Sheep and human studies have demonstrated the superiority of a single dose of hydroxocobalamin (OHB12) over cyanocobalamin (CNB12) in maintaining high levels of cobalamin in plasma and liver. However, limited data are available for cattle. The purpose of this study was to compare the pharmacokinetics of two forms of cobalamin—OHB12 and CNB12—as a single subcutaneous injection of 28 µg/kg BW at the same time of a trace mineral injection in six non‐cobalt/B12‐deficient Holstein‐Friesian steers. Plasma and liver samples were obtained to determine cobalamin concentration after treatment. Cyanocobalamin had lower retention in plasma and liver than OHB12 (p < .05). Cobalamin levels peaked in plasma by 8 h after treatment in both groups. However, OHB12 reached a higher peak compared to CNB12. Levels of cobalamin in plasma dropped closer to baseline levels 24 h after CNB12 treatment while OHB12 maintained higher concentrations. Hydroxocobalamin increased significantly hepatic concentration of cobalamin 28 days after treatment, while CNB12 did not increase liver levels relative to pre‐treatment (p < .05). These results confirm that a single subcutaneous OHB12 injection increases the level of cobalamin in the blood in the first 24 hours, and this increase is maintained in the liver for at least 28 days.Injectable vitamin B Cyanocobalamin is not the naturally occurring form of cobalamin in the animal's body; a reductive decyanation reaction is an enzymatically controlled process required to remove the cyanide group from CNB12  for the synthesis of vitamin BThis study was conducted with the approval of the Virbac Animal Ethics Committee (AEC 613/20) following the Australian Code for the Care and Use of Animals for Scientific Purposes National Health and Medical Research Council (2013). The study was conducted on a commercial farm in Gippsland, Victoria, Australia, between June and September 2020.A total of 6 Holstein‐Friesian steers  were included in this pilot study. On day ‐5, animals were weighed, and liver biopsies and blood samples were obtained to determine basal levels of cobalamin in plasma and liver. Blood samples (10 ml) were obtained from the caudal vein in lithium heparin tubes . On collection, the red cells were immediately separated from plasma by centrifugation to prevent any cobalamin exchanges. Liver biopsies were obtained following standard protocol under xylazine sedation  received one subcutaneous injection of a product containing 26.7 mg Zn/ml as disodium zinc EDTA, 6.7 mg Mn/ml as disodium manganese EDTA, 3.3mg Se/ml as sodium selenite, 10 mg Cu/ml as disodium copper EDTA and 1.4 mg cyanocobalamin/ml . The second group (OHB12) received one subcutaneous injection of a product containing 40 mg Zn/ml as disodium zinc EDTA, 10 mg Mn/ml as disodium manganese EDTA, 5 mg Se/ml as sodium selenite, 15 mg Cu/ml as disodium copper EDTA  and one subcutaneous injection of a product containing 2 mg hydroxocobalamin acetate/ml . All pharmaceutical products were injected as per label recommendation, and both treatment groups received the same amount of trace minerals and equivalent doses of cobalamin on a bodyweight basis (Table 2 receive12 was measured based on intrinsic factor with streptavidin and biotin (Roche cat#07212771); modifications to the assay are as per RLS 085 Issue B, Rev 4, and include a pre‐boiling step prior to the e411 assay, to eliminate assay interferences observed in non‐boiled assays. Full details of these modifications are commercial‐in‐confidence to RLS.Blood samples were obtained 8 h, 24 h, 7 days, 28 days and 56 days after treatment and liver biopsies were collected 7, 28 and 56 days after treatment. Plasma and liver samples were analysed for cobalamin levels by a commercial laboratory using the modified CEDIA GenII electrochemiluminescence immunoassay . Briefly, vitamin BAnimal health status was monitored daily, and initial and final bodyweights were obtained using a walk‐over scale. The six animals were kept in a group paddock of approximately 4 hectares of perennial ryegrass with an average yield of 2250 kg DM/ha and a target intake of 10 kg DM/day per animal, with ad libitum access to water, and without feed supplements or concentrate.Pasture samples were analysed for nutrients and Co content before the experiment (day ‐45) and once a month until the end of the experiment. Samples were sent to a commercial laboratory for analysis . Means were compared using Tukey’s all‐pairwise comparison test, for Treatment, Time and Treatment × Time. Model assumptions were checked via residual plots and were considered acceptable given the size of the data set (n = 3/group). Homogeneity of variance assumptions were checked and confirmed via Levene's test. Means and associated standard errors are presented in Table Data were collated by treatment group and time point. Treatment mean concentrations by time point and associated standard deviations and standard errors were calculated. Blood plasma and liver vitamin BCobalt levels in the pasture before and during the trial were within the recommended range to prevent cobalamin deficiency in cattle . Twenty‐four hours post‐injection, the cobalamin levels of OHB12 treated animals declined, while cobalamin levels in CNB12‐treated animals were not different from basal levels. By day 7, plasma cobalamin concentrations for both groups approximated the original baseline, and no further changes were observed at days 28 and 56 post‐treatment . By contrast, animals treated with CNB12 showed no difference from the baseline. At day 28, steers treated with OHB12 had peak levels of cobalamin in the liver that were higher than those treated with CNB12 (928 nmol/kg vs 685 nmol/kg). Although this difference was not statistically significant in this study, it may be of clinical and productive importance. A study with a larger number of cattle would be required to confirm those differences.Nevertheless, at day 28, steers treated with CNB12 showed no difference from baseline cobalamin liver levels. In contrast, those treated with OHB12 had significantly higher liver cobalamin relative to baseline . By day 56, both groups had liver cobalamin levels not different from baseline Table .12.The average daily gain (ADG) of steers receiving OHB12 was approximately 11 % higher than that observed in those treated with CNB12 . The ADG of > 1 kg/day demonstrates adequate nutrition for Holstein‐Friesian steers grazing improved pasture during winter in South Gippsland, Victoria. The numerically higher ADG for steers in OHB12 was not statistically significant than in those treated with CNB12. A study with a larger number of cattle would be required to demonstrate differences in ADG in pasture‐fed cattle supplemented with different forms of vitamin BThe results of this pilot study demonstrate that a single injection of OHB12 can be effective in increasing the level of cobalamin in the blood in the first 24 hours and this increase is maintained in the liver for at least 28 days in non‐Co/cobalamin‐deficient cattle, while CNB12 was unable to raise liver cobalamin reserves relative to basal levels at any time point. These results agreed with previous studies conducted in sheep and humans. The reduced response to CNB12 is based on its lower bioavailability, rapid mobilization from the injection site, and fast renal clearance resulting in a shorter duration of activity compared with OHB12  alone or in combination with butaphosphan, folic acid or other additives from 8 to 3 weeks before calving until 9 to 18 weeks of lactation were used to evaluate their effect on improving energy status, gluconeogenesis, lactation and/or preventing ketosis in different studies in dairy cows  in cattle. This study demonstrated that a single subcutaneous OHB12 injection increases the level of cobalamin in the blood in the first 24 hours and this increase is maintained in the liver for at least 28 days in non‐Co/cobalamin‐deficient cattle when used in combination with trace minerals and demonstrated that a single dose of CNB12 is not an effective therapeutic method to raise cobalamin levels in cattle as increased cobalamin levels in the blood were not sustained for more than 24 hours, and liver cobalamin storage levels were not elevated from baseline at any time point. The authors acknowledge that injectable cobalamin was not administered as a pure solution but associated with trace minerals which could affect animal metabolism. The experimental design did not allow the authors to infer the presence or absence of such effect, and therefore, further studies to clarify this are guaranteed. Furthermore, additional studies, including a larger number of animals and more frequent sampling after treatment, are recommended to evaluate the effect of CNB12 and OHB12 in Co/cobalamin‐deficient cattle on blood and liver pharmacokinetics and bodyweight change.1This study was conducted with the approval of the Virbac Animal Ethics Committee (AEC 613/20) following the Australian Code for the Care and Use of Animals for Scientific Purposes, National Health and Medical Research Council .® is a registered trademark of Virbac Australia Pty Ltd.Dr Paula A Gonzalez‐Rivas and Dr Jerry Liu are employees of Virbac Australia Pty Ltd. Multimin PAGR and JL designed the experiment; PAGR supervised the execution of the experiment; MC analysed the data; and PAGR wrote the manuscript in consultation with JL and MC.


Tinnitus disability is a heterogeneous and complex condition, affecting more than 10% and compromising the quality of life of 2% of the population, with multiple contributors, often unknown, and enigmatic pathophysiology. The available treatment options are unsatisfactory, as they can, at best, reduce tinnitus severity, but not eliminate its perception. Given the spread of tinnitus and the lack of a standardized treatment, it is crucial to understand the economic burden of this condition. We conducted a systematic review of the literature on PubMed/MEDLINE, Embase, the Cochrane Database of Systematic Reviews (CDSR) and Google Scholar, in order to identify all the articles published on the economic burden of tinnitus before 1 April 2021 . Out of 273 articles identified through our search strategy, only five articles from studies conducted in the United States of America (USA), the Netherlands and the United Kingdom (UK) provided data on tinnitus’s economic costs. Three studies provided mean annual estimates per patient ranging between EUR 1544 and EUR 3429 for healthcare costs, between EUR 69 and EUR 115 for patient and family costs and between EUR 2565 and EUR 3702 for indirect costs, including productivity loss. The other two studies reported an annual mean cost of EUR 564 per patient for tinnitus-related clinical visits, and total costs of EUR 1388 and EUR 3725 for patients treated with a sound generator and Neuromonics Tinnitus Treatment, respectively. Our comprehensive review shows a gap in the knowledge about the economic burden of tinnitus on healthcare systems, patients and society. The few available studies show considerable expenses due to healthcare and indirect costs, while out-of-pocket costs appear to be less financially burdensome. Comprehensive health economic evaluations are needed to fill the gaps in current knowledge, using a unified method with reliable and standardized tools. Tinnitus is a continuous auditory perception in the ears or head in the absence of a corresponding external stimulus . TinnituWhen disabling and impacting daily functioning, tinnitus is a heterogeneous and complex condition with multiple associated biological, psychological and contextual contributors, which often remain unknown . MoreoveConsequently, people suffering from this condition try out various possible treatments, one after the other , and refThus, tinnitus management carries a significant financial burden for both healthcare systems and society, including patients . ExpenseGiven the spread of tinnitus and the currently still relatively low investment in tinnitus research , which lThe following scientific databases were considered to conduct this systematic literature search: PubMed/MEDLINE, Embase and the Cochrane Database of Systematic Reviews (CDSR). A search on Google Scholar was also performed in order to identify relevant articles published in scientific journals not indexed in those databases. In addition, the databases of the World Health Organization (WHO), Eurostat and the Organisation for Economic Co-operation and Development (OECD) were consulted to retrieve possible additional relevant information about direct and indirect costs associated with tinnitus and related quality-adjusted life years. The search strategy was designed for PubMed and then adapted for use in the other databases. Search terms included terms for the condition and the considered outcome ). We did not apply any restriction on publication time, considering all scientific articles published in English before the search date. Articles published in a language other than English were excluded from the review. Reference lists of other reviews were also checked to identify other potentially relevant publications.On 1 April 2021, we applied the search strategy. Overall, through PubMed, 137 publications were identified, with an additional 14 on Embase, 28 on CDSR and 170 publications on Google Scholar. Excluding duplicates, we obtained a total of 273 publications .In order to be eligible, studies had to provide data on direct or indirect costs due to the management of tinnitus disability in human subjects. We excluded studies on animals or biological tissues. We did not apply restrictions on the type of study design; thus, studies considered for inclusion were: cross-sectional studies (including population-based surveys on specific sub-populations), case–control studies, cohort studies, clinical trials and case series. Both original articles and reviews were considered, while unpublished studies, conference abstracts and proceedings, dissertations, theses and, more generally, non-peer-reviewed papers were not considered.We combined those publications detected on various databases in an EndNote library . In the first screening, two reviewers evaluated titles and abstracts independently to identify publications that met our inclusion criteria. The reviewers independently assigned scores 1–5 to the articles as follows: 1 = Publication not pertinent or of limited interest for our review; 2 = Publication probably not pertinent or of limited interest; 3 = Not possible to evaluate based on title/abstract/keywords only; 4 = Publication probably pertinent or of interest; 5 = Publication pertinent or of clear interest for our review. After the first screening, the two reviewers’ scores were added, obtaining total scores ranging from 2 to 10. A publication with a combined score of 4 or less was not considered for further evaluation. In the second screening, the full text of all the potentially eligible studies was retrieved and independently assessed for eligibility by two reviewers.For each publication satisfying the eligibility criteria, we collected: (a) general information on the publication , (b) study characteristics , (c) data on direct and indirect costs for tinnitus management, and (d) the model used to compute the estimates (including adjustments). In order to allow for comparisons, all costs were converted from the local currency into EUR . Data extraction was performed using Microsoft Excel and EndNote X7.Out of 273 identified publications, 26 were included after the first screening of titles and abstracts. Of these, only five articles met the eligibility criteria and were included in the present systematic review. No relevant additional information was found in the databases of the WHO, Eurostat and OECD. A cross-sectional study from the Netherlands provided direct and indirect annual mean costs per patient. Total annual mean healthcare costs added up to EUR 1544, patient and family costs to EUR 69 and costs due to productivity loss to EUR 3702 [A randomized controlled trial (RCT) from the Netherlands conducted in 2007–2011 on 492 tinnitus patients estimated annual mean direct and indirect costs per patient divided by usual care (UC) and specialized care (SC). The study provided healthcare costs , patient and family costs  and costs due to a loss of productivity . Thus, estimates for the total annual costs  were EUR 5980 for UC and EUR 6283 for SC .The last eligible included study was a cohort study conducted in the UK, estimating mean annual direct costs expressed as healthcare costs only. The mean annual total healthcare costs were EUR 1938 . IndirecThe costs identified in studies that provided healthcare, patient and family and indirect costs are graphically presented in This is the first systematic review summarizing direct and indirect costs for tinnitus management in the current practice, based on the published scientific literature. Although there is a wide consensus among scientific and clinical professionals that tinnitus is associated with high direct and indirect healthcare costs, our systematic review identified only five studies that calculated costs for tinnitus management ,20,22,23One aspect that emerged from most studies in the scientific literature and other relevant databases was the lack of comprehensive and detailed information on tinnitus costs, except for two studies from the Netherlands. Cost categories ideally include both direct costs  and indirect costs, including societal costs  ,24. ThisIn our systematic review, two of the studies reporting information on the economic burden of tinnitus were conducted in the US ,23, two However, an attempt for a broad estimation of the total costs in the EU could pursue the following path: The first step would be to broadly translate expenses to the average income of these countries and then consider the average EU income. The second step would be to extend these estimates to the whole EU, taking into account that various epidemiological studies estimated a prevalence of tinnitus exceeding 10%, which means that at least 30 million people in the EU live with tinnitus.Additionally, comparisons between countries on the costs of various treatments for tinnitus management are lacking in the current literature. For future health economic evaluations of treatments for disabling tinnitus, consensus on a set of standardized and homogenous evaluative tools is of high importance, in order to facilitate cross-country and cross-study comparisons.Only one study conducted in the Netherlands investigated the impact of tinnitus severity on costs, showing that the more disabled patients had significantly higher healthcare costs compared to patients with mild to moderate complaints . In factTo our knowledge, this is the first systematic review providing a synthesis of the healthcare and societal costs of tinnitus at an individual level in the scientific literature. Strengths of this review include the comprehensive search strategy conducted on a large set of scientific databases, including PubMed/MEDLINE, Embase and the CDSR. Moreover, an additional search on Google Scholar and databases of the WHO, Eurostat and OECD was conducted in order to check for relevant articles published in scientific journals not indexed in the previously mentioned databases. The detailed procedure used for selecting articles and data extraction further assures the qualitative value of this systematic review. Limitations include the relatively small number of included studies, leading to low generalizability of results across countries and across patient groups.https://uniti.tinnitusresearch.net accessed on 1 April 2021). Within UNITI, a large multicenter randomized controlled trial will be carried out in five clinical centers across Europe. The cost analysis methodology of the studies from the Netherlands [This systematic review found only a few studies dealing with the economic burden of tinnitus, none of which were published in recent years (2018–2021). Thus, it is important to be able to make some updated and validated statements about the economic impact of tinnitus on the individual and society. Future studies are needed to provide new detailed data on the healthcare and societal costs for tinnitus. Such studies are useful today because of their implications for health policy . The framework for calculating tinnitus costs must include data from various countries see  and mustherlands ,19 will The current status of the scientific literature highlights a serious lack of studies estimating, in detail, the economic burden of tinnitus on healthcare systems, patients, their families and society. Available studies show considerable expenses due to healthcare costs and indirect costs—mainly costs for productivity loss—while out-of-pocket costs appear to be less financially burdensome. Findings also indicate a direct relationship between tinnitus severity and related costs. Insights from existing studies point to several under-investigated but tinnitus-relevant predictors, such as country of origin, socio-economic factors and individual patients’ sex, age, educational level and tinnitus severity and duration. Identifying the economic burden of tinnitus in various cost categories is crucial to better understand tinnitus healthcare organization and treatment implementation in current practice in various countries and consequently reduce unnecessary costly and ineffective treatment strategies for patients and healthcare systems.


Serology tests are extremely useful for assessing whether a person has been infected with a pathogen. Since the onset of the COVID-19 pandemic, measurement of anti-SARS-CoV-2-specific antibodies has been considered an essential tool in identifying seropositive individuals and thereby understanding the extent of transmission in communities. The Luminex system is a bead-based technology that has the capacity to assess multiple antigens simultaneously using very low sample volumes and is ideal for high-throughput studies. We have adapted this technology to develop a COVID-19 multi-antigen serological assay. This protocol described here carefully outlines recommended steps to optimize and establish this method for COVID-19-specific antibody measurement in plasma and in saliva. However, the protocol can easily be customized and thus the assay is broadly applicable to measure antibodies to other pathogens. The pathogen causing coronavirus disease 2019 (COVID-19) has been identified as a novel zoonotic coronavirus termed the severe acute respiratory syndrome coronavirus (SARS-CoV-2). Disease severity in individuals varies dramatically and ranges from asymptomatic disease to severe disease requiring hospitalization, oxygen supplementation, and intubation, and to death. IgG and IgM anti-SARS-CoV-2 antibodies are generated in most infected individuals, though mild and asymptomatic infections result in relatively low antibody levels ,4. DespiIgA is also detected in SARS-CoV-2-infected individuals, both in plasma and in saliva, and has been suggested to provide early neutralization of the virus. Similar to IgM, IgA antibodies have shorter longevity in plasma than IgG antibodies, although SARS-CoV-2-specific IgA antibodies in saliva have been described to persist for longer than in plasma . This suThe Luminex xMAP technology is based on commercially available COOH non-magnetic or magnetic beads that are read on a compatible Luminex instrument . Beads aThis protocol outlines steps required to optimize and set up a SARS-CoV-2 multi-antigen serological assay using magnetic carboxylated beads. The main experimental stages include coupling of SARS-CoV-2 protein antigens to the magnetic carboxylated beads  and evaluation of the coupled beads using either a dilution series of an antibody positive plasma or saliva pool or using antigen-specific monoclonal or polyclonal antibodies (time required approximately 3–4 h/plate depending on antibody isotype being measured and the compatible Luminex instrument used) summarized in Phosphate-buffered saline ,Monobasic sodium phosphate buffer pH 6.0 ,Sulfo-NHS ,N-Ethyl-N′-carbodiimide hydrochloride, Sigma, St. Louis, MO, USA, Cat # 03449-5G),EDC ,Tween-20 ,2O),MilliQ-water ,Bovine serum albumin ,7 bead/mL), Cat # MC100X01),Carboxylated microspheres ,R-PE AffiniPure F(ab’)2 Fragment, donkey anti-human IgM ,R-PE AffiniPure F(ab’)2 anti-human IgA-PE ,Goat F(ab’)Antigens ,Positive control ,Negative control ,96-well round-bottom plate, clear ,96-well flat-bottom plate, black ,Microcentrifuge tubes ,Falcon tubes ,Magpix Calibration Kit ,Magpix Verification Kit , andMagpix Drive Fluid .Magnetic plate washer ,Bio-Rad Sure Beads magnetic rack ,Ratek Platform shaker ,Vortex Sonicator (Branson 2200),BioSan Vortex V-1 plus,Table centrifuge (Eppendorf Centrifuge 5424), and® Multiplexing System .MAGPIXE. coli and Baculovirus-insect cells), supplier and relevant protein description. For the coupling of SARS-CoV-2 and control proteins, magnetic carboxylated beads were sourced from Bio-Rad  and stored at 2–4 °C.SARS-CoV-2 and control recombinant protein antigens included in this study were either obtained commercially or kindly provided by collaborators. See 5 pre-activated microspheres (6 pre-activated microspheres were used to prepare a larger quantity of coupled antigens (bulk coupling) that can subsequently be used to screen plasma or saliva from approximately 3000 individuals with unknown COVID-19 status.The optimal protein amount to couple to magnetic beads was first determined. All proteins were initially coupled to 6.25 × 10ospheres  at vario5 beads) and 200 µL beads for the bulk coupling phase (2.5 × 106 beads) to a microcentrifuge tube.Sonicate magnetic microsphere stocks for 15 s followed by 10 s vortexing, before transferring 50 µL beads in the trial coupling phase  by vortexing for 20 s.Remove the tubes from the rack and resuspend beads in MQ-HPlace tube back into magnetic separator rack for 30–60 s and while positioned in the rack remove supernatant, taking care not to disturb the beads.Remove the tubes from the rack and resuspend in 100 mM monobasic sodium phosphate buffer, pH 6.2  and vortex for 20 s.2O), mix by gentle vortex for 10 s and add EDC (50 mg/mL in MQ-H2O), mix again by gentle vortex for 10 s  and incubate while rotating in the dark for 20 min, to activate the beads. CRITICAL STEP Sulfo-NHS and EDC should be made fresh on the day of use.Add sulfo-NHS , vortex for 20 s and place in magnetic separator for 30–60 s and remove the supernatant.Remove tubes from magnetic separator and continue the washing with PBS one more time .Resuspend the activated and washed beads in PBS , vortex for 20 s and put aside for the protein coupling step.Minimize exposure of the magnetic beads to light by covering the beads in all steps as much as possible.PAUSE STEP Beads can also be left over night at 4 °C, on a rotator.Add the appropriate volume of each protein, which corresponds to the protein concentration being tested, to the activated beads and incubate while rotating in the dark for 2 h at room temperature. Place tubes again into a magnetic separator rack and allow separation for 30–60 s and remove supernatant without disturbing the beads.Resuspend the beads in 500 µL PBS-TBN , vortex for 20 s and repeat the washing two more times .Note: Bead loss is minimal when using magnetic beads and the magnetic separator racks and thus counting of beads after coupling are not required. However, a loss in bead numbers could occur due to the various centrifugation steps, when using non-magnetic beads. If non-magnetic beads are used, counting of the bead numbers under the microscope after final resuspension is recommended to achieve consistent final bead-concentration. PAUSE STEP Store the coupled beads at 4 °C in the dark.After the final removal of supernatant, resuspend the beads in PBS-TBN , vortex for 10 s. n = 10) was collected from healthy individuals in June 2019 prior to the pandemic samples. Depending on linearity and the relative MFI of each coupled protein, the protein amount used for the trial couplings needs to be adjusted. Previous qualitative assessments have confirmed the stability of protein-coupled beads by comparison of the MFI of the standard curve over a nine-month period [The reactivity of the coupled beads is evaluated by a multiplex antibody assay, using a dilution series of an antibody positive plasma or saliva pool to obtain a log-linear standard curve with a relative high mean fluorescence intensity (MFI) of preferably 10,000 or higher. The plasma and the saliva pool used in this study consisted of convalescent plasma or saliva from 10 SARS-CoV-2 qPCR-positive individuals, collected approximately 30 days after onset of symptoms (plasma) or within the three first months after diagnosis . The naïve control plasma .In the 96-well round-bottom dilution plate, we use one well of blank, consisting of 100 µL PBT buffer .One well of negative control, consisting of 99 µL PBT and 1 µL of the negative plasma or 90 µL PBT and 10 µL of the negative saliva .In wells A3–A12, we add 75 µL PBT to each well, except in well A3, in which a total of 147 µL PBT and 3 µL of the positive plasma pool or 135 µL PBT and 15 µL of the positive saliva pool will be added to achieve a 1:50 dilution or 1:10 dilution, respectively (S1). Two-fold serial dilution of the standard is achieved by transferring 75 µL after thorough mixing from A3 to A4, and consecutively from each well to the next thereafter until A12. This will provide a dilution series of the positive pool from 1:50 to 1:25,600  in the remaining wells of the 96-well round-bottom dilution plate. Plasma (1 µL/well) or saliva (20 µL/well) is added to yield a 1:100 dilution and 1:5 dilution, respectively.Magnetic beads are prepared and added to a 96-well flat-bottom plate (assay plate). The magnetic bead mix is prepared by vortexing the individual coupled bead regions for 30 s each, before adding 0.1 µL/well of each  to a total of 50 µL PBT/well in a master bead mix. A volume of 50 µL PBT, containing 0.1 µL of each of the coupled beads, are added to each well of the 96-well flat-bottom assay plate.After the addition of the bead mix to all wells, 50 µL of the plasma or saliva dilutions and control dilutions are added to each well on the black flat-bottom assay plate. This mix of coupled beads and plasma/saliva samples is incubated for 30 min (IgG and IgM) or 2 h (IgA) at room temperature on a shaker in the dark, followed by washing the plate 3× with 100 µL PBT, using a magnetic plate washer or a hand magnet.2 anti-human IgG, IgM or IgA to the plate, mix well and incubate for 15 min at room temperature on a shaker in the dark.After the washings, add 100 µL of a 1:100 dilution of PE-conjugated Donkey F(ab’)After a further washing of the plate, add 80 µL PBT to each well, mix well and keep on a shaker until plate is inserted and read on a compatible Luminex instrument.At least 15 beads of each region/antigen are then acquired and analyzed.To assess the reactivity of antibodies to the selected proteins, antigen-specific IgG, IgM or IgA was detected by incubating 500 beads of each antigen per well with plasma at a 1:200 final dilution or saliva at a 1:10 final dilution. ed beads  and to aThe results are expressed as the mean fluorescence intensity (MFI) of at least 15 beads for each antigen in a log-linear standard curve. The standard curve on each plate is used to convert each individual MFI value of the unknown samples to a relative antibody unit (RAU), which enables comparison between samples assessed in separate experiments and plates .The basis of the assay relies on the attachment of proteins to the beads, after these have been activated as described before. As proteins vary widely in their structure, purity and the methodology in which they have been expressed, careful optimization for each antigen is required. The optimum antigen concentration is established when a log-linear standard curve is achieved, aiming for a relatively high MFI value for the 1:50 dilution or 1:10 dilution , using the convalescence plasma or saliva pool. The range of protein concentrations tested, and the determined optimal concentration required is detailed in Optimal standard curves for SARS-CoV-2 antigens  and other antigens (influenza A (H1N1 and H3N2), influenza B (Phuket and Victoria), seasonal coronaviruses NL63 NP, OC43 Spike, 229E S1 and HKU1 Spike, as well as tetanus toxoid) are presented in We utilized a pool consisting of convalescent plasma from individuals with confirmed SARS-CoV-2 to establish the standard curves and a plasma pool consisting of plasma collected from individuals prior to the start of the COVID-19 pandemic in 2019 to determine background levels accounting for non-specific binding to antigen and beads. However, plasma may not always be readily available and human monoclonal antibodies can be used as an alternative approach to establish optimal standard curves. Here, we assessed the capacity of commercially available human monoclonal antibodies to recognize SARS-CoV-2 S1, S2 and NP antigens in our panel . The antThe MFIs of the bound antibody for both antigens were comparable, but S1 was recognized to a lesser extent d. None oConsidering that S1, RBD and Spike as well as S2 are overlapping regions, multiplexing a panel that consists of these closely related antigens may result in competition and antibody levels to certain antigens may incorrectly appear lower than the actual levels. Similarly, cross-reactivity to corresponding antigens of related pathogens  may affect accurate measurement of these antigen-specific antibody levels. To address this in the current panel, we compared the standard curves for SARS-CoV-2 antigens  and seasonal coronavirus antigens OC43 Spike and HKU1 Spike, when all the other antigens were included (multiplex) to when they were assessed separately (single-plex). There was no difference between the multiplex and the single-plex total IgG standard curves using human plasma for any of the antigens .IgM may be more relevant to assess in plasma collected at a relatively early time point after diagnosis. To test the adaptability of our panel to measure antigen-specific IgM levels, we used an anti-IgM detector antibody at various dilutions , usually require a sample volume equal to or larger than what is necessary for multiplex assays. In addition, it is well appreciated that measurements of multiple factors/cytokines/antigen-specific antibodies are more biologically relevant. In this way, multiplex assays save valuable patient/participant serum or plasma, while providing a comprehensive analysis of the sample. However, in antigen panels that include multiple domains or regions of a single protein, the signal for any given antigen of that protein may be diminished. This may occur if antibodies preferentially bind particular epitopes of one antigen, which is represented in several domains included in the panel. In the current panel described here, this would represent S1, S2, RBD and Spike. In these cases, it is recommended to compare the signal obtained from analysis of the individually coupled beads with the plasma pool and/or monoclonal antibody (single-plex) with the signal obtained from when all the beads are analyzed simultaneously (multiplex). We found that the signals for the antigens included in the described panel here were comparable when analyzed as single- or multiplex.Methods to measure SARS-CoV-2-specific antibodies in saliva are advantageous due to the ease of collecting the sample and have been used during the pandemic to support epidemiological surveillance or as alternative diagnostic tools ,24,25. N


A three-stage conceptual scenario is proposed to illustrate the formation mechanism of the PA layer in NF membranes, which is due to the variation of surface hydrophilicity of the supports. The overall findings specify how the surface hydrophilicity of the supports influences the formation of PA layers, which ultimately defines the separation performances of the corresponding NF membranes. Our current study experimentally evaluates the impacts of surface hydrophilicity of supports on the properties of polyamide (PA) thin-film composite (TFC) nanofiltration (NF) membranes. A series of “carboxylated polyethersulfone” (CPES) copolymers with an increasing “molar ratio” (MR) of carboxyl units were used to prepare supports with diverse surface hydrophilicities by the classical nonsolvent-induced phase separation (NIPS) method. Then, the PA-TFC NF membranes were finely fabricated atop these supports by conventional interfacial polymerization (IP) reactions. The linkages between the surface hydrophilicity of the supports and the characteristics of the interfacially polymerized PA layers as well as the permselectivity of NF membranes were investigated systematically. The morphological details of the NF membranes indicate that the growth of PA layers can be adjusted through increasing the surface hydrophilicity of the supports. Moreover, the separation results reveal that the NF membrane fabricated on the relatively hydrophobic support exhibits lower permeability (7.04 L·m Currently, the most predominant nanofiltration (NF) membranes and reverse osmosis (RO) membranes are prepared as polyamide thin-film composite (PA-TFC) membranes to produce freshwater from seawater and brackish groundwater ,2,3. A cFrom the lab bench to the market scale, the PA layer of TFC membranes is typically synthesized via the interfacial polymerization (IP) of diamine derivatives in the aqueous phase and trifunctional acyl chloride in the organic phase on the porous support membrane . During Recently, some studies have demonstrated that surface porous characteristics , a general and reliable understanding regarding the effects of surface hydrophilicity of supports drawn from independent research is still being debated. For example, Ghosh et al.  suggesteIn view of this, through the preparation of NF membranes on our self-made supports by an IP reaction, the influences of the surface hydrophilicity of supports on the properties of TFC membranes are investigated emphatically in what follows. Generally, supports with a wide variety of characteristics may be obtained via three different approaches: (i) bulk modification of the support materials (pre-modification) , (ii) blBased on the synthesis of a series of CPES copolymers with a different “molar ratio” (MR) of carboxyl units in the total molecule units (changed from 0% to 100%), which can be calculated via Equation (1), the supports were fabricated by the same NIPS method. Herein, the effects of the MR of carboxyl units in the CPES copolymers on the morphology, surface properties and separation performances of supports were studied in detail. Then, the NF membranes were prepared through IP on the above supports. The characteristics of the PA layers and the permselectivity of the corresponding TFC membranes were investigated thoroughly to shed light on the role of the surface hydrophilicity of the supports. Furthermore, the likely formation mechanism of the PA layers affected by the surface hydrophilicity of the supports during the IP process was elucidated. What needs to be pointed out is that the optimized parametric studies  of PA layers during the IP reaction are not reported in this paper.4) and hydrochloric acid (HCl) along with sodium hydroxide (NaOH), were purchased from Aladdin Reagent Co. Ltd.  and used as received without any further purification. Commercial polyester (PET) nonwoven fabrics with an areal density of 52.7 g/m2 were provided by Teijin Furuite Co. Ltd.  and preserved in a warehouse with constant temperature (25 °C) and humidity (<20%). Unless mentioned otherwise, laboratory-prepared deionized water with conductivity <1 μS/cm was used in all experiments.As shown in The supports were prepared by the NIPS method. Firstly, according to the concentration of the casting solutions (21 wt%), certain amounts of CPES copolymers with a different MR of carboxyl units and NMP were accurately weighed and then added to airtight glass bottles. After being stirred at 60 °C for 12 h, homogeneous casting solutions were obtained, which needed to be left still until all air bubbles disappeared before use. Next, the casting solutions were spread on the PET nonwoven fabrics with the initial film thickness of 230 μm using fully automatic equipment, and, subsequently, the casting films were immediately immersed into a coagulating bath (deionized water) at 15 °C to induce the phase inversion process. Finally, the resultant supports were washed thoroughly with deionized water and later stored in a refrigerator maintained at 4 °C.w/v) PIP at 25 °C for 3 min. Then, the wetted supports were taken out and subjected to the air (RH: 50~60%), and the excess PIP solution was removed from the surface of the supports using a clean rubber roller. After that, the PIP-soaked supports were brought into contact with a hexane solution of 0.1% (w/v) TMC at 25 °C for 1 min, leading to the formation of ultrathin PA layers over the supports. Moreover, after their exposure to the hexane–TMC solution, the membranes were immediately placed in an oven at 75 °C for 4 min. Ultimately, the resulting NF membranes were washed thoroughly with deionized water and stored in a laboratory refrigerator maintained at 4 °C prior to use.The NF membranes were synthesized on the top of the supports prepared previously via the IP process. Initially, the supports taped to the glass plates were impregnated with an aqueous solution of 0.2%  and the “total thickness of support and nonwoven fabrics” (δt) were measured using an oil-proof digital display thickness gauge  at eight different locations for each sample, and their average values were taken as the final result. Consequently, the “thickness of support” (δS) can be computed through Equation (2).The “thickness of nonwoven fabrics”  . To observe the cross-section morphologies, the wet samples needed to be freeze fractured in liquid nitrogen before drying. Then, an Apreo S SEM  was adopted to visualize the cross-section morphology of the supports, and a JSM-7500F FESEM was used to analyze the cross-section morphology of the PA layer in the TFC NF membranes.2 topographical surface image of the dried samples was scanned using the tapping mode in air. The measured results were reported in terms of root mean square  roughness. At least five random regions were scanned for each sample to obtain the final average roughness result.A quantitative surface roughness analysis of all the membranes was estimated by a Smart SPM AFM (AIST-NT). For each test run, a 5 × 5 μmThe relative surface hydrophilicity of all the membranes was evaluated by the sessile drop method using a DSA10 contact angle measuring instrument . At least five valid equilibrium contact angles defined by the average of the right and left contact angles of the deionized water on each dried surface of the samples were obtained. Finally, the average contact angle and the standard deviation of the membranes were calculated using the above test data.To reveal the surface structure of the PA layer in the TFC NF membranes, the detailed surface elemental information of the PA layers was assessed by a Kratos AXIS Ultra XPS  employing a monochromatized Al-Kα X-ray source (1.49 keV). Then, the Casa XPS software was adopted to perform the XPS analyses. (Note: the binding energy offset was corrected based on the binding energy of adsorbed carbon at 284.6 eV.)−3 m2). The dead-end filtration mode was adopted to measure the PWP. After the temperature of the deionized water in the storage tank was adjusted to 25.0 °C, the inlet pressure was slowly boosted to 0.150 ± 0.005 MPa along with a pre-pressure of 30 min. After that, the pressure was dropped to 0.100 ± 0.005 MPa, and then the test was run for 10 min in a steady state. Finally, we recorded the permeated volume of water that was collected in a small cylinder over 10 min since the beginning of the test. Equation (3) is the calculation formula of PWP.2) is the effective filtration area. Note: the end result of PWP for each support is the average of five test runs.The “pure water permeability” (PWP) and “rejection rate” (R) of the supports were evaluated via the standard permeation test with a lab-built filtration apparatus as illustrated in −1) was pumped into the storage tank and maintained at 25 ± 0.5 °C, the inlet pressure was slowly adjusted to 0.10 MPa. After stabilizing for 10 min, both the feed solution and the permeate solution were collected and later assayed by a LiquiTOC/TNb total organic carbon (TOC) analyzer  to obtain the concentration values. The following Equation (4) was employed to calculate the rejection rates:p (mgL−1) is the calculated concentration of PEG in the permeate solution, and Cf (mgL−1) is the average of the initial concentration and the final concentration of PEG in the feed solution. Note: the final result of rejection rate for each support is the average of five test runs.To lessen the impact of concentration polarization, the cross-flow filtration mode was selected to obtain the rejection rate. Once the aqueous PEG solution (1000 mgLp (μScm−1) is the measured conductivity of the collected permeate solution, and kf (μScm−1) is the arithmetic mean of the initial conductivity and the final conductivity of the feed solution.The above filtration apparatus was also used to evaluate the separation performances of the PA-TFC NF membranes. After starting the booster pump, the globe valves were slowly adjusted to meet the test conditions listed in A series of supports (S-0~S-100) were fabricated with CPES copolymers by adopting the NIPS method. From the overall cross-section SEM images in The surface pore morphology measurement of the supports was performed with FESEM . However, as the surface pores were easily covered by the gold nanoparticles sprayed during the sample preparation process, it would be unrealistic to measure and calculate the pore characteristics from the surface FESEM images. Here, we used the molecular weight cut off (MWCO) see  to estimS), roughness (RMS) and hydrophilicity (CA) of the supports, are also summarized in Other characteristic results, such as the thickness  of a material, the “apparent contact angle” (θA) of a porous surface  obtained through the static sessile drop method, as described in I < 90°, see With the increase in the MR of the carboxyl units in the CPES copolymers, the surface contact angle of the supports reduces from 90.43° for S-0 to 82.12° for S-100 (details about the data can be seen in −2·h−1·bar−1 to 64.82 L·m−2·h−1·bar−1 without impacting their rejection rates (46.49~47.86%). Further, the PWP of S-60~S-100 decreases substantially from 53.99 L·m−2·h−1·bar−1 to 32.12 L·m−2·h−1·bar−1, whereas their rejection rates increase from 73.83% to 99.30%. That is, a typical trade-off between permeability and selectivity is shown in the separation performances of S-60~S-100. In a previous study [The correlation between the pure water permeability (PWP) and rejection rate (R) of the supports is plotted in us study , Kim andus study .(1)S-0 is featured with a relatively hydrophobic surface (CA: 90.43°).(2)S-40 possesses an increasing surface hydrophilicity (CA: 87.56°).(3)S-100 has a more hydrophilic surface (CA: 82.12°).To investigate the interrelation between the surface hydrophilicity of the supports and the properties of the NF membranes, three representative supports were carefully chosen as the sublayers in the following IP reaction:What is remarkable about the selected range of hydrophilicity (82.12°~90.43°) is that a further raising of hydrophilicity will cause excessive swelling, leading to a nonporous membrane, a poor membrane compressive property etc.N) of the PA layers is visually characterized by the length of the red drawn lines using Image J software. As listed in w/v)) on the formation of PA layers corresponding to a fixed TMC concentration (0.1% (w/v)) and other reaction conditions by Saha et al. [w/v)), the apparent thickness of PA layers increased with the increase in PIP concentrations. With further increasing PIP concentrations (1.5~5.0% (w/v)), the apparent thickness of the PA layers remained nearly unchanged, but the density of the PA layers increased. Additionally, through a molecular simulation of PA synthesis [The cross-section and surface micrographs of the PA layer in the NF membranes are presented in a et al. , it was ynthesis , Nadler N, the randomly selected protuberances are measured on each surface FESEM image of PA layers in N: 282 nm). Increasing with the surface hydrophilicity of the supports, the PA layer surface of N-40 appears to have more small nodular features (λN: 195 nm). Eventually, many even smaller nodules (λN: 142 nm) are observed on the PA layer surface of N-100 with the further increase in the surface hydrophilicity of the supports. The surface morphological variation of the PA layers can be explained as follows. Generally, interfacial polymerization initiates immediately after the contact of the aqueous phase and the organic phase, leading to the formation of PA films near the organic phase side of the aqueous–organic interface [Besides, the obvious differences in the aspects of number and size (λnterface . Based onterface  observedThe surface element information of the PA layer in the NF membranes is assessed by X-ray photoelectron spectroscopy (XPS), and the spectrum analyses and statistical results are provided in Further, the C1s core level XPS spectra of different NF membranes a are sepI) of materials on the surface of PA layers and the surface porosity coefficient (f) and the surface roughness coefficient (ψ) of PA layers jointly determine the apparent contact angle (θA) of PA layers. Obviously, both the surface porosity factor , the PA layer of N-40 with the lowest surface porosity and the highest surface roughness among all the NF membranes does not show a relatively good hydrophilicity. So, we speculate that as the surface hydrophilicity of the supports increases, the improved hydrophilicity of the materials on the surface of PA layers  would play a dominant role in the surface hydrophilicity of PA layers. As discussed earlier though, the relative concentration of carbon atoms in C*=O on the surface of the PA layers is inconsistent with the surface hydrophilic result of the PA layers. This may be due to the fact that the concentration result of the elements obtained by XPS is a relative value expressed in percentage. Then, it is highly possible that the formation of the PA layer surface prepared on the support with a better hydrophilicity would undergo interfacial polymerization more times, causing a large absolute number of hydrophilic functional groups.However, as presented in −2·h−1·bar−1 (N-0) to 19.25 L·m−2·h−1·bar−1 (N-40), while the MgSO4 rejections decrease from 89.94% to 66.98%. Further, the water permeability of N-100 decreases to 14.64 L·m−2·h−1·bar−1, and the MgSO4 rejection increases to 73.48%.The surface hydrophilicity of the supports has a critical influence on the separation performances of the NF membranes as shown in For a long time, many studies focusing on the transport through osmotic membranes related the permselectivity of the TFC membranes to their distinct physicochemical properties when the solute materials were fixed . Some reFurthermore, noting the typical trade-off relationship between the permeability and selectivity of NF membranes, it is suggested that the compactness (represented in terms of cross-linking degree) of PA layers should be the major cause for the separation performances. However, our conjecture is inconsistent with the surface cross-linking degree of PA layers (i.e., the ratio of O/N shown in We noted that previous theoretical and experimental studies ,43 demonOverall, in In the first phase, immediately after the aqueous phase contacts with the organic phase, interfacial polymerization initiates, leading to the formation of nascent films ,7. With In this study, by adopting a series of carboxylated PES copolymers featuring an increasing molar ratio of carboxyl units, supports with different surface hydrophilicities were fabricated via the NIPS method. For the PA-TFC NF membranes, due to the fact that the concentration of PIP in the aqueous phase actually involved in the reaction decreases with the increase in the surface hydrophilicity of supports, thin PA layers with a low cross-linking degree of dense cores were formed on the relatively hydrophilic supports. On the contrary, a thick PA layer with a high cross-linking degree of dense core was synthesized on the relatively hydrophobic support. So, the NF membrane prepared on the hydrophobic support has a lower water permeability and a higher salt rejection than the ones prepared on the hydrophilic supports. Our direct experimental evidence has further revealed the impacts of the surface hydrophilicity of supports on the characteristics and permselectivity of the resulting PA-TFC NF membranes. In the future, this paper is bound to assist us in selecting suitable hydrophilic supports for achieving the desired NF membrane performances.


Although many studies have observed genome-wide host transposon expression alteration during viral infection, the mechanisms of induction and the impact on the host remain unclear. Utilizing recently published influenza A virus (IAV) time series data and ENCODE functional genomics data, we characterized virus induced host differentially expressed transposons (virus-induced-TE) by investigating genome-wide spatial and functional relevance between the virus-induced-TEs and epigenomic markers (e.g. histone modification and chromatin remodelers). We found that a significant fraction of virus-induced-TEs are derived from host enhancer regions, where CHD4 binding and/or H3K27ac occupancy is high or H3K9me3 occupancy is low. By overlapping virus-induced-TEs to human enhancer RNAs (eRNAs), we discovered that a proportion of virus-induced-TEs are either eRNAs or part of enhancer RNAs. Upon further analysis of the eRNA targeted genes, we found that the virus-induced-TE related eRNA targets are overrepresented in differentially expressed host genes of IAV infected samples. Our results suggest that changing chromatin accessibility from repressive to permissive in the transposon docked enhancer regions to regulate host downstream gene expression is potentially one of the virus and host cell interaction mechanisms, where transposons are likely important regulatory genomic elements. Our study provides a new insight into the mechanisms of virus-host interaction and may lead to novel strategies for prevention and therapeutics of IAV and other virus infectious diseases. The findings have been confirmed across multiple organisms, e.g. human, mouse, and drosophila2 and numerous viral species including HIV13 and SARS-CoV-24, which indicates that the host transposon expression alteration is a fundamental phenomenon of viral infectious diseases. Although hypotheses and speculation about possible roles of virus induced differentially expressed TEs (virus-induced-TEs) are emerging, understanding the mechanism of induction and impact of transposon expression alteration on the host remains largely unclear. A recent report shows that TEs are involved in chromatin looping and CTCF bindings5. In Arabidopsis thaliana plants, the DDM1 mutation causes significant reduction of DNA methylation in the TE loci, where Snf2 family member DDM1 as a chromatin remodeler plays an important role in suppression of TE transcription6. Igolkina, et al. also reported that the connection between TEs and histone markers may impact gene expression regulation7. These interesting findings are suggestive of possible gene expression regulation roles by TE transcripts, which prompted our investigation into the genomic spatial relationship between the virus-induced-TE and permissive and/or repressive epigenomic markers, such as histone markers, chromatin remodelers, and transcription factors.We recently demonstrated that host transposon element (TE) expression is largely impacted by viral infection9, including on histone occupancy/transcription factor binding sites (ENCODE project10) and computational algorithms for functional genomic element predictions11, facilitate a deep investigation into the virus-induced-TEs and their spatial relationship to the functional genomic elements. Taking advantage of our recently published studies on influenza A virus (IAV)12, we further characterized host virus-induced-TEs by examining their spatial relationship to known functional genomic elements such as chromatin histone markers. We have found that a significant number of virus-induced-TEs (~ 9%) are derived from the chromatin histone marker H3K27ac pre-occupied regions, of which a fraction are also chromatin remodeler CHD4 binding sites. In addition to the H3K27ac occupied genome regions, the chromatin histone marker H3K9me3 regions are also found to accommodate about 2.2% of virus-induced-TEs. In a parallel analysis, by querying a computational predicted human enhancer database with virus-induced-TEs, we discovered that about 9.6% of IAV-induced TEs overlap the predicted distal enhancers. Querying the same enhancer database with other viral datasets14, we revealed that about 7–9% of SARS-CoV-2-induced TEs overlap the predicted enhancers, with variation in different experiments; about 7.7–7.9% of RSV and 9.8% of HPIV3 induced TEs overlap the predicted enhancers, respectively.Advances in genome-wide enhancer RNA studies15,16,17. We found that ~ 9.2% of IAV-induced-TEs overlap eRNAs (TE eRNAs). Furthermore, we also found that about 44% of the TE eRNA targets (2144) are overrepresented in the differentially expressed gene list (4824 DE genes) of IAV infected samples, in which both upregulated and downregulated TE eRNA targets are significantly overrepresented in the upregulated DE genes (p < 0.05).To understand the potential functional role of virus-induced-TEs in the host, we carefully examined the genomic loci of IAV-induced-TEs to known enhancer RNAs (eRNAs). We did this due to the important regulatory roles eRNA plays in the genome for tissue specific gene expression regulationTaken together, our study is the first evidence of a connection between a subgroup of IAV-induced-TEs and host enhancer RNAs. Our results indicate that the enhancers docked by this group of IAV-induced-TEs are likely less permissive and/or poised prior to virus infection. However, such enhancers become more accessible and active during virus infection, resulting in host transposon expression changes and possible downstream gene expression alteration. Based on our data, we introduce a new virus-host interaction model, showing that viruses likely influence host gene expression by activating poised enhancers and promoting eRNA transcription, where host TEs may play complex roles in the virus host interaction and gene expression regulation. This model is based on IAV but may also apply to other RNA viruses including SARS-CoV-2, RSV and HPIV3. Our discovery provides new insight into the mechanisms of virus-host interaction and may lead to novel preventive and therapeutic targets for IAV and other RAN virus infection diseases.All virus infection data used for this study was obtained from the public database (e.g. GSE147832 is an accession number of the NCBI GEO database). The data analysis scheme is highlighted in Fig. We further analyzed and plotted the top differentially expressed TE families. The same TE family or subfamily can be presented in both up and down-regulated categories due to the nature of individual TE families spreading over hundreds or thousands of different genomic locations Fig. C. In gen10. Although the histone modification data was not available for IAV infected A549 cells at the time we conducted this study, data from non-infected A549 cells provided a good foundation to measure the genomic spatial relationship of virus-induced-TEs. After exploring the ENCODE data matrix10, we exploited 6 histone marker and 10 chromatin remodeler datasets for a genome-wide spatial relationship analysis of virus-induced-TEs. All ENCODE datasets were derived from control experiments in A549 cells without treatment, which is comparable to the mock samples of IAV time series data. The name and label of the ENCODE datasets used in this analysis are listed in Supplemental Table 18. Although only a small fraction of virus-induced-TEs (mostly upregulated TEs) were derived from CHD4 binding regions, this suggests that TE transcriptional repression by CHD4 was removed from these regions, which further indicates that the chromatin status changes from repressive to permissive in these genome regions to promote the TE transcription during IAV infection.The ENCODE database contains many high-quality datasets for us to study the spatial relationship between TE transcripts and histone markers as well as TE transcripts and chromatin remodelersOf the 6 ENCODE histone markers, we found that H3K27ac has a higher occupancy in the virus-induced-TE regions in non-infected A549 cells  of CHD4 binding sites are also occupied by H3K27ac , upregulated DE TEs, and downregulated DE TEs (FDR < 0.01). We also generated two control datasets; a random selection of 12,000 non-DE TEs , and a random selection of 15,000 TEs from the entire TE pool regardless of expression . Each of the test and control datasets overlap the known human eRNAs during analysis, obtaining a percentage of eRNA overlapped TEs, respectively. By comparing the eRNA overlapped TEs of test datasets to controls, we may determine whether the virus-induced-TEs are prone to eRNAs  , 244 overlapped the DE TEs that are derived from H3K27ac regions . These TE-eRNA targets were compared with differentially expressed genes (DE genes) of IAV time series samples. The hypergeometric test was subsequently applied for the overrepresentation significance assessment. We found that both down and up-TE-eRNA targets are enriched in the upregulated DE genes . Applying hierarchical clustering and GO TERM analysis for eRNA DE genes, we found that the IFN pathway, virus defense and JAK-STAT pathway genes are enriched in cluster 1, where the expression level increases as virus replication progresses  the host upregulated TEs temporally correlate with virus replication, (2) a significant number of IAV induced DE TEs are derived from host enhancer regions; the spatial relationship between DE TEs and enhancers also exists in SARS-CoV-2, RSV and HPIV3 infected samples, suggesting such a genomic relationship between virus-induced-TEs and enhancers is likely common in RNA virus-host interaction, (3) the IAV-induced-TEs are either eRNAs or part of eRNAs, which originate from the enhancers with three different epigenetic modifications, and (4) the TE-eRNA genes are significantly overrepresented in the DE genes of IAV infection time series samples, which indicates that during IAV infection the host DE genes or a proportion of DE genes are likely influenced through TE related enhancers and eRNA pathways. Our results suggest that IAV likely interacts with host cells by changing the chromatin accessibility from repressive to permissive in the enhancer regions, regulating host downstream gene expression, where TEs are docked and likely play important regulatory roles.1 and another group also found virus induced transposon expression in Drosophila somatic tissues2. These results from independent studies suggest that viral impact on host transposon expression is likely a common phenomenon across many organisms and viral species. However, many virus induced transposon transcripts are influenced by gene expression due to intron retention and gene readthrough27. In humans, only about 9–10% of transposon transcripts are from intergenic genome regions1, which makes study of this phenomenon even more challenging due to interference of gene transcripts. Even though TE transcription is real, many questions still remain, e.g. what function TE transcripts have, and what roles they might be playing besides canonical transpositions. Therefore, further characterization of virus induced transposon transcripts is necessary to fully understand their functionality.For a long period of time, transposon element expression and its potential influences on gene expression regulation have not been deeply studied. Our previous work revealed that the virus induced transposon expression in human and mouse29. Therefore, TE activity, in particular canonical activity, is in general repressed in the human genome by multiple mechanisms of chromatin condensation, epigenetic transcriptional silencing, and targeting by small interfering RNAs30. Investigating the epigenetic modifications in non-infected cells for the TE regions can build a foundation for understanding virus-induced-TEs. We selected six histone markers and ten chromatin remodelers from the ENCODE data matrix, which represent permissive and repressive chromatin status in the A549 cell line that was also used for the IAV infection time series study. Surprisingly, about 4.3% of non-differentially expressed transposons are derived from H3K9me3 occupied regions; however, only about 1.9% of randomly selected transposon genomes widely overlap H3K9me3 occupied regions 13. The fastq files were first mapped to human genome assembly (Homo.sapiens.GRCh38) using bowtie2 aligner (v.2.3.4)37 with parameters . After sorting with samtools (v.1.9)38, the mapped reads were used to summarize genes (Homo_sapiens.GRCh38.87.gtf) and TEs (GRCh38_Ensembl_rmsk_TE.gtf) using featureCounts (v.1.5.1)39 with either strand parameter on (reverse) for projects GSE147832 and GSE147507, or strand parameter off for the project CRA002606 due to non-strand specific protocol applied in the sequencing. The outputs were subsequently used for statistical assessment for the differentially expressed genes and TEs by R/bioconductor package (version 4.0.3/3.12) edgeR (v.3.32.1)41. The cutoff for the genes and TEs is FDR < 0.01 with one exception of FDR less than 0.001 for Cov2 24 h sample of CRA002606 dataset.All the data in this study was obtained from the GEO database The raw fastq files of IAV infected samples were mapped to in-house bowtie2 index of influenza A virus . The bowtie2 index file along with the fasta sequences and annotation files for IAV and SARS-CoV-2 viruses are available upon request. The featureCounts was used for virus reads assignment. The count per million (CPM) was introduced for normalization of virus reads crossing time series samples, in which the virus reads were normalized against overall library reads (mapped virus reads + host gene reads) per sample for IAV dataset.10 were used for this study as listed in Supplemental Table 42 was used for generating epigenetic marker distribution around DE TE and control TE regions. To create genomic regions for plotting, each TE was extended from the center of the TE to both up and down stream for 3000 bps. The peak file of epigenomic markers was used for counting peak frequency in the TE regions with default parameters of ChIPseeker.ENCODE functional genomic data files43. The 0 bp gap between DE TEs and epigenetic markers was used for determining the spatial relationship of DE TEs.To screen the DE TEs that overlap epigenetic markers , we used bedtools (v.3.5)  for measuring the distance of two genomic featuresThe randomly selected 10,000 TE expressors  were used as the control for either plotting and/or statistical testing.11. The enhancer coordinates were updated to genome version HG38 with UCSC liftover utility before further analysis44. The distal enhancer data was then overlapped with DE TE loci using bedtools (v.3.5) . To calculate the percentage of distal enhancer related (overlapped) DE TE loci, the number of distal enhancers overlapped DE TE loci were divided by total number of DE TE loci in the same category or same time point. The randomly selected 15,000 TEs are the controls for plotting and statistical testing. Of 15,000 randomly selected TEs, less than 300 have expression values bigger than 0. In addition to the randomly selected TEs, we also used a non-DE TE as a control. The difference between the randomly selected TEs and non-DE TE control is that non-DE TEs have the detectable expression values.The distal enhancer dataset was downloaded from the Enhancer Atlas website (version 2.0)9. The enhancer RNA coordinates were updated to genome version HG38 with UCSC liftover utility before further analysis. To generate the distance matrix, bedtools (v.3.5)  was used for calculating the distance between two genomic features. The randomly selected TEs and non-DE TEs were used as controls for this study to assess the statistical significance. To screen TE eRNAs, we allowed only 0 bp gap between two features.The enhancer RNA dataset of whole human tissues was downloaded from HeRA atlasTo obtain TE eRNA target genes, we downloaded eRNA targets from HeRA atlas. We grouped TE eRNA targets into overall DE TE, up DE TE, or down DE TE related and plotted a venn diagram for visualizing the TE eRNA targets and differentially expressed genes (DE genes). The 2144 genes that are common between TE eRNA targets and DE genes were further used for hierarchical clustering analysis.45 was used for gene clustering and heatmap visualization. For the GO TERM analysis, we used clusterprofiler R/bioconductor package (v.3.18.1)46. We used IGV genome browser for navigating and visualizing the individual eRNA related DE TEs and their spatial relationship to epigenetic markers. The R function chisq.test and phyper were used for either chi-square or hypergeometric test for the statistical significance assessment in our study.The ComplexHeatmap R/bioconductor package (v.2.6.2)14 and NGDC database (CRA002606)13. The Chipseq data were downloaded from ENCODE database and dataset names are listed in Supplement Table The RNAseq data were downloaded from GEO database Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


Bovine viral diarrhea (BVD) is controlled in many countries by detection and culling of persistently infected (PI) animals. The most important risk factor for BVDV introduction is purchase. An introduced cow can be PI and transmit the virus to other cattle in the herd. If she is not PI but is pregnant, there is still a risk because the subsequently born calf may be PI, when she encountered the virus in early pregnancy. To control this risk, all cows > 1 year from non-BVDV-free herds that are introduced in herds that participate in the Dutch BVDV control program are tested for virus and antibodies. Depending on the results, subsequent measures such as suspension of the BVDV-free status, removing the animals from the herd, or testing the off-spring of the cow for virus, are undertaken. The aim of this study was to evaluate the results of this risk mitigating measure. Data on cattle movements, calving's, herd-level BVDV status, and animal-level test data were available from all dairy herds that participated in the national BVDV control program  for the year 2019. The data were combined and parameters of interest were calculated, i.e., (i) the number of purchased BVD virus positive cattle and (ii) the number of BVD virus positive calves born from purchased cows within 9 months after introduction. In 2019, 217,301 cattle were introduced in Dutch dairy herds that participated in the BVDV control program. Of these, 49,820 were tested for presence of BVD virus and 27 (0.05%) cows introduced in 21 different herds tested BVD virus positive. Out of 46,727 cattle that were tested for antibodies, 20.5% tested positive. The seropositive cows produced 4,341 viable calves, of which 3,062 were tested for virus and subsequently, 40 (1.3%) were found BVD virus positive. These 40 BVD virus positive calves were born in 23 herds. The risk mitigating measure led to detection of 67 BVD virus positive animals in 44 unique herds in 2019. This study makes plausible that the probability and impact of re-introduction of BVDV can be minimized by testing introduced cattle and their subsequently born calves. Flaviviridae family  is a pestivirus belonging to the e family . It was e family . An impoe family , 4. These family . Besidese family . The dise family , 8 and he family , 9. There family  to contrThese control programs aim to detect and remove PIs, and results over time give insight into their success . When erIn the Netherlands, about 50 percent of dairy herds regularly purchase cattle . These cIn the Netherlands, a voluntary BVDV control program has been in place since 1997. In this control program, cattle herds can obtain a BVDV-free status after a full herd screening for BVD virus . In 2018Testing of introduced cattle is mandatory in all routes and independent of the BVDV status of the herd of destination. When cattle, unless from BVDV-free herds, are introduced into a herd, the herd's status will be suspended and only regained when the introduced cattle have a negative test result for BVD virus. Besides, cows older than 1 year need to be tested for the presence of antibodies. Cows that test positive for antibodies will cause further suspension of the BVDV-free status, even when they are BVD virus negative. When either (i) the cow produces a calf within 9 months after purchase that tests negative for BVD virus, (ii) if no calf is born within 9 months after introduction, or (iii) the cow is removed before calving, the herd will regain the BVDV-free status.Within the BVDV programs, test results are accepted from laboratories that have their BVD test accredited by Wageningen Bioveterinary Research (WBVR), the Dutch national reference institute.Diagnostic testing in the BVDV program is being supervised by the Dutch national reference institute  and all tests must meet specific requirements. Tests for virus must have a sensitivity of at least 99.5% and specificity of 99%. Antibody tests must have a diagnostic sensitivity and specificity of 98% or higher.To evaluate the risk of purchase, all data on purchased cattle and their subsequently born calves were assessed for 2019. Four datasets were available that provided census data on all cattle located on Dutch dairy herds that participated in the national BVDV program :Cattle movement data that is registered in the national identification and registration (I&R) database . These data contain movement level data with the unique herd number (UHI) of the herd of origin, the destination UHI, the unique animal ID, the date of movement, the reason of movement.Calving data, registered in the I&R database: these data contain the unique animal ID of dam and calf and the calving date.Herd-level data of the national BVDV program . These data contain the UHI, the chosen BVDV-free route, the status within that route  with the start and, if present, the end date.Animal-level test data  with the unique animal ID, the type of test (virus or antibodies), the matrix , the sampling date, the date the result was available, the test result.The data-validation and analyses were conducted in seven serial steps. First, the movement data were combined with the data of the BVDV control program and only movements of cattle introductions in dairy herds that participated in the BVDV control program were retained. Subsequently, the cattle were stratified into two groups indicating whether the introduction involved intracommunity cattle movements or intercommunity cattle movements. The movement data were combined with the BVD test results on the level of the animal and the date of introduction. Data from cattle without test results were removed from the data. These included (i) cattle originating from countries assumed BVDV-free in 2019 , (ii) cattle originating from herds that were classified as BVDV-free, (iii) cattle < 1 year old with an available virus result and (iv) cattle originating from a veterinary entity (VE). In the Netherlands, VEs are defined as dairy herds having one or two holdings in different locations with regular exchange of cattle (>5 times/year). Generally, in one of these locations, the young stock is housed. The BVDV status of these locations are considered the same. Therefore, in the BVDV program, cattle movements between locations of a VE will not require testing. In the fourth step the tested cattle were stratified in two groups according to their age: cattle < 1 year old that only needed to be tested for presence of BVD virus and cattle ≥ 1 year old that had to be tested for presence of antibodies and  for virus. This resulted in our first outcome of interest: the number of introduced cattle that appear a PI. These BVD virus positive cattle are removed from the destination herd shortly after detection.The data from cattle that tested antibody positive were subsequently kept for further evaluation. These data were combined with the calving data and the information of the first calving date  were retained. Data from seropositive cows of which no calving was registered within 9 months after the introduction were excluded from the analyses. The data of the cows with a calving date were divided into cows with a calving that did not result in a live born calf  and calving's that resulted in live born calves. The data of the live born calves were singled out and combined with the individual testing data. This resulted in our second result of interest: the number of BVD virus positive calves born from newly introduced seropositive dams. Additionally, we evaluated what happened to the calves that were not tested.In 2019, Dutch dairy farms introduced 217,301 cattle into their herds, of which 97% were female. These cattle originated mainly from within the Netherlands (84%). The remaining 16 percent originated from outside the Netherlands, of which 80% had German ear tags .The 217,301 cattle were introduced in 9,331 Dutch dairy herds, which comprises 52% of all Dutch dairy herds. The median number of cattle introduced per herd with introduction of cattle in 2019 was 13. On average herds in which cattle were introduced were larger with a median number of 98 cows (>2 years old) compared to herds without cattle introductions (median 83 cows > 2 years). More descriptive movement information for both herds with and without cattle introductions in 2019 can be found in Through antibody and virus testing of introduced cattle in 2019, in total 67 BVD virus positive cattle were found in 44 unique herds. From the 217,301 introduced cattle, virus results were available for 49,820 and antibody results were available for 46,727 cattle > 1 year old. For the remaining cows, no BVD diagnostics were required after the introduction because they originated from a BVDV-free herd, a VE, were previously tested for virus or originated from a BVDV-free country .Of the 49,820 introduced cattle of which a virus result was available 27 (0.05%) tested BVD virus positive. Of these, 23 originated from the Netherlands, and four had foreign ear tags. The 27 BVD virus positive animals were introduced into 21 dairy herds.A BVDV antibody test result was available for 46,727 cattle, of which BVDV antibodies were detected in 9,588 (20.5%) cattle. Cattle originating from the Netherlands tested antibody-positive more often than cattle originating from another country (mainly Germany), respectively, 28.1 and 13.6%. Of the BVDV antibody-positive animals, 4,527 (47.2%) produced a calf within 9 months after introduction. The 4,527 cows produced 4,737 calves, of which 4,341 were viable (91.6%). The remaining 393 calves were stillborn or died before being ear tagged .Of the 4,341 ear tagged calves, 3,062 (70.5%) were tested for BVD virus. Forty of the tested calves were BVD virus positive (1.3%). These calves were born in 23 unique dairy herds.n = 1,279), 976 were moved off the farm, the majority of these went to veal calf farms. Given that no result is available for these calves the BVDV status of these dairy herds is suspended for a period of 10 months and during this period all newborn calves have to be tested. Another 143 calves were still present in the herd and these farmers will have re-started in a route to become BVDV-free. Of the untested calves, 158 died, resulting in a mortality risk of 12.4% [95% confidence interval (CI) 10.7-14.4%]. Of the 3,062 calves that were tested significantly fewer calves died (proportion test P < 0.0001) compared to the untested group. In total 217 of the tested calves died, resulting in a mortality risk of 7.1% (95% CI: 6.2-8.1%)  .The BVDV national herd-level prevalence in dairy herds in the Netherlands declined from 26% (2004) to 8.7% (2016) and the number of BVDV-free and BVDV-unsuspected herds increased . TherefoAdditionally, the number of BVD virus positive calves detected may be an underestimation because not every calf born out of a Trojan cow was tested. The mortality risk in this untested group was higher than the mortality risk among the tested calves. It may be that part of the untested calves died because of a BVD virus infection. Furthermore, mortality risks in calves have been found to be higher in BVDV infected herds . Also inIn 2019, testing introduced cattle for BVD virus in the Netherlands led to the detection of in total 67 BVD virus positive animals in 44 herds. Out of these 67 positive cattle, 27 tested BVD virus positive right after the introduction, and 40 additional BVD virus positive calves were detected by screening of newborn calves born out of dams that tested antibody positive at the moment of introduction. This risk-mitigating control measure did not prevent the introduction of BVDV because the BVD virus positive cattle were already added to the herd or the BVD virus positive calf was born in the herd. However, the testing procedure for introduced cattle did result in early detection of BVDV in these herds. By early detection of BVD virus positive cattle, further spread in the farm can be prevented and actions can be taken to regain the free status as soon as possible. The BVDV-free status of these herds is suspended from the moment of introduction of the animal. Unless they prove to be virus negative (cattle < 1 year old) or both virus and antibody negative (cattle ≥ 1 year old), the herd status will remain suspended until 9 months have passed without the birth of a calf or the subsequently born calf is tested BVD virus negative. The implication of the suspended status is that the herd cannot longer trade cattle with a BVDV-free status. This will prevent further spread of the virus by cattle trade with other herds, that are often seronegative due to decreasing BVDV prevalence in the Netherlands .Early detection of introduced BVD virus positive cattle or of BVD virus positive calves is important because the Netherlands has a high cattle density. In a study by Veldhuis et al., , the oddIn Ireland the retention of PI calves is a risk for the progress of the control program . In the Within 9 months of introduction into the dairy herd, about half of the purchased cattle had not produced a calf. This proportion was higher in cattle that originated from other countries then the Netherlands (72.6%). In the Dutch program, all female cattle > 1 year of age have to be tested for antibodies, regardless whether the cow is pregnant or not. Other countries with a BVDV program in place, e.g., Ireland focus on detecting and eliminating of the virus  or do noThe risk of (re)introducing BVDV through purchase of cattle in herds that participate in the national Dutch BVDV-free program is limited. However, the (re)introduction of the virus can have a large impact and result in major economic losses for the individual herd. In a country or region that has a successful BVDV control program in place, the prevalence of BVDV will decrease, which leads to an increased proportion of susceptible cattle in the population. In such situation, the impact of a new outbreak and thus the importance of controlling the risk of purchase increases. Therefore, to support eradication of BVDV in the Netherlands, it remains important to limit the spread of new BVDV infections through introduction of cattle. The virus and antibody testing of purchased cattle has likely been beneficial in preventing spread of infection. This conclusion may also be true for other countries with a BVDV control program in place.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LV was the primary author of the manuscript. IS-B conducted the analyses, helped in writing the paper, and proof read the paper. IS-B, JM, FW, MB, and GS edited draft versions of the paper and provided advice for improvement. GS acted as supervisor of the group and provided scientific and epidemiologic input. All authors participated in the writing of this manuscript, have proofread the final version of the manuscript and agree with its contents.LV, IS-B, JM, FW, and GS are employed by Royal GD, which runs the national BVDV control program. The remaining author declares 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.


Rapidly ageing populations means that many people now die in advanced age. This paper investigated public hospital and long-term care home costs in the 12 months before death in Māori and non-Māori of advanced age in New Zealand.Data from an existing longitudinal study (LiLACS NZ) was used, in which 937 older New Zealanders were enrolled in 2010. At the time of this study, 213 Māori and 241 non-Māori in the cohort had died. National Health Index numbers were linked to the hospitalisation National Minimum Dataset to ascertain public hospitalisation and care home costs in the last year of life.The average total publicly funded hospital and long-term care home costs in the 12 months prior to death were $16,211 and $17,351 for Māori and non-Māori respectively. Non-Māori tended to have long lengths of stay in their last year of life, and non-Māori men had the highest proportion with high costs and long lengths of stay in care homes. Costs in the last year of life were 8.1 times higher in comparison to costs for individuals who did not die in the same time period.Despite New Zealand’s commitment to providing an equitable level of healthcare, this study illustrated that ethnic and gender disparities are still apparent at the end of life. This raises questions as to whether money at the end of life is being spent appropriately, and how it could potentially be more equitably targeted to meet the diverse needs of older people and their families.The online version contains supplementary material available at 10.1186/s12877-021-02458-6. Rapidly ageing populations means that many people now die in advanced age, and the number of deaths in advanced age is projected to increase substantially over the coming decades in most countries . CountriUnderstanding the costs of health services in the last year of life is important for several reasons. Firstly, there is evidence that the inverse care law applies at the end of life, meaning that people most in need of care often miss out . CrucialAlthough a high level of healthcare utilisation is common in the last year of life, this variation has not been sufficiently described in New Zealand. In light of population projections estimating a 88% increase in the number of older Māori , and a 55% increase in the number of older non-Māori in the next 10 years in NZ, as well an increase in the number of deaths per year in NZ by 30% in the next 15 years , 12, thiThis paper draws on data from an existing longitudinal study in New Zealand (LiLACS NZ), as well as data from matching participants National Health Index (NHI) numbers to the hospitalisation National Minimum Dataset (NMDS) held by the Ministry of Health (MoH). When LiLACS NZ was initiated in 2010, 937 older New Zealanders were enrolled from a population-based sample, including 421 Māori aged between 80 and 90, and 516 non-Māori all aged 85 . In thisCovariates on study participants were gathered in the first wave of data collection, including gender, age, region, district health board  of residence , and living arrangement . Deprivation (10 deciles regrouped into 3 categories) was based on the NZDep, an area level index ascertained by geocoding participants addresses at the time of enrolment . Health The NMDS contains admission data on acute public hospital admissions and long-term aged residential care admissions (henceforth termed care homes). Care homes in New Zealand consist of both high-level dependency  and low-level dependency . Based on a standard financial assessment, high dependency care home costs are fully subsidised in NZ, and low dependency care home costs are part subsidised based on a means test. In this paper, we report MoH (publicly) funded costs as recorded in the NMDS. Care home costs reported are those relating to high-level dependency  only.Acute public hospitalisation costs for each participant were calculated based on the Weighted Inlier Equivalent Separations (WIES) attached to each hospitalisation event. These WIES are calculated based on length of stay and diagnostic-related groups , with inCosts were reported in two categories, these being acute public hospital costs and long-term care home costs. As we are taking a health services costs approach to our study, we do not report other private costs such as those relating to primary care or out of pocket costs for care homes.Māori and non-Māori are reported separately to examine potential inequities and because care practices differ between cultural groups. Descriptive statistics illustrate costs in the last year of life stratified by sociodemographic and health variables Table a and b. In order to compare costs in those who died in the last 12 months against those who did not die in the same time period, participants were matched on birthdate and costs were compared between those who died and those still living. For example, if a participant died between their 85th and 86th birthdays, their last year of life costs were compared with the mean of all individuals who were still alive between their 85th and 86th birthdays. Age groups were grouped into 3-year age bands, and analysis was only carried out in each band if both groups had 10 or more participants. As individuals who did not die often had costs spanning multiple years, weighted averages were computed for this group. Comparisons between those who died and those still alive are presented as a ratio. Similar analysis was carried out for the number of admissions and length of stay variables.We used generalised linear regression models to explore variables statistically significantly associated with costs in the last year of life. Due to the skewed distribution of the cost data it was found that using a log link rather than identity link improved the model fit markedly, meaning that log (costs) was the dependent variable for regression analysis. Dummy variables were created with non-Māori males as the comparison group, and GLM regression models were built in stages; the 4 ethnic/gender combinations, the previous model plus all variables except health conditions, and the previous model plus health conditions. Analyses were conducted using SAS 9.4 for Windows .The average total publicly funded hospital and long-term care home costs in the 12 months prior to death were $16,211 and $17,351 for Māori and non-Māori respectively  and diabetes than Māori without these conditions  without care home costs 324, 71%), and 79 (17%) had no acute hospital or care home costs. Only 33 (7%) had any long-term residential care home costs reported in the NMDS  non-Māori men had high costs in their last year of life , whereas 7 (6.5%) Māori women had high costs Table , and 23 Costs in the last year of life were much higher in comparison to costs for individuals who did not die in the same time period Table . The difRegression analyses examining influences on costs in the last year of life showed that there were no variables statistically significantly associated with log (costs) and no differences between the gender/ethnic groups in unadjusted or adjusted models  , and a UWhile there was no overall disparity relating to ethnicity or gender in the adjusted regression models, there appears to be a small number of non-Māori who have very long lengths of stay in their last year of life. These results are largely in contrast to the international literature, where ethnic minorities have a higher degree of healthcare utilisation than whites in the last year of life –23. InteParticipants who died cost 8.1 times more than those living on in the same year, which is slightly lower than a ratio of 8.7 found in the USA . While aWe were unable to illustrate a strong trend toward older decedents costing less than their younger counterparts. Other literature has highlighted age disparities related to choice, care patterns, and care rationing , 39–41. Our regression model did not show any variables which were statistically significantly associated with costs. Although Hanchate and others  did not This is the beginning of an exploration to ensure equitable and appropriate end of life care is delivered in NZ. Higher healthcare costs at the end of life may not necessarily translate to improvements in health outcomes, quality, access, or satisfaction with care , 43. AccThe primary strengths of our study are that we enrolled a relatively large number of people of advanced age using a population-based strategy, had an acceptable response rate at the studies outset, and were able to access most participant’s administrative health records. This allowed us to calculate hospitalisation costs in the last year of life and compare these with individuals still living. Furthermore, the focus on advanced age and equal explanatory power for Māori make this study novel and of adequate size for conclusions to be drawn. As this data is part of the wider LiLACS study, the results here can be cross-checked in future against other findings from the wider study and can contribute to a much richer set of results than possible from purely administrative data.Conversely, the main limitation of our study is that we explored publicly funded healthcare costs exclusively and no other costs such as out of pocket care home costs, primary care costs, or informal care. We expect care home costs to be underestimated as we cannot be certain that all public funds for care home admissions are counted. As mentioned however, inpatient costs generally dominate expenditure at the end of life, so it is likely we captured the majority (or much more) of healthcare expenditure among our study population. Furthermore, it must be acknowledged that there is considerable variability in hospitalisation cost data and that results need to be interpreted with caution. While there was a suggestion that non-Māori men had higher costs and longer lengths of stay in their last year of life than other groups, it is possible that our results were driven by chance alone. The results from this study are suggestive of underlying differences between groups that warrant further investigation, such as using data from New Zealand’s Integrated Data Infrastructure. A further analysis of this administrative data would allow for a greater sample size and stronger regional lens as care access, utilisation, and underlying health structures may differ across the country.This study set out to investigate costs in the last year of life in Māori and non-Māori of advanced age in NZ. Acute hospitalisation and care home costs were 8.1 times greater for patients in their last 12 months of life compared to those still living. There were ethnic and gender disparities highlighted. This raises questions as to whether money at the end of life is being spent appropriately, and how it could potentially be more equitably targeted to meet the diverse needs of older people and their families.Additional file 1: Calculation of the costs of hospital-level care in residential settings in New Zealand.Additional file 2: Supplementary Table 1. Distribution of the cost, number of admissions, and length of stay in the last year of life variables. Supplementary Table 2. Distribution of the cost, number of admissions, and length of stay in the last year of life variables. Supplementary Table 3. Average costs in the last year of life vs costs for those still living. Supplementary Table 4. Average number of admissions in the last year of life vs admissions for those still living. Supplementary Table 5. Average length of stay in the last year of life vs length of stay for those still living. Supplementary Table 6. Generalised linear regression models predicting costs in the last year of life. Supplementary Figure 1. Distribution of the cost in the last year of life variable. Supplementary Figure 2. Distribution of the length of stay in the last year of life variable.