Sirenomelia in Bmp7 and Tsg compound mutant mice: requirement for Bmp signaling in the development of ventral posterior mesoderm

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.     

SERPINB11 Frameshift Variant Associated with Novel Hoof Specific Phenotype in Connemara Ponies

Horses belong to the order Perissodactyla and bear the majority of their weight on their third toe; therefore, tremendous force is applied to each hoof. An inherited disease characterized by a phenotype restricted to the dorsal hoof wall was identified in the Connemara pony. Hoof wall separation disease (HWSD) manifests clinically as separation of the dorsal hoof wall along the weight-bearing surface of the hoof during the first year of life. Parents of affected ponies appeared clinically normal, suggesting an autosomal recessive mode of inheritance. A case-control allelic genome wide association analysis was performed (n_cases = 15, n_controls = 24). Population stratification (λ = 1.48) was successfully improved by removing outliers (n_controls = 7) identified on a multidimensional scaling plot. A genome-wide significant association was detected on chromosome 8 (p_raw = 1.37x10^-10, p_genome = 1.92x10^-5). A homozygous region identified in affected ponies spanned from 79,936,024-81,676,900 bp and contained a family of 13 annotated SERPINB genes. Whole genome next-generation sequencing at 6x coverage of two cases and two controls revealed 9,758 SNVs and 1,230 indels within the ~1.7-Mb haplotype, of which 17 and 5, respectively, segregated with the disease and were located within or adjacent to genes. Additional genotyping of these 22 putative functional variants in 369 Connemara ponies (n_cases = 23, n_controls = 346) and 169 horses of other breeds revealed segregation of three putative variants adjacent or within four SERPIN genes. Two of the variants were non-coding and one was an insertion within SERPINB11 that introduced a frameshift resulting in a premature stop codon. Evaluation of mRNA levels at the proximal hoof capsule (n_cases = 4, n_controls = 4) revealed that SERPINB11 expression was significantly reduced in affected ponies (p<0.001). Carrier frequency was estimated at 14.8%. This study describes the first genetic variant associated with a hoof wall specific phenotype and suggests a role of SERPINB11 in maintaining hoof wall structure.     

Caenorhabditis elegans: A Simple Nematode Infection Model for Penicillium marneffei

Penicillium marneffei, one of the most important thermal dimorphic fungi, is a severe threat to the life of immunocompromised patients. However, the pathogenic mechanisms of P. marneffei remain largely unknown. In this work, we developed a model host by using nematode Caenorhabditis elegans to investigate the virulence of P. marneffei. Using two P. marneffei clinical isolate strains 570 and 486, we revealed that in both liquid and solid media, the ingestion of live P. marneffei was lethal to C. elegans (P<0.001). Meanwhile, our results showed that the strain 570, which can produce red pigment, had stronger pathogenicity in C. elegans than the strain 486, which can’t produce red pigment (P<0.001). Microscopy showed the formation of red pigment and hyphae within C. elegans after incubation with P. marneffei for 4 h, which are supposed to be two contributors in nematodes killing. In addition, we used C. elegans as an in vivo model to evaluate different antifungal agents against P. marneffei, and found that antifungal agents including amphotericin B, terbinafine, fluconazole, itraconazole and voriconazole successfully prolonged the survival of nematodesinfected by P. marneffei. Overall, this alternative model host can provide us an easy tool to study the virulence of P. marneffei and screen antifungal agents.     

Helicobacter pylori Infection in Thailand: A Nationwide Study of the CagA Phenotype

Background

The risk to develop gastric cancer in Thailand is relatively low among Asian countries. In addition, the age-standardized incidence rate (ASR) of gastric cancer in Thailand varies with geographical distribution; the ASR in the North region is 3.5 times higher than that in the South region. We hypothesized that the prevalence of H. pylori infection and diversity of CagA phenotype contributes to the variety of gastric cancer risk in various regions of Thailand.

Methods

We conducted a nationwide survey within Thailand. We determined H. pylori infection prevalence by detecting H. pylori, using histochemical and immunohistochemical methods. The anti-CagA antibody and anti-East-Asian type CagA antibody (α-EAS Ab), which showed high accuracy in several East Asian countries, were used to determine CagA phenotype.

Results

Among 1,546 patients from four regions, including 17 provinces, the overall prevalence of H. pylori infection was 45.9% (710/1,546). Mirroring the prevalence of H. pylori infection, histological scores were the lowest in the South region. Of the 710 H. pylori-positive patients, 93.2% (662) were immunoreactive with the anti-CagA antibody. CagA-negative strain prevalence in the South region was significantly higher than that in other regions (17.9%; 5/28; p < 0.05). Overall, only 77 patients (11.6%) were immunoreactive with the α-EAS Ab. There were no differences in the α-EAS Ab immunoreactive rate across geographical regions.

Conclusions

This is the first study using immunohistochemistry to confirm H. pylori infections across different regions in Thailand. The prevalence of East-Asian type CagA H. pylori in Thailand was low. The low incidence of gastric cancer in Thailand may be attributed to the low prevalence of precancerous lesions. The low incidence of gastric cancer in the South region might be associated with the lower prevalence of H. pylori infection, precancerous lesions, and CagA-positive H. pylori strains, compared with that in the other regions.     

Bmp6 Expression Can Be Regulated Independently of Liver Iron in Mice

The liver is the primary organ for storing iron and plays a central role in the regulation of body iron levels by secretion of the hormone Hamp1. Although many factors modulate Hamp1 expression, their regulatory mechanisms are poorly understood. Here, we used conditional knockout mice for the iron exporter ferroportin1 (Fpn1) to modulate tissue iron in specific tissues in combination with iron-deficient or iron-rich diets and transferrin (Tf) supplementation to investigate the mechanisms underlying Hamp1 expression. Despite liver iron overload, expression of bone morphogenetic protein 6 (Bmp6), a potent-stimulator of Hamp1 expression that is expressed under iron-loaded conditions, was decreased. We hypothesized that factors other than liver iron must play a role in controlling Bmp6 expression. Our results show that erythropoietin and Tf-bound iron do not underlie the down-regulation of Bmp6 in our mice models. Moreover, Bmp6 was down-regulated under conditions of high iron demand, irrespective of the presence of anemia. We therefore inferred that the signals were driven by high iron demand. Furthermore, we also confirmed previous suggestions that Tf-bound iron regulates Hamp1 expression via Smad1/5/8 phosphorylation without affecting Bmp6 expression, and the effect of Tf-bound iron on Hamp1 regulation appeared before a significant change in Bmp6 expression. Together, these results are consistent with novel mechanisms for regulating Bmp6 and Hamp1 expression.     

Genetic Requirements for High Constitutive SOS Expression in recA730 Mutants of Escherichia coli

The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5′–3′ exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5′–3′ exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo.     

Identification and Characterization of a Novel Allele of Caenorhabditis elegans bbs-7

Primary cilia play a role in the sensation of and response to the surrounding environment. Caenorhabditis elegans (C. elegans) have primary cilia only on the distal tips of some dendrites. In order to better understand the relationship between receptor localization to cilia, cilia structure and cilia function, we have characterized a mutation originally identified in a forward genetic screen for mutants with defective PKD-2 ciliary localization. Through behavioral assays and examination of the structure of cilia in the cil-5 (my13) mutant animals, we have found that my13 disrupts not only receptor localization, but also some cilia-mediated sensory behaviors and cilia structural integrity. We have identified the my13 lesion and found that it is a missense mutation in bbs-7, an ortholog of human BBS-7, a gene known to affect human cilia and to be involved in Bardet-Biedl syndrome. Finally, we show that bbs-7(my13) also affects the glia cells which support the cilia.     

A Novel Protein Kinase Localized to Lipid Droplets Is Required for Droplet Biogenesis in Trypanosomes

Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipid homeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved in lipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate its localization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydrophobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated with or inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestly affected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procyclic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This loss was dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid body biogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK is required for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specifically and predominantly associated with an intracellular organelle.Trypanosoma brucei is a single-celled eukaryotic pathogen responsible for human African trypanosomiasis (also known as African sleeping sickness) and nagana in domestic animals. More than 50,000 cases of human disease occur yearly, with over 70 million people at risk. No vaccine exists, and chemotherapy is difficult to administer and prone to pathogen resistance. As T. brucei transits between the mammalian bloodstream and the tsetse fly vector during its life cycle, the organism encounters and adapts to profoundly different environmental conditions. The parasite undergoes dramatic changes in both energy (7, 51) and lipid biosynthesis and metabolism (39, 47, 49) as it shifts between these environments.Protein kinases function in numerous regulatory aspects of the cell, including control of the cell cycle and morphology, responses to stress, and transmission of signals from the extracellular environment or between compartments of the cell. As is the case in other eukaryotes, protein kinases, particularly those associated with membranes, are expected to play pivotal roles in the cell''s ability to sense and appropriately respond to its environment. Trypanosoma brucei possesses over 170 protein kinases (16, 44). Most of these can be assigned to the standard groups of protein kinases based on sequence similarity within the kinase domain. However, sequence similarities with kinases from more well-studied organisms are rarely strong enough to allow one-to-one orthologous relationships to be determined (44), and even those which appear orthologous by sequence have sometimes shown functional divergence (46). Hence, an understanding of the roles of specific protein kinases of trypanosomatids requires an individualized assessment. The initial genome analysis of the trypanosomatids (16) showed a lack of receptor tyrosine kinases, but nine T. brucei predicted serine/threonine kinases were annotated as possessing transmembrane domains. One of these was recently shown to be strategically located at a key interface between the host and parasite: the flagellar pocket (38). This eukaryotic translation initiation factor 2α (eIF2α) family kinase was postulated to play a sensory role in monitoring protein transport.Only a very small number of protein kinases of various organisms have been observed to localize to the membranes of intracellular organelles, most of them to the endoplasmic reticulum (ER) (14, 27, 50). Lipid droplets (also known as lipid bodies, adiposomes, or oil bodies in plants) are thought to arise from the ER, although the routes of protein localization to them are not well understood. They are increasingly recognized as legitimate organelles due to their dynamic roles in energy metabolism (40), lipid trafficking (41), and protection against toxic effects of nonesterified lipids and sterols (18). Studies also suggest that they function as potential protein storage depots (12) and in antigen presentation (10). Although recent efforts to expand the lipid droplet proteome have resulted in a vastly increased and in many cases surprising catalogue of potentially associated proteins (3, 5, 11, 12, 23, 37), relatively little is known as to how these structures form and are regulated within the cell.We examine here a novel T. brucei protein kinase with a predicted transmembrane domain. Surprisingly, this protein is localized intracellularly in association with lipid droplets. RNAi-mediated knockdown of this newly identified kinase, dubbed LDK (for lipid droplet kinase), reveals a role in the formation or maintenance of lipid droplets in both mammalian bloodstream-form (BF) and insect procyclic-form (PF) stages of the parasite life cycle.     

DNMT3B7 Expression Promotes Tumor Progression to a More Aggressive Phenotype in Breast Cancer Cells

Epigenetic changes, such as DNA methylation, have been shown to promote breast cancer progression. However, the mechanism by which cancer cells acquire and maintain abnormal DNA methylation is not well understood. We have previously identified an aberrant splice form of a DNA methyltransferase, DNMT3B7, expressed in virtually all cancer cell lines but at very low levels in normal cells. Furthermore, aggressive MDA-MB-231 breast cancer cells have been shown to express increased levels of DNMT3B7 compared to poorly invasive MCF-7 cells, indicating that DNMT3B7 may have a role in promoting a more invasive phenotype. Using data gathered from The Cancer Genome Atlas, we show that DNMT3B7 expression is increased in breast cancer patient tissues compared to normal tissue. To determine the mechanism by which DNMT3B7 was functioning in breast cancer cells, two poorly invasive breast cancer cell lines, MCF-7 and T-47D, were stably transfected with a DNMT3B7 expression construct. Expression of DNMT3B7 led to hypermethylation and down-regulation of E-cadherin, altered localization of β-catenin, as well as increased adhesion turnover, cell proliferation, and anchorage-independent growth. The novel results presented in this study suggest a role for DNMT3B7 in the progression of breast cancer to a more aggressive state and the potential for future development of novel therapeutics.     

Stepwise Photoinhibition of Photosystem II : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the Q_B-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of Q_A reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified Q_B pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.     

PepExplorer: A Similarity-driven Tool for Analyzing de Novo Sequencing Results

Peptide spectrum matching is the current gold standard for protein identification via mass-spectrometry-based proteomics. Peptide spectrum matching compares experimental mass spectra against theoretical spectra generated from a protein sequence database to perform identification, but protein sequences not present in a database cannot be identified unless their sequences are in part conserved. The alternative approach, de novo sequencing, can make it possible to infer a peptide sequence directly from a mass spectrum, but interpreting long lists of peptide sequences resulting from large-scale experiments is not trivial. With this as motivation, PepExplorer was developed to use rigorous pattern recognition to assemble a list of homologue proteins using de novo sequencing data coupled to sequence alignment to allow biological interpretation of the data. PepExplorer can read the output of various widely adopted de novo sequencing tools and converge to a list of proteins with a global false-discovery rate. To this end, it employs a radial basis function neural network that considers precursor charge states, de novo sequencing scores, peptide lengths, and alignment scores to select similar protein candidates, from a target-decoy database, usually obtained from phylogenetically related species. Alignments are performed using a modified Smith–Waterman algorithm tailored for the task at hand. We verified the effectiveness of our approach using a reference set of identifications generated by ProLuCID when searching for Pyrococcus furiosus mass spectra on the corresponding NCBI RefSeq database. We then modified the sequence database by swapping amino acids until ProLuCID was no longer capable of identifying any proteins. By searching the mass spectra using PepExplorer on the modified database, we were able to recover most of the identifications at a 1% false-discovery rate. Finally, we employed PepExplorer to disclose a comprehensive proteomic assessment of the Bothrops jararaca plasma, a known biological source of natural inhibitors of snake toxins. PepExplorer is integrated into the PatternLab for Proteomics environment, which makes available various tools for downstream data analysis, including resources for quantitative and differential proteomics.Very often, groundbreaking discoveries with a significant impact on the biotechnological and biomedical fields have emerged from studying “non-canonical” organisms. For example, the study of Thermus aquaticus allowed us to ultimately pave the way to modern molecular biology with the characterization of that organism''s thermostable DNA polymerase (1). The characterization of the green fluorescent protein in Aequoria victoria led to a revolution in cellular biology and to a Nobel Prize being awarded to Osamu Shimomura, Martin Chalfie, and Roger Tsien. In Brazil, Sergio Ferreira''s work on the venom of the Brazilian poisonous snake Bothrops jararaca enabled the development of the first angiotensin-converting enzyme inhibitor drug (Captopril) for the treatment of hypertension (2).In scenarios such as these, proteomics has the potential to allow a better understanding of the complexity of biological systems and the process of evolution than the study of the genetic code alone. It enables the characterization of molecular processes according to their protein content, facilitating new discoveries. In proteomics, the most frequently used strategy for protein identification is so-called peptide spectrum matching (PSM),¹ or the comparison of experimental mass spectra obtained by fragmenting peptides in a mass spectrometer to theoretical spectra generated from a sequence database. In general, the identification process follows from the sequence whose theoretical spectrum yields the highest matching score according to some empirical or probabilistic function. Examples of search engines adopting this strategy are SEQUEST (3), X!Tandem (4), and Mascot (5).Back in the 1990s, establishment of a cutoff score for confident identification relied mostly on user experience; for example, given a specific charge state, Washburn et al. established cross-correlation and deltaCn cutoff values for SEQUEST in order to allow the selection of a subset of confident identifications from LCQ data. This has since been termed “the Washburn criterion.” In what followed, target-decoy databases were implemented to allow for more sophisticated refinements in filtering the data (6). In 2007, Elias and Gygi published a seminal paper on the target-decoy approach to shotgun proteomics (7) that ultimately firmed this approach as a standard and motivated the development of several statistical filters capable of converging to a list of confident identifications satisfying a user-specified false-discovery rate (FDR) with significantly more sensitivity than the conservative Washburn criterion. Such statistical filters include mixtures of probabilities (8), quadratic discriminant analysis (9), semi-supervised learning with support vector machines (10), and Bayesian logic (11) using a semi-labeled decoy analysis to account for overfitting (12). With so many advances, the PSM workflow has become the gold standard, as it is very sensitive and the least error-prone method when a database is available with the corresponding proteins. The latter factor limits the application of PSM to those organisms for which accurate sequence databases have been established. If a peptide''s sequence is not contained within the sequence database, it cannot be identified via the PSM method. However, efforts in developing error-tolerant PSM approaches such as implemented in Mascot have made it possible to handle minor sequence modifications constrained by a simple set of rules. Nevertheless, increasing the search space in the PSM approach leads to decreased sensitivity (13).Even though the concept of computer-aided de novo sequencing predates that of PSM (14), advances in the quality of mass spectrometry data and the power of computer hardware have allowed it to reemerge at the heart of a highly active field. De novo sequencing is unbiased insofar as it is not constrained by a sequence database, and it is therefore complementary to PSM. However, it has remained the most error prone of the two methods (15). The challenges of de novo sequencing notwithstanding, a few recent and notable improvements in computer-aided de novo analysis are PepNovo (16), which combines graph theory with machine learning; pNovo+ (17), which is optimized for high-resolution HCD data; NovoHMM (18), relying on hidden Markov models for increased sensitivity; and PEAKS (19), which creates a spectrum graph model by performing dynamic programming on the mass values regardless of the presence of an observed fragment ion. By considering the complementarities of different fragmentation strategies (e.g. collision induced dissociation, electron transfer dissociation (20), and electron capture dissociation (21)), computational proteomics scientists have also demonstrated significant advances in de novo accuracy (22). In particular, the Bandeira group has continually pushed the limits and redefined the notion of what de novo sequencing can do by introducing the spectral networks paradigm (23–25). Briefly, this strategy can assemble mass spectra into spectral pairs by joining overlapping spectra obtained from sample aliquots digested by different enzymes. As a consequence, it reduces noise and significantly improves protein coverage. Its latest version also combines data from different fragmentation techniques.These algorithm developments have improved de novo sequencing, shifting the bottleneck to post-sequence processing of data. This is because the output of de novo software is a long list of highly similar full and partial peptide sequence and scores. An initial attempt to overcome these limitations consisted of a tag approach that was a hybrid of de novo sequencing and database searching: short sequence tags were derived from tandem mass spectra and used to search a sequence database (26). In what followed, a modified version based on the FASTA homology search tool was proposed for homology-driven proteomics (27). This strategy was implemented as part of the CIDentify tool, whose novelty was to account, in the alignment score, for limitations of mass spectrometry sequencing such as switching between leucine and isoleucine or other combinations of amino acids having the same mass. The next steps were taken mainly by the Shevchenko group through the introduction of the MS-Blast algorithm, which relies on a different set of scores and uses the PAM30MS substitution matrix, itself tailored for mass-spectrometry-based proteomics (28, 29). For a complete review of de novo sequencing and homology searching, we suggest Ref. 30.The current de novo post-processing paradigm presents several limitations that are similar to those of the early PSM workflow. Output files generally consist of a peptide list with corresponding scores, demanding an experienced user to assess trustworthy identifications. If the same peptide is analyzed by different mass spectrometers, different scores might be generated, which makes data comparison between different groups a challenging task. In a sense, problems are similar to those encountered when adopting the early Washburn criterion. Assembling protein information from a list of peptides is not a simple task, and usually it is not performed using state-of-the-art de novo tools. Although there are great tools for doing this at the PSM level, there is still a lack of similar tools for de novo sequencing.To tackle the aforementioned shortcomings, and in line with our strong interest in diversity-driven proteomics (29), we present a methodology for post-processing de novo sequencing data that allows inference of protein identification through statistical mapping of de novo sequencing results to a protein sequence database. Our approach begins with the use of Gotoh''s version of the Smith–Waterman algorithm, based on affine gap scoring (31) for increased scalability, to align de novo sequences against those in a target-decoy database. Then a radial basis function neural network (RBF-NN) is used to rank results according to alignment score, de novo score, precursor charge state, and peptide length. Finally, a heuristic method is used to present protein identification results in a user-friendly, interactive report. The resulting algorithm was implemented as the software PepExplorer. In essence, its goal is somewhat similar to that of post-processing tools such as DTASelect (9), Percolator (10), and SEPro (11), but with an extra layer of complexity inherent from de novo sequencing. PepExplorer can handle the output of several widely adopted de novo tools, such as PepNovo, pNovo+, and PEAKS, and accepts a generic format to enable result analysis from a broader range of tools once results are run through simple parsers. Similarly, the software accepts a series of database formats for input analysis. These features are not found in other tools. PepExplorer is freely available to the scientific community and is provided with the necessary documentation.The effectiveness of our methodology has been verified in two distinct scenarios, the first a real but controlled experiment and the other pertaining to comprehensive profiling of the plasma components of Bothrops jararaca, a venomous viper endemic to Brazil, southern Paraguay, and northern Argentina. The first scenario''s purpose was to validate the effectiveness of the tool in analyzing a published Pyrococcus furiosus dataset (11). We note that this organism is recognized by the proteomics community as well suited for benchmarking, because it allows for the rigorous testing of identification algorithms at the peptide and protein levels (32, 33). We modified the P. furiosus sequence database in such a way that no more peptides were identified via the PSM approach or another widely adopted error-tolerant search tool, Mod-A (34). We then found that we could recover protein identifications using our tool. The B. jararaca scenario has allowed us to explore uncharted territory, as this organism has an incomplete sequence database and we were therefore required to rely on those of orthologous organisms. In particular, B. jararaca plasma was chosen because it is a main research model studied at the Laboratory of Toxinology (FIOCRUZ, Brazil), and several natural inhibitors of snake toxins have already been identified/characterized from this biological matrix (35–37).     

Identification of a Lifespan Extending Mutation in the Schizosaccharomyces pombe Cyclin Gene clg1 + by Direct Selection of Long-Lived Mutants

Model organisms such as budding yeast, worms and flies have proven instrumental in the discovery of genetic determinants of aging, and the fission yeast Schizosaccharomyces pombe is a promising new system for these studies. We devised an approach to directly select for long-lived S. pombe mutants from a random DNA insertion library. Each insertion mutation bears a unique sequence tag called a bar code that allows one to determine the proportion of an individual mutant in a culture containing thousands of different mutants. Aging these mutants in culture allowed identification of a long-lived mutant bearing an insertion mutation in the cyclin gene clg1 ⁺. Clg1p, like Pas1p, physically associates with the cyclin-dependent kinase Pef1p. We identified a third Pef1p cyclin, Psl1p, and found that only loss of Clg1p or Pef1p extended lifespan. Genetic and co-immunoprecipitation results indicate that Pef1p controls lifespan through the downstream protein kinase Cek1p. While Pef1p is conserved as Pho85p in Saccharomyces cerevisiae, and as cdk5 in humans, genome-wide searches for lifespan regulators in S. cerevisiae have never identified Pho85p. Thus, the S. pombe system can be used to identify novel, evolutionarily conserved lifespan extending mutations, and our results suggest a potential role for mammalian cdk5 as a lifespan regulator.     

Identification of a Novel Selenium-containing Compound,Selenoneine, as the Predominant Chemical Form of Organic Selenium in the Blood of Bluefin Tuna

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N_α,N_α,N_α-trimethyl-l-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H]⁺ ion of the compound was 533.0562 and the molecular formula was C₁₈H₂₉N₆O₄Se₂. Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound “selenoneine.” By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2–4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.     

Tetrahymena: An Alternative Model Host for Evaluating Virulence of Aeromonas Strains

An easier assessment model would be helpful for high-throughput screening of Aeromonas virulence. The previous study indicated the potential of Tetrahymena as a permissive model to examine virulence of Aeromonas hydrophila. Here our aim was to assess virulence of Aeromonas spp. using two model hosts, a zebrafish assay and Tetrahymena-Aeromonas co-culture, and to examine whether data from the Tetrahymena thermophila model reflects infections in the well-established animal model. First, virulence of 39 Aeromonas strains was assessed by determining the 50% lethal dose (LD₅₀) in zebrafish. LD₅₀ values ranging from 1.3×10² to 3.0×10⁷ indicated that these strains represent a high to moderate degree of virulence and could be useful to assess virulence in the Tetrahymena model. In Tetrahymena-Aeromonas co-culture, we evaluated the virulence of Aeromonas by detecting relative survival of Aeromonas and Tetrahymena. An Aeromonas isolate was considered virulent when its relative survival was greater than 60%, while the Aeromonas isolate was considered avirulent if its relative survival was below 40%. When relative survival of T. thermophila was lower than 40% after co-culture with an Aeromonas isolate, the bacterial strain was regarded as virulent. In contrast, the strain was classified as avirulent if relative survival of T. thermophila was greater than 50%. Encouragingly, data from the 39 Aeromonas strains showed good correlation in zebrafish and Tetrahymena-Aeromonas co-culture models. The results provide sufficient data to demonstrate that Tetrahymena can be a comparable alternative to zebrafish for determining the virulence of Aeromonas isolates.     

A Possible Role for Exocytosis in Aflatoxin Export in Aspergillus parasiticus

Filamentous fungi synthesize bioactive secondary metabolites with major human health and economic impacts. Little is known about the mechanisms that mediate the export of these metabolites to the cell exterior. Aspergillus parasiticus synthesizes aflatoxin, a secondary metabolite that is one of the most potent naturally occurring carcinogens known. We previously demonstrated that aflatoxin is synthesized and compartmentalized in specialized vesicles called aflatoxisomes and that these subcellular organelles also play a role in the export process. In the current study, we tested the hypothesis that aflatoxisomes fuse with the cytoplasmic membrane to facilitate the release of aflatoxin into the growth environment. Microscopic analysis of A. parasiticus grown under aflatoxin-inducing and non-aflatoxin-inducing conditions generated several lines of experimental evidence that supported the hypothesis. On the basis of the evidence, we propose that export of the mycotoxin aflatoxin in Aspergillus parasiticus occurs by exocytosis, and we present a model to illustrate this export mechanism.Secondary metabolites are chemically diverse natural products synthesized by plants, fungi, bacteria, algae, and animals. Secondary metabolites have an enormous impact on humans. Antibiotics, for example, are essential elements of the multibillion-dollar pharmaceutical industry, whereas mycotoxins cause hundreds of millions of dollars in damage to agriculture annually (11, 15). These chemicals help the producing organism to survive nutrient limitation (16). They also contribute to cellular defense mechanisms and development (11, 12), reduce cellular oxidative stress (10), and help maintain cellular homeostasis by regulating carbon flow in the cell (17).Many fungal secondary metabolites are exported outside the cell; examples include antibiotics and mycotoxins (3, 14). We and others conducted extensive studies on the regulation of fungal secondary metabolism at the molecular (11, 15) and cellular (3, 7) levels. However, little is known about the mechanisms that mediate secondary metabolite export or why export occurs.The filamentous fungus Aspergillus parasiticus produces aflatoxin, a secondary metabolite and the most potent naturally occurring carcinogen known. More than 90% of aflatoxin is exported to the cell exterior (3), making A. parasiticus an excellent model for studying secondary metabolite export. We recently demonstrated that specialized trafficking vesicles called aflatoxisomes play a key role in aflatoxin synthesis and export (3). As synthesis initiates, vesicle-vacuole fusion is downregulated by the global regulator Velvet, resulting in the accumulation of aflatoxisomes which contain at least the last two functional enzymes in the aflatoxin pathway and sequester aflatoxin (3). Treatments that block vesicle-vacuole fusion increase the number of aflatoxisomes, increase the quantity of aflatoxin accumulated in aflatoxisomes, and increase aflatoxin export to the cell exterior (3). On the basis of these previous observations, we hypothesized that aflatoxisomes play a direct role in aflatoxin export.Vesicle-mediated export could theoretically occur by one (or more) of at least three mechanisms (Fig. 1). (i) Vesicles pass across the cytoplasmic membrane intact and “shuttle” their contents into the external environment. This proposed mechanism mediates virulence factor release in Cryptococcus neoformans and Histoplasma capsulatum (1) during pathogenesis. (ii) Vesicles fuse to the cytoplasmic membrane and “pump” vesicle contents to the exterior using transporter proteins similar to those that mediate resistance to antifungal agents (4, 5). (iii) Vesicles fuse with the cytoplasmic membrane, which evaginates, bursts, and “blasts” vesicle contents to the exterior. This process is similar to exocytosis, a proposed secretory mechanism for specific proteins in filamentous fungi (18). We conducted the current study to determine which, if any, of these possible mechanisms most accurately reflects the process of aflatoxin export in A. parasiticus.Open in a separate windowFig. 1.Theoretical models for vesicle-mediated export. Aflatoxigenic vesicles (aflatoxisomes) arise due to downregulation of tethering complex (Tc) activity mediated by VeA (1). Aflatoxin synthesized in aflatoxisomes could theoretically be released to the cell exterior by one or more of three mechanisms: the shuttle (in which aflatoxisomes shuttle cargo across cytoplasmic membrane), pump (in which transmembrane transporter [Tp] proteins mediate the release of secondary metabolites as vesicles adhere to the inner surface of the cytoplasmic membrane), and burst-and-blast (in which vesicles protrude from the cell surface and blast their cargo into the medium) mechanisms. PM, plasma membrane.     

Metabolism of d-Glycero-d-Manno-Heptitol, Volemitol, in Polyanthus. Discovery of a Novel Ketose Reductase

Volemitol (d-glycero-d-manno-heptitol, α-sedoheptitol) is an unusual seven-carbon sugar alcohol that fulfills several important physiological functions in certain species of the genus Primula. Using the horticultural hybrid polyanthus (Primula × polyantha) as our model plant, we found that volemitol is the major nonstructural carbohydrate in leaves of all stages of development, with concentrations of up to 50 mg/g fresh weight in source leaves (about 25% of the dry weight), followed by sedoheptulose (d-altro-2-heptulose, 36 mg/g fresh weight), and sucrose (4 mg/g fresh weight). Volemitol was shown by the ethylenediaminetetraacetate-exudation technique to be a prominent phloem-mobile carbohydrate. It accounted for about 24% (mol/mol) of the phloem sap carbohydrates, surpassed only by sucrose (63%). Preliminary ¹⁴CO₂ pulse-chase radiolabeling experiments showed that volemitol was a major photosynthetic product, preceded by the structurally related ketose sedoheptulose. Finally, we present evidence for a novel NADPH-dependent ketose reductase, tentatively called sedoheptulose reductase, in volemitol-containing Primula species, and propose it as responsible for the biosynthesis of volemitol in planta. Using enzyme extracts from polyanthus leaves, we determined that sedoheptulose reductase has a pH optimum between 7.0 and 8.0, a very high substrate specificity, and displays saturable concentration dependence for both sedoheptulose (apparent K_m = 21 mm) and NADPH (apparent K_m = 0.4 mm). Our results suggest that volemitol is important in certain Primula species as a photosynthetic product, phloem translocate, and storage carbohydrate.Alditols (sugar alcohols or acyclic polyols) may be chemically described as reduction products of aldose or ketose sugars. The most prevalent plant alditols are the hexitols sorbitol, mannitol, and galactitol. However, as many as 17 different alditols occur naturally in higher plants (for review, see Bieleski, 1982; Lewis, 1984; Loescher and Everard, 1996). The lesser-known alditols are often restricted in their occurrence but still fulfill important functions in those plants where they do occur. Volemitol (Fig. ​(Fig.1) 1) is a good example of a less common but important alditol. This seven-carbon sugar alcohol seems to be confined to certain sections of the genus Primula, so much so that it has been suggested as a useful chemotaxonomical marker (Kremer, 1978). Very little is known about the physiology and metabolism of volemitol in primulas, except that it was an early photosynthetic product in cowslip (Primula veris) and oxslip (Primula elatior) (Kremer, 1978). Figure 1Fischer projections of volemitol and its four structurally related seven-carbon sugars. Nomenclature follows that of Collins (1987); trivial names are underlined.The physiological roles of alditols are manifold and largely resemble those of disaccharides and oligosaccharides. They include photosynthetic assimilation, translocation and storage of carbon, and reducing power, as well as protection against different types of stresses (for review, see Bieleski, 1982; Lewis, 1984; Loescher and Everard, 1996; Stoop et al., 1996). The biosynthetic pathways of the hexitols sorbitol (glucitol), mannitol, galactitol (dulcitol), and the pentitol ribitol have been established in higher plants. They generally use NADPH as a hydrogen donor and aldose phosphate as a hydrogen acceptor, in concert with the corresponding phosphatases. One exception might be galactitol, which was suggested to be formed directly from unphosphorylated Gal (and NADPH) (Negm, 1986). Although all foliar alditols are thought to be phloem-mobile (Lewis, 1984), this has only been demonstrated for sorbitol, mannitol, and galactitol (Zimmermann and Ziegler, 1975; Davis and Loescher, 1990; Moing et al., 1992; Flora and Madore, 1993).To expand our knowledge of alditol metabolism in higher plants beyond that of hexitols, we studied the carbohydrate metabolism of polyanthus (Primula × polyantha). This popular horticultural hybrid of primrose (Primula vulgaris), oxlip, and cowslip (Mabberley, 1997) was chosen because preliminary experiments showed that its volemitol content is very high, similar to that of the wild-type species, and because it may be easily grown both outdoors and indoors.We give a general overview on volemitol metabolism in polyanthus with special emphasis on the role of volemitol in plant development and phloem transport. We also report on a novel enzyme, a NADPH-dependent ketose reductase, which forms volemitol by the reduction of sedoheptulose.