dataesr/scientific-paragraphs-categorization huggingface dataset:
Immunofluorescence The immunofluorescence procedure was based on a published protocols  [12, 57]  with modifications. Skin tissues for immunofluorescent detection of involucrin were fixed for 2 h in 4% paraformaldehyde (Sigma-Aldrich) in 0. 1 M phosphate buffer (PB). Fixed tissues were stored in 18% sucrose (Sigma-Aldrich) in PB with 0. 01% sodium azide (Sigma-Aldrich). Cryostat sections (8 μm) were blocked in 10% normal horse serum (Vector Laboratories) in 0,1% BSA in PB for 1 h at RT and incubated with rabbit antiinvolucrin (1:1500; Covance) antibodies o/n at 4 °C. Tissue sections were subsequently incubated with Alexa Fluor 594 (Life Technologies) for 1 h at RT. Slides were mounted under coverslips using ProLong® Gold Antifade Mountant with DAPI (Life Technologies). Sections were visualized and photographed with an Olympus microscope (BX43) equipped with an Olympus digital camera (XC50) and analyzed with CellSens Dimension 1 Software (Olympus Soft Imaging Solutions GmbH).
Setting and study population We used data from a large, single-center prospective cohort of patients with meningitis, enrolled over a 31-year period at the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain), a 540bed tertiary university hospital serving an estimated population of 410,500 in a predominantly urban area. From 1982 through 2012, all consecutive adults (defined as patients of 14 years old or more) with a diagnosis of acute BM at the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) were prospectively identified and followed. The characteristics of this cohort have been described previously. [4, 8, 12] From 1999, information about the results of cerebral computed tomography (CT) scans, diagnostic sequence of CT in relation to lumbar puncture (LP), and start of antibiotic treatment were also registered.
Statistical analysis The gene diversity, number of alleles, and PIC value were calculated using the PowerMarker software (Ver. 3. 0;  Liu and Muse 2005). The glutenin relationship between cultivars was visualized as a dendrogram using the Pow-erMarker and MEGA5 software  (Tamura et al. 2011). The Neighbor-joining tree was constructed using the frequencybased distance for the shared allele.
8%, n = 13). The requirement for surgery after methotrexate was 21% (n = 21). The next pregnancy was classified as viable, non-viable or ectopic and time to next pregnancy was calculated as the number of months from discharge to conception. Minitab 17 Software was used for statistical analysis. A subsequent pregnancy was recorded in 65. 3% (n = 147; n = 11 required fertility treatment, n = 2 after methotrexate, n = 9 after salpingectomy). Subsequent pregnancy rates were 61. 5% (n = 48) for methotrexate, 66. 4% (n = 87) for salpingectomy, 100. 0% (n = 3) for salpingotomy and 69. 2% (n = 9) for conservative management (Table  1 ). There were no significant differences between the subsequent pregnancy rates after methotrexate or salpingectomy or between surgical and non-surgical management. The average time to next conception was 13.
Objective: To explore the phenotypic spectrum and pathophysiology of human disease deriving from mutations in the CNTNAP1 gene. Methods: In a field study on consanguineous Palestinian families, we identified 3 patients carrying homozygous mutations in the CNTNAP1 gene using whole-exome sequencing. An unrelated Irish family was detected by screening the GENESIS database for further CNTNAP1 mutations. Neurophysiology, MRI, and nerve biopsy including electron microscopy were performed for deep phenotyping. Results: We identified 3 novel CNTNAP1 mutations in 5 patients from 2 families: c. 2015G. A:p. (Trp672*) in a homozygous state in family 1 and c. 2011C. T:p. (Gln671*) in a compound heterozygous state with c. 2290C. T:p. (Arg764Cys) in family 2. Affected patients suffered from a severe CNS disorder with hypomyelinating leukodystrophy and peripheral neuropathy of sensory-motor type.
Primer sequences are available on request. Screening for CNTNAP1 patients using GEM. app/Genesis. Previous studies on CNTNAP1-associated disease have exclusively focused on cohorts with AMC. To capture the full phenotypic spectrum caused by CNTNAP1 in an unbiased approach, we screened a total of 2,599 families with various neurologic phenotypes and a family history compatible with autosomal recessive inheritance available in the GEM. app/Genesis database at the time  7  for rare homozygous or compound heterozygous CNTNAP1 mutations. Phenotypes included 492 families with neuromuscular phenotypes, 251 HSP families, 721 ALS families, and 168 families with hereditary ataxia. Electron microscopy. As described elsewhere,  9  samples of sciatic nerves were fixed for 3 hours in 2. 5% glutaraldehyde in Sorensen buffer and osmificated for 1 hour in 1% OsO4.
2:c. 2015G. A:p. (Trp672*). Sanger sequencing of all family members showed full segregation with the disease (figure  1 ). The parents were heterozygous for the mutation, and the healthy siblings were either heterozygous or homozygous for the wildtype. The mutation was not found in the ESP6500, 1000 Genomes, or the Exome Aggregation Consortium (ExAC) database. Further follow-up of the family revealed a 4-weekold child in a distantly related family branch (branch C in figure  1A ) who was born at term with a history of polyhydramnios, had similar dysmorphic features with low-set ears, micrognathia, and microcephaly and required controlled ventilation. However, there were no joint contractures or signs of AMC. Genetic testing revealed the identical homozygous CNTNAP1 mutation c. 2015G. A:p. (Trp672*).
Screening for CNTNAP1 mutations reveals further non-AMC cases. An unbiased screening approach using the GEM. app/Genesis database led to the identification     3 ). There were mega cisterna magna and thin corpus callosum, but clear cerebral hypoplasia or cortical malformations were not detected. Nerve conduction studies and EMG suggested sensory-motor neuropathy and denervation. Patient 5 was the brother of patient 4. Polyhydramnios and reduced fetal movements were diagnosed at 31-week gestation. At birth, he was profoundly hypotonic with no respiratory effort. He did not swallow and muscle reflexes were absent. No invasive treatment was initiated and he died at 4 hours of age. Sural nerve biopsy reveals paranodal abnormalities. Sural nerve biopsy of patient 4 revealed a significant axonal loss which was homogeneous between nerve fascicles.
2006 (Tixier et al. , 2008 (Tixier et al. , 2011 (Tixier et al. , 2012 (Tixier et al. , 2014)). Sequences of this species are not reported from Genbank. Remarks. Euseius fustis was collected in 62% of the cassava fields sampled in Kenya. This species was mostly observed in eastern, western and coastal areas covering most agro-ecological zones of upper and low midlands (UMs & LMs) as well as the coastal lowlands (CLs). This species was already reported from different plants in western Kenya including cassava  (Moraes and McMurtry 1988; Moraes et al. 1989; El-Banhawy and Kapp 2011). It is only present in Africa and has been reported on cassava also in Benin  (Moraes et al. 1989), Malawi and Democratic Republic of Congo (Gutierrez and Bonato 1994; Zannou et al. 2005). The high densities presently observed as well as its high frequency indicate this is the most common phytoseiid on cassava in Kenya, even after the introduction of T.
Neoseiulus asperisetatus Zannou,  Moraes & Oliveira, 2006  Specimens observed. 1 female: (01°12'25"S, 38°05'13"E, Alt. 1642 m Kyanika Kitui, 21-I-2011). Previous records. Benin, Cameroon, Democratic Republic of Congo, Kenya, Zambia. Remarks. his species is prevalent in Africa and has been previously reported from cassava in Benin  (Zannou et al. 2006). 3 ). DNA sequences of this species exist in Genbank but not for the fragment of COI herein sequenced. Remarks. This species was observed in 0. 7 % of the cassava fields sampled, with a total of 19 specimens. Based on the morphological details given by El-Banhawy and Knapp (2011) for specimens from Kenya reported as Neoseiulus onzoi Zannou Moraes & Oliveira, we consider that those mites instead belong to N. idaeus. The latter species has been introduced to Africa between 1985 and 1993 to control M.
Microarray Hybridization and Data Processing Total RNAs extracted from 3 biological replicates per condition were hybridized with 60-mer oligoarrays bearing a set of probes for 29,582 unigenes (NimbleGen Gene Expression 12 × 135K Arrays). Labeling, hybridization and scanning were carried out at the GeT-transcriptomic platform (GenoToul-Toulouse, France). Microarray data were analyzed using the R and R/Bioconductor software  (Gentleman et al. , 2004; R Core Team, 2013). Quality control was done using the arrayQualityMetrics package  (Kauffmann et al. , 2009) , one identified outlier (GHS14_1) was excluded from further analyses. Data were normalized using the Robust Multi-array Average algorithm (RMA)  (Irizarry et al. , 2003). Principal component analysis was done using the prcomp function, visualizations were made using ggplot2  (Wickham, 2009).
Differentially expressed genes between treatment and control samples for all stages and treatment durations were identified using Limma package  (Smyth, 2005). Differentials with absolute fold changes above 2 and BH  (Benjamini and Hochberg, 1995)  corrected P-values below 0. 05 were considered significant. Significantly affected gene categories based on the MapMan Ontology  (Thimm et al. , 2004; Usadel et al. , 2005)  were identified using a Chi-square test. MapMan mappings for the Cribi 12X grapevine genome were based on closest homologs regarding to the Arabidopsis thaliana genome. Empirical cumulative distribution function plots for selected categories were made using the latticeExtra package. Gene annotations were taken from  Grimplet et al. (2012). The raw microarray data were submitted to Gene Expression Omnibus (NCBI) and are accessible through GEO accession number GSE86551.
Alteration of Global Berry Transcriptome in Response to Localized Heat Treatment Principal component analysis of the whole normalized gene expression dataset showed that the 3 replicates of each experimental condition are well-grouped and therefore adequate for further analysis (Figure  3 ; Supplementary Figure  1 ). Principal FIGURE 1 | Sampling and fruit temperature recording during treatments. (A) Both control and HT were applied for 14 d periods, at middle-green, veraison (50% of berries turning to a visible red color) and middle-ripening. For transcriptomic analysis, samples were collected after 1, 7, and 14 d of treatment. For biochemical analysis, berries from each treatment condition were sampled at harvest. (B) Mean temperatures in control (opened symbols) and heated berries (filled symbols) during the herbaceous (green circles), veraison (pink triangles) and ripening (purple squares) periods were determined for each day of treatment.
MapMan analysis (Figure  6 ) shows the contrasted heat responses of DEGs related to the general phenylpropanoid and to the flavonoid biosynthesis pathways (Figure  6 ; Supplementary Figure  3 ; Supplementary Table  10 ). The transcriptomic responses of GHT samples clearly differed from both VHT and RHT samples. During the herbaceous stage, genes involved in the general phenylpropanoid pathway (phenylalanine ammonialyase PAL, cinnamate 4-hydroxylase C4H, 4-coumarate-CoA ligase 4CL) were induced by HT regardless to the stress duration. By contrast, in VHT and RHT samples, the same genes were up-regulated after 1 day, but strongly repressed after 7 and 14 days of treatment. Many transcripts related to the flavonoid biosynthesis pathway were repressed in GHT clusters. Two chalcone synthase transcripts (VviCHS1: VIT_14s0068g00920, VviCHS2: VIT_14s0068g00930) encoding the first committed enzyme in flavonoid biosynthesis  (Parage et al.
, 2008; Tarara et al. , 2008) , as well as separated treatments (daytime cooling or night-time heating) even though the effects were less marked in the latter case  (Cohen et al. , 2012). High temperatures promote the decrease of organic acid content observed after veraison, by exacerbating the malic acid breakdown  (Ford, 2012; Rienth et al. , 2016). Elevated temperatures accelerate the utilization of malate, enhancing the anaplerotic capacity of the TCA cycle for amino acid biosynthesis  (Sweetman et al. , 2014). By contrast to previous reports describing an increase in berry concentrations of various amino acids after exposure of whole grapevines to HT, the present data were obtained after berry-localized treatments. After a 14 d HT of whole grape fruiting cuttings,  Carbonell-Bejerano et al.
XETs contribute to cell wall expansion and loosening, through their action on xyloglucan frame. After whole grape exposure to heat, an increased level of XETs transcripts was measured in warmed green berries. It was postulated that enhanced expression could be related to the adaptation of berry volume to temperature and the need for more flexible cell walls  (Rienth et al. , 2014). Our data reinforce these observations with 19 up-regulated XETs in GHT clusters. Cell wall loosening through increase level of expansin might help to maintain cellular functions during HT (Le  Gall et al. , 2015).
ACKNOWLEDGMENTS The authors would like to thank  Pr. Laurent Torregrosa ,  Dr. Charles Romieu ,  Dr. Markus Rienth ,  Dr. ZhanWu Dai  and  Pr. Grant Cramer  for valuable discussions and scientific interactions. The authors also thank  Dr. Pablo Carbonell Bejerano  for critical reading of the manuscript. For the production of the fruiting cuttings and technical assistance during greenhouse experiments, we express our gratitude to  Jean Pierre Petit  and  Guillaume Pacreau.
Aberrant expression. Once normalized for technical biases, sex and biopsy site (Supplementary Method 1 and Supplementary Fig. 1 ), the samples typically presented few aberrantly expressed genes (median of 1, 90% of the samples with o10, Fig. 2a , Supplementary Data 3) with a large effect (|Z-score|43) and significant differential expression (Hochberg adjusted P valueo0. 05). Among the most aberrantly expressed genes across all samples, we found 2 genes encoding mitochondrial proteins, MGST1 (one case) and TIMMDC1 (two cases) to be significantly down-regulated (Fig. 2b-d  and Supplementary Fig. 2 ). For both genes, WES did not identify any variants in the respective patients, no variant is reported to be disease-associated and no case of potential bi-allelic rare variant is listed in our in-house database comprising more than 1,200 whole-exomes from mitochondrial patients and more than 15,000 whole-exomes available to us from different ongoing research projects.
3b ). Two genes encoding mitochondrial proteins, TIMMDC1 and CLPP, which were among the 20 most significant genes, caught our attention (Supplementary Data 6). Out of 136 exon-junction reads overlapping the acceptor site of CLPP exon 6 for patient #58955, 82 (per cent spliced in ref. 32, C ¼ 60%) skipped exon 5, and 14 (C ¼ 10%) showed a 3 0 -truncated exon 5, in striking contrast to other samples (Fig. 3c ). The likely genetic cause of these two splice defects is a rare homozygous variant in exon 5 of CLPP affecting the last nucleotide of exon 5 (c. 661G4A, p. Glu221Lys 1. 2 Â 10 À 5 frequency in the ExAC database  33  , Supplementary Fig. 5 ). Both detected splice defects result in truncated CLPP and western blots corroborated the complete loss.
112 and 1. 308, respectively) this emphasizes the influence of this variant in the creation of the new exon. Besides, the four other tools predicted no significant change in splicing  [40] [41] [42] [43]. We further discovered an additional family in our in-house WGS database (consisting of 36 patients with a suspected mitochondrial disorder and 232 further patients with UQCRC2 was used as loading control. CI, complex I subunit; CIII, complex III subunit; MW, molecular weight. (g) Blue native PAGE blot of the control fibroblasts re-expressing TIMMDC1 (NHDF-T), the control fibroblasts (NHDF), patient fibroblasts (#96687) and patient fibroblast re-expressing TIMMDC1 (#96687-T). Immunodecoration for complex I and complex III was performed using NDUFB8 and UQCRC2 antibodies, respectively. CI, complex I subunit; CIII, complex III subunit.
Moreover, MCOLN1 was missed by our WES variant filter since the intronic variant was not prioritized. Hence, the WES data could not be conclusive about MCOLN1. In contrast, the RNA-seq data demonstrated two loss-of-function alleles in MCOLN1 and therefore established the genetic diagnosis. RNA defects in exome-diagnosed patients. Here, we included genetically diagnosed patients in our RNA-seq analysis pipeline to increase the power for the detection of aberrant expression and aberrant splicing in fibroblast cell lines. However, when evaluating the results for 40 diagnosed cases with WES and RNA-seq available (Supplementary Table  1 ), aberrant splicing detected 7 out of 8 cases with a causal splicing variant, MAE recovered 3 out of 6 patients with heterozygous missense variants compound with a stop or frameshift variant, and aberrant expression recovered 3 out of 4 homozygous stop variants.
Methods Study cohort. All individuals or their guardians gave written informed consent before undergoing evaluation and testing, in agreement with the Declaration of Helsinki and approved by the ethical committees of the centres participating in this study, where biological samples were obtained. All studies were completed according to local approval of the ethical committee of the Technical University of Munich. The study cohort is described in detail in Supplementary Note 1. Exome sequencing. DNA from fibroblast cell lines was isolated from whole-cell lysates using the AllPrep DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Exonic regions were enriched using the SureSelect Human All Exon kit from Agilent (Supplementary Data 3) followed by sequencing as 100 bp paired-end runs on an Illumina HiSeq2000 and Illumina HiSeq2500 (AG_50MB_v4 and AG_50MB_v5 exome kit samples) or as 76 bp paired-end runs on the Illumina GAIIx (AG_38MB_v1 and AG_50MB_v3 exome kit samples)  54.
Exome alignment and variant prioritization. Read alignment to the human reference genome (UCSC Genome Browser build hg19) was done using Burrows-Wheeler Aligner  55  (v. 0. 7. 5a). Single-nucleotide variants and small insertions and deletions (indels) were detected with SAMtools 56,57 (version 0. 1. 19). Variants with a quality score below 90, a genotype quality below 90, a mapping quality below 30, and a read coverage below 10 were discarded. The reported variants and small indels were annotated with the most severe entry by the Variant Effector Predictor 58 based on The Sequence Ontology term ranking  59. The candidate variants for one patient are filtered to be rare, affect the protein sequence and potentially both alleles. Variants are rare with a minor allele frequency o0. 001 within the ExAC database  33  and a frequency o0.
We modelled the RNA-seq read counts K i,j of gene i in sample j with a generalized linear model using the R/Bioconductor DESeq2 package  63, 64  : K i;j $ NB s j Âq i;j ; a i À Á log 2 ðq i;j Þ ¼ b 0 i þ b condition i x condition i;j þ b batch i x batch i;j þ b sex i x sex i;j þ b hox i x hox i;j Where NB is the negative binomial distribution; a i is a gene specific dispersion parameter; s j is the size factor of sample j; b 0 i is the intercept parameter for gene i. The value of x condition i;j is 1 for all RNA samples j of the patient of interest, thereby allowing for biological replicates, and 0 otherwise. The resulting vector b condition i represents the log 2 fold changes for one patient against all others. Z-scores were computed by dividing the fold changes by the s. d. of the normalized expression level of the respective gene.
The P values corresponding to the b condition i were corrected for multiple testing using the Hochberg family-wise error rate method  65. Detection of aberrant splicing. The LeafCutter 66 software was utilized to detect aberrant splicing. Each patient was tested against all others. To adjust LeafCutter to the rare disease setting, we modified the parameters to detect rare clusters, capture local gene fusion events and to detect junctions unique to a patient (minclureads ¼ 30; maxintronlen ¼ 500,000; mincluratio ¼ 1e-5, Supplementary Data 9). Furthermore, one sample was tested against all other samples (min_samples_per_group ¼ 1; min_samples_per_intron ¼ 1). The resulting P values were corrected for multiple testing using a family-wise error rate approach  65. The significant splice events (Hochberg adjusted P valueo0.
05) detected in the undiagnosed patients were visually classified as exon skipping, exon truncation, exon elongation, new exon, complex splicing (any other splicing event or a combination of the aforementioned ones) and false positives (n ¼ 73, Fig. 3b ). However, due to LeafCutter's restriction to split reads it is difficult to detect intron retention events, since no split-read is present in a perfect intron-retention scenario. For further analysis, only reads spanning a splice junction, so called split reads, were extracted with a mapping quality of 410 to reduce the false-positive rate due to mapping issues. Each splice site was annotated as belonging to the start or end of a known exon or to be entirely new. For the reference exon annotation the GENCODE release 24 based on GRCh37 was used  67.
Hence, the components were modelled to have the same parameters a c , b c in all three groups but their mixing proportions p s,c to be group-specific. Fitting was performed using the expectation-maximization algorithm. For the initial step, the data points were classified as background (Co0. 001), weak spliced (Co0. 1) and canonical (C4 ¼ 0. 1). After convergence of the clustering the obtained parameters were used to estimate the probability for each junction side to belong to a given class. Detection of mono-allelic expression. For MAE analysis only heterozygous single-nucleotide variants with only one alternative allele detected from exome sequencing data were used. The same quality filters were used as mentioned in the exome sequencing part, but no frequency filter was applied. To get allele counts from RNA-seq for the remaining variants the function pileLettersAt from the R/Bioconductor package GenomicAlignments 62 was used.
The data were further filtered for variants with coverage of at least 10 reads on the transcriptome. The DESeq2 package  63, 64  was applied on the final variant set to estimate the significance of the allele-specific expression. Allele-specific expression was estimated on each heterozygous variant independently of others (that is, without phasing the variants). For each sample, a generalized linear model was fitted with the contrast of the coverage of one allele against the coverage of the other alleles. Specifically, we modelled K i,j the number of reads of variant i in sample j as:.
Acknowledgements We are grateful to the participating families. Further we thank  Dr Lina Florentin  for providing DNA samples of the parents of # 35791. This study was supported by the  German Bundesministerium fu ¨r Bildung und Forschung (BMBF)  through the  E-Rare  project  GENOMIT  ( 01GM1603  and  01GM1207 , H. P. and T. M. , FWF I  920-B13  for J. A. M. and J41J11000420001 for D. G. ), through the  Juniorverbund in der Systemmedizin 'mitOmics'  ( FKZ 01ZX1405A , D. M. B. and J. G. , and  FKZ 01ZX1405C , T. B. H. ), and the  DZHK (German Centre for Cardiovascular Research , L. S. K. , T. M. ). The study was furthermore supported by the  Deutsche Forschungsgemeinschaft (German Research Foundation ) within the framework of the  Munich Cluster for Systems Neurology  (EXC 1010 SyNergyDFG, T.