dataesr/scientific-paragraphs-categorization huggingface dataset:
The secondary structures are indicated by solid boxes (α-helices), checked boxes (3 10 helix) and arrows (β-sheets) above the sequence alignment and they are coloured light blue for the Mtase domain, orange for linker (residues 264-273) and blue for the fingers, green for the palm and red for the thumb of the RdRp domain. The bNLS sequence (residues 320-368)  [31] , the a/bNLS sequence (residues 369-389)  [29, 30]  and the priming loop sequence (residues 786-809)  [13]  are boxed in red. Within bNLS, K330 (red asterisk and bold) is important for NS3-NS5 interaction  [26]. NS5 residues that are not identical to DENV1 and DENV2 are highlighted in grey. Other highlighted residues Y838 and R888 are discussed in the text. The electron density of the last 12 amino acid residues (boxed in blue) are generally missing in most DENV NS5 crystal structures (PDB: 2J7U, 4C11 and 4V0Q).
The GenBank accession numbers of DENV1 and DENV2 are EU081230 and EU081177, respectively. doi:10. 1371/journal. ppat. 1005886. g001 Furthermore, the last 12 amino acid residues are thought to be highly flexible and either not modelled in crystallographic structures  [11, 13, 34] , or only modelled in a subset of molecules within the asymmetric unit where crystal packing may induce a stable conformation  [36]. To test the functional relevance of the C-terminal region, we truncated GFP-NS5 by introducing a stop codon at residue 882 or 883 of DENV1 and DENV2 NS5 respectively (Fig  3A , construct 15 and 17, respectively). Surprisingly, C-terminal truncated DENV2 NS5 protein, D2 1-882 (construct 17) that contains the previously characterized conventional a/bNLS  [29, 30]  was mostly localized to the cytoplasm (.
Data were fitted to one site specific binding equation using Graphpad Prism 6. 0f from triplicate measurements of two independent experiments. (B and C) Result of binding assay performed with glutathione beads that were incubated with GST-NLS fusion protein. (B) 2-fold serially diluted of Impα (starting from 800 nM) was incubated with GST-NLS fusion protein in a final volume of 50 ml. After incubation, beads were sedimented and protein was eluted using GST B buffer. (C) Elutled proteins were visualised on SDS-PAGE and analysis was performed using one site specific binding function in Graphpad Prism 6. 0f. Results are shown as the mean ± SD of duplicates from two independent experiments. To confirm this result in a different assay system, a GST pull down assay was performed using glutathione-agarose beads and serially diluted Impα, with visualisation and quantitation of bound Impα performed by SDS-PAGE and ImageJ respectively (Fig 4B  and 4C ).
The results confirm that DENV2 and 3 C-terminal residues bind Impα, whilst the binding observed in the NS5 for DENV1 and DENV4 NLSs are very weak. Similar binding affinity values were obtained, with strong binding for GST-DENV2 865-900 and GST-DENV3 865-900 (K d = 0. 27 ± 0. 11 μM and 0. 37 ± 0. 11 μM, respectively), whilst the binding of DENV1 and 4 were too weak to quantify. Overall, the affinity of DENV2 and 3 NS5 for importin by in vitro assay are similar while the affinity of DENV1 and 4 NS5 are weak. The differences in affinity measurement are within the expected range noted for well characterised targets such as SV40 T-ag NLS which can vary from 10-1000 nM depending on the methodolgy that is used  [38, 39]. Together, our biochemical binding data appear to be consistent with the subcellular localization in infected cells for DENV1, 2 and 3 where DENV1 NS5 is mainly cytoplasmic whilst the protein from DENV2 and 3 are mainly localized in the nucleus.
As expected the poor binding affinity of DENV1 and DENV4 C-terminal residues prevented their respective capture of Impα, and therefore precluded structural analysis. In contrast, the C-terminal region of DENV2 and 3 formed a stable complex with Impα throughout purification, and yielded well diffracting crystals (S4 Fig). Crystals of Impα:DENV2 and 3 C-terminal region diffracted to 2. 2 Å and 2. 1 Å resolution, respectively. Both crystals belong to the P2 1 2 1 2 1 space group, and the structures solved by molecular replacement using the structure of importin-α (PDB: 3UL1) as a search probe (Fig  5 ). The final R/Rfree were 0. 177/0. 216 and 0. 183/0. 207 for the Impα DENV2 and 3 C-terminal region respectively (see Table  1  for full data collection and refinement statistics). The nuclear import receptor Impα displayed the characteristic super-helical structure consisting of ten armadillo repeats (ARM), with each ARM repeat consisting of three α-helices.
To explore the lethality involved with mutations in the basic residues that form the monopartite NLS within Cter 18 ,in more detail we mutated DENV2 NS5 P884 to Thr (as found in DENV1 at position T883) in order to delineate the importance of presentation of the NLS for importin binding and its impact on infectivity and viral RNA replication. We also made a single mutation at DENV2 NS5 R888 to alanine, lysine or glutamate because this residue is completely conserved in all flaviviruses whilst K887 is not. Like the DENV2 NS5 K887A/ R888A, the R888A and R888E mutants were completely non-viable (Fig 7A  , 7C  and 7D ). R888K however was severely attenuated and no plaques could be observed although more than 2 log reduction in extracellular RNA could be detected by qRT-PCR. Interestingly, DENV2 NS5 P884T replicated like WT DENV2 NS5 but it showed a conspicuous difference in NS5 subcellular localization (Figs  7  and 8 ) similar to the data obtained for GFP-NS5 constructs bearing the same mutations (Fig  6A ).
Indeed CLSM studies confirmed this notion: GFP-DENV1 NS5 T883P with presumably restricted presentation of the Cter 18 became enriched mainly in the nucleus since the binding determinant is present in this sequence. In stark contrast GFP-DENV2 NS5 P884T showed the opposite phenotype and became predominantly cytoplasmic (Fig  6 ). More importantly, even in the context of DENV2 infectious clone the NS5 P884T mutation resulted in NS5 being mostly localized to the cytoplasm demonstrating that a single amino acid change can result in altered phenotype. Our data demonstrates that Cter 18 of DENV2 and 3 NS5s may directly interact with cellular importins to target the protein to the nucleus while the Cter 18 of DENV1 NS5 may have evolved to bind to other host factors as well and is mostly located in the cytoplasm.
Source protein IMPα2 Major cNLS binding site PDB code P1 P2 P3 P4 P5 Dengue 2 NS5 C-terminus M K R F R 5HHG (in this paper) Dengue 3 NS5 C-terminus M K R F R 5FC8 (in this paper) SV40T K K K R K 1EJL [60] , 1BK6  [61] , 1Q1S  [62] , 1Q1T  [62]  Human androgen receptor R  [67]  doi:10. 1371/journal. ppat. 1005886. t003 K L K K 3BTR [63] αIBB L K K R N 1IAL [50], 1IQ1 [64] Venezuelan Equine Encephalitis Capsid A K K P K 3VE6 [65] Ku70 S K R P K 3RZX [66] Ku80 A K K L K 3RZ9 [66] CLIC4 A K K Y R 3OQS NS5 in this environment. In this respect, it is possible that the Cter 18 of DENV1-4 NS5s may have undergone adaptive evolution to engage specific cellular proteins to shuttle the excess NS5s away from the replication factories. The fully functional DENV2 P884T mutant and the lethality of R888A mutation support this notion and suggests that the C-terminal region plays essential roles in the very early event in viral RNA replication and are independent of their role in nuclear localization (Figs  8C  and S10 ).
Cell lines and virus strains Huh-7 cells (ATCC) were cultured in DMEM medium (Gibco) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (1% P/S). Vero (ATCC) and HEK293T (ATCC) cells were cultured in DMEM high glucose medium (Gibco) supplemented with 10% FBS. A549 cells (ATCC) were cultured in Ham's F-12K medium (Gibco) supplemented with 10% FBS and 1% P/S. BHK-21 cells (ATCC) were cultured in RPMI1640 medium (Gibco) supplemented with 10% FBS and 1% P/S. All cells were maintained at 37°C, with 5% CO 2. C6/36 cells (ATCC) were cultured in RPMI1640 medium supplemented with 25 mM HEPES, 10% FBS and 1% P/S at 28°C, in the absence of CO 2. The four DENV serotypes (GenBank accession numbers for the relevant sequences are: DENV1 (EU081230), DENV2 (EU081177), DENV3 (EU081190) and DENV4 (GQ398256) that were used in the study was grown in C6/36 cells and titered in BHK-21 cells before storage at -80°C.
Plasmid construction His 6 -tagged DENV2 NS5 bacterial expression constructs: DENV2 NS5 gene was amplified by PCR from DENV2 cDNA clone (GenBank accession number: EU081177)  [17, 48]. The PCR fragment was cloned into pET28b (Novagen) that was pre-cut with NdeI and SacI by recombination with CloneEZ PCR cloning kit (Genscript) according to manufacturer's protocol. GST-tagged DENV1-4 NS5 peptide bacterial expression constructs: DENV1-4 NS5 gene fragments that corresponded to residues 865-900 of DENV2 NS5 were ordered from Genscript and amplified by PCR. The PCR products were digested with BamHI and EcoRI and cloned into pre-cut PGEX4T-1 vector. GFP-tagged DENV1 and 2 NS5 mammalian expression constructs: DENV1 and 2 NS5 genes were amplified by PCR from DENV1 (GenBank accession number: EU081230) and (same GenBank accession number as above), Japanese Encephalitis virus (JEV; M55506), West Nile virus (WNV; M12294), Murray Valley Encephalitis virus (MVE; AF161266), Tick-Borne Encephalitis Virus (TBEV; U27495), Yellow Fever virus (YFV; X15062) and Zika virus (ZIKV;KU497555)  [58].
(B) Model of the role of the C-terminal region of NS5 that is required for the formation of dimer and the initiation of de novo viral RNA replication and interaction with importin-α or NS3. On the left: de novo RNA synthesis by NS5 oligomers independent of NS3 interaction as implied by NS3 N570A mutation  [59]  and non-viability of NS5 R888A as identified in this study. On the right: NS3 interaction with NS5 thumb domain in the RC will support higher plus strand RNA synthesis or NS5 Cter 18 interacts with host importins or other factors to be localized to nucleus or cytoplasm in a regulated manner. doi:10. 1371/journal. ppat. 1005886. g009 DENV2 cDNA clone (same GenBank accession number as above)  [17, 48] , respectively. The PCR fragments were digested with XhoI and XmaI and cloned into pre-cut pEGFP-C1 vector (Clontech).
DENV1 and 2 GFP-NS5 domain-swapped and motif-swapped gene segments were generated by overlap extension PCR. DENV1 and DENV2 GFP-NS5 constructs (as described) were used as templates for overlap extension PCR. The PCR products were digested and cloned into pEGFP-C1 vector as described above. 2×GFP-tagged DENV1-4 NS5 mammalian expression constructs: To create a 2×GFP vector, GFP gene was amplified by PCR from pEGFP-C1 plasmid with the forward primer 5'-G TACAAGTCCGGAATGGTGAGCAAGGGCGAGGAGC-3' and the reverse primer 5'-CG CCGCCTCGAGCCTTGTACAGCTCGTCCATGCCGAG-3'. The PCR product was digested with BspEI and XhoI and cloned into pre-cut pEGFP-C1 vector. DENV1-4 NS5 gene fragments that corresponded to residues 883-900 of DENV2 NS5 were amplified by PCR from DENV1-4 cDNA clones (GenBank accession numbers for the relevant sequences are: DENV1 (same Gen-Bank accession number as above), DENV2 (same GenBank accession number as above), DENV3 (EU081190)  [48]  and DENV4 (GQ398256)  [49] ).
5 mg/ml for DENV2 C-NLS and 16 mg/ml for DENV3 C-NLS. All concentrated samples were assessed for purity using SDS-PAGE. Generation of in vitro transcribed m7-capped DENV 5'UTR-core-3'UTR RNA template The DENV2 RNA template used in de novo initiation/elongation assay comprised of the DENV2 5 0 -UTR sequence, 72nt of the capsid-coding sequence, followed by the 3 0 -UTR  [34]. Both the 5 0 -UTR-core and 3 0 -UTR sequences were obtained by PCR from DENV2 cDNA clone (same Gen-Bank accession number as above)  [17, 48]  and overlap PCR was performed to join the T7 promoter-5 0 -UTR-core PCR product to the 3 0 -UTR sequence. The final PCR product (DENV 5 0 -UTR-core-3 0 -UTR) was gel purified and was in vitro transcribed using the T7 mMESSAGE mMA-CHINE kit (Ambion) according to the manufacturer's protocol, with slight modification.
5, 10 mM KCl, 0. 5 mM MnCl 2 , 0. 01% Triton X-100, and 10 μM cysteine). The DENV2 3 0 -UTR-U 30 RNA was heated at 95°C for 3 min, and cooled to RT for pre-annealing. All reactions were allowed to proceed for up to 3 h at RT. At the indicated time-points, 10 μl of 2. 5× STOP buffer (200 mM NaCl, 25 mM MgCl 2 , 1. 5 M DEA, pH 10; Promega) with 25 nM calf intestinal alkaline phosphatase (CIP; New England Biolabs) was added to the wells of both assays to stop the reactions. The plate was shaken and centrifuged briefly at 1200 rpm, followed by incubation at RT for 60 min and the released AttoPhos was monitored by reading on a Tecan Infinite F200 PRO microplate reader at excitation max and emission max wavelengths of 422 nm and 566 nm, respectively. All data points were collected in duplicates in 384-well black opaque plates (Corning).
Image processing was performed with ImageJ software  [52]. For grey-scale image, the 8-bit full-colour image was converted to 16-bit grey-scale image. Image analysis was performed on digitized images with ImageJ software  [52]  to determine nuclear to cytoplasmic fluorescence ratio (F n/c ) of each cell using the following formula: F n/c = (F n -F b )/(F c -F b ) , where F n is the nuclear fluorescence, F c is the cytoplasmic fluorescence and F b is the background fluorescence  [28] [29] [30] 42]. F n/c value of > 1 indicates nuclear localization, < 1 indicates cytoplasmic localization and = 1 indicates equal distribution between the nucleus and cytoplasm. The mean F n/c ± SEM was calculated for 30 cells.
The beads were pelleted and the eluted proteins were analysed by SDS-PAGE. Densitometry and binding affinity analyses were performed using ImageJ and Prism Graphpad 6. 0f, respectively. In vitro transcription, RNA electroporation, plaque assay, real-time RT-PCR (reverse transcription-PCR) and immunofluorescence assay (IFA) The above experiment was performed as per described previously  [17]. IFA against NS5 protein by anti-NS5 human antibody 5R3  [34]  and dsRNA by anti-dsRNA mouse antibody J2 (Scicons) was performed. IFA images were captured on inverted fluorescence microscope (Olympus IX71, Center Valley, USA) at 40x magnification and image analysis was performed with ImageJ software  [52]. Crystallization, data collection and structure solution of Impα-NLS complexes Crystals of Impα in complex with DENV3 NS5 C-terminal NLS were obtained in a condition containing 1 M sodium citrate and 10 mM DTT at pH 7, and DENV2 NS5 C-terminal NLS were obtained in a condition containing 1 M ammonium sulfate and 0.
1 M sodium HEPES at pH 7. X-ray diffraction data were collected at the Australian Synchrotron on the MX1 macromolecular crystal beam line  [53]. Data were processed using iMosFlm  [54] , where the diffraction images were indexed and integrated, then scaled, phased and refined using AIMLESS  [55] [56] [57] , Phenix Phaser MR, and Phenix. refine, respectively  [57]. Structural modelling was performed using COOT to ensure all molecules were correctly positioned within the electron density map generated from the collected X-ray data. Once the best fit between the structural model and experimental data was achieved (measured by R-free, R-work), and other model validation procedures confirmed that the model was of good quality, the model and data was deposited into the protein data bank (PDB) for final validation.
This work was supported by the  Duke-NUS   Signature Research Program  (funded by the  Ministry of Health, Singapore) , the  National Medical Research Council , Singapore (http://www. nmrc. gov. sg)( NMRC/ MOHIAFCat1/0018/2014 ;  NMRC/CBRG/0073/2014 ;  NMRC/CBRG/0103/2016 ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
2004). Using a dataset collected during a 17-year period in northeast Greenland, a period with rapid warming (e. g. ,  Høye et al. 2013) , we investigated the inter-and intraannual variation in, and the interlinkages between the reproductive timing of a long-distance migratory high Arctic bird, the Sanderling (Calidris alba Pallas 1764, Fig. 1 ), clutch predation, chick growth, and arthropod prey abundance. We hypothesize that clutches have a larger risk to be found by predators early in the season, especially in snow-rich years, and that Sanderling chicks grow better if they hatched just before or during the peak abundance of their arthropod prey.
Nest searching and monitoring We searched for Sanderling nests and broods by foot in Zackenberg in June and July 1996-2013. In case the exact laying date was unknown, we estimated it by determining clutch age by flotation of two eggs in each clutch  (Liebezeit et al. 2007; Hansen et al. 2011). This also allowed us to predict the hatch date, and we always visited the clutches 2-3 days before expected hatch dates to check for signs of hatching (i. e. , cracks or holes in the eggs) and to visit the nests at hatch to measure and ring the hatched chicks. In 2007-2013, we equipped the majority (157 of 194) of nests with small temperature loggers (Tiny Tag, Gemini) with a probe placed between the eggs. The temperature loggers collected temperature data every minute for a minimum of 22 days. If clutches were lost, the temperature profiles accurately indicated when this occurred  (Reneerkens et al.
2011). Nests without temperature loggers were checked every 1-5 days until found depredated or until hatch. Clutches were considered successful if at least one chick hatched, and considered unsuccessful when temperature logger data indicated clutch loss, or when the nest was found empty before expected hatching. We considered clutches abandoned when cold eggs were encountered in the nest cup and the temperature profiles of the temperature loggers indicated that the clutch had not been incubated for more than 2 days. Abandoned nests (n = 13) or clutches which failed for other reasons than predation (n = 5) were not included in the analyses. The nest cup of each depredated clutch was examined for signs that could indicate the predator species or type. The smell of urine or the presence of fresh Arctic fox feces inside or near the empty nest cup was taken as signs of predation by Arctic fox.
Clutch survival analysis To evaluate the inter-and intra-annual trends in daily clutch survival, we used the nest survival model implemented in program MARK, version 7. 1, which takes biases in the detection of successful and nonsuccessful clutches into account  (Dinsmore et al. 2002). We only have detailed clutch information (i. e. , appropriate sample sizes and daily monitoring of nest success by use of thermologgers) for the period 2007-2013, where data were obtained on an average of 31 clutches (range: 25-46) annually. We tested a limited number of models in which clutch survival varied both between and within years and included date and year as covariates in our models. Within years, we expected positive linear or quadratic seasonal changes in clutch survival. Due to the highly synchronized egg laying within years, clutch age and date (day of year) were strongly correlated (Pearson's r = 0.
Chick growth Throughout the prefledging period (0-17 days), we captured and recaptured each chick within a family and measured their body mass using electronic scales with an accuracy of 0. 1 g. Growth data were fitted to both a Gompertz growth model, M = AÁexp (Àexp (ÀkÁ(t À T))), and a logistic growth model, M = A/ (1 + exp (ÀkÁ(t À T))), typically used to describe avian growth  (Starck and Ricklefs 1998). In these formulas, M is body mass (g), A is the asymptotic mass (g), k is the growth coefficient, t is the age of the chick, and T is the age at the point of inflection. Growth curves were fitted using maximum likelihood in the nlme package of R  (Pinheiro et al. 2015). The growth curves were based on 310 individual chicks of which 40 were measured at two ages and one was measured three times.
At hatch, shorebird chicks have energy stores in their residual yolk sac  (Starck and Ricklefs 1998)  and stay in the nest cup for at least some hours. Therefore, only chicks older than 1 day were used to study effects of hatch date in relation to food abundance on growth. Of 74 chicks (older than 1 day and excluding chicks measured in 2010 when no arthropod data were available), data were available for analyses of growth. All individuals were captured and measured only once (average age = 6. 5 days), but included chicks from the same families (family and year were initially included as random factors in the model, see "Statistical analyses"). Hatch date relative to the median date of food abundance (i. e. , the individual mismatch) may affect chicks' growth rate depending on the seasonal shape of the abundance of arthropods (i.
Statistical analyses We selected either linear or quadratic functions to describe the changing phenology of Sanderling hatch date and arthropods and of snow cover. Models with the best fit based on their F-values were selected to describe the data. For direct comparison of shifts in phenology between birds and arthropods, we also analyzed the linear relationship of arthropod phenology. The clutch survival analysis was performed in program MARK  (White and Burnham 1999). We scaled dates such that 10 June was day 1, the earliest date at which a clutch was found. In total, we considered 11 candidate models, for all of which we used a logit link function. Models were ranked with the corrected Akaike's information criterion for small samples (AIC c ), and DAIC c and Akaike weights (w i ) were used to infer support for models in the candidate set  (Burnham and Anderson 2002).
Acknowledgments We thank the  Greenland Ecosystem Monitoring program  for access to ecosystem data and  Aarhus University  for providing logistics at Zackenberg. This work was supported by an  International Polar Year grant  from the  Netherlands Organisation for Scientific research (NWO)  to TP and JR. Additional support for JR and TP came from the  Metawad  project awarded by  Waddenfonds  ( WF209925 , www. metawad. nl). JR also received two  INTERACT grants for Transnational Access  from the  European Community 's  Seventh Framework Programme  under grant agreement No  262693  and a grant from  World Wildlife Fund Netherlands. OG and JM were supported by the  French Polar Institute  ( IPEV; "Interactions" program   1036 ) and the  Groupe de Recherche en Ecologie Arctique (GREA). The  Government of Greenland (Ministry of Domestic Affairs, Nature and Environment ) in Nuuk granted access and research permits for field work in the National Park.
Funding Information This work was supported by an  International Polar Year grant  from the  Netherlands Organisation for Scientific research (NWO)  to TP and JR. Additional support for JR and TP came from the  Metawad  project awarded by  Waddenfonds  ( WF209925 , www. metawad. nl). JR also received two  INTERACT grants for Transnational Access  from the  European Community 's  Seventh Framework Programme  under grant agreement  No262693  and a grant from  World Wildlife Fund Netherlands. OG and JM were supported by the  French Polar Institute  ( IPEV; "Interactions" program   1036 ) and the  Groupe de Recherche en Ecologie Arctique (GREA).
g. New Zealand). A wide array of techniques (biological, chemical, physical and statistical) is available to palaeolimnologists to allow the deciphering of complex sedimentary records. Lake sediments are an important archive of how drivers have changed through time, and how these impacts manifest in lake systems. With a paucity of 'real-time' data pre-dating human impact, palaeolimnological archives offer the only insight into both natural variability (i. e. that driven by climate and intrinsic lake processes) and the impact of people. Whilst there is a need to acknowledge complexity, and temporal and spatial variability when deciphering change from sediment archives, a palaeolimnological approach is a powerful tool for better understanding and managing global aquatic resources.