Colobanthus quitensis and Deschampsia antarctica in Admiralty Bay, King George Island, Maritime Antarctic. Microarray and qPCR assays targeting 16S rRNA genes of specific taxa were used to assess microbial community structure, composition and abundance and analyzed with a range of soil physico-chemical parameters. The results indicated significant rhizosphere effects in four out of the six sites, including areas with different levels of bird influence. Acidobacteria were significantly more abundant in soils with little bird influence (low nitrogen) and in bulk soil. In contrast, Actinobacteria were significantly more abundant in the rhizosphere of both plant species. At two of the sampling sites under strong bird influence (penguin colonies), Firmicutes were significantly more abundant in D. antarctica rhizosphere but not in C. quitensis rhizosphere. The Firmicutes were also positively and significantly correlated to the nitrogen concentrations in the soil. We conclude that the microbial communities in Antarctic soils are driven both by bird and plants, and that the effect is taxa-specific.Understanding the environmental factors that shape microbial communities is crucial, especially in extreme environments, like Antarctica. Two main forces were reported to influence Antarctic soil microbes: birds and plants. Both birds and plants are currently undergoing relatively large changes in their distribution and abundance due to global warming. However, we need to clearly understand the relationship between plants, birds and soil microorganisms. We therefore collected rhizosphere and bulk soils from six different sampling sites subjected to different levels of bird influence and colonized by  Deschampsia antarctica Desv. (Poaceae) and Colobanthus quitensis (Kunth) Bartl. (Caryophyllaceae) are the only two native vascular plants in Antarctica. Both species are widespread, usually growing together and well adapted for life in the coastal Maritime Antarctic D. antarctica and C. quitensis in the western Antarctic has been reported and interpreted as a response to more favorable growing and reproductive conditions D. antarctica and C. quitensis did not differ significantly Through root exudation, plants produce a variety of chemical compounds that may be used by soil bacteria for energy and biomass production Firmicutes 16S rRNA gene sequences related to ornithogenic soils Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes from Adelie penguin guano.In the Maritime Antarctic, densely vegetated sites are often related to bird activities Firmicutes and Actinobacteria representing more than 70% of the total community Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria and Cyanobacteria, with a strong influence of the vegetation cover, highlighting the potential for indirect effects of global warming on Antarctic soils Acidobacteria, Bacteroidetes, Proteobacteria and Firmicutes were the main phyla recovered in nine different soils of Livingston Island, Maritime Antarctic Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria and FirmicutesRecent studies have provided a baseline knowledge of the microbial communities in terrestrial Antarctic environments D. antarctica and C. quitensis rhizosphere and in bulk soils from six different sampling sites around Admiralty Bay, King George Island, Maritime Antarctic. Soils with different levels of bird influence were compared to adjacent rhizosphere soils using 16S rRNA gene microarrays and phylum/class-specific quantitative PCR genes and the resulting patterns were analyzed together with soil physico-chemical parameters.Global warming is currently affecting both plant All necessary permits were obtained for the described field studies, from the Brazilian Antarctic Program, PROANTAR, as part of the IPY  Activity # 403 and MMA (Brazilian Ministry of Environment).The study was carried out at the Brazilian Antarctic Station Comandante Ferraz , located in Martel Inlet, Admiralty Bay, King George Island, Antarctic Peninsula, which is part of the South Shetlands Archipelago in Maritime Antarctica. During the austral summers of 2006–2007 and 2008–2009, the vascular plants D. antarctica or C. quitensis were sampled, where both plants were found, in triplicate at six different sites: A – Arctowski (E:423.807/N: 3.106.863–2006–2007), Q – Quimica (E: 427.335/N: 3.115.506–2006–2007), I – Ipanema (E: 426.570/N: 3.116.513–2006–2007), M – North Mountain (E: 426.63/N: 3.116.587- 2008–2009), D – Demay Point (E: 425.102/N: 3.100.854–2008–2009), C – Copacabana (E: 424.706/N: 3.105.149–2008–2009). Colobanthus quitensis were not found at points C and M. Points A, C and D were located inside an environmental protected area. Point A is close to the Arctowski Polish Station and next to a colony of Adelie penguins (Pygoscelis adeliae), point C is next to the USA summer station Copacabana in a Gentoo penguin (P. papua) colony, and point D is near to a Polish refuge next to a colony of Chinstrap penguins (P. antarcticus). At point I, there were no penguin colonies present, but this section was used as a nesting site by local species of flying birds. Point Q was located in the vicinity of the EACF; thus there has been (and continues to be) an intense anthropogenic influence on this spot, which is not the case at the other sampling sites. Besides, there is a transient presence of birds. Point M was located at the top of North Mountain, around 200 m altitude. This site has no influence from penguin colonies and only a few nests of skua (Catharacta sp.) were observed.At each sampling site, triplicate soil samples  were taken for chemical and biological analyses, with the exception of the Arctowski site (A) where we only take two replicates. Soil samples were preserved at 4°C for chemical and at −20°C for biological analyses. The plants occur in the same area, not necessarily growing one next to the other. Furthermore, we collected rhizosphere soil from isolated plant roots. Each vascular plant sample was frozen (−20°C) at the EACF. To assess the bacterial communities associated with the rhizospheres of both vascular plants, 5 g of the roots with aggregated soil was shaken in a 45 mL of saline solution (NaCl 0.85%) for 2 hours. Then, the supernatant (without roots) was centrifuged for 10 minutes at 9,000 rpm, and 0.5 g of the pelleted soil was used for DNA extraction using the Fast DNA Spin Kit for soil  following the manufacturer’s instructions. For the bulk soil, 0.5 g of each sample was used for the extractions also using the Fast DNA Spin Kit. The extracted DNA was quantified using the PicoGreen® dsDNA quantitation assay . The integrity of the DNA extracted from the soils was confirmed by electrophoresis on a 0.8% agarose gel in 0.5x TBE buffer .2+, Mg2+ and Al3+ extracted in 1 M KCl; P, Na and K by Mehlich-1 extractant (HCl 0.05 mol L-1 and H2SO4 0.0125 mol L-1)and pH ; Potential acidity: H+Al extracted with 1 N calcium acetate (pH 7), titrated with 0.0125 NaOH N, were analysed according to 2+, Mg2+ and Al3+, flame emission (K and Na) and photocolometry (for P) were used for nutrient determinations. Total organic C was determined by wet combustion Chemical analyses on air dried, sieved (2 mm), subsamples of bulk soil samples were performed. A single subsample/replicate was used for the following analyses. Exchangeable cations: Ca4+) and nitrate (NO3−) contents in the extracts were determined by use of an automatic flow injection (FIA) analysis system. The ammonium (NH4+) was quantified colorimetrically using the Solorzano method 3−) estimated by conductivimetry in the form of nitrite (NO2−), after being reduced with a cadmium base catalyst.For the nitrogen quantification, all the bulk soils replicates were analyzed. Mineral nitrogen forms were first extracted from the soil samples with a KCl (2N) solution. The ammonium (NH5 copies per µl) were mixed. When the recovery of lambda was below 100%, quantification values for all other genes were corrected accordingly.The primers and qPCR conditions used are summarized in 2, 2.5 U of Taq DNA polymerase (Fermentas), 0.2 mM of each dNTP and sterile filtered water to a final volume of 50 µl. The temperature profile included an initial denaturation step at 94°C for 5 min, 35 cycles of a denaturation step at 94°C for 30s, a primer annealing step at 50°C for 30s and an extension step at 72°C for 45s, followed by a final step of 7 min at 72°C. PCR products were purified using the PureLink ™ PCR purification Kit . A microarray platform targeting the 16S rRNA genes of Bacteria and Archaea found in cold environments was used as previously described For the microarray assays all the triplicate rhizosphere and bulk soil samples from the six sample sites were used. A 16S rRNA gene fragment was amplified using the universal primers F1-R13 C. quitensis, D. antarctica, no plant), site and nitrogen on the microbial community structure. Spearman linear correlation analyses (correlations on qPCR vs. soil data) were performed using the “cor.test” function. Levels of significance were corrected for multiple comparisons using the method of Bonferroni All statistical analyses were performed in R . For analysis of variance (ANOVA) of the qPCR data, normality was tested using the “shapiro.test” function and homoscedasticity was tested using the “bartlett.test” function. When necessary, data were log transformed to meet parametric ANOVA assumptions. ANOVA and post hoc Tukey HSD tests were then carried out using the “aov” and “TukeyHSD” functions, respectively. When transformations failed to normalize data, Kruskal-Wallis and associated multiple comparison tests were performed using “kruskal.test” and the “kruskalmc” functions of the “pgirmess” library, respectively. Spearman linear correlation analyses (correlations on qPCR vs. soil data) were performed using the “cor.test” function. For the microarray analysis, distance matrices were calculated using the ”vegdist” function of the ”vegan” library and principal coordinate analyses (PCoA) were carried out using the ”cmdscale” function based on the square root of 1-Jaccard. PerMANOVA 4+ and NO3−) were very high for the sites under the influence of penguins, very low in the sites under the influence of other birds (Q and I) and below the detection limit for the M site. For all sampling sites NH4+ values were higher than NO3− values. Site C had nitrogen values similar to the other sites under the influence of penguins, but lower values of exchangeable Ca and Mg.The three sites located in penguin colonies  had similar soil chemical characteristics, with low pH and high bioavailability of P . The twoC. quitensis and D. antarctica were detected for most sampling sites, with the exception of the A site where it was possible to see a clear segregation between the microbial communities associated with the rhizosphere of the two plant species  and the Firmicutes when considering all samples together. In the bulk soils from sites I, Q and M, Acidobacteria was also abundant , where this group were more representative.A PCoA analysis was also carried out using only the microarray profiles of bulk soils in order to compare the general microbial community at different sites around Admiralty Bay . Replicaer sites . The BacActinobacteria and the Firmicutes and the ammonia concentrations in soil  and NO3− .The statistical correlations between the soil physical and chemical composition and measured microbial communities were performed, but gave no significant result for most soil abiotic characteristics (data not shown). Only NH4 and NO3 presented some correlation. In order to check the relationship between nitrogen and soil bacterial communities in Admiralty Bay, correlation analyses were carried out . Using t in soil . A signiOur results showed that both plant and bird presence had significant influences on microbial community structure and on the relative abundance of several major bacterial taxa. The presence of birds was related to higher soil nitrogen concentrations, which was identified as significantly correlated with the abundance of several bacterial groups and as significantly influencing the microbial community structure. In the case of plants, it appeared that the effect observed was a general rhizosphere effect, as very little difference was seen between the rhizosphere of the two vascular plants sampled, in line with previous reports Catharacta sp), giant-petrels (Macronectes sp) and seagulls (Larus sp), leads to the development of weakly ornithogenic soils  while the presence of penguin colonies leads to highly ornithogenic soils (very high N and P concentrations and acidic pH). Based on the results from our soil analyses and from previous studies of Admiralty Bay soils As expected, we observed a strong relationship between microbial communities and soil characteristics, but we could not disentangle the effect of nutrient availability from any pH-related effect, as the sites with high nitrogen and phosphorus concentrations had also the lowest pH values. These characteristics are typical of ornithogenic soils as bird guano, feathers, eggshells and remains are rich in nitrogen and phosphorus Pygoscelis adeliae and P. antarctica  feed on krill, mainly Euphrasia superba, while P. papua (the species found in site C) eats krill but also fish, particularly Notothenia species P. papua, which colonizes site C, is at least partially responsible for the different microbial community patterns observed at this site.According to PCoA generated from the microarray data, the bacterial communities were strongly shaped by the nitrogen level in the soil, and consequently, to the bird influence. Samples of weakly ornithogenic soils generally grouped together (sites Q and I), while the non-ornithogenic soils (site M) formed a separate group. However, for the highly ornithogenic soils  two separate groups could be observed: one comprising samples from sites D and A, and another comprising only samples from site C. Interestingly, each of these sites is occupied by different penguin species which have different diets. Studies on penguin diet showed that Acidobacteria were more abundant in the non- and weakly ornithogenic soils and were consequently negatively correlated to ammonia. Similarly, Ganzert and colleagues Acidobacteria sequences were related to Antarctic soils with low C and N contents and neutral to alkaline pH values. In contrast, the Firmicutes and the Actinobacteria were positively related to ornithogenic soils, in line with previous studies Gammaproteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes directly from Adelie penguin guano. From our data it is not possible to deduce any functional information about the microbial communities studied, but Zhu and colleagues Corroborating the idea that bird presence has a fundamental role in shaping the microbial communities of Maritime Antarctic soils, due to the high N and P inputs in the soils, samples from the non-ornithogenic M site presented the most distinctive bacterial community structure and composition, probably due to the low availability of nutrients and higher pH. The C. quitensis and D. antarctica, in line with a previous report that used pyrosequencing and DGGE It was recently hypothesized that microbial communities were mainly driven by soil characteristics, but that within a soil type, plants shaped the microbial communities in their rhizosphere D. antarctica and C. quitensis rhizosphere samples from Admiralty Bay, it was shown that the Firmicutes were the most abundant phylum followed by the Proteobacteria and then the ActinobacteriaActinobacteria followed by Proteobacteria and Firmicutes. These discrepancies could be due to the fact that we did not use primers targeting the same positions in the 16S rRNA gene for both techniques. Furthermore, the primers used for the Actinobacteria in qPCR are not perfectly specific to this group and unspecific amplifications could artificially increase their abundance in qPCR assays Actinobacteria and the Proteobacteria are the main bacterial taxa present in the soils of Admiralty Bay. Similar results were found in several other studies on soil bacterial diversity around the world In a previous study from our group that used 16S rRNA gene pyrosequencing on D. antarctica and C. quitensis) and of birds play important roles in shaping the microbial communities of Antarctic soils. For the plant influence, the observed rhizosphere effect varied between the different sampling sites, probably due to differences in soil parameters, indicating that plant colonization will not have the same effect on microbial communities in all soils. Firmicutes and Acidobacteria were more abundant in bulk soils while Actinobacteria was more abundant in rhizosphere samples. For the bird influence, the effect varied with the level and the type of ornithogenic input and, in highly ornithogenic soils, seemed to be also modulated by the species of bird present, due to differences in diet. This indicates that the soils associated with bird colonies will influence microbial communities differently depending not only on the type of birds (flying birds vs. penguins), but also on the diet of the birds. Based on these results, we can hypothesize that not only will climate change directly influence Antarctic soil microbial communities, but it will also indirectly influence them through changes in plant and bird populations.Our results showed that the presence of vascular plants  of different penguins and even test the chemical attributes of exudates (or rhizosphere) of the two plants to confirm/deny the correlations, which may explain the resulting overlapping bacterial communities.


Substrates in medical science are hydrophilic polymers undergoing volume expansion when exposed to culture medium that influenced on cell attachment. Although crosslinking by chemical agents could reduce water uptake and promote mechanical properties, these networks would release crosslinking agents. In order to overcome this weakness, silicone rubber is used and reinforced by nanoclay.Attempts have been made to prepare nanocomposites based on medical grade HTV silicone rubber (SR) and organo-modified montmorillonite (OMMT) nanoclay with varying amounts of clay compositions.Incorporation of nanocilica platelets into SR matrix was carried out via melt mixing process taking advantage of a Brabender internal mixer. The tensile elastic modulus of nanocomposites was measured by performing tensile tests on the samples. Produced polydimetylsiloxane (PDMS) composites with different flexibilities and crosslink densities were employed as substrates to investigate biocompatibility, cell compaction, and differential behaviors.The results presented here revealed successful nanocomposite formation with SR and OMMT, resulting in strong PDMS-based materials. The results showed that viability, proliferation, and spreading of cells are governed by elastic modulus and stiffness of samples. Furthermore, adipose derived stem cells (ADSCs) cultured on PDMS and corresponding nanocomposites could retain differentiation potential of osteocytes in response to soluble factors, indicating that inclusion of OMMT would not prevent osteogenic differentiation. Moreover, better spread out and proliferation of cells was observed in nanocomposite samples.Considering cell behavior and mechanical properties of nanobiocomposites it could be concluded that silicone rubber substrate filled by nanoclay are a good choice for further experiments in tissue engineering and medical regeneration due to its cell compatibility and differentiation capacity. Cells within living tissues are sensitive to biochemical signals as well as to mechanical factors in their environments . PhysicoThe sensitivity of cells arises from mechanosensitive nature of cell adhesion . Cell-maRecent studies have reported that regulation of substrate elasticity in two-dimensional cell cultures affects the differentiation and behavior specifications of cells . MechaniHence in this study, we synthesized OMMT/SR nanocomposites in order to create a substrate with different stiffness and elasticity. Then the responses of endothelial cells and ADSCs to altered mechanical properties were analyzed.95) obtained from curing curves in a very low thickness.Medical SR was vulcanized at high temperatures and clay nanoparticles, Closite 15A, were supplied by Southern Clay Products, USA. Dicumyl peroxide was employed in preparation and cure of the samples. Initially, nanocomposites were prepared through melt mixing method. The rubbery compound was mixed in a Brabender mixer with OMMT mass ratios of 1%, 2%, and 3% at 60 rpm and 60°C for 20 minutes [19]. The samples were vulcanized at 160°C by compression molding at optimum cure time  and penicillin/ Streptomycin (concentration: 2%), and stirred continuously in an incubator for 30 minutes. Collagenase was neutralized by 5 ml of DMEM-20% FBS. The samples were extensively pipetted to separate connective tissue parts. Obtained samples, then, were centrifuged twice at 2000 rpm for 5 minutes. The cells were suspended in DMEM-10% FBS, transferred into 12-well plates and placed in an incubator for 72 hours to adhere to the plate . At 85% Mechanical properties, including tensile strength and elastic Moduli at 100 and 300 percent, were performed using at least three dumbbell shaped samples and a tensile testing device -Italy) according to ASTM D412 .2 incubator. To study cell morphology on membranes, the samples were placed firstly in a culture plate and 5 x 103 HUVECs with culture medium were added to each well. Subsequently, cells-included culture plate was incubated at 37°C in a CO2 incubator to adhere cells to the membranes after whichseveral images were captured via inverted microscope  overnight  were provided by the originator , and cultured in DMEM + Ham’s F12  containing 10% FBS  in a humidified 5% COvernight . Cell prt 570 nm .3 cells were put in the vicinity of the substrates as well as in a differential medium containing DMEM Ham’s F12,10% FBS, 10 mM ß-glycerophosphate, 50 µg/ml sodium ascorbate 2-phosphate, and 100 U penicillin/100 µg streptomycin/0.25 µg fungizone. The cells were incubated for up to 21 days, and osteogenic medium was replaced every 3 days. Afterwards, the cells were rinsed in 0.9 % sodium chloride and fixed by 70% alcohol. Alizarin red staining was performed to assess functional capacity of nanobiocomposites in the presence of growth factor (Confluent ADSCs (passage number = 2) were detached and counted; then 5 × 10h factor .t-test. Data was presented as mean values ±S.D. and P < 0.05 was considered statistically significant.The data were analyzed by using the There is a hypothesis expressing that mechanical properties of a substrate on which cells grow governs their behavior . To studIn tissue engineering, the cell fate and maintenance of stem cells to different lineage such as osteogenic would be manipulated by many factors including chemical stimulations of osteogenesis conducted by various proteins  and physP < 0.05). Moreover, addition of OMMT particles leads to statistically significant stiffening of the substrates (P < 0.05). This trend of increase in elastic modulus is consistent with tensile tests when performed in both 100% and 300% elongations.To study effects of substrate elasticity on cell behavior, nanocomposite substrates were used by dispersion of differing mass ratio of nanoparticles of OMMTs in silicone rubber. Previous studies have shown that composites based on HTV silicone rubber polymer and silicate platelets produced transparent nanocomposites with high surface energy originated from filler tactoids with thickness of about 10-15 nm in reference polymer matrix , 28. WelResults indicated significant influence in polymer matrix on mechanical properties of the substrate. Addition of OMMT resulted in stronger and stiffer matrix. Decrease of the crosslinking density which was evaluated from cure curves cited that nanofiller acts as an inhibitor. In other words, addition of nanoparticles into a polymer matrix would prevent chemical crosslinking and causes physical integrity  with a sIn addition to enhancement of mechanical properties, cytotoxicity and biocompatibility analyses revealed improved cell behavior on the modified substrates. Addition of nanoparticles did not result in toxic effects and the resultant substrates found to be biocompatible. Cell growth and alignment was smoothly enhanced in nanocomposite substrates. It has been shown that inorganic nanofillers have higher surface energy than polymer matrix . SiliconMechanical properties of substrate influences cell functionality. Cytoskeletal arrangement and orientation is highly dependent on mechanical and structural properties of the matrix such as elastic modulus, Poisson’s ratio, and roughness , 26. WheIn this work, we applied a comparatively easy method to fabricate a new type of substrate on which a cell could attach, proliferate and differentiate. It is also interesting to reveal that the mechanical properties have a very strong effect on cell morphology. Therefore, by controlling the elastic modulus of the composites, we can adjust cell compatibility, which may have the key role for applications in tissue engineering and regenerative medicine. This kind of alignment of the HUVECs on the rubber membrane could be of advantage for a more effective presence of OMMT. However, loading nanoparticles also enhances tensile strength and young’s modulus as evinced by the tensile experiments. Moreover, the results demonstrated that application of nanofiller into the polymer matrix could be a way to come by better proliferation and adhesion of cell lines. The elastic moduli of corresponding nanobiocomposites could be improved by increasing in the amount of OMMT, as their tensile strength could be done. In other words, adding certain ratios of nanoparticles resulted in substantial increase in the desirable mechanical properties. These results originate in good dispersion of the clay as well as strong interfacial interaction between polymer matrix and filler, which were best reflected in tensile parameters, because the strength values were remarkably promoted.


Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community  over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.Presently there is no vaccine against  Neisseria gonorrhoeae, a fastidious Gram-negative diplococcus, is an obligate human pathogen infecting mucosal surfaces and causes the gonorrhoea, the most predominant bacterial sexually transmitted infection worldwide. The spread of the organism occurs through direct person to person contact and infections occur exclusively in humans. In males, infections commonly cause urethritis and cervicitis in females. Rectal and pharyngeal infections can occur in both sexes and complications from adjacent spread of the microorganisms along mucosal surfaces may result in endometritis, salpingitis, and peritonitis in women, and bartholinitis in men. Both sexes may risk infertility if the infection is left untreated and the organism is one of the most common causes of female infertility worldwide N. gonorrhoeae and therefore preventive measures based on clinical and epidemiological analyses the identification of the organism and effective antimicrobial treatment are key strategies used to limit the spread of the infection. Thus, a desirable method for typing N. gonorrhoeae isolates so that isolate relatedness can be ascertained over long and short time periods should possess a high resolution power, reproducibility, comparability and objectivity.N. gonorrhoeae have been developed over the years, ranging from auxotyping N. gonorrhoeaeNeisseria gonorrhoeae multiangen sequence typing (NG-MAST) and porB DNA sequence analysis, target two hypervariable genes encoding outer surface proteins A number of typing methods for porB DNA sequence. Since these two groups of genetic markers, housekeeping and antigenic loci, have quite different genetic and evolutionary characteristics, we aimed to investigate their possible influence and outcome on genotyping analyses using the same panel of N. gonorrhoeae isolates collected worldwide. We examined 80 gonococcal isolates originating in China, North and South America that were collected over different time periods. In addition, we revealed a geographic population structure of tested N. gonorrhoeae isolates and their evolutionary relationship, using a combination of the concatenated sequence of the housekeeping loci and molecular evolutionary analyses.To evaluate the impact of different genetic markers, on the genetic relatedness of gonococcal isolates and subsequently on genotyping analysis of this human pathogen, we analyzed 10 housekeeping genes N. gonorrhoeae isolates from six geographic locations worldwide were analyzed. In 2008, 41 isolates were obtained from the Saskatchewan Disease Control Laboratory (SDCL) Canada, and 23 were collected at the STD Clinic of the Shanghai Skin Disease and STD Hospital, Shanghai, China. Another 14 N. gonorrhoeae isolates were collected between 1982 and 1986 in South America,  and were part of our culture collection N. gonorrhoeae isolates were American in origin and are well characterized laboratory isolates, F-62 and FA1090 80 PorB was typed as described previously and included an analysis of 85% of the entire gene PorB was amplified with porB-F and porB-R primers N. gonorrhoeae genes encoding enzymes for intermediary metabolism, we selected 10 housekeeping genes for analysis, including abcZ, adk, aroE, glnA, gdh, pyrD, pdhC, gnd, fumC and pgm. These loci were selected based on their polymorphisms to ensure an application of informative markers distributed throughout the gonococcal genome.Based on two previous studies, Bannett et al., http:/blast.ncbi.nlm.nih.gov/Blast.cgi) were used as templates to design oligonucleotide primer pairs , 0.5 µM of each primer and 2 U of Taq polymerase . Temperature conditions included an initial 95°C denaturation step for 4 min., followed by 30 cycles of: denaturation at 95°C for 60 sec., annealing at 55°C for 30 sec., amplification at 72°C for 30 sec. and a final extension step at 72°C for 10 min.Novel primers were designed for each housekeeping gene using the Primer 3 program er pairs  which brwww.mlst.net). Each unique combination of allele numbers was assigned as a different sequence type (ST). The average frequencies of synonymous (dS) and nonsynonymous (dN) substitutions per site were calculated for each locus using the Nei-Gojobori method porB sequences, respectively by using a combination of several methods including: unweighted pair group method with arithmetic mean (UPGMA) http://eburst.mlst.net and edited as required to produce the final figure. Editing did not change any of the links between the STs and was done to improve the clarity of the diagram.Amplicons were purified by a PCR purification kit  and their nucleotide sequences on both strands were determined using an Applied Biosystems 3730×1 DNA Analyzer . DNA sequences were analyzed using DNASTAR Lasergene 7 . All sequences for each locus were compared and every unique sequence was assigned as a distinct allele using Sequence Output  porB sequences ranged from HM481249–481254.Novel nucleotide sequences were deposited in GenBank. Accession numbers for the DNA sequences of the different housekeeping genes extended from HM359017 to HM359073 and the accession numbers for the pgm, pdhC, and adk loci to 17 for the glnA locus. The high polymorphism of the glnA locus owned to haplotype  7, where a cluster of 13 polymorphic sites was found over a short 55- bp region of the locus. Except for glnA where all variable sites were concentrated in a ∼130 bp gene segment, the polymorphic sites for all other loci were randomly distributed across the entire locus. The mean of the pairwise divergence was 0.002 with the most divergent locus being glnA (0.0037) followed by gnd (0.0033)  .pyrD locus showed the highest proportion of unique haplotypes (42.8%), followed by abcZ (33.3%) and glnA (28.5%). 26 haplotypes were uniquely distributed among specific geographic locations. The most numerous geographically distinct haplotypes, using the collection of 80 N. gonorrhoeae isolates, were found in Canada (n = 11), followed by China (n = 9), Venezuela (n = 2), the USA (n = 2), Chile (n = 1) and Argentina (n = 1).Overall, 58 haplotypes were identified among the 80 isolates with the 10 loci . The majSee Table S1. The index of discrimination (ID) for these 10 housekeeping loci was 0.953.The ratios of nonsynonymous and synonymous substitutions (dN/dS) for examined loci ranged from 0.026 to 0.633 , indicatN. gonorrhoeae isolates. ST22 was identified as a putative ancestral genotype of CC22, while ST24 and ST26 were identified as co-founders of the same clonal complex. Eight other STs - ST23, ST10, ST25, ST32, ST31, ST14, ST28, and ST27 - were found to descend from their ancestors ST22, ST24, and ST26. Another major CC, CC33, consisted of an ancestral genotype ST33, and four descent genotypes - ST3, ST34, ST18, and ST29. Minor CCs comprised two triplets  and one doublet , CC22 and CC33 and three minor CCs , among tporB gene nucleotide sequences, N. gonorrhoeae isolates were divided into two groups, porB1a (PIA) (n = 13) and porB1b (PIB) (n = 67). The majority of tested isolates from South America  were associated with the PIA group while most isolates originating from China and North America , belonged to the PIB group. Comparative analysis of porB nucleotide sequences revealed a profound variability of the PIB, accounting for 72 polymorphic sites, which resulted in a 0.0314 average pairwase distance  as a donor. Methods, number of individual recombination events and their P-values are shown in To determine how recombination events affected both N. gonorrhoeae isolates, providing accurate information for the identification of predominant strains, which, in turn, can be used to improve control measures and implement successful interventions. A typing method used for surveillance should be highly accurate and reliable so that epidemiologically unrelated individuals can be separated from each other by genetic fingerprinting and at the same time that highly predominant strains which circulate through population can be accurately detected by the same typing method Effective strain typing plays a pivotal role in the surveillance of N. gonorrhoeae isolates, we empirically validated the impact of these markers on genotyping of gonococcal isolates. Although there was a degree of consistency between the concatenated sequences of the housekeeping loci and porB locus, a most notable difference observed between the respective phylogenetic trees was a discordant topology observed in North American isolates. Strains that formed two major North American clusters in the housekeeping tree were mixed up and scattered across the porB tree  showed that these two STs represent two predominant strains, circulating within the province of Saskatchewan over a 4-year period (2005–2008). A possible explanation for the failure of the porB typing method to detect and separate these two circulating strains of N. gonorrhoeae is that some regions of the porB gene are under strong positive immune selection as shown by the Nd/dS ratio and recombination analysis. This would impose a distorting effect on the genetic relatedness of the isolates. Using Borrelia burgdorferi as a model organism, Margos et al., B. burgdorferi isolates analyzed by ospC, a hypervariable gene encoding the immunodominant outer surface protein C B. burgdorferi population. These results provide important evidence that the choice of genetic marker has a profound effect on the interpretation of epidemiological or population genetics results. Concatenated sequences of housekeeping genes can provide clonal stability further allowing discrimination of circulating strains or different bacterial ecotypes. In contrast, application of the commonly used genetic markers which are based on hypervariable DNA regions may result in an absence of phylogenetic signal indicating the presence of circulating strains or distinct bacterial ecotypes.In this study, using a sequence-based approach of two different types of genetic marker, housekeeping genes and an antigen encoding gene, of 80 geographically and temporally diverse isolates of orB tree . Using dporB was that the most numerous Asian cluster observed in the porB tree , ST3 (Canada), and ST18 (China). The eBURST analysis indicates that the smaller portion of the gonococcal population from Canada likely originated from somewhere else (South America and Asia). All minor clonal complexes were found in Canada, which most likely suggest that they represent an endemic gonococcal population. These endemic genotypes have been segregated for long enough to accumulate their own mutations and are diversifying and spreading over this geographic area.The low N. gonorrhoeae isolates will most likely have a profound effect on interpretation of genotyping results. Our study showed that the predominant N. gonorrhoeae strains from Saskatchewan, Canada, isolated in 2008 can be detected using the concatenated sequences of the housekeeping genes but not using a hypervariable genetic marker, porB. The concatenated sequences of the housekeeping genes retain the phylogenetic signals of the circulating strains and successfully identify them over a long period of time. On the other hand, the hypervaribale porB DNA sequence can be subjected to frequent mutation and recombination and therefore this type of genetic marker tends to introduce a distorting effect among genetically related isolates which may lead to a failure to detect the predominant strains of N. gonorrhoeae that circulate within a community over an extended period of time. Taken together, our data suggests that most likely a typing method based on antigen gene marker can be applied successfully only during most recent outbreaks. Application of these genetic markers in any other situation may result in more or less skewed picture. In addition, the concatenated sequences of the housekeeping genes can provide a high resolution power, discriminating unrelated strains. Using eBURST analysis, the study reveals that the gonococcal population from Canada was a mixture of endemic and introduced genotypes, with the endemic genotypes being predominant.The present study suggests that the choice of genetic marker, housekeeping or antigen genes, used for typing Table S1N. gonorrheae isolates used in this study.Details of (DOC)Click here for additional data file.


Background: Winter temperature inversions—layers of air in which temperature increases with altitude—trap air pollutants and lead to higher pollutant concentrations. Previous studies have evaluated associations between pollutants and emergency department (ED) visits for asthma, but none have considered inversions as independent risk factors for ED visits for asthma.Objective: We aimed to assess associations between winter inversions and ED visits for asthma in Salt Lake County, Utah.Methods: We obtained electronic records of ED visits for asthma and data on inversions, weather, and air pollutants for Salt Lake County, Utah, during the winters of 2003 through 2004 to 2007 through 2008. We identified 3,425 ED visits using a primary diagnosis of asthma. We used a time-stratified case-crossover design, and conditional logistic regression models to calculate odds ratios (ORs) and 95% confidence intervals (CIs) to estimate rate ratios of ED visits for asthma in relation to inversions during a 4-day lag period and prolonged inversions. We evaluated interactions between inversions and weather and pollutants.10 and maximum and mean temperatures were above median levels.Results: After adjusting for dew point and mean temperatures, the OR for ED visits for asthma associated with inversions 0–3 days before the visit compared with no inversions during the lag period was 1.14 . The OR for each 1-day increase in the number of inversion days during the lag period was 1.03 . Associations were only apparent when PMConclusions: Our results provide evidence that winter inversions are associated with increased rates of ED visits for asthma. Weather patterns and topographical features in Salt Lake County, Utah, are optimal for the formation of winter temperature inversions (hereafter referred to as inversions) , which oSeveral studies have evaluated associations between air pollutants and adverse respiratory symptoms, including nonadmitting emergency department (ED) visits, among asthmatics .We conducted a study of the effect of winter inversions on ED visits for asthma to provide data upon which doctors, patients, and public health officials can base inversion-specific asthma management plans and asthma-trigger prevention messages. Because inversions occur primarily during the winter months in Salt Lake County , we usedInversion criteria. The National Weather Service (NWS) office in Salt Lake City is one of approximately 90 sites in North America that routinely launch weather balloons (frequently referred to as upper-air soundings) as part of the NWS Upper-air Observations Program (NWS 2009). Weather balloons are launched twice each day, shortly after 0400 hours and after 1600 hours Mountain Standard Time; they gather temperature, humidity, and wind data as they ascend through the troposphere. Analysis of data gathered by weather balloons is one of the primary methods used by meteorologists at the NWS to identify the presence, and the strength, of inversions. We examined all data gathered from upper-air soundings released by the NWS office in Salt Lake City to identify inversion events in Salt Lake County during the study period (University of Wyoming 2012). The American Meteorological Society defines inversions as layers in which “temperature increases with altitude”  ( (ICD-9)  diagnosia = 17.625, b = 243.04. We obtained upper-air soundings data from the University of Wyoming (2012).We downloaded GSOD  measurements of daily maximum, mean (of maximum and minimum), and dew point temperatures (degrees Fahrenheit) for the Salt Lake City International Airport (one site) from the Climate Database server on the Utah Climate Center website  and conv2, two monitors), ozone , particulate matter (PM) with an aerodynamic diameter ≤ 2.5 µm  and ≤ 10 µm , and sulfur dioxide . Data on O3 were available only for 1 January 2006 through 29 February 2008 . For CO, NO2, PM2.5, PM10, and SO2, we assigned exposures to residents based on concentrations measured by the monitor nearest to the centroid of their residential ZIP code. Monitors were located where pollution levels were expected to be the highest  and to establish ambient levels of pollutants . Monitors were also placed next to specific industry sources to determine pollutant concentrations  , PM10 , and O3 . Thus, we imputed them by averaging concentrations measured on the day before and the day after each missing day.We obtained data on six air pollutants from the U.S. EPA AirData (2012) website: carbon monoxide , nitrogen dioxide (NOmonitor) , 2011. WStatistical analyses. We employed a time-stratified case-crossover design . We used constrained quadratic polynomial distributed lag models to estimate lag-specific and overall effects of inversions relative to noninversions on ED visits for asthma  and 95% confidence intervals (CIs) for the association between inversions and ED visits for asthma. We used an r asthma . We alsoWe modeled dose–response relations according to the length of inversions in three ways. First, we categorized the number of inversion days during lag 0–3 as zero (reference), 1, 2, 3, or 4 inversion days during the lag period. Second, we categorized inversions according to the day of inversion [the number of consecutive inversion days that had occurred on or before the case (ED visit) or corresponding control days] as not an inversion day (reference), 1st–3rd, 4th–6th, 7th–12th, or > 12th day of an inversion. Third, we categorized the inversion length based on the number of consecutive inversion days up to and including case or control days as not an inversion (reference), 1–3, 4–6, 7–8, or > 8 days. We tested linear trends by modeling ordinal versions of the three dose–response variables coded as the number of inversion days for the first variable and by using category medians for the day of inversion and inversion length variables.To evaluate interactions between inversions and age, sex, weather, and pollutants, we modeled dichotomized variables and product terms as any inversion days during lag 0–3 , age , sex , weather or pollutants . We tested the significance of multiplicative interactions using likelihood ratio tests comparing models with and without product terms with α = 0.1.3, which was measured at only one site); adjusted for relative humidity instead of dew point temperature; and adjusted for maximum temperature rather than daily mean temperature. As noted previously, we also repeated models after adjusting for individual air pollutants to determine whether adjusting for these potential causal intermediates altered associations between inversions and ED visits for asthma. We conducted all analyses with SAS software  using PROC LOGISTIC to fit conditional logistic regression models. We obtained Institutional Review Board approval from the Utah Department of Health; our study was exempted from informed consent requirements because our study was a secondary data analysis of preexisting data.We performed the following sensitivity analyses: excluded individuals who resided in Salt Lake County, but visited an ED for asthma outside of Salt Lake County (3% of ED visits); excluded individuals with ED visits associated with acute respiratory infections  in addition to asthma; used average air pollutant concentrations across all available monitors in Salt Lake County instead of using concentrations from monitors closest to residences (for pollutants other than Op < 0.0001) (p < 0.0001) and was higher after 12 inversion days (11.42 ± 2.76 visits/day) compared with the first 3 days of an inversion (7.98 ± 3.45 visits/day) .A mean (± SD) of 8.11 ± 3.40 ED visits for asthma occurred each day during our study period, and more ED visits for asthma occurred on inversion days than on noninversion days  . The meaits/day) . Femalesits/day) . O3 concy higher . ConcentO or SO2 . O3 concrom them . Inversirom them . Descriphttp://dx.doi.org/10.1289/ehp.1104349)].The OR for ED visits for asthma in association with inversions versus no inversions during lag 0–3, based on constrained quadratic polynomial distributed lag models adjusted for dew point and mean temperatures (Model 1), was 1.14  . The strWhen estimated according to the number of inversion days during lag 0–3, adjusted ORs were strongest for 3 and 4 inversion days  . The odd2.5, the air pollutant of greatest a priori interest . Adjusting for O3 also had little influence on associations when compared with ORs restricted to the time period when O3 data were available .Additional adjustment for PMinterest , had litinterest ; adjustm2, O3, PM2.5, SO2, dew point temperature, and relative humidity were not significant at α = 0.1 (data not shown), but interactions between inversions and PM10, maximum temperature, and mean temperature were significant  or who resided in Salt Lake County but visited an ED for asthma outside of Salt Lake County  gave nearly identical results (data not shown). Excluding 538 (16%) individuals who had acute respiratory infections at the time of their ED visits for asthma increased the ORs slightly . Using average pollutant concentrations across all available monitors in Salt Lake County instead of using concentrations from monitors closest to residences, adjusting for relative humidity instead of dew point temperature, and adjusting for maximum temperature in models instead of mean temperature gave nearly identical results (data not shown).Excluding those cases who returned to the ED for asthma on one of their control days , there was little change in ORs when individual air pollutant concentrations were included in models. Associations between inversion days and ED visits for asthma appeared to be limited to days with PM10, maximum temperature, and mean temperature above median levels based on stratum-specific ORs.Pollutant concentrations and the adjusted odds of ED visits for asthma were higher during inversions than noninversions, with stronger associations as the number of inversion days increased during the lag period. Results suggested that associations were stronger for longer inversions, although ORs did not increase monotonically. Although air pollutant concentrations changed during inversions  if they were experiencing worsening asthma symptoms at the time of the clinic visit or “stable” (n = 485) if their asthma symptoms were under control. After adjusting for age, smoking status, corticosteroid medication, surface temperature and relative humidity, a statistically significant increase in sputum cell counts, which is indicative of airway inflammation, was associated with the occurrence of inversions, but not with individual pollutant concentrations. Specifically, neutrophil and macrophage counts were 12.6% higher in the stable group and 2.5% higher in the exacerbated group on inversion days compared with noninversion days. Oxidative stress would be a plausible biological mechanism in the scenario where inversions act as indicators of high multipollutant atmospheres  in Oman . Monthlyospheres . FurtherResults from constrained quadratic polynomial distributed lag models suggested the association between inversions and ED visits for asthma was distributed over the entire 0–3 day lag, but strongest at lag 0. None of the lag-specific estimates were very precise, so these results should be interpreted cautiously. Dose–response relationships between inversions and ED visits for asthma were suggested when we estimated associations with the number of inversion days during the 0–3 day lag, the day of inversion, and the length of inversions, though clear monotonic relationships were not apparent.The occurrence of multiple, prolonged winter inversions is a unique characteristic of Salt Lake County that made it possible to estimate effects of inversions on rates of ED visits for asthma. The ED billing data used in this study are likely to reflect valid diagnoses of asthma . SilvermAn important limitation of our study was our inability to measure individual-level exposures to inversions or pollutants. Asthmatics may have self-medicated more often or remained mostly indoors during inversions. Despite the high concordance of asthma diagnoses between billing data and ED patient charts , ED visiOur study found an association between inversions, including prolonged ones, and ED visits for asthma. This result needs to be confirmed in other populations. Further research is needed to more fully investigate the extent to which inversions act as indicators of increased air pollutant concentrations that may lead to ED visits for asthma or other health effects. Finally, the potential value of incorporating inversion forecasts and/or warnings into existing air quality alert programs should be studied in the future.(180 KB) PDFClick here for additional data file.


Aspergillus nidulans.Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA::GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA::GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose.Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of  Lignocellulolytic fungi secrete a complex arsenal of enzymes that synergistically deconstruct plant cell wall polysaccharides. The capacity of these enzyme cocktails to release utilisable sugars from non-food lignocellulosic material represents an opportunity for the development of a new generation of biofuels, produced directly from plant biomass without the use of extensive pre-treatment. However, efficiencies in industrial enzyme production require dramatic improvement, as the presence of readily metabolisable carbohydrates strongly impedes cellulase and hemicellulase production via carbon catabolite repression (CCR) ,2. GenomAspergillus nidulans, Hypocrea jecorina and Neurospora crassa the orthologous repressors CreA/Cre1 have been shown to block the transcription of genes associated with the utilisation of alternate carbon sources when glucose is present, including cellulolytic and xylanolytic enzymes [A. nidulans CreA protein has two Cys2His2 zinc finger DNA binding structures that demonstrate high similarity to the zinc fingers of the Mig1 repressor involved in Saccharomyces cerevisiae CCR [In fungi, lignocellulolytic enzyme production is tightly controlled at the transcriptional level by the competitive action of transcriptional activators and repressors . In Aspe enzymes -10. The siae CCR  and has siae CCR .Aspergilli the ethanol utilisation pathway is tightly controlled by CreA-mediated CCR and positively induced by the regulon specific transcription factor AlcR. The positive regulator, AlcR, has overlapping binding sites with CreA, suggesting a competitive binding mode of action, while nucleosomal positioning and chromatin organisation has been shown to play a role [H. jecorina the cellulase activator, Ace2, has been shown to bind to the same promoter motif as XlnR, while the Hap2/3 complex opens the chromatin structure, promoting nucleosome reassembly and derepression [N. crassa and A. nidulans, two newly identified transcription factors, ClrA and ClrB, have been shown to be required for cellulase activity and expression [The transcription of genes involved in alternative carbon utilisation also requires the action of transcriptional inducers. In y a role . Other ay a role . Interespression . In N. cpression .S. cerevisiae has served as a model organism for the study of such cellular responses. However, many differences are known to exist in filamentous fungi, due to the adoption of different life styles. Protein phosphorylation state represents the most common form of post-translational modification. Protein kinases and phosphatases perform a central role in the transduction of such signals via modulating protein phosphorylation state and activity, thus coordinating subsequent responses. The importance of protein kinases and phosphatases is demonstrated by the fact that 30% of the S. cerevisiae genome is modified by these proteins, while collectively kinase and phosphatase genes represent only 6% of the genome [Sensing the external environment and intracellular energetic status ensures that a fungal organism can balance the requirements for growth and cell survival. e genome .Ras2 signalling that also induces the filamentous growth cAMP-PKA and MAPK pathways [S. cerevisiae pheromone response/filamentous growth MAPKs have been shown to influence the secretion of hydrolytic enzymes in various plant pathogenic fungi including; Alternaria brassicicola, Cochliobolus heterostrophus and Fusarium oxysporum[The most well studied examples included the mitogen-activated protein kinases (MAPK), which form the pheromone response, filamentous growth, the osmotic stress response and cell wall integrity pathways. The sensing of glucose or pheromones by the G-protein coupled receptors (GPCRs), results in the activation of the cAMP-protein kinase A (PKA) pathway and the MAPKs cascade, which influence filamentous growth . Intracepathways . Apart foxysporum-22.S. cerevisiae sensors of cellular energetic state Snf1 (sucrose non fermenting) and TOR (target of rapamycin) have been widely identified in filamentous fungi [. The Snf1 has been demonstrated to be required for growth on alternative carbon sources and also regulates the expression of 400 genes in response to carbon exhaustion [Cochliobolus carbonum, Ustilago maydis and F. oxysporum, has been shown to reduce hydrolytic enzyme production [A. nidulans genome contains fewer phosphatases (http://www.aspgd.org), suggesting that each phosphatase demonstrates less specificity and dephosphorylates multiple kinase targets. The known kinase/phosphatase signalling pathways have been proven to be highly conserved and closely interlinked, either regulating one another or the same gene sets, enabling the fine tuning of cellular responses to the extra- and intracellular environment.Homologues of the us fungi -28. The haustion . The absoduction -26. The oduction . TOR hasA. nidulans. The involvement of the identified NPKs in starvation/cellulose-induced responses and CreA derepression was assessed by transcriptomics and fluorescence microscopy of the CreA::GFP in the parental and NPK mutant backgrounds. Collectively, this study demonstrated the overlapping, synergistic roles of the NPKs schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production. In addition, a period of carbon starvation appeared paramount for CreA derepression and hydrolase induction.Despite recent advances in the understanding of hydrolytic enzyme regulation, less is known about how different carbon sources are sensed and which signalling cascades are subsequently activated, thus coordinating the adaptation of cellular metabolism and hydrolase secretion to the respective carbon source ,31,32. Sp < 0.01) . The function of these 11 NPKs was determined via homology (BLASTp) with S. cerevisiae, if not already characterised in Aspergilli, revealing NPKs involved in nutrient/cell energy status sensing and the regulation of cell growth and endocytosis , alternative carbon source usage , starvation responses  and endo- exo-cytosis, polarisation or morphogenesis .Eleven of the 103 NPKs screened demonstrated a reduction in total protein content when grown in liquid minimal media (MM) supplemented with AVICEL (synthetic cellulose) as the sole carbon source for 10 days, reflecting the reduction in fungal biomass. However, the 11 NPKs did not show a significant reduction in dry weight when cultured in complete media (CM) for 48 h (eglA/B) were determined in order to confirm that these NPKs were related to cellulase regulation and/or CCR. Both endocellulase activity and eglA/B transcription were dramatically reduced in 10 of the 11 NPKs after 5 days growth in MM plus AVICEL  and those which influenced only cellulase production including the NPKs involved in alternative carbon source usage .Subsequently, the endocellulase activity of these 11 NPKs and the transcription of two major endoglucanase genes , but grew on complete media. Subsequently, it was not possible to study the role of these NPPs in relation to growth on AVICEL as a sole carbon source and hydrolase production. The NPPs that could grow on MM plus glucose were screened for reduced growth on AVICEL. Seven NPPs showed reduced growth on MM plus AVICEL, in terms of total protein, and demonstrated no significant difference in growth on MM plus 2% glucose  and MAPK regulation . In S. cerevisiae Ppg1A is required for glycogen accumulation, while also associating with Tap42 and Sit4 which are involved in TOR signalling.The function of the identified NPPs, if not already characterised in ae Table . SeveralpkaC mutant on all carbon sources. In the absence of the ability to synthesis cAMP, assessed via utilising the adenylate cyclase ∆cyaA mutant [The identification of several NPKs involved in the cAMP signalling pathway as being required for both cellulase and xylanase production resulted in an interest to investigate PKA activity in the parental strain in the presence of multiple carbon sources. Overnight 1% fructose MM cultures were transferred to alternative carbon sources for 1–8 h and PKA activity determined. No substantial difference in PKA activity was noted between various carbon sources, except for when the mycelium was transferred to AVICEL or no carbon containing media for 8 h, in which PKA activity approximately doubled Figure . Only a A mutant , an inteG17V strain [G17V strain demonstrated an approximate eight-fold reduction in endocellulase activity after 5 days growth in MM plus AVICEL . This confirmed that the conventional route of PKA activation inhibits cellulase production, while suggesting that starvation induced PKA hyperactivation performed alternative function(s) that directly or indirectly contributed to growth on cellulose.Extracellular glucose is detected via extracellular GCPRs, while intracellular phosphorylated glucose results in RAS activation, with both routes accumulating with adenylate cyclase synthesising cAMP and PKA activation. To confirm that the conventional route of cAMP dependent PKA activation inhibited cellulase production during growth on cellulose, the endocellulase activity of constitutively activated RASV strain  was asseA. nidulans a CreA::GFP tagged protein under the control of the native promoter was constructed, enabling the study of CreA nuclear localisation under repression/derepression and the evaluation of the signalling components that led to CreA cellular compartmentalisation. The CreA::GFP strain constructed in the present work, demonstrated growth similar to the parental strain on various repressing and derepressing carbon sources  and then exposing it to carbon starvation for 5 h  enabled the study of the positive signals for CreA repression. The addition of 2-deoxyglucose which cannot be successfully metabolised, or 6-deoxyglucose that cannot be phosphorylated, to the carbon starved cultures demonstrated that the positive signal for CreA nuclear localisation required glucose phosphorylation Table .creA4 strain [schA and snfA were required for CreA derepression and endocellulase production. The absence of schA and snfA resulted in an inability to remove CreA from the nucleus upon growth on AVICEL , which is involved in glycogen homeostasis, autophagy and starvation stress responses, positive regulators of the ethanol regulon  and acetate utilisation (facB). In both of the parental and ∆schA transcriptional responses an amino acid starvation (AN1812) and the fatty acid utilisation farA transcription factors were also induced.The CAZy enzymes within these datasets were identified to create a profile of the hydrolytic capacity of each strain. In total, 54 glucoside hydrolases were induced by AVICEL in the parental strain, of which 31 were specifically induced in the parental strain, 8 were induced in all three strains, 14 induced in both the parental and ∆schA strains (33 transporters), in both the parental and ∆snfA strains (6 transporters), or specifically in the parental strain (41 transporters). Note the low number of transporters induced in the ∆snfA strain. The far majority of the transporters induced in the parental strain lacked a defined function in A. nidulans. However, the functionally defined genes included transporters of amino acid, sugar, iron, calcium and sodium , in both the parental and the ∆xprG starvation response transcription factor and hacA, which regulates the unfolded protein response, after 24 h growth on AVICEL, suggesting the existence of starvation induced stress. Accordingly, several starvation-related genes, atg8, hxkC and the GPCR gprH were also up regulated. However, despite the induction of xprG in all strains these three starvation-response genes were not induced in the ∆snfA strain, while the xprG-activator kinase gene atmA , was repressed at a low level. Other kinase sensors of energetic status including the sakA and the gal83 homolog, which are both required for Snf1 activation and nuclear localisation in S. cerevisiae, were only induced in the parental strain, while the TOR kinase (AN5982) was only not induced in the ∆schA strain.In all strains there was an induction of the schA and snfA are required to regulate the response to carbon limitation and growth on AVICEL. These two NPKs demonstrated a partially overlapping function in the modulation of CAZy enzyme, sugar and amino acid transporters, transcription factors and metabolism, with snfA appearing to be of paramount importance.Collectively, the transcriptomic data depicts how schA and snfA, controlled hydrolytic enzyme production  and Pkac1. Schuster et al.[A. nidulans PKA may perform additional functions during starvation and growth on cellulose, apart from the typical GPCR/Ras-PKA pathways , generates NADPH and acetyl CoA from the oxidative decarboxylation of pyruvate and facilitates uptake into the mitochondria. The phosphorylation state of the PDC controls the flux through this irreversible reaction, thus directing metabolism towards the consumption of glucose in respiration or the preservation of glucose for gluconeogenesis [. Under starvation the pyruvate dehydrogenase kinase phosphorylates and inactivates the PDC conserving glucose and promoting fatty acid utilisation [pkpA kinase suggests that a period of glucose deprivation is experienced by A. nidulans when grown on cellulose, which is supported by the observed up regulation of alternative carbon source usage, such as amino acid, ethanol, acetate, fatty acid and cellulose.Several additional NPKs involved in the starvation response of multiple organisms, such as the ogenesis . Under slisation . TherefoA. nidulans grown on cellulose could result in a similar AtmA, SakA and SnfA cascade of activation, thus regulating mitochondrial function, sugar uptake, fatty acid utilisation and hydrolytic enzyme production.The ATM kinase in mammalian cells has been identified as being involved in starvation, insulin signalling and the regulation of glucose homeostasis through the action of the p53 phosphorylation target, which in turn regulates metabolism, mitochondrial respiration and glucose transporters. In addition, a p53-independent ATM pathway has been demonstrated to activate AMPK  via LKB1  via dependent and independent routes -45. Low S. cerevisiae cells the SnfA homologs have been shown to interact with the essential TOR kinase, which is an integrator of nutrient and growth factor inputs that control cell growth [S. cerevisiae. Homologs of the SchA kinases have been shown to operate downstream of TOR [A. nidulans, possibly integrating the TOR signal. Similarly in S. cerevisiae and A. nidulans, the SchA kinase has also been shown to have synergistic and opposing functions with PKA, in the regulation of transcription [The presented study demonstrated how SnfA performed a key role in CreA derepression and the modulation of transcription, metabolism and hydrolytic enzyme secretion. In mammalian and l growth . Interesm of TOR . The ovecription ,47.Multiple NPKs and NPPs involved in filamentous growth, polarisation, and morphogenesis were also identified as being required for growth on cellulose and hydrolase secretion. Endo- and exo-cytosis are key processes for the sensing of, and interaction with, the environment. Such processes have been shown to regulate glucose transporters in mammalian cell . TherefoA. nidulans require further study prior to the reconstruction of relevant signalling cascades. Even so, it is clear that SnfA-mediated CreA derepression and transcriptional responses, which regulate metabolism, transport and hydrolytic enzyme secretion, are paramount for growth on cellulose, while the SchA kinase appeared to have an overlapping mechanism and synergistic function with SnfA.An initial period of carbon starvation appears to play a role in the activation of the nutrient/energy sensing kinase pathways and that CreA derepression is an essential prerequisite for hydrolytic enzyme induction. Through homology with more extensively studied systems, a model for how the identified kinases and phosphatases regulate hydrolytic enzyme production can be postulated. However, the genetic interactions between these kinases and phosphatases in A. nidulans TNO2a3  strain was used as a reference in all experiments. The null mutant collections of 103 non-essential protein kinases (NPKs) and 28 non-essential phosphotases, created by in vivo recombination in S. cerevisiae followed by A. nidulans protoplast transformation  wereson, USA . The ∆Crson, USA . All fun7 conidia and incubated on a rotary shaker (180 rpm) set at 37°C for 24 h. Liquid minimal media (MM) cultures containing 1% AVICEL, instead of glucose as a carbon source were prepared as with the CM cultures, but were incubated for 10 days under the same conditions. The mycelia of the CM and MM plus AVICEL cultures were filtered, frozen in liquid nitrogen and freeze dried prior to the determination of dry weight and total protein content, respectively. The fungal biomass (dry weight) within the MM plus AVICEL cultures cannot be measured directly, due to the presence of AVICEL. Therefore, total protein content was used as a relative measurement. The mycelia from the AVICEL cultures was ground in liquid nitrogen and added immediately to the protein extraction buffer ), vortexed for 5 min prior to centrifugation for 15 min at 14000 g. Protein content was measured using the Bio-Rad protein assay according to manufacturer’s instructions.Complete media (CM) cultures (50 ml) were inoculated with 1×107 conidia and incubated on a rotary shaker (180 rpm) set at 37°C for 24 h. Subsequently, the mycelia of the transfer cultures were washed with sterile water, resuspended in liquid MM plus 1% AVICEL or xylan and incubated under the same conditions for 5 or 3 days depending on the respective carbon source. The cultures were filtered and the mycelia frozen in liquid nitrogen prior to being freeze dried for RNA extraction. The culture supernatants were collected for endo- cellulase or xylanase activity assays (Megazyme) according to manufacturer’s instructions.Cultures of MM plus 1% fructose (50 ml) were inoculated with 1×10Media shift cultures were prepared as described previously. Post washing, the mycelia was transferred to a range of different carbon sources, as stated in the relevant figure and results section, for 8 h. The mycelia from the transfer cultures was collected by filtration, then frozen and ground in liquid nitrogen. Total protein was extracted as described previously and the Peptag cAMP dependent PKA activity assay (Promega) performed according to manufacturer’s instructions. Quantification of the intensity of the phosphorylated substrate was determined via densitometry analysis using the ImageJ software. Results are presented as the total PKA activity per culture.et al. [creA gene (minus the stop codon), the gfp gene plus a spacer, the pyrG gene and the 3’ UTR were co-transformed into S. cerevisiae. Homologous recombination within S. cerevisiae created the construct, which was subsequently amplified from pooled S. cerevisiae DNA, and 20 μg transformed into TNO2a3 according to Osmani et al., [schA and ∆snfA strains were confirmed by PCR, using the relevant external forward and exon reverse primers, while the absence of atmA was confirmed via increased camptothecin sensitivity (75 μM). The primers used are listed in Additional file The construction of the CreA::GFP strain was performed according to Colot et al. . Standaret al. . The 5’  et al., . Transfo4, 1.8 mM KH2PO4, pH 7.4). The mycelia were then stained with 100 ng/ml Hoescht 33258 (Molecular Probes) for 2 min. The mycelia were washed again in PBS and examined using a Zeiss epifluorescence microscope with excitations of 359, 498 nm and emissions 461, 516 nm for Hoescht and GFP respectively. Phase contrast bright-field and fluorescent images were captured with AxioCam camera (Carl Zeiss) and processed using the AxioVision software version 3.1.Strains were inoculated onto a coverslip and incubated for 12 h at 25°C in liquid MM plus various carbon sources (1% carbon w/v). During media shift experiments the coverslips were washed with MM lacking a carbon source prior to the addition of the following media and incubated at 25°C, for the duration stated in the text (dependent on carbon source). In the case of 2-deoxyglucose and 6-deoxyglucose, a final concentration 6 mM of either compound was added to 5 h carbon starved cultures and incubated for an additional hour prior to examination. Mycelia mounted on the coverslips were washed with phosphate buffered saline  and eglB (AN3418) [tubC (AN6838) was used as an endogenous control.Total RNA was isolated using TRIZOL (Invitrogen), treated with DNase (Promega) and purified using the RNeasy® Mini Kit (Qiagen) according to manufacturer’s instructions. RNA integrity was confirmed using the Bioanalyser Nano kit (Agilent technologies) and the Agilent Bioanalyser 2100. Purified RNA was used for cDNA synthesis using Superscript III (Invitrogen) according to manufactures instructions. Quantitative PCRs were performed as previously described . The Taq(AN3418)  are listschA and ∆snfA strains (1×107 conidia) were incubated in 50 ml CM on a rotary shaker (180 rpm) set at 37°C for 24 h. The mycelia were washed with sterile water and transferred to MM plus AVICEL for 8 and 24 h. Post incubation, the mycelia was collected by filtration and frozen in liquid nitrogen. Total RNA was extracted and integrity confirmed as described previously. The synthesis of cDNA from 200 ng of RNA and the array hybridisations were performed according to Souza et al.[http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE47810).The parental, ∆za et al. with a mt-test (p < 0.01) performed within the Mev software [http://www.broadinstitute.org/fetgoat/index.html). The differential regulation of gene expression between the three strains post transfer to MM plus AVICEL was determined via venn analysis and the functional profile of the gene lists determined using FunCats . Secreted proteins and hydrolytic enzymes were identified using TargetP (http://www.cbs.dtu.dk/services/TargetP/) and the CAZy enzyme database (http://www.cazy.org/) respectively.Genes were determined as differentially expressed between the CM and MM plus AVICEL via a software . The ovet-test , unless stated otherwise, using Prism 3.0 (GraphPad).Three biological replicates were performed for all experiments and the statistical tests for significance determined via a one-tailed ATP: Adenosine triphosphate; cAMP: Cyclic adenosine monophosphate; CAZy: Carbohydrate active enzymes; CCR: Carbon catabolite repression; CM: Complete media; GFP: Green fluorescent protein; GO: Gene ontology; GPCR: G-protein coupled receptor; MAPK: Mitogen activated protein kinase; MM: Minimal media; NADPH: Nicotinamide adenine dinucleotide phosphate; NPK: Non-essential protein kinase; NPP: Non-essential protein phosphatase; PKA: Protein kinase A complex; PDC: Pyruvate dehydrogenase complex; UTR: Untranscribed region.The authors declare that they have no competing interests.NAB contributed to the concept, design, acquisition and analysis of data, in addition to the preparation of the manuscript. PG, NK and MS contributed to the acquisition of data, and GHG contributed to the concept and design of the investigation in addition to the preparation of the manuscript. All authors have read and approved the final manuscript.The CreA::GFP strain demonstrated a similar phenotype to the parental strain. The CreA::GFP and parental TNO2a3 strains grown on complete media or minimal media for 4 days. Click here for filecreA strain. All strains were grown in MM plus 1% fructose overnight and then transferred to AVICEL as a sole carbon source for an additional 5 days. The comparison of the endocellulase activity (U/ml) of the parental, single ∆NPKs and the ∆creA strains with the double ∆NPK ∆creA strains is presented. Endocellulase activity was restored when the NPKs kinase mutants were crossed with the ∆Click here for filet-test p < 0.01) post transfer from CM to MM plus AVICEL for 8/24 h in the parental, ∆schA and ∆snfA strains. The total number of genes determined as differentially expressed (Click here for filep < 0.05) GO terms within the list of genes differentially expressed in the parental and ∆schA strains post transfer from complete media to media containing AVICEL as a sole carbon source for 24 h. Note that no GO terms were overrepresented in the ∆snfA strain post media transfer. The FetGOat analysis of overrepresented  post transfer from complete media to minimal media containing AVICEL as the sole carbon source for 24 h in the parental, ∆schA and ∆snfA strains. Genes are presented according to whether induction occurred in a single or multiple strains. The data presented includes; MIPS functional categorisation, TargetP prediction of secretion and CAZy identification. The genes significantly induced (Click here for fileA list of all the primers used in the presented investigation.Click here for file


ABCA1*Arg230Cys polymorphism were correlated. Further, we examined whether the evolutionary consequences of the agriculturally constructed niche in Mesoamerica could be considered as a gene-culture coevolution model. For this purpose, we genotyped 229 individuals affiliated with 19 Native American populations and added data for 41 other Native American groups (n = 1905) to the analysis. In combination with the SNP cluster of a neutral region, this dataset was then used to unravel the scenario involved in 230Cys evolutionary history. The estimated age of 230Cys is compatible with its origin occurring in the American continent. The correlation of its frequencies with the archeological data on Zea pollen in Mesoamerica/Central America, the neutral coalescent simulations, and the FST-based natural selection analysis suggest that maize domestication was the driving force in the increase in the frequencies of 230Cys in this region. These results may represent the first example of a gene-culture coevolution involving an autochthonous American allele.Culture and genetics rely on two distinct but not isolated transmission systems. Cultural processes may change the human selective environment and thereby affect which individuals survive and reproduce. Here, we evaluated whether the modes of subsistence in Native American populations and the frequencies of the  Homo sapiens sapiens, a particularly important event that triggered a new and striking gene-culture-coevolution cycle was the development of agriculture and animal domestication during the Neolithic period . Further, the human gene-culture coevolution mediated by the domestication of plants and animals has been argued to provide some of the clearest and most spectacular examples of niche construction. The Niche Construction Theory can be defined as a branch of evolutionary biology that emphasizes on the ability of organisms to modify the pressure of natural selection in their environment and thereby act as co-directors of their own evolution, as well as that of other directly associated species Human cultural practices have drastically modified environmental conditions and behaviors, promoting rapid and substantial genomic changes often associated with positive selection and adaptation , these first migrants had to adapt to an immensely wide variety of environments. In some regions during this evolutionary trajectory, as in Mesoamerica and the Andes, hunter-gatherer/forager societies gave rise to agriculturalist and urban communities, while others remained with a hunter-gatherer/forager subsistence system until the time of contact with Europeans or even until the present day. Thus, studies with Native American populations can provide useful information for better understanding gene-culture coevolution and the niche construction processes.et al.DRD4) alleles in several South Amerindian populations and found a significant difference in the allelic distributions between hunter-gatherers and agriculturalists, with an increase of the 7R allele among the former; and (2) Acuña-Alonzo et al. 230Cys allele  of the ATP-binding cassette transporter A1 (ABCA1) gene, which was previously associated with low HDL-cholesterol levels and obesity-related comorbidities, was exclusively present in Native American and mestizo individuals. These authors verified that cells expressing the ABCA1*230Cys allele showed a 27% cholesterol efflux reduction, confirming that this Native American autochthonous variant has a functional effect in vitro. Other investigations have shown that the presence of ABCA1*230Cys explains almost 4% of the variation in plasma HDL-C concentrations in Mexican admixed populations Based on studies with blood groups and other classical genetic polymorphisms, J. V. Neel and F. M. Salzano were pioneers in identifying complex population processes highly dependent on cultural factors in Native Americans  from Meso/Central America and South America , and a part of this information was used in some of our analyses. The populations were clustered according to their geographical location and ancient mode of subsistence as follows: (1) Mesoamerican agriculturalists, (2) Andean agriculturalists, and (3) South American hunter-gatherers/foragers. Of course, caution is needed regarding this classification, since subsistence modes are not stable over time and may not be unique. However, the two categories adopted here  represent general pre-Colombian subsistence conditions, providing a starting point for research related to gene-culture dynamics in Native Americans.New data for the  America . AdditioEthical approval for the present study was provided by the Brazilian National Ethics Commission (CONEP Resolution no. 123/98) for the Brazilian samples, as well as by ethics committee of: (a) Universidad de Antioquia, Medellin, Colombia (Colombian samples); (b) Universidad Nacional Autónoma de México, Ciudad de México, México (Mexican samples); (c) Universidad de Costa Rica, San José, Costa Rica (Costa Rican and Panamanian samples); (d) Universidad of Chile, Santiago, Chile (Chilean samples); (e) Université Paul Sabatier Toulouse 3, Toulouse, France (Bolivian and French Guianian samples). Individual and tribal informed oral consent was obtained from all participants, since they were illiterate, and they were obtained according to the Helsinki Declaration. The ethics committees approved the oral consent procedure as well as the use of these samples in population and evolutionary studies.Arg230Cys polymorphism was genotyped using TaqMan assays . Allele frequencies were obtained by direct counting. The level of the population structure observed within and between Mesoamerican agriculturalist, Andean agriculturalist, and South American hunter-gatherer/forager groups was estimated using F statistics and the Arlequin 3.5.1 software t-test  using the R Stats package .The et al., unpublished data). Based on this additional information   =  [−2p/(1−p)]ln(p), where E(t1)  =  expected age, time is measured in units of 2N generations, and p  =  population frequency ABCA1*230Cys, we considered the average of frequencies of all populations and only those from Mesoamerica/Central America . This program allows a Bayesian inference of the mutation age using an intra-allelic coalescent model to assess LD across the nineteen SNPs that occur around ABCA1*230 . Taken into account the historical information about our sampled populations, three parameters were introduced: (a) Generation time of 25 years; (b) Proportion of population growth of 0.005; and (c) Proportion of population sampled of 0.0002. The program was run in the haplotype mode using two million of iterations.Slatkin and Rannala Based on the long-range haplotype test STF) with that generated for a null simulated distribution under a hierarchical island model using a coalescent approach. In this model, demes exchange more migrants within groups than between groups to generate the joint distribution of genetic diversity within and between populations p value can be estimated from the joint distribution for the population heterozygosity (eH) and STF using a kernel density estimation procedure ABCA1*230 locus were known. Data from 20 other autosomal SNPs  from these same individuals were compiled from a major SNP panel  Mesoamerican agriculturalists (n = 68) vs. Andean agriculturalists (n = 35), (2) Mesoamerican agriculturalists vs. South American hunter-gatherers/foragers (n = 23), (3) Mesoamerican agriculturalists vs. South Americans , and (4) Andean agriculturalists vs. South American hunter-gatherer/foragers.To detect loci under selection, we used a method that contrasted the observed population differentiation  statistics–was compared to the observed genetic diversity of two genetic datasets: (1) that of the ABCA1*230 locus plus the same 19 flanking SNPs listed in item c.1; (The genetic diversity obtained in the simulations–summarized by intra- (heterozygosity) and interpopulation tem c.1; . The sumZea mays ssp. mays) had a single domestication origin from the wild grass teosinte (Zea mays ssp. parviglumis) in the Río Balsas region, southwestern Mexico, approximately 6,300–10,000 calendar years before present Zea (maize and/or teosinte) in the Americas and have been used to estimate the age of maize domestication and dispersion Zea pollen dates from several American archeological sites, and we selected this data set to perform our analysis. To test the connection between maize culture and the ABCA1*230Cys variant, we used allele frequencies from Mesoamerica/Central America populations  as well as Zea pollen dates obtained in archeological sites located geographically near these populations  were obtained using the Statistica 7.0 software .Genetic, archeological, botanical, and paleoecological data furnished evidence that maize . On the other hand, the highest STF among populations within subdivisions was observed in the South American hunter-gatherers/foragers , and a value 5 times lower was found among Mesoamerican agriculturalists ; no sign of structuration was found in the Andes area (p = 0.36).vs. agriculturalists) were compared using the student’s t-test . A comparison of South American hunter-gatherers/foragers and Andean agriculturalists revealed no significant differences (p = 0.351).No significant differences in allelic frequencies were found when subsistence modes , no significant departure from the expected under neutrality was found (data not shown). These results suggest that the ABCA1*230 allele frequencies in Mesoamerica are incompatible with a simple neutral model.Patterns of genetic diversity between populations can be used to detect loci under selection ABCA1*230 polymorphism and 19 flanking SNPs presented a slightly lower average heterozygosity than the putative neutral region dataset (0.32 vs. 0.34 respectively); but global and pairwise STF were higher for the ABCA1 region . Considering that both genomic regions were studied using the same quantity of markers and the same sampling strategy, in populations that were subjected to the same demographic history, the observed differences may occur due to diverse factors, one of them being natural selection. To test this hypothesis each dataset, summarized by the above-mentioned statistics, was also compared to each of 100,000 neutral simulations by means of Euclidian distances. The empirical dataset containing the ABCA1*230 polymorphism presented a poorer fit to neutrality than the putative neutral region dataset, showing Euclidian distances that were twofold higher than those of the neutral simulations (70.64 vs. 35.12). Interestingly, when the Mesoamerican agriculturalist subdivision was excluded from the analysis, this difference dramatically decreased (45.80 vs. 35.12). Thus, the poor fit to neutrality observed at the ABCA1*230 site and its flanking regions may be associated with the genetic pattern found in Mesoamerica.Our neutral demographic simulation analysis showed results in the same direction. The region containing the ABCA1*230Cys allele frequencies and the distribution of the Zea pollen relics in Mesoamerica (p = 0.002). It is important to note that the populations used in the correlation analysis performed here  were investigated for microsatellites and other genetic markers in previous studies conducted by our and other groups A significant correlation was observed between the ABCA1* Arg230Cys polymorphism.We can now examine some hypotheses in an attempt to explain the results and to draw the evolutionary scenario associated with the pattern of diversity of the Maize is considered the most important native crop of the Americas Other crops were also present in the pre-Columbian Mesoamerican civilizations , it is reasonable to suppose that ABCA1 alleles, since cholesterol plays an important role in various infectious processes, such as the entry and replication of Dengue virus type 2 and flaviviral infection ABCA1 gene deletions confer complete resistance against cerebral malaria in mice ABCA1*230Cys selective sweep associated with agricultural development. A sedentary village lifestyle with a corresponding growth in the density of the local population can promote an increase in the mortality rate, particularly in children under 5 years of age Rotavirus and Coronavirus , Streptococcus, Staphylococcus, Plasmodium , and HerpesvirusABCA1*230Cys variant in these infectious processes will require additional functional studies.Noteworthy is that other environmental factors may also have been involved in the distribution of the Arg230Cys presented a worse fit to neutrality than loci known to be neutral, indicating that selective mechanisms are necessary to explain the genetic diversity of Arg230Cys, especially when the Mesoamerican agriculturalist subdivision is considered in the analysis. This result also supports our hypothesis that maize domestication in Mesoamerica lead to changes in the gene pool of the natives from that region.In agreement with this historical scenario, the genetic variation in Arg230Cys polymorphism in South American groups, a situation different from that in Mesoamerica.South America presents much more diversity in relation to habitats, people, and culture than Mesoamerica. For instance, maize arrived in South America, but apparently the level of consumption seen in Mesoamerica/Central America was rarely found there. Archaeological data indicate that only during the implantation and expansion of the Inca Empire (800–500 YBP) was the level of maize consumption important, but the level of consumption was not comparable to that of Mesoamerica/Central America 230Cys allele, with a probable origin in America continent, seems have been the target for an ongoing directional selective sweep as a result of the origin and spread of the maize culture in ancient Mesoamerica.In conclusion, our analyses demonstrate for the first time a robust correlation between a constructed niche and a selected Native American autochthonous allele. The Figure S1Twenty SNPs selected based on their location (chromosome 9: from position 85252250 to 85317359) inside the putative neutral region, defined by Schroeder and colleagues, which comprises ∼76,000 bp around the D9S1120 locus.(JPG)Click here for additional data file.Table S1Populations included in the selection analyses.(DOC)Click here for additional data file.Table S2Allele frequency by region of 20 SNPs located around the D9S1120 locus.(DOCX)Click here for additional data file.Table S3Allele frequency by region of 19 SNPs located around the ABCA1*230 locus.(DOCX)Click here for additional data file.