Morphology and molecular taxonomy of Evlachovaea-like fungi,and the status of this unusual conidial genus

The entomopathogenic anamorphic genus Evlachovaea was described to differ from other fungi in forming its conidia obliquely to the axis of the conidiogenous cell and with successive conidia having alternate orientations with a zipper- or chevron-like arrangement resulting in flat, ribbon-like chains. Morphological and molecular studies of six Evlachovaea-like isolates baited from Central Brazilian soils using Triatoma infestans (a vector of Chagas disease) and of other entomopathogens with Evlachovaea-like conidiogenesis led to a re-evaluation of the status of this little known fungal genus. The Brazilian isolates formed two distinct groups based on gene sequences for both the internal transcribed spacer (ITS) and translation elongation factor (EF-1α) genes, morphology, and growth patterns; both groups also differed from the type species, Evlachovaea kintrischica. More detailed studies of these fungi indicated that the alternatingly oblique orientations of forming conidia are neither a stable nor invariant character (even on single phialides). Furthermore, the molecular cladistic analysis unambiguously placed the Evlachovaea isolates firmly within the genus Isaria (Hypocreales: Cordycipitaceae). The ITS sequences of E. kintrischica were very similar or even identical to those of Isaria amoenerosea and Isaria cateniobliqua, thereby suggesting that E. kintrischica is a synonym of one of these species, and that the genus Evlachovaea must be treated as a later synonym of Isaria, which must now be recognized to include several highly divergent modes of conidiogenesis. These taxonomic findings are discussed in the context of dramatic changes recently imposed on the nomenclatural standards used to determine the correct names of all pleomorphic fungi.     

The Role of Biliary Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 6 (CEACAM6) as a Biomarker in Cholangiocarcinoma

Objective

The aim of the present study is to determine if CEACAM6 can be detected in the bile of patients with biliary cancer and can serve as a diagnostic biomarker for cholangiocarcinoma.

Summary Background Data

Distinguishing bile duct carcinoma from other diagnoses is often difficult using endoscopic or percutaneous techniques. The cell surface protein CEACAM6 is over-expressed in many gastrointestinal cancers and may be selectively elevated in biliary adenocarcinoma.

Methods

Bile from patients with benign biliary disease and cholangiocarcinoma (hilar, intrahepatic and distal) was collected at the time of index operation. The concentration of CEACAM6 was quantified by sandwich enzyme-linked immunosorbent assay (ELISA) and correlated to pathologic diagnosis. Diagnostic capability of CEACAM6 was evaluated by Wilcoxon rank-sum, linear regression, multiple regression, and receiver operating characteristic (ROC) curve analysis.

Results

Bile from 83 patients was analyzed: 42 with benign disease and 41 with cholangiocarcinoma. Patients in the benign cohort were younger, predominantly female, and had lower median biliary CEACAM6 levels than patients in the malignant cohort (7.5 ng/ml vs. 40 ng/ml; p = <.001). ROC curve analysis determined CEACAM6 to be a positive predictor cholangiocarcinoma with a CEACAM6 level >14 ng/ml associated with 87.5% sensitivity, 69.1% specificity, and a likelihood ratio of 2.8 (AUC 0.74). Multiple regression analysis suggested elevated alkaline phosphatase and the presence of biliary endoprostheses may influence CEACAM6 levels.

Conclusion

Biliary CEACAM6 can identify patients with extrahepatic cholangiocarcinoma with a high degree of sensitivity and should be investigated further as a potential screening tool.     

Effects of Methadone on the Minimum Anesthetic Concentration of Isoflurane,and Its Effects on Heart Rate,Blood Pressure and Ventilation during Isoflurane Anesthesia in Hens (Gallus gallus domesticus)

The aim of this study was to measure the temporal effects of intramuscular methadone administration on the minimum anesthetic concentration (MAC) of isoflurane in hens, and to evaluate the effects of the isoflurane-methadone combination on heart rate and rhythm, blood pressure and ventilation. Thirteen healthy adult hens weighing 1.7 ± 0.2 kg were used. The MAC of isoflurane was determined in each individual using the bracketing method. Subsequently, the reduction in isoflurane MAC produced by methadone (3 or 6 mg kg^-1, IM) was determined by the up-and-down method. Stimulation was applied at 15 and 30 minutes, and at 45 minutes if the bird had not moved at 30 minutes. Isoflurane MAC reduction was calculated at each time point using logistic regression. After a washout period, birds were anesthetized with isoflurane and methadone, 6 mg kg^-1 IM was administered. Heart rate and rhythm, respiratory rate, blood gas values and invasive blood pressure were measured at 1.0 and 0.7 isoflurane MAC, and during 45 minutes after administration of methadone once birds were anesthetized with 0.7 isoflurane MAC. Fifteen minutes after administration of 3 mg kg^-1 of methadone, isoflurane MAC was reduced by 2 (-9 to 13)% [logistic regression estimate (95% Wald confidence interval)]. Administration of 6 mg kg^-1 of methadone decreased isoflurane MAC by 29 (11 to 46)%, 27 (-3 to 56)% and 10 (-8 to 28)% after 15, 30 and 45 minutes, respectively. Methadone (6 mg kg^-1) induced atrioventricular block in three animals and ventricular premature contractions in two. Methadone caused an increase in arterial blood pressure and arterial partial pressure of carbon dioxide, while heart rate and pH decreased. Methadone, 6 mg kg^-1 IM significantly reduced isoflurane MAC by 30% in hens 15 minutes after administration. At this dose, methadone caused mild respiratory acidosis and increase in systemic blood pressure.     

Antimicrobial Combinations against Pan-Resistant Acinetobacter baumannii Isolates with Different Resistance Mechanisms

The study investigated the effect of antibiotic combinations against 20 clinical isolates of A. baumannii (seven colistin-resistant and 13 colistin-susceptible) with different resistance mechanisms. Clinical data, treatment, and patient mortality were evaluated. The following methods were used: MIC, PCRs, and outer membrane protein (OMP) analysis. Synergy was investigated using the checkerboard and time-kill methods. Clonality was evaluated by PFGE. Based on clonality, the whole genome sequence of six A. baumannii isolates was analyzed. All isolates were resistant to meropenem, rifampicin, and fosfomycin. OXA-23 and OXA-143 were the most frequent carbapenemases found. Four isolates showed loss of a 43kDa OMP_. The colistin-susceptible isolates belonged to different clones and showed the highest synergistic effect with fosfomycin-amikacin. Among colistin-resistant isolates, the highest synergistic effect was observed with the combinations of colistin-rifampicin followed by colistin-vancomycin. All colistin-resistant isolates harbored bla_OXA-23-like and belonged to CC113. Clinical and demographic data were available for 18 of 20 patients. Fourteen received treatment and eight patients died during treatment. The most frequent site of infection was the blood in 13 of 14 patients. Seven patients received vancomycin plus an active drug against A. baumannii; however, mortality did not differ in this group. The synergistic effect was similar for colistin-susceptible isolates of distinct clonal origin presenting with the same resistance mechanism. Overall mortality and death during treatment was high, and despite the high synergism in vitro with vancomycin, death did not differ comparing the use or not of vancomycin plus an active drug against A. baumannii.     

Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.     

Trypanosoma cruzi Needs a Signal Provided by Reactive Oxygen Species to Infect Macrophages

Background

During Trypanosoma cruzi infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during T. cruzi infection and the results obtained are sometimes contradictory. T. cruzi has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress.

Methodology/Principal Findings

In order to investigate the contribution of ROS in T. cruzi survival and infection, we utilized mice deficient in the gp91^phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from Escherichia coli) or TcMTH (from T. cruzi), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and in vivo.

Conclusions

Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to T. cruzi growth inside the cells.     

Investigating the widespread introduction of a tropical marine fouling species

Little is known about the number and rate of introductions into terrestrial and marine tropical regions, and if introduction patterns and processes differ from temperate latitudes. Botryllid ascidians (marine invertebrate chordates) are an interesting group to study such introduction differences because several congeners have established populations across latitudes. While temperate botryllid invasions have been repeatedly highlighted, the global spread of tropical Botrylloides nigrum (Herdman, 1886) has been largely ignored. We sampled B. nigrum from 16 worldwide warm water locations, including around the Panama Canal, one of the largest shipping hubs in the world and a possible introduction corridor. Using mitochondrial (COI) and nuclear (ANT) markers, we discovered a single species with low genetic divergence and diversity that has established in the Atlantic, Pacific, Indo‐Pacific, and Mediterranean Oceans. The Atlantic Ocean contained the highest diversity and multilocus theta estimates and may be a source for introductions to other regions. A high frequency of one mitochondrial haplotype was detected in Pacific populations that may represent a recent introduction in this region. In comparison to temperate relatives, B. nigrum displayed lower (but similar to temperate Botrylloides violaceus) genetic divergence and diversity at both loci that may represent a more recent global spread or differences in introduction pressures in tropical regions. Additionally, chimeras (genetically distinct individuals sharing a single body) were detected in three populations by the mitochondrial locus and validated using cloning, and these individuals contained new haplotype diversity not detected in any other colonies.     

Depletion interactions and modulation of DNA‐intercalators binding: Opposite behavior of the “neutral” polymer poly(ethylene‐glycol)

In this work we have investigated the role of high molecular weight poly(ethylene‐glycol) 8000 (PEG 8000) in modulating the interactions of the DNA molecule with two hydrophobic compounds: Ethidium Bromide (EtBr) and GelRed (GR). Both compounds are DNA intercalators and are used here to mimic the behavior of more complex DNA ligands such as chemotherapeutic drugs and proteins whose domains intercalate DNA. By means of single‐molecule stretching experiments, we have been able to show that PEG 8000 strongly shifts the binding equilibrium between the intercalators and the DNA even at very low concentrations (1% in mass). Additionally, microcalorimetry experiments were performed to estimate the strength of the interaction between PEG and the DNA ligands. Our results suggest that PEG, depending on the system under study, may act as an “inert polymer” with no enthalpic contribution in some processes but, on the other hand, it may as well be an active (non‐neutral) osmolyte in the context of modulating the activity of the reactants and products involved in DNA‐ligand interactions. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 227–233, 2016.