Chronic Cadmium Treatment Promotes Oxidative Stress and Endothelial Damage in Isolated Rat Aorta

Cadmium is a highly toxic metal that is present in phosphate fertilizers, and the incidence of cadmium poisoning in the general population has increased, mainly due to cigarette smoking. Once absorbed, cadmium accumulates in the tissues, causing harmful effects including high blood pressure, endothelial damage and oxidative stress. Oxidative stress is known to efficiently produce oxidized low-density lipoprotein and consequently atherosclerosis, mainly in the aorta. However, the mechanisms through which endothelial damage is induced by cadmium have not been elucidated. Thus, the aim of this study was to investigate the effects of this metal in the isolated aorta and the possible role of oxidative stress. Rats received 100 mg.L⁻¹ cadmium chloride (CdCl₂) in the drinking water or distilled water alone for four weeks. The pressor effect of cadmium was followed throughout the exposure period by tail plethysmography. At the end of the fourth week, the blood cadmium content was established, and the vascular reactivity of the isolated aorta to phenylephrine, acetylcholine and sodium nitroprusside was analyzed in the context of endothelium denudation and incubation with L-NAME, apocynin, losartan, enalapril, superoxide dismutase (SOD) or catalase. We observed an increased response to phenylephrine in cadmium-treated rats. This increase was abolished by catalase and SOD incubation. Apocynin treatment reduced the phenylephrine response in both treatment groups, but its effect was greater in cadmium-treated rats, and NOX2 expression was greater in the cadmium group. These results suggested that cadmium in blood concentrations similar to those found in occupationally exposed populations is able to stimulate NOX2 expression, contributing to oxidative stress and reducing NO bioavailability, despite enhanced eNOS expression. These findings suggest that cadmium exposure promotes endothelial damage that might contribute to inflammation, vascular injury and the development of atherosclerosis.     

Oxidative stress parameters in blood, liver, and kidney of diabetic rats treated with curcumin and/or insulin

This study evaluated the effects of curcumin and/or insulin on antioxidant enzyme activity in blood, liver, and kidney, as well as on lipid peroxidation and delta aminolevulinic dehydratase (δ-ALA-D) activity, and a histopathological analysis of streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 6): control/saline (C); control/curcumin (CCur); diabetic/saline (D); diabetic/insulin (DIns); diabetic/curcumin (DCur); and diabetic/insulin/curcumin (DInsCur). After 30 days of treatment with curcumin and/or insulin, the animals were sacrificed and the liver, kidney, and serum were used for experimental determinations. Results of histopathological analysis showed that the treatment with insulin ameliorate renal and hepatic lesions from both DIns and DInsCur groups. TBARS levels were significantly increased in serum, liver, and kidney in D group and the administration of curcumin and insulin prevented this increase in DIns and DCur groups. The activities of catalase (CAT), superoxide dismutase, and δ-ALA-D presented a significant decrease in the liver and kidney D group when compared to C group (P < 0.05). The animals treated with curcumin and insulin presented an increase of CAT activity, revealing a positive interaction between both substances. The treatments with curcumin or insulin prevented oxidative stress in blood, through modulation of enzymatic antioxidant defenses. These findings contributed to the comprehension that antioxidants from medicinal plants could be used as adjuvant in the treatment of this endocrinopathy and not as single therapy.     

Golgi UDP-GlcNAc:Polypeptide O-α-N-Acetyl-d-Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of αGlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-α-N-acetyl-d-glucosaminyltransferases (ppαGlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased ppαGlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.     

Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to ≥90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.The family Poxviridae is a family of large, linear, double-stranded DNA viruses that carry out their entire life cycle within the cytoplasmic compartment of infected cells. Vaccinia virus (VACV) is a prototypical member of the genus Orthopoxvirus, which also includes the closely related cowpox virus (CPXV) (12, 52). The genomes of these viruses are approximately 200 kbp in length, with a coding capacity of approximately 200 genes. The genes involved in virus-host interactions are situated at both ends of the genome and are associated with the evasion of host immune defenses (1). These evasion mechanisms operate mainly extracellularly. For example, the secretion of soluble cytokine and chemokine receptor homologues blocks the receptor recognition by intercepting the cognate cytokine/chemokine in the extracellular environment. This mechanism facilitates viral attachment and entry into cells (1, 70). Therefore, decoy receptors for alpha interferon (IFN-α), IFN-β, IFN-γ, and tumor necrosis factor alpha play an important immunomodulatory role by affecting both the host antiviral and apoptotic responses.To counteract the host proapoptotic response, poxviruses have developed a number of antiapoptotic strategies, including the inhibition of apoptotic signals triggered by the extrinsic pathway (those mediated by death receptors such as tumor necrosis factor and Fas ligand) or the intrinsic pathway (mediated by the mitochondria and triggered upon viral infection) (1, 25, 70, 74). Many studies previously identified viral inhibitors that block specific steps of the intrinsic pathway. These include the VACV-encoded E3L, F1L, and N1L genes and the myxoma virus (MYXV)-encoded M11L gene, which block cytochrome c release (14, 20, 34, 39, 45, 75, 90), and the CPXV-encoded cytokine response modifier gene (CrmA) as well as the VACV-encoded SPI-2 gene, which inhibits both caspase-1 and caspase-8 (25, 58, 61, 74).An emerging body of evidence has also highlighted the pivotal role played by intracellular signaling pathways in Orthopoxvirus biology (18, 48, 92). We and others have shown that poxvirus manipulation of signaling pathways can be virus specific. For example, while both VACV and CPXV stimulate the MEK/extracellular signal-regulated kinase (ERK)/EGR-1 pathway during a substantial length of time of their infective cycle, the pathway is required only for VACV replication, whereas its role in CPXV biology has yet to be identified (71). MYXV, a rabbit-specific poxvirus, also activates the MEK/ERK pathway in a mouse model of poxvirus-host interactions. However, this stimulation led to the expression of IFN-β, which consequently blocked virus replication and possibly explains why MYXV has such a restricted host range (87).Another signaling molecule associated with viral replication is Akt kinase (also known as protein kinase B). The MYXV host range factor M-T5 is able to reprogram the intracellular environment, thereby increasing human tumor cell permissiveness to viral replication, which is directly associated with levels of phosphorylated Akt (88). In addition, M-T5 is functionally replaced by the host phosphatidylinositol 3-kinase (PI3K) enhancer A protein (92).The transmission of intracellular signals mediated by the serine/threonine kinase Akt to downstream molecules in response to diverse stimuli such as growth factors, insulin, and hormones is dependent upon the phosphorylation of serine 473 (S473-P) and threonine 308 (T308-P). This phosphorylation is mediated by mammalian target of rapamycin complex 2 (mTORC2) and phosphoinositide-dependent protein kinase 1 (PDK1), which act as downstream effectors of the PI3K/Akt/mTORC1 pathway (2, 66). PI3Ks are a family of enzymes (classes I to III) that generate lipid second messengers by the phosphorylation of plasma membrane phosphoinositides. Class IA PI3Ks consist of a catalytic subunit (p110, comprising the three isoforms α, β, and δ) and an adaptor/regulatory subunit (p85, comprising the two isoforms α and β) (for a detailed review, see reference 80).The Akt family of proteins is comprised of the three isoforms α, β, and γ, which are composed of an N-terminal pleckstrin homology domain, a central catalytic domain, and a C-terminal hydrophobic domain. Akt is recruited to the plasma membrane through the binding of its pleckstrin homology domain to the phosphatidylinositol 3,4,5-triphosphate (PIP3), which is a product of PI3K that is anchored to the plasma membrane. PDK1 is also recruited to the plasma membrane through interactions with PIP3. As both PDK1 and Akt interact with PIP3, PDK1 colocalizes with Akt and activates it by phosphorylating threonine 308 (T308-P) (2, 66). Following its activation, Akt phosphorylates a number of downstream substrates such as caspase-9, BAD, glycogen synthase kinase 3β (GSK-3β), and FKHR. This leads to the suppression of apoptosis, cell growth, survival, and proliferation (11, 16, 56).Another downstream target of PI3K/Akt is mTOR, a serine/threonine kinase that plays a central role in the regulation of cell growth, proliferation, survival, and protein synthesis (26). mTOR kinase has recently been found to be associated with two functionally distinct complexes in mammalian cells, known as mTORC1 and mTORC2 (63, 66). Although these multiprotein complexes share molecules in common, distinct adaptor proteins are recruited into each complex: regulatory-associated protein of TOR (raptor) is recruited into mTORC1, while rapamycin-insensitive companion of TOR (rictor) is recruited into mTORC2 (33, 64). While mTORC1 controls cell growth and protein translation and has proven to be rapamycin sensitive, mTORC2 regulates the actin cytoskeleton and is assumed to be rapamycin insensitive, even though under conditions of prolonged exposure to the drug, it appears to inhibit mTORC2 assembly (29, 64, 65). Additionally, it has been demonstrated that mTORC2 regulates the activity of Akt through the phosphorylation of S473 (S473-P). S473-P appears to be required for the full activation of Akt, since S473-P has been shown to enhance the subsequent phosphorylation of T308 by PDK1 (66, 67, 94). Moreover, the phosphorylation of both S473 and T308 results in a four- to fivefold increase in Akt activity compared to T308-P by PDK1 alone (66).The PI3K/PDK1/Akt(T308)/mTORC1 pathway regulates vital cellular processes that are important for viral replication and propagation, including cell growth, proliferation, and protein translation. This pathway is particularly important for the replication of DNA viruses, as their replication is cap dependent. However, the Akt signaling pathway can also negatively affect viral replication. The stress response downstream of Akt signaling, including hypoxia and energy and amino acid depletion, inhibits mTORC1 (5, 9, 69). Therefore, DNA viruses must overcome these constraints to translate their mRNAs.Pharmacological disruption of the PI3K/Akt pathway with the specific PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (2-morpholino-8-phenyl-4H-1-benzopyran-4-one) (82) has been reported to not only increase the cleavage of downstream molecules associated with proapoptotic activity [e.g., poly(ADP-ribose) polymerase (PARP) and the executioner caspase-3] (38, 41) but also promote microtubule stabilization, actin filament remodeling/cell migration, and bleb formation/viral infectivity (10, 35, 49, 54, 59).Because the PI3K/Akt and PI3K/Akt/mTOR pathways influence diverse cellular functions and possibly a healthy antiviral response, usurping these pathways could support an increase in viral replication. In support of this, a number of reports have demonstrated that either the PI3K/Akt or the PI3K/Akt/mTOR pathway plays a role in the replication of many viruses including flavivirus (38), hepatitis C virus (27), human immunodeficiency virus type 1 (93), human papillomavirus (44, 96), respiratory syncytial virus (77), coxsackievirus B3 (19), Epstein-Barr virus (17, 50, 73), human cytomegalovirus (36, 37, 72), herpes simplex virus type 1 (7, 83), varicella-zoster virus (60), Kaposi''s sarcoma-associated herpesvirus (89), adenovirus (55), and simian virus 40 (SV40) (95). With this in mind, we also investigated whether the PI3K/Akt pathway played a pivotal role in orthopoxvirus biology. In this study, we show that the VACV- and CPXV-stimulated PI3K/Akt pathway not only contributes to the prevention of host-cell death but also plays a beneficial role in the viral replication cycle.     

High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.     

Iterative taxonomy reveals a new species of Trichomycterus Valenciennes 1832 (Siluriformes,Trichomycteridae) widespread in Rio Doce basin: a pseudocryptic of T. immaculatus

This paper reports on a new species of Trichomycterus from the Rio Doce basin. Unusually for new taxa in the genus during the past few decades, the new species is not narrowly endemic but instead widely distributed in its major drainage, the Rio Doce. The species has been collected and deposited in scientific collections for some years, but has been systematically misidentified as the more abundant Trichomycterus immaculatus or, to a lesser degree, as other morphologically similar species from south-eastern Brazil such as T. nigricans and T. pradensis. A combination of several morphological characteristics, such as vertebral number, pectoral-fin ray counts, pigmentation pattern and barcoding distance, were iteratively used and unambiguously distinguish the new species from all congeners. The present case reveals a pattern of diversity-discovery in which rare and narrowly endemic morphologically conspicuous species are discovered and described before visually inconspicuous taxa, even when the latter are more abundant and widespread. The morphological similarities among south-eastern Brazilian species with a uniform dark-grey color serve as basis for a brief discussion about the concepts of cryptic and pseudo-cryptic species in Trichomycterus and their consequences for potentially hidden diversity in the genus.     

Savannas after afforestation: Assessment of herbaceous community responses to wildfire versus native tree planting

Afforestation and fire exclusion are pervasive threats to tropical savannas. In Brazil, laws limiting prescribed burning hinder the study of fire in the restoration of Cerrado plant communities. We took advantage of a 2017 wildfire to evaluate the potential for tree cutting and fire to promote the passive restoration of savanna herbaceous plant communities after destruction by exotic tree plantations. We sampled a burned pine plantation (Burned Plantation); a former plantation that was harvested and burned (Harvested & Burned); an unburned former plantation that was harvested, planted with native trees, and treated with herbicide to control invasive grasses (Native Tree Planting); and two old-growth savannas which served as reference communities. Our results confirm that herbaceous plant communities on post-afforestation sites are very different from old-growth savannas. Among post-afforestation sites, Harvested & Burned herbaceous communities were modestly more similar in composition to old-growth savannas, had slightly higher richness of savanna plants (3.8 species per 50-m²), and supported the greatest cover of native herbaceous plants (56%). These positive trends in herbaceous community recovery would be missed in assessments of tree cover: whereas canopy cover in the Harvested & Burned site was 6% (less than typical of savannas of the Cerrado), the Burned Plantation and Native Tree Planting supported 34% and 19% cover, respectively. By focusing on savanna herbaceous plants, these results highlight that tree cutting and fire, not simply tree planting and fire exclusion, should receive greater attention in efforts to restore savannas of the Cerrado.     

Unexpected Thiocyanate Adsorption onto Ferrihydrite Under Prebiotic Chemistry Conditions

The most crucial role played by minerals was in the preconcentration of biomolecules or precursors of biomolecules in prebiotic seas. If this step had not occurred, molecular evolution would not have occurred. Thiocyanate is an important molecule in the formation of biomolecules as well as a catalyst for prebiotic reactions. The adsorption of thiocyanate onto ferrihydrite was carried out under pH and ion composition conditions in seawater that resembled those of prebiotic Earth. The seawater used in this work had high Mg²⁺, Ca²⁺ and SO₄^2? concentrations. The most important result of this work was that ferrihydrite adsorbed thiocyanateata pH value (7.2?±?0.2) that usually does not adsorb thiocyanate. The high adsorptivity of Mg²⁺, Ca²⁺ and SO₄^2?onto ferrihydrite showed that seawater ions can act as carriers of thiocyanate to the ferrihydrite surface, creating a huge outer-sphere complex. Kinetic adsorption and isotherm experiments showed the best fit for the pseudo-second-order model and an activation energy of 23.8 kJ mol^?1forthe Langmuir-Freundlich model, respectively. Thermodynamic data showed positive ΔG values, which apparently contradict the adsorption isotherm data and kinetic data that was obtained. The adsorption of thiocyanate onto ferrihydrite could be explained by coupling with the exergonic SO₄^2? adsorption onto ferrihydrite. The FTIR spectra showed no difference between the C≡N stretching peaks of adsorbed thiocyanate and free thiocyanate, corroborating the formation of an outer-sphere complex. All the results demonstrated the importance of the artificial seawater composition for the adsorption of thiocyanate and for understanding prebiotic chemistry.