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Bacterial pathogens often show significant intraspecific variations in ecological fitness, host preference and pathogenic potential to cause infectious disease. The species of Listeria monocytogenes, a facultative intracellular pathogen and the causative agent of human listeriosis, consists of at least three distinct genetic lineages. Two of these lineages predominantly cause human sporadic and epidemic infections, whereas the third lineage has never been implicated in human disease outbreaks despite its overall conservation of many known virulence factors.
Here we compare the genomes of 26 L. monocytogenes strains representing the three lineages based on both in silico comparative genomic analysis and high-density, pan-genomic DNA array hybridizations. We uncover 86 genes and 8 small regulatory RNAs that likely make L. monocytogenes lineages differ in carbohydrate utilization and stress resistance during their residence in natural habitats and passage through the host gastrointestinal tract. We also identify 2,330 to 2,456 core genes that define this species along with an open pan-genome pool that contains more than 4,052 genes. Phylogenomic reconstructions based on 3,560 homologous groups allowed robust estimation of phylogenetic relatedness among L. monocytogenes strains.
Our pan-genome approach enables accurate co-analysis of DNA sequence and hybridization array data for both core gene estimation and phylogenomics. Application of our method to the pan-genome of L. monocytogenes sheds new insights into the intraspecific niche expansion and evolution of this important foodborne pathogen.
Listeria monocytogenes is a Gram-positive foodborne bacterial pathogen and the causative agent of the human and animal infectious disease, listeriosis. L. monocytogenes can thrive in diverse environmental reservoirs (e.g. soil, water, and sewage) and proliferate under unfavorable conditions (e.g. high osmolarity, low pH, and refrigeration temperature) that other bacterial pathogens cannot endure [1-4]. Its robust physiological characteristics, coupled with its ubiquity in food processing, distribution and retail environments, have made L. monocytogenes difficult to manage in food manufacturing, particularly for ready-to-eat food products. L. monocytogenes causes the highest rates of hospitalization (about 92%) and mortality (about 20%) among all foodborne bacterial pathogens in the United States [5], making the control of this bacterium in foods a high priority for both food safety and public health. Yet, the versatile lifestyle of L. monocytogenes both inside and outside its host, and its unique capability to invade and replicate in different host cell types (e.g. macrophages and nonprofessional phagocytes), have made this opportunistic pathogen a paradigm for studying host-pathogen interactions, pathophysiology, gene regulation, and stress adaptation [6,7].
Previous molecular subtyping studies have collectively suggested that the species of L. monocytogenes is composed of at least three major evolutionary or genetic lineages that notably differ in their prevalence in causing human and animal diseases [8-15]. Specifically, lineage I (or LI) and lineage II (or LII) of L. monocytogenes are frequently isolated from foods and implicated in the vast majority (> 95%) of both sporadic cases and epidemic outbreaks of human listeriosis [3]. Genetic lineage III (or LIII) strains are rarely reported in cases of human infections, but are sometimes associated with animal disease cases [3,14,16]. The mechanisms underlying the biased predominance of certain L. monocytogenes genetic lineages in human listeriosis remain largely unknown. Several recent studies have revealed elevated levels of genetic diversity among LIII isolates [12,15]. Multilocus sequence typing analysis on the basis of partial sigB and actA gene sequences have also suggested that LIII is polyphyletic, with the co-existence of at least three distinct subgroups (i.e. LIIIA, LIIIB, LIIIC) [14,16]. Atypical phenotypes of LIII isolates, such as deficiency in rhamnose fermentation [14], attenuated virulence potential [16], reduced resistance to heat and cold stresses [17] and lowered biofilm productivity [18], have collectively indicated that LIII may have followed a distinct evolutionary path from other L. monocytogenes lineages.
Compared to fairly extensive studies on LI and LII strains, little is known about LIII. Although it is documented that most listerial virulence factors such as the positive regulatory factor (or PrfA) are well conserved across the entire L. monocytogenes species, LIII strains are underrepresented in both food contamination and human listeriosis. This led us to speculate the existence of additional, yet-to-be-identified genetic factors in the predominant disease-causing L. monocytogenes lineages (i.e. LI and LII) that may mediate listerial niche adaptation, resistance to extra- or intracellular stresses, and pathogenicity. These unknown genetic factors may have been lost, mutated, or decayed in LIII as the genomes evolved, resulting in a defective phenotype for LIII isolates in certain ecological and host niches. To test our hypothesis, we combined in silico comparative genomic analyses with an array-based comparative genomic hybridization (CGH) approach to probe the genomic diversity of L. monocytogenes and to identify genomic features common in LI and LII but absent in LIII. Array CGH is a powerful yet cost-effective approach for genotyping and detecting intraspecies genomic diversity for many bacteria. Previous efforts on comparative genomic analyses underscore the usefulness of CGH in resolving genetic lineages and identifying strain- or lineage-specific genes in L. monocytogenes [10,19-22]. However, most of these studies targeted only a number of selected genes or partial listerial genomes, making an accurate assessment of intraspecies genomic diversity difficult.
It is recognized that a few sequenced genomes may not fully represent the entire genetic repertoire of a given organism [23-35]. For this reason, the pan-genome concept has triggered new investigations on genomic diversity for several bacterial species, including Streptococcus spp. [24,28,29], Haemophilus influenzae [25], Neisseria meningitides [30], Escherichia coli [31-33], and Lactococcus lactis [34]. Pan-genome refers to the total genetic repertoire of a given species, which is typically composed of "core" genes plus some "dispensable" or "accessory" genes [25,27,35]. Pan-genomic DNA arrays, which probe the full genetic repertoire, have recently gained increasing popularity for the systematic survey of diversity in prokaryotic species [31,36,37].
The availability of more than 20 L. monocytogenes full and draft genomes has made this pathogen an ideal candidate for pan-genomic study (Table (Table1).1). Our initial comparative analysis of 17 L. monocytogenes genomes indicated a "closed" pan-genome for this bacterial species. Species with a closed pan-genome typically share highly syntenic genomes with less frequent horizontal gene transfers (HGT) and genomic rearrangements. Therefore, the entire gene pool can be fully sampled by sequencing a small set of representative isolates, and the number of new genes to be discovered by sequencing additional genomes will quickly approach zero. This prompted us to design and construct a pan-genome CGH array that, in theory, accommodates the total genomic diversity of the L. monocytogenes species on a single DNA chip. Compared to several previous pan-genome microarrays that targeted either the conserved sequence of gene families with low probe density or no coverage of the intergenic regions, we utilized a novel probe selection algorithm (PanArray) to design a pan-genome tiling array that incorporates the genomes of 20 available L. monocytogenes strains [38]. This design provides unbiased coverage of the pan-genome, and also superior accuracy and resolution for data analysis.
Using integrated data obtained from both in silico whole-genome comparisons and pan-genome CGH analyses, we (1) explored the intraspecific genetic diversity of L. monocytogenes with a focus on the largely unexplored genetic lineage III; (2) estimated the core and pan-genome that define the L. monocytogenes species; (3) identified unique protein-coding genes and regulatory RNAs in the predominant disease-causing lineages, as they may relate to ecological fitness, host niche adaptation and pathogenicity; and (4) reconstructed phylogeny for different L. monocytogenes lineages and strains based on pan-genome characteristics.
Initial power-law regression analysis of 17 sequenced L. monocytogenes genomes (Table (Table1)1) suggested that this bacterial species exhibits a nearly closed pan-genome, which would yield rapidly diminishing returns of less than 7 novel genes per additional genome sequenced. Therefore, we presumed a single array could be designed to query the full genetic repertoire of the species, and be used to completely genotype currently unsequenced strains. For this purpose we designed a pan-genomic array comprising 385,000 50-mer in situ synthesized oligonucleotide probes that fully tile the sequences of 20 L. monocytogenes genomes (Table (Table1),1), with no gaps, at greater than 2-fold coverage of each genome. Shortly after we completed our chip design, four additional L. monocytogenes genomes were sequenced to closure, including strain Clip 80459 (LI), strain Finland 1988 (LII), strain R2-561 (LII) and strain HCC23 (LIII). These new L. monocytogenes genomes enabled us to evaluate the genomic coverage of our array design by individually mapping each of the 385,000 oligonucleotide probes to annotated genes to the four genomes. A 50-mer probe was mapped to a particular gene if it perfectly matched the gene sequence or contained only a single nucleotide mismatch. For each annotated gene, the probe coverage was calculated as the percentage of the gene length covered by mapped probes. Results in Table Table22 suggest that our design adequately represents the intraspecies diversity of L. monocytogenes, particularly for LI and LII genomes. However, due to the limited number of fully sequenced LIII genomes available at the time of design, the coverage for LIII specific genes is less optimal, as indicated by HCC23.
Genomic DNA of nine LIII strains were each co-hybridized on the pan-genome arrays with that of EGD-e (LII) as an internal reference. The nine LIII strains were carefully selected from a strain collection to represent 3 different serotypes (4a, 4b, and 4c) and 3 different subgroups (LIIIA, LIIIB, and LIIIC) of this lineage (Table (Table1).1). Individual probes were designated as present or absent in the sample based on statistical analysis of the normalized signal intensities (see Materials and Methods). Since the position of each probe is known for all sequenced L. monocytogenes genomes, genes were scored by the fraction of targeting probes with a positive signal, otherwise known as the positive fraction (PF). This yields a very flexible scoring scheme that can be readily applied to any intragenic or intergenic feature of the genome targeted by a sufficient number of probes. A high PF indicates a gene is likely present in the hybridized genome. Circular maps of all PF values for the nine LIII genomes in reference to an LI strain F2365 and an LII strain EGD-e are shown in Figure Figure11.
To select an appropriate PF threshold and test the accuracy of gene calls based on PF values, we examined the true-positive and false-positive rates of the PF criterion for 51,814 annotated L. monocytogenes genes, compared against genomes for which we had both sequence and CGH array data. True gene "presence" was determined by a tblastn search of the 51,814 predicted proteins against a six frame translation of the genome [39], requiring a minimum of 50% amino acid similarity and an E-value ≤ 10-5. Figure Figure22 shows the Receiver Operating Characteristics (ROC) curves for the PF criterion measured against the tblastn standard for two L. monocytogenes strains, EGD-e and J2-071. The PF measure is remarkably robust, as there appear to be very few genes near the classification threshold. Additional file 1 shows a density estimation of PF values for both present and absent genes, suggesting that the vast majority of present genes have PF > 0.9 and absent genes PF < 0.1. Based on the ROC analysis, a PF cutoff of 0.6 was chosen to best match the tblastn results and minimize the expected error rate. The seemingly higher false-positive rate for J2-071, in comparison to the closed EGD-e genome, is partially due to tblastn false-negatives incurred from the 78 gaps in the J2-071 draft genome. In these cases, a gene that is truly present, but overlapping a sequencing gap, is falsely reported as absent by the tblastn method which artificially increases the measured false-positive rate of the CGH array method.
Accuracy statistics for the chosen 0.6 PF cutoff versus the 50% alignment similarity cutoff are given in Table Table3.3. The array has perfect sensitivity for detecting the EGD-e and J2-071 control genes. Expected accuracy was estimated for detecting both individual gene variants from all other strains and for detecting homologous gene groups (HGs). Orthologous gene groups are typically preferred; however, the inability of CGH to accurately determine sequence identity and gene order makes it impractical to discriminate between highly similar paralogs. Alternatively, we tested for the presence of 3,560 strongly homologous groups, identified by clustering proteins with higher than 50% amino acid similarity. A gene group was marked as present in a genome if any gene from that group exceeded the BLAST or PF threshold. Figure Figure22 also displays the true- and false-positive rates of HG detection alongside the original ROC curves. In comparison to detecting individual gene variants, HG detection significantly increases the sensitivity of the array without increasing the false-positive rate. When analyzing only a single gene variant on the chip, high polymorphism in the sample genome can disrupt hybridization and lead to false-negatives. However, by considering an entire gene group, a sample only needs to hybridize with its nearest variant, thereby increasing the sensitivity [34]. To demonstrate the sensitivity of the array at detecting HGs in unsequenced strains, Table Table33 also lists accuracy statistics for EGD-e and J2-071 when the probes specific to those genomes are removed from the analysis. This simulates the accuracy of the array at calling genes in an unsequenced LII or LIII strain. The sensitivity of the array is only slightly affected, with a 0.2% true-positive rate drop for EGD-e and a 1.3% drop for J2-071. The drop is more pronounced for J2-071 because it is one of only two LIII genomes included on the array, so ignoring the J2-071 specific probes affects the sensitivity of calling HGs from that lineage.
The expected number of new genes to be discovered by sequencing additional L. monocytogenes strains, and the sizes of the core and pan-genomes, were estimated using methods adapted from Tettelin et al. [24]. Frequent gaps and sequencing errors in low-quality genome assemblies were found to cause many missed protein alignments, which affected the core genome estimation. For example, only 683 EGD-e proteins meet the alignment threshold in all 24 draft L. monocytogenes genomes, an unreasonably low number. Additionally, fragmented annotations in the low quality genomes artificially inflate the pan-genome size estimate. To avoid these artifacts, only 18 "high quality" L. monocytogenes genomes were used for the new genes and pan-genome estimation. Genomes sequenced to less than 10× coverage using 454 pyrosequencing were excluded from the sequence analysis (Table (Table1).1). Array CGH results for the 8 additional LIII genomes were included in the core gene estimate.
To estimate the L. monocytogenes core genome, the number of shared genes was computed for many random permutations of N genomes, and the mean number of shared genes was computed for each N. The number of core genes for the species was estimated by fitting an exponential decay function to the means. For the high-quality sequenced genomes, this analysis yielded an estimated horizontal asymptote of 2,467 ± 7 core genes. However, the sequenced genomes include only two LIII genomes. Repeating the analysis for all 26 genomes, including CGH results for the 8 additional LIII genomes, reduced the estimate by over 100 genes to 2,330 ± 5, emphasizing the importance of a balanced sample of diversity for estimating core genome size. Figure Figure3A3A displays the result of the 26 genome analysis including a smoothed density plot of the shared gene count distributions, the mean value for each N, and the best-fit exponential decay.
Imperfect detection sensitivity due to sequencing gaps makes it impossible to achieve convergence for real data, so an exact core genome cannot be determined. Any non-zero false-positive rate for detecting core genes will artificially shrink the core genome with each additional genome, violating the horizontal asymptote of an exponential decay. This is evident in the almost linearly decreasing means towards the tail of Figure Figure3A.3A. To account for these false-negatives, we introduced an additional parameter to the core genes model that adds a constant number of false-negatives upon the addition of each genome (see Materials and Methods). The revised model is a much closer fit to the data (residual standard error of 2.98 versus 10.68), accounts for noisy draft and CGH data, and yields an increased core genes estimate of 2,456 ± 4. This likely represents an upper bound on the core genome size. Considering results from both models, and the uncertainty caused by the draft genomes and CGH data, we estimate the core genome of L. monocytogenes to be between 2,330 to 2,456 genes (approximately 80% of a typical L. monocytogenes genome).
A major limitation of array CGH is that this method cannot detect novel genes contained in LIII genomes. For this reason, the pan-genome estimation was performed for only the high-quality sequenced genomes, of which two are from LIII. Again, the number of new genes identified by sequencing each additional genome was computed for many random permutations of N genomes. The number of new genes identified for each N was modeled by the power law function n = κN-α [26]. Using the median values, the power law exponent α was estimated to be 1.12 ± 0.02. This is slightly lower than our original estimate of 1.38 due to the recent sequencing of four additional genomes, an updated annotation, and a stricter similarity threshold. In both cases, an exponent α > 1 indicates a closed pan-genome, meaning the size of the pan-genome is a bounded function of the number of sequenced genomes. However, fitting a power law to the mean values of these distributions yields α = 0.85 ± 0.01, suggesting an open pan-genome (Figure (Figure3C).3C). This difference is caused largely by the diverse strains N1-017, HCC23, and J2-071, which contain many strain-specific genes and pull the mean values higher than the medians. For example, strain HCC23 contains 122 strain-specific genes not found in any of the other 17 strains. Removal of these three genomes from the analysis results in an α slightly greater than one for both the mean and median analyses. Two of these genomes are the only LIII strains in the analysis, indicating that additional sequencing from LIII may reduce the exponent even further. This regression analysis suggests L. monocytogenes has a significantly diverse gene reservoir, and additional sequencing of LIII genomes is necessary to resolve the exact size and nature of the L. monocytogenes pan-genome.
The estimated growth of the L. monocytogenes pan-genome with additional sequencing was also simulated using many random permutations of genomes. For open pan-genomes, the cumulative number of unique genes discovered with the sequencing of additional genomes can be modeled by Heap's law using the power law function n = κNγ [26]. This regression is illustrated by Figure Figure3B3B and γ was estimated as 0.12 ± 0.001. Since the growth of an open pan-genome is equivalent to the number of new genes added after sequencing each successive genome, the derivative of the pan genes function should be equal to the new genes function. That is Nγ-1 N-α and α = 1 - γ for α < 1. Although simulated separately, the pan and new gene functions do follow this property for the mean value regressions, with α = 0.85 and γ = 0.12 being in good agreement. For N = 18, the mean estimated pan-genome size is 4,052 and continues to grow, with diminishing returns, for larger N.
The above method is useful for estimating the size of the pan-genome, but because it depends on the order of the genomes analyzed, it does not yield a single representative set of pan genes for the analyzed strains. An alternative that does not depend on the order of genomes is to measure the number of gene groups identified by a similarity clustering method such as OrthoMCL [40]. We applied a similar approach, but for clustering strong homologs rather than orthologs, to be consistent with the other analyses. From a graph of 52,776 proteins with > 50% similar proteins connected by edges, 3,744 HGs were identified (Additional file 2) using the MCL graph clustering algorithm [41]. This provides a relative lower bound for the size of the currently sequenced L. monocytogenes pan-genome.
Lineage-specific genes refer to genes that are exclusively present in a single L. monocytogenes lineage based on the above defined similarity threshold. To maintain a stringent threshold, a gene is not considered to be lineage-specific if any member of its HG is present in another lineage. Annotated genes in F2365 (LI), EGD-e (LII), and J2-071 (LIII) were used to screen for gene lineage specificity against all genomes analyzed in this study. Table Table44 lists 4 LI- 5 LII- and 6 LIII-specific genes identified in our study. Most of these genes encode hypothetical proteins. It is notable that only 5 of the 21 LII-specific genes previously identified by Doumith et al [10] passed our lineage specificity threshold. We used colony polymerase chain reaction (PCR) assays to verify the lineage specificity for all LI- and LIII-specific genes identified by CGH analysis (except for LMOf2365-0409 due to the small size of this gene for proper PCR primer design). A total of 225 colony PCR assays were conducted for randomly selected L. monocytogenes strains in our collection, including 8 LI, 8 LII, and 9 LIII strains. The PCR results confirmed the lineage specificity for all genes analyzed, suggesting that the CGH approach was accurate for calling gene presence or absence and determining lineage specificity.
We identified 86 disparately distributed genes (or DDGs) as listed in Table Table5.5. DDGs refer to genes that are highly conserved (PF > 0.6 or protein similarity >50%) in LI and LII genomes but absent or highly divergent (PF < 0.6) in at least six of the nine LIII genomes. DDGs are of particular interest for us because the biased distribution and conservation of these genes in LI and LII genomes likely correlate to the enhanced ecological fitness and pathogenicity of L. monocytogenes in the host. The largest functional group of DDGs (41%) is associated with carbohydrate transport and metabolism. Figure Figure11 illustrates their distribution. L. monocytogenes harbors one of the largest bacterial carbohydrate phosphotransferase system (PTS) genes [42-44]. The abundance and diversity of the PTS system allows this soil saprophyte to utilize different carbon sources associated with the ecosystems it inhabits such as soil, silage and sediments. Fifteen PTS genes were identified as DDGs; most are associated with fructose-specific PTS enzyme II components (lmo0357-0358, lmo0631-0633, lmo2135-2137, and lmo2733). We surveyed the distribution of 978 annotated PTS genes and their homologs in all 26 L. monocytogenes genomes, and found 965 (99%) PTS genes are conserved in all LI and LII genomes and 7 (0.7%) are specific to LI. In contrast, 137 (14%) PTS genes are absent or divergent in LIII genomes. Diversity in PTS content is most noticeable among the three LIII subgroups, where 48 (4.8%), 137 (14%), and 136 (13.9%) PTS genes are absent in LIIIA, LIIIB and LIIIC, respectively. An interesting distinction among 3 subgroups is that LIIIA strains are capable of fermenting rhamnose, whereas LIIIB and LIIIC strains are deficient in rhamnose utilization [14]. We discovered a cluster of six genes (lmo2846-2851), which is likely to mediate rhamnose utilization, is missing from all LIIIB and LIIIC genomes. Five genes in this cluster [45] share protein similarities to the rhamnose catabolic pathway in Escherichia coli [46,47] and other Gram-positive bacteria such as Bacillus subtilius (Additional file 3).
The second-largest functional group of DDGs consists of 12 putative transcription factors representing 7 different regulatory gene families. Six are adjacent to PTS genes and possibly involved in regulating carbohydrate metabolism. Four are absent from the non-pathogenic L. innocua [43], L. welshimeri [48] and L. seeligeri [49], suggesting roles in virulence and pathogenicity. One Crp/Fnr (cyclic AMP receptor protein--fumarate and nitrate reduction regulator) family gene lmo0753 was found to be highly specific to LI and LII but absent in LIII. This Crp/Fnr factor is adjacent to a bile resistance gene btlB and shares similar functional domains with prfA, the master regulatory gene of L. monocytogenes virulence.
We found multiple DDGs associated with gastrointestinal (GI) tract adaptation. For instance, two bile-associated genes btlB (lmo0754) and pva (lmo0446) are absent in LIII. Both genes help L. monocytogenes resist the antimicrobial effects imposed by bile salts during its passage through human GI tract [50]. Loss of these genes lowered tolerance to bile and reduced persistence in murine GI tract [51]. The glutamate decarboxylase (GAD) system mediates the acid resistance in bacteria [52-54]. In L. monocytogenes gadD1 (lmo0447) is responsible for growth at mild acidic conditions (pH = 5.1) and gadD2 (lmo2363) primarily mediates the resistance to severe acidic stress (pH = 2.8) [55]. We found that gadD2 is conserved in all lineages, whereas gadD1 and its coupled glutamate: γ-aminobutyrate antiporter gadT1 (lmo0448) are absent in most LIII strains except for J2-071 and HCC23. An arginine deminase (ADI) system (lmo0036-0041) was recently characterized in L. monocytogenes [56]. The ADI system plays a role in listerial acid tolerance and may contribute to the enhanced adaptation to acidic conditions in the stomach. It was previously reported that this gene cluster is present in LI and LII but absent from LIII and non-pathogenic L. innocua and L. welshimeri [56]. Our results, however, showed that the ADI gene cluster is also highly conserved in LIIIB. An additional seventeen DDGs have no homolog in the genome of L. innocua, including three putative genes encoding LPXTG surface proteins (lmo0333, lmo1666 and lmo2085) and sepA, a putative virulence factor co-regulated by PrfA and σB [57,58].
Complete tiling of the L. monocytogenes pan-genome allowed us to survey the distribution of 100 non-coding small regulatory RNAs with specified 5' and 3' positions [45] in 9 LIII genomes. The majority (87%) of these sRNAs are conserved in LIII genomes, and only eight were found to be absent or divergent in LIII (PF < 0.6) (Table (Table6).6). Noticeably, all eight sRNAs are also absent from L. innocua, and five were differentially expressed in intestinal lumen or blood, suggesting roles in host niche adaptation. For example, ril38 contributes to listerial survival in human blood [45].
To reconstruct the phylogeny of all L. monocytogenes strains analyzed in this study, we surveyed the binary distributions of 3,560 HGs (Additional file 4) and 2,846 EGD-e protein-coding genes among the 26 L. monocytogenes genomes, respectively. We then constructed neighbor-joining (NJ) trees [59] based on a maximum-likelihood gene content distance measurement [60] (Figure (Figure4).4). The NJ trees based on 3,560 HGs (Figure (Figure4A)4A) and 2,846 EGD-e genes (Figure (Figure4B)4B) both clearly separated all L. monocytogenes strains into 3 main clusters (i.e. a LI cluster, a LII cluster and a LIII cluster)[61]. However, the EGD-e gene-based NJ tree showed a distorted topology, indicative of a bias caused by a restricted set of loci used for phylogenetic reconstruction [62].
Of note in LI, the serotype 4b strain N1-017 appears to be closely related to serotype 1/2b strains in the LI cluster, likely representing an evolutionary intermediate between the split of serotype 4b and serotype 1/2b [10]. Of note in LII, four strains F6900, F6854, J2818 and J0161 were previously traced back to a single food processing facility over a time span of 12 years [63]. These four isolates are clustered closely on a single branch, indicative of a recent common ancestry.
While the NJ trees based on gene content allowed some inference of L. monocytogenes phylogeny, the reliability of the tree topology can be compromised by reticulate events such as horizontal gene transfer (HGT). Therefore, a split network was constructed using the Neighbor-net algorithm [64] to evaluate the extent by which incompatible phylogenetic signals (e.g. HGT) might affect our estimation of phylogenetic topology. Split networks do not force the formation of a tree-like structure and are able to represent incompatible signals as parallel edges, indicating the possibility of HGT or recombination. The resulting split network (Figure (Figure4c)4c) shows a congruent topology with the NJ tree (Figure (Figure4a),4a), suggesting the majority of the 3,560 HGs have been vertically inherited.
Figure Figure5A5A shows a rooted NJ tree for the three LIII subgroups, using EGD-e as an outgroup. HCC23 appears to be most closely related to LIIIA. Further evidence that links HCC23 to LIIIA is the rhamnose utilization gene cluster. This gene cluster is conserved in LIIIA and HCC23 but absent in LIIIB and LIIIC. The rooted NJ tree also suggests that LIII is polyphyletic and HCC23 possibly resembles an ancestral state of LIII. The emergence of 3 LIII subgroups is likely to be concomitant with stepwise genome reduction as observed in some non-pathogenic Listeria species, including L. welshimeri [48] and L. seeligeri [49].
A total of 206 genes, that are highly conserved in LI and LII, are found to be phylogenetically informative for LIII (i.e. present or absent in at least one LIII strain) (see Additional file 5). Figure Figure5C5C shows a heat map of these genes in the ten LIII strains. Interestingly, gradual gene decay or diversification was observed in the order of LIIIA, LIIIC and LIIIB. Loss of select LI and LII core genes was most significant in LIIIB. This LIII subgroup forms a deep branch in a split network (Figures (Figures5B).5B). However, it should be noted that the contribution of novel LIII genes to the phylogenetic reconstruction is likely to be underestimated due to the limited number of fully sequenced LIII genomes available at the time of this study.
To access the inter-lineage diversity from a gene content perspective, we identified 576, 521 and 489 accessory genes in F2365 (LI), EGD-e (LII), and J2-071 (LIII), respectively and surveyed their distributions in 26 L. monocytogenes genomes (Additional file 6). Minimum spanning trees were then built to compare and visualize the different distributions of these accessory genes across the three lineages (Additional file 7). Accessory genes display similar distributions in most LI and LII strains, featured by one to two dominant subsets (shown as large circles) generated by genes present or absent in most strains of the same lineage. However, more complex and branched distributions were observed in LIII strains, demonstrating an elevated genomic diversity in this rare L. monocytogenes lineage.
Pan-genome CGH was used in this study to compare L. monocytogenes genomes in pursuit of novel genes that potentially promote the fitness and virulence of LI and LII strains in human, as these strains are predominantly associated with human listeriosis. We used phylogenomic concepts [65] to guide our search for DDGs and to infer the phylogeny for the species. Array CGH is suitable for the purpose of this study because it is relatively cost-effective compared to the sequencing and closure required to make accurate gene calls using whole-genome shotgun sequencing. Unlike whole-genome sequencing, however, the CGH approach has several inherent limitations in detecting novel genes or pseudogenes, inferring sequence-based phylogenies, and for a host of other analyses inaccessible with array data.
A particular challenge in this study was to unify the analysis of both genome sequence and CGH array data. The sensitivity of the two methods is fundamentally different. BLAST searches are capable of precisely measuring amino acid similarity and can identify orthologs and detect distant homologies. In contrast, DNA array hybridizations measure nucleotide conservation and are only capable of detecting highly conserved DNA sequences. In addition, hybridization gives no positional information and is non-specific, making it difficult to discriminate between paralogs. For this reason, we used homologous groups for gene content comparison, and permitted variant sequences to hybridize to their nearest neighbor in a group, rather than a single selected variant (see Methods). Prior to implementing this method, there was tremendous detection bias in the CGH data. The HG method greatly increased the agreement between the array and BLAST detection strategies, which was critical for the phylogenetic analysis of the combined data.
The low frequency of LIII in human listeriosis can be partially explained by its overall rarity in foods, lack of unrecognized virulence factors, or defective mutation in some known virulence factors. For instance, a novel streptolysin S-like hemolytic and cytotoxic virulence factor, listeriolysin S, was recently found to be exclusively present in LI strains [66]. This factor contributes to virulence of the pathogen in murine and human polymorphonuclear neutrophil-based assays [66]. Several studies also reported that premature stop codons are common in inlA in LIII strains [67-70]. Point mutations in inlA are presumably caused by localized recombination and lead to a truncated InlA protein and consequently a reduced invasion phenotype in human intestinal epithelial cells [67-70]. Our pan-genome study uncovered 86 DDGs and 8 non-coding small RNAs that are absent or mutated in the largely uncharacterized LIII genomes (Table (Table55 and Table Table6).6). Most of these genes fall into the functional categories of cell wall structure, transcription regulation, and carbohydrate metabolism and transport. Such functions are likely to play critical roles in ecological fitness of L. monocytogenes in different environment such as food processing facilities and host niches. Genes involved in carbohydrate metabolism and transport stand out as the largest functional group of DDGs, implying that the capability of utilizing different carbon sources in the transmission and infection cycle contribute most to the predominance of LI and LII strains in human infections. In particular, PTS systems that are likely to confer niche-specific metabolic advantages are conserved in LI and LII but decayed or lost in LIII. For example, the fructose-like PTS components (lmo2133---lmo2137) are conserved in all LI and LII genomes but completely lost in LIIIB and LIIIC (Figure (Figure1).1). This operon was postulated to have been acquired by L. monocytogenes through HGT from Enterobacteriaceae that cohabitate the GI tract of mammalian host [71]. A recent study of its homolog in extraintestinal pathogenic E. coli suggested that this operon promotes bacterial fitness against the stress in host serum and gut, and enhances bacterial invasion in eukaryotic cells [72]--both are integral parts of listerial pathogenesis.
L. monocytogenes possess extraordinary capabilities for sustaining harsh conditions during its residency in the environment (e.g. it can utilize limited carbon source), in foods (e.g. it can resist salts and grow at refrigeration temperatures), and in parasitized hosts (e.g. it can escape from immune defense). During its passage through the human GI tract, L. monocytogenes is able to resist the antimicrobial effects imposed by gastric contents. Multiple genes involved in combating GI tract-related stresses, primarily gastric acid (gadD1, gadT1 and the ADI system) and bile salts (btlB and pva), are missing in LIII. Loss of these genes may result in a defective phenotype in surviving the GI tract prior to invasive infection [50]. Also absent in most LIII genomes are a number of small regulatory RNAs (e.g. rli29 and rli48) and transcription factors (e.g. lmo2138 and lmo2851) that appear to be up-regulated in the murine intestine [45]. It is reasonable to speculate that the GI tract may act as a major barrier to prevent LIII strains from causing systematic infections. Epidemiological studies seem to support this speculation by collectively showing that gastroenteritis, rather than more severe listeriosis symptoms, is predominant among infected individuals [73-75]. Although intracellular strategies have been the primary focus in numerous studies of listerial pathogenesis, a few recent studies demonstrated that the GI passage has a fundamental impact on listerial pathogenicity [76,77]. Considering that most LIII strains possess virulence factors related to its intracellular lifestyle and are cytopathogenic [14], the inability to survive in the GI tract becomes a plausible explanation for the overall rarity of LIII in human listeriosis.
We estimate that the L. monocytogenes core-genome consists of 2,330 to 2,456 genes and the pan-genome encompasses over 4,052 genes (Figure (Figure3).3). Compared to several other bacterial species, L. monocytogenes has relatively higher proportions (about 80%) of core genes shared by individual genomes (Table (Table7),7), which in turn reflects lower intraspecies genomic variability. This is consistent with the low rates of recombination in this bacterial species [68]. Despite the perceived high genomic synteny, L. monocytogenes possesses considerably diverse pan gene reservoir and displays biased distribution of accessory genes across major evolutionary lineages (Additional file 7).
Some incompatible phylogenetic signals as indicated in the split network (Figure (Figure4C)4C) were traced back to prophage-associated genes. Notably, the comK prophage regions in different L. monocytogenes genomes display significant sequence variations (Additional file 8). Such variations may be a result of prophage decay, recombination that have accumulated in the remnants of common prophage ancestor(s), or multiple lysogenization of different bacteriophages at the same genomic location. Phages have been recognized as the major contributors of important biological properties (e.g. virulence factors) in many bacterial species [78,79]. The functional impact of bacteriophages on the biology of L. monocytogenes, if any, has yet to be determined.
Intraspecific variations in host preference, ecological fitness and virulence are common in many bacterial pathogens. This is exemplified by the species of L. monocytogenes which consists of multiple distinct genetic lineages. Two lineages of this species (i.e. LI and LII) predominantly cause human sporadic and epidemic infections, whereas the other (i.e. LIII) has never been implicated in human disease outbreaks for unclear yet intriguing reasons. Here we described a novel pan-genomic approach that combines in silico comparative analysis and high-density CGH arrays to explore the genomic diversity of L. monocytogenes. Our integrated approach allows vigorous core genome estimation and phylogenomic reconstruction, which in turn is nearly impossible for low-quality, short-read draft genome assemblies with hundreds of contigs. Exponential regression analysis predicts that L. monocytogenes has a core genome of between 2,330 to 2,456 genes (80% of each individual genome) and a pan-genome repertoire of over 4,052 unique genes. Comparison of all lineage strains reveals high genomic synteny with limited sequence drift associated with lysogenic bacteriophages. Phylogenomic reconstructions based on 3,560 homologous groups suggest a polyphyletic population infrastructure and gradual loss of metabolic genes as this saprophytic species diversified into the rare and probably defective lineage III. Based on our results, one L. monocytogenes strain carries about 75% of the pan genes of this species. That said, experiments based on a single reference strain may not adequately sample the total genetic repertoire and not fully interpret the versatile biology of L. monocytogenes. With a more defined species core genome, we may also be able to supplement new genomic criterion for taxonomic classification of L. monocytogenes, as some traditional methods are often inconclusive and controversial. The pan-genomic approach described here can be used to explore the genomic diversity in other pathogenic species, as such information would be extremely valuable for us to better understand the intraspecific variations in virulence, and the ecology, epidemiology and evolution of microbial pathogens.
Table Table11 lists the 31 L. monocytogenes strains analyzed in this study. As of November 2008, twenty sequenced L. monocytogenes strains were available and used for the pan-genome array design. CGH was performed for nine LIII strains representing 3 serotypes (4a, 4b, and 4c) and 3 subgroups (LIIIA, LIIIB, and LIIIC). Four additional isolates that were sequenced after the array design were incorporated in the pan-genomic and phylogenetic analysis. Bacterial strains were grown overnight in brain heart infusion (BHI) broth at 35°C. Genomic DNA was extracted and purified using MasterPure Gram positive DNA purification kit (EPICENTRE Biotechnologies, Madison, WI). Genomic DNA was labeled with Cy3 or Cy5 dye prior to array hybridization.
The pan-genome tiling array was designed using the PanArray software [38] to fully tile the 20 sequenced L. monocytogenes genomes (Table (Table1).1). PanArray employs a greedy probe selection algorithm to tile multiple whole genomes using a minimal number of probes. For this study, PanArray was used to design an array comprising 385,000 50-mer oligonucleotide probes that fully tile the 20 listerial genomes at 2.65 × coverage with no gaps. To avoid tiling low quality or contaminant sequence, contigs less than 2 Kbp in length were discarded, leaving 54,810,759 bp of tiled sequence. A full description of the array design is given in [38], and the array design is available from the Gene Expression Omnibus (GEO) [80] under accession number GPL8942. To incorporate newly sequenced strains that had not been included in the original array design, we aligned all probes on the array to the new genomes allowing one mismatch per probe, and added genes with probe coverage ≥ 90% of their length to the array annotation.
Genomic DNA of each LIII strain was co-hybridized with that of EGD-e on a Roche NimbleGen 385 K custom CGH array. Two dye-swap replicates were performed for each LIII strain/EGD-e pair to eliminate dye bias and test the array reproducibility. Genomic DNA labeling and array hybridization were performed at Roche NimbleGen according to the manufacturers specifications (Madison, WI). Technical details on DNA labeling and hybridization can be found at http://www.nimblegen.com/products/lit/cgh_userguide_v6p0.pdf . We designed a probe-based intensity classification scheme to provide the most flexibility for pan-genome array data analysis, allowing any locus to be classified based on the aggregated scores of its individual probes, without reference to control hybridization. Specifically, all raw signal intensities were first transformed to log values, then log intensities for replicate hybridizations were normalized using quantile normalization [81]. Replicates were combined at the probe level by taking the average of the normalized log intensities for each probe. Quantile normalization assumes similar intensity distributions, so to avoid cross-sample normalization bias. Each strain was normalized and processed independently.
Because there was no one single reference to operate on, and to preserve sensitivity for small polymorphisms, intensity data was not smoothed or segmented. Instead, individual probes were each classified as present or absent using a minimum kernel density (MKD) method. MKD methods have performed well for the binary classification of both genes and segments [31,82], and here we extended the idea to the classification of individual probes. Because the array contains the full genetic diversity of L. monocytogenes and 4,300 random control probes, there is expected to be a significant fraction of both present and absent probe intensities for any L. monocytogenes sample. Therefore, the distribution of probe intensities is generally bimodal, and the minima between the present and absent peaks can be used as an effective threshold for binary classification. For each sample, the probability density function of the observed intensities was estimated using kernel density estimation and the central minima of this function identified as the optimal cutoff (Additional file 9). This method was preferred because it is non-parametric, there is no potential normalization bias, it requires no training, and each sample can be processed independently without affecting the accuracy. It is also extremely flexible, in that a classification for any gene can be generated by aggregating the classifications of the probes targeting that gene. For this purpose, genes were scored by collecting all probes known to target a specific gene and computing the fraction of probes classified as present, the positive fraction (PF). A PF threshold of 0.6 was chosen by analysis of ROC curves for the EGD-e and J2-071 controls to minimize the total error rate (false-positive rate + false-negative rate) versus the tblastn 50% protein similarity threshold. PF was favored because it does not depend on cross-sample normalization, as would be necessary for an intensity threshold, and additional genomes can be analyzed independently without affecting accuracy. This makes it ideal for rapid and economical analysis of novel isolates, while maintaining comparable accuracy to alternative analysis methods [31,34].
Pan-genomic analysis was performed using the methods introduced by Tettelin et al. [24], with modifications on the conservation threshold and permutation sampling. Annotated proteins for each genome were aligned to the six frame translations of all other genomes using tblastn. Query proteins were marked as present in a subject genome if the corresponding amino acid sequences aligned at ≥ 50% similarity with an E-value ≤ 10-5, where "similarity" was defined as the number of positively scored residues divided by the length of the protein sequence. This threshold is more stringent than originally proposed in [24], but less stringent than those used in other studies (e.g. [32]). The 50% threshold was empirically selected as a compromise between tolerating draft genomes with fragmented annotations and avoiding false positive detections due to conserved domains and distant paralogs. A PF threshold of 0.6 was consequently chosen as an analogous threshold for the CGH results, as described above. Genomes sequenced to less than 10× coverage using 454 pyrosequencing were excluded from the analysis.
The addition of an Nth genome was simulated by examining ordered combinations of N genomes. Due to the large number of available genomes, it was not feasible to consider all possible permutations as originally suggested. Instead, a randomly selected subset of 100,000 permutations was considered for the addition of each N, and the mean (or median) values were computed from this subset. For each permutation, the number of new genes found in the Nth genome GN was computed as the number of proteins of GN not present in any genomes Gi for i = {1, ..., N-1}. The number of core genes was computed as the number of proteins of GN present in all genomes Gi for i = {1, ..., N}. Because gene sequences for the CGH strains are not known, EGD-e was set to be GN for all permutations. The number of pan genes in a permutation of N genomes was computed by examining the genomes Gi in order from 1 to N. A gene in Gi was identified as a pan gene if it was not present in any of the genomes Gj for all j < i.
The Gauss-Newton method implemented by the R function nls [83] was used to perform non-linear least squares regression on the mean and medians of the core genes, new genes, and pan genes distributions. According to [26], the number of new genes n expected to be discovered by sequencing an Nth genome was modeled by the power law function n = κN-α, and the number of pan genes also by a power law n = κNγ. According to [24], the number of core genes was modeled by the exponential decay function n = κe-N/τ + Ω, where Ω describes the horizontal asymptote and therefore the core genes estimate. In all cases, the functions were fit to the mean or median values for all N > 1.
To accommodate false-negative errors introduced by sequencing gaps and weak hybridization signal, the originally proposed exponential decay function was modified with the addition of a fourth parameter to model the effect of a constant number of false-negatives with the addition of each genome. The modified equation is:
where the linear parameter β represents the number of core genes lost to false-negative errors for each N. Core gene loss due to false-negatives is not a truly linear phenomenon (e.g. sequencing gaps are not independent and the core genome can never be negative), but for a large core genome and a modest N it is a reasonable approximation that is easy to fit. To assure convergence of the optimization algorithm, β was first estimated via linear regression for N ≥ 15, and this was used as the start estimate of β for the full model regression. The augmented model is useful in that the observed core genome size may be linearly decreasing (as is expected for draft genomes), but an estimate of the true core genome size Ω may still be recovered.
Homologous groups (HGs) were used for phylogenetic reconstruction and core genome estimation. HGs were identified by clustering a graph of protein similarity for all annotated protein-coding genes from the 18 high-quality L. monocytogenes genomes. A node was added to the graph for each one of the 52,776 annotated proteins. Edges were added between any two proteins with an alignment above the 50% similarity threshold. Unlike OrthoMCL, no orthology constraint was applied. Edges between any two similar proteins were added, including edges between proteins in the same genome. This was necessary due to the inability of CGH to accurately determine orthology. The MCL clustering algorithm was applied to this graph using an inflation parameter of 2.0. From this clustering, 3,744 HGs were identified, including strain-specific genes represented as singleton clusters (Additional file 2). Some HGs, mostly singletons, were not represented on the array because additional genomes had been sequenced after the array design. A total of 3,560 HGs, represented on the array by at least one member gene, were used for the phylogenetic analysis.
For sequenced genomes, an HG was called present if at least one member protein of the HG aligned above the 50% similarity threshold. For CGH genomes, an HG was called present if at least one member gene of the HG hybridized with PF ≥ 0.6. Results based on this threshold were converted to a unified binary table indicating gene presence or absence for all HGs in all genomes analyzed in this study (Additional file 4). These binary vectors were used for measuring evolutionary distance using the maximum-likelihood measure of [59], and Neighbor-net split networks [60] and neighbor-joining trees [65] were built using the SplitsTree program [84]. Alternative parsimony methods failed to build reasonable trees, most likely due to the large number of incompatible splits caused by both horizontal gene transfer and errors in the data.
PCR primers were designed for 3 LI and 6 LIII specific genes using the Primer3 software (available at http://frodo.wi.mit.edu/primer3/). Colony PCR for each gene was performed for 8 LI, 8 LII and 9 LIII strains using the Taq Mastermix PCR kit (Qiagen, Valencia, CA). PCR amplicons were confirmed by the proper size of the DNA bands after agarose gel electrophoresis.
Hybridization results have been deposited at the NCBI Gene Expression Omnibus under accession number GSE20367.
LI: genetic lineage I; LII: genetic lineage II; LIII: genetic lineage III; LIIIA: genetic lineage III subgroup A; LIIIB: genetic lineage III subgroup B; LIIIC: genetic lineage III subgroup C; CGH: comparative genomic hybridization; HGT: horizontal gene transfer; HG: homologous group; DDG: disparately distributed genes; PTS: phosphotransferase system; GI: gastrointestinal; GAD: glutamate decarboxylase; ADI: arginine deminase system; MKD: minimum kernel density; PF: positive fraction; ROC: receiver operating characteristics.
Conceived and designed the experiments: XD WZ. Designed the array and analysis methods: AMP. Performed the experiments: XD. Analyzed data: XD AMP ZL. Coordinated the project: SLS WZ. Wrote the paper: XD AMP WZ. All authors read and approved the final manuscript.
Density estimation of PF values for both present and absent genes. Barplot of the positive fraction probability densities for known present and absent genes demonstrates the vast majority of truly present genes have PF score greater than 0.9 and the vast majority of truly absent genes have PF less than 0.1. Green bars show the density of PF scores for genes found present by a tblastn search, and black bars show the density of PF scores for genes found absent by a tblastn search. PF labels give the minimum of each left-closed interval. For example, PF = 0.5 bars show the densities for the bucket PF = [0.5,0.6).
Homologous groups with locus tax IDs in 26 L. monocytogenes genomes. A total of 3,744 homologous groups were identified from 52,776 proteins annotated in 26 L. monocytogenes genomes. Each row in this file indicates the locus and tax IDs of proteins belonging to a specific homologous group.
Comparison of homologous genes in rhamnose metabolic pathways. Alignment of putative rhamnose utilization pathway in E. coli strain K-12, L. monocytogenes strain EGD-e, and B. subtilius strain 168. The percentage of amino acid sequence similarities is shown between homologous gene pairs. Genes encoding L-rhamnose isomerase, L-rhamnulose kinase, and rhamnulose-1-phosphate aldolase are located in the same orientation in L. monocytogenes EGD-e and E. coli K-12 genomes. The pathway is adopted from KEGG database.
Presence or absence of 3,560 HGs in 26 L. monocytogenes genomes. Binary distribution (i.e. present: "1"; absent or divergent: "0") of 3,560 HGs in 26 L. monocytogenes genomes. Each HG is designated by the tax and locus IDs of a representative protein.
Distribution of phylogenetically-informative LI and LII core genes in LIII. Binary distribution (i.e. present: "1"; absent or divergent: "0") of phylogenetically-informative LI and LII core genes in LIII strains were summarized in this table.
Binary distribution of accessory genes in 26 L. monocytogenes genomes. Binary distribution (i.e. present: "1"; absent or divergent: "0") of accessory genes in 26 L. monocytogenes genomes. These genes were used to generate the minimal spanning trees.
Minimum spanning trees that show the distribution of accessory genes in different L. monocytogenes lineages. A total of 576, 521 and 489 accessory genes were identified from F2365 (LI), EGD-e (LII), and J2-071 (LIII), respectively. The binary distribution of these accessory genes was surveyed in 28 L. monocytogenes genomes, including 4 newly sequenced strains. Each circle represents a group of accessory genes in F2365 (A, D, G), EGD-e (B, E, H), or J2-071 (C, F, I) that share a unique binary distribution (i.e. "1" for presence or "0" for absence) in all strains belonging to a specific lineage (i.e. I, II, or III). The size of each circle is proportional to the total number of genes that share the same binary distribution. Each circle is color-coded based on the number of L. monocytogenes strains (from 0 to 10, see color bar) that share the same distribution. This figure provides an overview of the genomic diversity of the three genetic lineages from a perspective of accessory gene presence or absence, where LIII displays the most diversified gene content.
Alignment of A118-like prophage in different L. monocytogenes lineages. The x-axis gives the location on the EGD-e chromosome, and for each strain, windowed alignment identity is given on a scale of 50-100% identity on the y-axis. Strains which show no homology to the EGD-e A118-like prophage are struck through in blue line. Strains which do show homology to the prophage, but the prophage is inserted somewhere other than comK, are struck through in red line (N1-017, HCC23). This plot illustrates some interesting phylogenetic incompatibilities. For example, based on whole-genome analysis, the nearest phylogenetic neighbor to EGD-e is R2-561. Yet the comK prophage in nearly all other strains appears more similar to EGD-e than does the prophage in R2-561, which has identity < 50% for most of its length.
Probe density versus mean log intensity of the CGH arrays. Histogram with overlaid kernel density estimation (red) of the distribution of probe intensities for sample J1-208, showing an optimal intensity cutoff of 8.82 at the minimum between the present and absent modes. Displayed distribution is for the mean intensities of the two normalized quantile replicates for strain J1-208.
We thank Henk den Bakker and Martin Wiedmann for providing the L. monocytogenes strains in this study. We thank Martin Loessner for helpful discussions on Listeria prophage biology, and Mary Lou Tortorello for helpful comments on the experimental design and the manuscript. We thank Brian Haas and Qiandong Zeng of the Broad Institute for investigating and correcting the mislabeling of two L. monocytogenes genome assemblies.
This study was supported by the U. S. Food and Drug Administration research fund to WZ at the National Center for Food Safety and Technology. XD is a recipient of a Fieldhouse research fellowship. AMP and SLS were supported in part by the U.S. Department of Homeland Security Science and Technology Directorate under award NBCH2070002. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Energy Crop Production Costs and Breakeven Prices Under Minnesota Conditions
Production costs and breakeven prices were calculated for four energy crops plus corn stover. The energy crop breakeven prices were calculated under two different scenarios regarding land costs and the opportunity costs of not utilizing the land for some other competing land use. One scenario is that the competing land use is pasture. The other is that the energy crops would compete with grain crops on more expensive land, and would need to provide a return over land costs equal to returns on the grain crops. Corn stover would be the cheapest of the energy biomass sources considered, at $50/ton to cover the additional machinery costs to shred, rake, bale, and transport 25 miles to a processing plant. Aside from stover, a grassland crop under high fertilization with a 4-ton yield has the lowest cost at $77/ton of dry matter. A grassland crop under low fertilization with a 2-ton yield but a longer stand life has the highest cost at $110/ton. Hybrid poplar comes in at $81/ton. Willow is at an early stage of development in Minnesota, but it would be the cheapest energy crop at $72/ton if it achieves a 5-ton yield with a 23-year stand life that has been reported in New York. These costs are based on the pasture land rental rate of $40/acre. The more expensive grain crop land translates directly into higher energy crop breakeven prices which are also presented in the paper.
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- Lazarus, William F. & Tiffany, Douglas G., 2009. "Economic Impacts of Establishing Short Rotation Woody Crops to Support Energy Production in Minnesota," Staff Papers 46869, University of Minnesota, Department of Applied Economics.
- Fruin, Jerry E. & Fortowsky, J. Keith, 2004. "Modal Shifts From The Mississippi River And Duluth/Superior To Land Transportation," Staff Papers 14057, University of Minnesota, Department of Applied Economics.
- Petrolia, Daniel R., 2006. "The Economics of Harvesting and Transporting Corn Stover for Conversion to Fuel Ethanol: A Case Study for Minnesota," Staff Papers 14213, University of Minnesota, Department of Applied Economics.
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Justified 6 Seasons 2015
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Boyd plans a daring heist in the Season 6 premiere. Meanwhile, Raylan tries to turn one of Boyd's old allies against him.TV-MA Language: English Runtime: 50 minutes Release date: January 20, 2015
Raylan's investigation leads to a conflict with a paramilitary crew.TV-MA Language: English Runtime: 44 minutes Release date: January 27, 2015
Raylan follows a trail of illegal explosives in an attempt to pin Boyd.TV-MA Language: English Runtime: 46 minutes Release date: February 3, 2015
Raylan declares war. Meanwhile, Boyd and Duffy search for a safecracker.TV-MA Language: English Runtime: 43 minutes Release date: February 10, 2015
Raylan turns to an old friend for help after Ava makes a drastic decision.TV-MA Language: English Runtime: 46 minutes Release date: February 17, 2015
Raylan investigates a murder. Meanwhile, Boyd is forced to revisit his mining days.TV-MA Language: English Runtime: 43 minutes Release date: February 24, 2015
Raylan is distracted by a visit from his baby daughter. Meanwhile, Boyd takes Ava on a hunting trip.TV-MA Language: English Runtime: 47 minutes Release date: March 2, 2015
Raylan makes an unexpected offer to Markham.TV-MA Language: English Runtime: 46 minutes Release date: March 10, 2015
Ava's loyalty is tested by Raylan and Boyd. Meanwhile, Markham's plans go awry.TV-MA Language: English Runtime: 41 minutes Release date: March 17, 2015
As Boyd looks to get the money he needs to leave Harlan for good, Ava offers Raylan one last chance to put him behind bars.TV-MA Language: English Runtime: 40 minutes Release date: March 24, 2015
Raylan confronts a crisis that threatens the marshals' careers; Duffy, Mike and Katherine mull the meaning of loyalty.TV-MA Language: English Runtime: 44 minutes Release date: March 31, 2015
Raylan and Boyd look for Ava in the mountains; Markham enlists an unlikely ally.TV-MA Language: English Runtime: 42 minutes Release date: April 7, 2015
In the series finale, one last battle between Raylan, Boyd and Ava will determine who--if anyone--gets out of Harlan alive.TV-MA Language: English Runtime: 47 minutes Release date: April 14, 2015
Graham Yost and Executive Producer Fred Golan sit down to discuss the upcoming final season of Justified. Yost and Golan provide insight into what will bring Raylan, Boyd and Ava together for their final, potentially deadly showdown and who will be there to witness it.TV-NR Language: English Runtime: 3 minutes Release date: January 12, 2015
Relive Raylan and Boyd's most definitive, action-packed moments from seasons 1-5.TV-NR Language: English Runtime: 2 minutes Release date: January 5, 2015
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It is 2555, more than two years after the Master Chief went missing in action following a decisive conflict on Installation 00 - the massive, extragalactic Forerunner construct known as the Ark - as part of the final chapter in humanity's bloody 30-year struggle against the overwhelming forces of the Covenant. Now, as a tenuous peace exists between the humans and the Elites, a startling scientific discovery is made - and the riddle behind its Forerunner origins could very well seal the fate of the entire galaxy within a matter of weeks.
In order to unravel these dangerous secrets, a heroic, hastily formed coalition of humans and Elites must attempt to overcome their differences as they embark on a covert mission back to the Ark - an astonishing, enigmatic place beyond comprehension from which few have returned and where mortal danger awaits them all....
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If this was not a Halo book it would be better...
This book decided it would have all it's characters be simple and single layered. It brought down already developed characters such as Sarah Osman and it didn't progress or add to the story of Halo in any positive way.
I am a huge Halo fan. I feel like this was a complete throw away novel. To change the story to something i would find more enjoyable i would make Luther Mann a more solid character and not have everything just magically line up to fix problems that should have better answers to, and saying that i would need to remove the Spartan 4s since one is a solve everything by just being there character and the other is a background soldier with no reason to be there.
Every character was its own. Dialogue was lacking but that's not the readers fault.
I feel like i'm being way to harsh on this novel, but as a Halo novel this falls way short. I would cut every scene with Sarah Osman do to the fact that they all make her seem incompetent.
First and foremost i am a crazy person who thought about this too long.
The worst books i ever read were the Innocent Mage / the Awakened Mage by Karen Miller because the dialogue was crap, along with the characters being single layered and felt the lack of progression.
Now this book is far from those but my complaints remain and to put the title Halo on this book that did nothing for the story except shit on already established characters just makes me question why they even tried.
With the upcoming Halo 5 and the audio dialogues Hunt the Truth, one would think the story would be in line with some part of the upcoming halo events, instead they threw away a part of the history they could have told better at a later time.
This book felt like cookie dough cutouts and very weak compared to my favorite books of all times Halo the Fall of Reach and Halo Ghosts of Onyx.
The one and only part of this book i found exciting was the monitor 000 Tragic Solitude but the minimal exposure to him as a monitor was irritating.
With all that said, i have listened to Halo the fall of reach through Halo Mortal Dictata, and out of the 9 i listened/ read this is the lowest halo story.
- Izerio
A very good read.
- Churi Nieves


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Pokemon – Pikachu 3D Model Papercraft Template; Review & Download
Check out LEARNIGAMI - 27 Origami Models!
This ebook has 27 fun origami models to learn with some free printable origami paper too! For a limited time, you can get this ebook for free by sharing with your friends! Also available as a printed book.read more


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1. Think beyond pies and jack-o-lanterns
All hail The Great Pumpkin! The pulp of this fall favorite is dense with vitamins A and C, and its tasty seeds, called pepitas, are rich in phytosterols, which may help to lower cholesterol. With all the health benefits of pumpkins, don't they deserve to be more than a craft project that rots a few weeks later?
2. Get your flu shot
Want to spend a week of autumn cooped at home, suffering from fever, fatigue and aches? Didn't think so. According to the U.S. Centers for Disease Control and Prevention, "The single best way to prevent the flu is to get a flu vaccine each season." And no, no, no the flu vaccine cannot give you the flu.
3. Fend of the common cold as well
Hydrate with tons of water throughout the day, and your immune system will thank you. The foods you eat can also help prevent the cold. Yogurt, with all its probiotic glory, has been shown to boost the immune system, and one serving of seaweed packs more vitamin C than an orange. Another immunity booster? Previously mentioned: pumpkin seeds.
4. Scare off those Halloween candy cravings
Maybe don't swear off every candy corn kernel - where's the fun in that? Just don't overdo it. If you buy candy for trick-or-treaters, buy only enough for the kids. Wait until Halloween day to buy the candy, so the in-house sweets aren't haunting you throughout October. If you buy the candy sooner, keep it out of sight in a cupboard.
5. Buy in-season veggies, such as beets, broccoli and Brussels sprouts
Not to mention cabbage, carrots, cauliflower, eggplant, kale and squash. An abundance of tasty autumn vegetables makes it easy to reap the benefits of a plant based diet, such as diabetes prevention, hypertension control, heart health and more.


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pyesis py·e·sis (pī-ē'sĭs)
See suppuration.
Tagged: p
Read Also:
- Pyg
Paraguay-guarani (currency)
- Pygarg
Heb. dishon, "springing", (Deut. 14:5), one of the animals permitted for food. It is supposed to be the Antelope addax. It is described as "a large animal, over 3 1/2 feet high at the shoulder, and, with its gently-twisted horns, 2 1/2 feet long. Its colour is pure white, with the exception of a short […]
- Pygidium
[pahy-jid-ee-uh m] /paɪˈdʒɪd i əm/ noun, plural pygidia [pahy-jid-ee-uh] /paɪˈdʒɪd i ə/ (Show IPA). Zoology. 1. any of various structures or regions at the caudal end of the body in certain invertebrates. /paɪˈdʒɪdɪəm; -ˈɡɪd-/ noun (pl) -ia (-ɪə) 1. the terminal segment, division, or other structure in certain annelids, arthropods, and other invertebrates
- Pygmaean
[pig-mee-uh n, pig-mee-] /pɪgˈmi ən, ˈpɪg mi-/ adjective 1. (defs 6, 7).


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This documentation is archived and is not being maintained.
setMonth Method
Visual Studio 2010
Sets the month value in the Date object using local time.
To set the month value using Coordinated Universal Time (UTC), use the setUTCMonth method.
If the value of numMonth is greater than 11 (January is month 0) or is a negative number, the stored year is modified accordingly. For example, if the stored date is "Jan 5, 1996" and setMonth(14) is called, the date is changed to "Mar 5, 1997."
The setFullYear method can be used to set the year, month, and day of the month.
The following example illustrates the use of the setMonth method.
function SetMonthDemo(newmonth){ var d = new Date(); d.setMonth(newmonth); var s = d.toLocaleString(); return(s); }
Applies To:


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The test for my black belt in taekwondo begins at 10 o'clock sharp this morning. I'm expecting it to run around three hours, give or take.
Among the things it will include:
-A timed 1/2 mile run
-2-1/2 minutes each of jumping jacks, sit-ups, and pushups
-Timed bagwork (hands, feet and combination)
-Demonstrating that I know the previous 11 (!!) belts' worth of forms, techniques and self-defenses (this will take the bulk of the test time)
-Seven belts' worth of step-sparring
-A so-called "chain of pain" (probably ten-fifteen minutes of straight sparring in gear, with a new opponent substituting in every minute; this is exhausting)
-Board breaking (two 1/2' boards with each foot strike, one board per hand strike), and
-Teaching the audience two self-defenses that I have come up with on my own, one each for an attack with a weapon and a bare-hand attack.
There will almost certainly be other stuff as well. The head instructor likes to change things up a bit with each test. Oh, and to make things even MORE fun, his mentor will be there observing as well! No pressure. My instructor is 3rd dan (third degree); the mentor is 5th dan. Oh, and as the icing on the cake, our dojo has changed over from taekwondo-based MMA to pure ITF taekwondo in the last year or so, and along with that has come a *lot* of terminology changes to keep straight. I can't even begin to tell you how happy I will be when this thing is finally over with!
As I stress, people keep reminding me that I wouldn't be testing if my instructor didn't think I was ready. I know they're right, but it is what it is. In the five-plus years I've been doing this, I've never seen someone test for a belt and not receive it even if they make a royal mess of things, so by early afternoon I'm pretty sure I'll have the black belt. At this point, I'm just hoping that I make a good show of earning it and that I don't get hurt while doing so!!
Wish me luck, please???


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My eldest son has just turned 5, so as of now we are officially doomed to book high season holidays.... Finding a charming place to stay in the South of France in August was not easy for 2 families with in total 5 kids (age: 1-7), who can best be characterised by the description: 'enthusiastic high energy level swimming pool addicts without swimming certificates (except for 1)'.
So we were searching for:
- a charming authentic house
- with a child friendly fenced swimming pool
- and lots of space to run around
- a great terrace with a view for endless wining and dining
- playground stuff not spoiling our view when wining and dining : )
Quite a wishlist....but we have found the ideal place.
Our stay at Le Mayne de Boulède was a 'totally relaxed, great for the kids, big chill for the parents, we will definitively come back' experience. Karen and Rolf, a Dutch couple with 3 daughters, have bought this little private hamlet ten years ago and turned it into this charming joie de vivre family paradise with 5 holiday houses and 2 swimming pools. You can see and feel the passion for their dream in everything. I love the little details like a the 'bread service' in and old Renault, hammocks for a little afternoon snooze, the playground with an old fishing boot sandbox, the massage room with the little chalkboard sign : pssst... massage.
Le Mayne de Boulede is situated just a few kilometres away from the lovely little medieval village Monflanquin and a 20km beautiful scenery roadtrip away from Monpazier. Both are charming little French villages with lots of restaurants and a typical place du village. Le Mayne de Boulede doesn't offer a table d'hote, but with the fully equiped kitchen and so many restaurant and eating/drinking market alternatives nearby you will hardly miss it.
The evening markets in July- August are a real thing in this area: venez faire un tour sur les marchés nocturnes et de producteurs de pays ! De 18h à 23h, dans une ambiance musicale, venez vous régaler : des tables et chaises seront mis à votre disposition, n'oubliez pas vos couverts ! Sans modération, pour les gourmands et les gourmets !
So we say: Au Revoir!


text: Human Rights Observatory US Immigration Agents Arrest 19 on Human Rights Violations U.S. immigration agents have arrested 19 fugitives sought for human rights violations committed in other countries, Immigration and Customs Enforcement said on Wednesday. Arrested last week during Operation No Safe Haven, the first nationwide crackdown of its kind, the 19 have outstanding removal orders and are subject to being deported, the agency said in a statement. Eight of the 19 are also convicted criminals. Those arrested and their home countries were not identified. They...Read complete article© Voice of America - Wednesday, September 24, 2014


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Housing pathway developed for ex-service personnel in Wales
Cabinet Secretary for Communities and Children, Carl Sargeant has thanked organisations in the housing and Armed Forces sectors who have worked with the Welsh Government to develop the National Housing Pathway for ex-service personnel.
The pathway aims to prevent homelessness amongst veterans and to help them find suitable accommodation.
"I take very seriously the responsibility we have to those members of our communities who have served, or are still serving, in the Armed Forces. Helping them to find suitable accommodation is an important part of the much broader and wider package of support which helps them to settle back into our communities," said Sargeant.
"We will continue our work to implement the Housing Pathway, particularly amongst organisations working on the front line of homelessness. We also aim to do more to raise awareness, with the publication of advice for veterans. This will build on the contents of the Pathway. It will be targeted at those due to be discharged from duty as well as those who were discharged at some point previously. It will include advice cards for those who are sleeping rough. The Pathway will also be shared widely amongst organisations providing support for the Armed Forces community in Wales.
"I am grateful to all the organisations who have worked with us on these developments. In collaboration with The Armed Forces Expert Group and partner organisations, we will continue to support and provide effective services for our Armed Forces community in Wales."
The Royal British Legion called for a pathway as one of the eight 'asks' of Welsh Government in its 2016 Wales Manifesto – Live On – Building a better future for the Armed Forces Community in Wales.
Antony Metcalfe, area manager for The Royal British Legion Wales added: "The Royal British Legion welcomes the new housing pathway for the Armed Forces community and the commitment the Welsh Government has shown in addressing the issues around housing support for the Armed Forces community in Wales.
"The Legion called for such a pathway in our Welsh Assembly election manifesto 2016, following a national conversation we held with the Armed forces community in Wales, who told us that accessing clear housing advice and support was difficult and that too often they were being given inconsistent advice which was leaving them confused about access to housing in their local areas.
"This document should now help provide clear information for the Armed Forces community but also be used as a training resource for housing professionals so that everyone is clear about housing support that can be expected and delivered."


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Warning: This post was published more than 12 years ago.
I keep old posts on the site because sometimes it's interesting to read old content. Not everything that is old is bad. Also, I think people might be interested to track how my views have changed over time: for example, how my strident teenage views have mellowed and matured!
But given the age of this post, please bear in mind:
- My views might have changed in the 12 years since I wrote this post.
- This post might use language in ways which I would now consider inappropriate or offensive.
- Factual information might be outdated.
- Links might be broken; embedded material might not appear properly.
Many thanks for your understanding.
I've started using this instead of Desktop Sidebar. It's rather good, clearly takes up less screen space than Desktop Sidebar, and has one of the smoothest scrolling actions I've seen on any RSS program. And it's free. If it had an option to shown only the last x stories on a particular feed, it'd be perfect.
This 313th post was filed under: Reviews, Technology.


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God I forgot how much I love street style.
In an age where personal style has been thrown out the window in favour of paid placements, it's so refreshing to go back to what women are actually wearing.
That's exactly what Women in this Town does. An attempt to snapshot what women are actually wearing in 2015, this book is total #stylegoals. Unlike so many street style photographers today, Giuseppe Santamaria hasn't captured the boldest outfits.
He knows the subtlety of style and it shows. His subjects span a wide range of ages, demographics and tastes, and yet each image is total style inspo.
Dare I even be so bold as to say he's giving Scott Schuman a run for his money? Yes.


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Nathan B.
Norfolk, VA, 23508
Joined on November, 2012
Gender: Male
Average Rating:
Weekends at any time
Lives Near:
Can Meet:
Up to 15 minutes away for no additional charge
EducationSenior Marine Biology Major at Old Dominion University
ExperienceI am intelligent in my area, and I love to help others out in learning more about the Sciences. I have been in a couple of hands on field trips actually working and doing experiments with organisms which I can use in teaching others about the research side of Biology.
HobbiesSoccer, Reading Scientific Papers, Skateboarding, Surfing, Snowboarding.
Preferred Subjects
Marine Biology, Biochemistry, Biology, Oceanography
Inquire about this tutor
The tutor will respond if they are available/interested.


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Hotel Tugu Bali
Indonesia: Bali
For those of you who enjoy luxury, the balinese culture and still like to surf all day, this ocean front boutique hotel in the surfer hotspot of Canggu is perfect.
"Experience balinese tradition & legends of Indonesia"
At the surfing hub Canggu, a village 20 minutes from Seminyak on the southwest coast of Bali, Hotel Tugu enables you to step out of bed every morning, check the waves out of your bedroom window, and come home from a hard day of surfing to the luxury of your own beautiful suite. The only 5 star resort in Canggu, your stay in this boutique, oceanfront hotel is an art lover's dream. With only 21 suites, each room is unique and represents legends of Indonesia, so you can immerse yourself in both waves and culture! Indulge in late breakfasts by the sea, heavenly massages at the spa, and ice cold beer(s) as you watch another perfect sunset.
"Iconic surf spots are on your doorstep"
You'll be staying right in front of Batu Bolong / Old Mans surf spot - perfect for beginner/intermediates, with famous Echo beach and some other hollow beach breaks within walking distance. A heaven for all you surfers, kite surfers and paddle boarders, you can ride all day and still relax in luxury.
"Breakfast served wherever you choose…"
Standard packages include bed and breakfast, and can be upgraded with surf lessons, massages and more. A 5 night package will provide you with 3 surfing classes, 4 massages, daily high tea, private airport transfers, welcome drink and in-room fruit and water.
Three reasons to choose Hotel Tugu
- Experience ancient tradition and relicts
- Ocean front location means you can check the waves from bed!
- Beautiful grounds and on point service
Quality and service comes first in this private tropical island sanctuary positioned in the centr...
Exclusive Sumba surf resort overlooking an uncrowded world class left. Immerse yourself in the...
Luxury resort overlooking the majestic waves of the Bukit Peninsula. Unparalleled accommodation a...


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We Love Stoke Newington is for residents living in the local area providing updates about local meetings, campaigns and useful information. This site is maintained by your local Labour team for Stoke Newington.
Wednesday, 10 November 2010
Set the priorities of our local police team - tonight
The Community Advisory Panel is meeting tonight at 7pm in the community flat on Clevedon Close, Smalley Road Estate.
This meeting happens every two months and is a chance for local residents to meet the Safer Neighbourhood Team for Stoke Newington and set their priorities for the months ahead.
More details of our local team are
Posted by
Louisa Thomson
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Currently 3.00/512345 Rating: 3.0/5 (Total votes: 1)
Rating: 3.0/5 (Total votes: 1)
LingvoSoft Suite is the complete language solution available only from LingvoSoft.
A unique collection of our most popular and useful applications that are Windows Mobile 6.0 ready, LingvoSoft suites provides a global solution for handheld devices that answers all your language management and translation needs reliably and accurately.
LingvoSoft Suite features a combination of advanced talking Dictionary, human voice enabled travel PhraseBook, and five effective and fun FlashCards learning games. Combining astonishing voice output capabilities that include both TTS (text-to-speech) and authentic native speaker voice narration with the world''s most advanced speech recognition accessed via an extremely user-friendly, customizable interface it is a fully integrated system that delivers precision you can depend on.
With unique User Dictionaries, extensively updated vocabularies, complete language resources and unparalleled compatibility across the entire platform it offers full integration with MS Word, Internet Explorer and Outlook for Pocket Office. And with superior search engines, instinctive cut and paste functionality between components as well as a choice of display language and other customizable elements it is fast, dependable and accurate.
Exclusive Features:
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Kundapur: Malathi B Shetty, a resident of Charukottige of Korgi village near Tekkatte in Kundapur taluk of Udupi district, had left her house around 1-30 pm on June 24. She has not returned home since and no clues are available about her whereabouts.
Latest Picture
A complaint was filed at the police station here. The family members and well-wishers have made enquiries all around but there has been no success. Her son Satish, who is the CEO of Codeproof Technologies Inc in Redmond, WA in the USA, made an airdash here on July 4.
Five year old Picture
Shetty met district superintendent of police Annamalai requesting him to fast-track the search and inquiry process. Until date, even after almost a month, nothing is known about her.
At the time of leaving the house, she wore a simple sari, a pair of old shoes and did not even take an umbrella and her mobile phone. It was obvious that she intended to visit a neighbour and not to travel very far. However, as usual, she wore jewellery worth about Rs 3-75 lakh. This has created fears that she might have been kidnapped.
Satish Shetty has appealed to the media and the public to spread the word around so that she can be traced.
Click Here to See video clip of Malathi B Shetty playing jokaley with her grandson Aryan 22nd days before she went missing
Kundapur: Elderly Woman Goes Missing – SP Assures Speedy Investigation


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Watch IRF's founder/administrator interviewed for regional TV news. Subjects in banned documentary, Positive Hell, to attend London screening (press release)
Tag Archives | joan shenton
Giveaway: 5 paperback copies of Positively False – 16th Anniversary Edition (US residents only)
If you haven't yet bought a copy of the second edition of Positively False (why not?!), you might be in luck if you're a US resident. We are giving away 5 copies of the paperback (with free delivery) to every lucky 7th entrant who also follows our shiny new Twitter account: @immunityorguk To enter, just click […]
Positively False 16th Ann. Ed. Kindle eBook available for pre-order now + 5 Feb launch event in London
IRF is proud to announce that the Amazon Kindle eBook edition of Joan Shenton's Positively False: Exposing the Myths around HIV and AIDS – 16th Anniversary Edition is now available internationally for pre-order for download on 4 February 2015 via the following links. UK http://www.amazon.co.uk/gp/product/B00QE80HNW US http://www.amazon.com/dp/B00QE80HNW Canada http://www.amazon.ca/gp/product/B00QE80HNW Australia http://www.amazon.com.au/gp/product/B00QE80HNW France http://www.amazon.fr/gp/product/B00QE80HNW Spain http://www.amazon.es/gp/product/B00QE80HNW […]
Delayed: Anniversary reprint of Positively False book
Although the reprint of Joan Shenton's 1998 book is completed and ready for publication in paperback and digitally, the release of it has been delayed until February due to needing sufficient time for promotional activities. A website for the book, however, should become active shortly. We apologise to anybody who was urgently anticipating this release.
In production: Positively False: Exposing the Myths around HIV and AIDS – 15th Anniversary Edition, book reprint
Joan Shenton's 1998 book Positively False is to be republished independently before year-end-2014 to celebrate the 15th anniversary of its release. The new edition will be exclusive to Amazon International and will be available in paperback and for Kindle. The original text will be bolstered with a myriad of retrospective contributions from key dissidents, plus Peter Duesberg's withdrawn […]
Trailer for 'Positive Hell' documentary by Reiss/Shenton
Positive Hell is the story of five individuals who have defied their doctors and lived on for nearly thirty years with a diagnosis of death. The film highlights a network of people diagnosed HIV-positive in the province of Galicia in Northern Spain.
Joan Shenton responds to cancellation of HIV sceptic film @ SOAS
NO FREEDOM OF SPEECH AT SOAS (London University's School of Oriental and African Studies) by Joan Shenton – Immunity Resource Foundation Anne Sono's film I won't go Quietly was due to be screened at SOAS Khalili Lecture Theatre on Friday 25th April at 6 pm. The film tells the story of 6 women who were […]


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A glimpse into our life. A handsome surf loving & photographer enthusiast Dad. A jewelry making Momma. A beautiful curly haired curious Babe. A tiny Pup. This is us. Hoping to once again grow our little family through adoption. Please help spread the word to your friends & family.
The City
Posted by SMALL and TALL at 10:10 PM 2 comments:
One last summer concert...
Just in time to catch the last concert in the park. And it sure was entertaining! The people watching was amazing from the dancing to the outfits people were sporting - I was impressed with the fashion I saw all over the place! This guy in front of us was really feeling the music, he was in his own world dancing away non-stop while not even noticing that everyone was watching him and photographing him! The music was great and so was the company.
Posted by SMALL and TALL at 9:55 PM 2 comments:
Exploring the City
Before moving back I had told myself I was going to get to know the city this year. There are actually a lot of interesting places to see and visit around here and I want to take advantage of them while I have the opportunity. This past week we discovered an Italian Market which also carries imported products from Spain! It was perfect since Tim was planning on making an authentic Spanish meal. The salad he made turned out great thanks to the imported products we stumbled across.
Posted by SMALL and TALL at 9:45 PM 1 comment:
an inexpensive meal
Our first week back has been busy. We seem to have something going on each day and each night. Our first weekday back we treated ourselves to a yummy meal... at Costco! Have you tried the pizza, polish dogs, mocha freeze or ice cream bars? They are all tasty and inexpensive. Our good friends Sandra and Brock joined us!
Posted by SMALL and TALL at 9:35 PM 1 comment:
To blog or not to?
So all week I have been debating wether to make my blog public v. private or to even have a blog at all. I have enjoyed finding many old friends, reading about friends lifes, getting recipes and staying in touch with everyone. I am not sure what I am going to do???
Posted by SMALL and TALL at 9:28 PM 1 comment:
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text: Human Rights Observatory Activists: HIV Treatment Lagging in West Africa The prevalence of HIV/AIDS is relatively low in West Africa compared to the rest of Africa, but treatment rates there are equally low. In Ivory Coast, about two-thirds of people living with HIV/AIDS are not on antiretroviral treatment. Emilie Iob has the story from Abidjan.Read complete article© Voice of America - Monday, July 11, 2016


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23 October 2017 - Corporate
10 July 2017 - Toiletries
23 June 2017 - Equipment
Mulben & Fearne No 1
Aslotel, drawing from the success of Mulben & Fearne London, has created Mulben & Fearne No 1 hotel toiletry line as a welcome addition to the Mulben & Fearne family.
Cleverly retaining the heritage and affordability of Mulben & Fearne London, this new generation of Mulben & Fearne elevates this genre of hotel toiletries to a new level, providing both you and your guests with a feeling of stylish chic.
In rich mocha chocolate brown and oatmeal packaging, this new line will add style and glamour to any bathroom - your guests feel pampered and special.
With an inspiring and invigorating fragrance Mulben & Fearne No 1 is certainly distinctive.
Accessories to complement the hotel line are available plus a selection of presentation trays.
Please click here to view brochure


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Christian Romero
Category: Uncut Men
Pornstars: Christian Romero
That's what Christian Romero said and he couldn't be more right! Who could resist sucking Christian's hard, thick, hooded, 8-inch shaft. He gets so hard and so turned on that the pre-cum is dripping off the tip of his dick-head. Robert Chandler couldn't resist sucking and tonguing the free-flowing pre-cum right off the the tip of Christian's meaty burrito!
Christian also loves touching his own cock, playing with it, stroking it and finally shooting his tasty load. Christian's foreskin workout will leave your mouth watering!
View other Fantastic Foreskin videos