dataesr/scientific-paragraphs-categorization huggingface dataset:
Crystal structures of representative PD-L1,VHH complexes To further characterize the interaction of the inhibitory VHHs with PD-L1, crystal structures were solved for representative VHHs (VHH1 bound to PD-L1 D1D2 and VHH6 bound to PD-L1 D1). As expected, VHH1 and 6 adopt the classical fold of a VHH  (43) , with four framework regions consisting mainly of β-strands and a conserved disulfide bond between framework regions 1 and 3. As described in detail later, residues within the CDRs were seen to form the majority of the contacts with PD-L1 D1. The overall structure of PD-L1 D1 was very similar in both complexes, with an average RMSD for Cα positions to free PD-L1 D1 (PDB: 3BIS (42)) of 0. 6 Å for both VHH1 and 6. The crystal structures obtained were entirely consistent with the VHH binding interfaces determined by the minimal shift analysis of NMR spectra, as shown in Figures  6C  and 7B.
Characterization of anti-PD-L1 affinity by BLI All BLI experiments were performed on an Octet QKe (ForteBio) at 25 C with a shaking speed of 1000 rpm. Proteins for analysis were prepared in sample buffer (PBS, pH 7. 4, 0. 01% (v/v) Tween-20 and 0. 1% (w/v) bovine serum albumin). The sensorgrams were obtained with a 60 s baseline, 180 s loading, 60 s baseline, 300 s association, and 300 s dissociation. PD-L1-Fc fusion protein (Invitrogen) was diluted to 5 μg/ml in sample buffer and loaded onto Protein G biosensors (Sartorius). Individual purified VHH proteins were diluted in sample buffer to between 10 μM and 0. 3 nM. Raw data were corrected with double referencing and analyzed using Prism9 (Graph-Pad). The dissociation constants (K D ) for PD-L1-VHH binding were derived from steady-state equilibrium analysis of the sensorgrams using the maximum response observed during association of the VHHs binding to PD-L1 Fc.
All NMR data were acquired on a Bruker Avance III 600 MHz spectrometer equipped with a TCI cryoprobe. Two-dimensional 15 N/ 1 H TROSY spectra were obtained at 25 C in the presence and absence of VHHs or CD80, with typical acquisition times of 80 ms for 1 H and 50 ms for 15 N. Data were processed in Topspin (Bruker Biospin Ltd) and analyzed using the NMRFAM-Sparky software  (66). Additional three-dimensional 15 N/ 13 C/ 1 H HNCO spectra were acquired for PDL1 D1 in complex with VHH1, 2, 4, 6, 9, 13, 17, 18, 20, and 22. Triple resonance acquisition times were 80 ms for 1 H, 25 ms for 13 C, and 18 ms for 15 N. All datasets were nonuniformly sampled to 25% and reconstructed using the iterative shrinking thresholding algorithm within NMRpipe  (67). Spectra were analyzed using the Sparky software package  (66).
Backbone assignments for PD-L1 D1 have been previously published (39) and were consistent with the NMR spectra collected. Minimal shift analysis was performed by comparing bound and unbound PD-L1 D1 spectra, for both two-dimensional and three dimensional experiments, as described previously  (40). The surface area affected by antibody binding was determined as the sum of the solvent accessible surface for each residue that experienced a major shift following binding of a VHH (>0. 08), with solvent accessibility calculated using the deposited crystal structure of PD-L1 PDB: 5C3T (6).
1 M Hepes:-Mops, pH 7. 5, 0. 1 M carboxylic acids, 10% (w/v) PEG 20000, and 20% (v/v) PEG 500-MME. Data collection and X-ray structure determination X-ray diffraction data from VHH1,PD-L1 D1D2 and VHH6,PD-L1 D1 crystals were collected by the autonomous European Synchrotron Radiation Facility (ESRF, Grenoble) beamline MASSIF-1 (68), using automatic protocols for the location and optimal centering of crystals. All diffraction data were processed using XDS  (69)  and AIMLESS (70) from the CCP4 suite  (71). Homology models of VHH1 and 6 were prepared using SWISSMODEL (72), using deposited VHH structures 5M3O  (73)  and 5IML  (74) , respectively. Structures of the VHH,PD-L1 complexes were determined by the molecular replacement program PHASER (75), using the VHH homology models and the published PD-L1 structure (PDB: 3BIK (42)) as search models.
CD80 AlphaFold model The amino acid sequences for human PD-L1 (18-234) and CD80  were submitted to AlphaFold-Multimer  (48, 49)  to produce a model of the complex. The top ranked AlphaFold model of CD80 bound to PD-L1 was compared to the solved crystal structure of PD-L1 bound to a high affinity variant of CD80 (PDB: 7TPS) (37) using DockQ  (80)  and the PyMOL Molecular Graphics System, Version 1. 2r3pre, Schrödinger, LLC.
In (Q), a ribbon representation of the PD-1,PD-L1 complex (PDB: 4ZQK (6)), with PD-1 in pink and PD-L1 D1 in blue is shown in the same orientation as (A-P). The sequence of human PD-L1 D1 is shown in (R) with the position of elements of regular secondary structure displayed above the sequence (42) and unassigned residues highlighted in yellow. This panel was produced with ESPript (81). PDB, Protein Data Bank.
The CD80 IgV domain (blue) is shown as a ribbon representation with selected glycosylation sites highlighted (orange). PD-L1 D1 is shown as a solvent accessible surface colored on the basis of the backbone amide minimal shifts induced by CD80 binding, as indicated in panel (B). D, superposition of the interacting domains from the PD-L1 D1D2,CD80 IgV complex solved by X-ray crystallography (orange/blue) (PDB: 7TPU) and predicted by AlphaFold (gray/yellow). The alignment was done on the basis of all Cα atoms and revealed an average RMSD of 1. 3 Å. E, comparison of the PD-L1,CD80 binding interface seen in the AlphaFold model and reported crystal structure, with the protein domains colored as depicted in panel (D). The side chains of key residues involved in intermolecular contacts are shown and labeled.
Acknowledgments-Tara Kang-Pettinger was supported by a PhD studentship from the  University of Leicester and LifeArc. Aspects of this work were supported by a research partnership award from  LifeArc  to the  University of Leicester. The naïve llama VHH library was kindly provided by  UCB Biopharma. Author contributions-T. K. P. , P. J. C. , M. D. C. , and G. H. conceptualization; T. K. P. , K. W. , L. C. W. , and F. W. M. methodology; T. K. P. , K. W. , L. C. W. , and F. W. M. formal analysis; T. K. P. , H. W. , R. B. , K. W. , L. C. W. , F. W. M. , R. C. , M. W. B. , G. H. , and K. S. investigation; T. K. P. , M. D. C. , and G. H. writing-original draft; T. K. P. , H. W. , R. B. , K. W. , L. C. W. , F. W. M. , R. C. , R. B. , M. W. B. , G. H. , P. J. C. , M. D. C. , and K. S.
The resulting sequences were assembled and manually edited using Sequencher 4. 5 (Gene Codes Corp. ). Sequence polymorphisms were checked and confirmed by visualizing the chromatograms of the raw sequences and then aligned with the reference sequences using MEGA software. Then, the sequences were grouped by species and gene and aligned with previously published sequences with accession numbers. For the Cox1 gene (~ 1200 bp), the reference sequences for Sh were JQ397330. 1 from Senegal and AY157209. 1 from Species-specific SNPs allowed us to differentiate them  [5]. Combining the species assignment of the mitochondrial and nuclear genes gives the following possible genotypes: Sb_SbxSb; Sh_ShxSh; Sc_ScxSc (considered pure) and Sb_SbxSh; Sb_SbxSc; Sh_ShxSb; Sh_ShxSc; Sc_ScxSh and Sc_ScxSb (considered hybrid).
Acknowledgements We thank  M. Mamadou Traore , chief of  Fangouné Bamanan  village, all the breeders,  M. Mamadou Sidibe , Director of  Diakalel School , and all the schoolchildren, school and health authorities and the population of Kayes for their appreciable contribution to the success of the study. The authors mainly thank  Amadou Dabo , called Boua, for his tireless efforts, the transport of the team We thank the  Malian government  for the funding and the  IHPE Laboratory, University Montpellier, CNRS, Ifremer, University Perpignan Via Domitia, Perpignan, France , for the welcome. This work was financially supported by  ARES Trading S. A. , an affiliate of  Merck KGaA, Darmstadt, Germany. This study is set within the framework of the  "Laboratoires d'Excellence (LABEX) "  TULIP  ( ANR-10-LABX-41 ) and the  HySWARM  ( ANR-18-CE35-0001 ) program of the  French Research National Agency.
4F ). The expressed tagged Cosmc (Fig. 4F , "HPC4" and "Cosmc" rows) and a small amount of restored T-synthase protein are shown (Fig. 4F , "T-Syn" rows). As an additional control, based on the presence of the C1GALT1C1 c. 58G>A (p. Ala20Thr) allele in the gno-mAD database of human variation, we generated an A20T-Cosmc-HPC4 construct. We similarly tested A20T-Cosmc for expression and chaperone function. The A20T-Cosmc variant was functional and well expressed, and was able to rescue T-synthase activity (SI Appendix, Fig. S8 ), in line with this substitution being a benign variant. We also studied different cell types from the same patients to see whether there are any differential impacts on expression of endogenous A20D-Cosmc. Interestingly, in our analysis on whole cell lysates of cultured fibroblasts from patient M2, we found that fibroblasts exhibited a higher level of A20D-Cosmc expression when compared to patient lymphoblastoid cells, but still substantially lower compared to control cells (HC) (Fig.
pnas. org Based on the lethal phenotype of complete Cosmc knockout in mice and the critical function of T-synthase in O-glycosylation, we had earlier assumed that in humans, a complete loss of Cosmc function would not be compatible with life. Of the 51 large deletions encompassing C1GALT1C1 in the human mutation databases ClinVar and DECIPHER, all occurred heterozygously in females. In line with this assumption, our functional studies of A20D-Cosmc cell lines show a substantial, but not complete reduction of T-synthase activity, and A20D-Cosmc itself was expressed at significantly reduced levels. The A20D-Cosmc expression was higher in patient fibroblasts than in lymphoblastoid cell lines. A complete loss of Cosmc function leads to an effectively complete loss of T-synthase function, as evident from C1galt1c1-KO mice and C1GALT1C1-KO human cell lines  (9, 25).
Variant annotation and filtering was done in the exome and genome analysis pipeline "Varbank2, varpipe v3. 4" (45) of the Cologne Center for Genomics (University of Cologne). We performed Sanger confirmation of the detected C1GALT1C1 variant in all probands. Variant Reporting. Variant reporting refers to the C1GALT1C1 transcript NM_152692. 5 in the human hg19 genomic reference. The variant has been submitted to the ClinVar variant database (accession ID VCV001675199. 1). X Inactivation Assay. One microgram of genomic DNA samples isolated from peripheral blood was predigested with 20 units of the methylation-sensitive restriction endonuclease HpaII (New England Biolabs) in a total reaction volume of 20 µL, and incubated at 37 °C for 12 h. The enzymatic reaction was stopped by heating the mixture to 80 °C for 20 min.
PCR amplification of the SLITRK4 locus was performed using 100 ng (from females) or 200 ng (from males) undigested or HpaII-digested DNA in a total reaction volume of 20 µL containing: Qiagen Multiplex 5 µL, 0. 5 µmol/L FAM-labeled forward SLITRK4 primer (5'-GCACACAAGCAGTCCTTCCT-3′), 0. 5 µmol/L reverse SLITRK4 primer (5′-TGGCTTCTTGGTTGCTCTCT-3′), and 2 µL Q-solution. Amplification was performed under the following conditions: 1 cycle for 12 min at 94 °C; 35 cycles for 30 s at 95 °C, 30 s at 72 °C, and 1 min at 60 °C; 1 cycle for 60 min at 60 °C. PCR products were analyzed on an ABI 3100er genetic analyzer (Applied Biosystems) using Certagen LS-600-ORN as an internal-lane size standard and GeneMapper software (Applied Biosystems). The X-inactivation ratios were calculated as previously described  (46).
Similarly, anti-Tn antibody conjugated with Alexa Fluor-633 prepared in house  (27)  was incubated with the cells (1 µL of the antibody per 10 6 cells in 50 to 100 µL of PBS containing 0. 5% albumin or fetal bovine serum (FBS), and 5% Human TruStain FcX™ in PBMCs) for 30 min on ice. The stained cells were washed twice with PBS and analyzed on a BD FACSCalibur (lymphoblastoid cells) or BD FACSCanto I (PBMCs) flow cytometer. Data analysis was performed using FlowJo and DIVA 5. 0 software. For comparison between patients lymphoblastoid and fibroblast cells, cells were stained as described above and analyzed on Attune NxT (Invitrogen). Hexosaminidase Activity Assay. Approximately 5 µg of the lysate in 10 µL were incubated with 40 µL of reaction mixture containing 610 µM of 4MU-GlcNAc, 0. 25% of Triton X-100 and 120 mM of cacodylate buffer pH 6.
Whole-cell lysates were prepared as described in transiently transfected cells. Proteasome Inhibition Experiment. Simple cells cultured in six-well plate (at 80 to 90% confluency) were transiently transfected with A20D-Cosmc-HPC4 and mock as described above. After 65 h of transient transfection, 5 and 10 μM Lactacystin (final concentration) or respective volume of DMSO was used and incubated for 6, 12, and 24 h (one plate for each time point). Cells were trypsinized and collected with 1ml of chilled PBS, pelleted (500 g), and washed once with 1 mL of PBS. Cell pellets snap frozen in dry ice and were lysed and processed at the same time. Lysates were quantified for protein concentration and stored at -20 °C. SDS-PAGE WB was performed as described above (WB analysis for HPC4tagged protein). Western blot data were quantified using ImageJ.
The highest value obtained in each time point was converted to 100% and the rest were normalized accordingly. Data, Materials, and Software Availability. Source data for T-synthase/hexosaminidase activity graphs and flow cytometry graphs (Figs. 4  and 5 ) and SI Appendix, Figs. S3, S6, S7, and S8) are available herein (Dataset S1). Human whole exome sequencing data of the study participants cannot be made publicly available due to data protection regulations.
ACKNOWLEDGMENTS. We thank the study's participants for their willingness to contribute to this project. We also thank  Aditya Ramanujan ,  Rachael Smith , and  Camille M. Bucci  for their technical assistance, and  Jamie Heimburg-Molinaro  for manuscript editing and review. This work was supported by the  Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) -Clinical research unit  ( KFO 329 ,  AL901/2-1  and  AL901/3-1 ) to J. A. and Clinical research unit ( KFO 329 ,  BE6072/2-1  and  BE6072/3-1 ) to B. B. B. Further support by the  Glycoscience Fund, BIDMC  to R. D. C.
6j ), suggesting that REG3A, by protecting gp130 from oxidative damage, releases the gp130-dependent AMPK activation pathway. Changes in REG3A expression in metabolic organs of obese and diabetic patients. We explored the human relevance of our findings by investigating whether altered glucose metabolism in obese/diabetic patients could be correlated with aberrant REG3A expression. We analyzed endogenous REG3A expression using publicly available large-scale microarray datasets from human tissues of individuals with different metabolic phenotypes. As expected, REG3A mRNA expression is mainly restricted to the intestine and pancreas, and low or nearly absent in liver, adipose tissue, and skeletal muscle under physiological conditions (Supplementary Fig. 10a ). We selected metabolic disorder datasets deposited in the Gene Expression Omnibus (GEO)  [59] [60] [61] [62] [63]  and examined REG3A gene expression in major metabolic tissues (i.
GUR: Whole-body glucose utilization rate. GIR: glucose infusion rate; EGP: endogenous glucose production. l Tissue sensitivity to insulin estimated by the quantification of glucose uptake in the indicated tissues. Data are averages ± SEM. * p < 0. 05; * * p < 0. 01; * * * p < 0. 005 by Anova test (c, e, f, g, right h, i, k, l) or one-way repeated measures Anova (left h, left j) both followed by post-hoc test, Wilcoxon rank sum test (right j). NS or no statistical indication, no significance. insulin sensitivity was found to be the effectiveness of REG3A in reducing oxidative stress and activating AMPK signaling in skeletal muscle. This phenomenon is not associated with changes in body fat levels for the recombinant REG3A protein. In contrast, in REG3A transgenic mice, improvement of metabolic phenotype was associated with changes in primary adiposity.
A. France). Animals were individually housed in a cage with lights on from 7 a. m. to 7 p. m. and an ambient temperature of 22 ± 1 °C. All animals were acclimated to their cages for 48 h before experimental measurements. Data regarding food and water consumption were collected every 40 min, and all ambulatory movements recorded during the entire experiment, with the aid of an automated online measurement system combining highly sensitive feeding and drinking sensors and an infrared beam-based activity monitoring system. Gas and movement detection sensors were operational during both light and dark phases, allowing for continuous recording. Animals were monitored for body weight and composition at the beginning and end of the experiment. Data analysis was carried out with Excel XP using extracted raw values of VO 2 consumption, VCO 2 production (ml/h), and energy expenditure (kcal/h).
The amounts of carbonylated and total proteins were determined by densitometry using Image J software; the results were presented as the ratio of carbonylated to total proteins. Oxidation of the GP130 and insulin receptor. DOC-insoluble protein fractions were precipitated with trichloroacetic acid (TCA), protein pellets were washed with ethanol/ethyl acetate solution (50/50) and resuspended 100 mM sodium acetate pH 6. 0, 20 mM NaCl, 1% SDS and the carbonyl groups were derivatized by reaction with biotin hydrazide for 2 h before reduction with sodium cyanoborohydride for 30 min at 4 °C. After derivatization, the proteins were precipitated with TCA, the protein pellets were washed with ethanol/ethyl acetate solution (50/50) and resuspended in SDS lysis buffer containing 1% SDS, 20 mM Tris-HCl pH 8.
2 µg/mL) and insulin receptor β (RRID:AB_631835; 1 µg/mL) antibodies (Santa Cruz Biotechnology), followed by HRP-conjugated goat anti-rabbit antibody (RRID:AB_390191; dilution 1:2000) to reveal proteins using chemiluminescent reagent (Pierce). The amount of proteins was determined by densitometry using Image J software; results were presented as the ratio of carbonylated proteins (biotinylated captured protein) on total proteins (input). In silico studies. Bulk tissue gene expression for REG3A was assessed from the GTEx portal (gtexportal. org; version V8). The Gene Expression Omnibus (GEO) data repository was used to study the expression profile of REG3A in liver, pancreas, white adipose tissue and skeletal muscle of patients with type 2 diabetes. We analyzed 21 publicly available datasets for normalized differential gene expression using limma and edgeR R packages.
Enrichment scores were calculated using the ssGSEA algorithm based on the KEGG and Reactome collections of the Molecular Signatures database (MSigDB). Pearson correlation coefficients were calculated between REG3A expression and enrichment scores to determine the relevant pathways associated with REG3A. Local false discovery rates were measured using the Benjamini-Hochberg method. All calculations were performed with the R programming language (v. 4. 1. 3). P values < 0. 05 were deemed statistically significant. Statistics and reproducibility. Differences between two sample groups were tested using Student's one-tailed t test or Wilcoxon test. Changes over time (cumulative food intake, energy expenditure, respiratory exchange ratio, fat oxidation, weight gain, OGTT, ITT) were assessed by two-way repeated measures ANOVA followed by Tukey's post-comparison test.
Acknowledgements The authors warmly thank  Dr. Bénédicte Chazaud  and  Dr. Rémi Mounier  of the  Neu-roMyoGène Institute, Lyon, France  for fruitful discussions and for providing primary myocytes,  Dr. Simone Niclou  of the  Neuro-Oncology lab, Luxembourg Institute of Health , for providing C2C12 myoblasts. We thank  Dr. Bernard Guigui  for histological analysis of liver tissues. For experimental assistance, we thank  B. Peuteman ,  G. Duvallet  and  C. Auriau  (animal care),  J. Castel  and  R. Denis  (metabolic cages, echoMRI),  E. Philippe  and  J. Denom  (glucose clamp), the  Metatoul-Lipidomic Facility Core Metabo-HUB  ( Inserm I2MC U 1297 ,  Toulouse, France)  and the  Functional Coprology Laboratory (Pitié-Salpétrière hospital, Paris, France ). This work was supported by grants from the  Fondation ARC pour la Recherche sur le Cancer , the  National Institute of Health and Medical Research (INSERM)  and  ALFACT Innovation.
DISCUSSION This 3-year study of all blood samples (25 469) processed in a regional tertiary hospital in Colombia showed that only 6% had a bacterial culture yield and WHO priority bacteria were isolated in patients from all hospital wards. The study strengths are that data covered a 3-year period, laboratory quality standards were ensured and reporting was in line with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines  (18). The study also had some limitations. We had no data on how blood sample collection, transport, and storage were done, and this might have influenced culture yields. We also used two blood specimens instead of the recommended three and this might have negatively influenced the culture yield. Laboratory turn-around times, which are important for clinical decision-making, were also not available in the database.
Going forwards, this element should be added to the database. Finally, we did not include Reserve antibiotics for resistance testing and this could be considered in future studies. The study findings have a number of implications. First, the finding of WHO priority bacteria in all general hospital wards is concerning. This may be explained by the reorganization of the hospital wards to accommodate severely ill patients without COVID-19 in the general wards as the ICU was overloaded with COVID-19 patients. Understandably, general wards do not have the same infection prevention and control standards as specialized ICUs, and acquisition and transmission of WHO priority pathogens become more possible in this context. Whatever the reasons for this finding, the priority now is to ensure high infection prevention and control standards and continued vigilance in the general wards.
Study design and participants A single-center retrospective cohort study including all children admitted with measles at VNCH between 01/01/2017 and 31/12/2019 was undertaken. During this time, individuals with suspected measles were admitted to VNCH, in addition to other hospitals in the region. Admitted children included either self-referrals (walk-in patients) or referrals from other health facilities. Patients with multiple missing data were excluded.
There was no information provided about the rash's features, dietary status, or whether the case was likely a main or secondary home case. Furthermore, to facilitate the analysis of factors associated with the risk of severe diseases, we defined severe patients as those requiring any form of respiratory assistance, namely oxygen cannula, continuous positive airway pressure (CPAP), conventional mechanical ventilation, highfrequency oscillatory (HFO) ventilation, or extracorporeal membrane oxygenation (ECMO). To build an anonymised dataset, selected data were extracted and personal information such as name, date of birth, and permanent address were removed.