Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments

Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin,Guaymas Basin

Adrien Vigneron1,2,3*,Perrine Cruaud1,2,3,Erwan G.Roussel7,Patricia Pignet1,2,3,Jean-Claude Caprais4, Nolwenn Callac1,2,3,5,Maria-Cristina Ciobanu6,Anne Godfroy1,2,3,Barry A.Cragg7,John R.Parkes7, Joy D.Van Nostrand8,Zhili He8,Jizhong Zhou8,9,10,Laurent Toffin1,2,3

1Ifremer,Laboratoire de Microbiologie des Environnements Extre?mes,UMR6197,ZI de la pointe du Diable,Plouzane′,France,2Universite′de Bretagne Occidentale, Laboratoire de Microbiologie des Environnements Extre?mes,UMR6197,ZI de la pointe du Diable,Plouzane′,France,3CNRS,Laboratoire de Microbiologie des Environnements Extre?mes,UMR6197,ZI de la pointe du Diable,Plouzane′,France,4Ifremer,Laboratoire Etude des Environnements Profonds,UMR6197,ZI de la pointe du Diable,Plouzane′,France,5Universite′de Brest,Domaines Oce′aniques IUEM,UMR6538,Place Nicolas Copernic,Plouzane′,France,6Ifremer,Ge′osciences Marines, Laboratoire des Environnements Se′dimentaires,ZI de la pointe du Diable,Plouzane′,France,7School of Earth and Ocean Sciences,Cardiff University,Cardiff,United Kingdom,8Institute for Environmental Genomics and Department of Microbiology and Plant Biology,University of Oklahoma,Norman,Oklahoma,United States of America,9State Key Joint Laboratory of Environment Simulation and Pollution Control,School of Environment,Tsinghua University,Beijing,China,10Earth Science Division,Lawrence Berkeley National Laboratory,Berkeley,California,United States of America

Abstract

Subsurface sediments of the Sonora Margin(Guaymas Basin),located in proximity of active cold seep sites were explored.

The taxonomic and functional diversity of bacterial and archaeal communities were investigated from1to10meters below the seafloor.Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches(Ribosomal Intergenic Spacer Analysis,16S rRNA libraries and quantitative PCR with an extensive primers set)and correlated to comprehensive geochemical data.Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates.The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems(Marine Benthic Groups B and D,Miscellaneous Crenarchaeotal Group,Chloroflexi and Candidate divisions)and remained relatively similar throughout the sediment section,despite defined biogeochemical gradients.However,relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin.Consistently,metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters,hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Citation:Vigneron A,Cruaud P,Roussel EG,Pignet P,Caprais J-C,et al.(2014)Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin,Guaymas Basin.PLoS ONE9(8):e104427.doi:10.1371/journal.pone.0104427

Editor:Jack Anthony Gilbert,Argonne National Laboratory,United States of America

Received May1,2014;Accepted July8,2014;Published August6,2014

This is an open-access article,free of all copyright,and may be freely reproduced,distributed,transmitted,modified,built upon,or otherwise used by anyone for any lawful purpose.The work is made available under the Creative Commons CC0public domain dedication.

Data Availability:The authors confirm that all data underlying the findings are fully available without restriction.Nucleic acid sequences are available in the EMBL database under the following accession numbers:HF543837–HF543861for archaeal,HF545450–HF545524for bacterial16S rRNA sequences and HF935025–HF935037for mcrA gene sequences.The raw GeoChip dataset is available at https://www.wendangku.net/doc/ab15581403.html,/4download/.

Funding:The oceanographic cruise and this study was funded by IFREMER and a IFREMER PhD grant.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*Email:avignero@https://www.wendangku.net/doc/ab15581403.html,

Introduction

Deep marine subsurface sediments are one of the most extensive microbial habitats on Earth,covering more than two-thirds of the Earth’s surface and reaching maximal thickness of more than 10km at some locations[1].Microbial populations are wide-spread in these sediments as deep as temperature permits[2]and cell numbers vary consistently ranging from1010to103cell-s per cm3of sediments according to their proximity from land, sedimentary rates and depth[3].In general,microbial abundance in subsurface sediments(below1mbsf)decreases exponentially with depth,as a probable consequence of the decreasing organic carbon quality and availability[4].Recent investigations based on NanoSIMS monitoring[5]or intact ribosomal RNA[6]and membrane lipid detection[6,7]demonstrate that sedimentary microbial communities are active as they can incorporate carbon and nitrogen.However,overall metabolic rates are very slow,with biomass turnovers ranging from years to millennia[8].Numerous of studies have focused on elucidating the microbial diversity of subsurface sediments[6,9–13].Specific lineages of Bacteria(for e.g.Chloroflexi,Candidate division JS1)and Archaea(for e.g. Miscellaneous Crenarchaeotal Group(MCG),Marine Benthic Group D(MBGD),South African Goldmine Euryarchaeotal Group(SAGMEG)[14,15],distinct from the surface biospheres (above1mbsf),appear to occur consistently in marine subsurface

sediments.However identification of the metabolism of these microbial populations remains challenging.Isotopic signatures of membrane lipids suggested that heterotrophic strategies dominat-ed in these ecosystems[6,7].Metagenomic and metatranscrip-tomic analyzes of subsurface sediments from the deep biosphere of the Peru Margin revealed metabolisms associated with lipids, carbohydrates and amino acids utilization.However detected genes and transcripts were mainly affiliated to Firmicutes, Actinobacteria,and Alpha-and Gammaproteobacteria rather than Archaea,Chloroflexi and candidate divisions[16,17].Finally, recent single cell genomic approaches indicated the capacity of peptides degradation for members of MCG and MBGD archaeal lineages[18].Despite these recent advances the metabolic pathways associated to the dominant microbial communities in subsurface sediments remain unclear.

The cold seeps of the Sonora Margin in the Guaymas Basin (Gulf of California),colonized by visible microbial mats and faunal assemblages,were previously characterized as highly active areas with abundant concentrations of methane and sulfur cycle microorganisms(Anaerobic methanotrophs,sulfate-reducing bac-teria)in the shallow sediments(0–20cmbsf)[19,20].However,the subsurface microbial communities and processes that occur in the deeper sediments of the Sonora Margin have not yet been explored.The aim of this study was therefore to estimate the phylogenetic and functional biodiversity of the Sonora Margin sediments by comparing the geochemical composition,the microbial taxonomic diversity and abundance,and the Geo-Chip-based metagenome from subsurface sediments sampled in proximity with active cold seeps of the Sonora Margin.We analyzed the archaeal and bacterial diversity,abundance and distribution in correlation with geochemical gradients and elementary composition of the sediments and compared with the Sonora Margin surface cold seep sediments.Furthermore,we identified the metabolic processes and the functional potentials in term of carbon utilization and energy for both bacterial and archaeal communities and present insights into the microorganism adaptability and capacity to use various substrates in marine subsurface sediments.

Materials and Methods

Core sampling and abiotic variables

Sediment samples were collected from Sonora Margin cold seeps in the Guaymas Basin,during the Ifremer‘‘BIG’’cruise on the research vessel L’Atalante in June2010.This cruise has benefited from a work permit in Mexican waters by the Mexican Secretariat of Foreign Relations(DAPA/2/281009/3803,Octo-ber28th,2009).Gravity core BCK1(N27u35.804,W 111u28.697),10meters in length,was recovered from an observed gas depression in methane plume fields,600meters distant from visible active cold seeps(WM14and EWM14in Vasconcelos area [20]),at1723meters water depth.In situ temperatures,measured using thermal sensors(THP,Micrel)attached to the core, increased gradually from3.5u C at the water-sediment interface to5u C in the bottom of the core(9mbsf).Immediately after retrieval,BCK1core was sectioned in1meter long sections and transferred into the cold room.The plastic core liner was opened every50cm for sub-sampling.Samples for molecular analysis were collected aseptically using cut-off sterile5mL syringes,and frozen at280u C.Sediment samples for activity rate estimations were taken using five cut-off sterile5mL syringes per section. These syringes were hermetically and anaerobically sealed with nitrogen in aluminum bags(Gru¨ber-Folien,Germany)and stored at4u C for processing back to laboratory.Methanogenic activity measurements from Acetate,Di-methylamines and CO2substrates were carried out at Cardiff University,UK,as detailed in Methods S1.

Pore water was obtained by spinning down approximately10 grams of crude sediment then was fixed as previously described [20].Sulfate concentrations were determined by ion exchange chromatography as previously described[21].Hydrogen sulfide and ammonium concentrations were measured by colorimetry [22].Methane concentrations were quantified using the headspace technique(HSS Dani86.50)and a gas chromatograph(Perichrom 2100)equipped with a flame-ionization detector[23].Total organic carbon(TOC)of the sediments were measured by combustion in a LECO CS125carbon analyzer,as previously detailed[24].Quantitative elemental chemical compositions of unfiltered pore waters were measured using Inductively Coupled Plasma-Atomic Emission Spectrophotometry(ICP-AES,Ultima2, Horiba,JobinYvon),as previously detailed[25].Effect of eventual particle contaminations was limited by normalization of the elemental concentrations by conservative element(Na)concentra-tions.The stable-isotope composition of methane was measured by ISOLAB https://www.wendangku.net/doc/ab15581403.html,pany(Neerijen,The Netherlands)in the first meter deep section(0.5mbsf)and in the deepest sediment layer (8.5mbsf)as previously described[20].

Nucleic acids extraction and amplifications

Total nucleic acids(DNA and RNA)were directly extracted in duplicate from2.5grams of sediments[26],then pooled and purified[27].Total RNA was purified from crude nucleic acids using Nucleospin RNA II Kit(Macherey Nagel,Du¨ren,Germany) prior to RT-PCR.Aliquots of rRNA were reverse transcribed using Quanta qScript kit according to manufacturer’s protocol (Quanta Bioscience,Gaithersburg,MD,USA).As control for DNA contamination,no amplification was obtained by PCR on RNA aliquots.All molecular experiments were carried out as previously monitored in surface cold seep sediments of the Sonora Margin[20].PCR primers and appropriate annealing tempera-tures are listed in Table S1.Sequencing of16S rRNA transcripts and their analysis including,taxonomic affiliations and phyloge-netic trees were performed as detailed in Methods S1.Automated ribosomal intergenic spacer analysis(ARISA)of the archaeal and bacterial communities and real-time(q)PCR experiments targeting various sedimentary microbial lineages(Archaea,ANME-1, ANME-2a,ANME-2c,ANME-3,Methanosarcinales,Methano-microbiales,Methanococcales,Methanobacteriales,Methanopyr-ales,MCG,MBGB,MBGD,Bacteria,Chloroflexi,Candidate division JS1,Desulfosarcina/Desulfococcus,Desulfobulbus,SEEP SRB2;Table S1)were carried out on purified DNA samples every 50cm from1mbsf to9mbsf as presented in Methods S1. Statistical tests were carried out using the software PAST[28]. Nucleic acid sequences are available in the EMBL database under the following accession numbers:HF543837–HF543861for archaeal,HF545450–HF545524for bacterial16S rRNA sequenc-es and HF935025–HF935037for mcrA gene sequences. GeoChip analysis

The GeoChip4.0microarray,containing83992oligonucleotide probes and targeting152414gene variants in401categories for different microbial functional and biogeochemical processes was monitored as previously detailed[29].Although the GeoChip was initially based on the genome of cultured microorganisms,the new generation of GeoChip has been extensively enriched with metagenome data from various environments and contains now an important number of relevant probes targeting genes from cultured and uncultured microorganisms involved in key biogeo-

chemical cycles.Total purified DNA samples were labeled then hybridized on GeoChip slides.Signal intensities were scanned and spots with signal-to-noise ratios lower than2were removed before analyses[29].The phylogenetic design of the data acquisition enabled confident assignment of metabolic capabilities to bacterial and archaeal phyla[30,31],thus dataset were sorted according to the taxonomic affiliation of the genes(Bacteria,Euryarchaeota and Crenarchaeota).Output was analyzed using the GeoChip4.0data analysis pipeline[32]and tested using the statistical software PAST[28].Relative signal intensity was normalized by the number of the probes for each indicated metabolic pathway.List of targeted genes for each category are provided in Table S2. Visualization of the bacterial and archaeal functional potential was achieved using spider dendrograms,where each arm of the plot corresponded to a metabolic pathway.The raw GeoChip dataset is available at https://www.wendangku.net/doc/ab15581403.html,/4download/.

Results

Geochemical description

The BCK1core,from the Sonora Margin sediments,showed the typical geochemical signatures of continental margin sediments (Figure1a),with a sulfate to methane transition zone(SMTZ) located around5mbsf.Sulfate pore water concentrations decreased from25mM at the sediment-water interface down to 2mM at5.5mbsf.Hydrogen sulfide concentrations were only detected in the deeper sediment layers with a maximum of32mM at5mbsf decreasing to around8mM at8mbsf.Methane pore water concentrations increased with depth reaching500m M at the bottom of the sediment core(8.5mbsf).and were positively correlated with the methanogenesis rates(Pearson correlation coefficient r=0.72,P=0.001;,45pmol/cm3/d at8.5mbsf) (Figure1d).Isotopic signature of methane was297.3%at the bottom of the core(9mbsf)and282%at1mbsf,confirming that most of methane produced was from biogenic origin and indicating that methane oxidation potentially occurred towards the sediment surface.Ammonium concentrations,likely resulting of organic matter degradation,increased with depth until reaching 2.5mM at5mbsf(Figure1b).Total organic carbon(TOC) content varied between 3.1and 4.3%(w/w)throughout the sediment with peaks at3.5,5and7.5mbsf(Figure1c).Analysis of the element composition of the pore water highlighted both a manganese reduction zone in the first meter of sediment and specific horizons(3.5,5–6,7and8mbsf)with significant enrichment of metallic elements(Fe,Al,Si,Mn,Ti)(Figure S1). These increases of metal concentrations in pore water suggest detrital terrigenous inputs in the sediment layers,as previously detected in the Guaymas Basin[33].

Microbial community structure and composition Microbial community structure variations with depth were compared from the Sonora Margin cold seep surface sediments using ARISA.The archaeal and bacterial community structures of the BCK1were significantly different from the surface sediments of both cold seep(WM14and EWM14samples[20])and outside active seepage areas(REF samples[20])as shown by clustering and ANOSIM(p,0.0008)on ARISA dataset(Figure2)Dendro-gram and Nonmetric Multidimensional Scaling(NMDS)analysis, based on Bray-Curtis similarity measure also indicated that BCK1 samples clustered according to sediment depths(1–4mbsf,4.5–6mbsf and 6.5–9mbsf).However this observation was not statistically supported by ANOSIM and seemed to rather reflect a difference in signal intensity more than in community composition.

Based on geochemical features,representative sediment depth horizons(1,4,5,7and8mbsf)were selected for the16S rRNA survey.A total of565partial16S rRNA sequences(303for Archaea and262for Bacteria)were obtained and used as a proxy for active microbial communities[34–36].Overall,statistical analysis of the microbial community structure of the samples indicated that the microbial community was nearly constant throughout the sediment core(SIMPER average similarities between paired samples above74.05%).

Archaeal16S rRNA libraries showed a very limited diversity throughout the sediment core(1-H Simpson=0.61560.08;Figure2, Figure S2,Figure S3),including three uncultivated phylotypes, mainly found in the deep biosphere:the Marine Benthic Groups B and D(MBGB,MBGD)and the Miscellaneous Crenarchaeotal Group(MCG),mainly represented by the MCG-8and MCG-10 sub-groups[37].Other groups such as South Africa Gold Mine Euryarchaeotal Group(SAGMEG),Marine Hydrothermal Vent Group(MHVG)and Terrestrial Miscellaneous Euryaechaeotal group(TMEG)were also detected in lower proportions in the deepest sediment layers.

In contrast,the bacterial16S rRNA libraries indicated a larger diversity(1-H Simpson=0.71260.06),dominated by Chloroflexi and diverse bacterial candidate divisions including JS1,OP11,OP1, OP8and OP3(Figure2,Figure S2,Figure S4).The Chloroflexi lineage included different sub-groups and most of the amplified sequences were relatives to Dehalococcoidetes or subphylum IV groups.A few Deltaproteobacteria,usually related to sulfate-reducers and hydrocarbon degraders in cold seep sediments were detected in4,5and7meters depth sediment horizons. Microbial16S rRNA gene abundance and distributions Depth distributions and relative abundance of microorganisms were analyzed every50cm by real-time PCR(Figure3).16S rRNA gene abundance of Bacteria was around10fold higher than Archaea throughout the sediment core,and decreased with depth from4610916S rRNA gene copies per gram of sediment in the top of the core to2.86108copies at the bottom.Bacterial relative abundance showed elevated concentrations in particular at5,7 and8meters below the seafloor with1.956109,1.456109and 1.1610916S rRNA gene copies g21respectively.As sequences affiliated to Chloroflexi and candidate division JS1dominated bacterial16S rRNA gene libraries,the16S rRNA genes of these groups were specifically quantified.Chloroflexi16S rRNA gene abundance was estimated by subtracting JS116S rRNA gene copy numbers from quantifications with JS1and Chloroflexi groups specific primers[38].Chloroflexi16S rRNA gene abundance appeared to mirror the bacterial distribution profile(Pearson correlation coefficient r=0.914,P,0.0001)and strongly domi-nated the bacterial community throughout the sediment core.In contrast,JS116S rRNA gene copy numbers increased with depth until reaching maximum values between2.5and5mbsf with 4.626108copies g21.No cold seep sulfate-reducing bacteria (Desulfosarcina/Desulfococcus and Desulfobulbus groups)were detected.

Total archaeal16S rRNA gene copy numbers,represented4–10%of the total number of16S rRNA gene and decreased with depth,from1.9610816S rRNA gene copies g21at1mbsf to 2.67610716S rRNA gene copies at the bottom of the sediment core.However,specific horizons(1mbsf,5mbsf,7and8mbsf) showed peaks of elevated archaeal16S rRNA gene concentrations with4.86108,16108,1.16108and6.8610716S rRNA gene copies respectively.Within the Archaea,uncultivated groups MBGD,MBGB and MCG were detected throughout the sediment core.Their distributions were correlated with the

archaeal distribution (Pearson correlation coefficient r =0.98,P ,0.0001)and no specific niche repartition was detected along the sulfate and methane concentration gradients.Assuming the same 16S rRNA copy number for each microbial lineage,MCG were fivefold less abundant than marine benthic groups except at 7mbsf with 4.8610716S rRNA gene copies g 21.Consistently with 16S rRNA library results,ANME lineages were below the detection limit (,10416S rRNA gene copies g 21)and methan-ogens were only represented by Methanosarcinales at 1mbsf with 2.4610616S rRNA gene copies g 21.

Functional gene diversity and GeoChip array

In order to investigate the ecophysiology of the microbial community associated to subsurface Sonora Margin sediments,an array targeting functional genes was used for sediments collected at selected depths (1,2.5,5,7and 8mbsf).The microarray results indicated a small but significant variation between the metabolic potential of microbial communities from each sediment horizon (ANOVA:F =5.64,P =0.002).Similarity percentages (SIMPER)and clustering analyses using Bray-Curtis similarity measure showed that the microbial communities associated with the 2.5and 5mbsf sediment horizons and the two deeper sediment horizons (7and 8mbsf)shared the greatest number of functional genes (93.3%and 91.92%similarity respectively),and that divergence between these metabolic potentials increased with sediment depth.These analyses indicated that this divergence was mainly due to the highest presence,in deepest sediment layer communities,of genes involved in hydrocarbon degradation (13%of variation)and in the upper sediment layers the predominance of genes involved in cellulose degradation (6.79%of variation,Figure 4).Using the taxonomic nature of the GeoChip probes [31,32],putative metabolic functions were sorted according to specific taxonomic ranks:Archaea (3%of the total prokaryotic signal)or Bacteria (97%)super kingdoms and Euryarchaeota or Crenarchaeota phyla.Crenarchaeota phylum was recently revised to include only thermophilic lineages,excluding lineages such as MCG and MBGB [39].However,GeoChip array was designed on the former phylogeny,thus the crenarchaeotal metabolic

pathways detected in this study are likely to include MCG and MBGB lineages.

Carbon metabolism

A large variety of bacterial genes for carbon utilization were identified (Figure 4).Genes coding for the RuBisCo,the propio-nyl-CoA/acetyl-CoA carboxylase (ppc ),the ATP citrate lyase (acl

B )and the carbon-monoxide dehydrogenase (CODH)were detected throughout the sediment core,indicating an autotrophic carbon fixation potential for both bacterial and archaeal lineages.Genes involved in heterotrophic metabolic pathways were also detected,indicating an important potential to transform a large variety of organic compounds.Bacterial genes associated with metabolic pathways for carbohydrates degradation (starch,cellu-lose,hemicellulose,chitin;lignin and pectin degradation),notably with extracellular enzyme genes,were detected in slightly higher proportion in the surface sediments.Hydrocarbon degradation pathway genes such as chnA ,involved in ethylphenol and ethylbenzene catabolism,the tut operon,involved in toluene degradation and alk genes in the alkane degradation pathway [40]were also detected in increasing proportion with depth.The ability to degrade chlorinated,aromatic,polycyclic and xenobiotic compounds were also detected for bacteria,particularly with genes involved in the superpathway of aromatic compound degradation via 2-oxopent-4enoate and in the metacleavage of aromatic compounds [41].Finally,the bacterial potential to use methylated amines was also identified throughout the sediment core.Archaeal metabolic genes for carbon utilization involved in carbohydrates and complex organic matter degradation as well as autotrophic metabolisms associated with Euryarchaeota and Crenarchaeota -related lineages were also detected.Finally,mcrA euryarchaeotal genes,involved in both methane production and anaerobic oxidation [42]were detected in increasing proportion with depth consistently with methane concentrations (Pearson correlation coefficient r =0.832,P =0.08;Figure 1e).

Sulfate and Nitrogen metabolisms

The elevated ammonium concentrations measured in the sediments suggested that nitrogen cycle might be significant

in

Figure 1.Geochemical depth profiles,putative methanogenesis activity rates and GeoChip genes detection of the sediment core BCK1.1a)Dissolved methane (grey square,m M),sulfate (white circle,mM)and sulfide (grey cross,mM)concentrations in pore waters.1b)Dissolved ammonium concentrations (mM)in pore waters.1c)Total organic carbon (TOC)content in the sediments (%w/w).1d)Methanogenesis activity rates from acetate (white circle),bicarbonate (black square)and di-methylamine (grey triangle)in the sediments (pmol/cm 3/day).1e)Relative signal intensity of the GeoChip microarray for sulfate-reduction (circle),methanogenesis (grey square),carbohydrates degradation (triangle)and hydrocarbon degradation (black square)pathways,normalized by the number of the probes for each indicated metabolic pathway.doi:10.1371/journal.pone.0104427.g001

the Sonora Margin sediments.Analyses of the functional gene array detected essential genes involved in the major pathways of the nitrogen cycle (Figure 5).Genes suggesting metabolic poten-tials for nitrogen fixation and mineralization (Glutamate dehy-drogenase and urea amidohydrolase genes),allowing nitrogen input to the microbial ecosystem,were observed in both bacterial and euryarchaeotal lineages,while nitrification genes were detected in Bacteria and Crenarchaeota .Denitrification potential was identified in Bacteria and in higher proportion in Archaea .Hydrazine oxidoreductase genes involved in the anaerobic oxidation of ammonium (anammox)were also detected through-out the sediment core and in higher proportion (1.5times)at 5mbsf.Finally,genes involved in sulfate-reduction (dsrAB,aprAB )were identified throughout the sediments and in higher intensity at 1,2.5and 5mbsf sediment horizons,which coincided with the sulfate-rich sediment layers (Figure 1).

Discussion

Microbial community structure

In this study,we document the taxonomic and functional diversity of the microbial community associated with subsurface sediments from a site adjacent (600m)to cold seep sediment sites of the Sonora Margin [19,20].Although identical molecular methods were used in both studies,the microbial diversity associated with the subsurface sediments (0.5–9mbsf)was different from the surface cold seeps (0–0.2mbsf)of the Sonora Margin.For example,anaerobic methanotrophs and associated sulfate-reducing bacteria,observed in high concentrations in the cold seep surface sediments [19,20]were not detected in subsurface sediments despite presence of a sulfate and methane transition zone.In contrast,the subsurface bacterial community was strongly dominated by members of Chloroflexi and candidate

division

Figure 2.Microbial diversity.Clustering analyses using unweighted pair-group average (UPGMA)and Bray-Curtis Similarity measure of the a)archaeal and b)bacterial community structures visualizing the ARISA dataset.Depth distribution of the c)archaeal and d)bacterial phylogenetic affiliations of the 16S rRNA-derived sequences at 1,4,5,7and 8mbsf sediment layers of BCK1.WM14(White Microbial mat),EWM14(Edge of White Microbial mat)and REF (reference outside active seepage area)samples were previously analyzed with the same material and method in Vigneron et al 2013and corresponded to archaeal community structure of the surface sediments of the Sonora Margin.TMEG,Terrestrial Miscellaneous Euryarcheotal Group;MBGD/B,Marine Benthic Group D/B;MG I,Marine Group I;MCG,Miscellaneous Crenarchaeotic Group;MHVG,Marine Hydrothermal Vent Group;Hua1,Huasco archaeal group 1;DHVE3,Deep-Sea Hydrothermal Vent Euryarchaeotal Group 3;SAGMEG,South Africa Gold Mine Euryarchaeotal Group.

doi:10.1371/journal.pone.0104427.g002

phyla (JS1,OP8,etc.),and the major archaeal lineages detected were MCG,MBGB (also known as DSAG [43])and MBGD.All these microbial populations have been frequently encountered in continental margin sediments and in the deep subsurface marine biosphere [9,10,13],but only in minor proportion in highly active ecosystems (hydrothermal vent,cold seeps)[20,44]and in low carbon environments (open ocean sediments)[45].Interestingly,no significant variation of the microbial community structure,excepted for the candidate division JS1,was detected throughout the sediment core,despite the presence of marked geochemical gradients (sulfate,methane).These results suggest that dominant microbial lineages were probably not directly involved in these biogeochemical cycles,as previously proposed for archaeal lineages [6,37].Overall,estimated cell abundance decreased with depth as commonly observed in marine sediments [3,4].In the Sonora Margin,elevated amounts of organic matter,derived from both marine production and continental inputs,sedimented in the seafloor with an estimated rate of 2mm/y [46].The accumulation of these sedimented particles led to an elevated sedimentary TOC content (3.5,4%).Distance to land,geochemical gradients and organic carbon quality and abundance can control the microbial

community structure and abundance in marine sediments [3,7,14],thus the high cellular abundance in the Sonora Margin sediments could be a consequence of the high concentrations of organic carbon.Elevated Q-PCR-based cell abundance estima-tions in the first meters of sediment could be due to higher concentrations of several electron acceptors (oxygen,nitrate,manganese and sulfate).Furthermore,significant correlations were found between TOC percentage and total Bacteria ,Chloroflexi ,candidate division JS1and MBGD cell abundance estimations below 1.5mbsf (Pearson correlation coefficients r =0.58,0.66,0.75and 0.66respectively;P ,0.04),which are consistent with reports of correlation between TOC and subsur-face microbial biomass [7,47].Likewise,fluctuations below 3mbsf of all microbial lineage cell abundances,appeared to be positively correlated with the local elementary composition of the sediments (Fe,Ti and Al,Pearson correlation coefficients r .0.67,P ,0.04;Table S3).These results clearly indicate that in subsurface margin sediments microbial communities are influenced directly or indirectly by the geochemical composition of the sediments and suggest that the microbial abundance in margin ecosystems could be enhanced by the continental detrital inputs rather than

by

Figure 3.Q-PCR estimations.Q-PCR estimation of 16S rRNA gene copy numbers per gram of sediment for a)total Bacteria and bacterial groups of Chloroflexi ,candidate division JS1and b)total Archaea and archaeal groups of Marine Benthic Group B (MBGB),D (MBGD),Miscellaneous Crenarchaeotal Group (MCG),from BCK1sediment core.Methanosarcinales were only detected at 1mbsf with 2.4610616S rRNA gene copies g 21but were not represented in the figure.ANaerobic MEthanotrophs (ANME),Desulfosarcina /Desulfococcus (DSS),Desulfobulbus (DBB)and other methanogens orders were not detected in analyzed samples.doi:10.1371/journal.pone.0104427.g003

oceanic production,as indicated the correlations with terrigenous-derived metallic elements [48,49].This result is congruent with recent model calculations in subsurface sediments,indicating that buried organic carbon is sufficient to fuel microbial communities over turnover of millions of years [50].

Organic matter degradation

Based on single cell genomics,it was recently proposed that archaeal MCG and MBGD lineages could degrade detrital organic matter [18].Moreover,genes and transcripts,involved in anaerobic metabolism of amino acids,carbohydrates and lipids have been previously detected in the deep subsurface biosphere [16,17].However,it remains unclear how the microbial commu-nity is organized to degrade the detrital inputs and which microbial processes are involved.Although the GeoChip cannot be considered to be a comprehensive array with respect to marine sediment environments,it does contain an important number of relevant probes targeting genes involved in key biogeochemical

cycles and represents an interesting approach to analyze the genomic potential in environments.The microbial metabolic potential analyzed using the GeoChip showed that the majority of the genes detected were related to various bacterial metabolic pathways for the transformation and the anaerobic degradation of simple and complex organic matter (Figure 1e).The high ammonium concentrations in these sediments could therefore be a consequence of the degradation of large amounts of organic matter by microbial communities associated to the Sonora Margin subsurface sediments.Genes associated with several metabolic pathways including extracellular and intracellular enzymes involved in the degradation and assimilation of decaying wood were detected,supporting the importance of subsurface microbial communities degrading organic matter such as plants and starch.For example,genes for transformation of lignin and complex organic aromatic substrates were also identified,notably involved in the superpathway of the aromatic compound cleavage,indicating that even the more recalcitrant wood particles could

Figure 4.Carbon-cycling methabolic pathways detected by GeoChip.Carbon-cycling metabolic pathways identified for a)Bacteria and b)Archaeal Euryarchaeota (Blue)and Crenarchaeota -related (Green)lineages at different depths for BCK1sediment core.Relative signal intensity was normalized by the number of the probes for each indicated metabolic pathway.List of targeted genes for each category are provided in Table S2.

doi:10.1371/journal.pone.0104427.g004

Figure 5.Nitrogen-cycling metabolic pathways identified at different depths for BCK1sediment cores.Bacterial metabolic pathways are not underlined while Euryarchaeota and Crenarchaeota -related pathways are underlined with solid and dotted line respectively.Relative signal intensity was normalized by the number of the probes for each indicated metabolic pathway.List of targeted genes for each category are provided in Table S2.

doi:10.1371/journal.pone.0104427.g005

potentially be degraded by the bacterial community in the Sonora Margin(Figure4a).This wood-based degradation metabolism appeared to be predominant in the upper sediment layers while hydrocarbon catabolism predominated the deeper sediment horizons.The Guaymas Basin sediments are well known to harbor various C1to C8hydrocarbon compounds such as ethane, butane,pentane and other alkanes[51].Thus the bacterial community may be able to degrade this upward migrating organic carbon source as well as sedimented particles.

Other genes implicated in metabolic pathways for carbon assimilation have also been identified,indicating that different strategies for carbon assimilation occur amongst the different bacterial lineages(Figure4a).For example,potential for degrada-tion of chlorinated compounds was present,which is congruent with previous detection of dehalogenase enzymes and dehalogena-tion activities in similar deep biosphere sediments dominated by Chloroflexi lineages[52].Degradation of chlorinated compounds derived from decaying marine phytoplankton pigments[53], suggests that in addition to terrestrial input the Sonora Margin bacterial community,(e.g.Chloroflexi members)could catabolize marine production and phytoplankton[54].This metabolic specialization,which is energetically more favorable than sulfate reduction,may also explain the overall abundance of Chloroflexi representatives in marine sediments[45].In addition,part of the bacterial community could also decompose decaying macrofauna with metabolic pathways involved in chitin and methylamine degradation.Finally,genetic potential for autotrophic metabolism was identified in both Bacteria and Archaea domains,suggesting that carbon dioxide could be either assimilated by specific microbial groups or that some subsurface microorganisms might be facultative heterotrophs,as previously suggested[5].

In contrast to bacterial lineages,the detected metabolic potential of Archaea appeared to be less diverse,maybe due to the more limited number of genes targeted by the GeoChip.Even if we could not exclude that our representation of the archaeal metabolic potential may be biased by unknown or non-targeted archaeal genes that escape to the microarray detection,various archaeal functional genes were identified.Crenarchaeotal-related lineages,likely including MCG and MBGB phyla,appeared to have the metabolic potential for complex organic carbon degradation(cellulose and aromatic polymers;Figure4b).This result is supported by single cell MCG genomes[18]and distribution[37]suggesting heterotrophic metabolisms,possibly linked to aromatic compounds degradation[55].Likewise, euryarchaeotal lineages,dominated by MBGD(95%based on Q-PCR estimations),appeared to have mainly the potential to degrade wood detrital polymers like starch,cellulose and aromatic compounds(Figure4b).Hence,MBGD members could be anaerobic and heterotrophic degraders of complex organic matter, as previously suggested[18].Resulting peptides from enzymatic degradations could be further assimilated by MBGD cells via peptidases and oligopeptide transporters,recently detected in their genome[18].

Methane and Sulfate cycles

Interestingly,the low GeoChip signal intensity for the mcrA gene,a gene coding for an enzyme involved in production and anaerobic oxidation of methane[42]was correlated with methane concentrations and methanogenesis rates measured in the sediments(Figure1).However,Q-PCR quantification and mcrA gene clone libraries(data not shown)only detected putative methane cycling Archaea related to Methanococcoides in sediments at1mbsf.Detection of these methanogens degrading noncom-petitive substrates,such as methylated amines[56]is consistent with the presence of methanogenesis from dimethylamine and the detection of euryarchaeotal genes involved in methylamine degradation(Figure4b).In deeper sediments with low methano-genesis rates(10–100fold lower than in cold seeps[57]),relative abundances of known methanogens were probably below the PCR and Q-PCR detection limits(,100016S rRNA gene copy per gram of sediment)or escape amplification due to primer deficiencies[13].As suggested by the changing d13-CH4 signature,anaerobic methanotrophs could also be present in extremely low abundance or with altered key genes that would escape molecular detection[58].These methanotrophs might be coupled directly or indirectly with sulfate-reducing Deltaproteo-bacteria,detected between4and7mbsf by16S rRNA libraries and dsrAB AprAB GeoChip probes and thereby,lead to the formation of the SMTZ in these sediments(Figure1). Nitrogen cycle

Key bacterial metabolic genes involved in the nitrogen cycle were also detected with the microarray approach in the Sonora Margin sediments(Figure5).In addition to nitrogen fixation, denitrification and anammox by bacterial communities,Eur-yarchaeota showed genetic potential for nitrogen fixation.Nitrogen assimilation is an important metabolic process for deep subsurface sediment microbial communities[5]and various members of the Euryarchaeota such as methanogenic lineages[59,60],ANME-2 [61]and ANME-1[62]were previously found to anaerobically fix nitrogen.The detection of euryarchaeotal nitrogen fixation genes in our results suggested that members of MBGD,representing 95%of the Euryarchaeota could also be diazotrophic Archaea. Nitrification(ammonium oxidation)genes(amoA)were identified as a potential metabolism in crenarchaeotal-related lineages. Although ammonium,a potential electron donor,is abundant in the Sonora Margin sediments,probably due to organic matter microbial degradation,the presence of such oxygenase enzymes in this anoxic environment remains enigmatic[47,63].It was therefore suggested that ammonium oxidation could be performed using an alternative electron acceptor[47]or that amo genes in anoxic environments could have an alternative function[64]. Consistently with the detection of nar transcripts in deep marine sediments[16],archaeal and bacterial denitrification genes were present throughout the sediment core,which could contribute to the elevated ammonium concentrations.Anaerobic ammonium oxidation was previously suggested for the nitrate origin in the deepest sediments as it could potentially be produced as a by-product of the process[16].This would be supported by the detection of the hzo genes by the GeoChip probes,as well as the previously report of anammox process in the Sonora Margin sediments[65].

Conclusion

This study clearly indicated that Sonora Margin sub-surface sediment microbial communities,probably controlled by terrige-neous inputs,are composed of deep biosphere-related microor-ganisms,distinct of the Sonora Margin surface cold seep communities.Consistently,genetic potentials for the catabolism of complex organic matters(decaying wood,macrofauna,phyto-plankton and hydrocarbon)were identified,suggesting that various heterotrophic strategies occur amongst sedimentary microbial communities.Further specific measurements of rates of degrada-tion these different substrates could confirm these results and lead to a better understanding of the biochemical processes driving subseafloor microbial communities.

Supporting Information

Figure S1Geochemical depth profiles of:total iron,aluminum, potassium,manganese,total sulfur,silica and titanium concentra-tions in the unfiltered pore waters of the BCK1core.The blue shade represent important changes in elemental composition profiles.

(TIF)

Figure S2Rarefaction curves for A)archaeal and B)Bacterial 16S rRNA gene libraries.

(TIF)

Figure S3Maximum Likelihood phylogenetic tree of the archaeal16S cDNA sequences amplified from sections1,4,5,7 and8mbsf(labeled S1,S4,S5,S7and S8respectively)of the BCK1sediment core.Phylogenetic tree was performed using RAxML7.2.8.and GTRCAT model approximation with1000 replicates.Only bootstrap values up to70%are shown.Only one representative sequence(.97%identical)per sediment horizon is shown.Number in brackets shown the number of clones analyzed from RNA clone libraries.MBG-D/B,Marine Benthic Group D/ B;TMEG,Terrestrial Miscellaneous Euryarcheotal Group; MCG,Miscellaneous Crenarchaeotal Group;MHVG,Marine Hydrothermal Vent Group;SAGMEG,South Africa Gold Mine Euryarchaeotal Group.

(TIF)

Figure S4Maximum Likelihood phylogenetic tree of the bacterial16S cDNA sequences amplified from sections1,4,5,7 and8mbsf(labeled S1,S4,S5,S7and S8respectively)of the BCK1sediment core.Phylogenetic tree was performed using RAxML7.2.8.and GTRCAT model approximation with1000 replicates.Only bootstrap values up to70%are shown.Only one representative sequence(.97%identical)per sediment horizon is shown.Number in brackets shown the number of clones analyzed from RNA clone libraries.

(TIF)

Table S1Primer sets and annealing temperatures used for real-time PCR of16S rRNA gene.

(DOC)

Table S2Details of GeoChip-targeted genes,related proteins and processes corresponding to each identified metabolic path-ways.

(DOC)

Table S3Correlation statistical tests and associated P values for microbial lineages and elementary composition of the sediment pore-waters.

(DOC)

Methods S1Detailed methods for methanogenesis activity measurements,gene library constructions and phylogenetic affiliations,Q-PCR and ARISA experiments.

(DOCX)

Acknowledgments

We would like to thank the crew of L’Atalante for their help on core preparation,Franc?ois Harmegnies for temperature gradient recovery and Olivier Rouxel for geochemical interpretation.

Author Contributions

Conceived and designed the experiments:AV LT JP JZ.Performed the experiments:AV PC EGR PP JCC NC MCC BAC JVN ZH.Analyzed the data:AV PC EGR BAC JVN ZH.Contributed reagents/materials/ analysis tools:NC MCC.Contributed to the writing of the manuscript:AV PC EGR JP JZ LT AG.

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脐带干细胞综述

脐带间充质干细胞的研究进展 间充质干细胞（mesenchymal stem cells,MSC S ）是来源于发育早期中胚层 的一类多能干细胞[1-5]，MSC S 由于它的自我更新和多项分化潜能，而具有巨大的 治疗价值 ,日益受到关注。MSC S 有以下特点：(1)多向分化潜能，在适当的诱导条件下可分化为肌细胞[2]、成骨细胞[3、4]、脂肪细胞、神经细胞[9]、肝细胞[6]、心肌细胞[10]和表皮细胞[11, 12]；(2)通过分泌可溶性因子和转分化促进创面愈合；(3) 免疫调控功能，骨髓源（bone marrow ）MSC S 表达MHC-I类分子，不表达MHC-II 类分子，不表达CD80、CD86、CD40等协同刺激分子，体外抑制混合淋巴细胞反应，体内诱导免疫耐受[11, 15]，在预防和治疗移植物抗宿主病、诱导器官移植免疫耐受等领域有较好的应用前景；(4)连续传代培养和冷冻保存后仍具有多向分化潜能，可作为理想的种子细胞用于组织工程和细胞替代治疗。1974年Friedenstein [16] 首先证明了骨髓中存在MSC S ，以后的研究证明MSC S 不仅存在于骨髓中，也存在 于其他一些组织与器官的间质中：如外周血[17]，脐血[5]，松质骨[1, 18]，脂肪组织[1]，滑膜[18]和脐带。在所有这些来源中，脐血(umbilical cord blood)和脐带(umbilical cord)是MSC S 最理想的来源，因为它们可以通过非侵入性手段容易获 得，并且病毒污染的风险低，还可冷冻保存后行自体移植。然而，脐血MSC的培养成功率不高[19, 23-24]，Shetty 的研究认为只有6%，而脐带MSC的培养成功率可 达100%[25]。另外从脐血中分离MSC S ，就浪费了其中的造血干/祖细胞(hematopoietic stem cells/hematopoietic progenitor cells,HSCs/HPCs) [26, 27]，因此，脐带MSC S (umbilical cord mesenchymal stem cells, UC-MSC S )就成 为重要来源。 一．概述 人脐带约40 g, 它的长度约60–65 cm, 足月脐带的平均直径约1.5 cm[28, 29]。脐带被覆着鳞状上皮，叫脐带上皮，是单层或复层结构，这层上皮由羊膜延续过来[30, 31]。脐带的内部是两根动脉和一根静脉，血管之间是粘液样的结缔组织，叫做沃顿胶质，充当血管外膜的功能。脐带中无毛细血管和淋巴系统。沃顿胶质的网状系统是糖蛋白微纤维和胶原纤维。沃顿胶质中最多的葡萄糖胺聚糖是透明质酸，它是包绕在成纤维样细胞和胶原纤维周围的并维持脐带形状的水合凝胶，使脐带免受挤压。沃顿胶质的基质细胞是成纤维样细胞[32]，这种中间丝蛋白表达于间充质来源的细胞如成纤维细胞的，而不表达于平滑肌细胞。共表达波形蛋白和索蛋白提示这些细胞本质上肌纤维母细胞。 脐带基质细胞也是一种具有多能干细胞特点的细胞，具有多项分化潜能，其 形态和生物学特点与骨髓源性MSC S 相似[5, 20, 21, 38, 46]，但脐带MSC S 更原始，是介 于成体干细胞和胚胎干细胞之间的一种干细胞，表达Oct-4, Sox-2和Nanog等多

全国翻译价格

全国翻译价格 关于全国各地区翻译价格我们根据客户的不同需求和具体情况，提供多种等级和特色的翻译服务，供客户选择：（注：以下报价均为参考价格，精确报价将根据稿件内容的难度、技术处理的复杂程度和时限要求的缓急而定。

品质控制 坚持高端定位是外语通翻译的核心要素，追求高品质翻译需要译员具备深厚的语言功底和专业背景知 识，更需要严格的质量控制体系来管理这一过程： 外语通六阶梯质量控制体系 第一阶梯：译文评估承接 分析稿件性质、用途要求、商务背景、专业术语、数量和交稿时间等，确定是否有100%的把握承接， 否则坚决放弃，以免因质量或交稿时间耽误客户和影响品牌形象。 第二阶梯：专业译员翻译 专业背景的译员只专注于一个行业领域的精准翻译，项目经理根据译文评估，从外语通全球译员库中 分析挑选多名此行业的专业译员成立项目组，统一专业术语和标准，协同翻译。 第三阶梯：翻译质量监控 项目经理监控翻译进展，每日集中疑难词汇，请签约专家释疑。每日抽查译文质量，及时解决译文质 量问题。 第四阶梯：译文校对排版 汇总所有译文，查错补漏，进一步统一术语，按原文进行排版，形成完整初稿。 第五阶梯：专家译审修改 专家译审对翻译初稿进行翻译准确性审核，确保译稿忠于原文，专业词汇纯正地道。 六阶梯：外籍母语润色第 在华外籍翻译（外译中稿件由中文功底深厚的编辑）对译稿的语法、词汇进行修正和润色，确保译稿 纯正、地道，达到母语品质。 外语通翻译严格执行《ISO译文质量体系》，《翻译质量国家标准GB/T 19682-2005》： 译文质量标准Ⅲ类通用笔译Ⅱ类专业笔译Ⅰ类高级笔译译文用途内容概要、参考资料一般文件和材料正式文件、法律文书、出版物错漏译率小于5‰小于2‰0‰ 译员经验3年以上5年以上8年以上 译员学历硕士以上硕士以上硕士以上 行业背景常识业内资深 海外背景无/短期中期长期 译文校对有有有 专家译审无有有 母语润色无无有 译文排版简单排版详细排版出版级别

脐带血造血干细胞库管理办法(试行)

脐带血造血干细胞库管理办法（试行） 第一章总则 第一条为合理利用我国脐带血造血干细胞资源，促进脐带血造血干细胞移植高新技术的发展，确保脐带血 造血干细胞应用的安全性和有效性，特制定本管理办法。 第二条脐带血造血干细胞库是指以人体造血干细胞移植为目的，具有采集、处理、保存和提供造血干细胞 的能力，并具有相当研究实力的特殊血站。 任何单位和个人不得以营利为目的进行脐带血采供活动。 第三条本办法所指脐带血为与孕妇和新生儿血容量和血循环无关的，由新生儿脐带扎断后的远端所采集的 胎盘血。 第四条对脐带血造血干细胞库实行全国统一规划，统一布局，统一标准，统一规范和统一管理制度。 第二章设置审批 第五条国务院卫生行政部门根据我国人口分布、卫生资源、临床造血干细胞移植需要等实际情况，制订我 国脐带血造血干细胞库设置的总体布局和发展规划。 第六条脐带血造血干细胞库的设置必须经国务院卫生行政部门批准。 第七条国务院卫生行政部门成立由有关方面专家组成的脐带血造血干细胞库专家委员会（以下简称专家委

员会），负责对脐带血造血干细胞库设置的申请、验收和考评提出论证意见。专家委员会负责制订脐带血 造血干细胞库建设、操作、运行等技术标准。 第八条脐带血造血干细胞库设置的申请者除符合国家规划和布局要求，具备设置一般血站基本条件之外， 还需具备下列条件： （一）具有基本的血液学研究基础和造血干细胞研究能力； （二）具有符合储存不低于1 万份脐带血的高清洁度的空间和冷冻设备的设计规划； （三）具有血细胞生物学、HLA 配型、相关病原体检测、遗传学和冷冻生物学、专供脐带血处理等符合GMP、 GLP 标准的实验室、资料保存室； （四）具有流式细胞仪、程控冷冻仪、PCR 仪和细胞冷冻及相关检测及计算机网络管理等仪器设备； （五）具有独立开展实验血液学、免疫学、造血细胞培养、检测、HLA 配型、病原体检测、冷冻生物学、 管理、质量控制和监测、仪器操作、资料保管和共享等方面的技术、管理和服务人员； （六）具有安全可靠的脐带血来源保证； （七）具备多渠道筹集建设资金运转经费的能力。 第九条设置脐带血造血干细胞库应向所在地省级卫生行政部门提交设置可行性研究报告，内容包括：

结婚证(英文翻译模板)

THE PEOPLE’S REPUBLIC OF CHINA MARRIAGE CERTIFICATE Ministry of Civil Affairs of the People’s Republic of China (The Special Seal of Management of Marriage Certificate) This application conforms to the Marriage Law of the People’s Republic of China. We hereby give them the permission to register and issue this marriage certificate. Authorized by: Special Seal for Marriage Register of XX District, Civil Affairs Bureau of Beijing Undertaker (signature): XX XXX Certificate Holder: X XX Registration Date: xx xxx 2009 Marriage Certificate No.: Jing Chao Jie Zi 050920381 Note: Name: X XX Sex: Female Nationality: Chinese Date of Birth: xx xxx 1980 ID No.: 110105xxxxxxxxxxxx Name: X X Sex: Male Nationality: Chinese Date of Birth: xx xxx 1978 ID No.: 120104xxxxxxxxxxxx

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翻译公司收费标准 1.客户需要翻译的目标语言的普遍性和稀缺性可能导致非常 不同的费用。英语比较普遍，需求大，市场专业的英语翻译人 才也很多，翻译公司无论是从降价到抢占市场，还是成本核算 来考虑，英语收费都比较合理和透明。 其他诸如法语、德语、日语、俄语排在第二梯队，翻译公司收 费标准一般都是200-280元，视稿件专业度和数量略有调整； 意大利，西班牙，越南，泰文等东南亚语种已经接近稀有语 种了，翻译报价至少300元千字起。 2.根据翻译项目类型 常见的翻译方法主要包括翻译翻译、同声传译、本地翻译、口译翻译等，翻译项目自然是不同的收费。 3.根据翻译项目时长 这一时期的持续时间主要是指项目长度：同声传译、会议翻译、商务洽谈、双语主持人、口译、护送翻译、展览翻译，当然，视频翻译、音频翻译按时间计算的时间和会议类型是一个重要因素，是翻译时间决定翻译价格的一个重要因素。 4.根据翻译项目字数

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结婚证英文翻译模板

The People’s Republic of China Marriage Certificate Marriage Certificate of Ministry of Civil Affairs of the People’s Republic of China (seal) Under the supervision of Ministry of Civil Affairs of the People’s Republic of China Your Marriage application is in accordance with the regulations of <> and is allowed to register, and this certificate is thus issued. Issued Organ: XXX Marriage Registrar: XXX (signature) (Photo) Certificate Holder: xxx Registration Date: xx.xx, xx Marriage Certificate No.: XXXX Remark: The original Marriage Certificate is lost, this is a renewed one. xx/xx/xx Name: xxx Sex: Male Nationality: China Date of Birth: xx/xx/xx ID Card No.: xxxxxxxxx Name: xxx Sex: Female Nationality: China Date of Birth: xx/xx/xx ID Card No.: xxxxxxxxxxxx As requested by marriage law, the dual parities of man and woman to be married must personally come for the marriage registration. If they are in accordance with the marriage law, they will be registered and issued the marriage certificate. Once they have obtained marriage certificate, they are confirmed for conjugal relationship.

卫生部办公厅关于印发《脐带血造血干细胞治疗技术管理规范(试行)

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脐带血造血干细胞 治疗技术管理规范（试行） 为规范脐带血造血干细胞治疗技术的临床应用，保证医疗质量和医疗安全，制定本规范。本规范为技术审核机构对医疗机构申请临床应用脐带血造血干细胞治疗技术进行技术审核的依据，是医疗机构及其医师开展脐带血造血干细胞治疗技术的最低要求。 本治疗技术管理规范适用于脐带血造血干细胞移植技术。 一、医疗机构基本要求 （一）开展脐带血造血干细胞治疗技术的医疗机构应当与其功能、任务相适应，有合法脐带血造血干细胞来源。 （二）三级综合医院、血液病医院或儿童医院，具有卫生行政部门核准登记的血液内科或儿科专业诊疗科目。 1.三级综合医院血液内科开展成人脐带血造血干细胞治疗技术的，还应当具备以下条件： （1）近3年内独立开展脐带血造血干细胞和（或）同种异基因造血干细胞移植15例以上。 （2）有4张床位以上的百级层流病房，配备病人呼叫系统、心电监护仪、电动吸引器、供氧设施。 （3）开展儿童脐带血造血干细胞治疗技术的，还应至少有1名具有副主任医师以上专业技术职务任职资格的儿科医师。 2.三级综合医院儿科开展儿童脐带血造血干细胞治疗技术的，还应当具备以下条件：

英文合同翻译价格 英文合同翻译需要多少钱

英文合同翻译价格英文合同翻译需要多少钱 在企业的经营过程中，有时候可能会涉及到翻译这个问题，但是一般的小企业并没有专门的人去做这件事情，大部分都是外包。那么对于企业来讲，翻译一份英文合同需要多少钱呢？作为浙江省最大的翻译公司，以琳翻译就在这里为大家解读一下。 一般来讲，翻译这项服务都是以字数来计价的，市场上的一般的价格是50-80元/千字，这是一个基本的价格。但是不同的公司的专业性质不一样的话，所给出的价格也是不一样的。对于公司的衡量标准来讲，影响价格的因素主要有：公司的资历、翻译人员的专业性、翻译文件的种类、难度等。所以，如果你需要去找翻译公司去服务，那么就需要考虑这些方面的东西。而对于合同这种文件，对于公司来讲是十分重要的，所以也需要去找专业的公司去进行翻译，如果是找一个资质不够的公司或者团队，那么就可能产生一些意想不到的问题，从而影响到公司的最终利益。 下面，我们来看看以琳翻译给出的翻译的价格。 从上面的价格可以看出，以琳翻译给出的价格是高于一般市场上的价格的，最低级别的翻译是160元/千字，然后分为A、B、C三级。C级译稿为普通中籍译员+中籍译员审核，满足客户对译文的普通要求。这是对于一般的合同而言的，但是如果是部分专业性质较强或者要求比较高的译文的话，那么可以选择更高级别的翻译，当然价格还是相对比较高的。 那么以琳翻译的资质是怎么样呢？我们再来看一下。 杭州以琳翻译有限公司是浙江省最大的实体翻译公司、中国翻译协会单位会员、美国翻译协会会员、全国翻译专业硕士研究生教育实习基地、西博会指定合作伙伴、以琳杭州翻译公司翻译团队成员均具有五年以上专业翻译、项目管理经验，绝大部分成员具有十年以上行业翻译经验。翻译服务涵盖英语、法语、韩语、日语、德语、俄语、西班牙语、葡萄牙语、

新版、旧版结婚证英文翻译模板.(优选)

精品word. 1 / 5 Ministry of Civil Affairs of the People ’s Republic of China (seal) Supervised by the Ministry of Civil Affairs of the People ’s Republic of China The application for marriage is in accordance with the Marriage Law of the People ’s Republic of China and allowed to register. Hereby this certificate is issued. Registration Authority: Department of Civil Affairs of XXX City XXXXXXX Province (special seal for marriage registration) Marriage Registrar: (signature) (unclear )

精品word. 2 / 5 Certificate Holder XXXXXXXX Registration Date xxx. xxth, xxxx Marriage Certificate Zi No. XXXXXXXXX Remark No Name Xxx xxxxxx Sex Male Nationality China Date of Birth xxx,xx 19xx ID card No. 0123456789900000000 Name Xxx xxxxxx Sex Female Nationality China Date of Birth xxx,xx 19xx ID card No. 0123456789900000000

专业英文翻译中文收费标准

精诚英语翻译报价50-80元千字（市场价格100左右 精诚英语翻译工作室是由众多英语方面精英组成的翻译团队,一直致力于为广大中小企业和个人提供专业低价中英文翻译服务。价格是我们永远的优势！！！！最低价格支付宝担保交易，让你省钱又放心接受试译！！自信源于专业可以百度搜索精诚英语翻译找到我们 选择我们的理由：可以百度搜索精诚英语翻译找到我们 1.保证价格最低，团队网络化运作，无需经营成本，可以通过低价让利于客户。（有些客户看到这么低的价格还不敢相信，但是对于我们来说是完全可以接受的。） 2.保证准时、保密、准确 3.接受淘宝交易，让您没有任何担忧。 4.长期翻译经验，保证质量让您满意。 龚如心遗产案虽然告一段落，「遗产」二字仍然成为近日香港的焦点。新春期间，民政事务局局长曾德成表示，政府将展开全港非物质文化遗产首期普查，希望市民为遗产清单提出建议。 「遗产」是「资产」？ 近五、六年间，香港对保护本地小区和文化传统的意识高涨，现在政府带头要列一个「非物质文化遗产」清单，理应是很受欢迎之举。不过普查尚未展开，就引来学者争议，其中单是「非物质文化遗产」这个译名，就引起不少误会。 「非物质文化遗产」的原文是intangible cultural heritage（英文）或patrimoine culturel immateriel （法文），是联合国在1997年以尊重多元文化为大原则而提出的概念，并由联合国教科文组织制定「保护非物质文化遗产公约」，2006年生效。 「非物质文化遗产」是中国大陆的翻译，香港有学者不约而同就「非物质」和「遗产」二字提出质疑。香港城市大学中国文化中心主任郑培凯早在2005年就大声疾呼译名不妥。他认为原文heritage/patrimoine的意义是「传承」而非资产，不容易引发出财产的概念。而现在约定俗成译作「遗产」，容易令人觉得祖宗留下的东西，是可以变卖和投资的生财工具，与联合国提出的文化传承精神背道而驰。郑教授认识，正确的译名是「非物质文化承继」或「非实物文化传承」。另一位民俗学研究者陈云进一步指出，intangible「乃触摸不到的事，无形无相之事」，应用「精神价值」代之，「非物质」有消灭了精神之嫌，所以中国人应堂堂正正将之翻译为「无形文化传承」。 姗姗来迟的「遗产」 不论是「非物质」还是「无形」，「遗产」还是「承传」，即使公约成员国中国曲译甚至错译，香港特区政府还是只能照单全收。而且，随之而来的不止是字面的斟酌，而是「遗产」的搜寻和管理问题。 中国自2005年起，就开始非物质文化遗产（由于这个名称已约定俗成，故下文仍沿用之，并简称为「非遗」）普查，并陆续列出清单。香港也在翌年提出编制非遗清单，但却延至去年才聘专家普查，估计最快要2012年才完成。 非遗普查尚未展开，在国家文化部的再三邀请（或是说催促？）下，去年九月，香港终于申请将长洲太平清醮、大澳端午游涌、大坑舞火龙和香港潮人盂兰胜会列为第三批国家级非遗，预计今年六月有结果。 其实香港已错过了2006和2008年首批和第二批的申报机会，所以，至目前为止，在中国的文化版图上，香港是唯一没有任何有形和无形「遗产」的主要城市／特区，就连比邻的澳门也凭神像雕刻工艺获得2008年国家级非遗之「奖项」。 有人说非遗不过是人有我有，纯粹锦上添花；也有人说，中国在维护主权和领土完整的概念下，又怎能在文化层面少了香港一席？香港能够「出产」一个非遗，中国在全球的文化图谱中就多一个筹码。 姑勿论背后原因为何，由于「保护非物质文化遗产公约」也适用于香港，香港特区政府就有责任找出和保护濒危失传、与社会关系密切及具香港独特性的文化传统。现在起步虽迟，但为时未晚。 「遗产」的管理问题 不过，既然政府要展开普查，另一个问题来了。民间传统应该是属于民间的，并由民间自行发展，还是属于官方，由政府承担保护与管理？ 据政府委聘负责首期普查的香港科技大学华南研究中心主任廖迪生表示，政府至今仍未有任何政策配合或承诺给予全面的保护，所以，即使清单出炉，有些遗产仍有可能难逃「破产」的命运。他强调制作非遗清单只是第一步，更重要的是如何保护这些项目。 再问民政事务局，曾德成局长除了曾向立法会议员表示，制定清单是向国家文化部申请列为国家级非遗的第一步，进而再向联合国教科文组织提出申报为世界非物质文化遗产，他所提出的，就是以遗产作招徕吸引外地游客，「以提升香港作为旅游目的地的吸引力」。 这才是令人担心的地方。「非遗」这个金漆招牌在中国许多地方都有点石成金之效。戴上这个冠冕，民俗文化很容易沦为生财工具、游客的消费品，连婚嫁仪式也可用来表演，完全违背了保护非遗的原意。曾德成之言，是否意味着香港也要跟着祖国一起走上同一条路？

卫生部关于印发《脐带血造血干细胞库设置管理规范(试行)》的通知

卫生部关于印发《脐带血造血干细胞库设置管理规范(试行)》的通知 发文机关：卫生部(已撤销) 发布日期： 2001.01.09 生效日期： 2001.02.01 时效性：现行有效 文号：卫医发(2001)10号 各省、自治区、直辖市卫生厅局： 为贯彻实施《脐带血造血干细胞库管理办法（试行）》，保证脐带血临床使用的安全、有效，我部制定了《脐带血造血干细胞库设计管理规范（试行）》。现印发给你们，请遵照执行。 附件：《脐带血造血干细胞库设置管理规范（试行）》 二○○一年一月九日 附件： 脐带血造血干细胞库设置管理规范（试行） 脐带血造血干细胞库的设置管理必须符合本规范的规定。 一、机构设置 （一）脐带血造血干细胞库（以下简称脐带血库）实行主任负责制。 （二）部门设置 脐带血库设置业务科室至少应涵盖以下功能：脐带血采运、处理、细胞培养、组织配型、微生物、深低温冻存及融化、脐带血档案资料及独立的质量管理部分。 二、人员要求

（一）脐带血库主任应具有医学高级职称。脐带血库可设副主任，应具有临床医学或生物学中、高级职称。 （二）各部门负责人员要求 1．负责脐带血采运的人员应具有医学中专以上学历，2年以上医护工作经验，经专业培训并考核合格者。 2．负责细胞培养、组织配型、微生物、深低温冻存及融化、质量保证的人员应具有医学或相关学科本科以上学历，4年以上专业工作经历，并具有丰富的相关专业技术经验和较高的业务指导水平。 3．负责档案资料的人员应具相关专业中专以上学历，具有计算机基础知识和一定的医学知识，熟悉脐带血库的生产全过程。 4．负责其它业务工作的人员应具有相关专业大学以上学历，熟悉相关业务，具有2年以上相关专业工作经验。 （三）各部门工作人员任职条件 1．脐带血采集人员为经过严格专业培训的护士或助产士职称以上卫生专业技术人员并经考核合格者。 2．脐带血处理技术人员为医学、生物学专业大专以上学历，经培训并考核合格者。 3．脐带血冻存技术人员为大专以上学历、经培训并考核合格者。 4．脐带血库实验室技术人员为相关专业大专以上学历，经培训并考核合格者。 三、建筑和设施 （一）脐带血库建筑选址应保证周围无污染源。 （二）脐带血库建筑设施应符合国家有关规定，总体结构与装修要符合抗震、消防、安全、合理、坚固的要求。 （三）脐带血库要布局合理，建筑面积应达到至少能够储存一万份脐带血的空间；并具有脐带血处理洁净室、深低温冻存室、组织配型室、细菌检测室、病毒检测室、造血干/祖细胞检测室、流式细胞仪室、档案资料室、收/发血室、消毒室等专业房。 （四）业务工作区域应与行政区域分开。

英文翻译价格

英文翻译价格 根据以英文作为母语的人数计算，英文是最多国家使用的官方语言，英语也是世界上最广泛的第二语言，也是欧盟，最多国际组织和英联邦国家的官方语言之一。但仅拥有世界第二位的母语使用者，少于标准汉语。上两个世纪英国和美国在文化、经济、军事、政治和科学上的领先地位使得英语成为一种国际语言。如今，许多国际场合都使用英语做为沟通媒介。英语也是与电脑联系最密切的语言，大多数编程语言都与英语有联系，而且随着网络的使用，使英文的使用更普及。英语是联合国的工作语言之一。 为了方便大家了解英文翻译价格，小编在目前汇集最多翻译团队的高校译云上面获得了不同翻译精英团队所展示的价格。 暨南大学翻译中心：中英---普稿---150---千字英中---普稿---250---千字 武汉理工大学-外国语学院MTI翻译中心 ：中英互译中英130-150 英中100-130 华中科技大学-翻译研究中心 ：中英互译中英120-150 英中100-120 湖南科技大学MTI中心：中英---普稿---150---千字 上海师大外国语学院翻译中心： 中英---普稿---200元---千字英语普通文本译成汉语---120元---千字西南大学翻译中心：中英---普稿----300---千字英中---普稿---200---千字 上海理工大学MTI翻译中心：中英---普稿---100---千字 南京财经大学外国语学院翻译研究中心：中英---普稿---100---千字 一般英文翻译价格是是在100—300元每千字，根据译员质量、翻译内容、需要的时间等都会有一定的波动，所以以上价格供大家参考，具体的可以准备好稿件了去问，这样会更加准确一些。

新版结婚证英文翻译模板

命运如同手中的掌纹，无论多曲折，终掌握在自己手中。 你今天的日积月累，终会变成别人的望尘莫及。 11 Ministry of Civil Affairs of the People ’s Republic of China (seal) Supervised by the Ministry of Civil Affairs of the People ’s Republic of China The application for marriage is in accordance with the Marriage Law of the People ’s Republic of China and allowed to register. Hereby this certificate is issued. Registration Authority: Civil Affairs Bureau of XXX District, XXX City (special seal for marriage registration) Marriage Registrar: XXX (signature)

命运如同手中的掌纹，无论多曲折，终掌握在自己手中。 你今天的日积月累，终会变成别人的望尘莫及。 22 Certificate Holder XXXXXXXX Registration Date xxx. xxth, xxxx Marriage Certificate No. XXXXXXXXX Remark No Name Xxx xxxxxx Sex Male Nationality China Date of Birth xxx,xx 19xx ID card No. 0123456789900000000 Name Xxx xxxxxx Sex Female Nationality China Date of Birth xxx,xx 19xx ID card No. 0123456789900000000

脐带血间充质干细胞的分离培养和鉴定

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胎盘干细胞与脐带血干细胞的区别

胎盘干细胞与脐带血干细胞的区别 干细胞是一类具有自我复制和多向分化能力的原始的未分化的细胞，它可以向多种类型细胞分化，并具有相应的功能，可以用来修复和替代受到损伤，病变的组织和器官。目前主要的成体干细胞来源有胎盘来源、脐带来源、脐带血来源和骨髓来源的干细胞，本文主要介绍一下胎盘来源的干细胞和脐带血来源的干细胞的区别： 1、分离部位不同：胎盘干细胞是从胎盘组织中分离提取的干细胞，脐带血干细胞是从脐带里面血液中分离提取的干细胞。 2、种类不同：胎盘中的干细胞主要指的是间充质干细胞，而脐带血干细胞主要指的是造血干细胞。 3、分化能力不同：1）胎盘干细胞分化能力强，在特定的诱导条件下可以分化成血管干细胞、神经干细胞、肝干细胞等多种类型的干细胞，从而修复受损和病变的组织和器官2）脐带血干细胞可以在体内向红细胞、血小板等各种血液细胞分化。 4、数量不同：1）胎盘体积大，从中提取的干细胞数量丰富，并可以在体外培养扩增，扩增培养的子细胞数量达十亿个，可以供成人多次使用2）脐带血干细胞的数量要根据抽取的脐带血多少而定，不可以在体外培养扩增，一份脐带血干细胞可以供40kg以下患者一次使用。 5、治疗使用：1）胎盘干细胞目前在治疗脑瘫、糖尿病、肝硬化、心血管疾病等诸多疾病都显示了良好的效果2）脐带血干细胞可以治疗白血病，再生障碍性贫血等血液系统疾病。不过对于儿童白血病多以先天性为主，所以对于这种情况，自己的脐带血干细胞是不能使用的，需要配型使用捐献的脐血干细胞。 6、配型方面：二者自体使用都不需要配型，如果异体使用，胎盘干细胞由于免疫源性低的特点，所以配型成功率非常高，有血缘关系的亲属都可以使用；脐带血干细胞与父母配型有1/2的几率，与兄弟姐妹有1/4的几率。