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α-Amylase inhibitors are attractive candidates for the control of seed weevils, as these insects are highly dependent on starch as an energy source. In this study, we aimed to reveal the structure and diversity of dimeric α-amylase inhibitor genes in wild emmer wheat from Israel and to elucidate the relationship between the emmer wheat genes and ecological factors using single nucleotide polymorphism (SNP) markers. Another objective of this study was to find out whether there were any correlations between SNPs in functional protein-coding genes and the environment.
The influence of ecological factors on the genetic structure of dimeric α-amylase inhibitor genes was evaluated by specific SNP markers. A total of 244 dimeric α-amylase inhibitor genes were obtained from 13 accessions in 10 populations. Seventy-five polymorphic positions and 74 haplotypes were defined by sequence analysis. Sixteen out of the 75 SNP markers were designed to detect SNP variations in wild emmer wheat accessions from different populations in Israel. The proportion of polymorphic loci P (5%), the expected heterozygosity He, and Shannon's information index in the 16 populations were 0.887, 0.404, and 0.589, respectively. The populations of wild emmer wheat showed great diversity in gene loci both between and within populations. Based on the SNP marker data, the genetic distance of pair-wise comparisons of the 16 populations displayed a sharp genetic differentiation over long geographic distances. The values of P, He, and Shannon's information index were negatively correlated with three climatic moisture factors, whereas the same values were positively correlated by Spearman rank correlation coefficients' analysis with some of the other ecological factors.
The populations of wild emmer wheat showed a wide range of diversity in dimeric α-amylase inhibitors, both between and within populations. We suggested that SNP markers are useful for the estimation of genetic diversity of functional genes in wild emmer wheat. These results show significant correlations between SNPs in the α-amylase inhibitor genes and ecological factors affecting diversity. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs, and the SNPs could be classified into several categories as ecogeographical predictors. It was suggested that the SNPs in the α-amylase inhibitor genes have been subjected to natural selection, and ecological factors had an important evolutionary influence on gene differentiation at specific loci.
Wild emmer wheat, Triticum dicoccoides, the progenitor of bread and pasta wheats, presumably originated in and adaptively diversified from, northeastern Israel into the Near East Fertile Crescent [1]. In this center of diversity, wild emmer wheat harbors rich genetic diversity and resources [1]. Previous studies in T. dicoccoides and other cereals have shown significant nonrandom adaptive molecular genetic differentiation at single and multilocus structures in either protein-coding regions or randomly amplified polymorphic DNAs among micro-ecological environments [2,3]. It was also determined that wild emmer wheat is genetically variable and that the genetic differentiation of populations included regional and local patterns with sharp genetic differentiation over short distances [4]. Genetic polymorphisms of α- and β-amylase in wild emmer wheat have been characterized, and it was found that diversity of climatic and edaphic natural selection, rather than stochasticity or migration, was the major evolutionary force driving amylase differentiation [5].
The estimates of molecular diversity derived from PCR-based techniques such as amplified restriction fragment length polymorphism (AFLP), microsatellites (short sequence repeats or SSR), single nucleotide polymorphism (SNP), and sequence comparisons are several-fold higher than enzymatic diversity [6]. A substantial private and public effort has been undertaken to characterize SNPs tightly associated for genetic diversity. SNPs are identified in ESTs (expressed sequence tags), thus the polymorphisms could be directly used to map functional and expressed genes, rather than DNA sequences derived from conventional RAPD and AFLP techniques, which are typically not functional genes [7-9]. The majority of SNPs in coding regions (cSNPs) are single-base substitutions, which may or may not result in amino acid changes. Some cSNPs may alter a functionally important amino acid residue, and these are of interest for their potential links with phenotypes [10].
α-Amylase is a family of enzymes that hydrolyze α-D-(1,4)-glucan linkages and play an important role in the carbohydrate metabolism of many autotrophic and heterotrophic organisms [11]. Heterotrophic organisms use α-amylase primarily to digest starch in their food sources [12]. Several kinds of α-amylase and proteinase inhibitors in seeds and vegetative organs act to regulate the numbers of phytophagous insects [13-15]. α-Amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source [16]. In cereal seeds, α-amylase inhibitor proteins with 120–130 amino acids, which include trypsin inhibitors, as well as α-amylase inhibitors, can be grouped into one large family on the basis of the homology between their amino acid sequences [17]. In this family, the dimeric α-amylase inhibitor has been well characterized. For weevil control, α-amylase inhibitors could be manipulated through plant genetic engineering. However, many insects have several α-amylases that differ in specificity, and successful utilization of a food source is dependent on the expression of a α-amylase for which there is no specific inhibitor [12]. The dimeric α-amylase inhibitor genes were located on chromosome 3BS and 3DS; there was no known evidence of a homoeologous locus or loci on chromosome 3AS of the polyploid wheats [18,19]. Therefore, the tetraploid wheats, which are lacking the D genome, have only the inhibitor genes on chromosome 3BS [19].
Evolutionary pressures of various kinds have often been hypothesized to cause active and rapid evolutionary changes. In a co-evolving system of plant-insect interactions, plants synthesize a variety of toxic proteinaceous and nonproteinaceous molecules for their protection against insects [20,21]. Proteinase inhibitors are therefore a potential model system in which to study basic evolutionary processes, such as functional diversification [22].
It is well established that multiple forms of proteins are active on exogenous or endogenous α-amylases in the wheat kernel, and proteinaceous dimeric α-amylase inhibitors could function against α-amylase from various origins [23]. It is known that the bulk of wheat albumins consist of a few amylase iso-inhibitor families that are very likely phylogenetically related and coded by a small number of parental genes [24]. The α-amylase inhibitors have long been proposed as possible important weapons against pests whose diets make them highly dependent on α-amylase activity. In vitro and in vivo trials using α-amylase inhibitors, including those made under field conditions, have now fully confirmed their potential for increasing yields by controlling insect populations [16].
Two conflicting views confront ecologists and evolutionary biologists on the degree of symmetry in interactions between plants and phytophagous insects [25]. The symmetrical view holds that insects and plants have strong effects on one another's evolutionary and ecological dynamics. The asymmetrical view acknowledges that plants have major effects on insects but claims that insects seldom impose significant effects on plants [25]. Plant defense mechanisms have been the subject of intense investigation [26]. The genome shaping events and processes occurring at dimeric α-amylase inhibitor gene loci from the B and S genomes of wheat and Aegilops section sitopsis, respectively, have been characterized. A Phylogenetic Median-Joining network of the haplotypes and a neighbor-joining tree analysis have indicated that the inhibitor gene sequences from common wheat and T. dicoccoides are closely related to those from Ae. speltoides [27]. However, little is known about their evolution under the influence of ecology. The molecular diversity of α-amylase inhibitor genes, as well as their divergence among 16 populations of wild emmer wheat from Israel, was investigated to gain insight into the correlation between plant defense proteinaceous inhibitors and ecological factors.
Isolation of the ORF of dimeric α-amylase inhibitors
Using two cloning primers, genomic PCR amplifications were conducted, and one desired DNA band was detected in each accession of wild emmer wheat. Cloning the fragments yielded 244 positive clones from 13 accessions (randomly selected from 10 populations), which were subsequently sequenced (data not shown). Only three out of 244 dimeric α-amylase inhibitor genes had a common three bp deletion, and those three genes were obtained from one accession derived from Mt. Hermon, whereas the other cloned fragments had 426 bp long (data not shown). It was predicted that all of the 426-bp sequences would encode functional dimeric α-amylase inhibitors. Alignment of the gene sequences from emmer wheat with sequences from the species of Aegilops section Sitopsis (including Ae. speltoides, Ae. bicornis, Ae. longissima, Ae. searsii, and Ae. sharonensis), Ae. tauschii, einkorn wheats, and common wheat clearly indicated that the emmer wheat sequences were derived from the B genome [27].
SNP and haplotype analyses of dimeric α-amylase inhibitor genes
The frequency of SNPs in the dimeric α-amylase inhibitor genes in emmer wheat was 1 out of 5.7 bases, which was higher than the SNPs observed for kunitz-type α-amylase inhibitor and α-amylase/subtilisin inhibitor genes in barley and dimeric α-amylase inhibitor genes in common wheat [28-30]. Among the 426 nucleotides, there were 351 conserved positions and 75 variable positions among the 244 α-amylase inhibitor genes sequenced from 13 accessions.
A total of 74 haplotypes were revealed by sequence analysis (Figure 1); 53 of these were each found in only a single sequence. Haplotype 41 was observed at the highest frequency, i.e., in 38 gene sequences, followed by haplotype 27 in 33 sequences (Figure 1).
Figure 1. Haplotypes of 244 dimeric α-amylase inhibitors obtained from 13 accessions (10 populations) in Israel.
The relationship between SNPs and amino acid changes in the α-amylase inhibitor proteins is summarized in Table 1. The 75 SNPs resulted in 38 amino acid substitutions. The position of each SNP in the sequence, whether the predicted change was synonymous (silent) or non-synonymous (replacement), was determined. Forty percent of SNPs were found to occur at the third codon position, and as expected, most of these were synonymous (Table 1). A number of changes were also identified in codon positions 1 and 2, and these accounted for more than 95% of the non-synonymous changes (Table 1). In total, 60% of the SNPs resulted in non-synonymous changes.
Table 1. The variation of amino acids caused by the nucleotide changes in genes. The SNPs were detected by primers at the position with bold numbers in the Site column.
Primer design and SNP mining of wild emmer wheat
Using the information from the 75 SNPs identified in the α-amylase inhibitor genes, 16 primers (combined with the reverse cloning primer, R, as SNP markers) were successfully designed to detect the SNPs in 205 accessions from 18 populations. The primers, with the SNP (bold letters) at the 3' end and an extra mismatched nucleotide (underline) on the third nucleotide from the end are listed in Table 2. A total of 14 SNPs were detected with the 16 SNP markers from position 19 to 288 of the α-amylase inhibitor gene, and the size of the amplified fragments ranged from 158 to 426 bp. The data was then organized in terms of genotypic frequencies ("0" or "1") to assess the population structure.
Table 2. Specific primers designed from SNPs in the dimeric α-amylase inhibitor genes
There were only 5 and 2 accessions from Yehudiyya and Achihood, respectively. Thus, the data for Yehudiyya and Achihood were not used in further analyses. Positive fragment frequency for each primer in the 16 populations is listed Additional file 1.
Additional file 1. Positive fragment and the frequency of each primer in 16 Population of wild emmer wheat. This data showed the frequency of each specific primer in the 16 populations calculated by POPGENE 1.32.
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Genetic diversity and distance of α-amylase inhibitor genes
Some genetic parameters of the 16 populations of wild emmer wheat are summarized in Table 3. The proportion of polymorphic loci P (5%), the expected heterozygosity He, and Shannon's information index of the 16 populations of wild emmer wheat were 0.887, 0.404, and 0.589, respectively. The values of He ranged from 0.182 to 0.437, and the population of Kokhav Hashahar had the highest value of He (0.437), followed by the population of Rosh-Pinna, whereas the population from Daliyya was characterized by the lowest He value of 0.182.
Table 3. Genetic diversity of wheat dimeric α-amylase inhibitor genes, based on SNPs in 16 populations of wide emmer wheat.
The genetic distances (D) were calculated for comparisons of all 16 populations based on the positive fragment of SNP markers among all population pairs (see Additional file 2). The highest genetic distance (0.263) was obtained between populations of Kokhav Hashahar and Daliyya, whereas the most related populations were Qazzrin and Gamla with a genetic distance of 0.017. However, lower D values (< 0.050) were observed between some populations from different areas, and, for the most part, the estimates of D value were geographically independent. Large genetic distances and sharp genetic differentiation over long geographic distances could be found. For example, Kokhav Hashahar in southern Israel had higher D values with the populations from Gamla (0.221), Nahef (0.247), Beit-Oren (0.215), Daliyya (0.263), and Bat-Shelomo (0.224) in northeast Israel.
Additional file 2. Nei's genetic distance of the inhibitor genes in the 16 populations. This data showed the genetic distances (D) based on the positive fragment of SNP markers among all population pairs.
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Principle components & multiple regression analysis of environmental variables and SNPs
To assess if some of the ecological factors are correlated to each other, principle components analysis (PCA) was carried out using 23 ecological factors as variables. A combination of the first four components could give us a high cumulative percent (88.81%) according to the eigenvalues of the correlation matrix (see Additional file 3; 4), which could be used to explain the ecological associations. The main ecological factors of the first component were Rd and Ev (two water availability factors) that could give 40.60% eigenvalues, and the second component could give 28.66% eigenvalues (see Additional file 3). And from PCA analysis, it was known that the accessions from Mt. Hermon were affected the most by ecological factors (see Additional file 3).
Additional file 3. Principal components analysis. This data showed the eigenvalues of correlation matrix, eigenvectors and factor coodinates of 16 populations.
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Additional file 4. Principal components analysis of the ecological factors. This data showed the projection of the variables on the ecological factor-plane and eigenvalues of correlation matrix.
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After analyzed of the factors by projection of the variables on the factor-plane (see Additional file 4) and consulted the correlations of these factors (see Additional file 5), 11 independent ecological factors were chosen. And then, multiple regression analysis was done using these 11 factors to investigate the relationship between environmental variables and SNPs.
Additional file 5. Correlations of the Factors. This data showed the correlation of the 20 ecological factors that separated into four groups.
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The geographical, temperature, water, and solar radiation factors in Table 4, singly or in combination, explained a significant proportion of the diversity in the SNPs (Table 5). The best variable predictors of P, He, and Shannon's information index, significantly explaining 0.264 – 0.355 of the variance, was the water availability factor Hu-an. The combination of three variable predictors accounting for geographic and water availability factors Hu-an, Ev, and Lt (mean annual humidity, mean annual evaporation and latitude) accounted significantly (p < 0.05) for about 0.55 of the genetic diversity. The addition of a fourth variable predictor, Rad (total solar radiation per year), or Sh (mean number of Sharav days) to the first three factors accounted for approximately 0.65 of the diversity (significant at p < 0.05) (Table 5).
Table 4. The eco-geographical background of populations in this study
Table 5. Coefficient of multiple regressions of genetic indices and allele frequencies and environmental variables in 16 populations of wild emmer wheat as independent variables. *** = p < 0.001; ** = p < 0.01; * = p < 0.05; @ = p < 0.10; ns = p > 0.10. The definitions of factors were in Table 4.
SNPs could be classified into several categories in terms of their prime ecogeographical predictors. The best single variable predictors of SNP marker allele frequencies were: (1) water (Hu-an, Rr, Th, Rd, Ev): W24A, W46A, W47AT, W127G, W190C, W276A; (2) temperature (Td, Tm, Sh): W19G, W35A, W207T, W263TC, W263TA, W288CG; (3) geographic (Ln, Lt): W125G, W288AG; and (4) solar radiation (Rad): W195T; W259A. It was obvious that water factors were the best variable predictors, singly or in combination, with other ecological factors (Table 5).
Spearman rank correlations of SNP positions with environment
The average of genetic indices (P, He, and Shannon's information index) and He of each of the SNP positions with ecogeographical variables appear in Table 6. We recorded the ecological variables for the populations. The P, He, and Shannon's information index were negatively correlated with the three water factors: mean annual humidity (Hu-an), mean humidity at 14:00 h (Hu-14), and mean number of dew nights in summer (Dw). However, they correlated positively with other factors: latitude (Ln), seasonal temperature difference (Td), estimate of population size (Sz), marginality (Ma), mean inter-annual variability of rainfall (Rv), and total solar radiation per year (Rad). The correlation matrix between He in the SNPs and geographic variables contained 30 significant (p < 0.05) correlations. Five SNPs (e.g., W47AT, W125G, W127G, W263TA, and W288AG) positively correlated with Sz (rs = 0.556–0.687), and four SNPs (e.g., W19G, W47AT, W127G, and W288AG) positively correlated with Ln (rs = 0.508–0.567). Four SNPs: W24A, W127G, W190C, and W263TA negatively correlated with two water factors, Hu-an or Rn (mean annual rainfall or mean annual humidity) or both of these factor (Table 6).
Table 6. Spearman rank correlations of genetic indices and the genetic diversity of each SNP sites.
SNPs in the α-amylase inhibitor genes
In sequence comparisons, the 244 dimeric α-amylase inhibitor genes from wild emmer wheat, had a high level of similarity, indicating that the primary structure of these genes was similar to those of known dimeric α-amylase inhibitors 0.19 (WDAI-0.19) and 0.53 (WDAI-0.53). The predicted protein sequence of the 244 cloned α-amylase inhibitor genes from wild emmer wheat showed the presence of 10 Cys, which were the amino acids most important to the structure and function of the mature protein [31]. Changes in structure of α-amylase inhibitor proteins would affect their specificity and activity against different mammalian and insect α-amylase [32]. A comparison of sequence between members of the α-amylase inhibitors 0.19 group indicated that not only the 10 Cys residues were of importance, but also Asp110, Lys116, Asn29, Glu35, Ser94, Leu90, Trp51, His47, and Gln13 were important to form the structure of those inhibitors [33]. Most of the SNPs did not occur at highly conserved positions, which ensures that the α-amylase inhibitors keep their correct 3D structure to combine with the α-amylase. However, Gln13, His47, Ser49, Leu90, Val105, and Asp110 were changed by SNPs in some of the cloned α-amylase inhibitor genes (Table 1). It is noteworthy that only the α-amylase inhibitors from the D genome of Ae. tauschii and common wheat, which were closely related to inhibitor 0.19, had the His47 [30], whereas the His47 was replaced by Asp or Tyr in 98% of the inhibitor genes from wild emmer wheat.
Genetic diversity of the α-amylase inhibitor genes in wild emmer wheat
Genetic diversity of the α-amylase inhibitor genes of 198 wild emmer wheat accessions from 16 populations in Israel were revealed by 16 SNP markers. Individual accessions from different populations could not be distinguished clearly by the sequences of their α-amylase inhibitor genes; whereas, using the SNP-specific primers, all wild emmer wheat populations were distinguishable, even within closely related populations originating in proximate geographic locations (Table 3). Our results demonstrated that the polymorphism of α-amylase inhibitor genes in wild emmer wheat correlated with the ecogeographic distribution of the accessions. The results suggest that the gene was subjected to strong natural selection. The observations were consistent with previous results obtained with high- and low-molecular-weight glutenin subunits, which are also seed storage proteins [34-36]. In other studies, DNA diversity of glutenin subunits was shown to be correlated to environmental factors and variation [34].
The genetic diversity profiles in this study were compared with earlier allozyme studies [1], RAPD loci [37], and with the microsatellite studies [38] in wild emmer wheat populations. Although the SNP markers in the protein-coding genes yielded lower values of diversities than other methods, the results in this study were able to reveal the correlations of SNP variations in specific functional genes with ecological factors.
Central populations used in this study were collected in warm, humid environments on the Golan Plateau and near the Sea of Galilee. Marginal steppic populations were collected across a wide geographic area on the northern, eastern, and southern borders of wild emmer distribution involving hot, cold, and xeric peripheries, while marginal mesic (Mediterranean) populations were collected from the western border of wild emmer distribution [1,39]. The present study included 198 accessions collected from 16 different sites in Israel and covered a wide range of ecogeographical conditions across the distribution range of the species. Specific SNP positions detected in the α-amylase inhibitor genes were found to be highly effective in distinguishing genotypes and populations of wild emmer wheat originating from diverse ecogeographic sites in Israel. High levels of polymorphic loci (P), expected heterozygosity (He), and Shannon's information index (Table 3) with high genetic distance values between populations could be found (see Additional file 2). These results suggest that the genetic variation at these SNP positions in the dimeric α-amylase inhibitor genes was somewhat ecologically determined for these populations.
Genetic distance versus geographical distance
The relationship between SNPs' genetic distance and geographical distance was investigated, and it was found that the estimates of genetic distance (D) were geographically independent, as was previously found for allozymes, RAPD loci, and microsatellite analyses [1,37,38]. Quite often it is easier to find a greater genetic difference between proximal populations than among populations that are far apart. This was clearly demonstrated by local short transects of different soil types at Tabigha [40] and by the micro-differences of sun-shade differentiation at Yehudiyya [41]. Sharp genetic divergence (large D) over very short geographic distances against small genetic divergence (small D) between large geographically distances were observed in wild emmer populations (see Additional file 2). For example, it was shown that the genetic distance obtained between the population at Gitit and the population at Kokhav Hashahar (located only about 10 km apart), with D = 0.1513, was 2.66 times higher than the genetic distance between the population at Mt. Hermon and the population at J'aba (separated by 160 km, with D = 0.0569). In other words, the distance between the first 2 populations was 1/16 of the distance between the second 2 populations (Figure 2 and Additional file 2).
Figure 2. Geographic distribution of the tested populations of wild emmer wheat. The numbered populations are according to Nevo and Beiles (1989) [1] and details about the populations can be found in Table 4.
The genetic structure of wild emmer wheat populations in Israel is mosaic [36]. This patchy genetic distribution appears to reflect the underlying ecological heterogeneity at both micro- and macro-scales [1,37,38,40,41]. Thus the higher polymorphisms and genetic variations of dimeric α-amylase inhibitor genes within and between populations could be explained by natural selection.
Ecological genetics of SNPs in dimeric α-amylase inhibitor genes
Natural populations of wild emmer are highly polymorphic in morphological characters, as well as for various economically important traits [3,5,34]. Although major collection areas such as Mt. Hermon, Rosh Pinna, Gamla, Bat-Shelomo, and Tabigha are at similar longitude and latitude, they differ significantly in altitude. These locations, for example, are respectively at 1300, 700, 200, 75, and 0 m above sea level (Table 4). Along with these features, several other environmental factors differ for these locations [1,39].
In this study, the mean number of P, He, and Shannon's information index were negatively correlated with the three water factors and positively correlated with the other six factors (Table 6). It was noteworthy that the significant ecological factors (Ln, Ta, Td, Hu-14, Hu-an, Dw, and Rv), revealed by a Spearman rank correlations matrix between allozyme and climate, were similar to the results in this study [1]. This similarity might be because the correlation between ecological factors and coding sequences or proteins (allozyme) is different from the non-coding sequences. Moreover, the correlation between photosynthetic performance and ecogeographical variables indicated that ecological factors, e.g., sharav (Sh), dewy nights (Dw), radiation (Rad), rainy days (Rd), altitude (Al), and latitude (Lt) were distinctly correlated with photosynthetic factors [42]. Photosynthetic efficiency needs specific ecological factors, especially light.
In this study, the SNP variations showed significant correlations with ecological factors (Table 5; Table 6). Geographical, temperature, water availability, edaphic, biotic and solar radiation factors (Sz, So, Rad, Al, Rn, Lt, Sh, Rv, Ln, Td, Hu-14, Hu-an, Dw, and Ma), singly or in combination, explained a significant proportion of the diversity in the SNPs of α-amylase inhibitor genes. The association of these factors with SNPs was similar to the association of latitude/altitude with RAPD and microsatellite diversity [37,38]. It could be explained by the change in ecological factors, i.e., Al, the sharp gradient of climatic conditions from north to south in Israel, with increasing temperatures and decreasing water availability towards the semiarid zones in southern Israel. Also, the ecological factors used in this study were not representative of all the possible components involved in the determination of the real climate.
The SNPs that could determine the amino acid changes in the mature protein of α-amylase inhibitors were of great importance. Six specific primers (W125G, W127G, W190C, W259A, W263TC, and W263TA) were designed, based on the SNPs at five positions associated with amino acids changes (Table 1). It was shown that these SNPs were significantly correlated with water availability factors (Rd, and Hu-an), temperature factors (Sh, and Td), geographical factors (Ln, Al, and Lt), and solar radiation (Rad) better than the other factors (Table 5 and Table 6). Environmental stress can greatly influence plant susceptibility to herbivores and pathogens, and drought stress can promote outbreaks of fungal diseases and plant-eating insects [43,44]. Louda and Collinge (1992) reported guild-specific insect responses following soil water manipulations, and Larssou (1989) has clearly articulated why the actual response of insect herbivores to plant stress should be feeding-guild specific [45,46]. The results in this study indicated that water availability is the main factor that could affect the dimeric α-amylase inhibitor genes and, thus, the concordance between insect and plant. Recently, based on SNP analysis, highly significant correlations were also found between diversity at the barley Isa locus (coding for a bi-functional α-amylase/subtilisin inhibitor) and key water variables (evaporation, rainfall, and humidity) plus latitude [47]. The soil fungi may influence the survival of wild barley seed in soil and the subsequent establishment of plant populations. The higher diversity of soil fungi in dry environments may select for a higher diversity of defense proteins encoded by the Isa locus in the seed [47].
The herbivore insect and the level of herbivore pressure may vary with ecological factors, so that the wheat is under different herbivore-related selection pressures at each site. Different environmental pressures at each site directly related to the climate, but the wheat alpha-amylase inhibitors responded indirectly to those factors. There might be some evolutionary mechanisms that underlie the differences in diversity of α-amylase inhibitors and water factors. Historical events may have given rise to diversity patterns that correlate coincidentally with the ecogeographical variables tested in this study. However, probability would suggest it is far more likely that the variation in genetic diversity of this gene between populations is a product of selective forces. Selection pressure at this locus is likely to be caused by insects.
The populations of wild emmer wheat showed great diversity in dimeric α-amylase inhibitors, both between and within populations. We suggest that SNP markers are useful for the estimation of genetic diversity of protein-coding genes in wild emmer wheat. These results show significant correlations between SNPs in the α-amylase inhibitor genes and ecological diversities. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs, and the SNPs could be classified into several categories as ecogeographical predictors. A sharp genetic divergence (large D) over very short geographic distances against small genetic divergence (small D) between large geographical distance was found in wild emmer populations. It was suggested that the SNPs in the α-amylase inhibitor genes were subjected to natural selection, and ecological factors had an important evolutionary role in gene differentiation at the gene loci.
Plant material and ecological background of wild emmer wheat
Wild emmer wheat is a tetraploid and predominantly self-pollinated wheat, which is distributed over the Near East Fertile Crescent (Israel, Jordan, Lebanon, Syria, eastern Turkey, northern Iraq, and western Iran) [48]. The center of distribution and diversity of emmer wheat was found in the catchment area of the upper Jordan Valley (Golan Heights, eastern Upper Galilee Mountains, etc.) in Israel and its vicinity [1]. Wild emmer wheat covers wide ranges of eco-geographical conditions in Israel. However, towards their marginal and peripheral areas, both in Israel and Turkey, wild emmer wheat became semi-isolated or isolated, and smaller in size. This distributional pattern has a dramatic effect on their population genetic structure and differentiation [1]. Individual plants of emmer wheat were collected at random, at least 1 m apart, from populations differing in major ecological properties. These collection sites and populations have been described in detail elsewhere [1,39]. The genotypes used for the present study are conserved in the cereal gene bank of the Institute of Evolution, University of Haifa.
In this study we examined 205 T. dicoccoides accessions representing 18 populations collected from various locations in Israel, which represent a wide range of ecological conditions of soil, temperature, altitude, and water availability. The populations used in this study, along with their geographic origin and climatic conditions, are listed in Table 4. A full description of these populations was reported in Nevo et al. [1,39], and the map location of these populations were provided in Figure 2.
DNA isolation and PCR amplification
Ten seeds of each accession were germinated in the dark at room temperature. Genomic DNA was extracted from plant leaves at about 2 weeks of age with a modified CTAB protocol, as described in Murray and Thompson [49]. Two primers, F (5'-CTATGTATGCTCGTGGCGAC-3') and R (5'-ACTCATTT/CGCTTGACTAGGC-3'), were used to amplify the gene coding sequences of dimeric α-amylase inhibitors [30]. PCR amplification was performed with PTC-240 cycler (Bio-Rad) in 50 μL volume, which consisted of about 100 ng of genomic DNA, 100 μM of each dNTPs, 1 μM of each primers, 1U Taq polymerase, 1.5 mM Mg2+, and 1×PCR buffer. The cycling parameters were 95°C for 5 min to pre-denature, followed by 35 cycles of 95°C for 1 min, 60°C for 30 sec and 72°C for 1 min, and a final extension at 72°C for 5 min.
Sequence analysis of α-amylase inhibitor
Amplification products were separated in 2% agarose gels. The desired DNA fragments were recovered from gels and ligated to the pBluescript SK (+) T-vector plasmid (Stratagene), and then the positive clones were screened and sequenced. The analysis of full-length sequence and the construction of subsequent nucleotide sequence were carried out under DNAman 5.2.2 [50], and the multiple sequence alignment software Clustal W [51] was used for the SNP assessment. The α-amylase inhibitor ORFs were translated into amino acid sequences using the ORF Finder program at the NCBI [52]. The polymorphic positions were used instead of all of the mutation positions, including the positions with change that observed only once in the dataset, in the subsequent analysis.
Specific primer design and analysis of SNP
Polymorphic positions were identified by MEGA version 3.1 [53]. Sixteen specific PCR forward primers (combined with the cloning reverse primer R), were designed based on the alignments of dimeric α-amylase inhibitor gene sequences obtained from wild emmer wheat (Table 2). The SNPs were positioned at the 3'-end of the primers, based on the fact that a 3' mismatch makes PCR more specific at the selected annealing temperature [54,55]. The power of the oligonucleotide for allele discrimination was enhanced by introducing an artificial mismatch at the 3'-terminal base [56]. Sequences for the specific primers for dimeric α-amylase inhibitor genes and the basic cycling conditions are listed in Table 2. PCR was performed on genomic DNAs from all accessions of the 18 populations.
Data acquisition and analysis
The gels were scored for the presence or absence of bands that showed a reproducible pattern among genotypes, and for each band with a SNP position with two alternative alleles: present (1) or absent (0). For wild emmer wheat, which is a self-pollinating species with a quite limited rate of outcrossing (estimated t approximately 0.005), we assumed 100% homozygosity. The identification of 16 SNP positions led to the construction of a 198 accessions (two populations with less than 8 accessions were not used in this analysis) × 16 loci data matrix, which was analyzed for diversity within and between populations.
POPGENE 1.32 [57] was used to compute genetic polymorphism (P), expected heterozygosity (Nei's gene diversity) (He), and Shannon's information index (I) for each SNP position and population. Spearman rank correlation coefficients were used to assess differences in genetic indices P, He, and Shannon's information index and climatic variables in 16 populations. STATISTICA version 6.0 [58] was used to do the PCA analysis and conduct stepwise multiple regression (MR). Multiple regression analysis was conducted to test the best predictors of P, He, and Shannon's information index in the 16 populations using these genetic indices as dependent variables and the eco-geographic factors as independent variables at each of the polymorphic SNP loci.
Authors' contributions
WJR designed and carried out the experiment and wrote the manuscript. WYM designed the experiment, formulated the questions, and contributed to writing the manuscript. LXY participated in the experiment. YZH carried out sequence alignment analysis. BB contributed to the data analysis and writing. EN formulated the questions, retrieved and analyzed the data, and planned the experiments. ZYL planned the study and participated in the design of the experiments.
The authors thank Drs. George Fedak and Thérèse Ouellet (Agriculture and Agri-Food Canada), and Dr. Manyuan Long (Department of Ecology and Evolution, University of Chicago, USA) for critical review of the manuscript. We also express our gratitude to the four anonymous reviewers for helpful comments to improve the manuscript. This work was supported by the National High Technology Research and the Development Program of China (863 program), the Key Technologies R&D Program of China (2006BAD01A02-23 and 2006BAD12B02), and the FANEDD project (200357 and 200458) from the Ministry of Education, China. Y.-M. Wei was supported by the Program for New Century Excellent Talents in the University of China (NECT-05-0814). Y.-L. Zheng was supported by the Program for Changjiang Scholars and Innovative Research Teams in University of China (IRT0453).
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Join the 'Carbidopa / Levodopa' group to help and get support from people like you.
Carbidopa / Levodopa News
New Clues to Easing Side Effects From Parkinson's Drug
Posted 8 days ago by Drugs.com
WEDNESDAY, Nov. 18, 2015 – The drug levodopa is a leading treatment for Parkinson's disease, but for most patients the medication also brings debilitating side effects. Now, scientists say animal studies are pointing to a compound that might reduce those unwanted effects. "If clinical trials confirm our preliminary findings, the eventual drug developed could make a significant improvement in the quality of life for people with Parkinson's disease," lead researcher D. James Surmeier, chair of physiology at Northwestern University School of Medicine in Chicago, said in a university news release. According to the researchers, Parkinson's disease affects more than one million Americans, and that number is expected to double by 2030. Levodopa is widely used to treat the stiffness, tremors and poor muscle control caused by the illness. But the drug can also trigger uncontrolled movement – ... Read more
Related support groups: Parkinson's Disease, Sinemet, Levodopa, Stalevo, Carbidopa/Levodopa, Sinemet CR, Stalevo 100, Parkinsonian Tremor, Parkinsonism, Rytary, Parcopa, Carbidopa/Entacapone/Levodopa, Stalevo 200, Dopar, Stalevo 50, Stalevo 125, Atamet, Larodopa, Duopa, Stalevo 75
Parkinson's Drug Shows Promise Against Macular Degeneration
Posted 14 days ago by Drugs.com
THURSDAY, Nov. 12, 2015 – A common Parkinson's disease medication might hold potential for preventing or treating macular degeneration, the leading cause of vision loss in the elderly, new research suggests. At this stage, no one is recommending that patients take the drug, levodopa (L-dopa), to thwart eye disease. But the findings are intriguing, researchers said. "Patients taking L-dopa for any reason are much less likely to develop age-related macular degeneration. If they do, they develop the disease much later in life than those not taking L-dopa," said study lead author Brian McKay, an associate professor of ophthalmology and vision science at the University of Arizona. However, the study doesn't actually prove that levodopa causes a lower incidence of age-related macular degeneration. It only uncovered an association between the two. Age-related macular degeneration affects ... Read more
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Exercise Benefits People With Parkinson's Disease: Study
Posted 24 Jun 2015 by Drugs.com
WEDNESDAY, June 24, 2015 – Parkinson's disease patients who begin regular exercise earlier have a much slower decline in quality of life than those who start exercising later, a new study finds. National Parkinson Foundation (NPF) researchers looked at information from nearly 3,000 patients. More than 1,300 reported doing little regular exercise before taking part in the study. Over two years, 500 of the inactive patients began to exercise more than 2.5 hours a week. The researchers compared patients who exercised regularly for the entire two years to people who were inactive at the start of the study, but then began a regular exercise routine. The study didn't note the type of workouts, just the total amount of exercise. After two years, scores on a questionnaire that measured the impact of Parkinson's on daily life in a number of areas – including mood, movement and social ... Read more
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Ex-Baseball Star Kirk Gibson Has Parkinson's Disease
Posted 28 Apr 2015 by Drugs.com
TUESDAY, April 28, 2015 – Kirk Gibson, who played 17 seasons of Major League Baseball and is perhaps best known for a dramatic pinch-hit, ninth inning home run to win a World Series game in 1988, said Tuesday he has Parkinson's disease. Gibson, 57, a Michigan native who played 12 seasons with the Detroit Tigers, said in a statement that "I have faced many different obstacles in my life, and have always maintained a strong belief that no matter the circumstances, I could overcome those obstacles. "While this diagnosis poses a new kind of challenge for me, I intend to stay true to my beliefs," he added. "With the support of my family and friends, I will meet this challenge with the same determination and unwavering intensity that I have displayed in all of my endeavors in life. I look forward to being back at the ballpark as soon as possible." According to the U.S. National Institutes of ... Read more
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FDA Approves Duopa (carbidopa and levodopa) Enteral Suspension for Parkinson's Disease
Posted 12 Jan 2015 by Drugs.com
NORTH CHICAGO, Ill., Jan. 12, 2015 /PRNewswire/ – The U.S. Food and Drug Administration (FDA) has approved AbbVie's (NYSE: ABBV) Duopa (carbidopa and levodopa) enteral suspension for the treatment of motor fluctuations for people with advanced Parkinson's disease. DUOPA is administered using a small, portable infusion pump that delivers carbidopa and levodopa directly into the small intestine for 16 continuous hours via a procedurally-placed tube. Duopa was approved by the FDA as an orphan drug, a designation granted to products intended for the treatment of rare diseases or conditions affecting fewer than 200,000 patients in the U.S. "There is unmet need for treatment options for patients with advanced Parkinson's disease. As the disease advances, it can be difficult to control motor features," said C. Warren Olanow, M.D., Professor, Department of Neurology and Department of ... Read more
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Impax Pharmaceuticals Announce FDA Approval of Rytary (carbidopa and levodopa) for Parkinson's Disease
Posted 12 Jan 2015 by Drugs.com
HAYWARD, Calif., Jan. 8, 2015 /PRNewswire/ – Impax Pharmaceuticals, a division of Impax Laboratories, Inc. (NASDAQ: IPXL), today announced that the U.S. Food and Drug Administration (FDA) approved Rytary, an extended-release oral capsule formulation of carbidopa-levodopa, for the treatment of Parkinson's disease, post-encephalitic parkinsonism, and parkinsonism that may follow carbon monoxide intoxication and / or manganese intoxication. Rytary is not for use in patients using nonselective monoamine oxidase inhibitors (MAO) inhibitors. "The FDA approval of Rytary (pronounced rye-TAR-ee) is an important new development for the treatment of Parkinson's disease and provides an extended-release carbidopa-levodopa product that treats Parkinson's disease," said Fred Wilkinson, president and CEO, Impax Laboratories. "Rytary is designed to address one of the most significant unmet needs for ... Read more
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Levodopa May Beat Newer Meds for Long-Term Parkinson's Care: Study
Posted 11 Jun 2014 by Drugs.com
WEDNESDAY, June 11, 2014 – When it comes to which drug works best for patients with newly diagnosed Parkinson's disease, older may still be better, a new study finds. Research published June 10 in The Lancet finds that the dopamine drug levodopa still outperforms newer medications for the long-term care of people newly diagnosed with Parkinson's. "This study lays to rest lingering questions among both people with Parkinson's disease and their doctors about which drug is most beneficial when first beginning treatment for the disease," said James Beck, vice president of scientific affairs at the Parkinson's Disease Foundation (PDF). In the largest-ever trial of Parkinson's disease treatment, levodopa offered patients better mobility and a higher quality of life than the two main alternatives – drugs called dopamine agonists and monoamine oxidase type B (MAO-B) inhibitors. The study ... Read more
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Experimental Gel May Help Those With Advanced Parkinson's
Posted 18 Apr 2012 by Drugs.com
WEDNESDAY, April 18 – A gel form of two common Parkinson's drugs delivered via a feeding tube-like device may help people with advanced disease reduce medication side effects and possibly avoid brain surgery. That's the report from researchers who found the experimental levodopa-carbidopa intestinal gel works better than a standard pill regimen in reducing "off" time in people with advanced Parkinson's disease. About 1 million people in the United States are living with Parkinson's disease, a progressive movement disorder marked by tremor, slowness and/or rigidity. The disease slowly destroys the nerve cells in the brain that produce the chemical dopamine, which controls muscle movement. Treatment with oral levodopa-carbidopa – brand names include Sinemet, Sinemet CR and Parcopa – helps replace dopamine levels, but higher doses and long-term use of the oral drugs can cause ... Read more
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Parkinson's Drugs Linked to Behavior Problems in Study
Posted 30 Mar 2011 by Drugs.com
TUESDAY, March 29 – Parkinson's disease drugs called dopamine agonists appear to cause impulse control problems in almost one-quarter of patients, says a new study. Previous research has linked dopamine agonists, which include Mirapex (pramipexole) and Requip (ropinirole), to impulse control disorders, such as gambling addiction and hypersexuality, and to compulsive behaviors, such as binge eating, overspending and excessive computer use. In this study, Mayo Clinic researchers analyzed Parkinson's disease patient records over two years. "What we found was that as many as 22 percent of patients during that two-year period had a new-onset impulse control disorder," lead investigator and neurology fellow Dr. Anhar Hassan said in a Mayo Clinic news release. The higher the dose of dopamine agonist, the more likely a patient was to develop an impulse control disorder, the researchers found. ... Read more
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Parkinson's Drugs Tied to Compulsive Behaviors
Posted 10 May 2010 by Drugs.com
MONDAY, May 10 – Medicines used to treat Parkinson's disease may increase the risk of impulse control disorders, such as problem gambling, compulsive shopping and binge eating, researchers warn. In a new study that included 3,090 patients being treated for Parkinson's at 46 movement disorder centers in the United States and Canada, the researchers found that 13.6 percent of the patients had impulse control disorders. These impulse disorders included gambling (5 percent), compulsive sexual behavior (3.5 percent), compulsive shopping (5.7 percent) and binge eating (4.3 percent), and nearly 4 percent of the patients had two or more of these disorders. Impulse control disorders were more common among patients taking dopamine agonist medications (17.1 percent) than in those not taking the drugs (6.9 percent), the study authors found. Other factors associated with impulse control disorders ... Read more
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Cutting Parkinson's Drug Dose Linked to Withdrawal Effects
Posted 14 Jan 2010 by Drugs.com
WEDNESDAY, Jan. 13 – Reduced dosages of dopamine agonists, drugs routinely used to treat Parkinson's disease, can cause symptoms similar to those experienced by addicts in withdrawal, such as anxiety, panic attacks, pain, dizziness and drug cravings, researchers say. The symptoms of what the researchers have dubbed "dopamine agonist withdrawal syndrome" have been linked to a disruption in levels of dopamine in the brain, according to the study published in the Jan. 12 issue of the Archives of Neurology. "Like cocaine and methamphetamines, dopamine agonists work by stimulating the reward pathways in the brain," senior study author Dr. Melissa J. Nirenberg, said in a news release from Weill Cornell Medical Center. "For this reason, it makes sense that they would engender similar withdrawal symptoms, particularly in those with high cumulative drug exposure," explained Nirenberg, associate ... Read more
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Maritime Today
The Maritime Industry's original and most viewed E-News Service
The Singapore Shipping Association (SSA) has welcomed the decision by Singapore's Minister for Trade and Industry to extend the Competition (Block Exemption
China's Hong Kong Nicaragua Development (HKND) Co. said it is delaying the start of construction on a controversial $50 billion inter-ocean canal across Nicaragua until late 2016.
Dan-Bunkering (Middle East) DMCC is pleased to announce that Lamin Bara has joined the office in Dubai as Bunker Trader as per 1 November 2015.
Lamin was
Owners are rapidly laying up containerships as the market slows. The size of the idle fleet will get bigger while rates and profits slide, says Drewry Shipping Consultants Limited.
Egypt plans to complete a side channel in East Port Said, near the Suez Canal, that would speed up shipping and allow ships direct entry into the port by the end of June 2016,
Concordia Maritime has signed a contract for the charter of an IMO2/3 class MR tanker, the company announced today. The vessel will be chartered jointly with Stena Weco,


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What's The Most Important Warcraft Movie Casting Decision?
Blizzard is making a movie based on its rather popular Warcraft property. Sam Raimi is directing it, meaning one actor's involvement is almost assured. So which actor and which role? »
Blizzard is making a movie based on its rather popular Warcraft property. Sam Raimi is directing it, meaning one actor's involvement is almost assured. So which actor and which role? »
The creators of Castle Crashers brought their latest game, right now known as Game 3, to Comic-Con, an Xbox Live Arcade title heavy on the "TBA." Platforms, release date, final game name and just about everything is still "TBA." »
Heading to Comic-Con? Yeah, me too. If you don't already have plans for your Thursday night and wouldn't mind have your face melted clean off, join us at the Brutal Legend Metal Meltdown show. It's free. GWAR will be there. »
We're making the annual trek to the San Diego Convention Center for Comic-Con 2009, which kicks off with a preview night on Wednesday and wraps up on a lazy Sunday. This year, we're doing things a little different. »
The Comic-Con comic book news explosion continues, as Activision and Twistory announce plans for a graphic novel based on the upcoming time-twisting shooter Singularity. »
Add BioWare's Mass Effect to the growing list of video games properties becoming comic book series. Electronic Arts and Dark Horse revealed Mass Effect: Redemption today, a series set before the events of Mass Effect 2. »
Capcom shows off the cover to its Marvel VS Capcom 2 hip-hop mixtape today, with a limited-edition vinyl version of the soundtrack up for grabs at the San Diego Comic-Con this week. »
Series creator Jordan Mechner once again plumbs the depths of the Prince of Persia mythology, penning an anthology graphic novel to serve as a prequel to the upcoming feature film. »
EA and DC Comics have teamed up to create a comic book mini-series based off of the upcoming Dante's Inferno video game, with preview copies available at the San Diego Comic-Con later this week. »
The San-Diego Comic-Con is all about exclusive goodies, and in that spirit Mad Catz is hawking a limited number of exclusive Street Fighter IV arcade sticks and pads to show attendees. »
2K introduces the BioShock 2 Artists Series today, with a rendition of sisters both big and little by Jhonen Vasquez, creator of Invader Zim and Johnny the Homicidal Maniac. »
The annual San Diego Comic-Con isn't just about comic book culture. It's the mecca for all things nerdy and obsessive, including video games. This year's gaming presence at the convention is impressive, not to mention regularly double-booked. »
Electronic Arts and Valve will be revealing all-new Left 4 Dead 2 content at this year's San Diego Comic-Con, including the new campaign "Swamp Fever," darkly pictured above. Where and when can you play it? »
Match the lips to the character in this teaser image from the San Diego Comic-Con exclusive poster for DC Universe Online. »


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Chicago, IL (Sports Network) - Andrew Shaw scored a pair of goals while Corey Crawford turned away 25 shots as Chicago stayed alive with a 4-1 victory over Detroit in Game 5 of this Western Conference semifinal series.
The Blackhawks won Game 1 of this series, but then dropped the next three games, their first three-game losing streak of the season, to fall into a 3-1 hole.
Bryan Bickell and Jonathan Toews also scored while Duncan Keith added two assists for the Blackhawks, who went 2-for-3 on the power play to keep the series going.
"We had nothing to lose tonight because there was no tomorrow," said Bickell. "Going into the game we had a plan and really stuck to it, so we'll see where it goes from here."
Daniel Cleary had the lone goal while Jimmy Howard gave up all four goals on 45 shots for the Red Wings, who will have another chance to close out this set on Monday when Game 6 will be played in Detroit.
"We just weren't good enough tonight at all," said Detroit head coach Mike Babcock. "As far as our plan and the way we have to be successful, they had way too much space to skate around and that's not the way we want to play them."
Detroit had the first power play of the game when Viktor Stalberg was whistled for hooking at 7:20, but Crawford kept the Wings off the board thanks to a nice stop on a Niklas Kornwall blast through traffic.
Patrick Kane was stopped by Howard on a short-side attempt down low at the right side with just over nine minutes to play, but the Hawks went up three minutes later.
A shot on net was stopped by Howard, but the rebound came out to the left side where Michal Handzus got a stick on it before Kane threw it on net. Howard again made the stop, but this time the rebound came out to the right where Bickell slammed it home for his fourth of the playoffs and a 1-0 lead with 5:52 left in the frame.
Crawford finished the first period with 11 saves to keep the Red Wings out of the net.
Pavel Dastyuk had a shot hit off the left post about 6 1/2 minutes into the second, but Detroit was able to get on the board three minutes later.
A backhand saucer shot from the right wing by Henrik Zetterberg got on net and Cleary snuck home the rebound from the left for his fourth of the playoffs.
Chicago, though, got the goal back on the power play as a one-timer from Keith in the high slot was redirected into the net by Shaw at 13:08.
Another power play chance saw the Blackhawks add to their advantage as a clearing attempt was kept in at the left point by Keith. He fired the disc over to the right where Marian Hossa fed it down low. Toews kicked it from the skate to the stick and snapped it high past Crawford at 15:47 for his first of the playoffs.
"It is a relief and a confidence builder to know that what you're doing out there will lead to something," said Toews. "I feel like that if I keep shooting the puck it will go in or force the goaltender to do something so it's a good feeling to have."
At 6:58 of the third, Chicago put the game out of reach. Michal Rozsival sent a pass from the right wing to the slot where Stalberg's shot missed the net. However, Shaw snuck around behind the net from the right side to the left and stuffed it in for his third of the playoffs.
This is the 16th meeting in the postseason between these Original Six squads,
with Chicago taking eight of the previous matchups ... Detroit went 0-for-4 on
the power play as Chicago has killed off 35 of the 36 shorthanded situations
it has faced in the playoffs ... Zetterberg has eight assists in the playoffs
... Detroit had just four shots in the third period.
Pope Francis' visit to Philadelphia in September has altered the date of the Penn-Villanova football game.
Kyle Busch won Sunday's road course race in Sonoma, California, but he still has a long way to go to become eligible for a spot in the Chase for the Sprint Cup championship.
NFL players have a very short shelf life to make significant amounts of money so it's tough to fault anyone who has outperformed a contract when they go knocking on the door to ask for more.
Pete Mackanin will remain interim manager of the Philadelphia Phillies for the remainder of the season, the team announced Tuesday.
The Oklahoma City Thunder have reportedly retained restricted free agent forward Kyle Singler with a five-year contract extension.
The Detroit Red Wings signed veteran forward Brad Richards a one-year contract on Wednesday.


text:
|Setting the story straight
Dear Lucas Papaw Customers
There is currently a lot of interest in our products on social media and in the press. Over the past 105 years, we are honored to have become a household name and a staple item in many medicine cabinets across Australia, and indeed around the world. We are very proud of the benefits of our products, of the healing nature of what we do and our ability to help people of all ages in their everyday lives.
|Read more...


text: Featured comments + Add comment Comments (1) Featured GraveLord "LMFAO! So misinformed. "Having recently been dissected the sleek machine boasts a quad-core processor, 2 GB of RAM (half of which is currently reserved by Nintendo's OS) and modern architecture pumping out advanced features such as increased eDRAM and Direc..." 1102d ago


text:
Continuation of Part 1…
Written on the evening of Thursday, 12 March 2015
After lugging all our stuff, my husband decided that Mama should come over and spend the night with me. I myself could not envision going through labour alone, and though I was sad that my designated birth companion (my husband), the one who has been attending birth classes with me, who knows all the right words to say, couldn't be there until morning, I was still thankful.
But the time between my husband leaving to fetch my mum and coming back (which was about an hour) was honestly one of the worst experiences of my life. My surges' intensity took such a turn to the point where both my thighs felt the "zaps" every time; my legs trembled like crazy. At the time I was not aware that surges may be felt in the thighs. Only later on, reading articles, did I find out that that can be the case:
"…may be characterized by pains in your abdomen or lower back (or both) and may be accompanied by pain in the upper thighs."
I don't know how to describe surges other than, you can tell that it's coming, it's like a tightening all around your abdomen (unlike period cramps which you can usually pinpoint to a certain area and massaging it or curling into certain positions tends to ease the discomfort). Being alone left me with the most negative thoughts possible. Lying on the bed trying to massage my thighs while remembering to breathe correctly and keep good thoughts was a true struggle. I don't remember whether I started to vocally cry out with each surge at this point, but I certainly did that a lot towards morning and still feel guilty at disturbing my wardmates' peace and quiet. (Though I still did hear them snore sometimes.)
Mama was such a champ throughout. The massages were epic, and she kept repeating reassuring phrases to me, "Mama sayang Nchah," "Sikit je lagi, ni semua nak bagi baby keluar," "Breathing, breathing". She did not sleep a wink. I think I managed some form of short naps, though mostly I did keep my eyes shut as much as possible anyway throughout both labour and delivery.
I was not aware of the time, but remember it being time for Subuh prayer, and letting my mum pray first. My husband came back at some point earlier (he spent the night in the hospital's surau downstairs) and again I felt so grateful. I managed to take wudhu' at the toilet and pray while lying down on the bed, but not before vomitting on myself right after drinking a cup of warm water. I have been feeling wind and burping urges all night but they did not come out. I hardly ate anyway other than one date and plain water. Throwing up made me feel better though.
(Wanted to also mention this: more of the mucus plug was discharged throughout labour, so toilet experiences were already such a sight. )
I think we called the nurses before Subuh as my surges were getting unbearable (though still not very frequent — they said to inform them if it gets to five surges per ten minutes, but my mum still counted about two surges per ten minutes). I cannot remember the details of events, but there was another VE, and I was four cm dilated, and they said they would bring me into the labour room after 7am. I was in a wheelchair again, though this time in a green hospital gown, head haphazardly covered in a towel and my mind already in a "separuh akal" state, not caring or noticing anything around.


text:
Cozy Up at a Spa Resort Inside Beautiful Catskill Park
The Emerson Resort and Spa • Mt. Tremper, NY
Escape Kit
- A One-Night Stay for Two in a Regal Suite, Sundays Through Thursdays from Now to November 8, 2015
- Combine Vouchers to Stay for Two Nights
- After Purchase, Follow Your Voucher's Redemption Instructions to Complete Your Reservation
A sophisticated take on rustic styling, this Catskills resort contains an award-winning spa and toasty in-room fireplaces.
The Destination
Mt. Tremper is located in the verdant Hudson Valley inside Catskill Park, a world apart from the big city. Perhaps that's what has lured generations of vacationers here from New York City. Natural beauty abounds in the form of towering waterfalls cutting through beds of crumbling shale, and dense forests crisscrossed with hiking trails and bike paths. Mt. Tremper is also home to the Kaatskill Kaleidoscope—housed in a converted grain silo—which is officially the world's largest kaleidoscope.
The Digs
The Emerson Resort & Spa captures the historic ambiance of a Catskills resort. The regional feel of the resort extends to the suites, where guests are greeted with warm fireplaces and polished wood furnishings. Each suite features oversize windows, a flat-screen TV, and a bathroom with a Swiss shower with multiple shower heads. For even more pampering, The Emerson Spa contains ten treatment rooms and a large fitness center with a yoga studio. Services include massages, facials, salon services, and more—all designed to make your stay here an unforgettably blissful retreat.
The Details
• The 10% room tax and $12 service fee are not included and are payable directly to the property
• Be advised: This resort is currently undergoing some renovations, particularly in the restaurant
• Take note of the blackout date on October 11, 2015 when making plans
The place is so beautiful and so relaxing that I fell in love with it. If you love hiking and photography like I do, this is the place for you. The room that I stayed in was so beautiful. This is a great place to visit. I had a wonderful time.-- LivingSocial Member
It was an awesome deal. I had a terrific massage and the room was beautiful and spacious. We also ate at the dining room and the meal was fabulous. I would highly recommend this to everyone.-- LivingSocial Member
escapes 30-day promise
LivingSocial gives you up to 30 days to return your unused voucher for a full refund. Once you've made a reservation, you are subject to the merchant's cancellation and refund policy. If you want to secure your preferred dates of travel, we strongly recommend that you complete reservation with the merchant within 30 days of your purchase—after that, LivingSocial will not issue any refunds.
the fine print
- Limit 2 per customer, up to 2 additional as gifts; up to 2 vouchers may be combined to extend stay
- Only valid for stays now to November 8, 2015
- Nights must be spent consecutively; all inclusions must be redeemed during one stay
- Reservations required and subject to availability
- Merchant cancellation/re-scheduling policy of 3 days in advance of scheduled arrival; voucher subject to forfeiture
- Not valid toward existing or room block reservations
- 10% room tax $12 service fee not included; payable directly to the property
- Incidental charges, charges for additional services and gratuity at the property are not included
- Voucher valid for 2 guests
- The promotional value is valid only for date range purchased and may not be exchanged or redeemed for any other dates
- Blackout date(s): October 11, 2015
- Other conditions apply • PAID AND PROMOTIONAL VALUES EXPIRES ON November 8, 2015 • PAID VALUE DOES NOT EXPIRE • PAID VALUE EXPIRES ON November 8, 2020 • PROMOTIONAL VALUE EXPIRES ON November 8, 2015 • Enjoy By November 8, 2015
Other conditions apply
Except where noted in the fine print:
• Cannot be combined with any other offer or promotion
• Tax and gratuity are not included
• Merchant is responsible for the quality of the products or services provided to you at redemption
• LivingSocial Terms and Conditions - https://www.livingsocial.com/terms
Original Price:
- 0% savings
- remaining


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- Filed Under
There will be an all-you-can-eat fish fry and sloppy joe dinner from 4 to 7 p.m. Saturday at the Pentecostal Tabernacle of Oceola, 3938 Marion Melmore Road.
Cost is $8 for adults and $5 for kids 12 and younger. For take out, call 419-562-6304 during the fish fry. For details, call 419-561-0098.


text:
2002 Ford Ranger • 135,000 miles
Have a 4.0 that misses on number 6 have removed the head had it reworked and replaced springs reienstalled the head an it still misses on number 6 the coil has been replaced. Themoter has 135000 miles on it I have checked the compressone on that side all three cylender have 120 lb on all three cylinders running compressione
October 19, 2013.
October 19, 2013.
I have a 1996 Saturn SL1. The engine has a misfire and alternates between running fine for a few weeks and then running rough for a few weeks. When the car is running rough it has less ...
3 answers • 1996 Saturn SL1 • 183,173 miles
Can a compression test be done if engine will not turn? Car will not start. Mechanic wants to tow car to his workshop and have compression checked. All parts under bonnet have been ...
2 answers • 2000 Ford Ranger
In completing a dry compression test my results were as follows: 1 - 140 2 - 10???? 3 - 100 4 - 170 I repeated the test twice cranking the car for 5 seconds on each cylinder...
7 answers • 1996 Toyota Paseo • 165,000 miles
I just started the car and put it in drive and took off when i heard a puff noise and the car stalled. no it wont turn over. everything electrical works but it sound like no compression...
2 answers • 1998 Plymouth Breeze • 100,000 miles


text:
A virtual atmosphere manages all applications, information and data resources. It consists of a virtually hosted atmosphere allowing the users to connect the services being hosted over the internet. A cloud computing basically provides three types of services:
1. Infrastructure-
2. Platform-as-
3. Software-as-
Cloud computing has many features that can be of huge advantage to the companies and the clients. They are:
1. The Cloud server provides you all the required application and support for your work and it greatly reduces your burden.
2. A user friendly interface is provided to the user. You only need to have an internet connection to start work and remaining all requirements will be fulfilled by the cloud server.
3. The risk of data loss is never a problem in the cloud server because the data are stored in multiple locations and servers are placed at remote locations.
4. You can access your data and applications from anywhere and at any time. Along with that multiple users can work simultaneously on the same application at the same time.
5. Very much cost efficient and you don't need special persons to deal with the infrastructure and the hardware.
6. On the clients' demand capacity of the user to access and storage can be increased at any point of time.
Hiring a good cloud computing company http://www.cloud-
Depending on the client's requirements, cloud computing companies provide services that can be profitable to a large extent. And also significantly the cloud services are device independent;
About Author:


text:
Press Release Distribution
Filter NewsShow All Results
News By Country
News By TagYoutube
Related Tags
youtube Press Releases+ XML/RSS
By Carriage House Entertainment
(Los Angeles, CA – July 2014) Recording artist Tae Brooks debuts the highly anticipated official music video for his original ballad, 'It's Always Been You' on YouTube http://youtu.be/GIsDEbiOD8w .
By BLOOPIT
Tens of Thousands of Downloads in Pre-Release Prompts Major Upgrades, Partnerships & Collaborations
By Waterlogg Productions
Joe Bev & Lorie Kellogg's 2-minute video shows off the married couple's 103 audio titles and 34 hours per month of radio and podcasts. Includes radio drama and comedy, documentary, interview, biography, autobiography, and archival rarities.
By North Carolina Department of Cultural Resources
The North Carolina State Archives' YouTube channel now offers a series of online tutorials on how to preserve your family documents and photographs and how to protect them from weather damage and normal deterioration.
By Bridget O'Brien PR and Events
Lenore and Joseph Luca are young adults making an impact for other teens by hosting their TV series Keeping Up With Cool, but that's not all. These two ambitious youngsters don't and won't stop motivating their generation.
By Robert Lucy Creative
The accomplished comedian adds another video parody to her arsenal of humor.
By FINE Success Impact Media
June 2014 F.I.N.E. Success Magazine features Diamond Dallas Page (DDP). Page is an actor, author, motivational speaker, and founder of DDP Yoga, but what he's most recognized for is his professional wrestling career.
By VideoMakerFX
Over 20,000 copies sold in less than 30 days! All of the top businesses out there are using video! The most successful businesses have proved that video is what captures the attention of buyers and makes that sale!
By Bridget O'Brien PR and Events
Join Jenna Rose as she kicks off her concert for the summer starting on May 24th and May 31st
By GCFLearnFree.org
User-Friendly Mobile App Allows Users To Create, Curate Their Own Playlists
By Eclipse Trade and Investments
Helping viewers find humor in life situations and pictures is the goal of the website. A wide collection of funny images and jokes can be accessed on the website and youtube.com.
By IECRM
DENVER, Colorado -- May 6, 2014 -- IECRM (www.iecrm.org), the non-profit energy trade association and electrical school based in Denver, today released a new video that features personal interviews with ten members of the association.
By Bond Twins Productions
The YouTube channel "Watch Twinzies" (http://www.youtube.com/watchtwinzies) is launching its first online series, "Couples Therapy". This comedic show centers on identical twins Jennifer (Jennifer Bond) and Lauren (Lauren Bond).
By PMD-Partners
Powerup TV enters consumer's homes and Content Creators' hearts this week as it is adopted by its first one thousand users just in time for YouTube to cut out tens of thousands of partners channels from their once lucrative ad revenue stream.
By J Blair Brown Consulting
The series, aptly titled "Shine," opens doors for serious actors in/around the Central PA region
By BoredomBreak.com
Relying solely on social media, new media production company BoredomBreak plays with the idea of utilizing the significance of social media in the 21st century instead of operating a traditional website.
By PodcastOne
The man who made the honey badger a household name with over 67 million YouTube views now has a podcast. Randall, the narrator of YouTube's World Famous Honey Badger video joins PodcastOne with Honey Badger's Badass Animal Podcast.
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Ecuador Freedom Bike Rental Partners with VideoTov to Produce Customer Motorcycle Videos that People Will Want to Watch
By MY LIFE AS A VIDEO GAME
Kickstarter innovation leaps forward with the direct to fan business and release of MY LIFE AS A VIDEO GAME webseries by Britain's Petros L. Ioannou.
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Featuring Dynamic Personalities from Africa and the Diaspora.
By Martan Productions
Four Episodes Now Available on the Marvelless Martan YouTube Channel
By Netaim LLC
Marc Bullard reveals what to do in order to have a successful video marketing campaign on YouTube.
By Have Fun Teaching
The "What Do The Letters Say?" video by Have Fun Teaching is an educational alphabet and phonics parody of the What Does The Fox Say? video.
By Lime Jam PR
Huddersfield's very own Brit hop hero Savvy is set to release the new single 'Powerskills' on 6th April.
By Sockeye Media LLC
New York, NY - Feb 28, 2014 –Sockeye Media LLC, the creator of the Mother Goose Club and Mother Goose Club Playhouse children's series, has surpassed a distinguished one billion combined views on its YouTube channels after less than three years of...
By Solopress
A Flippin' Brilliant Way To Celebrate Pancake Day And Support A Great Cause
By J Blair Brown Consulting
In a twist from the norm, it's the women whose lives are the central theme of the show
By Treycent.com
Company: Treycent.com Contact: Alex Jones Address: Treycent, Sarasota, FL Telephone Number: 941-363-1751 Email: email@example.com Treycent.com Good news for all music lovers! Now you can use Treycent's mobile web app to play SoundCloud tracks or...
By Doylestown Gold Exchange
Doylestown Gold Exchange & Jewelers, in Bucks County, is excited to announce the launch of their new YouTube series, highlighting the Doylestown area.
By RK Chevrolet
"Things You Didn't Know About Your Chevy" provides informative tips and tricks for RK Chevrolet customers.
By A&M Entertainment Inc.
Motivational speaker and TV host Ugo Lord has announced the new schedule for his popular YouTube-based talk show.
By Buy Here Pay Here NJ
February 3, 2014 - http://bhphcardealers.net has announced that they added a new video to their main page. The video is simple and provides a clean visual introduction to the site.
By Modernism Week
Introducing "MAN ABOUT MODERNISM" - An Exclusive Insider's Guide to the Sold-Out Midcentury Celebration in Palm Springs, CA
By ScaleLab LLC
Santa Monica-based multi-channel network, ScaleLab, has become the first YouTube MCN to permanently abolish 'lock-in' contracts. The result is that all YouTube creators in ScaleLab's MCN may come and go as they please.
By TLR Publicity & PR
Jessica Lewis, best known as 'MahoganyCurls™', host premier LIVE Q&A show and is inundated with more than 800 natural hair questions in less than 30 minutes!
By CareSync
"I'm a patient and I need to be heard" video attracts thousands of views and national media attention as hospital bed-bound Gleason tells providers what she needs.
By Lesson That Rock
Youtube is a great website for entertainment purposes. But did you know there is another whole side to YouTube that includes How-To Videos. How to play Guitar is very popular and watching videos has many advantages!
By Featured Music Videos
It just easier for Indie Artist to get their music videos featured and extend their fanbase.
By MacSolutions Plus
Garret Cleversley leads informational how-to video guides for Apple users
By Jenn Page
Talented filmmaker-on-the-rise Jenn Page has found herself working with some notable Hollywood names in the past few years, including actor Corey Feldman, Academy Award Nominee Eric Roberts, Academy Award Winner Shirley Jones, and most recently Billy Ray.
By The Pinnock Agency
Author and YouTube Personality, JStillz Takes Entertainment to Higher Level In 2014.
By VIPGIF.com
VIPGIF.com is a free web app that's allows users to convert Instagram, YouTube and Liveleak videos into GIFs. This easy to use service quickly converts video and allows for GIFs to be downloaded or shared with friends and family.
By Talk Fusion
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By Apex Feline
Do you work on improving yourself and your skills? Or do you waste time competing against others?
By alotta.edu, inc.
Today, alotta.edu, inc. reveals the most AMAZING "alien helps human encounter" since ET met Drew Barrymore.
By Advanstar Automotive
The Advanstar Automotive Group, a leading provider of integrated media solutions to the automotive aftermarket and collision repair industry, has announced that the Motor Age YouTube channel has surpassed one million views.
By Harmony Soul Entertainment
Harmony Soul Entertainment and it's Star Hip Hop artist Brooklyn Sarden has inked a multi level Publishing Deal with Mega social media site YouTube.
By New World Films
New World Films, Samuel Palomin, collaborated with Fashion News Live on their new video episodes. There are five new episodes airing each week.
By 11:24 Design,Inc.
Los Angeles, September 23, 2013 – Hyundai Hope On Wheels selected Art Sims' 11:24 Design as the ad agency to create the official Hyundai Hope On Wheels Videos campaign for children with cancer 2013.
By MRB Public Relations
For the season of giving, you can give ten children the ability to walk
Page updated every 10 minutes


text:
Zack's Kernel News
Speeding Up Boot-Time
Nathan Zimmer noticed that on large-memory systems, booting up could take more than an hour, just due to initializing memory. He posted a patch written by himself and Mike Travis, which would cause the CPUs to start up first – that way the memory initialization could be offloaded to multiple CPUs at the same time, thus speeding up the whole process.
Greg Kroah-Hartman replied, pointing out that Nathan and Mike had made this patch a configuration option. But, Greg said, there really wasn't any reason to make it optional, because it was a pure win. Surely no one would ever choose to have a slower system for no reason.
H. Peter Anvin joined Greg in that assessment, saying that probably any system with more than a certain amount of memory – Peter guessed 128GB – would want to enable this feature by default.
Peter also wanted to know how much code this patch added to the kernel. Nathan said it was only about 400 lines of source code, but Peter clarified – he meant he was curious how many bytes of machine code the patch would add to the actual binary. To which Nathan said it was about 32KB. Peter was surprised that the patch could really be that large. He said, "Is this init code? 32K of unconditional runtime addition isn't completely trivial." Nathan replied that perhaps 24KB of it was real run-time code.
There was some back-and-forth about alternative implementations for Nathan's and Mike's idea. Yinghai Lu had some ideas, and Ingo Molnár had a proposal for a whole different concept that relied on the "buddy allocator" – a mechanism of dividing up system memory into easily handled chunks.
One way or another, though, this feature will soon be accepted into the official tree. Whether Nathan's and Mike's patch gets in, or something by Ingo, the patch will be implemented somehow. The speedup is simply too great to ignore.
Named Anonymous Memory
Colin Cross felt that certain features were just languishing in linux-next, and he wanted to see them migrate into the official tree, if possible. Specifically,
ashmem (Android Shared Memory) had some very cool features. Among them, Colin said, were the features implementing various types of anonymous memory.
Colin especially liked "named" anonymous memory – a region of memory that would show up as a file in
/proc/pid/maps. He said, "The Dalvik VM likes to use this feature extensively, even for memory that will never be shared and could easily be allocated using an anonymous mmap, and even malloc has used it in the past. It provides an easy way to collate memory used for different purposes across multiple processes, which Android uses for its 'dumpsys meminfo' and 'librank' tools to determine how much memory is used for java heaps, JIT caches, native mallocs, etc."
In addition to migrating the code out of linux-next, Colin wanted to enhance it with new system calls and other behaviors to make it more generally useful.
Christoph Hellwig was not convinced that this feature was really valuable, though. He didn't see "what ashmem buys over a shared mmap of /dev/zero." Colin replied, "ashmem ties the memory to an fd, so it can be passed to another process and mmaped to get to the same memory, but /dev/zero does not. Passing a /dev/zero fd and mmaping it would result in a brand new region of zeroed memory. Opening a tmpfs file would allow sharing memory by passing the fd, but we don't want a world-writable tmpfs."
At this point, Christoph said he was surprised that
/dev/zero didn't already do what Colin attributed to ashmem – but after looking at the
/dev/zero code, he had to affirm that ashmem represented something new.
Alex Elsayed joined the discussion, as did John Stultz, and proceeded to explore implementation details with Colin. A solid design didn't emerge in the discussion, but it seems as though at least in theory, ashmem may have some valuable qualities, worth migrating out of linux-next and into the main tree.
Zack Brown
The Linux kernel mailing list comprises the core of Linux development activities. Traffic volumes are immense, often reaching 10,000 messages in a week, and keeping up to date with the entire scope of development is a virtually impossible task for one person. One of the few brave souls to take on this task is Zack Brown.
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