Proteomic Differences between Tellurite-Sensitive and Tellurite–Resistant E.coli

Tellurite containing compounds are in use for industrial processes and increasing delivery into the environment generates specific pollution that may well result in contamination and subsequent potential adverse effects on public health. It was the aim of the current study to reveal mechanism of toxicity in tellurite-sensitive and tellurite-resistant E. coli at the protein level.In this work an approach using gel-based mass spectrometrical analysis to identify a differential protein profile related to tellurite toxicity was used and the mechanism of ter operon-mediated tellurite resistance was addressed. E. coli BL21 was genetically manipulated for tellurite-resistance by the introduction of the resistance-conferring ter genes on the pLK18 plasmid. Potassium tellurite was added to cultures in order to obtain a final 3.9 micromolar concentration. Proteins from tellurite-sensitive and tellurite-resistant E. coli were run on 2-D gel electrophoresis, spots of interest were picked, in-gel digested and subsequently analysed by nano-LC-MS/MS (ion trap). In addition, Western blotting and measurement of enzymatic activity were performed to verify the expression of certain candidate proteins.Following exposure to tellurite, in contrast to tellurite-resistant bacteria, sensitive cells exhibited increased levels of antioxidant enzymes superoxide dismutases, catalase and oxidoreductase YqhD. Cysteine desulfurase, known to be related to tellurite toxicity as well as proteins involved in protein folding: GroEL, DnaK and EF-Tu were upregulated in sensitive cells. In resistant bacteria, several isoforms of four essential Ter proteins were observed and following tellurite treatment the abovementioned protein levels did not show any significant proteome changes as compared to the sensitive control.The absence of general defense mechanisms against tellurite toxicity in resistant bacteria thus provides further evidence that the four proteins of the ter operon function by a specific mode of action in the mechanism of tellurite resistance probably involving protein cascades from antioxidant and protein folding pathways.     

Heterotrophic Compensation: A Possible Mechanism for Resilience of Coral Reefs to Global Warming or a Sign of Prolonged Stress?

Thermally induced bleaching has caused a global decline in corals and the frequency of such bleaching events will increase. Thermal bleaching severely disrupts the trophic behaviour of the coral holobiont, reducing the photosynthetically derived energy available to the coral host. In the short term this reduction in energy transfer from endosymbiotic algae results in an energy deficit for the coral host. If the bleaching event is short-lived then the coral may survive this energy deficit by depleting its lipid reserves, or by increasing heterotrophic energy acquisition. We show for the first time that the coral animal is capable of increasing the amount of heterotrophic carbon incorporated into its tissues for almost a year following bleaching. This prolonged heterotrophic compensation could be a sign of resilience or prolonged stress. If the heterotrophic compensation is in fact an acclimatization response, then this physiological response could act as a buffer from future bleaching by providing sufficient heterotrophic energy to compensate for photoautotrophic energy losses during bleaching, and potentially minimizing the effect of subsequent elevated temperature stresses. However, if the elevated incorporation of zooplankton is a sign that the effects of bleaching continue to be stressful on the holobiont, even after 11 months of recovery, then this physiological response would indicate that complete coral recovery requires more than 11 months to achieve. If coral bleaching becomes an annual global phenomenon by mid-century, then present temporal refugia will not be sufficient to allow coral colonies to recover between bleaching events and coral reefs will become increasingly less resilient to future climate change. If, however, increasing their sequestration of zooplankton-derived nutrition into their tissues over prolonged periods of time is a compensating mechanism, the impacts of annual bleaching may be reduced. Thus, some coral species may be better equipped to face repeated bleaching stress than previously thought.     

Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.     

IL-10 Inhibits the NF-κB and ERK/MAPK-Mediated Production of Pro-Inflammatory Mediators by Up-Regulation of SOCS-3 in Trypanosoma cruzi-Infected Cardiomyocytes

Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease.     

SERS Properties of Different Sized and Shaped Gold Nanoparticles Biosynthesized under Different Environmental Conditions by Neurospora crassa Extract

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these ‘green’ synthesized gold nanoparticles might have potential applicability in the field of biological sensing.     

2-Bromopalmitate Reduces Protein Deacylation by Inhibition of Acyl-Protein Thioesterase Enzymatic Activities

S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT), while deacylation requires acyl-protein thioesterases (APT), with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP) is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.     

Environmentally Enriched Male Mink Gain More Copulations than Stereotypic,Barren-Reared Competitors

Wild carnivores in zoos, conservation breeding centres, and farms commonly live in relatively small, unstimulating enclosures. Under these captive conditions, in a range of species including giant pandas, black-footed ferrets, and European mink, male reproductive abilities are often poor. Such problems have long been hypothesized to be caused by these animals'' housing conditions. We show for the first time that rearing under welfare-improving (i.e., highly valued and stress-reducing) environmental enrichments enhances male carnivores'' copulatory performance: in mate choice competitions, enriched male American mink (Neovison vison) mated more often than non-enriched males. We screened for several potential mediators of this effect. First was physiological stress and its impact on reproductive physiology; second, stress-mediated changes in morphology and variables related to immunocompetence that could influence male attractiveness; and third, behavioural changes likely to affect social competence, particularly autistic-like excessive routine and repetition (‘perseveration’) as is reflected in the stereotypies common in captive animals. Consistent with physiological stress, excreted steroid metabolites revealed that non-enriched males had higher cortisol levels and lower androgen levels than enriched conspecifics. Their os penises (bacula) also tended to be less developed. Consistent with reduced attractiveness, non-enriched males were lighter, with comparatively small spleens and a trend to greater fluctuating asymmetry. Consistent with impaired social competence, non-enriched males performed more stereotypic behaviour (e.g., pacing) in their home cages. Of all these effects, the only significant predictor of copulation number was stereotypy (a trend suggesting that low bodyweights may also be influential): highly stereotypic males gained the fewest copulations. The neurophysiological changes underlying stereotypy thus handicap males sexually. We hypothesise that such males are abnormally perseverative when interacting with females. Investigating similar problems in other taxa would be worthwhile, since many vertebrates, wild and domestic, live in conditions that cause stereotypic behaviour and/or impair neurological development.     

Venezuelan Equine Encephalitis Viruses (VEEV) in Argentina: Serological Evidence of Human Infection

Venezuelan equine encephalitis viruses (VEEV) are responsible for human diseases in the Americas, producing severe or mild illness with symptoms indistinguishable from dengue and other arboviral diseases. For this reason, many cases remain without certain diagnosis. Seroprevalence studies for VEEV subtypes IAB, ID, IF (Mosso das Pedras virus; MDPV), IV (Pixuna virus; PIXV) and VI (Rio Negro virus; RNV) were conducted in persons from Northern provinces of Argentina: Salta, Chaco and Corrientes, using plaque reduction neutralization test (PRNT). RNV was detected in all studied provinces. Chaco presented the highest prevalence of this virus (14.1%). Antibodies against VEEV IAB and -for the first time- against MDPV and PIXV were also detected in Chaco province. In Corrientes, seroprevalence against RNV was 1.3% in the pediatric population, indicating recent infections. In Salta, this was the first investigation of VEEV members, and antibodies against RNV and PIXV were detected. These results provide evidence of circulation of many VEE viruses in Northern Argentina, showing that surveillance of these infectious agents should be intensified.