dataesr/scientific-paragraphs-categorization huggingface dataset:
5 or BV510, CD21 FITC, CD38 PE-Cy7, CD24 PerCP-Cy5. 5, and anti-IgD PE (BD Biosciences). Natural killer (NK) cell samples were stained with a mixture of antibodies against the following proteins, at optimal concentrations: CD3 FITC and CD56 PerCP 5. 5. The cells were washed and resuspended in 0. 2 mL 13 PBS for acquisition in a BD FACSCelest cell analyzer. Data were analyzed by FlowJo v10. 8. Events were then gated on a lymphocyte gate based on forwardscatter (FSC-A) versus side-scatter area (SSC-A). Initial gating involved the use of a forward-scatter width (FSC-W) versus height (FSC-H) plot and side-scatter width (SSC-W) versus height (SSC-H) plot to remove doublets. Events were further gated on CD3, CD4, and CD8 for T cells and on CD19 for B cells. After their identification, T cells were analyzed and classified on the basis of their expression of CD45RO, CD45RA, CD25, CD127, CXCR3, and CCR6 into regulatory T, T H 1, T H 2, and T H 17 cells, and on the basis of their expression of CD45RO, CD45RA, and CD31 for thymic naive and central naive CD4 1 T cells.
The cells were collected after 6 days and labeled with CD3 BV510 Vio-Green (BD Biosciences), CD4 BV395 (BD Biosciences), and CD8 PercP (Miltenyi Biotec) or without fluorochromes for the measurement of cell autofluorescence. After 20 minutes' incubation, the cells were washed with PBS and resuspended in 0. 2 mL PBS for acquisition in a BD FACSCelesta cell analyzer. A magnet was used to remove the beads before acquisition for cells stimulated with human T-activator CD3/CD28 beads. On day 0, the CFSE signal was measured by flow cytometry, to adjust the settings. Data were analyzed by FlowJo.
Calcium flux EBV-B cells from the patient and healthy donors were loaded with Fluo-4 (Molecular Probes; Thermo Fisher Scientific) by electroporation. Some of the patient's cells were left untransfected or were transfected with pcDNA3. 1-ezrin or pcDNA3. 1 (mock) and then stimulated with 2. 5 ng/mL ionomycin (Sigma-Aldrich). Fluorescence was measured in a BD FACSCelesta flow cytometer, and the data were analyzed by FlowJo (Kinetics). The histograms show the populations gated on the basis of forward scatter versus side scatter and singlets. The data shown are representative of 3 different experiments.
Statistical analysis Data were collected from a minimum of 3 experiments and are expressed as means 6 SDs. As a result of the small sample size, statistical significance was assessed by Kruskal-Wallis (nonparametric) tests comparing the differences between healthy donors and the patient in the same conditions. Differences between samples were considered statistically significant if P <. 05. Statistical analysis was performed by GraphPad Prism v8. 4. 0.
HLA class I expression was normal. High-resolution typing of HLA by next-generation sequencing revealed heterozygosity. Whole-exome sequencing identified a biallelic missense variant in exon 4 of EZR on chromosome 6 that segregated with the disease in this kindred as an autosomal-recessive trait (Fig 1  , B ). This mutation replaces an alanine with a threonine at position 129 (g. 158785391G>A [GRCh38. p12], c. 385G>A, p. A129T) (Fig  1, C ). This variant (rs528409234) is present in the Genome Aggregation Database (gnomAD; gnomad. broadinstitute. org) in the heterozygous state, with a minor allele frequency of 0. 0001803, or 0. 000268 in the non-Finnish European population, and exclusively in the heterozygous state and 0. 00002 in the Trans-omics for Precision Medicine (TOPMed; imputation. biodatacatalyst.
nhlbi. nih. gov) database, consistent with the prevalence of B-cell deficiency in the general population. The variant was validated by Sanger sequencing of genomic DNA from peripheral leukocytes, which confirmed that the patient's mother and 2 sisters were heterozygous for this mutation (Fig  1, A  and B ). It was not possible to test the father. No other mutations were found in the EZR coding region. Ezrin is composed of 586 amino acids forming 3 principal domains: an N-terminal FERM (4. 1 ezrin, radixin, moesin) domain that binds to transmembrane and adaptor proteins, an a-helical domain, and a C-terminal domain that binds to filamentous actin (Fig  1, C ). 20 y aligning the ezrin protein sequences of humans and other species, we were able to show that A129 was highly conserved throughout the evolution of vertebrates, including agnathans (Fig  2, A ).
The replacement of an alanine residue with a threonine residue is predicted to modify the polarity of the region as a result of the replacement of a nonpolar amino acid with a polar amino acid, and to generate steric hindrance as a result of the larger size of the threonine residue (Fig 2  , B ). The combined annotation-dependent depletion (CADD) score for this variant is way above the mutation significance cutoff, suggesting that this mutation may be deleterious (Fig  2, C ). 21 Furthermore, according to the f parameter, EZR is under strong purifying selection, indicating that mutations of this gene would be likely to have severe functional consequences (Fig 2  , D ). 22 In total, 17 EZR variants in the homozygous state have been reported in public databases, all with a CADD score below that of the A129T variant (Fig 2  , E  and F ).
Alvarez 2022 FIBHULP , and  MINECO RTI2018-095673-B-I00. B. G. S. is supported by  PEJD2019-PRE/ BMD-16556  Predoctoral Fellowships CAM and  ESID Bridge Fellowship. A. V. D. R. received support from  Instituto de Salud Carlos III (ISCIII)  through the project  PI17/00543. J. J. P. C. was funded in part by  National Institutes of Health (NIH)  training fellowship T32GM139800 and the  Initiative for Maximizing Student Development at Vanderbilt. R. M. B. was funded in part by  Clinical and Translational Science Award (CTSA)  award  UL1 TR002243  from the  National Center for Advancing Translational Sciences  and the  National Institute of Allergy and Infectious Diseases of the NIH  (grant  R21AI171466 ). The  Laboratory of Human Genetics of Infectious Diseases  is supported by the  Howard Hughes Medical Institute, Rockefeller University , the  St Giles Foundation , the  NIH CTSA program  ( UL1TR001866 ), and the  French National Research Agency (ANR)  under the  ''Investments for the Future'' program  ( ANR-10-IAHU-01 ), the  Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence  ( ANR-10-LABX-62-IBEID ), the  French Foundation for Medical Research  ( EQU201903007798 ), the  SCOR Corporate Foundation for Science , the  Institut National de la Sant e et de la Recherche M edicale (INSERM) ,  REACTing-INSERM , and the  University of Paris  Cit e.
Unit costs (reference year 2018) were obtained from national databases and published literature. Mean costs and uncertainty intervals were calculated via bootstrapping. Data were extrapolated to the Belgian population. Generalized linear models were used to determine associated covariates with total direct costs and out-of-pocket expenditures. Results: Mean annual direct costs amounted to e4618 (95% CI e4070-5152), of which 49. 5% were out-of-pocket expenditures. Mean annual indirect costs amounted to e36 081 (e31 312-40 923). Direct and indirect costs at the population level were estimated at e19. 4 and 151. 5 million, respectively. A sickness or disability benefit as source of income was associated with higher direct and out-of-pocket costs. Conclusions: The economic burden associated with PTLDS on patients and society is substantial, with patients consuming large amounts of non-reimbursed healthcare resources.
22  A recently published Belgian study estimated e193 per patient with EM and e5148 per patient with disseminated LB. 24  The same Belgian study included few patients with PTLSD and called for more research in this patient population. 24  Hence, data on direct and indirect costs attributable to persistent symptoms are still lacking for Belgium. Information is also limited on possible non-reimbursable healthcare utilization, as such data are not included in administrative databases. Thus, this study estimated the direct and indirect costs of confirmed PTLDS patients in Belgium, thereby also focussing on outof-pocket expenditure. A cross-sectional design using an online selfreporting questionnaire was applied to collect data. The results provide more insight in patients' healthcare seeking behaviour in a reality where guidance regarding diagnosis and treatment of PTLDS is lacking.
Data collection Cross-sectional data collection was assimilated using an online selfreporting questionnaire. Self-reporting was chosen to capture all healthcare utilization, even reimbursed care, because of the limited use of administrative data in not well-defined patient populations, such as the PTLDS population at hand. Moreover, we aimed to identify LB-related healthcare utilization data only, which would have been difficult, if not impossible, using administrative claims data. Questions were related to: (i) sociodemographic information (i. e. sex, age, marital status, education and type of income), (ii) disease characteristics (i. e. diagnosis, testing and disease status), (iii) healthcare resource utilization [i. e. general practitioner (GP) visits, medical specialist visits, medical examinations, home-based nursing care, hospital care, pharmaceuticals and complementary treatment] and (iv) impact on work situation (i.
e. employment status and absenteeism). To limit recall bias, data collection on healthcare resource utilization covered a retrospective period of 6 months before the survey was completed, except for hospitalization, which covered a 1-year period. Employment-related questions also covered a 1-year period. The questionnaire was launched mid-September 2018 via Time for Lyme (a patient organization), and remained open until the end of October 2018. A reminder to participate was sent after 5 weeks. At the time of the survey, the patient organization had 260 members, 2510 followers on Facebook and 835 newsletter subscribers.
Cost categories: data sources and calculation of costs Direct medical costs were calculated by applying unit costs to different types of healthcare resource utilization. Unit costs were obtained from the publicly available Nomenclature of Medical Performances database of the National Institute for Health and Disability Insurance (NIHDI). These costs were related to GPs, medical specialists, paramedics (other than psychologists), imaging and home-based nursing care. This database contains unit costs of all (partly) reimbursable performance that is included in compulsory health insurance. 28  Unit costs for visits to psychologists could not be derived from this source, because the NIHDI did not consider reimbursement for visits to psychologists in 2018. The cost of a visit to a psychologist was obtained from a published report on the organization and reimbursement of psychological and orthopedagogical care in Belgium.
29  An out-of-pocket cost of e47. 02 for a 45-min consultation was derived. Hospitalization costs were based on the average costs for hospital stays at the all-patient diagnosis related groups (APR-DRG) level. These costs were obtained from the publicly available National Database of Medical Diagnosis, Care and Cost. 30  Participants were asked to indicate the reason why they were hospitalized during the reference period. Subsequently, the National Database of Medical Diagnosis, Care and Cost was consulted to obtain the average cost at the APR-DRG level. For example, 'intestinal infection' was reported as a reason for hospitalization. For this item, we used the APR-DRG code 245 'inflammatory bowel diseases'. Costs related to a day hospitalization (without an overnight stay) were obtained from a report on the financing of day hospitalization in Belgium.
31  The unit costs for prescribed medication were derived from the publicly available Drug Repository database of the Belgian Centre for Pharmacotherapeutic Information. 32  For nonprescription (or over-the-counter) pharmaceuticals (mainly vitamins, other nutritional supplements and herbal medicines), cost information was obtained from the product websites. The price/dose/ day for each drug was calculated and multiplied by the number of days medication was used in the 6-month period. For complementary treatments (e. g. acupuncture and osteopathy), participants were asked to report the type of treatment, number of sessions in the 6month period and cost per session. All unit costs were reported separately by both the patient and a third party (public health insurance). NIHDI and the Drug Repository database stratify between out-of-pocket and reimbursed costs, but hospitalization and day hospitalization costs were not stratified.
Data analysis Data were analyzed with SPSS 27. 0 software. A P-value of 0. 05 was considered statistically significant. Standard summary statistics were used to describe participant characteristics. Categorical variables were presented as frequencies and percentages. Continuous variables were presented as means with standard deviations or medians with interquartile ranges (IQRs). Mean direct medical costs were calculated for each category of healthcare utilization. 95% bias-corrected and accelerated confidence intervals were estimated using a bootstrap procedure with 1000 iterations. Missing data on healthcare utilization costs were interpolated using the mean value for a given category. Generalized linear models with log-link function and gamma distribution (Tweedie distribution for indirect costs), reflecting the positively skewed distribution with high kurtosis and a heavy tail, were applied to investigate potential predictors of four dependent variables: (i) total direct costs, (ii) out-of-pocket costs of patients, (iii) total costs and (iv) indirect costs.
Patients have a good recall concerning hospitalization, whereas outpatient healthcare utilization recall is less reliable. 40  This study had several limitations inherent to retrospective selfreporting over long periods. First, information on the number of PTLDS patients in Belgium was lacking, which was required to extrapolate the results from the individual patient level to population level. Thus, we used data from a study in the Netherlands 22 as a proxy, while accounting for differences in EM and disseminated Lyme cases between the two countries. 36, 37  Second, Lymeattributable costs related to emergency department visits were not included, because of a lack of data. These two limitations relate to missing information, which is interesting as the aim of cost-of-illness studies is to examine the socio-economic impact of diseases and identify data gaps and desirable refinements in national registration systems.
Patients and methods The research was approved by the ethics committee of the medical faculty of LMU Munich (project number 20-0932). Written informed consent was waived because of the retrospective and anonymous data collection. Inclusion criteria were as follows: for this multicenter retrospective study, patients with HLH-associated ICD-10 codes (D76. -) were searched by the medical statistics departments of the participating hospitals in their respective digital patient documentation systems. The patient files of all suspected HLH cases were reviewed manually, and the data of interest were extracted. Sixty-one adults (≥18 years) who met at least 5 of the HLH-2004 criteria  11  or had a HScore 12 > 200 and 4 positive HLH-2004 criteria were included for further analysis. In addition, in accordance with the HLH-2004 criteria, 1 male patient with a mutation in the XIAP gene was included as well, despite only fulfilling 4 of the diagnostic criteria and reaching 107 points in the HScore.
A CONSORT (Consolidated Standards of Reporting Trials) diagram of the included patients per clinic is shown in Figure  1. For the calculations of the diagnostic criteria, the maximum/ minimum values of the corresponding laboratory findings up to a maximum of 14 days before treatment initiation were used. When no data were available for this period, the first value after treatment initiation was used. For patients who had more than 1 HLHassociated hospital stays, only the data from the first stay were included. Exceptions are genetic and NK cell activity tests, which were incorporated independent of the time of diagnosis. Five patients had been reported to the German HLH register up to July 2017, and part of their data were published by Birndt et al. 25.
The laboratory values measured on the day of the beginning of a specific HLH-directed therapy were used for the calculations of the Cox regression models. When no data were available, primarily the first value up to 72 hours before or secondarily 72 hours after treatment initiation was noted. If the same laboratory parameter was conducted more than once per day, only the first available value, usually from the morning's blood sample, was taken into account for further calculations. Laboratory data that were not determined exactly above or below a specific cutoff point were transformed by adding or subtracting 1 from the last decimal number (eg, for sCD25 values >7500 U/mL, the value 7501 U/mL was used). The time span from the beginning of an HLH-specific therapy to death by any cause or date of last follow-up until 31 January 2021 was used for calculations on overall survival.
Acknowledgments The authors thank  Andreas Humpe  and  Doris Schmidbauer  of the  Department of Transfusion Medicine, Cell Therapeutics and Hemostaseology at University Hospital LMU Munich  for their help in providing selected data for the study (not part of this publication). T. W. is a doctoral student at  Ludwig Maximilian University of Munich (LMU). This manuscript is submitted in fulfillment of the requirement for his medical doctoral thesis.
Indeed, mRNA prophylactic tools have already shown comparable polyfunctional antibody responses to proteins in nonhuman primates  [31] , and other studies have confirmed the ability to elicit tier 2 neutralizing antibodies able to reduce the risk of heterologous infections  [32] [33] [34]. Those preclinical successes permitted the National Institute of Allergy and Infectious diseases to launch three experimental HIV vaccine clinical trials in 2022 (NCT05217641). Despite this new hope, HIV vaccine history has been punctuated by succes-sive failures, reminding us to maintain our effort in the development and optimization of effective vaccines; comparing routes of mRNA delivery could be very helpful in designing new mRNA formulations. In the current study, LNP-based vaccines were formulated with different ionizable lipids: D-Lin-MC3-DMA (DLin) and GenVoy TM (GV), for the production of DLin-and GV-LNP, respectively.
Statistical Analysis All statistical analyses were performed using GraphPad Prism version 8. 0 software (San Diego, CA, USA). All data were collected and expressed as the mean ± standard deviation (SD). Lipid nanoparticle DLS characteristics were analyzed with a multiple unpaired t-test, the luciferase transfection assay was analyzed using a two-way ANOVA parametric test, the humoral immune response monitoring, before and after protein heterologous boost, was analyzed using the Wilcoxon or Mann-Whitney U nonparametric test; and correlation analysis was performed using the Pearson or Spearman test, for parametric and nonparametric data, respectively.
Acknowledgments: The authors would like to thank  Precision Nanosystem 's team ( Serra Gurcan ,  Natalie Orr ,  Samuel Clarke ,  Martin Rabel , and  Angela Zhang ) for loaning us the NanoAssemblr Ignite apparatus and their kind donation of the GenVoy-ILM TM nonviral LNP delivery kit. The authors also want to thank them for their technical contribution during our collaboration. Lastly, the authors want to acknowledge the staff of the  Plateau de Biologie Expérimentale de la Souris (PBES, ENS Lyon, France) facility  for their help and advice.
The gradient was developed from 4% to 22% of CH3CN supplemented with 0. 1% formic acid over 50 minutes and then from 10% to 32% over 20 minutes. Mass spectrometry was performed in a data-dependent acquisition mode following a Top20 strategy with full MS scans acquired from 350 to 1,500 m/z at a 60,000 resolution. After each scan of precursors, the 20 most abundant ions were sequentially selected for fragmentation and MS/MS acquisition at a 15,000 resolution. An intensity threshold of 8. 3 × 10 4 was applied. A 10-second dynamic exclusion was used to increase the detection of low-abundance peptides. Only double-and triple-charged ions were selected for MS/MS analysis. The MS/MS data were searched against LSDV and Bos taurus protein sequences (127,411 sequences) using Mascot software, version 2.
6. 1 (Matrix Science, Boston, USA). The search parameters included only 2+ and 3+ peptide charges, 5 ppm mass tolerance for the parent ion, 0. 02 Da mass tolerance for MS/MS ions, and a maximum of two missed cleavage sites for trypsin. For the database search, the carbamidomethyl (C) modification was considered as a fixed modification, and the following variable modifications were considered: oxidation (M) and deamidation (NQ). All peptide matches with a peptide score associated with a Mascot P-value of less than 0. 05 were retained. Proteins were considered valid when at least two distinct peptides were detected in the same sample. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD037293 and 10. 6019/ PXD037293.
Purification and quality control of LSDV particles Figure  1  shows the experimental workflow adopted to decipher the proteome of LSDV particles. The viral particles were obtained from infected MDBK cells and purified either on sucrose or tartrate continuous gradients. Continuous sucrose gradients allow nonequilibrium rate zonal velocity sedimentation to be used, while continuous tartrate gradients allow for equilibrium buoyant density isopycnic banding. First, the identity of the virus strain provided by the manufacturer, namely LSDV KSGP-0240, was confirmed using PCR and sequencing (data not shown). At the development stage of both methods, virions collected from the sucrose or tartrate bands were tested for infectiousness (titration) and morphology (electron microscopy). Titration of these viral suspensions demonstrated that purified LSDV particles were still infectious (data not shown).
ACKNOWLEDGMENTS We thank  Julien Soms  for his active contribution to the initial setting-up phase of the viral purification workflow. We thank  Andrew Lewer  for reviewing the English language. This work was carried out with the financial support of (1)  French Ministry of Agriculture and Food (DGAL convention)  for National activities of the  French National Reference Laboratory for Ruminant Poxviruses , (2)  Agence Nationale de la Recherche , Grant/Award Number:  F2E NewSy-LSD ,  French Ministry of Agriculture and Food , and ( 3) Région Occitanie (Direction de la Recherche, du Transfert Technologique et de l'Enseignement Supérieur).
NMR structure of U 9 peptide To better understand the mode of action of U 9 at the molecular level, the 3D-structure of U9 was determined by nuclear magnetic resonance (NMR). A total of 346 nuclear overhauser effect (NOE)-derived distance restraints, 9 hydrogen bonds and 30 dihedral angles were considered (Table  S1 ). amphipathic helical structure because its five lysine residues side chains are exposed to the solvent and spread on the same side of the molecular surface including the convex side of the hinge (Figure  S3B ). Hydrophobic amino acids are segregated on the opposite side of U 9 (Figure  S3A ). A similar fold of U 9 was obtained in sodium dodecyl sulfate (SDS) micelles (100 mM SDS-d25 at 318K) (data not shown). Because the activity and pharmacology of U 9 are compared throughout the present study with those of M-Tb1a whose 3D structure was determined previously,  34  we have a refined 3D-structure of M-Tb1a.