Bacteria and protozoa differentially modulate the expression of rab proteins

Phagocytic cells represent an important line of innate defense against microorganisms. Uptake of microorganisms by these cells involves the formation of a phagosome that matures by fusing with endocytic compartments, resulting in killing of the enclosed microbe. Small GTPases of the Rab family are key regulators of vesicular trafficking in the endocytic pathway. Intracellular pathogens can interfere with the function of these proteins in order to subvert host immune responses. However, it is unknown if this subversion can be achieved through the modulation of Rab gene expression. We compared the expression level of 23 distinct Rab GTPases in mouse macrophages after infection with the protozoan Plasmodium berghei, and the bacteria Escherichia coli and Salmonella enterica. We found that P. berghei induces an increase in the expression of a different set of Rab genes than E. coli and S. enterica, which behaved similarly. Strikingly, when one of the Rab proteins whose expression was increased by P. berghei, namely Rab14, was silenced, we observed a significant increase in the phagocytosis of P. berghei, whereas Rab14 overexpression led to a decrease in phagocytosis. This suggests that the parasite might induce the increase of Rab14 expression for its own advantage. Similarly, when Rab9a, whose expression was increased by E. coli and S. enterica, was silenced, we observed an increase in the phagocytosis of both bacterial species, whereas Rab9a overexpression caused a reduction in phagocytosis. This further suggests that the modulation of Rab gene expression could represent a mechanism of immune evasion. Thus, our study analyzes the modulation of Rab gene expression induced by bacteria and protozoa and suggests that this modulation could be necessary for the success of microbial infection.     

Development of protein biomarkers in cerebrospinal fluid for secondary progressive multiple sclerosis using selected reaction monitoring mass spectrometry (SRM-MS)

ABSTRACT: BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). It involves damage to the myelin sheath surrounding axons and to the axons themselves. MS most often presents with a series of relapses and remissions but then evolves over a variable period of time into a slowly progressive form of neurological dysfunction termed secondary progressive MS (SPMS). The reasons for this change in clinical presentation are unclear. The absence of a diagnostic marker means that there is a lag time of several years before the diagnosis of SPMS can be established. At the same time, understanding the mechanisms that underlie SPMS is critical to the development of rational therapies for this untreatable stage of the disease. RESULTS: Using LC coupled mass spectrometry; we have established a highly specific and sensitive multiplex selected reaction monitoring (SRM) assay. Our SRM assay has facilitated the simultaneous detection of surrogate peptides originating from 28 proteins present in cerebrospinal fluid (CSF). Protein levels in CSF are generally ~200-fold lower than that in human sera. A limit of detection (LOD) was determined to be as low as one femtomole per uL. We processed and analysed CSF samples from a total of 22 patients with SPMS, 12 patients with non-inflammatory neurological disorders (NIND) and 10 age-matched healthy controls in parallel for the levels of 28 selected potential protein biomarkers, followed by principal component analysis (PCA) for clustering protein biomarkers. Our SRM data suggested different levels of agrin, kallikrein and putative myosin-XVB in SPMS patients as compared to healthy controls. PCA reveals that these proteins are correlated, can be grouped into four principal components. Overall, we established an efficient platform to verify protein biomarkers in CSF, which can be easily adapted to other proteins of interest related to neurodegenerative diseases. CONCLUSIONS: A highly specific and sensitive multiplex SRM-MS assay was established for verifying CSF protein biomarkers in SPMS. Three proteins were found to be expressed significantly differently in SPMS patients as compared to health controls, which will help further our current understanding of SPMS disease pathology and/or therapeutic intervention.     

Molecular characterization of Corynebacterium pseudotuberculosis isolated from goats using ERIC-PCR

Corynebacterium pseudotuberculosis, the infectious agent of caseous lymphadenitis (CLA), is responsible for substantial economic losses in goat and sheep production. Molecular characterization of C. pseudotuberculosis isolates by enterobacterial repetitive intergenic consensus (ERIC)-PCR has shown promising results in genotyping strains isolated from sheep with CLA. We evaluated the genetic diversity of C. pseudotuberculosis isolates collected from the Sert?o region of the Pernambuco (PE) State, Brazil, and investigated the potential of ERIC-PCR as a tool for the molecular typing of strains of C. pseudotuberculosis isolated from goats. Thirty-two C. pseudotuberculosis strains isolated from goats in the municipalities of Floresta and Ibimirim, PE, C. pseudotuberculosis type strain ATCC 19410, the 1002 vaccine strain, and a field isolate of Rhodococcus equi were fingerprinted using the primers ERIC-1R and ERIC-2 and the primer pair ERIC- 1R+ERIC-2. Using 100% similarity as the cutoff, 8, 10, and 7 genotypes were obtained with ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively. The Hunter-Gaston discriminatory index calculated for the ERIC-1-PCR was 0.75. The index for the ERIC-2-PCR was 0.88, and the index for the ERIC-1+2-PCR was 0.79. Among goat isolates of C. pseudotuberculosis, three, two and four genotypes (found by ERIC-1-PCR, ERIC-2-PCR, and ERIC-1+2-PCR, respectively) had been previously described among sheep isolates from Minas Gerais State, Brazil. These results showed that ERIC-PCR has good discriminatory power and typeability, making it a useful tool for discrimination among C. pseudotuberculosis isolates from goats.     

Human brain aminopeptidase A: biochemical properties and distribution in brain nuclei

Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.     

Yarrowia lipolytica lipase production enhanced by increased air pressure

Aims: To study the cellular growth and morphology of Yarrowia lipolytica W29 and its lipase and protease production under increased air pressures. Methods and Results: Batch cultures of the yeast were conducted in a pressurized bioreactor at 4 and 8 bar of air pressure and the cellular behaviour was compared with cultures at atmospheric pressure. No inhibition of cellular growth was observed by the increase of pressure. Moreover, the improvement of the oxygen transfer rate (OTR) from the gas to the culture medium by pressurization enhanced the extracellular lipase activity from 96·6 U l^?1 at 1 bar to 533·5 U l^?1 at 8 bar. The extracellular protease activity was reduced by the air pressure increase, thereby eliciting further lipase productivity. Cell morphology was slightly affected by pressure, particularly at 8 bar, where cells kept the predominant oval form but decreased in size. Conclusions: OTR improvement by total air pressure rise up to 8 bar in a bioreactor can be applied to the enhancement of lipase production by Y. lipolytica. Significance and Impact of the Study: Hyperbaric bioreactors can be successfully applied for yeast cells cultivation, particularly in high‐density cultures used for enzymes production, preventing oxygen limitation and consequently increasing overall productivity.     

Bacterial and fungal communities in bulk soil and rhizospheres of aluminum-tolerant and aluminum-sensitive maize (Zea mays L.) lines cultivated in unlimed and limed Cerrado soil

Liming of acidic soils can prevent aluminum toxicity and improve crop production. Some maize lines show aluminum (Al) tolerance, and exudation of organic acids by roots has been considered to represent an important mechanism involved in the tolerance. However, there is no information about the impact of liming on the structures of bacterial and fungal communities in Cerrado soil, nor if there are differences between the microbial communities from the rhizospheres of Al-tolerant and Al-sensitive maize lines. This study evaluated the effects of liming on the structure of bacterial and fungal communities in bulk soil and rhizospheres of Al-sensitive and Al-tolerant maize (Zea mays L.) lines cultivated in Cerrado soil by PCR-DGGE, 30 and 90 days after sowing. Bacterial fingerprints revealed that the bacterial communities from rhizospheres were more affected by aluminum stress in soil than by the maize line (Al-sensitive or Al-tolerant). Differences in bacterial communities were also observed over time (30 and 90 days after sowing), and these occurred mainly in the Actinobacteria. Conversely, fungal communities from the rhizosphere were weakly affected either by liming or by the rhizosphere, as observed from the DGGE profiles. Furthermore, only a few differences were observed in the DGGE profiles of the fungal populations during plant development when compared with bacterial communities. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Cerrado bulk soil revealed that Actinomycetales and Rhizobiales were among the dominant ribotypes.     

Malaria liver stage susceptibility locus identified on mouse chromosome 17 by congenic mapping

Host genetic variants are known to confer resistance to Plasmodium blood stage infection and to control malaria severity both in humans and mice. This work describes the genetic mapping of a locus for resistance to liver stage parasite in the mouse. First, we show that decreased susceptibility to the liver stage of Plasmodium berghei in the BALB/c mouse strain is attributable to intra-hepatic factors and impacts on the initial phase of blood stage infection. We used QTL mapping techniques to identify a locus controlling this susceptibility phenotype (LOD score 4.2) on mouse chromosome 17 (belr1 locus). Furthermore, analysis of congenic mouse strains delimited the belr1 locus boundaries distally to the H2 region. Quantification of parasites in the liver of infected congenic mice strongly suggested that the belr1 locus represents a genetic factor controlling the expansion of P. berghei in the hepatic tissue. The mapping of belr1 locus raises the hypothesis that host gene variation is able to control the progression of Plasmodium liver stage infection and opens the possibility that the human genomic region orthologue to belr1 may contain genes that confer resistance to the human malaria liver stage.     

A variational constitutive model for soft biological tissues

In this paper, a fully variational constitutive model of soft biological tissues is formulated in the finite strain regime. The model includes Ogden-type hyperelasticity, finite viscosity, deviatoric and volumetric plasticity, rate and microinertia effects. Variational updates are obtained via time discretization and pre-minimization of a suitable objective function with respect to internal variables. Genetic algorithms are used for model parameter identification due to their suitability for non-convex, high dimensional optimization problems. The material behavior predicted by the model is compared to available tests on swine and human brain tissue. The ability of the model to predict a wide range of experimentally observed behavior, including hysteresis, cyclic softening, rate effects, and plastic deformation is demonstrated.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

Song Frequency Does Not Reflect Differences in Body Size among Males in Two Oscine Species

Large animals, having large vocal organs, produce low sound frequencies more efficiently. Accordingly, the frequency of vocalizations is often negatively related to body size across species, and also among individuals of many species, including several non‐oscine birds (non‐songbirds). Little is known about whether song frequency reveals information about body size within oscine species, which are characterized by song learning and large repertoires. We asked whether song frequency is related to body size in two oscines that differ in repertoire size: the dark‐eyed junco (Junco hyemalis) and the serin (Serinus serinus). We also asked whether the extent to which receivers sample repertoires might influence the reliability of their assessment of body size. We found that none of the frequency traits of song that we investigated was related to male body size, nor did more extensive sampling of repertoires lead to any relationship between frequency and body size. Possible reasons for these results are the small range of variation in size within species, or the elaborate vocal physiology of oscines that gives them great control over a wide frequency range. We discuss these results as they relate to female preferences for high‐frequency song that have been previously reported for oscine species.