Differences in the Detection of BrdU/EdU Incorporation Assays Alter the Calculation for G1, S,and G2 Phases of the Cell Cycle in Trypanosomatids

Trypanosomatids are the etiologic agents of various infectious diseases in humans. They diverged early during eukaryotic evolution and have attracted attention as peculiar models for evolutionary and comparative studies. Here, we show a meticulous study comparing the incorporation and detection of the thymidine analogs BrdU and EdU in Leishmania amazonensis, Trypanosoma brucei, and Trypanosoma cruzi to monitor their DNA replication. We used BrdU‐ and EdU‐incorporated parasites with the respective standard detection approaches: indirect immunofluorescence to detect BrdU after standard denaturation (2 M HCl) and “click” chemistry to detect EdU. We found a discrepancy between these two thymidine analogs due to the poor detection of BrdU, which is reflected on the estimative of the duration of the cell cycle phases G1, S, and G2. To solve this discrepancy, we increase the exposure of incorporated BrdU using different concentrations of HCl. Using a new value for HCl concentration, we re‐estimated the phases G1, S, G2 + M, and cytokinesis durations, confirming the values found by this approach using EdU. In conclusion, we suggest that the studies using BrdU with standard detection approach, not only in trypanosomatids but also in others cell types, should be reviewed to ensure an accurate estimation of DNA replication monitoring.     

Molecular phylogeny of the Drosophila tripunctata and closely related species groups (Diptera: Drosophilidae)

We suggest a new phylogenetic hypothesis for the tripunctata radiation based on sequences of mitochondrial genes. Phylogenetic trees were reconstructed by parsimony, maximum likelihood and Bayesian methods. We performed tests for hypotheses of monophyly for taxonomic groups and other specific hypotheses. Results reject the monophyly for the tripunctata group whereas monophyly is not rejected for the tripunctata radiation and other specific groups within the radiation. Although most of the basal nodes were unresolved we were able to identify four clusters within the tripunctata radiation. These results suggest the collection of additional data before a proper taxonomic revision could be proposed.     

Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.     

Antifungal and physicochemical properties of Ocimum essential oil loaded in poly(lactic acid) nanofibers

Poly(lactic acid) (PLA) nanofibres containing different proportions of the essential oils from Ocimum basilicum L. and Ocimum gratissimum L. were prepared by solution blow spinning method. The essential oils were extracted by hydrodistillation and characterized by gas chromatography. MEV, contact angle, DSC and FTIR were used to characterize the nanofibres. The effect of bioative nanofibres on the growth of the fungus and on the production of ochratoxin A were evaluated using the fumigation test. Linalool, 1·8-cineole and camphor were the principal components of the essential oil from O. basilicum, and eugenol was the principal constituent in the oil from O. gratissimum. An increase in the average diameter of the nanofibres was observed with the addition of the essential oils. The essential oils acted as a plasticizer, resulting in a reduction in the crystallinity of the PLA. The encapsulation of essential oils in PLA nanofibres was verified by FTIR. An effective antifungal and antimicotoxygenic activity against Aspergillus ochraceus and Aspergillus westerdjikiae was observed for the bioative nanofibres. These results confirm the potential of PLA nanofibres containing the essential oils for the control of toxigenic fungi that cause the deterioration of food and are harmful to human health.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

Hospital costs associated with delirium in older medical patients

Introduction

Delirium is a common and serious complication in older patients, associated with increased, potentially preventable, morbidity and mortality. The aim of this study was to evaluate the associated costs of delirium during hospitalization in a university affiliated hospital in Chile.

Materials and methods

Prospective cohort study of consecutive patients 65 years and older, admitted to a medical ward. A psychogeriatric team assessed patients during the first and every 48 h until discharge using the Confusion Assessment Method (CAM-S), length of hospital stay, pharmacy and total hospitalization costs were analyzed. Statistical analysis was performed using bivariate and multivariate analysis according to delirium diagnosis.

Results

Data from 454 patients was analyzed, 160 of them in a delirium cohort (35.2%) and 294 in a non-delirium cohort (64.8%). The delirium cohort had a longer hospital stay (DATA) and higher mortality (7.0% versus 1.7%). The median of total costs of delirium during hospital stay was 38.7% higher than the non-delirium cohort (P < .001). Total costs were significantly higher in the delirium cohort after adjustment of covariables (P = .01).

Conclusions

This study confirms that delirium is associated with significantly greater costs. Considering that effective delirium prevention is possible, the knowledge of associated costs can help health care providers to justify prevention strategies and finally give better care for older patients.     

Lipid bilayer stabilization of the Na,K-ATPase reconstituted in DPPC/DPPE liposomes

Different subunit aggregates of the Na,K-ATPase may be formed depending on the method used to solubilize and purify the enzyme. We have studied the thermal unfolding of detergent-solubilized and dipalmitoylphosphatidylcholine/ dipalmitoylphosphatidylethanolamine liposome-reconstituted forms of the Na,K-ATPase by circular dichroism (CD) spectroscopy and p-nitrophenylphosphatase activity. The ellipticity at 222 nm of the solubilized and reconstituted forms showed a sigmoid decrease in the absolute value of the signal of 36 and 31% with T(50%) of 44 and 42 degrees C, respectively. The catalytic activity was reduced in two steps with T(50%) of 32 and 52 degrees C in the detergent-solubilized enzyme and T(50%) of 25 and 53 degrees C in the reconstituted enzyme. The reduction in catalytic activity of the detergent-solubilized enzyme was bi-exponential with t(1/2) of 8.3 and 67.9 min, resulting in the total loss of activity after 120 min. However, under the same conditions, the ATPase activity of the reconstituted enzyme was reduced by approx 35% with a t(1/2) of 145 min. The results suggest that the alpha- and beta-subunits present different thermal stability that may be modulated by the nature of the co-solvent (detergent or lipid) used in the preparations of the Na,K-ATPase. In addition, distinct processes of beta-subunit displacement and alpha-alpha-subunit aggregate formation may also contribute to the changes in both the CD spectra and the enzyme activity. Furthermore, we have demonstrated the protective role of the phospholipid bilayer in the reconstituted enzyme compared with the detergent-solubilized enzyme.