 3  and 75 . This cumulative incomemeasure was divided into low, medium or high income by tertiles.The parental educational achievement variable distinguished between girls whohad, or had not, at least one parent with a tertiary education (i.e. high vs.low education).The parental country of birth variable was categorised into native, mixed orimmigrant based on whether both, only one or neither of the girls’ parents wereborn in Sweden. Parents with missing information were considered as immigrants,as all those born in Sweden are registered as such.Place of residence was based on the location where the vaccination wasadministered, according to categories provided by Statistics Sweden (1–9) as bigcity (1–3), small city (4–5) and rural (6–9).The multi-categorical variable was constructed through all possible combinationsof the explanatory variables (3×2×3×3), forming 54 multi-categorical strata.Girls whose parents had a high income and education and were born in Sweden,living in a big city, were used as the reference stratum in the analyses.In the multilevel analysis, we identified the municipality and county where thevaccination was administered.Following a stepwise analytical approach described previously , we perfAssociations were expressed as odds ratios (ORs). We also computedstratum-specific prevalence rates or absolute risks (ARs). We calculated 99%confidence intervals (CIs) to minimise the problem of multiplecomparisons.We assessed the DA of the regression models, that is, the predictive accuracyor the ability of the categorisations used in the models to distinguishbetween individuals who received HPV vaccination or not, by computing thearea under the receiver operator characteristics curve (AUC) . The curWe calculated the incremental change in the AUC value (Δ-AUC), whichquantifies improvements in DA yielded by a model compared to a previous one.If any statistical interaction of effects were identified in themulti-categorical variable, the AUC of Model 2 would be higher than that ofModel 1 and the Δ-AUC>0 . In ModeIBM SPSS Statistics for Windows v22  for PC and MLwiNv3.00 called from within Stata v14.1  wereused to conduct the analyses. The multilevel estimations were performedusing Markov chain Monte Carlo methods.Overall, as shown in Supplemental Tables S1, S2 and S3). The highest risk, 3.5 times higher than that of girls withnative parents with high income and education residing in big cities (AR=0.11), wasfound among girls whose parents had a low income, high education and an immigrantbackground, living in a rural area (AR=0.39).The multi-categorical analysis , Model 2Among girls with parents born in Sweden, a social gradient was present, asnon-vaccination was less common among those with high parental income and educationcompared to those with a medium or low income and low education. Among girls withparents born elsewhere, the distribution was more complex. Of the 18 high-educationstrata, in 13, non-vaccination was more common than in the corresponding  low-education strata. Of those comprising parental immigrationbackground and high or medium income, three strata showed a higher non-vaccinationprevalence than the corresponding medium- or low-income strata. The five strata withthe highest prevalence  compriseHowever, the DA of Model 2 was weak (AUC=0.610). In fact, while the non-vaccinationprevalence was 2.7 times higher in the five highest-risk strata than in the fivelowest-risk strata, the number of cases was 2.9 times higher in the lowest-risk thanin the highest-risk strata. The five lowest-risk strata included 19% of allnon-vaccination cases, whereas the corresponding number for the five highest-riskstrata was 7%. Half of the non-vaccination cases were found in eight strata .The geographical analysis  and V, aThe AR of 25.19 in the highest-risk municipality was 5.2 times higher than the AR of4.88 in the lowest-risk municipality. The five highest-risk municipalities included1.8% of all the adjusted number of cases in the population, while the correspondingnumber for the five lowest-risk municipalities was 0.5%. Of all the 290municipalities, 37 shared 50% of all adjusted cases of HPV non-vaccination.This nationwide register study shows between-group disparities in the averageprevalence of HPV non-vaccination in Sweden. Girls with parents born outside ofSweden and with a lower income or education had a higher probability ofnon-vaccination than those whose parents had a high income and education and wereborn in Sweden. This is in alignment with previous research indicating a higherprobability of non-vaccination among groups with low income, low education andimmigration background . HoweverIn a Swedish study of disparities in cervical screening associated with place ofbirth, these were largely explained by socio-economic rather than cultural orlanguage factors .The resMeanwhile, a central contribution of the AIHDA approach is the assessments of the DAof the categorisations used, without which measures of average risk may convey arisk of stigmatisation of ‘high-risk’ groups, of creating false expectations in‘low-risk’ groups and of ineffective interventions due to over- or undertreatment. Our anaThis low DA co-exists with a universal vaccination programme which has alleviatedpreviously greater disparities by largely benefitting less privileged groups inSweden . In thatIt should furthermore be noted that an increased uptake of HPV vaccination among boyshas been predicted to improve the resilience of HPV infection prevention overall.Being observational, this study does not enable the drawing of conclusions aboutcausal relationships. In addition, some multi-categorical strata were rathersmall, which is reflected in the wide CIs conveying a limited reliability ofsome point estimates. Furthermore, the categorisation based on country of birthcan be seen as simplistic and insufficient, as it disregards largeheterogeneities within the group  by conflThis study corroborates previous findings by indicating a higher prevalence of HPVnon-vaccination among girls with immigrated parents with low income and loweducation who live in rural areas, while also showing geographical differencesbetween municipalities. We provide, however, a more precise mapping ofsocio-economic disparities in HPV non-vaccination in Sweden. We also observed thatthe DA of the categorisations used was low, and that many cases of non-vaccinationare found in low-risk groups. These findings suggest that interventions aiming toincrease HPV vaccination should be directed towards the whole population, whilesimultaneously considering characteristics of particular groups. We underline theimportance of providing adequate information about HPV vaccination in Swedish andother languages, and of health-care professionals displaying sensitivity topatients’ questions or needs while avoiding forms of communication which maydiscourage trust and confidence.Click here for additional data file.Supplemental material, sj-docx-1-sjp-10.1177_14034948221075410 for Mappingsociodemographic and geographical differences in human papillomavirusnon-vaccination among young girls in Sweden by Maria Wemrell, Raquel PerezVicente and Juan Merlo in Scandinavian Journal of Public HealthClick here for additional data file.Supplemental material, sj-docx-2-sjp-10.1177_14034948221075410 for Mappingsociodemographic and geographical differences in human papillomavirusnon-vaccination among young girls in Sweden by Maria Wemrell, Raquel PerezVicente and Juan Merlo in Scandinavian Journal of Public Health