dataesr/scientific-paragraphs-categorization huggingface dataset:
However, whatever the marker, a same pattern can be observed with higher richness for the Toulon samples. Then, the COI and 18S rDNA datasets showed distinct clusters based on different localities (Figure  7 ). The NMDS ordination plot showed three clusters based on location (PERMANOVA, p < 0. 0001) for the 18S rDNA data and two clusters based on databases for the COI, with Mediterranean samples clustering together (Figures  7A, B ). In addition, long vectors for Banyuls, Saint-Nazaire and Toulon in ordination plots revealed that location was strongly associated with the variability of eukaryote assemblages (envfit for 1 st and 2 nd axes, p < 000. 1 for 18S rDNA and p < 0. 05 for COI), while primers and databases were statistically not related to variation in biofouling community structure (envfit for 1 st and 2 nd axes, p = 0.
999 for 18S rDNA and p = 0. 5930 for COI). A total of 597 genera were detected with the five regions of 18S rDNA against 52 with the COI dataset (Figure  S11 ). For each marker, the number of exclusive genera was highest at Toulon, and lowest at Banyuls and Saint-Nazaire. Most genera found at Toulon and Banyuls were shared. With the regions of 18S rDNA, some nematodes such as the genera Pellioditis, Prochaetosoma and Dracograllus, constituted a major exclusive group in Banyuls while the Florideophyceae group was exclusive in Saint-Nazaire, e. g. , the genera Heterothamnion, Bonnemaisonia, and Asparagopsis. Moreover, only two genera were unique in the COI dataset from Saint-Nazaire, Electra (Bryozoa) and Platynereis (Annelida). In Toulon, more diverse groups were found such as Bacillariophyta, Phaeophyceae, Arthropoda and Bryozoa, with the last two being the most abundant groups.
Only a small part of genera remained unassigned within the 18S rDNA dataset. For COI, however, a large proportion of A B FIGURE 3 Heat map based on the relative abundance [in log_trans (4)] of: (A) the 33 most abundant eukaryote genera across five hypervariable regions of 18S rDNA selected if > 0,5%. (B) The 10 most abundant eukaryote species across COI using two databases selected if > 0,1%. Samples used are the three panels (PVC, PA, and HDPE) from Toulon extracted with the PMS method and amplified with five hypervariable regions of 18S rDNA (n = 45 samples). sequences remained unassigned, especially for the Mediterranean sites, Banyuls and Toulon. The proportions of ASVs assigned to small metazoans were notably higher with 18S rDNA than with COI (annelids: 23 ± 9%, nematodes: 18 ± 7%). Arthropods were abundant in all localities, especially in Saint-Nazaire which included nearly 62% of the sequences for 18S rDNA and 94% for COI data, exclusively represented by the genus Jassa.
18S and COI are complementary but differences in taxonomic identification reflect current gaps in assignment databases Several studies have highlighted the difficulties encountered to elucidate the biodiversity of marine eukaryotes as a major issue is the markers choice. (Drummond et al. , 2015; Valentini et al. , 2016; Clarke et al. , 2017; Alberdi et al. , 2018; Wangensteen et al. , 2018). Consistent with the latter, our results showed that each primer recovered a distinct biofouling community with some taxa being associated with a given primer. Moreover, the COI analysis yielded a high number of ASVs assigned (≈ 400 ASVs) compared to some regions of 18S rDNA. A similar result has been reported by  Wangensteen et al. (2018)  who investigated the suitability of the 18S and COI markers to detect marine hard-bottom communities.
Primer specific detection is probably due to the much lower variability of the 18S gene, which underestimates the actual number of taxa  (Hadziavdic et al. , 2014). A second major difference was found in the taxonomic assignment performance. Among affiliations with COI, a larger discrepancy between the amounts of unassigned ASVs from BOLD and from MIDORI databases, even at high-level taxa, and with more unassigned ASVs from BOLD dataset, was observed. While leaving out ASVs that could not be assigned to a eukaryotic kingdom or lower, a higher proportion of genera was found unassigned with COI. Conversely, 18S rDNA marker, irrespective of the region targeted, recovered 47 eukaryotic classes (among which 12 were metazoans) while COI recovered only 8 and 14 classes with BOLD and MIDORI databases, respectively.
Therefore, both markers resulted in different taxonomic profiles. Although the BOLD database is based on COI barcodes and currently contains over 5 million sequences from over 5 million different species  (Zhao et al. , 2021) , only 9% were assigned of read sequences at species and genus level while 36% read sequences were recovered with the MIDORI database, which is specific to marine taxa. For the MIDORI database, there were 335,177 sequences classified into Diversity and composition of top eukaryote genera within each site for COI gene. Barplots were represented in relative abundance (> 0. 1%) of each taxonomic group of eukaryotes. Above the barplot, graph shows chao1 index at each site (Banyuls, Toulon, Saint-Nazaire). Letters above boxplots represent pairwise comparisons between sites.
29 phyla representing 47,113 species whereas the BOLD database was built with 2,425,004 sequences into 6 phyla representing 190,318 species. Despite the high number of sequences, the high proportion of unassigned sequences highlights significant gaps in the reference databases for marine biofoulers. Our results were in line with a previous study on marine biofouling showing that almost all ASVs remained unassigned when using the BOLD database  (Ammon et al. , 2018a). This can also be explained by the high intraspecific variability and the high substitution rates within the COI gene  (Djurhuus et al. , 2018). In addition, taxa identified by MIDORI were exclusively assigned to marine organisms, so excluding terrestrial groups such as Diplopoda or Amphibia. Although we did find that the COI region allowed to identify the metazoans diversity with BOLD database, we observed a high proportion of organisms unlikely to be present in a marine sample, including terrestrial metazoans like organisms belonging to the Insecta and Arachnida classes.
Due to the more conservative nature of 18S rDNA, it is possible to identify a sequence at a lower taxonomic rank as soon as a related sequence is present in the reference database, even if it is from a distant relative. Conversely, with its greater variability, identification with the COI marker requires a closely related sequence to match the sequences present in the reference database. Moreover, the degeneration of the COI primer sets may partly explain the production of nontargeted sequences which correspond to taxa in the database. This problem has been raised by  Horton et al. (2017) , who demonstrated the lack of COI region primers specificity to identify metazoans from soil samples. Berry et al. (2015)  have identified a similar issue with COI primers, which failed to detect most mollusk taxa in the stomachs of several Australian fish species.
Nevertheless, such an issue could also be due to a lower number of marine sequences in the database. Finally, COI primers have considerable potential because the rate of evolution of the COI gene is much faster than for the 18S rRNA gene, usually facilitating the identification at the species level  (Hebert et al. , 2003). In this sense, we showed that the COI gene can recover more ASVs with a large number of them unidentified but with reliable assignment at low taxonomic levels while the 18S rDNA gene allowed to assign more ASVs but poorly identified at the species level because several taxa can share the same sequence. Similar conclusions were observed to detect the NIS in biofouling communities, and increasing evidence showed that the use of two genes provides a more complete view of diversity and identification of broad marine eukaryote taxa  (Borrell et al.
However, it is important to note that regardless of the marker or the assignment database, spatial variation of biofouling community structure can be assessed using a metabarcoding approach which shows specific communities for each locality. Future investigations should include a multigene approach to obtain the more complete coverage of fouling diversity. Pochon, X. , Bott, N. J. ,  Smith, K. F. , and Wood, S. A. (2013). Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests. PloS One 8 (9), e73935. doi: 10. 1371/ journal. pone. 0073935 Pochon, X. ,  Zaiko, A. , Hopkins, G. A. , Banks, J. C. , and Wood, S. A. (2015). Early detection of eukaryotic communities from marine biofilm using high-throughput sequencing: An assessment of different sampling devices.
tumor immunity in preclinical models  11, 12. These studies led to a clinical trial protocol NCT04426669. CISH has been also shown to be a critical immune checkpoint in NK cells  13  and we recently contributed to show the importance of CISH in NK cell homeostasis using a Cish KO germline mouse model  14. However, we still do not fully appreciate mechanistically how CISH regulates NK anti-tumor functions. These are essential requirements to comprehensively uncover the safety of genetically or pharmacologically targeting of an intracellular protein in NK cell immunotherapy. To further understand how CISH regulates NK cell activity, we developed and investigated a new conditional mouse model that depletes CISH specifically in NK cells (Cish fl/fl Ncr1 iCre ). In conditional Cish-deficient mice (Cish fl/fl Ncr1 iCre ), we found no change in homeostatic NK cell numbers, no alterations in maturation or cycling.
6F ), in accordance with the in vitro observations (Fig. 2D ). One of the main causes of relapse in immunotherapy treatment is the exhaustion of cytotoxic cells due to inhibiting receptor expression. We thus tested for expression of the PD-1, TIM-3 and TIGIT receptors that are all suggested to be a part of NK cell exhaustion  18  (Fig. 6G ). Interestingly, only TIGIT expression was strongly decreased in infiltrated CISH KO NK cells compared to WT NK cells (Fig. 6H-J ). We then wanted to see if this observation was confirmed in infiltrated NK cells in human breast cancer patients. For this, we used previously published Database of Single cells mapping of immune cells infiltrated in human breast tumors  19. Using loupe cell browser software, infiltrated NK cells (CD3-, NCAM1+) were gated then TIGIT gene expression was compared in CISH + NK cells versus CISH -cells.
ACKNOWLEDGEMENTS This work is supported in France by institutional grants from the  Institut National de la Santé et de la Recherche Médicale (Inserm) ,  Centre National de la Recherche Scientifique (CNRS)  and  Aix-Marseille Université  to CRCM; by project grants from the  Fondation ARC pour la recherche sur le Cancer  ( PJA20161204835 ,  PJA20191209406 ),  Groupement des Entreprises Françaises dans la Lutte contre le Cancer (GEFLUC) Marseille-Provence , then the  Janssen Horizon Fonds de dotation. P-L. B. is supported by a doctoral fellowship from  Ligue Nationale contre le Cancer  and V. L. by a  doctoral fellowship  from the  Ministère de l'Education et la Recherche , then from the  Fondation ARC pour la recherche sur le Cancer. G. G. was supported by a post-doctoral fellowship from the  Fondation ARC pour la recherche sur le Cancer  and is currently supported by the  Janssen Horizon Fonds de dotation.
Background Prior studies have revealed remarkable phenotypic heterogeneity in KCNQ2-related disorders, correlated with effects on biophysical features of heterologously expressed channels. Here, we assessed phenotypes and functional properties associated with KCNQ2 missense variants R144W, R144Q, and R144G. We also explored in vitro blockade of channels carrying R144Q mutant subunits by amitriptyline. Methods Patients were identified using the RIKEE database and through clinical collaborators. Phenotypes were collected by a standardized questionnaire. Functional and pharmacological properties of variant subunits were analyzed by whole-cell patch-clamp recordings. Findings Detailed clinical information on fifteen patients (14 novel and 1 previously published) was analyzed. All patients had developmental delay with prominent language impairment.
2  In addition to membrane voltage, Kv7 channel opening requires phosphatidylinositol 4,5-bisphosphate (PIP 2 ), a membrane lipid which binds to distinct channel regions at each VSD gating state. 3 CNQ2/3 pathogenic variants exerting loss-of-function (LoF) effects are associated with a spectrum of neonatal-onset phenotypes ranging from self-limited familial neonatal epilepsy (SLFNE, MIM:121200) to KCNQ2 developmental and epileptic encephalopathy (KCNQ2-DEE, MIM:613720) with therapy-resistant seizures and intellectual disability (ID). 4  Most KCNQ2-DEE pathogenic variants exert dominant-negative LoF effects when studied in vitro. 5  Seizures in these children tend to respond well to sodium channel blockers, now recommended as first-line treatment,  6  and a randomized, double-blind, placebo-controlled trial with the potassium channel opener retigabine has recently been initiated (ClinicalTrials.
gov Identifier:NCT04639310). 7  Few heterozygous KCNQ2 missense variants enhance channel function (gain-of-function, GoF) by causing a hyperpolarizing shift in voltage-dependent activation. [8] [9] [10] [11]  Phenotypes associated with GoF variants differ from the classical KCNQ2-DEE phenotype. They range from profound ID without neonatal seizures but with a characteristic nonepileptic myoclonus and poor prognosis for the R201C variant with strong GoF effect,  8  to epileptic spasms with hypsarrhythmia and severe developmental delay (DD) for the R198Q variant with milder GoF effect. 10  Interestingly, KCNQ3 variants at R230, the homologous residue of KCNQ2 R201, also have strong GoF in vitro effects; patients carrying these variants display global developmental delay within the first 2 years of life, sleep-activated near-continuous multifocal spikes, and autistic features, but no neonatal seizures.
The absence of neonatal seizures has emerged as a robust differentiator between the (rarer) KCNQ2 GoF and (more common) LoF variants. This distinction is clinically very important, as the entities present in very different clinical settings and require development of different therapeutic approaches. Raising awareness about these distinct phenotypes has become especially important now that an international randomized clinical trial with a pediatric formulation of the potassium channel opener retigabine (ClinicalTrials. gov Identifier: NCT04639310; https:// clinicaltrials. gov/ct2/show/NCT04639310) for patients with KCNQ2-encephalopathy has recently been initiated. In this trial, only patients with clear LoF variants can be included, and the "presence of a known GoF variant in the KCNQ2 gene, or clinical characteristics consistent with previously reported pathogenic GoF variants in the KCNQ2 gene" are explicitly listed among exclusion criteria for enrolment in the trial.
Patient series collection We collected detailed clinical information of patients carrying a KCNQ2 R144W (NM_172107. 3:c. 430C>T), R144G (NM_172107. 3:c. 430C>G), or R144Q (NM_172107. 3:c. 431G>A) variant. Patients were recruited through the Rational Intervention of KCNQ2 Epileptic Encephalopathy (RIKEE) database (http:// www. RIKEE. org), a curated database aggregating (un) published patient data provided by physicians or families after parental informed consent. The site is hosted at Baylor College of Medicine under an institutional review boardÀ approved research protocol. Additional patients were directly referred for inclusion in the study by collaborating paediatric neurologists and medical geneticists. A standardized phenotyping sheet, including data fields on technical exams as EEG and MRI of the brain, was sent to the referring physician of all unpublished patients (n=14) and of Patient 11, who was previously reported with minimal clinical details as part of an Epi4K epileptic encephalopathy cohort.
Whole-cell electrophysiology Currents from CHO cells were recorded at room temperature (20À22 °C) 1-2 days after transfection, using the whole-cell configuration of the patch-clamp technique with glass micropipettes of 3À5 MV resistance. The extracellular solution contained (in millimolar): 138 NaCl, 2 CaCl 2 , 5. 4 KCl, 1 MgCl 2 , 10 glucose and 10 HEPES, pH 7. 4 with NaOH. The pipette (intracellular) solution contained (in millimolar): 140 KCl, 2 MgCl 2 , 10 EGTA, 10 HEPES, 5 Mg-ATP, pH 7. 3À7. 4 with KOH. The pCLAMP software (version 10. 0. 2) was used for data acquisition and analysis. Current densities, conductance-voltage curves, and current activation and deactivation kinetics were obtained and analysed as previously described. 15  stock solution (10 mM) of amitriptyline (Sigma-Aldrich, Germany) was prepared in dimethyl sulfoxide (DMSO); extracellular solution was used for subsequent drug dilutions.
Cohort description and genotypes Fourteen previously unpublished KCNQ2-DEE patients were included in our study: 9 carrying the p. R144W variant, and 5 the p. R144Q variant (eTable 1 and 2). We also collected clinical data on a sixth p. R144Q patient (patient 11) who was previously published with limited clinical information. 13 We defined this cohort of 15 wellphenotyped patients as our "study cohort". Eight of the 15 were male. Age at last follow-up of R144W patients and R144Q patients ranged from 3. 5 to 27 years (median 7. 5 years), and from 3 years 9 months to 26 years (median 9 years), respectively. In 14 patients, the pathogenic variant occurred de novo. For patient 13, the variant was shown to be absent in mother, but paternal DNA was not available. We also summarized published phenotypical data of 5 patients: 3 carrying the p.
16 ere, through identification of a larger study cohort and inclusion of functional analysis, we provide evidence supportive of a single gene with phenotypic heterogeneity model. The R144W/Q alleles are recurrently associated with a neurodevelopmental disorder without neonatal seizures. ] [11] [12] 28  Recognizing the clinical characteristics of pathogenic KCNQ2 GoF variants, with the absence of neonatal seizures as robust differentiator between KCNQ2 GoF and LoF variants, is of clinical importance since individuals with KCNQ2 GoF variants are likely to need different therapeutic approaches compared to individuals with KCNQ2 LoF variants. Recently, an international randomized clinical trial with a pediatric formulation of the neuronal KCNQ channel opener retigabine (Clinical-Trials. gov Identifier: NCT04639310; https://clinical trials.
gov/ct2/show/NCT04639310) has been initiated. In this trial, eligibility is restricted to patients with pathogenic KCNQ2 LoF variants. Individuals with known GoF KCNQ2 variant or individuals showing clinical characteristics consistent with previously reported pathogenic GoF KCNQ2 variants, are excluded. This highlights the continued need for description of rare patients such as performed by Mary et al. and in the current study. The present data also reveal novel genotype-phenotype correlations in the GoF spectrum of KCNQ2-DEE, as the severity of the phenotype seems to correlate with the electrophysiological effect of the variant in vitro, with larger functional changes of R144Q-carrying channels paralleling the more severe neurodevelopmental outcome (Figure  6 ). Patients carrying the KCNQ2 R144 GoF variants are also less severely affected than those carrying the strong KCNQ2 GoF variants at R201 in S 4.
Limitations This study has limitations that are intrinsic to our approach: data were collected retrospectively, and as a result not all desired clinical information was available for every patient (e. g. , not all had sleep-EEG recordings capturing sleep), and some reports were impossible to further validate (e. g. , the etiology of reduced responsiveness episodes). Sampling bias may be arise due to recruitment via a voluntary family registry and referral to specialized centres. In addition, the small size of the two subgroups within our cohort (R144W and R144Q), limits the strength of conclusions drawn about phenotypic differences between these subgroups. Despite these limitations, this study strengthens available evidence that KCNQ2 GoF variants cause DD in the absence of neonatal seizures and that disease severity is correlated with the extent of the V 1/2 shift in vitro.
Acknowledgments The authors thank the families and the children for their participation in this study. SW received funding from  FWO  ( 1861419N  and  G041821N );  Queen Elisabeth Medical Foundation for Neurosciences (GSKE) ;  KCNQ2-Cure ;  Jack Pribaz Foundation ;  KCNQ2e. v. ; European Joint Programme on Rare Disease JTC 2020 (TreatKCNQ). MT received funding from the  Italian Ministry for University and Research (MIUR)  ( PRIN 2017ALCR7C ); the  Italian Ministry of Health  (Project  RF-2019-12370491 ); the  European Commission H2020  ( UNI-COM À 875299 );  European Joint Programme on Rare Disease JTC 2020 (TreatKCNQ ). ECC received funding from  NINDS   NS49119  and  NS108874 ; The  Jack Pribaz Foundation ; The  Chalk Family Foundation and The Miles Family Fund. FM  received funding from the  Italian Ministry for University and Research (MIUR)  ( PRIN 2017YH3SXK ).
0001. (F) shRNA-treated melan-Ink4a cells or HEK293T cells as a negative control were analyzed by quantitative melanin content assay. Data are presented as mean ± SD of five independent experiments as a percentage of the signal from shNC-treated cells and were analyzed by Welch's ANOVA relative to shNC-treated cells; ****, P < 0. 0001; *, P < 0. 05. and PI4KIIβ -GFP localized predominantly to cytoplasmic puncta (PI4KIIα) and to the perinuclear region or a few cytoplasmic puncta (PI4KIIβ), PI4KIIα-L61,62A-GFP (Fig. 3, E q ) and PI4KIIβ-L29,30A-GFP (Fig. 4 , E q) were predominantly cytoplasmic. These data suggest that (1) both PI4KIIα and PI4KIIβ require both their kinase activity (and hence PtdIns4P generation) and their interaction with APs to promote TYRP1 localization, and (2) recruitment to endolysosomal membranes requires binding of PI4KIIα to AP-3 and of PI4KIIβ to AP-1.
01% BSA and 0. 02% Saponin in PBS), washed three times 5 min with PBS, incubated with secondary antibodies (RT, 30 min) diluted in blocking buffer, washed again, and mounted onto slides using Prolong Gold (Thermo Fisher Scientific). Fixed cell images were acquired using a Leica DMI8 inverted microscope (Leica Biosystems) equipped with a CSU-WI spinning-disk confocal scanner unit (Yokogawa), a 100X total internal reflection fluorescence objective lens (Leica 1. 47 NA) or a 63X objective lens (Leica, 1. 40 NA) a Hamamatsu Photonics ORCA-Flash 4. 0 sCMOS digital camera, and VisiView software (Visitron Systems). Images were further processed and analyzed using Fiji-ImageJ. Live-cell imaging was done essentially as described  (Bowman et al. , 2021). Briefly, melanocytes were seeded onto matrigelcoated 35-mm glass-bottomed dishes, incubated in Leibovitz's CO 2 -independent medium without phenol red (Invitrogen) supplemented with 10% FBS in an environmental chamber set to 37°C, and imaged using the same microscopic system.
g. , 10 pixels for GFP-VAMP7 and PI4KIIβ-GFP). Pigment granules from the bright field channel were processed using either the "Invert" function first and then "Threshold" function by ImageJ or, for Fig. 1  C, the plugin "Spot Detector" by Icy software (https://icy. bioimageanalysis. org/). After background subtraction, images were converted to binary images using either the "Threshold" function (TYRP1, STX13, LAMP2, and OCA2-HA) or the Bernson algorithm of the "Auto Local Threshold" function with a 15-pixel radius (GFP-VAMP7, PI4KIIα, PI4KIIβ, PI4KIIα-GFP, PI4KIIβ-GFP) or both (TfR). Structures of <5 pixels in area were considered background and filtered out using the "Analyze Particles" function. To generate an overlap image, two binary images were multiplied using the "Image Calculator" function, and the areas of the individual and overlap binary images were calculated using the "Analyze Particles" function.