dataesr/scientific-paragraphs-categorization huggingface dataset:
Study design We retrospectively reviewed all patients referred to our university hospital ophthalmology department with chorioretinitis, chorioretinal diseases in other infectious diseases (code H320 in the French PMSI database), and herpetic eye diseases from January 1, 2012 to December 31, 2021. An official waiver of ethical approval was granted from the IRB of Toulouse University Hospital given the retrospective nature of the study as asserted by French Jardé law (study reference: RnIPH 2021-110). All procedures performed were part of routine care, in accordance with institutional guidelines and with the principles and regulations of the Declaration of Helsinki. Patients or their legal guardians/relatives received clear written information and gave free and written informed consent to participate.
Patient selection and data collection Patients with inflammatory diseases of the posterior segment (foci, optic disk edema, vasculitis) testing positive for viral retinitis after anterior chamber paracentesis were included for study. The baseline data concerning patient demographic and clinical characteristics, ophthalmologic characteristics and outcomes, and antiviral treatments were collected. Clinical characteristics included history of herpes infection, onset of NHR, delay between NHR onset and consultation/diagnosis, presence of immunodeficiency and type, and follow-up time. Ophthalmologic characteristics and outcomes included initial and final visual acuity using the logarithm of the minimum angle of resolution (logMAR) scale, fundus features (retinography capturing the posterior pole/periphery were available for all patients included), complications, surgical interventions, and recurrences.
Statistical analyses Qualitative variables are presented by frequency/ percentage and quantitative variables by average ± standard deviation. Comparisons between continuous variables from two groups were made using the Mann-Whitney U test, and the Kruskal-Wallis test was used for comparisons between more than two groups. Each eye was considered as independent for statistical analyses. The significance threshold retained was the classic 5% threshold (p < 0. 05). Statistical analyses were performed using STATA 11. 2 software (StataCorp, Texas).
Finally, Fardeau et al. [2]  reported final visual acuity strictly > 1 log-MAR for 50-75% of patients versus 27% of patients with ARN in our series. These differences in reports on final visual acuity could be explained by (1) the performance of intensive intravitreal injections, (2) differences in retinal detachment/break rate, (3) differences in initial visual acuity. We cannot rule out that differences could also be partly related to a former lack of a single and clear description of disease type and severity rendering inter-study comparisons difficult. This issue should be solved in the future given the new SUN Working Group classification criteria. Finally, limitations to our study are the retrospective nature, meaning we can also expect some variation in the data collected given the multiple different operators over the 10-year period performing the ophthalmologic examinations.
Therefore, 825 patients were eligible for the study. A group of 245 subjects with a normal CBC and differential referred to our institution from September 29, 2019, to February 9, 2020, when the prevalence of SARS-CoV-2 was very low in France, without evidence of infectious diseases, was used as controls to define Fig. 1. Selection of study patients. Patients were recruited from the emergency department. To be included, they had to have both a positive SARS-CoV-2 RT-qPCR and a complete blood count. Exclusion criteria were pregnancy, because a possible influence of pregnancy on cellular population data (CPD) can be hypothesized, immunosuppressive and antineoplastic treatments, which modify leukocyte counts and hemopathies, because abnormal blood cells interfere with the determination of the CPD of the normal lineages.
Statistical analyses As most of the parameters tested were non-normally distributed, results were expressed as the median and the 25 th and 75 th percentiles. MedCalc software, version 19. 2 (MedCalc Software Ltd. , Ostend, Belgium) was used. Mann-Whitney tests were used for the comparison of two groups of patients, and the Kruskal-Wallis test and Dunn post hoc tests were employed for multiple group comparisons. For correlation, we used the rank correlation test. To evaluate the performance of individual parameters to discriminate different subgroups of COVID-19 patients, we used receiver operating characteristic (ROC) analysis to obtain the area under the curve (AUC), which demonstrates the discriminative ability of each individual parameter. We considered that a parameter had a discriminating interest when the AUC was above 0.
Discussion Cellular population data are parameters used by Beckman Coulter analyzers for differential determination. They correspond to a morphological analysis of leukocyte subtypes  [18]  and several studies have shown that their analysis could constitute a useful aid for the diagnosis of viral, parasitic, or bacterial pathologies  [19] [20] [21]. Among the different CPD, @SD-V-MO and its derivative, MDW, was shown to be useful for the detection of early sepsis associated to bacterial infections  [4, 6, 7] , and was regulatory cleared. Very quickly, it appeared that @SD-V-MO or MDW was increased in patients with COVID-19, and this increase was correlated with the severity of the disease  [8, 9, 12, 13]. However, none of these studies, often because the number of patients included was small or because the design did not have this purpose, defined CPD thresholds for triaging ED patients according to their need for care: hospitalization in conventional wards or ICU, or in contrast to be discharged.
PAM screening in a validation cohort revealed multiple likely pathogenic singlenucleotide variants by in silico prediction We next determined whether predicted pathogenic PAM variants are associated with other types of PAs, both hereditary and sporadic. To do so, we screened a diverse group of 326 germline and 60 tumor DNAs from PA patients for PAM SNVs (see the Methods section and Table  S1  for details). Sequencing covered the PAM CDS and exonintron junctions. Germline CNVs at the PAM locus were analyzed by ddPCR in a cohort of 137 individuals with PAs (16 with gigantism and 121 with different types of PAs). Germline DNA sequencing detected 51 SNVs (Tables  2, S4 ). There were no germline CNVs or rare/ pathogenic somatic DNA SNVs (data not shown). A subset of the germline SNVs (15 of 51) was prioritized for further screening together with the p.
His778fs were accompanied by at least a twofold decrease in PHM activity. Normal levels of both PHM and PAL activity were detected for p. Gly641Arg. The maturation of N-linked oligosaccharides is generally completed only when the newly synthesized protein exits the Golgi  Variants were annotated using the NC_000005. 9(NM_000919. 3) reference sequence. All variants were observed in heterozygosis. Unless specified, MAFs were retrieved from the gnomAD database ver. 3. 1. 2. "European" excludes the Finnish population; "African" includes African-American; and "Latino" includes Admixed American. For in silico predictions, the computational verdict was based on the combined outputs of 19 softwares available in Varsome (within parentheses, the first number represents the pathogenic verdicts, the second number represents the uncertain verdicts, and the third number represents the benign verdicts) for all missense variants.
MW, molecular weight marker. (B) The identity of the PCR products (panel A) was confirmed by Sanger sequencing. The arrows point to the variant-specific peaks present only in the carriers. MUT, mutated; WT, wild type. (C) Minigene assay. After transfection into HEK-293AD cells, mRNA synthesis from the plasmids using the cells' own transcription and splicing machinery led to mRNA products containing (WT) or lacking (variant) exon 21 of PAM flanked by two exons from the pSPL3 vector. The RT-PCR analysis of the minigene transcripts was conducted using vector-specific primers. MOCK, cDNA from empty vector-transfected cells consisting of a 260 bp band made up of fragments of pSPL3 exons; -, negative control (RT-PCR without cDNA). The lines separating MOCK and negative control from WT and c. 2332-2A>T indicate that the data for intervening samples were removed.
PAM single-nucleotide variants are significantly associated with a hyperfunctioning pituitary gland Having detected multiple SNVs with deleterious effects on PAM protein function in our cohort, we decided to test whether SNVs within the PAM locus are statistically associated with a pituitary disease phenotype in a larger and more heterogeneous cohort. To this end, we interrogated the UK Biobank (UKBB), a powerful resource for genetic association analyses that contains the exome sequencing data of 200,643 adult individuals with different and well-annotated pathologies. First, we performed a gene-based association study using the sequence kernel association test (SKAT)  (23, 24). For this analysis, we considered ICD-10 codes associated with sellar lesions (D352, E220, E221, E222, E229, E237, and E240, covering the following conditions: benign neoplasm of pituitary gland; acromegaly and pituitary gigantism; hyperprolactinemia; syndrome of inappropriate secretion of antidiuretic hormone; hyperfunction of pituitary gland, unspecified; disorder of pituitary gland, unspecified; and pituitary-dependent Cushing's disease, respectively) and SNVs at the PAM locus for which we assigned a weight based on their detrimental effect on protein (see the Methods for details).
Subjects for the study were recruited from the Centre Hospitalier Universitaire (CHU), University of Liège (231), the NIH (90), and the Bicetre Hospital and Foch Hospital Paris  (38). UDP patients provided written informed consent under clinical protocol 76-HG-0238, "Diagnosis and Treatment of Patients with Inborn Errors of Metabolism and Other Genetic Disorders," approved by the NHGRI Institutional Review Board. Other individuals from the NIH were recruited between 1997 and 2017 under research protocol 97-CH-0076 (ClinicalTrials. gov: NCT00001595). The study was approved by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Institutional Review Board and by the Ethics Committee of the University of Liège, Belgium. Affected subjects or their relatives signed informed consent or assent forms approved by the local ethics committee.
Alignments were made to human reference assembly GRCh38 using BWA-MEM  (56). Variant calling and joint genotyping were performed using GATK Best Practices (GATK ver. 4. 2. 3. 0, HTSJDK ver. 2. 24. 1, Picard ver. 2. 25. 4) (57). The VCF files were annotated with population data from the gnomAD database (ver. 3. 1. 2). Functional and predictive annotations were attached using Ensembl Variant Effect Predictor (ver. 102)  (58)  and the dbNSFP (ver. 4. 3a) (59) database. Pathogenicity predictions used in filtering came from SIFT, ClinPred, PolyPhen-2, FATHMM, and Combined Annotation Dependent Depletion (CADD) score. No known disease-causing or damaging biallelic/hemizygous variants segregating to the affected status were identified. Slivar  (60)  was Biological processes and cellular components that might be affected by PAM haploinsufficiency in pituitary hormone-secreting cells.
Several in vitro studies have shown that PAM misexpression can affect a variety of steps in the regulated secretory pathway; these are highlighted in red in the cartoon. ER, endoplasmic reticulum; GH, growth hormone; MVB, multivesicular body. used to filter the variants and apply pedigree analysis. We prioritized rare heterozygous damaging variants with non-zero pituitary median expression from GTEx that appear in affected individuals for downstream validation and analysis, excluding variants with a population minor allele frequency (MAF) > 1%. Variants reported in the ClinVar and/or the UniProt databases as benign or likely benign were also excluded. PCR (GoTaq Green Master Mix, M7123, Promega) and Sanger sequencing (BigDye Terminator 3. 1 Cycle Sequencing Kit, 4337456, ThermoFisher Scientific) were used to confirm the PAM variant identified by WES.
Validation cohort WES data on germline and tumor DNA samples from subjects with CD were generated as described previously  (54). These WES datasets were manually assessed using the Integrative Genomics Viewer ver. 2. 12. 3 platform (Broad Institute)  (61). For all other subjects in the validation cohort, genomic DNA was extracted from peripheral blood and screened for PAM variants by Sanger sequencing. Whenever a PAM pathogenic/likely pathogenic variant was identified and family members were available for analysis, cascade screening was initiated. This led to two instances of genotype-positive family members. In 38 individuals with sporadic acromegaly, tumor DNA was available for analysis. However, due to the limited amount of DNA available, Sanger sequencing was targeted to specific regions of PAM where the majority of likely pathogenic variants were previously observed (Figure  2 ), i.
Single-cell RNA sequencing analysis Figure  S2A  was derived from data published in the NCBI Gene Expression Omnibus (GEO) Series (accession number GSE132224) using methods described in detail (62). Briefly, scRNA-seq data were derived from freshly dispersed rat anterior pituitary cells from postpubertal males (3,562 cells) and diestrus females (3,334 cells). Cell type clusters were identified using known genetic markers for secretory cell types, folliculostellate cells, erythrocytes, leukocytes, and endothelial cells. Here, we examined the expression levels of the Pam gene and the percentage of cells of each type expressing this gene, plotted as a tSNE map (63) and percent-expressing heatmaps using Matlab (R2018b).
The region of interest was pulled down using a pool of 400 attomoles of each of the 86 probes described above. The resulting material was amplified, and SMRTbell adapters were ligated to the ends. This library was sequenced on a Sequel (Pacific Biosciences) on a SMRT Cell 1M v2 cell with Sequencing kit 2. 1 and a 10 h acquisition time. The resulting reads were analyzed using the program "Targeted Phasing Consensus" from Pacific Biosciences (https://github. com/ WenchaoLin/targeted-phasing-consensus) to perform the read phasing and alignment using CCS reads (circular consensus sequences from 3+ passes), basic subreads, and the hg38 genome for the target region. A male with sporadic acromegaly harbored PAM variants c. 1654G>A (p. Gly552Arg) and c. 1688A>G (p. Asp563Gly), both located in exon 16.
Bioinformatic analyses Variant pathogenicity and gene damage predictions Aside from the bioinformatic tools used in the variant prioritization process, Alamut Visual ver. 2. 9 software (Interactive Biosoftware) was used for annotation, for in silico prediction, and for determining the frequency in public databases of all the PAM variants identified. For non-synonymous variants, unless specified, the computational verdict was based on the combined outputs of 19 software packages available in Varsome (accessed on 11/24/2022). Five algorithms (Splice Site Finder, MaxEnt, NNSplice, GeneSplicer, and Human Site Finder) integrated in Alamut were used to evaluate candidate splicing-affecting variants. Variants were considered probably damaging or affecting splicing when most of the algorithms agreed.
Full-length alignments are available in the Supplemental Methods. The ribbon diagrams of the crystal structures of the catalytic cores of rat PHM (PHMcc) (PDB: 1OPM) and PAL (PALcc) (PDB: 3FW0) were used to contextualize the identified variants. Three-dimensional models were generated using the PyMOL molecular graphics system (Schrödinger, LLC). Rat PHMcc and PALcc are 93% and 92% identical to human PHMcc and PALcc, respectively. Analysis of PAM single-nucleotide variant enrichment in pituitary diagnoses from the UK Biobank cohort.
UK Biobank dataset and disease annotation The UK Biobank (UKBB) is a prospective study that recruited 502,611 participants aged 38-73 years from 22 sites across the UK with baseline measures collected between 2006 and 2010 (71). This research was conducted using the UKBB Resource (Application Number 48008). The UKBB obtained ethics approval from the North West Multi-centre Research Ethics Committee (approval number: 11/NW/0382) and obtained informed consent from all participants. The diagnoses of hyperfunctioning pituitary diseases were retrieved from hospital inpatient diagnosis fields 41270, 41202, and 41204, with International classification of diseases (ICD-10) codes E220 (acromegaly and pituitary gigantism), E221 (hyperprolactinemia), E222 (syndrome of inappropriate secretion of antidiuretic hormone), E229 (unspecified hyperfunction of the pituitary gland), E240-E248-E249 combined as E240 (pituitarydependent Cushing disease), D352-D359 combined as D352 (benign neoplasm of pituitary gland), E237 (disorder of pituitary gland), or as self-reported medical condition with codes 1237 (disorder of pituitary gland combined with ICD-10 code E237), 1238 (PA/tumor combined with ICD-10 code D352), 1239 (Cushing's syndrome combined with ICD-10 code E240), 1431 (hyperprolactinemia combined with ICD-10 code E221), and 1429 (acromegaly combined with ICD-10 code E220).
Analysis of single-nucleotide variants in the UK Biobank The analysis of SNVs in UKBB individuals with pituitary diagnoses was performed with PLINK and a custom code in Python ver. 3. 7. The CADD v1. 660 VEP plugin was used to provide prediction scores for their detrimental effect. A CADD_PHRED score of ≥15 and an MAF < 1% were considered as pathogenicity criteria.
Vectors and mutagenesis All cloning reactions were performed using the In-Fusion cloning system (Clontech). Cloning primers are reported in Table  S2. Tissuespecific alternative splicing generates multiple PAM isoforms (73). The longest human isoform is a 974 amino acid integral membrane protein identified as P19021-5 in UniProt. Since a GenBank search revealed that this isoform is more prevalent than the one chosen as the canonical sequence (UniProt P19021-1/NM_001177306. 2, 973 amino acid-long), we regarded it as human PAM-1, corresponding to what was previously extensively characterized as rat PAM-1, a 976 amino acid integral membrane protein. The ORF expression clone for WT human PAM-1 (NM_000919. 3) cloned into pReceiver-M02 (EX-A3104-M02-GS, Genecopoeia) was subcloned into the pCMV-FLAG-C vector (635688, Clontech) using the EcoRI and XhoI restriction sites.
Statistical analysis All graphs were plotted as mean ± standard error of the mean (SEM). Data distributions were assessed for approximate normality. Differences between experimental groups were analyzed by two-tailed Student's t-test or one-way ANOVA with Dunnett's post hoc test, or corresponding non-parametric tests, as appropriate. PAM SNV allele frequencies in the study population were compared with frequencies in the general population reported in the gnomAD public database using Fisher's exact test or the chi-square test, as appropriate. Data were analyzed using GraphPad Prism (GraphPad, San Diego, CA, USA). P-values < 0. 05 were considered statistically significant.
(B) Protein sequence alignments for five of the variants with deleterious effects. Conserved affected residues are shown in yellow. PAL activities, indicated in red, refer to functional experiments in PEAKrapid cells. (C) The crystal structure of rat PALcc (PDB entry 3FW0) was used to contextualize the missense variants categorized as likely pathogenic based on in silico analyses; the WT residue is shown on the left and the mutant residue on the right. PAL folds as a b-propeller, with six blades (numbered 1-6) positioned around a central cavity. The calcium and mercury ions are depicted as yellow and orange spheres, respectively. The mercury ion was used instead of zinc to capture the binding of a non-peptide substrate, a-hydroxyhippuric acid, depicted in blue. The affected residues are highlighted in purple (Ser or Trp 539), cyan (Gly or Arg 552), red (Asp or Gly 563), green (Arg or Gln 703), and orange (Phe or Ser 759), along the ribbon visualization of WT rat PALcc in gray.
Acknowledgments This work was partially supported by  "Ricerca Corrente " funding from  Italian Ministry of Health  to  IRCCS Humanitas Research Hospital. The authors would like to thank  Steven Coon  of the  NICHD's IRP Molecular Genomics Core  for conducting the phasing experiment, the late  Mario Amzel  ( Johns Hopkins University School of Medicine ) for useful comments on the impact of PAM variants on its 3D structure,  Lyssikatos Charalampos  and  Marıá de la Luz Sierra  ( NICHD/NIH ) for their help with the collection and preparation of DNA and histopathological samples used in this study;  Emilie Castermans  and  Leonor Palmeira  ( CHU de Liège ) for genetic and bioinformatic analyses of FIPA kindreds, and  Jean-Francois Bonneville  ( CHU de Liège, Belgium ) for discussions on the neuroradiological images.
2 2 FIGURE 2 Location and evolutionary conservation of the missense and frameshift PAM SNVs that were functionally tested. (A) Schematic representation of the PAM gene (GenBank: NM_000919. 3, PAM-1, 25 exons) and encoded protein (NP_000910. 2, 974 amino acids), including functional domains, with 15 missense and one frameshift SNVs. Gene and protein structures were drawn with the Gene Structure Display Server (GSDS ver. 2. 0) (12). Variants found to have deleterious effects on PAM function/expression (p < 0. 01) are shown in yellow lettering, while those without major effects are shown in white lettering. Brackets identify the non-catalytic regions that precede PHMcc (N-terminus) and follow PALcc (C-terminus); CD, cytosolic domain; CDS, coding sequence; PALcc, catalytic core of peptidyl-a-hydroxyglycine a-amidating lyase; PHMcc, catalytic core of peptidylglycine a-hydroxylating monooxygenase; TMD, transmembrane domain; UTR, untranslated region.
Actimetry test Locomotor activity was performed in rats in weeks one and two after a stereotaxic surgery. A 15-minute test, using an infrared probe, records the rat's total movements in the CPP apparatus. The data were analyzed with the IMétronic software (Bordeaux, France). The sum of all movements was made to obtain the overall data on the locomotor activity of the rat  (Ouachikh et al. , 2013).
TBS containing 1% BSA and 0. 3% Triton X-100 was used to dilute all antibodies. The sections were then rinsed several times in PBS before being placed on slides Scienti c's Superfrost® plus), dried, and then mounted between slides and coverslipped in mounting medium (Dako Fluorescence Mounting Medium, Dako). Slides were examined using a uorescent microscope (Nikon Eclipse Ni) and the TH-positive cells were counted using ImageJ software. The number of brain sections (each SNc side) used for TH count was n = 29 for for sham, n = 35 for 6-OHDA-lesioned, and n = 39 for the CEO-treated group.
Bone parameter analyses. After fixation, hind paws were scanned in a micro-computed tomography system (µCT SkyScan 1176, Bruker). Each scan was performed according to the following parameters: 0. 5 mm Aluminium filter, 50 kV, 500 µA, pixel size of 18 µm and 0. 5° rotation angle. Images were then reconstructed using NRecon software (SkyScan NRecon version 1. 7. 4. 6, Bruker). Misalignment compensation, ring artefacts and beam-hardening were adjusted to obtain a correct reconstruction of entire knee joint. Bone degradation was quantified in subchondral bone plate and epiphysis region on medial and lateral plateaus of each tibia using CTAn software (version 1. 17. 7. 2, Bruker). Osteophyte formation and meniscal/ligament calcification were quantified on entire knee joints. Reconstructed 3D images of knee joints were obtained using Avizo Lite software (version 2019.