Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) are used in a variety of consumer products and have been detected worldwide in human blood. Recent studies mainly of highly exposed populations have indicated that PFOA and PFOS may affect serum cholesterol levels, but the magnitude of the effect may be inconsistent across exposure levels. The aim of the present cross-sectional study was to investigate the association between plasma PFOA and PFOS and total cholesterol in a general, middle-aged Danish population. The study population comprised 753 individuals (663 men and 90 women), 50–65 years of age, nested within a Danish cohort of 57,053 participants. Blood samples were taken from all cohort members at enrolment (1993–1997) and stored in a biobank at -150°C. Plasma levels of PFOA and PFOS and serum levels of total cholesterol were measured. The associations between plasma PFOA and PFOS levels and total cholesterol levels were analysed by generalized linear models, both crude and adjusted for potential confounders. We observed statistically significant positive associations between both perfluorinated compounds and total cholesterol, e.g. a 4.4 [95% CI  =  1.1–7.8] higher concentration of total cholesterol (mg/dL) per interquartile range of PFOA plasma level. Sex and prevalent diabetes appeared to modify the association between PFOA and PFOS, respectively, and cholesterol. In conclusion, this study indicated positive associations between plasma PFOA and PFOS levels and total cholesterol in a middle-aged Danish population, although whether the observed pattern of results reflects a causal association is unclear. Perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) are synthetically produced perfluorinated chemicals (PFCs) known for their antiwetting and surfactant properties. They are widely used in commercial applications such as nonstick cookware, waterproof, breathable textiles and protective coatings for paper, food packing materials, and carpetsPFOA and PFOS have in some studies been positively associated with serum lipid levels such as cholesterol, which may increase the risk of cardiovascular diseases. Most human studies investigating the association between PFCs and cholesterol have been carried out in populations with high PFC levels in blood, such as occupationally exposed workers or highly exposed communities  50–65 years of age, born in Denmark and with no previous cancer diagnosis, were enrolled in the prospective Danish Diet, Cancer and Health (DCH) cohort 30 mL non-fasting blood samples were drawn from each participant at enrolment, spun and divided into 6×1 mL of plasma, 4×1 mL of serum and 2×1 mL of erythrocytes and 2×1 mL of buffy coat and stored at -150°C. For the present study we used the PFOA and PFOS plasma levels that were measured in the previous study L) for the method was 1.0 ng/mL for both PFCs. In our analysis, PFOS was measured above L in all samples and two PFOA values were below the L and were assigned the value 0.71 ng/mL, according to the formula L/√2 for replacement of non-detectable values Concentrations of PFOA and PFOS in plasma were determined using high performance liquid chromatography/tandem mass spectrometry at the 3M Toxicology Laboratory, using the methods described by Ehresman et al. Non-fasting total cholesterol (mmol/L) was determined in whole blood using a Lipotrend® C device with Lipotrend test strips (Boehringer Mannheim). Lipotrend® C is a reflectance photometer for the determination of total cholesterol from capillary blood or plasma in the range of 100–500 mg/dl (2.6–13.0 mmol/L). The sample was applied onto the test strip by means of a plastic capillary. Cholesterol determination was automatically begun upon correct insertion of the test strip into the instrument. The optical system measures the color intensity of the test strip’s reaction zone at two different times, and the difference is converted into the cholesterol concentration using the Lipotrend® C software and shown in the display. For our analyses, the SI unit (mmol/L) was converted to mg/dL by multiplying by 38.67.2), smoking status , alcohol intake (g/day), egg intake (g/day), animal fat intake (g/day) and physical activity . Some of the adjustment variables were selected based on what is previously shown to be associated with PFC levels in this population The association between PFOA and PFOS plasma levels and total cholesterol were analyzed by generalized linear models. Linearity of the associations between PFCs and cholesterol was verified by linear splines, and the primary analyses were conducted using PFOA and PFOS as continuous variables. In addition, for visualization of the results we investigated cholesterol levels in relation to categorization of PFOA and PFOS concentrations into eight groups, with approximately 100 persons in each exposure group, using the lowest exposure group as reference. Regression analyses were performed (with adjustment for sex only) and adjusted regression analyses were performed with adjustment for sex, age, years of school attendance , body mass index . Mean plasma PFOA and PFOS levels were 7.1 ng/mL and 36.1 ng/mL, respectively. Men had significantly higher levels of both compounds with a difference of 1.5 ng/mL (95% CI: 0.9–2.2) for PFOA and a difference of 6.1 ng/mL (95% CI: 3.0–9.2) for PFOS.Linearity of the associations between PFOA and PFOS and total cholesterol was verified in spline analyses showing that the associations did not deviate significantly from linearity .The distributions of PFOA and PFOS values are shown in Sex significantly modified the association between PFOA and cholesterol, with a stronger association among women, and prevalent diabetes significantly modified the association between PFOS and cholesterol, with a stronger association among diabetics. BMI did not modify the associations .Risk factors for high cholesterol level include diet high in saturated animal fats, low physical activity, and a family history of the condition, but increasing evidence indicates that environmental chemicals may also contribute. In this study, we found positive associations between plasma PFOA and PFOS levels and total cholesterol in a general, middle-aged Danish population. Total cholesterol levels were about 4 mg/dL higher per IQR of plasma PFOA and PFOS levels, and the association seemed to follow a dose-response pattern.The mechanism by which perfluorinated compounds are related to cholesterol in humans is unknown, but the finding of positive associations between perfluorinated compounds and cholesterol levels contradicts what would be expected from animal studies. In animals, PFOA is a strong peroxisome-proliferator-activated receptor-alpha in the liver and this proliferation has been shown to alter lipid metabolism with decreases, not increases, serum lipids PFOA and PFOS are considered sensitive and accurate biomarkers of internal exposure to these substances Our study is one of the first to show a positive association between PFC levels and cholesterol levels in a low-level exposed general population, which is supported by previous studies of mainly highly exposed populations In conclusion, this study indicated small but significant associations between plasma PFOA and PFOS levels and total cholesterol in a middle-aged, general Danish population.


Flavobacteriales and Sphingomonadales, appeared to be particularly susceptible to the presence of NPs. Together, the migration of NPs through soil matrix and the ability of these potential pollutants to influence the composition of microbial community in this field study, cannot help but raise some environmental concerns.It is not known if the annual production of tonnes of industrial nanoparticles (NPs) has the potential to impact terrestrial microbial communities, which are so necessary for ecosystem functioning. Here, we have examined the consequences of adding zero valent copper and zinc oxide NPs to soil in pots that were then maintained under field conditions. The fate of these NPs, as well as changes in the microbial communities, was monitored over 162 days. Both NP types traveled through the soil matrix, albeit at differential rates, with Cu NPs retained in the soil matrix at a higher rate compared to ZnO NPs. Leaching of Cu and Zn ions from the parent NPs was also observed as a function of time. Analysis of microbial communities using culture-dependent and independent methods clearly indicated that Cu and ZnO NPs altered the microbial community structure. In particular, two orders of organisms found in rhizosphere,  Engineered metal nanoparticles (NPs) have numerous applications in a variety of consumer goods and industrial processes due to their unique physical, chemical and biological properties. As a consequence, the elevated production to meet this demand will result in an increase in NP release into the environment. To date, there is very little understanding on how such discharged NPs will influence microbial biodiversity. Microorganisms play important roles in geologic, hydrologic and ecological cycles, and any change in microbial diversity can potentially influence environmental quality and health, and even human development ZnO NPs are used in electronics, personal care products, biosensors, food additives, pigments and rubber manufacture 2+ orZn2+ in the untreated soil were adsorbed to a mineral or organic surface. To elucidate this further, reference spectra of Cu and Zn adsorbed to ferrihydrite, alumina γ-Al2O3, bentonite, and humic acid  were used as analogs for organic matter and mineral phase present in soils and to model the Cu and Zn XANES data using Linear Combination Fitting (LCF) , total Kjeldahl nitrogen (TKN) and phosphorus with values of 13100 mg/kg, 980 mg/kg and 357 mg/kg, respectively. The average soil pH was 7.5±0.2 (n = 5). Low levels of metals including Cu and Zn, as determined by acid digestion, were present in the soil . K-edge ng (LCF) . ResultsNPs were analyzed using several techniques, with transmission and scanning electron microscope (TEM and SEM) images showing that both the Cu and ZnO NPs were aggregated. Attempts to de-aggregate the material were unsuccessful, and as a result, obtaining the true particle size distribution was not possible. Aggregation also prevented the use of dynamic light scattering as a means of estimating particle size. TEM data obtained for the Cu NPs revealed a wide range of particle sizes (2O), and tenorite (CuO). Based on X-ray peak intensities the most abundant phase was metallic Cu followed by CuO and Cu2O   identifiand Cu2O . The preand Cu2O . NP spec and CuO . Consist CuO  for the Cu NPs from alkalimetric titration was at a pH of 9.4, and close to previous values . TitratiCu and ZnO NPs were deposited on the soil surface and allowed to freely migrate through the soil while exposed to environmental conditions over 160 days. Daily temperature and precipitation  were monitored over the field study period .0; shoulder), and a post-edge feature associated with adsorbed Cu . The first soil pots were sampled 24 h after NP addition and prior to any precipitation. There was no translocation of the Cu or Zn, and no detectable change in Cu and Zn soil concentrations as a function of depth or NP speciation based on XANES analysis , 4, 5. Ind horizon at 24 h, by 7 days there was a significant increase in the Cu and Zn concentration at 3–8 cm, and detectable levels in the lower 3rd horizon. For Cu NP treatment pots, the emergence of the Cu0 shoulder in the 2nd horizon . XANES analysis at the surface of the day 7 samples showed that Cu and ZnO NPs were the only phases present, but there was a significant decrease in the Zn concentration and little change in the Cu concentration . Althoug horizon . For ZnO section . It is u horizon . When thnd-horizon was partially due to translocation. However, since the Cu and ZnO NPs only accounted for 50% of the Cu and Zn species identified , gave no indication that recovered NP mass was associated with instrument error. Our soil-nanoparticle experimental pots were open systems, and no attempt was made to collect the leachate or prevent the erosion of nanoparticles caused by wind, which would be necessary for determining the relationship between NPs and soil on a mass basis. In the future, consideration must be given to controlling wind erosion as well as the collection of soil leachate. Not withstanding these caveats, the analyses presented here clearly demonstrate that both Cu and ZnO NPs are mobile in agricultural soils and have the potential to escape beyond an initial ‘spill’ site.in vitro, the added NPs would have been carried over with the soil samples explaining the observed high toxicity, as has been previously reported for Cu and ZnO NPs in vitro physiological assays, may be necessary for acute bacterial toxicity. A previous study showed that when Cu NPs were homogenously mixed with the NPs and incubated under laboratory conditions only marginal changes in the microbial community were observed The impact of Cu and ZnO NPs on the soil microbial community was measured using culture-idependent (Biolog® ecoplates) and culture-independent methods . Physiological profiles obtained using Biolog® ecoplates showed that NP additions had an immediate impact on the culturable microorganisms . SubstraCulture-independent FAME analysis showed no significant impact of the addition of either NP on the overall microbial soil profile across all three horizons and over time. Gram-negative bacteria were more prevalent (65%) than Gram-positive bacteria in control soil and this proportion remained the same throughout the horizons at all time points with minor changes irrespective of the NP treatment. Likewise, diatom and actinomycete fatty acid signatures showed no impact due to neither the NPs nor the metallic ion species.Enterobacteriales and Lactobacillales in some of the samples  and extensively characterized prior to use. NP crystal structure was determined by powder XRD using a PANalyticalXpert pro MPD  with CuKα radiation and a scan rate of 0.02° θ, from 10 to 85° 2θ. Six scans were collected and averaged prior to analysis. The PZC for Cu and ZnO was determined by measuring the point of zero net proton charge in a NaCl background electrolyte The soil used for the field study was obtained from an agricultural soil-distribution center , from a single mound of topsoil. When the experiments were initiated, the first 15–16 cm of the soil was frozen, and thus soil was taken from deeper in the mound. The soil was transported in plastic bags and any visible debris  was removed manually before soil was added to the pots. A portion of the soil was sampled to determine the pH by measuring the supernatant of a 1∶1 soil to Milli-Q water mixture. Similarly, the chemical composition of the soil was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) after carrying out microwave assisted nitric/hydrochloric acid digestion nd horizon (2.5–7.5 cm) and 3rd horizon (7.5–20 cm). The soil from each horizon was mixed for 5 min to decrease soil heterogeneity. One pot each of control, Cu NPs, and ZnO NPs were removed at each time point. Time points were chosen as follows: short term incubation within a month of NP inoculation for the analysis of acute, local effects , and long-term incubation (160 days) for long-term transport investigation. Weather data were recorded using Davis-Vantage Pro2 . The instrument was located no more than 2 m from the pots, ensuring it was not directly over them.Plastic planting pots  containing a rolled rim and a saucer at the bottom were filled with soil to a height of 20.3 cm and transported to the field site . The area was secured to prevent unauthorized access. No specific permits were required from any agencies as the contaminated soil was never directly introduced into the environment. In addition, as Dowling College houses The Center for Estuarine, Environmental, and Coastal Oceans Monitoring (CEECOM) at this location, no specific permission was required to place the pots at this location. The field study did not involve endangered or protected species. After transport, the soil was allowed to stabilize for 7 days prior to adding the Cu or ZnO NPs  by sprinkling over a 5 cm diameter at the center of the soil surface. Control pots received no NPs. At specified times (see results) sample pots were transported to the lab, and a ‘core sample’ was taken from the center by inserting a poly vinyl chloride (PVC) pipe (5 cm diameter). The cored soil sample was separated based on the depth with the top horizon (0−2.5 cm), 2After sampling, samples were freeze dried , fractionated based on particle size  and stored in airtight containers prior to analysis. The concentration of Zn and Cu in each of the soil particle size fractions was determined by microwave-assisted acid digestion using the previously described methods.2O, CuO, CuCO3, aqueous Cu2+, CuCl2, metallic Zn, ZnO, ZnCO3, aqueous Zn2+, and ZnCl2. Reference scans were also collected for Cu and Zn adsorbed to ferrihydrite, γAl2O3, smectite, birnesite, and humic acid. Initial Cu and Zn concentrations were 500 mg L−1 and conducted at pH 6, in 0.01 M NaCl, and a suspension density of 2 g L−1.The oxidation state and local bonding environment of the NPs in the soil were examined using XAFS spectroscopy as described except that PVP was not added. K-edge spectra were collected for the <75 µm NP fraction. In addition to the soil samples, mineral reference compounds were analyzed. Reference compounds included: metallic Cu, CuXAFS data processing and analysis were conducted using the Athena software package in the IFEFFIT computer program Microbial community physiological profiles were obtained in triplicate using Biolog® Ecoplates  using previously established methods Fatty acids were extracted from soil samples and analyzed by Microbial ID .www.researchandtesting.com). Subsequently, all failed sequence reads, low quality sequence ends, tags and primers were removed and any non-bacterial ribosomal RNA (rRNA) sequences and chimeras were removed using B2C2 www.krakenblast.com) against the database of high quality 16S rRNA bacterial sequences. Using a. NET and C# analysis pipeline, the resulting outputs were compiled and data reduction analysis performed as described previously Soil samples (0.5 g) were first treated with ethidiummonoazide (EMA) as described Based upon the sequence identity determined as the percent of the total length query sequence, aligned with a known sequence in the database, each sequence was assigned to the appropriate taxonomic level based upon the following criteria. Sequences with identity scores, to known or well characterized 16S rRNA sequences, greater than 97% identity were resolved at the species level, between 95–97% at the genus level, between 90–95% to the family and between 85–90% to the order level 80–85% to class and 77–80% to phyla. The percentage of sequences at each bacterial level were then used to determine the relative abundance within and among the individual samples based upon relative numbers of reads within each. Evaluations presented at each taxonomic level, including percentage compilations represent all sequences resolved to their primary identification or their closest relative Figure S1Cu (A) and Zn (B) K-edge XANES spectra for the untreated soil and Cu and Zn adsorbed to reference materials. The dashed red line represents the LCF best fit. For Cu the LCF results were 43% Cu adsorbed to alumina and 57% Cu adsorbed to humic Acid. For Zn the LCF results were 27% Zn adsorbed to ferrihydrite, 32% Zn adsorbed to smectite, and 41% Zn adsorbed to alumina.(TIFF)Click here for additional data file.Figure S2Visualization of the NPs showing A) a representative TEM micrograph of Cu NPs B) a representative SEM image of ZnO NPs.(TIFF)Click here for additional data file.Figure S3st derivative XANES spectra, and C) extended X-ray Adsorption Fine Structure (EXAFS) χ(k)k2 data. Relative percentage values are presented in Linear combination fitting results for Cu K-edge A) normalized XANES spectra, B) normalized 1(TIFF)Click here for additional data file.Figure S43 reference materials.Normalized and the first derivative of the normalized XANES spectra for ZnO nanoparticles, compared to ZnO and ZnCO Dashed grey lines in the figures highlight the energies corresponding to spectral features present in the ZnO NPs.(TIFF)Click here for additional data file.Figure S5Alkalimetric titration curves for Cu and ZnO nanoparticles. The dashed lines indicate the point of zero charge. The grey-circled area indicates the pH region where metallic Cu is oxidized to Cu1+ and Cu2+.(TIFF)Click here for additional data file.Figure S6X-ray diffraction pattern of pre- and post- titration Cu nanoparticles. Different colored arrows indicate specific Cu phases. A break in the Y-axis occurs at an arbitrary value and was used to illustrate changes in the finer features of the XRD pattern.(TIFF)Click here for additional data file.Table S1Elemental composition of the untreated soil determined by EPA Method 3051, at horizons 1–3 , expressed as mg/kg showing the relative standard deviation (RSD).(XLS)Click here for additional data file.Table S2Bacterial population present in the soil classified at order level. .(XLS)Click here for additional data file.


Yersinia species most pathogenic for mammals. Although factors encoded on this plasmid enhance the ability of Yersinia to thrive in their mammalian hosts, the loss of this virulence plasmid does not eliminate growth or survival in host organs. Most notably, yields of viable plasmid-deficient Yersinia pseudotuberculosis (Yptb) are indistinguishable from wild-type Yptb within mesenteric lymph nodes. To identify chromosomal virulence factors that allow for plasmid-independent survival during systemic infection of mice, we generated transposon insertions in plasmid-deficient Yptb, and screened a library having over 20,000 sequence-identified insertions. Among the previously uncharacterized loci, insertions in mrtAB, an operon encoding an ABC family transporter, had the most profound phenotype in a plasmid-deficient background. The absence of MrtAB, however, had no effect on growth in the liver and spleen of a wild type strain having an intact virulence plasmid, but caused a severe defect in colonization of the mesenteric lymph nodes. Although this result is consistent with lack of expression of the type III secretion system by Wt Yptb in the mesenteric lymph nodes, a reporter for YopE indicated that expression of the system was robust. We demonstrate that the ATPase activity of MrtB is required for growth in mice, indicating that transport activity is required for virulence. Indeed, MrtAB appears to function as an efflux pump, as the ATPase activity enhances resistance to ethidium bromide while increasing sensitivity to pyocyanin, consistent with export across the inner membrane.A highly conserved virulence plasmid encoding a type III secretion system is shared by the three  Yersinia genus: Yersinia pseudotuberculosis, Yersinia pestis, and Yersinia enterocolitica. Yersinia pestis, the causative agent of plague, has had profound effects on human civilization, killing one out of three people in Europe during one epidemic, the Black Death Y. pseudotuberculosis (Yptb) and Y. enterocolitica (Ye) usually cause self-limiting gastrointestinal infections. Although they differ in the route of infection and disease outcome, all three Yersinia species share a tropism for growth in lymph nodes. Infection with Y. pestis often results in dramatically inflamed lymph nodes, while Yptb or Ye infections are associated with acute mesenteric lymphadenitis due to their tropism for colonization of the mesenteric lymph nodes Yersinia species also share a conserved virulence plasmid, which encodes a Type III Secretion System and its associated translocated substrate proteins, called Yops There are three mammalian pathogens in the Yptb growth in a variety of mammalian organs, including the small intestine, cecum, Peyer's patches, liver, spleen, and lung Yptb in the mesenteric lymph node, as measured by colony forming units Yptb virulence factors appear to be sufficient for growth in the MLN.The virulence plasmid is required for optimal Yersinia species in pursuit of virulence factors required during animal infection Yptb virulence factors yet to be discovered.Several genetic screens have been performed in pathogenic mrtAB, encoding a previously uncharacterized heterodimeric ABC transporter that is critical for the growth and persistence of plasmid-cured Yptb in mice. Intriguingly, mrtAB is only necessary for Wt Yptb (P+) to colonize a single organ, the mesenteric lymph node.In this study, we describe the screening of over 20,000 plasmid-deficient sequence-identified transposon insertion mutants in mice, and the identification of a number of candidate virulence factors. We identified Yersinia pseudotuberculosis (Yptb (P−)) persists in various host organs Yptb virulence factors. Previous studies indicated that the number of clones that colonize the small intestine or successfully invade internal organs after oral inoculation was small Yptb (P−) to infect mouse spleens and livers following intravenous (IV) injection. IV injection of 105Yptb (P−)revealed that approximately 10% of the inoculum were present in the liver or spleen at 4 hours post-infection . This is a critical distinction whenever performing a genetic screen in an animal, as it determines how many mutants one can screen per mouse.Although we were able to get a rough estimate of the number of clones that colonized the spleen and liver by looking at viable counts of bacteria in these organs at 4 hours post-infection, it was unclear how many of these clones would survive after facing the full onslaught of the host immune system for days. For example, the increase in CFU in the liver from 10Yptb(P−), the number of clones in the liver and spleen that initially entered these organs, and whether the clonal number is reduced over time, we used TnSeq, an insertion mutagenesis procedure that allowed us to follow the fate of individual insertions by deep sequencing of the entire pool of insertion sites before and after inoculation 4 clones establishing residence in the liver or spleen, we generated libraries of approximately 104 mariner transposon mutants, which should represent the maximum number of clones present in each organ . One of the pools of 104 mutants was inoculated into mice; bacteria were isolated from the liver and spleen at 4 hours, 3 days, or 6 days post-infection; and genomic DNA was isolated from the bacterial colonies derived from these organs . We then performed deep sequencing on the insertion sites To determine the size of the bottleneck for Based on the clonal analysis , we chosAll the data for insertions in a given gene were then analyzed, and average ratios for output/input was determined for each gene, using colonies isolated from each liver as a separate output and colonies isolated from the injection dose as input . Genes oYersinia models of disease (candidate virulence factors). Identification of 5 genes known to encode proteins implicated in virulence, including the genes for pH 6 antigen (mutations in 3 genes), invasin, and SufI Yptb(P+) background  Geneckground . MutatioYptb(P−) was generated. Three days after IV inoculation, this strain gave yields between 102 and 103 lower than the parental strain in the liver and spleen, respectively   . These eYptb (P+) background. Surprisingly, even though the absence of the predicted ABC transporter lowered yields of the P− strain in the liver and spleen, there was no apparent defect in these organ sites after IV inoculation when the same mutation was made in the Yptb(P+) background (Yptb (P+) resulted in a defect in only one organ, the mesenteric lymph nodes (MLN). Oral infection of mice with WT or the deletion mutant revealed that bacteria lacking the putative ABC transporter were fully capable of persisting in the small intestine and exponentially increasing their numbers in the Peyers patches as rapidly as Wt, but had almost a 100-fold defect in the colonization or early growth in the MLN ΔmrtAB in the spleen (mrtAB (p+B+-flagmrtA) rescued growth of the ΔmrtAB strain in the spleen to the same extent as a wild type version of the gene (mrtAB-flag complementation construct (p+B*-flagmrtA) resulted in a six-fold decrease in yield in the spleen 3 days post-inoculation (Yptb(P−) in vivo. We attempted to perform an analogous experiment with MrtA, except we placed a peptide tag (HA) at the N terminus of the protein in an effort to avoid disruption of the mrtAB operon, where the 3′ end of the mrtA coding region overlaps with the 5′ end of the mrtB gene. While we observed that mutation of the MrtA Walker A box had no negative effect on the rescue of Yptb (P−)ΔmrtAB by +HA-mrtA*mrtB in the spleen (data not shown), we were also unable to detect either the HA-tagged MrtA or the mutated HA-tagged MrtA proteins, possibly due to cleavage of HA along with the signal sequence, complicating any interpretation of this result.The ATPase activity of ABC family transporters is the driving force behind either the export or import of cargo across the membrane e spleen . The FLAthe gene , 5A. Disculation , withoutculation . These eYptb(P−) in the MLN are indistinguishable from a virulence plasmid-containing strain even though Yptb(P−) exhibits a growth defect in every other organ tested Yptb do not express the plasmid-encoded TTSS and Yops, making MrtAB essential for growth in this organ site. A second hypothesis is that the bacteria interact with different sets of innate immune cells in the MLN and the spleen, creating two distinct selective environments for Yptb in these organ sites.Based on the results detailed above, it appeared that the virulence plasmid bypassed a requirement for MrtAB in all organs except the MLN. It is known that after oral inoculation, yields of yopE on the virulence plasmid. The yopE-mCherry construction was regulated in the same fashion as yopE during growth in broth culture, as bacteria encoding the fusion displayed thermally-induced mCherry expression that required the transcription factor LcrF +yopEmCherry infections, we compared spleens from mice infected 2 days post IV inoculation, to MLN from mice 2 days post oral inoculation. Contrary to our hypothesis, we found that the expression of the YopE reporter was identical in the spleen and MLN  and the mrtAB mutant in the small intestine, PP, and MLN 4 days post-infection. MrtAB was not required for Yptb(P+) to persist in the small intestine or the PP following oral infection (mrtAB Yptb(P+) in the MLN compared to Wt, the mutant had largely caught up to the Wt Yptb(P+) numbers in this organ by 4 days post-infection (compared to 100 fold less of the mutant at 1 day post infection), suggesting that the primary role of MrtAB may be during the initial colonization of the MLN rather than growth after the bacteria establish a replication site in this organ  (Yptb(P+) to colonize internal organs such as the spleen, Wt Yptb(P+) and the mrtAB mutant caused an equivalent rate of lethality during acute Y. pseudotuberculosis oral infection with 109 bacteria  . Consistbacteria .E. coli genes revealed that a strain lacking the E. coli mrtB ortholog showed enhanced sensitivity to pyocyanin, an antimicrobial produced by Pseudomonas aeruginosamrtAB on this plasmid resulted in increased susceptibility to pyocyanin. The point mutation predicted to interfere with the ATPase activities of MrtB removed this pyocyanin sensitivity , increasing the minimal inhibitory concentration (MIC) by 4 fold  as the genetic background for the screen. While we anticipate that some of the Yptb genes identified in this study will only be required in the P− background, we present the 5 hits in known virulence factors as evidence that many of the genes in aroA was shown to be essential for growth of Yersinia enterocolitica in mice aroA and purine synthesis genes are essential for Salmonella typhimurium pathogenesis The 5 known virulence factors Yptb chromosomal virulence factors. Yptb, and many of which have not been investigated in any pathogen. One of the few characterized virulence factors in this list is apaH, which is required for both invasion and adherence of Salmonella enterica to mammalian cells flgD and flgC, but it is unclear why these genes, which are essential for hook assembly, would play a role in this animal model when none of the other insertions in flagellar genes had any effect , predicted to be involved in initiating synthesis of the O subunit, and waaL (YPK_3646), predicted to encode the ligase that attaches O-Ag to the lipid A core outer saccharide Yptb unfit to grow at elevated temperatures (Yptb(P−) in deep tissue sites. YPK_3177, the predicted O-Antigen chain length regulator (wzz), on the other hand, is not essential for growth in mouse infection model we have used, as transposon insertions in this gene located at the end of the operon had no effect on growth in the liver  operon is highly conserved in most Enterobacteriaceae, with the predicted protein sequence similarity being 85% conserved for mrtA in E. coli, Shigella flexneri, S. enterica, and Klebsiella pneuomoniae. One study showed that high levels of expression of mrtAB homologs in S. enterica correlated with increased resistance to a fluoroquinolone antibiotic, although deletion of these genes had no effect on fluoroquinolone resistance mrtAB expression on resistance of E. coli to a variety of toxic compounds, and saw no effect We decided to focus on mrtAB or of the individual genes faithfully recapitulated the phenotypes from the screen, without any noticeable effect on growth in vitro  (Yptb (P+) in these same organs as well as the small intestine and Peyer's patches  . To our  patches . Furtherph nodes .Yptb requires the same set of virulence factors in a variety of organ sites, such as the Peyer's patches, spleen, liver and lung Yptb infection of the MLN is the anomaly, in that it is the only organ in which the virulence plasmid is not required mrtAB is essential for infection of MLN provides additional evidence for the unique nature of the MLN interaction with Yptb. This raises the possibility that fully virulent Yptb persists in an entirely different selective environment in the MLN than in other organ sites. Since it is known that Yptb interacts with and preferentially translocates Yops into neutrophils in vivo, we first tested if there were altered neutrophil co-localization with bacteria in the spleen relative to MLN. In almost all bacterial foci in the MLN or the spleen, neutrophils similarly surrounded the bacterial microcolonies  infection of the spleen and MLN did not reveal any obvious differences that could explain the differential requirement for MrtAB in the colonization of these two organs  ΔmrtAB in the spleen  to survive in the liver and spleen  from the intestine, we also tested the ability of the mrtAB mutant to colonize the spleen following oral infection. Interestingly, the mrtAB mutant was capable of colonizing the spleen at a level equal to Wt Yptb(P+), indicating that MrtAB is specifically required for transit to the MLN , to inflammation of the mesenteric lymph nodes  Y. pestis, 91–93% ID in Y. enterocolitica), we predict that MrtAB will play a role in the colonization of lymph nodes by all pathogenic Yersinia species, including Y. pestis and Y. enterocolitica. It will also be important to test the role of MrtAB in strains of Y. pseudotuberculosis that do not share the phoP mutation present in the YPIII strain used in this study Salmonella enterica serovar Typhimurium (76–79% ID), suggesting that transport mediated by MrtAB may be a common mechanism by which bacterial pathogens colonize this immune organ.All pathogenic Y. pseudotuberculosis. MrtAB is the first mesenteric lymph node specific virulence factor identified in Yersinia species. Further study of this ABC transporter and its substrate(s) should provide valuable insight into the interaction of Y. pseudotuberculosis with the mesenteric lymph node and its unique requirements for establishing bacterial replication in this site.In conclusion, this study identified a number of candidate virulence factors in Yersinia pseudotuberculosis (Yptb) strains used in this study were derived from YPIII Yptb has been previously described mrtAB knockout construct were the following: mrtAB FOR1: attaGCATGCTTGCTGGAAACGTTTAAAGCGTTTGG, mrtAB REV1: attaGAATTCTAATTGTGCAAACAATCTCACGCAGTTT, mrtAB FOR2: AGGAGGTCGAAGC CGATGAATAACattaGAATTC , mrtAB REV2: attaGAGCTCTTGAAA TCAGCGCCATCCGCCAAT. For HA tagging of mrtA (YPK_3222), the HA sequence was inserted directly downstream of the ATG start codon of the operon. For the FLAG tagging of mrtB (YPK_3221), the FLAG sequence was inserted just upstream of the stop codon. The coding regions of the two genes are overlapping, which is why we avoided making any tags in the C terminus of MrtA or the N terminus of MrtB. Yptb were tagged with GFP by driving expression of GFP off the constitutive Tet promoter on pACYC184. The tetA::GFP promoter-gene fusion from pDW5 SphI end sites and moved it into pACYC184 cut with SphI. Forward primer: 5′ gatcgcatgcgaattctcatgtttgacagcttat 3′ Reverse primer: 5′ gccgccgcaaggaatggtgcatgc. This plasmid is very stable in vivo. For the construction of the mrtAB complementation plasmid (pmrtAB), pACYC184 was digested with EcoRV and SalI, and the mrtAB operon was PCR-amplified with EcoRV and SalI end sites. The entire intergenic sequence in between YPK_3223 and YPK_3222 (101 bps) was included upstream of the mrtA start codon, and the mrtB terminator was included after the gene. The primers used for the complementation vector were: CompFor: attaTCTAGAATAATTCACTAAAAAATCTGTTTATCAATGGT, and CompRev: attaGTCGACAAGTGA GTGAGTGAGTGAGTGAGT. A YopE reporter strain was constructed with a FLAG-mCherry sequence immediately following the yopE stop codon. An isogenic, unmarked T3SS reporter strain was constructed that contains FLAG-mCherry sequence immediately after the yopE stop codon (see yopE stop codon was constructed by PCR and cloned into the SacI and BamHI sites of pSR47s. The resulting plasmid (pSR47s-yopE-FLAG-mCherry) was introduced into E. coli DH5α λpir and integrated onto the Y. pseudotuberculosis virulence plasmid via triparental mating using the helper strain HB101(RK600).All odon see , panel AYptb cultures were grown in 2XYT, except for determination of the MIC (below). Kanamycin (30 µg/mL) for selection of transposon and Irgasan (2 µg/mL) for selection for Yptb were used in the production of Yptb transposon mutant libraries. Chloramphenicol (25 µg/mL )was used in selection for pACYC184 derived complementation plasmids. For IV, oral, or IP infections, Yptb cultures were grown at 26°C overnight, rolling, prior to infection. For in vitro growth for measuring MrtB protein levels by Western blot, bacteria were grown overnight in LB, and backdiluted 1∶40 the following morning in LB. Cultures were allowed to grow for 90 minutes at 26°C, then half the samples were switched to 37°C and half were left at 26°C, and the cultures were growth for an additional 90 minutes prior to protein isolation. Mouse anti-FLAG was used as a primary antibody, overnight at 4°C, and Cy5 Goat anti-Mouse was used as a secondary antibody. Westerns were visualized on a Fuji FLA-9000.All MmeI restriction site, as described Yptb strain, the Himar1(MmeI) transposon was introduced into YPIII(P−) by mating with SM10λpir. Briefly, 25 mL of YPIII(P−) was grown overnight (O/N) in 2XYT broth at 26°C, and 75 mL of SM10λpir(pSC189Himar1(MmeI) was grown O/N at 37°C in LB containing 30 µg/mL Kan and 100 µg/mL Amp. The SM10λpir cultures were washed 3X with PBS, pelleted, and resuspended in the YPIII(P−) culture. Mating was allowed to proceed for 16–24 hours at 37°C in the spent Yptb culture, standing. Bacteria were then pelleted, resuspended in 5 ml 2XYT, and spread on 10 LB plates containing 30 ug/ml kanamycin and 2 µg/ml irgasin. Libraries of approximately 10,000 colonies were scraped off plates, pelleted, resuspended in 50% glycerol and stored at −80°C.The vector pSC189 containing Himar1 5 bacteria were introduced into mice, organs were isolated and homogenized at various times post infection, and bacteria were isolated by plating for all bacteria, on LB Medium containing 30 ug/ml kanamycin and 1 ug/ml irgasan. Colonies were scraped off plates and genomic DNA was isolated using Qiagen DNeasy kit. Samples were prepared for Illumina sequencing, as described Libraries of 10,000 Himar1 mutants were adjusted to yield a total of 200,000 colonies on LB medium, scraped and resuspended in 2XYT, and small aliquots were used to start O/N cultures in 2XYT at 26°C. Tail vein injections of 1×102 value of this ratio was used for further statistical analysis, including determining the average ratio and standard deviation (s.d.).Each overnight culture of an individual library of 10,000 transposon mutants was plated out for over 200,000 colonies, which were used to generate genomic DNA (above). This is defined as an Input sample. For each library, there were at least 2 Input samples, with a corresponding set of Output samples, defined as the colonies from an individual infected liver derived from a given Input injection dose. After Illumina sequencing, the number of reads for each gene in the Output sample was normalized for amount of DNA added to sequencing run  and normalized for the number of unique insertions in a particular pool. This provides a value for each gene, for the relative abundance of clones containing a transposon insertion in gene X within the pool. Insertions in the first 5% or last 10% of a gene were discarded and all remaining values for insertions within a single gene were summed. Finally, the values for each gene in the Output liver samples were divided by the values in the corresponding Input sample. This provides a ratio of the relative abundance of clones containing a transposon insertion in Gene X in the Output liver sample, divided by the relative abundance of clones containing a transposon insertion in Gene X in the Input sample. The LogYptb (P−), mice were infected IV with 1×105 bacteria. For WT, mice were infected IV with 1×103 bacteria. All oral infections were performed in BALB/c mice for ease of Peyer's patch isolation. Intraperitoneal infections were also performed in BALB/c mice. For oral infections, food was removed from cages 16 hours before oral inoculation with 2×109Yptb. For Peyer's Patch quantification of CFU, all visible PP from a single animal were combined prior to homogenization and plating. For small intestine CFU quantification, the 5 cm of small intestine upstream of the caecum were removed. For both PP and small intestine, homogenates were plated on LB with 1 µg/mL Irgasan. For histology, inoculations with GFP tagged bacteria were performed as all other inoculations, and organs were fixed in 4% paraformaldehyde for 3 hours, then flash frozen in Sub Xero freezing media . 10 µm sections were cut using a cryostat microtome, and stained with Hoechst . For neutrophil staining, monoclonal anti-Ly6G clone 1A8 (BD Pharmingen) was used at 1∶100. For quantification of the YopE reporter strain, tissue sections were prepared as described above, and imaged using a Nikon A1R confocal. Images were quantified using ImageJ, with each microcolony analyzed for median mCherry fluorescence, normalized to median GFP, which was constitutively expressed.All mice used were 8–10 weeks old. All intravenous (IV) infections that were analyzed by CFU were performed in C57BL/6 mice. For 2 asphyxiation followed by cervical dislocation.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Tufts University Institutional Animal Care and Use Committee (IACUC). Our approved protocol number is B2010-100. All efforts were made to minimize suffering: animals were carefully monitored following infection and were euthanized prior to or directly upon exhibiting substantial signs of morbidity. Animals were euthanized by C0Yptb cultures were grown in 2XYT overnight at 26°C, with chloramphenicol 25 µg/mL if they contained a pACYC184 derivative. Bacteria were diluted in LB broth, and used to inoculate 96 well plates containing 2 fold serial dilutions of Acridine Orange, Ethidium Bromide, or Pyocyanin, with chloramphenicol 25 µg/mL if they contained a pACYC184 derivative. Bacteria were grown overnight at 37°C without shaking, and OD600 was measured 18 hours later. The MIC is defined as the lowest concentration of toxic compound that results in half maximal growth , and the values presented in Figure S1Construction and characterization of YopE reporter strain.A) yopE reporter strain (yopE-STOP::FLAG-mCherry) construction. FLAG-mCherry sequence was inserted immediately after the yopE stop codon to serve as a reporter for yopE expression. B) yopE reporter expression is properly regulated. Bacteria were grown at 37°C (Wt and ΔlcrF containing yopE-STOP::FLAG-mCherry) or 26°C (Wt containing yopE-STOP::FLAG-mCherry) and bacteria were visualized by Phase contrast and fluorescence microscopy. C) Reporter expression does not affect endogenous yopE expression. Wild-type, two Wt yopE-STOP::FLAG-mCherry isolates, and an ΔlcrF yopE-STOP::FLAG-mCherry isolate were grown in yop inducing conditions. Bacteria were lysed and proteins analyzed by Western blotting. D–E) Reporter strain fluorescence decreases in the absence of yop expression. Bacteria were grown in yop-inducing conditions , washed, and shifted to non-inducing conditions . Samples were taken every two hours and imaged by phase and fluorescence microscopy (D). Fluorescence was quantified on a per-bacterium basis (E).(PDF)Click here for additional data file.Table S1Screen data for genes hit in both libraries.Column C–F: The number of reads for each gene in the Input samples normalized for the amount of DNA added to sequencing run  and normalized for the number of unique insertions in a particular pool. Column G–AC: All the Output Liver samples, normalized as in C–F, then divided by the values in the corresponding Input sample. Column AD–AM: Statistical analysis and annotation, including the average ratio of Output/Input, the Log2 value of this ratio, the number of Standard Deviations away from the mean, and a reference to the 26° vs 37° growth (° growth .(XLS)Click here for additional data file.Table S2Screen data from genes only hit in Library A. Similar analysis as in Column B, H: The number of reads for each gene in Library A Input sample normalized for the amount of DNA added to sequencing run  and normalized for the number of unique insertions in a particular pool. Column C–G, I–M: Output Liver samples, normalized as in B and H, then divided by the values in the corresponding Input sample. Column O–U: Statistical analysis, including the average ratio of Output/Input, the Log2 value of this ratio, and the number of Standard Deviations away from the mean.(XLS)Click here for additional data file.Table S3Screen data from genes only hit in Library B. Similar analysis as in Column B, H: The number of reads for each gene in Library B Input sample normalized for the amount of DNA added to sequencing run  and normalized for the number of unique insertions in a particular pool. Column C–G, I–P: Output Liver samples, normalized as in B and H, then divided by the values in the corresponding Input sample. Column Q–Y: Statistical analysis, including the average ratio of Output/Input, the Log2 value of this ratio, and the number of Standard Deviations away from the mean.(XLS)Click here for additional data file.Table S426° vs. 37° growth in vitro. Libraries A and B were combined and grown overnight at 26°, diluted into 2XYT the following day, and grown overnight at either 26°C or 37°C. Column B, C: The number of reads for each gene from samples grown at 26°C, normalized for the amount of DNA added to sequencing run  and normalized for the number of unique insertions in a particular pool. Column D, E: Similar analysis to Column B and C, only from samples grown at 37°C. Column F–N: Statistical analysis, including the ratio of 26°C vs. 37°C values, the Log2 value of this ratio, and the number of Standard Deviations away from the mean.(XLSX)Click here for additional data file.


Ranunculus laetus from the cold regions of Northwest Himalayas. One accession showed the presence of 14 bivalents at diakinesis and regular segregation of bivalents at anaphase I which lead to normal tetrad formation with four n microspores and consequently n pollen grains and 100% pollen fertility. Second accession from the same locality revealed the erratic meiosis characterized by the presence of all the 28 chromosomes as univalents in meiocytes at metaphase I. Univalent chromosomes failed to segregate during anaphases and produced restitution nuclei at meiosis I and II. These restitution nuclei resulted into dyads and triads which subsequently produced two types of apparently fertile pollen grains. On the basis of size, the two types of pollen grains were categorized as n  and 2n . The estimated frequency of 2n pollen grains from dyads and triads (61.59%) was almost the same as that of the observed one (59.90%), which indicated that 2n pollen grains in R. laetus were the result of dyads and triads. The present paper herein may provide an insight into the mechanisms of the formation of various intraspecific polyploids through sexual polyploidization in R. laetus.Two accessions were studied for male meiosis in  Ranunculus laetus Wall. ex Royle (family: Ranunculaceae), a highly polymorphic species [n = 14) [n = 16) [n = 28), [n = 32) [n = 42) [n = 56) [ species , 2, has [n = 14) , Cangshan = 28), , 6, Kashn = 28), , 8, Eastn = 28), , Northwen = 28), , 11, Rusn = 28), , 13, Garn = 28), , 16, Cha[n = 32) , Indian [n = 32) , Kashmir[n = 32) , 20, Wes[n = 32) , 22 Pakin = 28) showed normal bivalent formation and equal segregation of chromosomes at anaphases. However, these accessions depicted some irregularities during male meiosis such as cytomixis, chromosome stickiness, pycnotic chromatin material, out of plate bivalents at metaphase I, nonsynchronous disjunction of bivalents, and laggards at anaphases/telophases which resulted into PMCs (pollen mother cells) with abnormal microsporogenesis and 9–31% pollen sterility [n (unreduced) pollen grains.Despite these intraspecific chromosomal variations and levels of ploidy  nothing is known about the origin of various polyploids in the species. All the previous studies carried out in the species were restricted either to count the chromosome number or to study the karyotype or DNA content. Previous communications , 16 fromn pollen grains. The study may also provide an insight into the mechanisms of the formation of various intraspecific polyploids in R. laetus.The present study herein aims to analyze the detailed meiotic course, microsporogenesis and to elucidate the cytological mechanism that lead to the formation of 2Material for male meiotic studies was collected from the wild plants growing on open moist slopes around the apple orchards in Bharmour in Chamba district  of Himachal Pradesh in June-July of 2009. The cytologically worked-out plants were identified using regional floras and compared with the specimens deposited at the Herbarium of Botanical Survey of India, Northern Circle, Dehra Dun. The voucher specimens  were deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).For meiotic chromosome counts, unopened floral buds of suitable sizes were fixed in a freshly prepared Carnoy's fixative  for 24 h. These were subsequently transferred to 70% alcohol and stored in refrigerator at 4°C until used for meiotic analysis. Meiocytes were prepared by squashing the developing anthers, and stained with acetocarmine (1%). Chromosome number was determined at M-I from freshly prepared slides with light microscope Olympus. 500−600 pollen mother cells were analyzed for meiotic behaviour at different stages, M-I/II, anaphase I/II (A-I/II), telophase I/II (T-I/II).n  pollen in diameter was taken as 2n (unreduced) pollen. Estimation of the theoretical frequency of 2n pollen grains was made from the number of observed dyads, triads, and tetrads during microsporogenesis. Generally a dyad resulted into two unreduced pollen grains, a triad produced one unreduced pollen grain and two reduced pollen grains, and each tetrad gave rise to four reduced pollen grains. The frequency of 2n pollen grains was calculated following Xue et al. [Pollen fertility was estimated through stainability tests using glycerol-acetocarmine (1 : 1) mixture and aniline blue (1%). Up to 450−800 pollen grains were examined for pollen fertility and size frequencies. Well-filled pollen grains with stained nuclei were taken as apparently fertile while shriveled and unstained pollen were counted as sterile. In each case, the size of 200 pollen grains was measured using an occulomicrometre. As per Xue et al.  pollen ge et al. :Leica QWin. Photomicrographs from the freshly prepared desirable slides having clear chromosome counts, dyads, triads, tetrads, and pollen grains were taken with a digital imaging system of n microspores  and 2n  compared to the n pollen grains (40.10%).Although pollen fertility was not affected significantly 92%, , the errn microspores whereas a triad produced only one 2n microspore and two n microspores. The frequency of apparently fertile 2n pollen grains which was estimated from different types of sporads found to be 61.59%. The frequency of 2n pollen grains estimated collectively from dyads and triads was almost near the observed one, 59.90% which indicated that the 2n pollen grains in R. laetus were resulted from dyads and triads at sporad stage which originated from the restitution nuclei observed during meiosis I and II.Each dyad give rise to two 2The chromosome number in sexually reproducing eukaryotes does not get doubled at each generation which is ensured through a precise, systematic, and specialized process of meiosis . Vital e Synaptic variation resulting in complete and partial failure of chromosome pairing of homo/homeologous chromosomes has been studied in a large number of species , 34–38. n pollen grains in plants. Owing to the relatively close correlation between larger pollen grains and 2n status, the presence of large-sized pollen grains had been frequently used as a criteria for the indication of 2n pollen [n  and 2n (unreduced). The pollen grains which were 1.5-times larger than the normal pollen were regarded here as 2n pollen grains. Similar criteria to distinguish between n and 2n pollen grains had been used earlier by Peng [n pollen formation in Chinese jujube. The exact chromosome number of such double-sized pollen grains could not be ascertained during the present investigations but these were surely of the unreduced in their genetic constitution as is clearly depicted from their size, as increasing nucleus and cytoplasm content may in turn influence pollen diameter [n pollen grains from dyads and triads was almost the same as that of the observed one, which indicated that the 2n pollen grains in R. laetus were the result of dyads and triads at sporad stage which originated from the restitution nuclei formed during meiosis I and II. Another interesting phenomenon in the presently investigated species is the formation of restitution nuclei. Restitution nuclei were formed because univalent chromosomes/daughter chromatids failed to distribute themselves uniformly at the poles during anaphases. These restitution nuclei resulted into the formation of dyads and triads which subsequently produced two types of pollen grains. Different methods had been used to detect production of 2n pollen , 46–52.  by Peng  and Xue  by Peng  while stdiameter , 54–58. n pollen grains were observed to be well filled, stained, and apparently fertile; therefore, it is very much possible that fertilization by these 2n gametes can produce intraspecific polyploids [n gametes is a common phenomenon in the plants which may result from a variety of different meiotic irregularities [n pollen grains) or gametes with somatic chromosome number are considered one of the main processes for natural polyploidization of plants. These 2n pollen grains may play an important role in the establishment of new polyploid genotypes as suggested by Dewitte et al. [n pollen grains offer over asexual polyploidization is the transmission of the parental heterozygosity to the offspring. The 2n gametes produced through restitution nuclei can transfer at least 75–80% heterozygosity [ The large-sized 2lyploids , 59–63. larities . Unreduce et al.  and Silve et al. . Unreduce et al. . The maizygosity , 68.Allium amplectens had been made by Levan [ In a number of plants earlier workers have reported that synaptic mutation causes pollen sterility , 69–72. by Levan . The pren pollen grains. Furthermore, authors safely conclude that 2n pollen grains may have played a role in the evolution of species by forming the polyploid genotypes through sexual polyploidization as has been suggested by others [Presently studied accession with erratic male meiosis is a spontaneous asynaptic mutant in which univalent chromosomes behaved in a highly irregular manner resulting into restitution nuclei and consequently 2y others , 74, 75.


Cerebral MRI performed on preterm infants at term-equivalent 30 weeks' gestational age (GA) is increasingly performed as part of standard clinical care.We evaluated safety of these early MRI procedures.We retrospectively collected data on patient safety of preterm infants who underwent early MRI scans. Data were collected at fixed times before and after the MRI scan. MRI procedures were carried out according to a comprehensive guideline.A total of 52 infants underwent an MRI scan at 30 weeks’ GA. Although no serious adverse events occurred and vital parameters remained stable during the procedure, minor adverse events were encountered in 26 infants (50%). The MRI was terminated in three infants (5.8%) because of respiratory instability. Increased respiratory support within 24 h after the MRI was necessary for 12 infants (23.1%) and was significantly associated with GA, birth weight and the mode of respiratory support. Hypothermia (core temperature < 36°C) occurred in nine infants (17.3%). Temperature dropped significantly after the MRI scan.Minor adverse events after MRI procedures at 30 weeks GA were common and should not be underestimated. A dedicated and comprehensive guideline for MRI procedures in preterm infants is essential.The online version of this article (doi:10.1007/s00247-012-2426-y) contains supplementary material, which is available to authorized users. In preterm infants, early recognition of neonatal brain injury and assessment of risks of later impairment is a challenging goal of current neuroimaging studies –3. MagneMRI is considered a safe imaging technique. No evidence exists of serious harm to human tissue, besides loud acoustic noise, tissue heating and peripheral nerve stimulation –16. PerfOur aim was to study the safety of routine MRI scans in preterm infants at a postmenstrual age of 30 weeks. To accomplish this, we collected data of these infants regarding safety incidents and (avoidable) adverse events over a long time period: 24 h before and 24 h following the MRI scan.A tailored, centre-specific guideline for MRI procedures in VLBW infants was developed in collaboration with representatives from the radiology and neonatology departments as well as a patient safety officer from the Erasmus Medical Centre, Sophia’s children’s hospital. The guideline was based on the MR safety literature and our own experiences and was adjusted using the principles of the ‘‘plan-do-study-act’’ quality improvement , an iterAs part of standard clinical care practices, MRI scans were performed on VLBW infants that were born at a gestational age (GA) < 29 weeks. These scans were performed at a postmenstrual age of 30 weeks (29 4/7 to 30 4/7 weeks). In all patients, the MRI procedure was carried out according to our multi-disciplinary guideline  adverse events, were retrospectively collected from our electronic patient data management system. These data were sampled at fixed times: 24, 16 and 8 h before the MRI scan, during the MRI procedure itself and 8, 16 and 24 h after the MRI scan. The definitions of major and minor adverse events are listed in Table The study was approved by the medical ethical committee of the Erasmus Medical Centre Rotterdam, The Netherlands.All MRI scans were performed using a 1.5-T GE Echo Speed scanner . The standard imaging protocol included the following: axial T1-weighted spin echo, axial T2-weighted dual spin echo, sagittal T1-weighted spin echo, axial 3-D T1-weighted SPGR and echo planar diffusion tensor imaging. The acquisition times were approximately 5-6 min per sequence.P value of < 0.05 (two-sided) was considered statistically significant.Statistical analysis was performed using SPSS v17.0.2 . A repeated measures ANOVA using Wilks lambda test was conducted to test the stability of vital parameters during the MRI procedure. Correlations of adverse events with GA, birth weight, weight at image acquisition, gender, temperature drop, mode of respiratory support and total acquisition time were tested. Pearson correlation coefficients were used for continuous variables. Pearson chi-squared test was performed for proportional differences between two categorically scaled variables. One-way ANOVA was used for mean differences among three or more groups holding the groups as a factor variable categorically scaled. A A total of 158 infants were eligible for inclusion in the study. Among these, 32 infants died before term-equivalent 30 weeks’ GA, 36 infants were transferred to other hospitals before the MRI scan could be performed and in 38 infants the MRI scan was postponed because the infants were not haemodynamically stable enough for MRI scanning at 30 weeks’ GA. Therefore, 52 infants (30 boys) underwent a cerebral MRI scan. Patient characteristics are listed in Table Generally, compared with 24 h before the MRI scan, vital parameters  remained stable during the 24 h after the scan Fig. .Fig. 2ViEven though vital parameters remained stable during the MRI scan itself, increased haemodynamic instability occurred within the following 24 h in some infants.No adverse events occurred in 26 infants (50%). However, in 26 infants (preventable) incidents and minor adverse events were encountered Table . The MRIOur study stresses the importance of providing a controlled environment for early MRI procedures for preterm infants. Despite the presence of a multi-disciplinary guideline specifically designed for preterm infants, minor adverse events, such as hypothermia and the need for increased respiratory support after the scan, were encountered regularly: these events occurred in 26 infants, 50% of our study population. In total, 39 minor adverse events occurred. Therefore, caution needs to be taken regarding the safety of VLBW infants during MRI procedures. Critical incident review and continuous re-evaluation of the guidelines are essential in this process.MRI is becoming increasingly important for accurately evaluating brain injuries and the consequent effects on neurodevelopment in preterm infants , 21, 22.Because of the increased risk of respiratory and circulatory compromise, no sedation was used in this study. To reduce motion artefacts, we use other strategies to comfort the infant, such as those according to the principles of the Newborn Individualised Developmental Care and Assessment Program , 30.Safety incidents in  health care are generally related to poor preparation, equipment failure and human error , 32. StuWe have shown that adverse events related to MRI scans in this vulnerable population are common. This is in contrast to other studies –19, in wAlthough it is reasonable and logical to assume that a longer total acquisition time is likely to increase the number of adverse events, we were unable to demonstrate this relationship in our study, possibly related to the small sample size.The limitations of this study include selection bias, as our data consist only of infants considered haemodynamically stable enough for an MRI scan. In our setting, the medical team decided whether the infants were haemodynamically stable enough to undergo an MRI scan.Perhaps if more strict criteria for haemodynamic stability were applied, the incidence of adverse events might be less frequent. In contrast, the incidence of adverse events might increase if more critically ill preterm infants were scanned, emphasising the importance of a comprehensive guideline with strict contraindications and staff training to ensure the safe execution of MR procedures.Another limitation could be the retrospective design and the lack of temperature measurement during the MRI scan. Despite the use of an MR-compatible incubator, which provides controlled temperature and humidity, we encountered an increased incidence of hypothermia after the MRI scan. This could be explained by the mode of respiratory support: the infants were supported with cold air or oxygen during the procedure, which is in contrast to the setting at our wards, where infants are supported with pre-heated (40°C) air or oxygen. In order to decrease the high incidence of hypothermia after the MRI scan, we propose using an optical temperature probe to measure temperature continuously during the scan. Although minor adverse events were encountered more frequently after the MRI scan, it is not with certainty established that this in fact can be attributed to having undergone an MRI scan. Being transported from the NICU alone could in fact be stressful enough. However, due to the lack of evidence against causality and in the context of patient safety, we argue that each adverse event should be considered as a result of the procedure. Moreover, in order to avoid this possible bias, vital parameters, mode of respiratory support and the number of episodes of bradycardia, apnea or oxygen desaturation that occurred within 24 h before the MRI scan were compared with the same details occurring within 24 h following the MRI scan of each infant individually.n = 12) in that group.Logistical regression to weigh gestational age, birth weight and mode of respiratory support with the increased need for respiratory support was not performed given the small sample size Below is the link to the electronic supplementary material.


Acropora hemprichii after 1 and 16 weeks of in-situ inorganic nutrient enrichment (via fertilizer diffusion) and/or herbivore exclusion (via caging) in an offshore reef of the Central Red Sea. Simulated eutrophication and/or overfishing treatments did not affect coral physiology with respect to coral respiration rates, chlorophyll a content, zooxanthellae abundance, or δ 15N isotopic signatures. The bacterial community of A. hemprichii was rich and uneven, and diversity increased over time in all treatments. While distinct bacterial species were identified as a consequence of eutrophication, overfishing, or both, two bacterial species that could be classified to the genus Endozoicomonas were consistently abundant and constituted two thirds of bacteria in the coral. Several nitrogen-fixing and denitrifying bacteria were found in the coral specimens that were exposed to experimentally increased nutrients. However, no particular bacterial species was consistently associated with the coral under a given treatment and the single effects of manipulated eutrophication and overfishing could not predict the combined effect. Our data underlines the importance of conducting field studies in a holobiont framework, taking both, physiological and molecular measures into account.Coral reefs of the Central Red Sea display a high degree of endemism, and are increasingly threatened by anthropogenic effects due to intense local coastal development measures. Overfishing and eutrophication are among the most significant local pressures on these reefs, but there is no information available about their potential effects on the associated microbial community. Therefore, we compared holobiont physiology and 16S-based bacterial communities of tissue and mucus of the hard coral  Coral reefs are threatened by global  and local  anthropogenic stressors The basic functional unit of coral reefs is the coral holobiont Acropora hemprichii in the Central Red Sea after 1 and 16 weeks of in situ artificial nutrient enrichment , in situ herbivore exclusion (using cages with a mesh size of 4 cm to exclude larger herbivores), and a combined treatment to simulate effects of eutrophication and overfishing. While the latter two processes generally refer to ecosystem scale effects, we were investigating effects on the scale of the holobiont in this study. To our knowledge, this is the first study from the Red Sea that evaluates top-down (i.e. herbivory) and bottom-up (i.e. nutrient availability) factors on coral holobiont functioning and provides first insights into the effects on coral physiology and associated bacterial community changes.A number of previous studies investigated the bacterial diversity in coral reef organisms via 16S amplicon sequencing Acropora hemprichii is a common reef building coral present in the Red Sea and the Western Indian Ocean Acropora hemprichii is listed as vulnerable on the ICUN Red List (http://www.iucnredlist.org/details/132981/0).The herbivore exclusion and nutrient enrichment experiments were carried out at Al-Fahal reef about 13 km off the Saudi Arabian coast in the Central Red Sea  were collected from the same water depth in the area where the experiment was conducted. A total of nine frames were deployed  each holding four coral fingers (for collection after 1 and 16 weeks) from each of the three mother colonies  yielding a total of 18 samples for physiological as well as for microbial analyses . Experimental treatments started after attachment of coral fingers to the PVC frames via cable ties on stainless steel screws. After one and sixteen weeks, respectively, coral fingers were collected in sterile plastic bags and brought to the surface, where half of them were rinsed with filtered seawater (FSW) and subsequently shock-frozen in liquid nitrogen for microbial analyses and stored at −80°C until further processing. The other half of the coral fragments was used for incubations . Water samples for microbial and nutrient analyses were collected with large ziplock bags directly above each frame prior to coral fragment collection during both sampling time points. These samples were transported on ice and 500 mL were subsequently filtered on 0.22 µm Millipore Durapore membrane filters for microbial analysis. Filters were stored at −80°C. Additionally, 50 mL seawater were filtered using 0.7 µm fiberglass filters (Whatman-GF/F) and stored at −20°C until inorganic nutrient analysis was carried out. The inorganic nutrients were photometrically analyzed by segmented debubbled continuous flow analysis (FlowSys - Alliance Instruments) according to The experimental setup included nine polyvinyl chloride (PVC) frames equipped with temperature loggers and stainless steel screws for coral finger attachment. Frames were deployed at 5–6 m water depth along a 40 m transect on a slightly sloped reef wall. Treatments were arranged in alternating order and separated from each other approximately by 2–5 m. Treatments were: (1) caging (CA), with a cage mesh size of 4 cm to simulate overfishing of larger herbivores; (2) fertilizer (FE), perforated PVC tubes filled with Osmocote fertilizer standard 15+9+12  embedded in 3% agarose to simulate nutrient enrichment; (3) a combination of caging & fertilizer (CF) . The fer2 concentrations were measured with an O2 probe  inside each box after evenly distributing the water, with open incubation jars. The jars were then sealed and incubated over a period of 90–100 min before O2 concentrations in each jar were measured as described above. During incubations the boxes were carefully moved every 5 minutes to cause stirring of water inside the jars. Onset HOBO temperature loggers  in each box assured that water temperature during incubation remained constant (temperature differences between incubation jars and in-situ temperatures measured at PVC frames ranged between 0.5 and 1.6°C). All samples were subsequently stored on ice until further processing. Net O2 consumption was calculated subtracting end concentrations from the start concentrations. Normalization of data to µg O2 cm−2 h−1 was carried out by measuring the coral finger surface area using a cylinder as approximation according to the “simple geometry” model described by Naumann et al. a concentration, and δ15N isotopic signatures, tissue of each coral finger was washed with filtered sea water (FSW) before tissue was removed from the skeleton by air-blasting, collected in FSW, and homogenized using an Ultra-Turrax . Zooxanthellae abundance was counted using an Improved Neubauer hemocytometer . Cell numbers were counted in 6 grid squares à 4 µm3 with 6 counts per sample (average count: 29 cells per grid square). To determine isotopic signatures of coral tissue and zooxanthellae, a modified version of −2 with surface area (see method described above). Homogenized coral tissue was first filtered on a GF/F filter and washed with MilliQ water to remove remaining salt. Subsequently, the filters were dried for 2–4 d at 40°C until constant weight and δ15N signatures were determined relative to atmospheric nitrogen in an isotope ratio mass spectrometer . Isolated zooxanthellae were freeze-dried and directly measured with the mass spectrometer. For Symbiodinium-typing, DNA was extracted using the DNeasy Plant Mini Kit  according to the manufacturer’s instructions. The ITS2 rDNA region was amplified with the primer pair ITSintfor2 and ITS2CLAMP Symbiodinium types were determined by DGGE fingerprint profiles and sequencing. For sequencing, prominent band(s) were excised from the DGGE gel and re-amplified as described in Symbiodinium ITS2 sequences. Coral respiration rates, chlorophyll a, zooxanthellae abundance, and δ15N isotopic signatures of corals and zooxanthellae were analyzed with Sigmaplot 12  by two-way ANOVA. To meet parametric assumptions, δ15N coral data for colony-time factorial analysis were exponential transformed, and chlorophyll a and respiration data for genotype-time factorial analysis were square transformed.Incubation measurements were performed as follows: coral fingers were transferred to 1 L incubation glass jars in three opaque polyethylene (PE)-boxes filled with reef water (∼ 70 L) to keep samples at constant ambient temperatures during incubations. One jar per PE-box, filled only with reef water, served as a control. All PE-boxes were placed in the shade to avoid warming of the water. Dark incubation started after acclimatizing the samples for 1–2 hours in the dark PE-boxes. Initial OE. coli position 781 to 1,060 CTATGCGCCTTGCCAGCCCGCTCAGtaAGGATTAGATACCCTGGTA3′ (784F) and 5′CGTATCGCCTCCCTCGCGCCATCAG(N)8ctCRRCACGAGCTGACGAC3′ (1061R). Primers include 454 LibA library adapters (underlined), a barcode (shown as N) http://www.ncbi.nlm.nih.gov/sra) under accession number SRA062645. Mann-Whitney U test was conducted in Sigmaplot 12 . To determine distinct Operational Taxonomic Units (OTUs) that were significantly associated with A. hemprichii under treatments of overfishing, eutrophication, or both, we used the statistical package indicspecies P<0.01) associated with one or several sample groups.DNA from flash frozen coral fragments and water filters was extracted using the Qiagen DNeasy Plant Mini Kit  according to the manufacturer’s instruction. Coral tissue was separated from skeleton using high-pressure air and extraction buffer, while filters were bead-beaten with extraction buffer for 1 minute. For water samples, DNA of all filters from week 1 and of all filters from week 16 were pooled resulting in two water samples that represent a comprehensive reef water microbial composition from week 1 and week 16. For PCR amplification of the 16S rRNA gene, we used the primers 784F and 1061R that amplify in-situ treatments and those represented biological variance (a measurements and colony B differed from C in zooxanthellae abundance after week 16 (Symbiodinium clade over the duration of the experiment (clade C41), banding profile designation and sequence analyses were congruent  and in soluble reactive phosphate (SRP) . A paralvariance . More sp week 16 . All corongruent . Hence, P = 0.011, median number of OTUs week 1 = 120, median number of OTUs week 16 = 432, all specimens were subsampled to 3,283 reads). This observation is also supported when counting the number of distinct OTUs that we detected. We identified a total of 4,442 distinct OTUs of which 3,290 were found in corals and 1,432 in water. After week 1 we found 818 OTUs in all coral samples, after week 16 we identified 2,780 OTUs in all coral samples. Accordingly, bacterial diversity of corals increased over time. The Inverse Simpson Index for diversity varied from 2.14  to 16.41 . Overall, bacterial diversity between corals was highly variable but lower than in the water column. However, Simpson Evenness estimates ranged from 0.01 to 0.04 indicating few dominant bacterial taxa.We sequenced a total of 166,741 reads, of which 112,414 were left after error correcting, chimera detection, and undesirable mitochondrial and chloroplast sequence removal. We retrieved the highest number of reads from the water sample from week 1 , and the lowest number of reads from coral colony C, week 1, cage treatment . One coral fragment was dead after 16 weeks and left out from the remainder of the analyses . Good’s estimator of coverage A. hemprichii, irrespective of time point or treatment. Most interestingly, only two OTUs made up>99% of all Endozoicomonas bacteria (these OTUs could not be classified to the species level). In total, seven OTUs  were consistently found across all coral samples and on average accounted for >72% of the total number of bacteria  and two bacteria in the phylum Bacteroidetes (family Flavobacteriaceae) that made up 3% of all reads. Hence, in contrast to the substantial microbial richness identified in A. hemprichii , and also, but not significantly, according to coral colony and/or treatment . Interestingly, specimens from week 1 separated mainly along axis 2, whereas specimens from week 16 separated mainly along axis 1, indicating that the factor(s) driving community composition were different for both time points.To further analyze differences in bacterial community composition between colonies, treatments, and time points (beta diversity), we summarized unweighted UniFrac distances between samples with principal coordinate analysis (PCoA) P<0.01  was significantly associated with the cage & fertilizer treatment in week 1, but was also present in the cage & fertilizer treatment in week 16 and the cage treatment in week 1. After week 16, we identified nine OTUs that were significantly associated with an experimental treatment or a combination thereof. Two OTUs were overrepresented upon cage and fertilizer treatments, but interestingly not in the combined cage & fertilizer treatment. Note that samples from only two colonies were analyzed for the combined treatment in week 16. The two OTUs belonged to the phylum Proteobacteria in the families Desulfovibrionaceae (Desulfovibrio capillatus) and NB1-i (not further classified) and were not identified in week 1. Seven OTUs were significantly enriched upon fertilizer exposure. Of these OTUs, a bacterium from the genus Defluvibacter showed a very strong increase in abundance in comparison to all other treatments and time points. OTU counts ranged from 111 to 285 in all three coral specimens in week 16. In comparison, in all other treatments this OTU was either absent or present in low numbers . The remaining six OTUs were exclusively identified after week 16 and belonged to the families Balneolaceae, Marinilabiaceae, and Saprospiraceae (phylum Bacteroidetes) and Phyllobacteriaceae, Rhodobacteraceae, Campylobacteraceae (phylum Proteobacteria).In addition to community level patterns, we analyzed the association of single OTUs to time points and experimental treatments. We identified ten OTUs that were significantly associated to one or more experimental treatments at a significance level of P<0.01 . One OTUWith a complex experimental design that entails single and combined treatments, we were aiming at better separating the relative contribution of nutrient enrichment and overfishing to a combined manipulation. Interestingly, neither coral physiology data nor bacterial community data indicated that the combined treatment of cage & fertilizer is a mere combination of the effects of each treatment on its own. Rather, each treatment produced a distinct and complex response that was not necessarily identified in the simultaneous and combined treatment. Furthermore, as a consequence of conducting a time course study during the summer months, all samples were subject to an increase and subsequent decrease in water temperature over the course of the project. While the average water temperature of week 1 was 28.7°C , the average water temperature after 10 weeks was 31.8°C , and the average water temperature in week 16 was 30.2°C . We see that the microbial community increases over time in all coral samples and this could be attributable to the variable temperature regime all samples were exposed to. Secondly, the coral fingers where subjected to the experimental conditions after being harvested from the respective mother colony without a time of acclimation. However, coral holobiont performance did not show a dependence on time, as physiological measures were similar after week 1 and week 16. Rather, physiology had a strong association with the coral colony the samples were taken from. Furthermore, all coral fingers were sampled at the same time, so that differences between treatments did still reflect biological variation as a response to experimental treatment.a levels, zooxanthellae abundance, or coral and algal isotopic nitrogen ratios. Hence, the consequences of overfishing and eutrophication on coral holobiont physiology in this study may have been too subtle to affect measured parameters. At the same time, however, high nutrient loads do not necessarily have negative effects on holobiont performance (e.g. photosynthetic performance) as long as they are balanced Acropora hemprichii may have proven more resilient to our experimental treatments than other coral species that have not been investigated here. Last, our experiment lasted for a total of 16 weeks. Overfishing and nutrient enrichment are often anthropogenically induced processes that can last much longer than the here chosen time frame, so that we may also have to consider a temporal component. Nonetheless, we were able to detect differences in the microbial community structure upon treatments and this underlines the importance of conducting field studies in a holobiont framework, taking both physiological and molecular measures into account.A study by A. hemprichii harbors hundreds of different OTUs and our results are in accordance with previous estimates of coral microbial diversity While we did not see significant differences in holobiont physiology as a result of either of the treatments, we could clearly pinpoint differences in the microbial community after treatments and in both time points. Most notably, the majority of bacteria (>72%) did not change over time or treatment and were comprised of only seven OTUs. Accordingly, microbial changes were proportionally small and overall holobiont function  may have been ‘conserved’. Nonetheless, our data indicate that Endozoicomonas have now been identified in a number of studies and seem to be present in many marine invertebrates Endozoicomonas is predominant in A. hemprichii across its range, given its strong presence irrespective of time or treatment in our study. Furthermore, it is interesting to note that only two OTUs made up>99% of all Endozoicomonas bacteria. This indicates that the association of Endozoicomonas to A. hemprichii is very specific. Different members of the Oceanospirillales order (to which Endozoicomonas belong) include obligatory heterotrophic rod-like bacteria known for biofilm production that allows other bacteria to colonize surfaces Endozoicomonas suggesting that they may play a key symbiotic role as an “architect microbe” that structure and contribute to microbial community function.UniFrac distance significantly separated samples according to time. However, it seems that the factors driving sample separation were not the same between both time points as samples from week 1 show little differentiation on axis 1 but high differentiation on axis 2. In contrast, and to a lesser degree, samples from week 16 did separate along axis 1 and showed less variance on axis 2. In line with a longer treatment exposure we would expect a stronger separation by treatment after week 16, but the phenotypic reaction may be complex and the clustering of samples from week 16 a result of origin, treatment effect, and time. The results from our microbial species analysis however do support a stronger treatment effect with time.Nautella representing marine bacteria belonging to the Roseobacter lineage of the Alphaproteobacteria. Up to date little is known about this type of bacteria but one member, Nautella sp. R11, has been shown to cause bleaching in the temperate-marine macroalga Delisea pulchra while switching from an opportunistic to a pathogenic lifestyle Defluvibacter was highly abundant. Defluvibacter are aerobic denitrifiers, and members of this family are found in activated sludge from wastewater treatment plants 2 fixation still occurs at high ambient concentrations of nitrate 2 fixation in eutrophic environments may be attributed to protecting the enzyme nitrogenase from inactivation In the analysis of bacterial OTUs specifically associated with treatments, we found more OTUs in single treatments after week 16 (nine OTUs vs. one OTU after week 1), and we also found a higher abundance of significantly associated OTUs after week 16. The finding that coral associated microbial communities respond to treatments supports previous findings that show that bacterial assemblages vary under different conditions such as increased temperature, elevated nutrients, dissolved organic carbon loading, and reduced pH in situ fertilizer diffusion, caging, and both over a period of 16 weeks on a coral reef in the Central Red Sea. While the physiology of the coral holobiont did not show significant differences in regard to O2 consumption, zooxanthellae counts and identity, chlorophyll a, or nitrogen isotopic ratios in the coral tissue as a function of experimental treatment, the bacterial communities derived from the different treatments illustrate that the microbial assemblage of the coral holobiont is variable and a consequence of mother colony, environmental conditions, and time. This underlines the importance of conducting field studies in a holobiont framework, taking both, physiological and molecular measures into account. Secondly, the functional associations of overrepresented bacteria in the treatments corresponded well with the environmental footprints of a given treatment . However, they were not stable over time indicating that the presence of potential indicator bacterial species may vary. Notably, the majority of bacterial cells of A. hemprichii were provided by a few OTUs of the genus Endozoicomonas that formed a stable association.This study analyzed the distinct and combined effects of nutrient enrichment and herbivore exclusion via Figure S1Study site. Right panel shows position of the study area in the Red Sea. The circle on the left panel indicates study site at the Northern tip of Al Fahal-reef about 13 km off the Saudi-Arabian coast.(EPS)Click here for additional data file.Figure S2Treatment scheme. Coral fingers were attached to stainless steel screws on PVC frames with cable ties. A: cage treatment imitating overfishing pressures, frame with coral fragments underneath. B: fertilizer treatment imitating eutrophication pressures with slow releasing fertilizer diffusing out of the red bars. C: combined treatment of cage & fertilizer as in A and B.(EPS)Click here for additional data file.Figure S3Physiological parameters of corals and zooxanthellae. The left column shows the comparisons of treatments, and the right column the comparisons of coral colonies. Given P-values are from a 2-factorial ANOVA. See (EPS)Click here for additional data file.Figure S4DGGE banding pattern of ITS2 from all coral samples. Please note that the coral samples are not depicted in a temporal order.(PDF)Click here for additional data file.Table S1Overview over sequence counts, taxonomic classification, and 16S reference amplicon sequence for all OTUs identified.(XLSX)Click here for additional data file.


To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008–2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondônia State, leading to the establishment and spread of this pathogen. VACMato Grosso State is partially inserted into the southeastern Amazon biome  and has From June 2008 through June 2010, we investigated outbreaks of poxvirus-related disease on 56 dairy farms of Mato Grosso and Rondônia . A proba,,,,,,A total of 52 samples of scabs were collected from cattle on 23 farms. DNA was isolated as described ,,,,,, were poAn epidemiologic investigation was conducted by using data available from the 56 epidemiologic investigation forms, analyzing cattle movement forms issued during this period, and interviewing farmers and animal health agency veterinarians. In addition to the 23 farms with positive samples, 33 other farms had epidemiologic links to CTGV-positive farms. Therefore, our data suggest that CTGV infection was the cause of all outbreaks.>100 cattle (mean=253), in contrast to 90% of southeastern farms, which had <100 cattle  . These rBR-364 is a 4,141.5-km highway with a major veterinary inspection station, the Amazon Gate, at the Mato Grosso–Rondônia border. Affected farms were mostly located along this main road connecting Mato Grosso with the Amazon region , panel A,2 in contrast to 17.44 cattle/km2 in Mato Grosso. The occurrence of new CTGV outbreaks in 2010 supports a nonsporadic pattern of infection in Rondônia, highlighting the establishment of this zoonotic infection within the state.Cattle trade during the auction launched virus spread throughout the state, which was amplified by subsequent trading and human translocation . Animal –This study investigated the appearance, establishment, and spread of CTGV infection into the Amazon biome. Our findings suggest that animal movement was the main cause of virus long-distance dissemination in Rondônia, whereas the migration of dairy workers was involved in the focal spread. Animal movement plays an essential role in cross-border spread of several disorders worldwide, including bovine tuberculosis, foot-and-mouth disease, and bovine diarrhea viruses –, panel B