bfb,a Novel ENU-Induced blebs Mutant Resulting from a Missense Mutation in Fras1

Fras1 is an extracellular matrix associated protein with essential roles in adhesion of epithelia and mesenchyme during early embryonic development. The adhesive function of Fras1 is achieved through interaction with a group of related proteins, Frem 1–3, and a cytoplasmic adaptor protein Grip1. Mutation of each of these proteins results in characteristic epithelial blistering and have therefore become known as “blebs” proteins. Human Fraser syndrome presents with a similar phenotype and the blebs mice have been instrumental in identification of the genetic basis of Fraser syndrome. We have identified a new ENU-induced blebs allele resulting from a novel missense mutation in Fras1. The resulting mouse strain, blood filled blisters (bfb), presents with a classic blebs phenotype but does not exhibit embryonic lethality typical of other blebs mutants and in addition, we report novel palate and sternal defects. Analysis of the bfb phenotype confirms the presence of epithelial-mesenchymal adhesion defects but also supports the emerging role of blebs proteins in regulating signalling during organogenesis. The bfb strain provides new opportunities to investigate the role of Fras1 in development.     

Candidatus Bartonella merieuxii,a Potential New Zoonotic Bartonella Species in Canids from Iraq

Bartonellae are emerging vector-borne pathogens infecting erythrocytes and endothelial cells of various domestic and wild mammals. Blood samples were collected from domestic and wild canids in Iraq under the United States Army zoonotic disease surveillance program. Serology was performed using an indirect immunofluorescent antibody test for B. henselae, B. clarridgeiae, B. vinsonii subsp. berkhoffii and B. bovis. Overall seroprevalence was 47.4% in dogs (n = 97), 40.4% in jackals (n = 57) and 12.8% in red foxes (n = 39). Bartonella species DNA was amplified from whole blood and representative strains were sequenced. DNA of a new Bartonella species similar to but distinct from B. bovis, was amplified from 37.1% of the dogs and 12.3% of the jackals. B. vinsonii subsp. berkhoffii was also amplified from one jackal and no Bartonella DNA was amplified from foxes. Adjusting for age, the odds of dogs being Bartonella PCR positive were 11.94 times higher than for wild canids (95% CI: 4.55–31.35), suggesting their role as reservoir for this new Bartonella species. This study reports on the prevalence of Bartonella species in domestic and wild canids of Iraq and provides the first detection of Bartonella in jackals. We propose Candidatus Bartonella merieuxii for this new Bartonella species. Most of the Bartonella species identified in sick dogs are also pathogenic for humans. Therefore, seroprevalence in Iraqi dog owners and bacteremia in Iraqi people with unexplained fever or culture negative endocarditis requires further investigation as well as in United States military personnel who were stationed in Iraq. Finally, it will also be essential to test any dog brought back from Iraq to the USA for presence of Bartonella bacteremia to prevent any accidental introduction of a new Bartonella species to the New World.     

SERPINA2 Is a Novel Gene with a Divergent Function from SERPINA1

Serine protease inhibitors (SERPINs) are a superfamily of highly conserved proteins that play a key role in controlling the activity of proteases in diverse biological processes. The SERPIN cluster located at the 14q32.1 region includes the gene coding for SERPINA1, and a highly homologous sequence, SERPINA2, which was originally thought to be a pseudogene. We have previously shown that SERPINA2 is expressed in different tissues, namely leukocytes and testes, suggesting that it is a functional SERPIN. To investigate the function of SERPINA2, we used HeLa cells stably transduced with the different variants of SERPINA2 and SERPINA1 (M1, S and Z) and leukocytes as the in vivo model. We identified SERPINA2 as a 52 kDa intracellular glycoprotein, which is localized at the endoplasmic reticulum (ER), independently of the variant analyzed. SERPINA2 is not significantly regulated by proteasome, proposing that ER localization is not due to misfolding. Specific features of SERPINA2 include the absence of insoluble aggregates and the insignificant response to cell stress, suggesting that it is a non-polymerogenic protein with divergent activity of SERPINA1. Using phylogenetic analysis, we propose an origin of SERPINA2 in the crown of primates, and we unveiled the overall conservation of SERPINA2 and A1. Nonetheless, few SERPINA2 residues seem to have evolved faster, contributing to the emergence of a new advantageous function, possibly as a chymotrypsin-like SERPIN. Herein, we present evidences that SERPINA2 is an active gene, coding for an ER-resident protein, which may act as substrate or adjuvant of ER-chaperones.     

A Unique Pool of Compatible Solutes on Rhodopirellula baltica,Member of the Deep-Branching Phylum Planctomycetes

The intracellular accumulation of small organic solutes was described in the marine bacterium Rhodopirellula baltica, which belongs to the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. Sucrose, α-glutamate, trehalose and mannosylglucosylglycerate (MGG) are the main solutes involved in the osmoadaptation of R. baltica. The ratio and total intracellular organic solutes varied significantly in response to an increase in salinity, temperature and nitrogen content. R. baltica displayed an initial response to both osmotic and thermal stresses that includes α-glutamate accumulation. This trend was followed by a rather unique and complex osmoadaptation mechanism characterized by a dual response to sub-optimal and supra-optimal salinities. A reduction in the salinity to sub-optimal conditions led primarily to the accumulation of trehalose. In contrast, R. baltica responded to salt stress mostly by increasing the intracellular levels of sucrose. The switch between the accumulation of trehalose and sucrose was by far the most significant effect caused by increasing the salt levels of the medium. Additionally, MGG accumulation was found to be salt- as well as nitrogen-dependent. MGG accumulation was regulated by nitrogen levels replacing α-glutamate as a K⁺ counterion in nitrogen-poor environments. This is the first report of the accumulation of compatible solutes in the phylum Planctomycetes and of the MGG accumulation in a mesophilic organism.     

Candidatus Rickettsia andeanae,a spotted fever group agent infecting Amblyomma parvum ticks in two Brazilian biomes

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.     

Schistosoma japonicum Eggs Induce a Proinflammatory,Anti-Fibrogenic Phenotype in Hepatic Stellate Cells

Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of S . japonicum on HSCs are lacking. Disease caused by S . japonicum is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of S . japonicum eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast, S . japonicum eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to S . japonicum eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation.     

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.     

Proteolysin,a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus

Through genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus. The gene was functionally expressed in Escherichia coli, and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required.     

The Role of Courtship Behavior and Size in Mate Preference in the Sex‐Role‐Reversed Gulf Pipefish,Syngnathus scovelli

In sex‐role‐reversed species, sexual selection acts more strongly on females than on males, a situation that can result in the evolution of secondary sexual traits in females and strong mating preferences in males. While some research exploring mating preferences in sex‐role‐reversed species has been conducted, overall, this topic remains relatively unexplored. The Gulf pipefish, Syngnathus scovelli, is a highly polyandrous pipefish species. Sexual selection is significantly stronger in females than in males, which has led to the evolution of both morphological and behavioral female secondary sexual traits. However, because males gestate the offspring in specialized pouches and make a substantial investment in embryos during development, females may also benefit from being choosy. The goal of this study was to examine both male and female mating preferences in this species. We found that male mating preference was significantly associated with female courtship behavior. Larger females were also able to maintain these behaviors for longer intervals than smaller females. No evidence of female mating preference in regard to male size was observed but the data suggest that male behaviors may be providing positive reinforcement to courting females. This research provides further insight into how mate preferences vary among sex‐role‐reversed species.     

Functional Consequences of a Novel Variant of PCSK1

Background

Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity.

Methodology/Principal Findings

A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3.

Conclusion/Significance

Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.     

Effect of Starvation on the Endocytic Pathway in Dictyostelium Cells

Dictyostelium discoideum amoebae have been used extensively to study the structure and dynamics of the endocytic pathway. Here, we show that while the general structure of the endocytic pathway is maintained in starved cells, its dynamics rapidly slow down. In addition, analysis of apm3 and lvsB mutants reveals that the functional organization of the endocytic pathway is profoundly modified upon starvation. Indeed, in these mutant cells, some of the defects observed in rich medium persist in starved cells, notably an abnormally slow transfer of endocytosed material between endocytic compartments. Other parameters, such as endocytosis of the fluid phase or the rate of fusion of postlysosomes to the cell surface, vary dramatically upon starvation. Studying the endocytic pathway in starved cells can provide a different perspective, allowing the primary (invariant) defects resulting from specific mutations to be distinguished from their secondary (conditional) consequences.Dictyostelium discoideum is a widely used model organism for studying the organization and function of the endocytic pathway. In Dictyostelium, the organization of the endocytic pathway is similar to that in higher eukaryotes. The pathway in Dictyostelium can be divided into four steps (see Fig. S1 in the supplemental material): uptake at the plasma membrane of particles and medium, transfer through early acidic endocytic compartments (lysosomes), passage into less acidic postlysosomes (PLs), and finally, exocytosis of undigested materials (17, 20). Thus, Dictyostelium recapitulates many of the functions of the endocytic pathway in mammalian cells, including some features observed in most cell types (lysosome biogenesis) and some observed only in specialized cells (phagocytosis, macropinocytosis, and lysosome secretion).Dictyostelium amoebae live in the soil, where they feed by ingesting and digesting other microorganisms. In addition, axenic laboratory strains can macropinocytose medium to ensure their growth. Accordingly, both in natural situations and in laboratory settings, the endocytic pathway plays a key role in the acquisition of nutrients by Dictyostelium cells. In agreement with this notion, several observations suggest that the physiology of the endocytic pathway is sensitive to nutrient availability. In particular, starvation induces secretion of lysosomal enzymes by an unknown mechanism (11). The morphology of the endocytic pathway is also sensitive to nutritional cues, as shown for example by the observation that formation of multilamellar endosomes is enhanced in cells fed with bacteria (18).Here, we analyzed the effect of starvation on the organization as well as the dynamics of the endocytic pathway. We found that, while the overall organization was not extensively modified in starved cells, the dynamics of endocytic compartments were altered. Moreover, analysis of two specific knockout mutants, the apm3 (6) and lvsB (8) strains, revealed that their phenotype was profoundly altered upon starvation, providing further insight about the role of Apm3 and LvsB in the endocytic pathway.     

Transformation Frequency of a mariner-Based Transposon in Rickettsia rickettsii

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.     

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background

Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin.

Methodology/Principal Findings

We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin.

Conclusions/Significance

PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.     

Kretzoiarctos gen. nov., the Oldest Member of the Giant Panda Clade

The phylogenetic position of the giant panda, Ailuropoda melanoleuca (Carnivora: Ursidae: Ailuropodinae), has been one of the most hotly debated topics by mammalian biologists and paleontologists during the last century. Based on molecular data, it is currently recognized as a true ursid, sister-taxon of the remaining extant bears, from which it would have diverged by the Early Miocene. However, from a paleobiogeographic and chronological perspective, the origin of the giant panda lineage has remained elusive due to the scarcity of the available Miocene fossil record. Until recently, the genus Ailurarctos from the Late Miocene of China (ca. 8–7 mya) was recognized as the oldest undoubted member of the Ailuropodinae, suggesting that the panda lineage might have originated from an Ursavus ancestor. The role of the purported ailuropodine Agriarctos, from the Miocene of Europe, in the origins of this clade has been generally dismissed due to the paucity of the available material. Here, we describe a new ailuropodine genus, Kretzoiarctos gen. nov., based on remains from two Middle Miocene (ca. 12–11 Ma) Spanish localities. A cladistic analysis of fossil and extant members of the Ursoidea confirms the inclusion of the new genus into the Ailuropodinae. Moreover, Kretzoiarctos precedes in time the previously-known, Late Miocene members of the giant panda clade from Eurasia (Agriarctos and Ailurarctos). The former can be therefore considered the oldest recorded member of the giant panda lineage, which has significant implications for understanding the origins of this clade from a paleobiogeographic viewpoint.     

In Silico Analysis of the Metabolic Potential and Niche Specialization of Candidate Phylum "Latescibacteria" (WS3)

The “Latescibacteria” (formerly WS3), member of the Fibrobacteres–Chlorobi–Bacteroidetes (FCB) superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs) representing the “Latescibacteria” were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada), and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece). Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta) and brown (Phaeophycaea) algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins), storage molecules (starch and trehalose), and extracellular polymeric substances (EPSs). The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC) to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed “Latescibacteria” mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.