dataesr/scientific-paragraphs-categorization huggingface dataset:
-Reconstruction values and classification was also performed on random data obtained by reconstructing the licking activity of a session with the neural activity of a subsequent session. This made it possible to establish the distribution of accuracy for randomized data. -The accuracy of classification was compared between the true data and the randomized data sets and a p-value was calculated by counting the number of permutations showing better accuracy for the randomized data than the true data. -We progressively removed units, starting with those with highest classifier weights, which reduced the accuracy of classification, until the p-value of population classification rose above 0. 4. This indicated that the remaining units contained no more information about lick events than randomized data.
Reference/target asymmetry is the difference of the distance of reference and target projected data to a given baseline. We examine three cases: (i) passive evoked responses, distances calculated relative to engaged spontaneous activity; (ii) engaged evoked responses, distances calculated relative to passive spontaneous activity; (iii) engaged evoked responses, distances calculated relative to engaged spontaneous activity. These values are shown during the sound (top) and the silence (bottom). In all three cases, the engaged decoding axis was used for projections. Decoding axes determined during sound presentation and post-stimulus silence are respectively used for projections in the top and bottom rows. Error bars represent 95% confidence intervals (n=400 cross validations; sound: p(col1,col3)=0.
Following this, the recording could be commenced at any point as in regular electrophysiological experiments in excised patch configuration. Recorded activity was quantified by performing a single-channel search analysis using the Clampfit-10 (pClamp software suit, Molecular Devices, Sunnyvale, CA, USA) and QuB 2. 0 programs. Statistical analyses, t-tests and Gaussian fits to amplitude distributions were carried out using Microcal Origin software. Values are expressed as mean ± s. e. m. , unless indicated otherwise.
Acknowledgements This work was supported by grants from  INSERM, la Ligue Nationale Contre le Cancer, le Ministère de l'Education Nationale , the  Région Nord/Pas-de-Calais  to NP and from the  Research Foundation -Flanders  ( FWO; G. 0C91. 14 ) to GB. George Shapovalov  was supported by fellowships from  FRM. Gabriel Bidaux  was supported by  SIRIC OncoLille and CHRU Lille. NP and GB are partners within the  FWO Scientific Research Community  "Ca 2+ signaling in health, disease and therapy" ( W0. 019. 17 ). We would like to thank  Prof. J. B. Parys ,  Giovanni Monaco  and  Hristina Ivanova  ( KU Leuven, Belgium ) and  Prof. C. W. Distelhorst  ( Case Western Reserve University, Cleveland, OH ) for technical assistance and providing WEHI7. 2 cell lines and IP 3 R antibodies.
Results: A new stwintron was encountered in Aspergillus nidulans and Aspergillus niger. It is present across three classes of Leotiomyceta in the transcript of a well-conserved gene encoding a putative lipase (lipS). It occupies the same position as a standard intron in the orthologue gene in species of the early divergent classes of the Pezizomycetes and the Orbiliomycetes, suggesting that an internal intron has appeared within a pre-extant intron. On the other hand, the stwintron has been lost from certain taxa in Leotiomycetes and Eurotiomycetes at several occasions, most likely by a mechanism involving reverse transcription and homologous recombination. Another ancient stwintron present across whole Pezizomycotina orders-in the transcript of the bifunctional biotin biosynthesis gene bioDA-occurs at the same position as a standard intron in many species of non-Dikarya.
Nevertheless, also the bioDA stwintron has disappeared from certain lineages within the taxa where it occurs, i. e. , Sordariomycetes and Botryosphaeriales. Intriguingly, only the internal intron was lost from the Sordariomycetes bioDA stwintron at all but one occasion, leaving a standard intron in the same position, while where the putative lipase stwintron was lost, no intronic sequences remain. Conclusions: Molecular phylogeny of the peptide product was used to monitor the existence and fate of a stwintron in the transcripts of two neatly defined fungal genes, encoding well conserved proteins. Both defining events-stwintron emergence and loss-can be explained with extant models for intron insertion and loss. We thus demonstrate that stwintrons can serve as model systems to study spliceosomal intron evolution.
The two-step splicing process resulting in stwintron excision demonstrated that splice sites pair via intron definition (cf. [6] ) in filamentous ascomycota (Pezizomycotina). For the [D1,2] stwintron we found in the alternative oxidase transcript of Helminthosporium solani, we demonstrated a complex structure that could undergo two alternative pathways of sequential splicing. An internal intron could be envisaged as interrupting the donor sequence or the acceptor sequence of an external intron. In every case where a [D1,2] stwintron is extant (which implies that the complex intervening sequence starts with two successive Gs at its 5′ terminus), a stwintron in which the acceptor sequence of the external intron is disrupted between the penultimate and ultimate nucleotides-an [A2,3] stwintron-will occur every time that the 5′ base of the downstream exon is also a G.
Only the [D2,3] stwintron in the biotin-biosynthetic bifunctional gene bioDA was found across a complete class of fungi, the Sordariomycetes. This stwintron thus appears evolutionary stable: loss of any of the internal splice sites will inevitably result in the inability to properly remove the complete intervening sequence from the primary transcript and hence, in biotin auxotrophy. Nevertheless in the bioDA gene of Nectria haematococca, a standard U2 intron is present at the position occupied by a [D2,3] stwintron in other Sordariomycetes. To facilitate molecular and mutational study of mechanisms involved in the emergence and disappearance of spliceosomal introns using stwintrons as model systems, we initiated a search for bona fide stwintrons in the amenable species Aspergillus nidulans, in which molecularand classical genetic tools are readily developed  [7] [8] [9] [10] [11] [12] [13].
We devised a crude informatics tool that enabled us to identify putative stwintrons in whole genome sequences, the principle and basics of which were described in detail in  [5]. Here, we describe a stwintron we have uncovered in a putative lipase gene after a preliminary screen of the A. nidulans genome sequences for [D1,2] stwintrons. We also experimentally verified its presence in Aspergillus niger. This stwintron is present across three classes of Pezizomycotina in the transcript of a well-conserved gene without paralogues of note, sufficiently ancient to monitor its emergence and evolution.
A new [D1,2] stwintron in Aspergillus nidulans and Aspergillus niger An informatics method to detect putative [D1,2] stwintrons has been detailed elsewhere as auxiliary methodology  [5]. We screened the genome sequences of A. nidulans (whole genome shotgun master accession AACD00000000: annotated scaffolds CH236920-CH236936) and detected, amongst dozens of others, a candidate [D1,2] stwintron at locus AN7524. This locus is predicted by auto-annotation to harbour two intervening sequences. At 69 nt from the ATG, there would be a small canonical intron of 52 nt (5′-GUACGU-33-nt-ACUGAC-4-nt-CAG). 497 nt further downstream, a 289-nt-long, phase 2 intron (5′-GUAUGC-268-nt-GCUAAU-6-nt-CAG) would split a CGA (Arg) codon, while the mature messenger would code for a peptide of 824 amino acids. However, our stwintron search tool suggested the presence of a [D1,2] stwintron at the 5′ end of this large auto-annotated second intron, of which the proposed donor sequence would actually serve as the donor of the 53-nt long internal intron (5′-GUAUGC-33-nt-GCUAAC-5-nt-UAG).
According to our predicted intron-exon structure of locus AN7524 (Fig. 1a ), there would thus be four exons. The A. nidulans gene is best expressed growing on complete medium as compared with minimal medium conditions used for high-throughput RNA sequencing (JBrowse module at AspGD;  [8] ). Nevertheless, there are no RNASeq reads from which the complete stwintron was absent nor reads from which the large auto-annotated intron sequence was removed (data not shown). We therefore sought to confirm excision of all three intervening sequences we predicted by sequencing of cDNA generated from total RNA isolated from a 16-h cultivation in complete medium (see "Methods" section for details). The cDNA (GenBank Accession number MF612150) specifies a reading frame of 2616 nt that codes for a.
c The existence of the splicing intermediate from which the internal intron has been removed by the first excision, has been confirmed experimentally (GenBank MF612152). The newly formed donor splice site sequence of the retained external intron, (5′-gugagu), is underlined peptide of 871 amino acids. The first intron separates the codons for His23 and Tyr24, the [D1,2] stwintron splits the codon for Pro189 while the third intron splits the codon for Ser236 (Fig. 1a ). Next, we confirmed the existence of the predicted splicing intermediate of the [D1,2] stwintron (Fig. 1b ) with our previously published RT-PCR strategy using a reverse primer that terminally overlaps the 3′ distal junction with the predicted external intron to avoid amplification off fully spliced RNA. Upon cloning and sequencing, the smaller amplified fragment was shown to lack the predicted internal intron (53 nt) with the 5′-GUGAGU donor of the external intron reconstructed (Fig.
1c ). We deposited the determined sequence of this splicing intermediate at GenBank (MF612152). The four exon gene model is supported by Expressed Sequence Tag (EST) clone Asn_02874 from the related fungus A. niger (Accession DR703192), covering the stwintron position and the last intron position, 142 nt downstream. We independently verified the positions of the [D1,2] stwintron and the two other extant introns in the orthologue gene (miscalled locus ASP-NIDRAFT_53020) by sequencing cDNA (GenBank MF612151) including the complete coding region encoding a peptide of 909 amino acids in A. niger ATCC 1015 (see Fig. 2a  for the gene model). The 109-nt long [D1,2] stwintron splits the CCU codon for Pro184 in phase 1 (Fig. 2b ). A 52-nt long internal intron (5′-GUAAGA-31-nt-GCUAAC-6-nt-CAG) would be nestled in the donor of a 57-nt long external intron between the first and second nt (5′-G|UGAGU-35-nt-GCUAAU-7-nt-CAG).
We collected hunderds of lipS orthologue genes in both phyla of Dikarya upon TBLASTN screening of the DNA databases with the A. nidulans protein as the query (data not shown). We noticed that in 52 Basidiomycota orthologue genes-a selection of genome-sequenced species representing all three subphyla-the stwintron position was not occupied despite the wealth of introns in certain taxa: Basidioascus undulatus, for instance, has 22 introns in its lipS orthologue gene and Mrakia frigida, 18. In the Taphrinomycotina subphylum (Ascomycota), the orthologue genes in Taphrina species have one intron at a unique position, 21 nt behind the start codon, while the gene is intronless in five other genome-sequenced species (including Saitoella complicata). On the other hand, both the stwintron position and the downstream third intron position in the DNA coding for the Pfam07859 domain were occupied in certain taxa of Pezizomycotina (as specified further below): the stwintron position thus appears to be specific to that subphylum.
Remarkably, in Nectria haemotococca (formerly known as Fusarium solani f. sp. pisi) a standard intron occupies the position of the stwintron in the other Sordariomycetes, including in other Fusarium species. Since the spring of 2013, hundreds of new Pezizomycotina genome sequences have been added to publicly accessible databases. We took the opportunity to update our analysis of the bioDA stwintron, to compare defining aspects of its evolution with those of the (new) stwintron in the putative lipase lipS gene (see above). We screened the databases with TBLASTN using the A. nidulans protein (protein accession number ACR44943) as the query and collected hundreds of fungal bioDA genes for which we manually deduced the intron-exon structure and the cognate protein (results not shown). From the updated collection from non-Pezizomycotina taxa, we can now confirm that the intron position at which the stwintron occurs (in Sordariomycetes and Botryosphaeriales, see below) is deeply rooted in fungal evolution (see, e.
terreus, A. apis, C. variabilis and "G. candidum 3C") (Figs. 4, 5 ). On the other hand, in Sordariomycetes, the loss of the stwintron from the bioDA gene has resulted in the retention of a standard U2 intron in all but one instance (i. e. , that involving the separate clade of five Diaportales, see above) (Fig. 7 ). It would thus appear that in most Sordariomycetes bioDA, the event(s) leading to the emergence of an internal intron was/were reversed to return to the primordial situation. Despite this rather striking difference between the two ancient stwintrons, all observed instances of accurate intron loss are compatible with one single mechanism, involving reverse transcription of spliced transcripts and homologous recombination of cDNA at the genome locus (e. g. ,  [25] ; reviewed by  [26] ).
cDNA sequencing Double strand cDNA was gel-purified (NucleoSpin Gel and PCR Clean-up, Macherey-Nagel) and cloned (pGEM-T Easy Vector System I, Promega). Plasmid DNA was isolated using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). Three independent clones were sequenced over both strands using universal primers hybridising to the vector (MWG-Biotech AG, Ebersberg, Germany) or gene-specific oligonucleotide primers where appropriate. cDNA sequences were deposited at Gen-Bank under accession numbers MF612150-MF612153.
Phylogenetic analyses For the analysis of the putative lipase orthologue, 292 proteins were aligned with Multiple Alignment using Fast Fourier Transform (MAFFT; version 7)  [27] , applying the E-INS-i algorithm and a BLOSUM 62 similarity matrix. The MAFFT alignment was trimmed with Block Mapping and Gathering with Entropy software (BMGE version 1. 12)  [28]  using BLOSUM 30 and a block size of 5. A Maximum Likelihood tree was then calculated online from the curated alignment by PhyML 3. 0  [29]  using the WAG substitution model. For the study of the bifunctional BioDA protein, 298 proteins were aligned using a BLOSUM 45 similarity matrix in the MAFFT E-INS-i module. The alignment was BMGE trimmed using BLOSUM 55 and a block size of 5, before a Maximum Likelihood phylogeny was inferred using the WAG substitution model.
The DNA was amplified in an Applied Biosystems 1 (Applied Biosystems, Foster City, CA) for All positive kDNA qPCR specimens were systematically amplified by ITS1-PCR as previously described  [15]  using LITSR (5 0 -CTGGATCATTTTCCGATG-3 0 ) and L5. 8S (5 0 -TGATAC CACTTATCGCACTTA-3 0 ) primers. Analysis on a 2% agarose gel was used to verify the amplified product size. ITS1 PCR products were purified by using ExoSAP (ThermoScientific, EU) and Leishmania species identification was done by DNA sequencing using an ABI Prism 1 Big Dye™ Terminator, Cycle Sequencing Ready Reaction Kit and AB1 3130 sequencing system (ABI, PE Applied Biosystems), with the same primers used for PCR. DNA sequences from both strands were aligned and edited using Staden software package (http://staden. sourceforge. net/ ).
MEGA version 7 software (www. megasoftware. net) was used to conduct multiple sequence alignments (ClustalW option) and to construct phylogenic tree. Relationships between specimens and reference isolates (L. infantum isolates from Mediterranean countries as well as Tunisian isolates of L. tropica and L. major) were inferred based on genetic distances using the Neighbor Joining (NJ) method using the Kimura 2 parameters model. Statistical support for tree distances was evaluated using bootstrapping (2000 replicates).
Sandfly molecular typing Positive female specimens were molecularly typed based on the cytochrome b method. Amplicons were generated by PCR using (5 0 -CAT/CATTCAA CCA/TGAATGATA-3 0 ) and N1N-PDR (5 0 -GGTAC/TA/TTTGCCTCGAT/ATTCG T/ATATGA-3 0 ) primers  [16]. PCR products were purified and cycle-sequenced as described previously. DNA sequences from both strands were aligned and edited using Staden software package (http://staden. sourceforge. net/). MEGA version 7 software (www. megasoftware. net) was used to conduct multiple sequence alignments (ClustalW option) and to construct phylogenic tree. Sequences were aligned with Phlebotomus perniciosus sequences already obtained from Tunisia and other countries  [17] [18] [19]. Neighbor Joining (NJ) tree was performed using the Kimura 2 parameters model with homologous sequences from Phlebotomus longicuspis as the least ambiguous outgroup.
Leishmania infection in phlebotomine sandfly female specimens From end-June to end-October, 1715 female specimens (208 from IH and 1507 from AS) were collected and stored individually. According to the period of capture, a dozen to thirty specimens from each monthly IH and AS collection were randomly sampled for DNA extraction (Table  1 ). Among the 190 analyzed specimens, 28 were positive by kDNA qPCR. Among them, 24 (12. 6%) were confirmed by ITS1-PCR and were considered for result analysis (Table  1 ). ITS1 amplification showed a band of expected size (360 bp). Purified PCR products from the 24 specimens corresponded to one unique sequence identified by inspection of the input data matrix and deposited into GenBank database (http://www. ncbi. nlm. nih. gov/) under the accession numbers MF597933 and MF597934.
Sandfly molecular typing For the 24 positive female sandflies infected by L. infantum, the last 279 nucleotides of Cytochrome b (Cyt b) were sequenced. Two unique sequences (haplotypes KHF8 and KHF12) were identified by inspection of the input data matrix and deposited into GenBank database (http://www. ncbi. nlm. nih. gov/). KHF8 haplotype (accession number MF682976) was identified in 5 specimens. Whereas haplotype KHF12 (accession number MF682977) was shown in 19 specimens. Neighbor joining analyses showed that the two sandfly haplotypes fell in the P. perniciosus branch. KHF12 which was the predominant haplotype in our study fell in the P. perniciosus branch that included haplotype pern01 whereas the second haplotye KHF8 defined a new divergent branch (Fig  6 ). According to place of capture, the 5 infected specimens caught in house in June belonged to the same P.
Discriminative delay conditioning and peak procedure To evaluate timing behavior, after a different discriminative delay conditioning experiment lasting only four days, mice were subjected for four more consecutive days to two randomly assorted trial types: trial type 1 (40 trials) and trial type 2 (20 trials) -the peak procedure task (Fig. 1C ). Trial type 1 was exactly as described above, and used in order to maintain mice responding; while trial type 2 was as trial type 1 but differing on CS duration in the way that it did not stop after 20 s but kept going for a total of 40 s and no food was presented. This is called a peak procedure trial. Data from the peak procedure trials were collected and then each 1-s bin was divided by the highest value of response (the peak value), in order to normalized the average response, and then averaged over 5 s.
Functional magnetic resonance imaging (fMRI) Brain imaging was performed in naı¨ve Gdi1 WT (n = 7) and Gdi1-null (n = 11) mice (age 22-27w) under shallow etomidate anesthesia as described previously  (Petrinovic et al. , 2016). In brief, anesthesia was induced with 3% isoflurane (Abbott, Baar, Switzerland) in oxygen and air (1:5 v/v), and maintained with etomidate (B. Braun Melsungen AG. Melsungen, Germany) administered intravenously at a rate of 0. 75 mg/kg/min. Body temperature was maintained at 37 °C and breathing rate and concentrations of inhaled and exhaled oxygen and CO 2 were continuously monitored on a PowerLab data acquisition system (ADInstruments, Spechbach, Germany). The fMRI study was conducted on a 9. 4 T Bruker Biospec, 20 cm bore animal scanner (Bruker Biospin, Ettlingen, Germany), equipped with a 7-cm-diameter birdcage coil for signal excitation and a surface coil for signal reception.
Perfusion imaging as a proxy readout of neural activity was conducted based on the continuous arterial spin-labelling (CASL) method (Centered RARE, TR/TE = 3. 75 s/5. 4 ms, RAREfactor = 32, 20 mm Â 20 mm field-of-view, 128 Â 64 matrix, 0. 6-mm slice thickness, 2 averages, 3-s labeling pulse, 0. 4 s post labeling delay). Three sets of perfusion images were collected over 12 min from eight coronal slices positioned at +1. 9, +1. 0, +0. 1, À0. 8, À1. 7, À2. 6, À3. 5 and À4. 4 mm relative to the bregma. fMRI data were processed and analyzed as described previously  (Bruns et al. , 2009). Absolute perfusion values obtained from the CASL-images were normalized for each brain area to the mean perfusion of each slice in order to account for possible systemic changes affecting brain perfusion as a whole, and to eliminate part of the inter-individual variability.
Paired-pulse protocols were applied using dual stimuli at 50-through 250-ms inter-stimulus intervals in 50-ms increasing steps. Each stimulus lasted 0. 2 ms, while current intensity was previously adjusted in order to obtain steady, reliable EPSCs with no failures during low-frequency stimulation (0. 1 Hz). Paired-pulse ratios (PPRs) were calculated by dividing the second EPSC peak amplitude by the first one for any paired-pulse sequence. High-frequency stimulus trains (20 Hz, 4 s) were applied to assess SV readily releasable pools (RRPs)  (Bianchi et al. , 2009). Cumulative EPSC peak amplitudes were plotted and the late phase (20-25 points) of the dataset was linearly fit. Back-extrapolation of the fit line to the Y-axis intercept provided an estimate of the RRP  (Schneggenburger et al. , 1999).
The probability of release (Pr) of any given vesicle in the RRP was calculated as the ratio between the first-evoked EPSC in the train and the RRP size. All recordings were performed using a MultiClamp 700B amplifier interfaced with a PC through a Digidata 1440A (Molecular Devices, Sunnyvale, CA, USA). The series resistance was partially compensated (40-50%) using the amplifier control circuit. Data were acquired using pClamp10 software (Molecular Devices) and analyzed with Origin 9. 1 (Origin Lab, Northampton, MA, USA). Voltage-clamp traces were sampled at a frequency of 30 kHz and low-pass filtered at 2 kHz.
Notwithstanding, the current fMRI data are in alignment with the notion that prelimbic and infralimbic cortices might be more relevant in the temporal and reward processing underlying Pavlovian conditioning  (Galtress et al. , 2012). To deeply investigate the role of fronto-striatal circuitry we then performed whole-cell recordings, which revealed significantly smaller individual glutamatergic EPSCs evoked by brief cortical stimulation and recorded in striatal MSNs. Such weaker transmission was not accounted for by a reduced probability of neurotransmitter release, but rather by a smaller sized pool of readily releasable synaptic vesicles as previously also described in the hippocampus. As negative control for the validation of the experimental conditions in our model, we went then to investigate the release of dopamine in the amygdala because of the key role of dopamine in the above behavioral paradigms and the implication of the amygdala in associative and reward learning.