dataesr/scientific-paragraphs-categorization huggingface dataset:
Recently, a subdivision into 51 subfamilies has been implemented to improve correspondence between specificity and sequence  (7). In this work, we present a detailed structural and enzymatic characterization of one of the GH5 enzymes encoded by Saccharophagus degradans , named SdGluc5_26A (formerly Cel5F)  (8). Sde2-40 is a marine bacterium known mainly for its capacity to degrade different polysaccharides including agar, cellulose, and chitin  (9). The genome analysis of Sde2-40 revealed 139 GHs including 19 in family GH5  (10). In the CAZy database (4), SdGluc5_26A belongs to GH5 subfamily 26 (GH5_26). This subfamily contains mainly ␤(1,3;1,4)-glucanases with weak capacity to degrade cellulose  (8, (11) (12) (13) (14). Herein, we show that SdGluc5_26A is a lichenase with ␤(1,3; 1,4)-glucanase and side cellobiohydrolase activity.
The purification protocol was the same as for SdGluc5_26A. Biochemical Characterization-The protein concentration was determined using a Thermo Scientific NanoDrop 2000 spectrophotometer (Thermo Scientific, Illkirch, France) by measuring absorbance at 280 nm. The oligomerization state was confirmed by performing size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS, Wyatt technology, Santa Barbara, CA) experiments. For this purpose, 100 l of enzyme at 5 mg Ϫ1 ml Ϫ1 were dialyzed over- night in 50 mM Tris-HCl, pH 7. 0, and 300 mM NaCl. After overnight equilibration of the column (Shodex TM column KW803), 30 l of sample were injected. The elution was carried out during 47 min at a flow rate of 0. 5 ml Ϫ1 min Ϫ1. Results were analyzed by the ASTRA software (Wyatt Technology).
1 M Tris-HCl buffer, pH 7. 5, and 0. 2 M MgCl 2. These crystals belong to space group P2 1 with unit cell parameters 72 ϫ 60 ϫ 130 Å, ␤ ϭ 104°and three molecules per asymmetric unit. A second crystal form in space group C2 and unit cell dimension of 188 ϫ 132 ϫ 131 Å, ␤ ϭ 134°was Structure-Activity Relationships of a GH5_26 Glucanase obtained in the same crystallization condition. All crystals were cryo-protected with mother liquor containing 25% (v/v) glycerol prior to flash-cooling in liquid nitrogen. X-ray diffraction data for all crystals were collected on beamline Proxima1 at the Synchrotron SOLEIL (Gif-sur-Yvette, France). Diffraction data were indexed with XDS (17) and scaled with the program SCALA  (18). Structure Determination and Refinement-The sub-structure of selenium-labeled SdGluc5_26A was determined with the program ShelxD  (19) , and phase calculations and solvent flattening were carried out with ShelxE  (20).
The protein chain was traced automatically with the program Buccaneer  (21) , and refinement and model adjustment were carried out with the programs Refmac  (22)  and Coot  (23) , respectively. Random sets of ϳ5% reflections were set aside for cross-validation purposes. The composition of cross-validation data sets was systematically taken over from the parent data set of the equivalent space group. Model quality was assessed using the MolProbity server  (24). Figures representing structural renderings were generated with the PyMOL Molecular Graphics System (W. L. DeLano, Schro ¨dinger, LLC, New York), and Fig. 3B  was drawn with ChemDraw 9. 0 Pro (CambridgeSoft Corp. , Cambridge, MA). Atomic coordinates and structure factors have been deposited within the Protein Data Bank http://www. rcsb.
Interestingly, both tryptophan residues are well conserved in the N-terminal extension of some GH5_26 members (Fig. 1D ). A search for structural homologues of SdGluc5_26A with the DALI server identified a large number of GH5 enzymes with significant structural similarity. The highest Z-scores are associated with the subfamily GH5_26 metagenome-derived endoglucanase Cel5A (Z-score 51. 8; Protein Data Bank (PDB) entry 4HTY (  13 )), and with Thermobifida fusca TfCel5A (Z-score 32. 6; PDB entry 2CKR) and BaCel5A from Bacillus agaradhae-    34 )). The other most closely related structures are endoglucanases from subfamilies GH5_1 and GH5_2, pointing toward a strong structural relationship with these two subfamilies. The structure of metagenome-derived Cel5A has previously been solved in complex with a cellotetraose molecule bound to subsites Ϫ2 to ϩ2 (PDB entry 4HU0 (  13 )), and the structure of a TfCel5A mutant with a cellopentaose molecule spanning subsites Ϫ3 to ϩ2 is available in the PDB (accession code 2CKR).
Cellotetraose G4 binds in subsites Ϫ2 to ϩ2 of the enzyme, by-passing the catalytic machinery in a non-productive manner (Figs. 2A  and 3 ). Similar non-productive binding modes evading the catalytic machinery have been observed in the cellopentaose complex of TfCel5A (accession code 2CKR) and in the complex of BaCel5A with a thio-cellopentaoside (PDB entry 1H5V  (36) ). In the E291Q-G4 complex, the individual pyranoside units are all in the standard 4 C 1 chair conformation and refine with average temperature factors of 28, 29, 27, and 44 Å 2 , going from subsites Ϫ2 to ϩ2. The glucose unit in the Ϫ2 subsite stacks against the fully conserved Trp 336 and interacts with Ser 337 main-and sidechain atoms via its O 2 hydroxyl group, and with the side chain of His 107 by means of the O6 hydroxyl.
Due to the displacement of the glucose in subsite Ϫ1 from a catalytically relevant position, this subsite is characterized by a paucity of direct interactions with the protein. Only the O 2 hydroxyl establishes direct hydrogen bonds with the carboxyl unit of the catalytic acid/base Glu 189 and the side chain of the fully conserved Tyr 256. Also, as a consequence of the linearized bypass binding mode of cellotetraose, the glucose units at the aglycon side are shifted away from the true ϩ1 and ϩ2 binding sites, as inferred from comparison with other family GH5 enzyme substrate complexes. However, a structural comparison with metagenome-derived Cel5A containing a cellotetraose molecule bound to subsites Ϫ2 to ϩ2 (PDB entry 4HU0 (  13 )) suggests that in a catalytically relevant binding mode, pyranose units in subsites ϩ1 and ϩ2 could be stabilized by stacking interactions with residues Trp 232 and Pro 305 and by hydrogen bonds with the side chains of Lys 259 and Lys 261.
All monomers of both crystal forms showed clear electron density in subsites Ϫ3 to Ϫ1 (Fig. 2, D  and E ), and a trisaccharide with all pyranose units in the low-energy 4 C 1 chair conformation could be modeled, with the ␤(1,3) linkage between subsites Ϫ3 and Ϫ2 (Fig. 2 ). Average temperature factors for glucose units in the individual subsites are in the order Ϫ3 Ͼ Ϫ1 Ͼ Ϫ2. In the tetrasaccharide G4A, the (1,3) linkage is situated at the non-reducing end, and for an intact substrate, one would expect the chain spanning the substrate-binding cleft from subsites Ϫ3 to ϩ1. Nonetheless, no residual density could be observed in subsite ϩ1, indicating that the E291Q retained sufficient residual activity for cleaving the substrate during the prolonged incubation necessary for crystal growth. In both crystal forms, the glucose units in subsites Ϫ1 and Ϫ2 adopt positions almost identical to the ones observed in the Acidothermus cellulolyticus endocellulase E1 in the Michaelis complex with cellotetraose (PDB entry 1ECE (37)).
In subsite Ϫ1, the O1 hydroxyl group forms a hydrogen bond with the side chain of the catalytic acid/base, and the O3 group contacts the conserved His 142 (Fig. 3B ). The O6 hydroxyl is found in intermediate states between syn-and anti-positions with respect to the endo-cyclic O5 and contracts accordingly a short hydrogen bond of 2. 3-2. 65 Å in length with His 303. This latter residue is located on loop ␤7-␣7, structurally and sequence-wise very conserved within subfamily GH5_26, but very distinct from members of other GH5 subfamilies. Interestingly, a spatially overlapping His residue is delivered in the T. maritima enzymes Cel5A (subfamily GH5_25) and Cel5B (subfamily GH5_36) from loop ␤6-␣6 into the active site cleft, and His 205 of T. maritima Cel5A (PDB accession code 3AZT) interacts equally with the O6 hydroxyl of a pyranose located in subsite Ϫ1.
The three-dimensional structure of SdGluc5_26A adopts a (␤/␣) 8 topology, classical for GH5 members and clan GHA enzymes in general. Surprisingly, a unique N-terminal extension projects away from the main structural core and protrudes into the substrate-binding groove of a neighboring catalytic subunit, thereby conferring a trimeric quaternary architecture to SdGluc5_26A. To ascertain that this novel structural feature was not a crystallographic artifact, we confirmed the oligomeric nature of SdGluc5_26A by solution studies. Interestingly, the only other known three-dimensional structure of a GH5_26 member, metagenome-derived Cel5A (PDB code 4HTY (  13 )), exhibits an identical trimeric assembly, generated through the three-fold axis of the cubic space group. Nevertheless, in this case, the N-terminal extension, sequence-wise quite conserved with respect to that of SdGluc5_26A, adopts a different conformation and does not protrude into the active site of a neighboring subunit.
where F o and F c are observed and calculated structure factors, respectively. e r. m. s. , root mean square. f There are no Ramachandran outliers. Structure-Activity Relationships of a GH5_26 GlucanaseMARCH 25, 2016 • VOLUME 291 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 7187 rans (Z-score 32. 0; PDB entry 1A3H (.
* This work was supported by the  French National Research Agency  ( Funlock  project  ANR-13-BIME-0002-01  and the  Microbio-E A*MIDEX  project number  ANR-11-IDEX-0001-02 ). This work was also supported by a grant from the  French Infrastructure for Integrated Structural Biology (FRISBI)  ( ANR-10-INSB-05-01 ). The authors declare that they have no conflicts of interest with the contents of this article. The atomic coordinates and structure factors (codes  5A8N ,  5A8M ,  5A8O ,  5A8P ,  5A8Q ,  5A94 , and  5A95 ) have been deposited in the Protein Data Bank (http://wwpdb. org/).
009 0. 009 0. 011 0. 007 0. 007 0. 007 Bond angles (°) 1. 28 1. 282 1. 39 1. 191 1. 138 1. 217 Chiral volumes (Å 3 ) 0. 078 0. 072 0. 089 0. 069 0. 066 0. 074 Average B-factors (Å 2 ) Main/side chain 29. 32/31. 98 22. 49/23. 88 15. 27/17. 28 24. 77/26. 34 21. 97/24. 39 18. 94/20. 59 Ligands/solvent 49. 02/39. 37 24. 19/29. 71 31. 64/28. 62 41. 33/32. 86 43. 16/32. 71 33. 36/32. 85 Sugars 26. 03 31. 12 43. 69 20. 92 r. m. s. e deviations on B-factors (Å 2 ) Main chain 0. 855 0. 651 0. 586 0. 633 0. 945 0. 527 Side chain 1. 621 0. 828 1. 103 0. 941 1. 703 0. 796 Ramachandran plot statistics (%) f Residues in favored regions 98. 81 98. 80 98. 65 98. 53 98. 53 98. 56 Residues in allowed regions 1. 19 1. 2 1. 35 1. 47 1. 47 1. 44 PDB code 5A8N 5A94 5A8M 5A8O 5A8P 5A8Q i I i(hkl) , where I is an individual reflection measurement and ͗I͘ is the mean intensity for symmetry related reflection.
Study Site and Study Design In this cross-sectional survey, baseline data from the LUPROGAR study were used. LUPROGAR was implemented at Apsara Garment Co. Ltd. , Phnom Penh, Cambodia, an export-oriented garment factory located in the suburban commune Chom Chau in Cambodia's capital Phnom Penh, approximately 10 km west of the city center. Baseline data were collected in April 2015. The factory was previously not operating a canteen, but a canteen was installed specifically for the LUPROGAR study. Enrolled study participants received either free lunch provision through the newly-established canteen during workdays (intervention arm) or an equal monetary compensation at the end of the study (control arm). At the time of study implementation, the factory employed about 1300 workers, the majority of which were young unmarried women from low-income rural households.
Sample Size The LUPROGAR study used an explorative approach (two tail) to estimate an appropriate sample size, as data on the nutritional and micronutrient status of Cambodian garment workers are rare, as well as data on the effects of lunch provision in this context. Calculations were carried out using G*Power software (Version 3. 1. 9. 2, University of Kiel, Kiel, Germany). Assuming a 5% level of significance (alpha = 0. 05) and a statistical power of 80% (beta = 0. 20) to detect a small to medium standardized effect size of 0. 35  (Cohen's d)  between both arms using a two-sided test  [15] , 130 subjects in each group were required. To allow for about 20% loss to follow up, it was initially aimed at recruiting a total of 330 subjects, 165 subjects in each arm.
Enrolment of Study Participants Firstly, the factory management, superintendents and union representatives were informed in detail about the objectives and procedure of the LUPROGAR study. Subsequently, the study was announced during a meeting to all factory employees. Written informed consents, including a study description in lay language, were obtained (signature or fingerprint) at lunch breaks and after end of work, prior to any data collection, by trained local project assistants. Workers who signed the informed consent were invited to the baseline assessment, which took place in a separate room during working hours and included a clinical screening performed by trained local nurses. Inclusion criteria for the LUPROGAR study were: female, nulliparous, non-pregnant and <31 years at date of enrolment.
Questionnaires Trained local project assistants applied a semi-structured questionnaire, collecting data on background information and the socio-economic status of study participants and their respective households. Obtained data on age of workers was cross-checked with reference data from the factory's personnel department. Trained local project nurses administered a semi-structured health questionnaire, collecting data on present intake of medications, as well as on illness history and sick leave in the 14 days preceding the interview. Both questionnaires were similar to those from the CDHS 2014  [8]  and were pre-tested under field conditions.
Data Management and Statistical Analysis Data entry and validation by double entry of questionnaires and anthropometry sheets was performed by trained project assistants using EpiData software (Version 3. 1, EpiData Association, Odense, Denmark). Data management and statistical analyses were executed using SPSS software (Version 22. 0. 0. 1, IBM Corp. , Armonk, NY, USA). Normality of distributions was evaluated using the Shapiro-Wilk test. As most continuous variables (background characteristics, anthropometry and micronutrient status) were skewed, descriptive statistics for continuous variables are therefore represented by the median and interquartile range (IQR). Categorical variables are expressed as frequency and percentage. To assess if BMI is associated with Hb and micronutrient status, bivariate correlations between BMI and Hb, serum FER, serum sTfR, serum RBP and serum VitB12 concentrations were calculated with non-parametric Spearman's correlation.
Ethics The LUPROGAR study was approved by the Institutional Review Board of the Faculty of Medicine at Justus Liebig University, Giessen, Germany (14 November 2014) and the National Ethics Committee for Health Research (NECHR) at the Ministry of Health, Phnom Penh, Cambodia (29 December 2014). Written informed consent was collected from all study participants prior to enrolment by signature or fingerprint. The ethics committees approved the consent format prior to data collection. The study was registered at the German Clinical Trials Register (9 January 2015, Identifier: DRKS00007666).
Acknowledgments: The LUPROGAR study was conducted with financial support from the  Federal Ministry of Economic Cooperation and Development, Bonn, Germany. H. F. received a travel grant from the  Fiat Panis Foundation, Ulm, Germany. Costs to publish in open access were covered by the open access publication fund of the  Justus Liebig University, Giessen, Germany. We thank  Khov Chhay  (Managing Director) and the management of  Apsara Garment Co. Ltd. , Phnom Penh, Cambodia , for helping to implement the study;  Juergen Erhardt ,  VitMin Laboratory, Willstaett, Germany , and the  Pasteur Institute, Phnom Penh, Cambodia , for conducting our serum biomarker analyses;  Johannes Herrmann ,  Justus Liebig University, Giessen, Germany , for statistical advice; and staff at the  Department of Fisheries Post-Harvest Technologies and Quality Control, Fisheries Administration, Ministry of Agriculture, Forestry and Fisheries, Cambodia , for their overall support.
Implementation requires a full range of competent and motivated health professionals, including midwives and specialists, within functioning health systems. Major bottlenecks of quality care around birth include health financing, workforce and service delivery. 67 ncentives for demand creation and strengthening implementation must also be explored. 70, 71   orporating stillbirths into the investment cases for women's and children's health strengthens the cost-effectiveness argument;  25  however, often supporting programmes do not use stillbirth rates to monitor progress and document the full return on investment. 72 Limited reporting on stillbirth in the Organisation for Economic Cooperation and Development's database of donor funding reflects another missed opportunity to integrate stillbirth into existing programmes for women's and children's health.
Acknowledgements We thank  Ingrid Friberg  for review and inputs on this paper,  Aleena Wojcieszek  for her extensive coordination and technical support for the Lancet Ending Preventable Stillbirths Series,  Zachariah J. Falconer-Stout  and  Kelsey Simmons  for their contributions to the organisational network analysis, and the  Lancet Ending Preventable Stillbirths Series advisory group  for their valuable inputs to the concepts of this paper.
Study Design and Sampling In 2014, The Cambodian Demographic Health Survey (CDHS) conducted a nationallyrepresentative survey of women and men between the ages of 15 and 49 years of age, in 16,356 households  [31]. The sample participants were selected in two stages. In the first stage, 611 villages (also known as clusters or enumeration areas) were selected with probability proportional to village size. Village size is the number of households residing in the village. Then, a complete mapping and listing of all households existing in the selected villages was conducted. The resulting lists of households served as the sampling frame for the second stage of sample selection. Households were systematically selected from those lists for participation in the survey. The 2014 CDHS included a micronutrient component which was implemented in one-sixth of the clusters selected for the main survey, collecting data only in women from the survey and adding children under five years of age.
In these clusters, urine and blood samples were collected from women who had given birth in the five years preceding the survey and from their children aged 6-59 months. Only clusters where data on anemia was collected were eligible for inclusion in the micronutrient survey. The sample size for this study was calculated to obtain a margin of error of 3% at the national level and 5% at urban and rural level. Anticipating up to 20% refusal/dropouts, 935 mothers and 1096 children were recruited. The number of blood samples that could not be collected from women was 210 and from children 315 samples, resulting in a net number of samples from women of 725 and 781 from children. The women of reproductive age were selected according to the following criteria: (i) mother of the child 6-59 months surveyed; (ii) from households having participated in CDHS 2014 the previous week(s); (iii) no evidence for severe or chronic illness; and (iv) having agreed to, and signed the informed consent form to participate.
Statistical Analysis Data analysis was performed with SPSS software (SPSS, V20; IBM, Chicago, IL USA). We defined vitamin D status as: According to the recommended values from the Endocrine Society  [32]  (and expert assessment for severe deficiency  [33] , but other values are recommended by other organizations)  [34] [35] [36]. Descriptive statistics were used to examine 25(OH)D relationships. Vitamin D concentration for mothers was not normally distributed according to the test of Kolmogorov-Smirnov (p = 0. 000) and, therefore, only non-parametric tests were performed. Median and interquartile range values of the vitamin D concentrations are presented and disaggregated by wealth index, and urban/rural status. Vitamin D concentration (continuous variable) was compared by groups by using the non-parametric Mann-Whitney U-test (two groups) or Kruskal-Wallis for independent variables.
For children, vitamin D concentrations followed a normal distribution (Kolmogorov-Smirnov test, p = 0. 669) and, therefore, parametric tests were performed to compare groups (i. e. , t-test and ANOVA test). Odds analysis was also performed to examine the association between having an insufficient vitamin D status (<75 nmol/L) and several factors. Results were considered significant at p < 0. 05. Socio-economic status was calculated using the Demographic Health Statistic (DHS) Wealth Index  [37]  to divide households surveyed into five socio-economic groups: the "extreme poor" (category 1), the" poor" (category 2), the "intermediate" (categories 3 and 4), and the "wealthiest" (category 5). The Wealth Index was constructed from recorded data on household assets, such as tables, chairs, refrigerator, air conditioners and beds, and also from housing conditions (materials of house floor, house roof, main wall) and facilities (energy for cooking, electricity and latrines); income and expenditure were not used.
Acknowledgments: The Micronutrient survey was supported by several organizations ( UNICEF, IRD, ILSI SEAR, World Vision , and WFP) and collected by the  Department of Fisheries Post-harvest Technologies and Quality control (DFPTQ) ,  Fisheries Administration, Ministry of Agriculture, Forestry and Fisheries, Cambodia. The development of the paper was supported by  UNICEF National committees (Australian, Korean , and  Hong Kong ). We would like to thank the  Fisheries Administration of Cambodia and its team  for their support.
Study Population Subjects were selected from the "SUppl ementation en VItamines et Min eraux AntioXydants" (SU. VI. MAX) study. The SU. VI. MAX study was a randomized, double-blind, placebo-controlled primary prevention trial conducted between 1994 and 2002. It included 12 741 French adults (women aged 35-60 years and men aged 45-60 years). Its primary aim was to evaluate the potential efficacy of daily supplementation with antioxidant vitamins and minerals (ascorbic acid, vitamin E, b-carotene, selenium, and zinc) delivered at nutritional (ie, nonpharmacologic) doses on the prevention of cancer, ischemic heart disease, and overall mortality. 21 The trial phase ended in 2002 and health events monitoring was pursued until September 2007. The SU. VI. MAX study was registered at clinicaltrials.
gov (number NCT00272428) and was conducted according to the guidelines laid down in the Helsinki Declaration of 1975 as revised in 1983, and was approved by the Ethics Committee for Studies with Human Participants of Paris-Cochin Hospital (CCPPRB no. 706) and the Commission Nationale Informatique et Libert e (CNIL no. 334641), which ensures that medical information be kept confidential. All subjects provided written informed consent.