Proteome changes in platelets activated by arachidonic acid,collagen, and thrombin

Background  

Platelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS.     

Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease

Introduction

Fibromyalgia is a chronic pain syndrome with unknown etiology. Recent studies have shown some evidence demonstrating that oxidative stress may have a role in the pathophysiology of fibromyalgia. However, it is still not clear whether oxidative stress is the cause or the effect of the abnormalities documented in fibromyalgia. Furthermore, the role of mitochondria in the redox imbalance reported in fibromyalgia also is controversial. We undertook this study to investigate the role of mitochondrial dysfunction, oxidative stress, and mitophagy in fibromyalgia.

Methods

We studied 20 patients (2 male, 18 female patients) from the database of the Sevillian Fibromyalgia Association and 10 healthy controls. We evaluated mitochondrial function in blood mononuclear cells from fibromyalgia patients measuring, coenzyme Q₁₀ levels with high-performance liquid chromatography (HPLC), and mitochondrial membrane potential with flow cytometry. Oxidative stress was determined by measuring mitochondrial superoxide production with MitoSOX™ and lipid peroxidation in blood mononuclear cells and plasma from fibromyalgia patients. Autophagy activation was evaluated by quantifying the fluorescence intensity of LysoTracker™ Red staining of blood mononuclear cells. Mitophagy was confirmed by measuring citrate synthase activity and electron microscopy examination of blood mononuclear cells.

Results

We found reduced levels of coenzyme Q₁₀, decreased mitochondrial membrane potential, increased levels of mitochondrial superoxide in blood mononuclear cells, and increased levels of lipid peroxidation in both blood mononuclear cells and plasma from fibromyalgia patients. Mitochondrial dysfunction was also associated with increased expression of autophagic genes and the elimination of dysfunctional mitochondria with mitophagy.

Conclusions

These findings may support the role of oxidative stress and mitophagy in the pathophysiology of fibromyalgia.     

Young-of-the-year fish assemblages as an alternative to adult fish monitoring for ecological quality evaluation of running waters

Studies on the Upper Mississippi River, particularly over the last 15 years, have contributed to our understanding of trophic processes in large rivers. The framework established by earlier population-specific studies, however, cannot be overlooked. Examination of the feeding habits of fish ranging from planktivores to piscivores gave the first indication that trophic processes were influenced by the spatial complexity and annual hydrological patterns of river-floodplain ecosystems. Experimental studies, which have often been considered impossible or impractical in large rivers, demonstrated the potential for biotic controls of system dynamics through predator–prey and competitive interactions. Such studies have been particularly helpful in understanding the potential impact of non-native species, including zebra mussels and Asian carp, to biodiversity and secondary production. Our understanding of riverine ecosystem function expanded greatly as food web studies began the application of a new tool—natural stable isotopes. Studies employing stable isotopes illustrated how food webs in a number of large rivers throughout the world are supported by the autochthonous production of microalgae. This study, coupled with other studies testing the prevailing models of riverine ecosystem function, has brought us to a point of better understanding the nature of river ecosystem functions. It is through looking back at the earlier studies of fish diet that we should realize that the temporal and spatial complexities of river ecosystem function must still be addressed more fully. This and a better grasp of the significance of the arrangement of patches within the riverine landscape will prove beneficial, as we assess the appropriate scale of river rehabilitation with an eye on how rehabilitation promotes productivity within complex ecosystems, including the Upper Mississippi River.     

Cochaperone Interactions in Export of the Type III Needle Component PscF of Pseudomonas aeruginosa

Type III secretion (T3S) systems allow the export and translocation of bacterial effectors into the host cell cytoplasm. Secretion is accomplished by an 80-nm-long needle-like structure composed, in Pseudomonas aeruginosa, of the polymerized form of a 7-kDa protein, PscF. Two proteins, PscG and PscE, stabilize PscF within the bacterial cell before its export and polymerization. In this work we screened the 1,320-Ų interface between the two chaperones, PscE and PscG, by site-directed mutagenesis and determined hot spot regions that are important for T3S function in vivo and complex formation in vitro. Three amino acids in PscE and five amino acids in PscG, found to be relevant for complex formation, map to the central part of the interacting surface. Stability assays on selected mutants performed both in vitro on purified PscE-PscG complexes and in vivo on P. aeruginosa revealed that PscE is a cochaperone that is essential for the stability of the main chaperone, PscG. Notably, when overexpressed from a bicistronic construct, PscG and PscF compensate for the absence of PscE in cytotoxic P. aeruginosa. These results show that all of the information needed for needle protein stabilization and folding, its presentation to the T3 secreton, and its export is present within the sequence of the PscG chaperone.Many Gram-negative bacteria are endowed with a specialized secretion machinery called the type III secretion (T3S) system (T3SS) that allows a set of bacterial proteins (effectors) to be injected directly into a eukaryotic cell cytoplasm. The effectors carry versatile enzymatic activities and target the main host defense functions, such as phagocytosis (14, 19). The T3S nanomachinery is composed of three main subassemblies: the basal body, the needle, and the translocon (5, 15). The basal body, which is in composition and structure similar to a flagellum base, is embedded within two bacterial membranes and is composed of several protein rings made up of identical subunits with 12-fold symmetry (20, 33). Protruding from the surface and in continuum with the base, the needle is formed by a low-molecular-weight protein that polymerizes into a 50- to 80-nm-long and 8-nm-wide structure whose length is highly regulated (22, 24, 27). It is widely accepted that the secretion of effectors takes place through this 2-nm-wide needle channel and is continued through a three-protein pore complex called the translocon. In related T3S systems of pathogens Pseudomonas aeruginosa and Yersinia spp., the translocon is composed of one hydrophilic (PcrV and LcrV in Pseudomonas and Yersinia, respectively) and two hydrophobic (PopB/PopD and YopB/YopD, respectively) proteins, which allow crossing of the host plasma membrane (16, 18, 25).A highlight of the T3S systems is a class of intrabacterial helper proteins, called chaperones, which are proposed to participate in several steps of substrate stabilization and export. The sequence identity between chaperones is notably low, but they possess common features such as small size (100 to 150 residues) and a tendency toward an acidic pI (26). T3S chaperones have been classified into three categories according to their partners and their modes of interaction. Class I chaperones act as dimers and bind one (class IA) or several (class IB) effectors. Crystal structures of several class IA and IB molecules show that they share a similar 5β/3α fold, the central α helix being responsible for dimerization (3, 34). They act mainly as “bodyguards” preventing their substrates from generating premature or nonspecific interactions with other proteins but are also thought to play a role in secretion. The class II chaperones bind to hydrophobic translocators and keep them in a soluble state (13, 31). SycD of Yersinia binds YopB and YopD translocators, while PcrH from Pseudomonas is responsible for recognition of PopB and PopD (4, 9, 13, 21). These chaperones display all-helical structures with three tetratricopeptide repeat (TPR) motifs, with a single TPR module being composed of two antiparallel α helices; the overall structure forms a concave substrate-binding groove (4, 21, 23).The third class consists of chaperones interacting with needle proteins. Until now, they have been documented only in the Ysc/Psc subclass of T3SSs (29, 35, 36). We have previously demonstrated that in P. aeruginosa, an opportunistic pathogen, the type III needle component PscF is maintained in its monomeric form within the bacterial cytoplasm by a bimolecular chaperone, PscE-PscG (29, 30). The 2-Å crystal structure of the ternary complex revealed that PscE is a 67-amino-acid protein which folds into three α helices (Ha, Hb, and Hc) and interacts directly only with PscG. PscG is composed of seven α helices (H1 to H7) organized into a TPR-like domain harboring a concave region which binds to the C-terminal helix of PscF (30). The interacting surface between PscG and PscF is essential for needle formation and bacterial cytotoxicity (30).In this work, we investigate the role of two chaperones in needle protein stabilization and T3S function. We define interaction hot spots of the PscE-PscG surface by site-directed mutagenesis and then show that PscE is required for stabilization of PscG both in vivo and in vitro. Moreover, we show that when PscG is overproduced in concert with PscF in P. aeruginosa, the absence of PscE does not affect T3S functionality. These data demonstrate that PscG is the main needle chaperone, being sufficient to maintain PscF in a secretion-prone fold, and that PscE is a cochaperone needed to ensure stability of PscG.     

Bifidobacteria in the digestive tract of bumblebees

Bifidobacteria and other bacterial groups (lactobacilli, facultative anaerobes, anaerobes) from the digestive tract of three bumblebee species (Bombus lucorum (34 samples), Bombus pascuorum (18 samples) and Bombus lapidarius (9 samples)) were enumerated and characterised. Counts of facultative anaerobic bacteria and lactobacilli (5.41 ± 2.92 and 2.69 ± 3.02 log CFU/g of digestive tract content) were lower than those of anaerobes (7.66 ± 0.86 log CFU/g). Counts of bifidobacteria were determined using two selective media: MTPY (Modified Trypticase Phytone Yeast extract agar) and a new medium with pollen extract. There was no significant difference between the counts of bifidobacteria from both media, 5.00 ± 2.92 log CFU/g on MTPY and 5.00 ± 2.87 on the pollen medium. Subsequently, 187 bacterial strains of the family Bifidobacteriaceae (fructose-6-phosphate phosphoketolase-positive) were isolated from three different localities and from all three species of bumblebees. Bifidobacteria were found in 42 out of 61 specimens (69%). Twenty-three (38%) specimens had counts of bifidobacteria higher than 7.0 log CFU/g. Bifidobacteria represented the dominant group of anaerobes (>70% of total anaerobes), i.e., the principal group of bacteria in the bumblebee digestive tract, in only fourteen specimens (23% of total). For the first time, bifidobacteria were isolated from the digestive tract of bumblebees. In addition, we suggest, on the basis of biochemical tests (API 50 CHL and RAPID ID 32) and genetic methods (PCR and DGGE), that these bacteria may represent new species within the family of Bifidobacteriaceae.     

Interactions of the Hemiparasitic Species Rhinanthus minor with its Host Plant Community at Two Nutrient Levels

For root hemiparasites, host plants are both the source of water and nutrients below-ground, but competitors for light above-ground. Hemiparasites can reduce host biomass, and in this way considerably affect the whole plant community. To investigate these effects, we carried out two experiments in an oligotrophic meadow with a native population of Rhinanthus minor. In the first experiment, removal of R. minor was combined with fertilization in a factorial design, and in the second one, we manipulated R. minor density by thinning. The presence of R. minor decreased the biomass of its host community, mostly by suppressing grasses. In this way, the species was able to counterbalance the effect of fertilization, which increased community biomass and in particular that of grasses. Neither the presence of R. minor nor fertilization affected the total number of species or the Shannon-Wiener diversity index (H’) of the host community. However, H’ of grasses was higher and H’ of forbs (non-leguminous dicots) was lower in the presence of R. minor. Reduction of grasses by R. minor favored mainly the dominant forb Plantago lanceolata, which partly acquired the role of a competitive dominant. Effects of R. minor on community diversity seem to be highly dependent on the relative sensitivities of dominant and subordinate species. Fertilization increased the mortality of seedlings, resulting in a lower number of flowering plants. However, surviving individuals on average produced more flowers. Thinning resulted in lower mortality of R. minor plants. This indicates that intraspecific competition in R. minor populations results in negative density dependence.     

Determination of doxazosin and verapamil in human serum by fast LC-MS/MS: application to document non-compliance of patients

A rapid and sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) for simultaneous determination of doxazosin and verapamil in human serum has been developed. Trimipramine-d₃ as an isotopic labelled internal standard was used for quantification. Serum samples were prepared by simple liquid–liquid extraction with mixture of tert butyl methyl ether and ethyl acetate (1:1, v:v). The analytes and internal standard were separated on C18 column using an isocratic elution with 5 mM ammonium formate with 0.02% formic acid and 0.02% formic acid in acetonitrile (55:45, v:v) at a flow rate of 1.1 mL/min. Positive TurboIonSpray mass spectrometry was used with multiple reaction monitoring of the transitions at: m/z 455.3 → 165.2 and 150.2 for verapamil, m/z 452.2 → 344.4 and 247.4 for doxazosin, m/z 298.2 → 103.1 for trimipramine-d₃. Linearity was achieved between 1 and 500 ng/mL (R² ≥ 0.997) for both analytes. An extensive pre-study method validation was carried out in accordance with FDA guidelines. This assay was successfully applied to determine the serum concentrations of doxazosin and verapamil in suspect non-compliance patients.     

The phylogenetic position of <Emphasis Type="Italic">Obolarina dryophila</Emphasis> (Xylariales)

The inconspicuous inner-bark parasite Obolarina dryophila is reported from wood of Quercus petraea and as an endophyte of Salix alba. In addition, viable ascospores of O. dryophila have been found in the gut of the oak bark weevil Gasterocercus depressirostris, suggesting a possible dissemination mechanism for the fungus. A phylogenetic analysis based on three genes (nrDNA, actin, β-tubulin) placed Obolarina inside the genus Biscogniauxia as a close relative of the oak pathogens B. atropunctata and B. mediterranea.