dataesr/scientific-paragraphs-categorization huggingface dataset:
LKO is a LEDGF-null cell line, generated via TALENmediated PSIP1 gene disruption in HEK293T cells  52. Cells additionally null for HRP2 (LHKO) were generated from LKO cells using CRISPR-Cas9 guide RNAs targeting exon 2 (5′-ACCCAACAAGTACCCCATCTTTTTC) and exon 15 (5′-CG ACCGGCAGGAGCGCGAGAGGG), resulting in deletion of most of HDGFL2 (26 kb). Gene disruption was verified by genomic DNA sequencing, and the absence of detectable HRP2 protein was verified by immunoblotting. For ectopic expression of ovine LEDGF/p75, LHKO cells were transduced with murine leukemia virus virus-based retroviral vector pQ-OaLEDGF-IRES-PuroR and selected in the presence of 1 μg/mL puromycin. To construct this vector, a DNA fragment encoding unaltered full-length ovine LEDGF/p75 (GenBank accession code FJ497048), PCR-amplified from a sheep peripheral blood mononuclear cell cDNA library  33  using primers 5′-GCGCATGCGGCCGCAGACACCATGACTCGCGAC TTCAAACCTGGGGACC and 5′-GGCGGGATCCCTAGTTATCTAGTGTAGA ATCCTT CAGAG, was ligated between NotI and BamHI restriction sites of pQCXIP (Clontech).
5 μM), 0. 2 μM TaqMan probe 5′-[6FAM]-CGAGACGCTAC CATGGCTATCGCTGTAG-[TAMRA], 5 U RiboLock RNase inhibitor, 100 ng phage MS2 phage RNA (Sigma-Aldrich), and 2 μL diluted viral lysate in a final volume of 25 μL. Reactions were assembled in MicroAmp Optical 96-well reaction plates and carried out in a QuantStudio-7 Flex real-time PCR instrument (Applied Biosystems). PCR conditions were as follows: 42 °C for 20 min (reverse transcription step), 50 °C for 2 min, 95 °C for 10 min (activation of the host start Taq DNA polymerase), followed by 40 cycles of PCR (denaturation at 95 °C for 15 sec and extension at 60 °C for 1 min). All PCR reactions were performed in triplicate. Standard curves were generated using recombinant HIV-1 RT (Merck Millipore). The relative viral quantities were calculated based on the standard curve generated using QuantStudio-7 systems software (Applied Biosystems).
13 (Perkin Elmer) was normalized by the total protein content in each precleared lysate. Flow cytometry was used to count cells infected with GFP reporter viruses. Cells were harvested by trypsinization, fixed in 6. 5% (w/v) formaldehyde in PBS and analyzed for GFP expression using a Fortessa flow cytometer (BD Biosciences). GFP was excited at 495 nm and emission was detected with a 530/30 band pass filter. Data were acquired using BD Diva software version 6. 0 (BD Biosciences) and analyzed using FlowJo version 13. Live cells (population P1) were initially gated using the area plot of forward scatter (FSC-A) versus side scatter (SSC-A), to separate them from cell debris. Singe cells (population P2) were then differentiated from doublets in population P1 by gating on FSC-A vs forward scatter height (FSC-H).
The nested PCR primers contained Illumina adaptor sequences appended at the 5′ ends. The PCR products, multiplexed on a single lane of a flow cell, were subjected to 150-bp paired-end sequencing on a HiSeq-4000 Illumina instrument (GENEWIZ, Boston, MA, USA). Paired-end Illumina sequence reads, cropped to remove linker-and viral U5 DNA-derived sequences, were aligned with the hg38 version of human or the oviAri4 version of sheep genome using BWA version 0. 7. 12-r1039  106. The results were parsed using SAMtools version 1. 2  107  to extract unique, high-confidence alignments (samtools view -F 4 -F 256 -q 1). Only reads matching the host genome sequence immediately downstream of the processed vDNA U5 end were considered for further analysis. Genomic coordinates of the unique integration sites were converted to the browser extensible data format (with each interval corresponding to the middle dinucleotide of an integration site).
Distributions of the integration sites with respect to various genomic features (http://genome. ucsc. edu/cgi-bin/hgTables), cLADs  55  , SPADs  56  as well as local gene density and nucleotide content at the sites of integration were calculated using BEDtools version 2. 25. 0  108. Expression data averaged across sheep brain tissues  109  was used to approximate gene activity in CPT-Tert cells; HEK293T gene expression data were from Gene Expression Omnibus entry GSE11892  110. Local gene density and nucleotide content comparisons were computed using Wilcoxon rank sum test, and significance of all other comparisons were calculated with Fisher's exact or Chi-Square test in R version 4. 0. 4. Sequence logos were generated using WebLogo version 3  111. HIV-1 integration sites in HEK293T and LKO cells were from published datasets  57, 112.
Acknowledgements We thank  Massimo Palmarini  for CPT3 cells,  Didier Trono  for a generous gift of pMD2. G,  Ron Vale  for His 10 -PS-SNAPf vector,  Goedele Maertens  for sharing the luciferase assay protocol and critical reading of the manuscript,  Massimo Pizzato  for advice on RT assays and nucleofection,  P. Walker  and  A. Purkiss  for computer and software support, and  M. Singer  for help with tissue culture. This work was funded by  US National Institutes of Health  grants  P50 AI150481  (P. C. and A. N. E. ),  R01 AI070042  (A. N. E. ), and  U54 AI150472  (D. L. );  US National Science Foundation   CAREER MCB-2048095 , the  Margaret T. Morris Foundation , and the  Hearst Foundations  (D. L. ); the  Spanish Ministry of Science and Innovation   PID2019-108850RA-I00  (JV); and the  Francis Crick Institute  (P.
Data availability The data that support this study are available from the corresponding authors upon reasonable request. The final cryo-EM reconstructions were deposited with the EMDB under accession codes EMD-26322 (CSC) and EMD-14453 (STC) and fitted coordinates with the PDB under accession codes 7U32 (CSC) and 7Z1Z (STC). In addition, our Illumina sequencing data along with locations of mapped MVV integration sites were deposed with the Gene Expression Omnibus database under accession code GSE196042. Source data are provided with this paper as a Source Data file. Source data are provided with this paper.
Results of minimum inhibitory concentrations were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints  [21]. All data were collected, stored, and managed using REDCap (Research Electronic Data Capture) by an ID specialist. REDCap is hosted at Lausanne University Hospital. REDCap (Research Electronic Data Capture) is a secure, webbased software platform designed to support data capture for research studies  [22, 23].
Statistical Analyses SPSS version 26. 0 (SPSS, Chicago, Illinois) software was used for data analysis. Categorical variables were analyzed using the χ 2  or Fisher exact test and continuous variables with Mann-Whitney U test. Univariate logistic regression models were assessed with 14-mortality as dependent variable. Covariates were tested for multi-collinearity through variance inflation factor assessment; those clinically relevant and not collinear were used in multivariate analysis. After checking Cox assumptions, a multivariate Cox proportional hazards regression model was performed with 14-day mortality as the time-to-event. For the multivariate analysis, episodes for which no source control was warranted were imputed as appropriate early source control. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of any association.
RESULTS According to the database of the microbiology laboratory, 255 episodes of candidemia were identified, from which 222 episodes of candidemia in 209 patients were included (Figure  1 ); the 13 subsequent episodes of candidemia occurred at a median of 5 months from the previous episode (range, 2-41 months). A total of 229 Candida spp were isolated (2 different species were isolated in 7 episodes). The median time from blood culture sampling to positivity was 38 hours (range, 4-96 hours) with 68 (31%) episodes for which blood cultures were positive after 72 hours. Time to positivity did not significantly differ according to the source of candidemia. Candida albicans predominated (112 [49%]), followed by Candida glabrata (67 [29%]), Candida tropicalis  (19 [8%] ), and Candida parapsilosis  (11 [5%] ).
Confirmed cases of COVID-19 were defined by a positive reverse transcriptase-polymerase chain reaction (RT-PCR) assay of a specimen collected by a nasopharyngeal swab. Pandemic waves were defined in time as follows: January-June 2020 (n=108), July-December 2020 (n=144), January-June 2021 (n=62), July-January 2022 (n=52). Each institutional review board independently approved the study. The study was conducted following the Declaration of Helsinki. Researchers at each center collected data using an online questionnaire hosted at www. clinicalsurveys. net (EFS Fall 2018, Questback, Cologne, Germany). EPICOVIDEHA is registered at http://www. clinicaltrials. gov, with the identifier NCT 04733729. Only deidentified data were entered and analyzed. We obtained demographic data, comorbidities, and underlying hematological disease including clinically significant outcomes (hospital admission and intensive care unit [ICU] admission, vital status) and COVID-19 management strategies.
The severity of COVID-19 at admission was graded according to the China Centers for Disease Control and Prevention definitions: mild (non-pneumonia and mild pneumonia), severe (dyspnea, respiratory frequency ≥30 breaths per min, SpO2 ≤93%, PaO2/FiO2 50%), and critical (respiratory failure, septic shock, or multiple organ dysfunction or failure). SPSSv25. 0 was used for statistical analyses (SPSS, IBM Corp. , Chicago, IL, United States). Categorical variables were presented as frequencies and percentages, while continuous variables by the median, interquartile range (IQR), and absolute range. Additionally, overall mortality was evaluated by a Cox proportional hazard model. A Univariable Cox regression model was performed including variables that potentially played a role in the mortality of patients.
04. 03. 65603, Grenoble, France; ref20-ANGE-01) and the French National Agency for Medicines and Health Products Safety (ANSM). It was supervised by an independent data and safety monitoring board. All authors and contributors are listed in S1 Supplemental Appendix. The study is reported as per the Consolidated Standards of Reporting Trials (CONSORT) guideline (S1 CONSORT Checklist).
Outcomes and follow-up The primary outcome was overall mortality within 14 days after randomization. Secondary outcomes were overall mortality within 28 days after randomization, mortality due to COVID-19 at 14 and 28 days, and between-group comparison of safety. Data on vital status were available for all participants at day 14 and were missing at day 28 in one participant from the high-dose vitamin D3 group and in one participant from the standard-dose vitamin D3 group. Safety was assessed according to a list of protocol-specified adverse events of interest by means of participant report and by onset at day 7 of hypercalcemia or severe kidney failure.
09), a landmark approach was also used with day 6 set as the cutoff point of the time window for survival analyses. Analyses were performed on each of the 2-time windows. The same methods were used to analyze the secondary outcomes of the 28-day overall mortality and the 14-day and 28-day mortality due to COVID-19. Finally, exact chi-squared tests were used to examine the between-group differences in the protocol-specified adverse events of interest. All P values are 2 sided. The full database is held by the trial team, which collected the data from trial sites and performed the analyses at the University Hospital of Angers, France, using SAS, version 9. 4, and R, version 3. 4. 0. The trial was registered on ClinicalTrials. gov on April 14, 2020, NCT04344041.
Material of the Analysis Our primary data sources were all publicly available documents and documentation regarding the 2 algorithms from verified sources that were actually involved in research and development. Sources were collected from January 2021 to March 2022, with an update in August 2022. The source collection included scientific publications and presentations, technical documents and manuals, and user documentations. Data were analyzed from a qualitative perspective with the general idea to understand the algorithms and their creation as product and social process of sense and meaning making  [45, 49]. Hence, our aim was to learn how the creators of the algorithms provide a rationale for their calculations; make sense of their parts; and explain their inner workings, purpose, and functioning.
These authorities then gather additional data and share the patients' mobility information with the operators of the mobile phone grid under a nondisclosure agreement. Mobile phone owners can request a clarification of potentially risky encounters or allow clarification and information in advance. Mobile operators will gather the locational information about the requesting user (second aspect) and will evaluate the risk of a contact. On the basis of the outcome of the mobile operator's inquiry, the probability of contact can be calculated and provided to the potential contact person to recommend further follow-up with the health authorities and a fine-grained risk assessment using a standardized data structure called Patients' Locational Open Data (PLOD). PLOD provides manually gathered locational information in a standardized form and, hence, allows an assessment of the encounter based on the names of places visited or exact time of stay.
FRAP analysis Fluorescence recovery after photobleaching was performed on the Leica SP5 confocal microscope using the FRAP wizard of Leica AF software. Generally, the middle plane (or brightest z-plane) of a single BMC was selected (x25 zoom, 15. 5x15. 5 µm). A bleach area (region of interest ROI) of 20x20 pixels within the selected BMC was set for bleaching. We used the same ROI size for all our FRAP experiments. Photobleaching was done using a 405nm argon laser at 60% intensity for 6 frames. The bleaching session was preceded by 2 prebleach frames every 1. 293s. Fluorescence recovery was images through 2 postbleach session: one with 10 frames every 3 seconds and a second one with 78 frames every 5 seconds. BMCs photobleaching has to reach at least 50% of fluorescence intensity to be used for analysis.
ACKNOWLEDGEMENT We wish to thank the Snow Foundation for their generous support towards establishing the transgenic zebrafish facility at Macquarie University and continued support of the researchers. We also wish to thank the zebrafish facility staff (past and present) for assistance in zebrafish care. This work was supported by the ALS Foundation Netherlands, FightMND, MNDRA, a Snow Foundation Fellowship, and donations towards the MND research at Macquarie University. examples and quantification of fission and fission events characterizing LLPS and BMC in the nucleus (2. 5 events ± 0. 4 for WT, 1. 8 events ± 0. 4 for G294V, 4. 2 events ± 0. 7 for K136R, 2. 7 events ± 0. 5 for G294V_K136R, Kruskal-Wallis test, n=14-25, ns). Scale 5 µm.
Study Design A modified Delphi study was conducted with a panel of international experts belonging to the MDS Task Force on Postural Abnormalities. 2] [13] [14]  The Delphi methodology is based on multiple rounds of anonymous and structured questionnaires. Eleven movement disorders specialists (neurologists, neurosurgeons, and physiotherapists) were selected to answer questionnaires through several rounds of two different surveys, one related to nosology and the other to cut-off values of postural abnormalities. Four additional members (M. T. , C. G. , R. B. , and C. A. A. ) who belonged to the steering committee organized and distributed the questionnaires and sent to the panelists a personal web survey link created with Google Forms (freeware software) via e-mail. Disagreements were solved by web-based meetings to promote an interactive discussion before reaching final consensus.
Data Analysis Aggregated results obtained from survey 1 and 2 were analyzed, presented, and discussed in a web-meeting among panelists. For both surveys, a consensus for each question was pre-defined as ≥70% agreement among the panelists. 14 In case of disagreements after two rounds, a real-time and web-based anonymous polling was performed at the end of the discussion and an agreement consensus was obtained for all remaining questions. Relevant aspects and issues that emerged during the meeting were recorded and used for a more comprehensive evaluation of the consensus criteria.
To obtain a reliable evaluation of postural abnormalities, patients should be evaluated with adequate exposure of affected body parts and degrees of spine flexion calculated on both coronal and sagittal plane. Degrees of spine flexion and fulcra should be separately assessed, according to the currently accepted evaluation methods. 15 In particular, we recommend to calculate posture degrees by analyzing photographs using the NeuroPostureApp, a freeware software-based measurements (https://www. neuroimaging. uni-kiel. de/NeuroPostureApp/). 16 all goniometer or smartphone can be used instead, but possible  underestimation of the degrees with these methods should be considered. 15 omorbidities should always be carefully assessed when approaching patients with postural abnormalities to exclude the presence of other diseases potentially causing the abnormal posture, with relevant implications in terms of treatment and prognosis.
Here, we first demonstrate the virtual 100% accuracy and reproducibility of this approach when sequencing three separate H37Rv cultures, as well as the ability of the assembly tool to reveal ~6. 4 kb of new sequence that is absent from the GenBank (NC_000962. 3) published reference, including 10 major regions of difference. Together, this work reveals an important update to the H37Rv references sequence and shows the utility of Bact-Builder for assembling highly accurate bacterial genomes, both for use as genomic references or applied to other metagenomic and pangenome studies of bacterial species.
Building and validating Bact-Builder Bact-Builder (Fig. 1a ) was designed to generate highly accurate gapclosed de novo bacterial genomes. Bact-Builder steps were first designed using in silico-generated reads created with BadReads 23 and ART  24  , which removed common experimental variables that can affect sequencing output and quality (Supplementary Note 1, Figure  S2a-d ). Further testing was then performed by experimentally sequencing three independent cultures of the same H37Rv stock, extracting genomic DNA, preparing sequencing libraries and sequencing each culture separately. Sequencing was performed on both Oxford Nanopore Technologies (ONT) MinION and Illumina NovaSeq (paired-end 2×150) platforms (Table  S1 ), and the raw sequencing data was used to assemble each of the three replicates (H37Rv.
We further tested the impact of polishing the individual assemblies and saw that although polishing improved some assembly sizes, there remained inconsistencies across assembly sizes producing genomes of substantially different lengths and incorrectly called indels (Supplementary Note 2, Table  S4 , Table  S5 , Figure  S5 ). We worked to reconcile the differences observed across assemblers using Trycycler  19  a program that generates a consensus sequence from the outputs of the four individual assemblers. Trycycler produced contigs that were more similar to the reference than any individual assembly and further polishing with both long and short reads generated even more accurate assemblies (Fig. 1c ), although some remained too fragmented or could not be reconciled and were dropped by the program (Fig.
3) except for one sample (H37Rv. 1) that was one nucleotide shorter than the other two (Table  1 ). Further investigation into the H37Rv. 1 indel (compared to H37Rv. 2 and H37Rv. 3), showed that it was an assembly error (Supplementary Note 2, Figure  S5 ). Updating relevant components of Bact-Builder corrected the single basepair indel (Supplementary Note 3, Figure  S6 ). The complete updated genome of H37Rv. 1 is reported here as H37Rv (new) and the full genome sequence has been deposited (Supplementary Data 2, 3; Genbank accession: CP110619).
All 10 H37Rv regions are expressed As further confirmation that none of the regions found in H37Rv(new) were caused by sequencing or assembly artifacts, we tested whether published H37Rv RNA sequencing studies contained reads that mapped to each region. This investigation also allowed us to determine whether the newly discovered esxN. 2 and esxJ. 3 genes were expressed differently comparted to their esxN and esxJ paralogs as one possible indication of significant functional differences between these genes. We downloaded all public M. tuberculosis H37Rv RNA sequencing datasets on the NCBI SRA database. Of the 908 datasets available at the time of our analysis, 905 datasets used Illumina sequencing chemistry, passed our quality control metrics, and were therefore included in these analyses. We aligned these raw reads to both the existing H37Rv1998 reference genome and to H37Rv(new) and computed the feature counts for each genomic region of difference following batch correction.
We discovered that raw RNA reads from public data sets did indeed map onto the newly discovered regions in H37Rv(new) (Figure  S4 ). However, both esxN. 2 and esxJ. 3 showed significantly different levels of expression across the 905 datasets compared to their esxN and esxJ paralogs (Fig. 3c, d ). We also found that on average, 99. 8% of the reads mapping to esxN. 2 and 46% of the reads mapping to esxJ. 3, did not map back to H37Rv1998 (Figure  S4k ), indicating that esxN. 2 reads were likely discarded in previous studies and that esxJ. 3 expression was likely ascribed mistakenly to esxJ. 1. Expression levels of both esxN paralogs and of both esxJ paralogs were only moderately correlated (r = 0. 557; p = 7. 02e-75 and r = 0. 516; p = 1. 41e-65 respectively), suggesting that the paralogs were under different regulatory control (Fig.
Discussion The development of new NGS tools and pipelines to generate accurate complete genomes is vital to our understanding of drug resistance evolution, pathogenesis and virulence of clinically relevant pathogens such as M. tuberculosis. The first complete whole genome sequence of the H37Rv strain generated using shotgun sequencing was published in 1998  4  and ushered in a revolution in M. tuberculosis research. The published sequence led to the discovery of novel protein families responsible for fatty acid and polyketide biosynthesis, drug efflux pumps, PE/PPE proteins and transposon elements  4, 5, 30. Minor divergences among subcultures of these strains leading to differences in SNPs, indels and transposable elements have also been described  5. H37Rv is the workhorse of M. tuberculosis research and its Genbank sequence is widely used for aligning and analyzing DNA, RNA, and Tn-seq studies.
Thus, the importance of establishing a fully accurate reference genome for this strain cannot be overstated. Our current study indicates that there are substantial regions of difference between the Genbank H37Rv reference sequence and what is likely to be the true complete genome sequence of H37Rv isolates, including NR-123, TMC 102 and a culture of the original 1998 isolate (   (rv1038c) gene is a member of the QILSS subfamily and is a paralog of esxB (CFP-10)  31. Canonical esxA and esxB proteins interact to form a heterodimer that is secreted from M. tuberculosis. These proteins are known to be frequently recognized T-cell antigens  32. Several esx gene pairs are contained within the conserved ESX-1 -ESX-5 loci which together encompass a type VII secretion system (T7SS). ESX-5 has been linked to PE and PPE protein secretion during macrophage infection  33, 34.