text:
Volume 13 Supplement 5

Proceedings of the International Conference of the Brazilian Association for Bioinformatics and Computational Biology (X-meeting 2011)

Open Access

The Corynebacterium pseudotuberculosis in silico predicted pan-exoproteome

  • Anderson R Santos1,
  • Adriana Carneiro2,
  • Alfonso Gala-García1,
  • Anne Pinto1,
  • Debmalya Barh3,
  • Eudes Barbosa1,
  • Flávia Aburjaile1,
  • Fernanda Dorella1,
  • Flávia Rocha1,
  • Luis Guimarães1,
  • Meritxell Zurita-Turk1,
  • Rommel Ramos2,
  • Sintia Almeida1,
  • Siomar Soares1,
  • Ulisses Pereira1,
  • Vinícius C Abreu1,
  • Artur Silva2,
  • Anderson Miyoshi1 and
  • Vasco Azevedo1Email author
BMC Genomics201213(Suppl 5):S6

DOI: 10.1186/1471-2164-13-S5-S6

Published: 19 October 2012

Pan-genomic studies aim, for instance, at defining the core, dispensable and unique genes within a species. A pan-genomics study for vaccine design tries to assess the best candidates for a vaccine against a specific pathogen. In this context, rather than studying genes predicted to be exported in a single genome, with pan-genomics it is possible to study genes present in different strains within the same species, such as virulence factors. The target organism of this pan-genomic work here presented is Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis (CLA) in goat and sheep, which causes significant economic losses in those herds around the world. Currently, only a few antigens against CLA are known as being the basis of commercial and still ineffective vaccines. In this regard, the here presented work analyses, in silico, five C. pseudotuberculosis genomes and gathers data to predict common exported proteins in all five genomes. These candidates were also compared to two recent C. pseudotuberculosis in vitro exoproteome results.

The complete genome of five C. pseudotuberculosis strains (1002, C231, I19, FRC41 and PAT10) were submitted to pan-genomics analysis, yielding 306, 59 and 12 gene sets, respectively, representing the core, dispensable and unique in silico predicted exported pan-genomes. These sets bear 150 genes classified as secreted (SEC) and 227 as potentially surface exposed (PSE). Our findings suggest that the main C. pseudotuberculosis in vitro exoproteome could be greater, appended by a fraction of the 35 proteins formerly predicted as making part of the variant in vitro exoproteome. These genomes were manually curated for correct methionine initiation and redeposited with a total of 1885 homogenized genes.

The in silico prediction of exported proteins has allowed to define a list of putative vaccine candidate genes present in all five complete C. pseudotuberculosis genomes. Moreover, it has also been possible to define the in silico predicted dispensable and unique C. pseudotuberculosis exported proteins. These results provide in silico evidence to further guide experiments in the areas of vaccines, diagnosis and drugs. The work here presented is the first whole C. pseudotuberculosis in silico predicted pan-exoproteome completed till today.

Reverse Vaccinology (RV) [1] analyses the genome sequence of a pathogen, which is an expected coded sequence for all the possible expressed genes in the pathogen's life cycle. All Open Reading Frames (ORF's) derived from the genome sequence can be evaluated using a computer program to determine their ability as vaccine candidates, giving special attention to exported proteins, as these are essential in host-pathogen interactions. Examples of such interactions include: (i) adherence to host cells, (ii) invasion of the cell to which there is compliance, (iii) damage to host tissues, (iv) environmental stresses resistance from the defense machinery of the cell being infected, and (v) mechanisms for subversion of host immune response [25].

Regarding exported proteins, these can distinguish between those that are exported to the cell wall, and after cleaved, release the mature portion into the extracellular milieu, which are referred to as secreted proteins (SEC), and those proteins exported to the cell wall which, even after cleaved, do not release the mature portion to the extracellular milieu, due to one or more hydrophobic motifs causing anchoring to the cell wall, and which are referred to as potentially surface exposed proteins (PSE). Different PSE subcategories exist according to the presence of a carboxy (C) or amino (N) terminal portion anchored to the cell wall, lipoproteins (E), end terminal loops (L), retention signals-like such as LGxTG, LysM, GW, Choline binding and PG binding (R), in combination or not with other PSE subcategories [6].

The term 'Reverse' from RV can be explained by the reverse genetics (RG) technique. Before the dawn of genomic, there were attempts to discover the responsible genes from a phenotype, reversing the research path of Crick's Central Dogma [7] (DNA → RNA → Protein) discovery. Holding the likely gene sequence, several techniques can be used to identify gene sequence modifications responsible for changes in the organism's phenotype. Crick's Central Dogma principle is also used for RV, as this technique searches within a gene sequence for possible proteins that could act as antigens capable of stimulating an immune response in a host organism [8].

The concept of RV was adapted to fit a new reality of widespread availability of genomic data [9]. With this technique, instead of searching for targets in a single strain or subspecies of an organism, it is now possible to simultaneously research in dozen of genomes, exploring potential joint antigens or exclusive ones to multiple genomes [10]. The availability of a large number of genomes to implement RV has lead to the emergence of the pan-genomics reverse vaccinology concept [11], which can also apply to the concepts of core, extended (dispensable) and character (unique) genomes. While the core genome is composed of exported genes (genes that transcribe for exported proteins) that are common to these multiple strains and could represent candidates for a vaccine, the dispensable genome consists of genes that are absent in at least one of the strains of the studied species and the unique genome consists of genes that are specific to only a particular a strain [10]. From the standpoint of vaccines, the core genome represents to be a good candidate to compose a vaccine that is suitable for all studied strains. In this regard, the first step to enable any pan-genomic reverse vaccinology study is to predict the core genome, along this work denominated in silico predicted pan-exoproteome (ISPPE). The model organism here analyzed (C. pseudotuberculosis) is a Gram-positive (GRAM+) bacterium, intracellular facultative parasite that affects small ruminants causing a chronic infectious pyogranulomatous disease characterized by the formation of abscesses in lymph nodes [12]. This pathogen infects mainly goats and sheep causing caseous lymphadenitis, but can also infect a huge variety of hosts throughout the world such as camels, horses, cattle, buffaloes, llamas, alpacas and, more rarely, humans [1318], causing different diseases with different degrees of severity in each of them [12, 19].

Results and discussion

In silico exoproteome prediction schema

As shown in our proposed prediction schema (Figure 1), the software SurfG+ (Surface Gram positive), specially configured for GRAM+ bacteria, is responsible for most of the sub-cellular classifications, which vary between cytoplasmic (CYT), membrane (MEM), SEC and PSE (Figure 2). SurfG+ was configured for GRAM+ bacteria. Figure 1 represents the prediction schema using SurfG+ and three additional software, TatP 1.0 [20], SecretomeP 2.0 [21] and NclassG+ [22], which are specialized in non-classical secretion prediction. SurfG+ incorporates SignalP 3.0 predictor, responsible for identification of classical putative secreted proteins or exported proteins by the SEC pathway [23].
Figure 1

C. pseudotuberculosis pan genomic prediction schema. Software used, identified sub-cellular compartments and flow scheme to create the final pan genomic data sets.

Figure 2

Predicted gene quantities by sub-cellular compartment from full C. pseudotuberculosis genomes. Classification of more than 10,000 distinct genes from the five different C. pseudotuberculosis strains in the four sub-cellular categories: cytoplasmic (CYT), membrane (MEM), potentially surface exposed (PSE) and secreted (SEC). Predictions were made using the schema presented in Figure 1.

The results obtained after running SurfG+, TapP, SecretomeP and NClassG+ have gave rise to two gene data sets labeled as SEC and PSE, which correspond to the C. pseudotuberculosis ISPPE. These ISPPE data sets are composed of putative proteins present fivefold (5x), fourfold (4x), threefold (3x), twofold (2x) or onefold (1x), where fivefold means that a gene was predicted in all five strains, four fold meaning that a gene was predicted in four strains, and so on. A gene fold was obtained by reciprocal blast results, as described in the methods section. Since not all predicted genes are named, it was necessary to create a pan genome identifier, here denominated pan locus, to nominate each unique gene fold. The pan locus is unique within a pan genome and is shared by all homologous genes. For example, when a putative exported protein was found within the five strains, each gene copy received the same pan locus to facilitate further data processing and identification. Following, it was necessary to confirm these results by systematical manual curation of each gene using the ACT tool from the Artemis software package [24]. Once completed this manual curation, it was possible to answer several questions regarding the correctness of each blast result and, as a consequence, it was possible to identify, for instance, that a gene formerly classified as 1x was indeed a 5x, as the other four gene copies were created starting beyond the signal peptide motif. After initial methionine correction, and also taking into account homologous genes, a new prediction step indicated all remaining putative proteins to be exported, composing the core ISPPE. However, gene's start positions incorporating a less probable signal peptide motif were also observed. In general, genes formerly predicted as Nx proved to be correct by manual curation as the remaining (5-N)x genes were predicted as cytoplasmic, PSE or pseudogenes. These results are particularly interesting because they compose the dispensable and unique ISPPE data sets. These genome annotation corrections, as a consequence of these analyses, were incorporated into the official annotation of the five C. pseudotuberculosis strains deposited at GenBank in August, 2011. This genomes are also available in the additional file 1, as EMBL files.

Classical and non-classical secreted putative proteins

Figure 3 exhibits the in silico predicted pan secretome results for C. pseudotuberculosis, which comprise 150 genes, out of 377 from the whole ISPPE, representing 750 locus_tags in the five studied C. pseudotuberculosis strains. However, despite representing 750 locus_tags, not all were predicted as secreted. If at least one gene copy, within a specific pan locus, was not predicted as secreted, it still received the same pan locus but was not classified as part of the predicted core secretome. There are 122 genes composing the predicted core secretome (5x), followed by 25 genes constituting the predicted dispensable secretome (4x, 3x and 2x) and just 3 genes as the predicted unique secretome (1x). These results were obtained applying the prediction schema from Figure 1; however, different contributions were obtained from different predictors, as shown in Figure 4.
Figure 3

Predicted C. pseudotuberculosis pan secretome. Predictions for 150 genes from strains 1002, C231, I19, FRC41 and PAT10 made by SurfG+ 1.0, TatP 1.0 Server and SecretomeP 2.0 Server.

Figure 4

Predicted C. pseudotuberculosis pan secretome by predictor software. Predicted secreted genes coverage in the predicted pan secretome of the five bacterial strains separated by predictor software SurfG+, TatP and SecretomeP.

SurfG+ predicted 104 genes, corresponding 85, 18 and 1 to the predicted core, dispensable and unique secretome respectively. On the other hand, TatP predicted 25 genes, of which 17, 7 and 1 corresponded to the predicted core, dispensable and unique secretome respectively. Finally, SecretomeP and NClassG+ predicted 21 genes, corresponding 20 and 1 to the predicted core and unique secretome respectively. It can be easily observed that the main predicted portion is originated by SurfG+, as it predicts putative proteins possibly secreted by the SEC pathway. A considerable portion of genes (~31%), only within the predicted core secretome, comes from non-classical secretion predictors that cannot be ignored when the subject is about vaccine candidates.

The dispensable and unique C. pseudotuberculosis predicted secretomes contain ~8%, or 58 locus_tags, not predicted as secreted. Putative proteins predicted as CYT, PSE and putative frame shifts (pseudogenes) account for 22, 24 and 10 locus_tags respectively. In the dispensable and unique C. pseudotuberculosis in silico predicted secretomes, the numbers of genes identified as membrane integral or absent in a genome are insignificant. Nevertheless, the manual curation step ensured no annotation errors in these predictions, making it possible to claim the hypothesis that these differences could be due to environment adaptations. A table containing the complete list of C. pseudotuberculosis secreted proteins is available in the additional file 2.

Potentially surface exposed (PSE) putative proteins

The SurfG+ software was calibrated by the cell wall thickness for each C. pseudotuberculosis strain. Figure 5 shows 184 genes, out of 377 from the whole ISPPE, comprising the predicted core surfaceome (5x), 34 genes composing the predicted dispensable surfaceome (4x, 3x and 2x) and just 9 genes as predicted unique surfaceome (1x). These 227 genes account for 1135 locus_tags in all five strains. In this set, homologous genes within a pan locus do not ever share the same sub-cellular prediction. Genes predicted as MEM, CYT, SEC and putative pseudogenes account for 29, 23, 20 and 17 distinct locus_tags, respectively. Genes predicted as MEM (~3%) compose the second major group. This could be explained by the fact that membrane proteins already contain hydrophobic extension and could be more susceptible to expose or occult parts of a protein to the extracellular milieu. However, the same reasoning does not suit to explain the third major group of locus_tags with surfaceome pan locus that correspond to proteins predicted as secreted ones. These 20 locus_tags that were predicted as secreted, but also received surfaceome pan locus, raise a question; do these fit SEC or PSE labels? There exist no simple paths to estimate their sub-cellular compartment by software, since some locus_tags were predicted as PSE receiving surfaceome pan locus and other were predicted as SEC and also received secretome pan locus. Ten pan locus (plcppse193, plcppse194, plcppse205, plcppse218, plcppse226, plcpsec096, plcpsec097, plcpsec098, plcpsec100, plcpsec101) faces this question, as some genes appear in both the predicted secretome and surfaceome.
Figure 5

Predicted C. pseudotuberculosis pan surfaceome. Pan surfaceome predictions for 227 genes from strains 1002, C231, I19, FRC41 and PAT10, performed by SurfG+ 1.0.

The PSE subcategories show predominance of genes, as presented in Figure 6. Most of the 1045 genes predicted as PSE are cell wall anchored outward C-terminal (~40%) (≥ 50 AA long), followed by lipoproteins (~24%), outward loops (~11%) (≥ 100 AA long) and outward N-terminal (~17%) (≥ 50 AA long), whereas genes containing retention signals (PSE R) account only for ~8%.
Figure 6

Predicted C. pseudotuberculosis pan surfaceome by PSE subcategories. PSE categories are distributed in outward C-terminal or N-terminal portion greater than or equal 50 AA. Outward N or C terminal greater than 100 AA are classified as L. Lipogenes identified by LipoP are classified as E and retention signals identified by HMMSEARCH profiles are classified as R. These labels can also be conjugated to create other PSE subcategories.

The PSE results of all strains were analyzed considering that a significant cell wall thickness difference between strain I19 and the other ones was observed (~34 nm versus ~24 nm). Despite the significant cell wall thickness difference, a small difference was predicted in the genome, which accounts for a decrease in the number of PSE and an increase in the number of MEM genes in C. pseudotuberculosis strain I19. A table containing the complete list of C. pseudotuberculosis PSE proteins is available in the additional file 3.

Revised in vitro exoproteome results

The 104 observed genes in both TPP/LC-MSE [25] and 2-DE-MALDI-TOF/TOF, (Silva WM, Seyffert N, Castro TLP, Santos AV, Pacheco LGC, Santos AR, Ciprandi A, Zurita-Turk M, Dorella FA, Andrade HM, Pimenta AMC, Silva A, Miyoshi A, Azevedo V, unpublished observations) experiments were compared with the ISPPE results here presented. This comparison, explained in the methods section, brought novel insights into the in vitro exoproteome and showed the possibility of having additional genes in the main C. pseudotuberculosis in vitro exoproteome. In Table 1 are listed all 35 proteins of the variant in vitro exoproteome (strains 1002 and C231), that correspond to ~23% of the total amount. These proteins were found to be highly conserved in the five compared C. pseudotuberculosis strains and comprise the core ISPPE. Moreover, it was verified that three proteins (ADL20466, ADL20097 e ADL19973), previously classified as belonging to the variant in vitro exoproteome of strains 1002 [25], did actually belong to the main in vitro exoproteome. These findings give raise to the possibility that more proteins of the variant in vitro exoproteome indeed make part of the main in vitro exoproteome.
Table 1

Core C. pseudotuberculosis in silico predicted pan-exoproteome found in the variant in vitro exoproteome

Protein identifier

Gene name

Predicted local sub-cellular

GenBank organism identifier

Hypothetical protein

Surface layer protein A

Hypothetical protein

L,D-transpeptidase catalytic domain, region YkuD

Maltose/maltodextrin transport system substrate-binding protein

Carbonic anhydrase

Hypothetical protein

Resuscitation-promoting factor RpfB

Hypothetical protein

Amino deoxychorismate lyase

Hypothetical protein

Cytochrome c oxidase subunit II

Hypothetical protein

Secretory lipase

Phospholipase D

Penicillin-binding protein A

Hypothetical protein

Penicillin binding protein transpeptidase

Oligopeptide-binding protein oppA

Hypothetical protein

Maltotriose-binding protein

Transcriptional regulator lytR

Hypothetical protein

Iron ABC transporter substrate-binding

Protein yceI

Zinc metallopeptidase

Serine/threonine protein kinase

Corynomycolyl transferase

Hypothetical protein

Hypothetical protein

Membrane protein

Hypothetical protein

Glycerophosphoryl diester phosphodiesterase

The 35 proteins listed in this table were not found in the experimental main in vitro exoproteome [47; 48] but were found in the in silico predicted pan-exoproteome of all five C. pseudotuberculosis strains.

This comparison also served as a rebuttal argument against some specific genes. The Cp1002_0369 gene, classified under the plcpsec100 pan locus as a pseudogene, was identified by the in vitro exoproteome experiment. Interestingly, this gene copy also suits the plcppse226 pan locus. Both pan locus make part of previous related genes that already showed difficulties to be classified, by software, into any potential sub-cellular compartment, as some genes within the pan locus fit both SEC and PSE labels. The in silico predictions enforces that there are at least three secreted proteins, inspite of the other two gene copies being predicted as having PSE and CYT labels.

Furthermore, the genes plcppse180, plcppse192, plcpsec077, plcpsec095 and plcpsec099 also had both genes found in the main in vitro exoproteome of strains 1002 and C231, but were not classified in the ISPPE. The plcppse180 pan locus holds a putative pseudogene (CpPAT10_0459), and is therefore not present in the in silico predicted core surfaceome. Other genes were predicted as cytoplasmic. It is possible that these genes were wrongly assembled since there is evidence that at least two homologous genes, from strains 1002 and C231, are exported to the extracellular milieu.

Core C. pseudotuberculosis ISPPE candidates homologous to Mtb

Within the core C. pseudotuberculosis ISPPE, homologous genes to those of the previously studied Mycobacterium tuberculosis H37Rv (Mtb) were observed. In this work we present some of these homologous genes featuring at least 90% protein alignment and 50% identity within this alignment. These cut-offs were obtained during the search for C. pseudotuberculosis homologous genes in the Mtb genome.

The core C. pseudotuberculosis ISPPE, that accounts for ~81% of the total, is composed of 306 genes or 1,530 distinct locus_tags, being ~40% predicted as SEC and ~60% predicted as PSE proteins, of which 20 genes present high similarity to Mtb's genes (Table 2); however, not all of these Mtb genes have known functions.
Table 2

Core C. pseudotuberculosi s in silic o predicted pan-exoproteome homologous to Mtb's proteins

Corynebacterium pseudotberculosis

pan locus

Reference genome locus_tag

ORF size

% of amino acid alignment's identity

ORF size

Gene name

protein ID

Annotated product

uroporphyrinogen decarboxylase

transmembrane protein

propionyl-CoA carboxylase beta chain

D-alpha-D-mannose-1-phosphate guanylyltransferase MANB

hypothetical protein

bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase

50S ribosomal protein L35

membrane NADH dehydrogenase

NADH dehydrogenase

hypothetical protein

Fmu protein (SUN protein)

bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate synthase

hypothetical protein

hypothetical protein

hypothetical protein

Rieske iron-sulfur protein QcrA

tRNA/rRNA methyltransferase

C4-dicarboxylate-transport transmembrane protein DctA

bifunctional penicillin-binding protein 1A/1B

Related C. pseudotuberculosis's proteins containing at least 50% amino acid identity and 90% alignment size to the Mtb H37Rv's proteins.

In this regard, here we only discuss some of these Mtb's genes with experimental evidence. The plcppse174 pan locus shows 51% protein identity with Rv3915 (YP_178027.1), a gene named cwlM that was the first autolysin gene identified and cloned from Mtb. This finding offers a new drug target class that could alter the permeability of the mycobacterium cell wall and enhance the effectiveness of treatments for tuberculosis [26]. Applying principles of in vivo expression technology (IVET), it was possible to identify upregulated genes from Mtb in an in vitro simulation of anaerobic persistence condition. The upregulated genes under hypoxic condition (dissolved oxygen <1%) include Rv0050 (ponA1), a penicillin binding protein that has 52% protein identity to the plcppse165 pan locus and 90% alignment extension [27]. The plcpsec122 pan locus shows ~58% protein identity with Rv2752c (NP_217268.1), a unique bi-functional Mtb gene that owns both β-lactamase and RNase activities. Both activities are lost upon deletion of the 100 AA long C-terminal 100 tail, which contains an additional loop when compared to the RNase J of Bacillus subtilis [28]. As it can be observed, the plcppse080 pan locus appears twice in Table 2, as it is homologous to both NADH dehydrogenase gene copies of Mtb, ndh (NP_216370.1) and ndhA (NP_214906.1), with ~57% protein identity. In Mtb, energy generation is mainly performed by type II dehydrogenases ndh and ndhA, being both, as such, essential genes [29].

The plcpsec113 pan locus is homologous to the glmU gene (NP_215534.1), holding ~59% protein identity and more than 90% alignment extension. This gene is essential in Mtb, being required for optimal bacterial growth, and has been selected as a possible drug target for structural and functional investigation [30]. GlmU is a bifunctional acetyltransferase/uridyltransferase that catalyses the formation of UDP-GlcNAc from GlcN-1-P. UDP-GlcNAc is the substrate for two important biosynthetic pathways: lipopolysaccharide and peptidoglycan synthesis. Due to its important roles, glmU had its conformational structure solved [30]. The plcpsec113 pan locus for C. pseudotuberculosis is an interesting putative drug candidate since it is predicted to be secreted, part of the core ISPPE and is able to infer its conformational structure by homology modeling using Mtb glmU.

Several genes involved in mannoglycoconjugate biosynthesis have shown to be involved in virulence, due to their central role in biosynthesis of major surface-associated glycoconjugates. Within these genes, the Mtb gene manB (Rv3264c) is defined as a GDP-mannose pyrophosphorylase (GDPMP) and disruption of its activity leads to decrease of surface-associated mannosylated lipoglycans. For GDPMP, this decrease correspond directly to reduced virulence in both BALB/c mice and cultured human macrophages [31]. The Mtb manB gene holds 69% protein identity to the plcpsec110 pan locus and more than 90% alignment extension, making plcpsec110 a considerable putative drug target.

Mycolic acids and multimethyl-branched fatty acids are found uniquely in the cell envelope and are essential for survival, virulence and antibiotic resistance of Mtb. Acyl-CoA carboxylases (ACCases) commit acyl-CoAs to the biosynthesis of these unique fatty acids. Previous studies indicate that AccD5 is important for cell envelope lipid biosynthesis and its disruption leads to pathogen death [32]. The Mtb gene accD5 (NP_217797.1) had its structure determined and also shows ~74% protein identity to the plcppse045 pan locus in more than 90% alignment extension, making it also a promising candidate for further vaccine candidate evaluations.

Moreover, it was demonstrated that Mtb can use heme as an iron source, suggesting that Mtb contains a yet-unknown heme acquisition system [33]. We found that the C. pseudotuberculosis plcpsec076 pan locus holds ~52% protein identity to the Mtb gene hemE (NP_217194.1) and more than 90% alignment size, therefore also representing an interesting drug target for C. pseudotuberculosis.

Candidates filtering

The here presented results provide a plethora of putative vaccine candidates never seen before for C. pseudotuberculosis. However, genes predicted as MEM and CYT account respectively for 18% and 65% of the in silico predicted pan genome. Despite the 227 surfaceome and 150 secretome genes here presented, these only represents ~16% of the C. pseudotuberculosis in silico predicted pan genome. Most of the genes remain inaccessible for the current in silico prediction techniques and it is possible that these neglected genes could also be good candidates against C. pseudotuberculosis. These findings raise the need for more elaborated and driven software or prediction schemas capable of uncovering these major genome neglected portions. Using the prediction schema here presented, it was possible to include more than ~2% of non-classic secreted putative proteins that compose putative vaccine candidates. However, this low income amount of vaccine candidates is due to the optional parameter selected in our prediction schema, the non-classic secreted score greater than or equal 0.90. If using the default parameter from the software secretomeP and NClassG+, this income would be increased up to ~6% and the final income of putative vaccine candidates would be ~20%, using a couple of motifs predictors as depicted in Figure 1. The current reverse vaccinology software allows obtaining a number of candidates closer to 20% of the C. pseudotuberculosis genome. These considerations raise a question: supposing that novel software for unexplored secretion pathways come into scenario, what is the genome's percentage that could be selected as putative vaccine candidates? Supposing that this percentage reaches 40%, how could the problem of choosing between almost one thousand putative vaccine candidates to be used for the next vaccine production stage for C. pseudotuberculosis be solved? This dilemma could be solved by using further software prediction just like those addressing epitopes MHC class I and II allele affinity [34]; however, this could be just a part of the solution. There are chances of solving this dilemma by means of broader vaccine projects, which would take into account particular variables for each target organism in order to minimise research efforts and the number of possible vaccine candidates [35].

In silico versus non-in silico

It is broadly known that in silico genome investigations could give evidence about the genome's function and structure. It is also known that such in silico investigations could only be proved or denied by non-in silico experiments. Therefore, such reasonable thinking is not a single-hand avenue. Non-in silico experiments could be improved by means of more comprehensive or specific approaches with the objective of getting a closer answer to the reality for biological questions. The fact is that in silico analyses cannot vary when executed over and over again and no matter how many folds are run. We know that exactly 122 genes will be always predicted as having classical exportation motifs; on the other hand, we cannot expect the same behavior from non-in silico analysis. Some real proteins could be or not be found in an in vitro or in vivo exoproteome result, due to an uncountable number of factors [21]. Therefore, we suggest that the core C. pseudotuberculosis ISPPE could be composed of a larger number of predicted genes, but such confirmation could only be affirmed with additional non-in silico exoproteome experiments.

The in silico pan-exoproteome prediction methodology applied to the pathogen C. pseudotuberculosis helps to raise new insights into putative vaccine candidates against CLA. Additional investigations of the in vitro exoproteome of two strains of C. pseudotuberculosis, 1002 and C231, showed evidence that the major part of the variant in vitro exoproteome is contained in the core ISPPE. A simultaneous curation of the in silico predicted core secretome and surfaceome within the five C. pseudotuberculosis strains also contributed to homogenize the genome annotations and it was possible to fix the most probable putative methionine proteins. Moreover, putative miss assembled genes, formerly classified as pseudogenes by in silico analyses, were also revised. The efforts to create a C. pseudotuberculosis ISSPE catalogue proved to be necessary and computationally viable to ensure a uniform set of putative vaccine candidates free of annotation errors.

The analyzed C. pseudotuberculosis genomes were obtained from the GenBank according to the following accession numbers: EMBL: CP001809 (strain 1002), EMBL: CP001809 (strain C231), EMBL: CP002251 (strain I19), EMBL: CP002924 (strain PAT10) and EMBL: NC_014329 (strain FRC41).

Prediction schema

Predicted genes from all five C. pseudotuberculosis strain genomes were exported as amino acid fasta files using the Artemis software. These fasta files were passed as parameters to SurfG+ 1.0 (Figure 1), and lists of genes predicted as CYT, SEC, PSE and MEM were created by this software. Genes formerly predicted as CYT by SurfG+ were then submitted to the TapP 1.0 predictor; when a Tat motif was found, the putative protein was automatically classified as SEC, otherwise, another prediction round would took place using two other non-classic secretion predictors, SecretomeP 2.0 and NclassG+ 1.0. With a positive prediction from both software and a prediction score greater than or equal to 0.90, the genes were automatically classified as SEC. The SEC and PSE data sets were finally submitted to a reciprocal blastp processing and posterior filtering, giving rise to the fivefold categories according to folds occurring in each strain: 5x, 4x, 3x, 2x and 1x. The results were then manually curated using the ACT software and strain 1002, the first to be sequenced and annotated. The strain 1002 was disposed, in ACT software, in the middle of two pairs of the other two genome strains, facilitating to exhibit differences among all of them.

SurfG+ 1.0

Sub-cellular localization prediction of C. pseudotuberculosis putative proteins was made by in silico analysis using the SurfG+ 1.0 software [6]. SurfG+ is a pipeline for protein sub-cellular prediction that incorporates common software, such as SignalP, LipoP and TMHMM to search for motifs. It also creates novel HMMSEARCH profiles to predict cell wall retention signals. SurfG+ starts searching, in the following order for: retention signals, lipoproteins, SEC pathway export motifs and transmembrane motifs. If none of these motifs are found in a protein sequence, it is then characterized as CYT. A novel possible characterization introduced by SurfG+ is its ability to better distinguish between MEM and PSE, by informing an expected cell wall thickness in amino acids. Using the literature or an electronic microscopy it is possible to estimate a reasonable cell wall thickness value for prokaryotic organisms. By means of this last option, C. pseudotuberculosis genes were classified into four different sub-cellular locations: CYT, MEM, PSE, or SEC.

TatP 1.0 Server

Twin-arginine signal peptide motifs were predicted using the on line server hosted by http://www.cbs.dtu.dk/services/TatP/[20]. Only putative proteins formerly classified as CYT by SurfG+ were submitted to the TatP analyses. There were no intersections between SignalP and TatP predictions.

SecretomeP 2.0 and NClassG+ 1.0

Non-classical secreted putative proteins were predicted using the online server hosted by http://www.cbs.dtu.dk/services/SecretomeP/[21]. NClassG+ [22], a second non-classical secreted protein predictor, was also used; however, the predictions were directly performed contacting the software authors. This double check prediction ensured greater accuracy. Only those genes formerly classified as CYT by SurfG+ and without the twin-arginine signal peptide motifs were submitted to a non-classical secreted analysis. Despite the significant scores of SecretomeP and Nclass+, ranging between 0.5 and 1.0, only those genes with a score greater than or equal to 0.9 were selected, in order to ensure a minimal false positive in future wet lab experiments, the focus of our research group.

Pan genome

To predict the C. pseudotuberculosis pan genome, reciprocal blastp results were used. All the putative proteins predicted as SEC were put apart in a single amino acid fasta file to make a reciprocal blast. A similar file was also created for the proteins predicted as PSE. To avoid homologous mismatches, the blastp results obtained using the PAM70 substitution matrix and the 10-6 e-value were manually filtered. In this regard, the first step was to establish the alignment size and identity percentages of cut-offs, being 89.58 and 50.00%, respectively, for SEC putative proteins, whereas for PSE putative proteins, these cut-offs were 88.16 and 48.80%, respectively. Identity percentages closer to 50% are explained by frame shifts not annotated until this work. All the putative proteins from the five strains (query) with alignment size and identity percentages higher than these cut-offs had no more than one group of blast hits (subject) against the others strains. Moreover, within each of these blast hits groups, there was a blast hit from the query protein against it self as subject. The results were manually curated using the ACT software, from the Artemis package [24], using the strain 1002 as reference strain for the other two strains. This ACT view was composed by strains C231-1002-I19 and FRC41-1002-I19. Each putative protein predicted as SEC and PSE was compared against their other four homologues for correct initial methionine, frame shifts and finally annotating the correct sub-cellular location.

Revised in vitro exoproteome results

In lists 1 and 2 of the annex are both gene locus present in the C. pseudotuberculosis ISPPE, together with the quantity of homologous genes present in the all five genomes. These results were inserted in a relational database, denominated C. pseudotuberculosis Data Base (CpDB) [36], in a specific table called 'exopred'. The list of the in vitro exoproteome proteins was also inserted to the CpDB into a table called 'exo' that discriminates the identification of each protein regarding GenBank (protein id), as well as in which strains it is found. To make a relationship between the 'exopred' and 'exo' tables, a third table of the CpDB, called 'gene', which contains all the functional annotation of the genomes of C. pseudotuberculosis, was created. The CpDB is the repository of the pan genome of C. pseudotuberculosis, harbouring the genomes since their initial genomic prediction, deposited in the GenBank, as well as the annotation corrections for future deposits. For this last purpose, the CpDB stores the identification of each protein according to the GenBank. In this way, it is possible to make a link between the three tables in the form of a clause of JOIN of the SQL: "... WHERE gene.locus_tag = exopred.locus_tag AND gene.protein_id = exo.protein_id AND exopred.pangenome_coverage = '5x' ...". This clause returns the registries of the CpDB whose locus_tag in the gene table is equal to the locus_tag of the explored table, being this same gene in the protein_id field in the exo table with prediction of belonging to all five genomes. Other conditions can also be included, such as for example, restraining the results to specific genes of a C. pseudotuberculosis strain or simultaneously present in the exoproteome of specific strains.

We would like to thank Daniel Restrepo-Montoya, Camilo Pino, Luis F Nino, Manuel E Patarroyo and Manuel A Patarroyo for their support with NGClass+ predictions. We would also like to thank to LISI, ALGOS-UN and FIDIC bioinformatics staff which were funded supported by Asociación Investigación Solidaria SADAR, Caja Navarra (CAN) (Navarra, Spain) and the Spanish Agency for International Development Cooperation (AECID).

Funding: CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brazil.

This article has been published as part of BMC Genomics Volume 13 Supplement 5, 2012: Proceedings of the International Conference of the Brazilian Association for Bioinformatics and Computational Biology (X-meeting 2011). The full contents of the supplement are available online at http://www.biomedcentral.com/bmcgenomics/supplements/13/S5.

Authors’ Affiliations

Molecular and Celular Genetics Laboratory, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais
DNA Polimorfism Laboratory, Universidade Federal do Pará
Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology

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© Santos et al.; licensee BioMed Central Ltd. 2012

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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The Motley Fool Discussion Boards

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Subject:  Re: Oldest Fridge Contest Date:  11/6/2011  5:23 PM
Author:  SeattlePioneer Number:  37871 of 99355

<<Really? You'd pay an extra $160/year in power costs to avoid paying $500 for a new refrigerator?

I rest my case.

intercst >>

I just got my electric bill from the city owned electric utility that gets most of it's power from city owned dams built in the 1920s-1940s.

$36.92 for 632 KWH. That's for two months.

Seattle Pioneer
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1st-timer I just started up Roth IRAs w/a famous online disc. broker, for my wife & myself. I should be able to trade on both eff. Jan. 20th. I intend to utilize the RP4 variation. FOOL: Thank you for making it so simple!

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Cocktail Reception Menu Kind of (w/out open bar) I need out of the box ideas

posted 4 years ago in Food
  • poll: Which Menue
    Menu 1 : (14 votes)
    38 %
    Menu 2 : (20 votes)
    54 %
    You need more choices and better ideas : (3 votes)
    8 %
  • Post # 3
    1867 posts
    Buzzing bee
    • Wedding: March 2013

    I say Menu 2 – you sold me with BBQ and chicken and waffles!

    Post # 4
    497 posts
    Helper bee
    • Wedding: March 2013

    Either of these menus sound great and I would be quite pleased as a guest.

    Post # 6
    5877 posts
    Bee Keeper
    • Wedding: October 2010

    @JJSoon2bwife14:  As long as you spread the word that there is a cash bar, you should be fine. (Will they be able t take credit or debit cards?) Most people dont carry a lot of cash anymore.

    Post # 8
    3696 posts
    Sugar bee
    • Wedding: August 2013

    I think the menus look great. If at all possible, I would try to have a couple of free kegs (and if you’re feeling really generous, both beer and wine available for those of us who don’t drink beer). This may just be me, but I’d rather have less variety of food and be able to have a couple glasses of wine that I don’t have to pay a lot of money for.

    Post # 9
    10219 posts
    Sugar Beekeeper
    • Wedding: December 2012

    Etiquette Snob here… lol

    Your Question about whether a Cash Bar is rude or not, really has a lot more to do with what is NORM in your area and for your Guests more than anything else.

    For example, in Canada, many parts of Country and Cash Bars are the norm **… mind you most of us offset that with some sort of COMPLIMENTARY Drinks as well…

    Be that a Welcome Cocktail – His & Hers Signature Cocktails – Wine with Dinner – Wine & Beer Bar – White Bar (all “white” things are free… Gin, Vodka, White Wine)… – or something here we like to call a Loonie or Twoonie Bar, where we offset the costs by a $ 1 or $ 2.

    Mr TTR and I had a Back Home Reception Party after our Honeymoon… we served up a Complimentary First Drink of the Guest’s Choice (no maximum) – FREE Bottled Water, Juices, Pop, Tea & Coffee all night – and all the Bubbly you could drink !! (The Bubbles were extremely popular with the women…)

    ** Cash Bars or Mix & Match Bars are quite popular in Canada, because of our Laws around Alcohol which are a lot more restictive than in the USA … and the cost here is also a lot higher.  Infact, most Guests come to a Wedding / Party here expecting to have to pay for alcohol because it is such an expensive item (A simple domestic Beer here can be close to $ 10 when tax & tip is added in).  Most Guests are very appreciative when they discover that something is covered for them / FREE.

    Hope this helps,

    Post # 10
    497 posts
    Helper bee
    • Wedding: March 2013

    @JJSoon2bwife14: there aren’t many things that I think are rude at a wedding, because, after all it’s your day and things get expensive. The exception to this for me is a cash bar. I just don’t think when you are hosting people you would ask them to pay for cocktails. I think an alternative would be to offer one drink complimentary. Whether that’s wine or one signature cocktail. Your menu sounds so amazing that I would consider removing an item to be able to accomodate a free drink.

    Post # 12
    497 posts
    Helper bee
    • Wedding: March 2013

    @JJSoon2bwife14: Naw, that sounds awesome. I don’t think the menu will be boring at all. With your options and a signature drink/shot, it will be great and guests will be happy.

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  Good Morning,  Nutrition and Hormonal Balance As an acupuncturist in the area of fertility, I realize tha...

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Tuesday, November 8, 2011

[AlternativeAnswers] Re: My 7 yr old cold/respiratory infection

I called the doctor at day 12
He said my son didnt need to be seen, do fluids, steam, rest etc.
That it should take 2-4 weeks to recover
My son has such a hunky cough, and not sleeping well.

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Blog Tour: ‘Virgins’ by Caryl Rivers Review

Blog Tour: ‘Virgins’ by Caryl Rivers Review

I received this book for free from blog tours in exchange for an honest review. This does not affect my opinion of the book or the content of my review.

Blog Tour: ‘Virgins’ by Caryl Rivers ReviewVirgins by Caryl Rivers
Published by Diversion Books on May 17th, 2012
Genres: Contemporary, Friendship, Young Adult
Pages: 275
Format: eBook
Source: blog tours
For the seniors at Immaculate Heart High, hormones triumphed over the State of Grace - and everyone's vocation was to DO IT.

The Map of Forbidden Sexual Delights extended its boundaries nightly in the back seats of tail-finned cars.

And nothing - not even the Anti-Smut League - could keep the boys and the girls from a rowdy, raunchy romp through those heart-stopping, heart-wrenching days of growing up!

The Book DepositoryChapters/IndigoGood Reads

Warning: The book includes white supremacists, mild racial slurs, and casual sexual acts.

Peggy walks the fine line of being a good Catholic school girl and a young woman who wants to become independent and live her own life, instead of the one that is taught by the strict Catholic church. It makes it that much harder on Peggy when her girl best friend Con is a confident flirt with boys, and her childhood best friend Sean starts to become more than just her friend. When Sean promises to go into the priesthood, what happens to Peggy?

I would have loved to have this one as a teenager, and writing an essay about it would have been indeed wonderful since I am Catholic and went to a Catholic school. Boy has society changed! Set in the 1950s, the book has the old school way of thinking where the woman stays at home and makes everything comfortable for the husband (vomits). Virgins also brought a lot of other themes into being, like religion being such a huge part of anyone’s life, and its restrictions. How does one struggle with their religion’s beliefs and live a life where they have to do everything the Bible says? There’s a certain amount of close-minded thinking that some Catholics in this book think about and I can’t help but get angry at it. For example, everyone who isn’t Catholic will go straight to hell. People will believe what they want to believe. I always had a problem with narrow-minded people, and if I ever came across a person like Mr. McCaffrey in person, a healthy debate will ensue. The point of my rant: forcing religion upon someone is morally wrong.

The characters in this book are fully developed and their personalities emerge straight off the page. Peggy, as any young teenage girl is suddenly inundated with teenage hormones, and being Catholic makes it that much harder for to stay within the Church’s teachings. She ends up questioning a lot about her sexuality, and her thoughts are understandable and highly relatable for any teenage girl. Sean is a character who is wise beyond his years. His quotes were beautiful and I found myself agreeing with him whole heartedly. His faith in God and what He claims to do, is just so beautifully written. I found his character the easiest to like and fall in love with. These characters get into such crazy acts and say the funniest things that I couldn’t help but quote some of them. I find them quite hilarious, even being Catholic myself. Mr. McCaffrey, Sean’s close-minded father is rather unlikeable and I felt myself wanting to slap him at some points. I have society to thank that times have changed, and women are no longer just housewives who cater to the whim of every male that we encounter. I do pity the people who think like this character and hope for the best that they can respect other people’s religions and beliefs.

In this humourous parody on the Catholic Church, this coming-of-age story will get your panties in a twist, and make you laugh and cry. Highly recommended for people who want a good laugh and can poke fun at religion. Not recommended for people who are wishing for a plot, because I don’t believe there is one except the lesson that our main character learns.

Sean: “And I think maybe God doesn’t do a lot. I think he set up the rules, and once He did that, even He can’t break them.”
Peggy: “He can do everything.”
Sean: “No he can’t. God can’t make a stone so big He can’t lift it. That’s impossible. Even God can’t exist and not exist at the same time. He made the rules of nature, and can’t break them.”
Peggy: “So He just—watches?”
Sean: “Maybe. He doesn’t want bad things to happen, but He knows they have to. Maybe the universe can’t exist without bad things to balance the good things. So He has to let them both happen.”
Sean just kept kissing me, hard, and I felt the Natural Wonder getting very stiff and pressing against my thighs.
When I met Con, freshman year, I thought an orgasm was some kind if big monkey.
And they said the pope ate babies. There said there was an underwater tunnel from the Vatican and one day the pope and his army were going to invade America and murder all the Protestants.
The part of the church that drew Sean like a magnet was the hard part, the dirty part—his heroes were men who worked in filth and disease, who cared for people no one else cared about, who went where the rest of us refused to go.
Peggy: “I don’t think I’d want one. I mean, they’re nice and all, but what would I do with it?”
Con: “Go around sticking it into people, I suppose. That’s what it’s for.
Well, I was thinking about how I wanted to do something good with my life. Something really good. Not like my father, who tries to make people do what they don’t want to do. I wanted to do something to help people, you know how I mean.
What’s so great about being a virgin? Unless you’re the Blessed Virgin, and you know what? I think she got gypped. She had to have a baby and didn’t have sex. Whose idea is that of a good deal?
I love hot dogs,” Sister Immelda said. “We don’t them much in the convent.
All these years and I barely knew what a penis looked like and now they seemed to be sprouting all around me like mushrooms.
He would always believe that if he could just try harder, if he could just be better, he could change things.

  1. Jaime Lynn

    August 20, 2013 at 10:04 PM

    This sounds funny. Like a good weekend, laugh out loud book!

  2. Drennan

    October 24, 2012 at 7:40 AM

    Hi Giselle! I’m so glad that someone besides me thought this novel was completely hilarious. I literally laughed out loud many times. And I feel like Rivers is just such a good writer! If you’re interested, here’s a link to my review:

    Happy Reading,

  3. amel armeliana

    October 22, 2012 at 2:21 AM

    Oh, wow!! Don’t know what to say. Since I’m not a catholic, there’s some parts that don’t understand. But review is great. Thanks for the review.

  4. Heather J. @ TLC Book Tours

    October 19, 2012 at 2:35 PM

    I grew up in the Catholic Church and though I still love and appreciate some aspects of it I can also appreciate a bit of humor about it as well. I’m sure this would be a fun read for me!

    Thanks for being on the tour.

  5. Pingback: Caryl Rivers, author of Virgins, on tour October 2012 | TLC Book Tours

  6. The Bumble Girl

    October 14, 2012 at 11:08 AM

    Oh my, this really does sounds hilarious! As a Catholic, I can totally picture some of the things in this book as well. This would be a book that my MIL would frown upon too – I want it just to show it to her 😉
    Great review, and thanks!

    • Giselle

      October 14, 2012 at 12:36 PM

      Haha it was hilarious! I guess someone who isn’t a Catholic wouldn’t get the jokes lol.

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text:
Titch, Paperback

Titch is the youngest and smallest in his family. His brother Pete and his sister Mary have bigger bikes, kites that fly higher, and instruments that make bigger noises.

But when Titch plants a tiny seed, they all find that starting off small is just the first stage of growing.

  • Format: Paperback
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  • ISBN: 9780099262534

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Saturday, March 08, 2008

I didn't do it, I didn't mean to do it, and I'll never do it again...

So, after getting caught with its hand in the cookie jar, the FBI offered up a flurry of explanations that would be familiar to the parent of any grade-schooler. Let's parse them, shall we?
The breach occurred before the FBI enacted broad new reforms in March 2007 to prevent future lapses, FBI Director Robert Mueller said.
Translation: We're big kids now and we don't do that any more. That was back when we were just little kids.
And it was caused, in part, by banks, telecommunication companies and other private businesses giving the FBI more personal client data than was requested.
Plus, it was the other kids' fault, too. Everyone was doing it. We didn't really want to; they made us.
The new audit, which examines use of national security letters issued in 2006, "will identify issues similar to those in the report issued last March," Mueller told senators. The privacy abuse "predates the reforms we now have in place," he said.

"We are committed to ensuring that we not only get this right, but maintain the vital trust of the American people,"
...and we'll never do it again. Honest!

(Hey, Mueller, you can only "maintain" what you actually have. The word you were looking for is "regain". Honest.)

Earl said...

Pause-ible denial, anyone?

Anonymous said...

What has the FBI done lately? Hoover was a monster. They wiretapped Martin Luther King, then his wife after he was gone. They missed the fall of the Berlin Wall. They burned the Davidians. They missed arabs learning to fly. They haven't done squat in the WOT besides increase their budget. Now they can't seem to wiretap without breaking the law.

Why do we have these people again?

Anonymous said...

No I think he may have used the term maintain correctly. We can trust that they have us by the vitals, especially if they can tell the media that we're the criminals. This works because the media reposts without checking their facts or figures, and just making the small type to small to be seen on anything but the largest HD TVs. You know that's what some of us spent all our hard whined dollars on.

ColtCCO said...

Perfectly analyzed. It wasn't us, it was that other guy, and we were somewhere else when we did it, and anyway, no one saw it.

Rob said...

I love your interpretation! Spot on translation I would say.


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We've moved to http://www.booksellingonlineblog.com

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UpBeat Audio, LLC. :: Audio Amplifiers :: AC Powered Headphone and PC Speaker Amplifier R234-TV

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How do I create a new mail folder?

First make sure the folder list is displayed (see this topic). Then:

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  • The New Folder dialog box will appear. Type the name of your new folder in the "Name:" box, and then click OK. The new folder will appear in the appropriate place in the folder list.


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Truck Carrying 38,000 Pounds Of Cheese Crashes, Drops Power Lines On I-93 In MiltonThe Southeast Expressway was shut down in both directions early Thursday morning after a tractor-trailer crash brought down power lines on the highway in Milton.
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Family Of Teacher Killed In Tragic Accident Keeps Her Memory AliveThe loss of Caitlin Clavette is devastating for her family, her soon to be fiancé and those who knew her best.
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Thursday, September 4, 2014

A New Acrylic Display for Our Bookmark Calendar, A Waffle Maker for Daniel Shumski, The Mystery of the Two "Found" Covers, and A Selfie with Lisa McMann.

1. A bookseller asked me what we were going to do with our bookmark calendars, but we were using the plexy for another sideline, so we decided to buy a new plexy. These multi-tiered acrylic displays are very, very helpful at Boswell, and only occasional fall and break.

We decided that the only thing wrong with our existing displays is that tended to be limited to two or three tiers, and so we upgraded to four. And then the craziest thing happened. Without measuring, it turned out that the display exactly fit the twenty styles of bookmark calendars that Amie brought in, meaning no rotation of titles on her part. How the heck did the happen when we were just winging it? Sometimes things just work out.

2. It took longer than we hoped, but we finally put together a program to backup our event with the dynamic Dan Shumski, author of Will it Waffle: 53 Irresistible and Unexpected Recipes to Make in a Waffle Iron. At first we hoped to have a restaurant help us with a dinner, but we couldn't get any takers. A lot of breakfast places do not make waffles, it turns out, and a lot of waffle places are franchises (meaning it's tough for them to do something out of the box) or are located too far from the store. While we sometimes go a half hour away for things like a library event, this seemed too complicated to do too far afield, as our best hope was to get our customers engaged.

In the end,, we found the perfect companion in Julie Pandl, author of Memoir of the Sunday Brunch, and marketing person for Boelter SuperStore. Pandl documents her time on the brunch line in her memoir, but I suspect they rarely used it to make, say, waffled meatballs, except perhaps as a prank. Ms. Pandl agreed to make samples and demo a recipe at our event, and all we had to do was find her a waffle maker. The only problem? It's not as easy as you think to find a classic (that reads "non-Belgian", where the indentations are too dep), rectangular (that reads "not round") waffle maker. We finally picked one up at the Bayshore Boston Store, with the help of Carla in the housewares department.

3. I bring up a mystery that has not yet been solved in her current series of Margaret Peterson Haddix, "The Missing." For one thing, we long thought that the series would have seven installments, and it turns out with the release of Revealed, there are now eight. It's sort of like the fourth book in a trilogy, which is not that unsuaul. But stranger than that, when we got our lrage order of volume one, Found, it turns out our stock was equally divided between bluish covers and purplish covers. And we had a lot of copies to sample.

It's probably just an issue of dye lots, but I couldn't help thinking there was some sort of secret code involved.

Speaking of our event with Haddix and McMann, there's no consistency with when we get author photos and when we don't, but for some reason, the camera was flashing a lot during our two days (four events, including the school visits) with our authors. I have some nice photos of Ms. Haddix and Ms. McMann with students, with principals, in front of their Powerpoints, and so forth, but for sheer amusement, nothing beats this silly selfie with Lisa McMann, right?

That sort of catches me up on the posts I hoped to write last week. And now I'm already behind for this week! So goeth life in a bookstore.

No comments:


text:
[The View]
"Should I practice what I preach, man? Should I just do what I'm told? Because the clock keeps ticking, let's hope we don't grow old."

The only thing that I thought could come out from a placed called Dundee is crocodiles, but apparently that's the wrong Dundee. No, these teenage Dundonian upstarts root themselves in Scotland and were plucked from their patch into the hazy world of British rock during their youth. Now, the hooligans continue to grow in their new patch and have already got two albums under their belt with a third slated for release sometime next year. The quartet carries a scruffy, strident sound that's gritty and quick. Shock Horror, in particular, just packs a wallop that hits me hard right in my tum-tum. From where I'm standing, the View looks good.

From The View's Which Bitch?:
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Learn more at their site.
Be their friend!

Otherwheres - I've got plans to switch over into the Mac camp, and I've been nesting by getting it some goodies. My latest purchase? An overlay that changes up the color of the apple logo at this etsy shop. I'm on the fast track to being broke...

Otherwheres - Nathaniel over at IGIF's got Crystal Castles' vid for Celestica...

Otherwheres - Get a taste of LA sound as Aquarium Drunkard posts up L'Aventure, a set of tracks provided by various LA artists covering tracks from Television's Adventure...
Anonymous chris said...
Sorry I don't comment (ever), but I just wanted to chime in and let you know how much I appreciate the drawings you guys do for your posts. They're so damn creative... leaves me feeling inspired and jealous at the same time ;-)


text:
Local Components

Now we have a way to publish and use Bower components. But you probably also develop your own front-end code: we call these “local components”. BowerStatic also helps with that. For this it is important to understand that locally developed code has special caching requirements:

  • When you release a local component, you want it to be cached infinitely just like for Bower components.

    When later a new release is made, you want that cache to be invalidated, and not force end-users to do a shift-reload to get their browser to load the new version of the code.

    We can accomplish this behavior by using a version number in the URL, just like for Bower components.

    XXX one way to release a local component would be to release it as a bower component at this point. But this may be cumbersome for code maintained as part of Python package.

  • When you develop a local component, you want the cache to be invalidated as soon as you make any changes to the code, so you aren’t forced to do shift-reload during development. A simple reload should refresh all static resources.

    A way to look at this is that you want the system to make a new version number for each and every edit to the local component.

To have local components, you first have to create a special local components registry:

local = bower.local_components('local', components)

You can have more than one local components registry, but typically you only need one per project.

The first argument is the name of the local components registry. It is used in the URL.

The second argument is a components object for a bower_components directory, created earlier with bower.components(). This makes all those bower components available in the local component registry, so that the local components can depend on them.

Note that the local components registry does not point to a bower_components directory itself. Instead we register directories for individual local components manually.

Here’s how we add a local component:

local.component('/path/to/directory/mycode', version='1.1.0')

The /path/to/directory/mycode directory should have a bower.json file. BowerStatic uses name and main for local components like it uses them for third party Bower components. The name of the component should be unique within the local registry, as well as not conflict with any component in the Bower components registry.

As with bower.components, you can use bowerstatic.module_relative_path to create a path relative to the calling module:

components = local.component(

dependencies is also picked up from bower.json, but unlike for third party components these dependencies are not automatically installed.

The version number is not picked up from bower.json. Instead it is passed through to the local component. This will make it possible to support the right caching behavior. We go into detail about this later.

If you have a local component called mycode, and there is a file app.js in its directory, it is published under this URL:

To be able to include it, we can create an includer for the local registry:

include = local.includer(environ)

This includer can be used to include local components, but also the third-party components from the registry that the local components registry was initialized with.

You can now include app.js in mycode like this:

Let’s consider versioning in more detail.

The version number is passed in when registering the local component. We want it to do the right thing with caching:

  • When the application is deployed, we want the version number to be the version number of that application (or sub-package of that application), so that infinite caching can be used but the cache is automatically busted with an application upgrade.
  • When the application is under development, we want the version number to change each time you edit the local component’s code, so that the cache is busted each time.

Versioning deployed applications

You can use the version of a Python application easily, as long as it is packaged using setuptools ( pip, easy_install, buildout, etc). You can retrieve its version number like this:

import pkg_resources

version = pkg_resources.get_distribution('myproject').version

This picks up the version given in setup.py of myproject.

Using this to obtain the version and passing it into local.component() is enough to make sure the cache is busted when you make a release of your application.

Versioning during development

We have to make sure the cache is busted automatically during development as well. For that we have to turn on BowerStatic’s development mode. You can do this by passing None as the version into local.component.

This causes the version to be automatically determined from the code in the package, and be different each time you edit the code. Since the version is included in the URL to the package, this allows you to get the latest version of the code as soon as you reload after editing a file. No shift-reloads needed to reload the code!

Putting it all together

Development mode is relatively expensive, as BowerStatic has to monitor the local directory for any changes so it can update the version number automatically. You should therefore make sure it is only enabled during development, not during deployment. When your application is deployed you need to pass in a real version number, for instance the one you pick up using pkg_resources as described before.

If your application has a notion of a development mode that you can somehow inspect during run-time, you can write a version function that automatically returns None in development mode and otherwise returns the application’s version number. This ensures optimal caching behavior during development and deployment both. Here’s what this function could look like:

def get_version():
    if is_devmode_enabled():  # app specific API
        return None
    return pkg_resources.get_distribution('myproject').version

You can then register the local component like this:

local.component('/path/to/directory/mycode', version=get_version())