Anti-Sporothrix Antibody Detection in Domestic Cats as an Indicator of a Possible New Occurrence Area for Sporotrichosis in North Brazil

Mycopathologia - Feline sporotrichosis has emerged as an important public health issue in some countries, especially Brazil. Currently, zoonotic transmission of Sporothrix brasiliensis by domestic...     

Quantification of Metabolic Rearrangements During Neural Stem Cells Differentiation into Astrocytes by Metabolic Flux Analysis

Proliferation and differentiation of neural stem cells (NSCs) have a crucial role to ensure neurogenesis and gliogenesis in the mammalian brain throughout life. As there is growing evidence for the significance of metabolism in regulating cell fate, knowledge on the metabolic programs in NSCs and how they evolve during differentiation into somatic cells may provide novel therapeutic approaches to address brain diseases. In this work, we applied a quantitative analysis to assess how the central carbon metabolism evolves upon differentiation of NSCs into astrocytes. Murine embryonic stem cell (mESC)-derived NSCs and astrocytes were incubated with labelled [1-¹³C]glucose and the label incorporation into intracellular metabolites was followed by GC-MS. The obtained ¹³C labelling patterns, together with uptake/secretion rates determined from supernatant analysis, were integrated into an isotopic non-stationary metabolic flux analysis (¹³C-MFA) model to estimate intracellular flux maps. Significant metabolic differences between NSCs and astrocytes were identified, with a general downregulation of central carbon metabolism during astrocytic differentiation. While glucose uptake was 1.7-fold higher in NSCs (on a per cell basis), a high lactate-secreting phenotype was common to both cell types. Furthermore, NSCs consumed glutamine from the medium; the highly active reductive carboxylation of alpha-ketoglutarate indicates that this was converted to citrate and used for biosynthetic purposes. In astrocytes, pyruvate entered the TCA cycle mostly through pyruvate carboxylase (81%). This pathway supported glutamine and citrate secretion, recapitulating well described metabolic features of these cells in vivo. Overall, this fluxomics study allowed us to quantify the metabolic rewiring accompanying astrocytic lineage specification from NSCs.     

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL^-1, including 15.7% with an MIC of 8 μg mL^-1 and 2% with an MIC of 16 μg mL^-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.     

Microstructural morphology in early dermal denticles of hammerhead sharks (Elasmobranchii: Sphyrnidae) and related taxa

Mello, W.C., de Carvalho, J.J., Brito, P.M.M. 2011. Microstructural morphology in early dermal denticles of hammerhead sharks (Elasmobranchii: Sphyrnidae) and related taxa. —Acta Zoologica (Stockholm) 00 : 1–7. This study uses scanning electron microscopies to investigate and describe the microstructural diversity of dermal denticles in the family Sphyrnidae, which comprises all living hammerhead shark species, comparing them to other related taxa (i.e. Carcharhinus dussumieri, Carcharhinus plumbeus, Carcharhinus acronotus, Rhizoprionodon acutus, Negaprion brevirostris and Hemigaleus microstoma). The results reveal that sphyrnids present noticeable microstructures in the dermal denticles, distinguishing them from the other related species investigated. Additionally, scale patterns are the same in three distinct body regions (i.e. cephalic, branchial and dorsal fin). Species of Sphyrnidae that reach bigger total lengths and that are widely distributed (i.e. Sphyrna lewini and Sphyrna mokarran) presented more, smaller and nearly hexagonal microstructures that do not cover the entire scale surface, unlike species reaching smaller sizes and restricted to coastal habits (i.e. Sphyrna tiburo, Sphyrna tudes, Sphyrna media and Eusphyra blochii). The sphyrnid scales are similar to R. acutus and C. dussumieri rather than to the other species, but it is not possible to identify the sphyrnid species only by scale features. It is clear that a similar morphology of scales is not necessarily related to similar life habits, and that they are candidates to provide new characters in phylogenetical studies among sphyrnids.     

Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite-driven apoptotic death

Peroxynitrite (ONOO-) is a potent oxidizing and nitrating agent produced by the reaction of nitric oxide with superoxide. It readily nitrates phenolic compounds such as tyrosine residues in proteins, and it has been demonstrated that nitration of tyrosine residues in proteins inhibits their phosphorylation. During immune responses, tyrosine phosphorylation of key substrates by protein tyrosine kinases is the earliest of the intracellular signaling pathways following activation through the TCR complex. This work was aimed to evaluate the effects of ONOO- on lymphocyte tyrosine phosphorylation, proliferation, and survival. Additionally, we studied the generation of nitrating species in vivo and in vitro during immune activation. Our results demonstrate that ONOO-, through nitration of tyrosine residues, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after PHA- or CD3-mediated activation but not upon phorbol ester-mediated stimulation. We also provide evidence indicating that peroxynitrite is produced during in vitro immune activation, mainly by cells of the monocyte/macrophage lineage. Furthermore, immunohistochemical studies demonstrate the in vivo generation of nitrating species in human lymph nodes undergoing mild to strong immune activation. Our results point to a physiological role for ONOO- as a down-modulator of immune responses and also as key mediator in cellular and tissue injury associated with chronic activation of the immune system.     

New occurrence of microchromosomes B in Moenkhausia sanctaefilomenae (Pisces, Characidae) from the Paraná River of Brazil: analysis of the synaptonemal complex

The Moenkhausia sanctaefilomenae specimens showed a karyotype consisting of 2n = 50 chromosomes with 12 metacentrics, 36 submetacentrics and two subtelocentrics. In addition to the basic karyotype, all the males specimens have cells ranging from zero to two B microchromosomes in mitotic metaphases. These chromosomes were not observed in the female specimens. C-band analysis showed a distribution pattern of characteristic heterochromatin with interstitial and centromeric blocks. However, the B chromosomes were faintly stained with C-banding and were not fluorescent with CMA₃ staining. The meiotic studies showed the formation of bivalents in metaphase I and in pachytene under an optical microscope. Through synaptonemal complex analysis with an electron microscope, the pachytene showed 25 bivalents completely paired and a small bivalent corresponding to the B chromosomes. In the same preparation, one of the B chromosomes was observed in a univalent form. On the basis of pairing behavior and morphology it is assumed that B chromosomes of M. sanctaefilomenae show homology between them and their evolutionary aspects are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet

We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.     

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II

One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.     

Azilsartan Reduced TNF-α and IL-1β Levels,Increased IL-10 Levels and Upregulated VEGF,FGF, KGF,and TGF-α in an Oral Mucositis Model

Oral mucositis (OM) is a common complication of treatments for head and neck cancer, particularly radiotherapy with or without chemotherapy. OM is characterised by oral erythema, ulceration, and pain. The aim of this study was to evaluate the effect of azilsartan (AZT), an angiotensin II receptor antagonist, on 5-fluorouracil (5-FU)-induced oral mucositis (OM) in Syrian hamsters. OM was induced by the intraperitoneal administration of 5-FU on experimental days 1 (60mg/Kg) and 2 (40mg/Kg). Animals were pretreated with oral AZT (1, 5, or 10 mg/kg) or vehicle 30 min before 5-FU injection and daily until day 10. Experimental treatment protocols were approved by the Animal Ethics Committee Use/CEUA (Number 28/2012) of the UFRN. Macroscopic analysis and cheek pouch samples were removed for histopathologic analysis. Myeloperoxidase (MPO), Malonyldialdehyde (MDA), interleukin-1 beta (IL-1β), interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF-α) were analysed by Enzyme Linked Immuno Sorbent Assay (ELISA). Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), and transforming growth factor (TGF)-α were measured by immunohistochemistry. Analysis of variance followed by Bonferroni’s test was used to calculate the means of intergroup differences (p ≤ 0.05). Treatment with 1 mg/kg AZT reduced levels MPO (p<0.01), MDA (p<0.5) and histological inflammatory cell infiltration, and increased the presence of granulation tissue. AZT treatment at 1 mg/kg reduced the TNF-α (p<0.05) and IL-1β (p<0.05) levels, increased the cheek pouch levels of IL-10 (p<0.01), and upregulated VEGF, FGF, KGF, and TGF-α. Administration of AZT at higher doses (5 and 10 mg/kg) did not significantly reverse the OM. AZT at a dose of 1 mg/kg prevented the mucosal damage and inflammation associated with 5-FU-induced OM, increasing granulation and tissue repair.     

Anti-herpesvirus bovine type 5 activities of extracts obtained from Plocamium brasiliense

Bovine herpesvirus type 5 (BoHV-5) is an important etiologic agent of meningoencephalitis in cattle and has been frequently identified in outbreaks of neurological disease in bovine in the southern hemisphere including Brazil. This study aimed to evaluate the cytotoxic effect and the antiviral properties of extracts obtained from Plocamium brasiliense (Greville) Howe and Taylor in BoHV-5 RJ42/01 replication. The cytotoxic effects were measured in Madin-Darbin bovine kidney cells (MDBK) and cytotoxic concentration (CC₅₀) values have been determined for acyclovir (ACV) (223 μg?±?2.0), ethyl acetate extract from P. brasiliense (2,109 μg?±?10), hexane extract from P. brasiliense (7.181 μg?±?5), dichloromethane extract from P. brasiliense (2.356 μg?±?6.5), and hydroalcoholic extract from P. brasiliense (1.408 μg?±?5.8). As a first approach to characterize the action of these extracts on BoHV-5 RJ42/01, a virucidal assay activity was performed. A virus suspension containing 1?×?10⁵ plaque-forming units (PFU) of the BoHV-5 RJ42/01 was mixed with 600 μg of extract and acyclovir and kept at room temperature (24 °C) for 3 h. Meanwhile, a control of untreated infected virus was performed in the same conditions. Then, treated virus suspension and untreated control were diluted, and percentage of inhibition of infectivity was determined by plaque assay: ethyl acetate extract (99 %), hexane extract (90 %), dichloromethane extract (99 %), and hydroalcoholic extract (27 %). Acyclovir had a slight virucidal activity on viral particle. The inhibition of attachment was performed in MDBK cells inoculated with 100 PFU of BoHV-5 RJ42/01 in the presence or absence of various concentrations of extracts (0.3, 0.9, and 1.5 μg mL^?1). Acyclovir was also assayed at 2.8 and 11.25 μg mL^?1. The inhibition of adsorption was also tested in MDBK cells treated with the same concentrations of the extracts before virus inoculation. Results: hexane extracts inhibited virus attachment in pre-treated cell 0.9 μg (55 %) and 1.5 μg (71 %) and untreated MDBK cell only with 1.5 μg (63 %). Ethyl acetate extract on cell pre-treated with 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 μg (91 %). Ethyl acetate extract on pre-treated cell 0.3 μg (67 %), 0.9 μg (81 %), and 1.5 (91 %) but discrete inhibition on cell untreated. Dichloromethane extract and acyclovir slightly inhibited virus binding on MDBK cell.