The effect of environmental factors on the growth rate of Karenia brevis (Davis) G. Hansen and Moestrup

The red tide dinoflagellate Karenia brevis (Davis) G. Hansen and Moestrup is noted for causing mass mortalities of marine organisms in the Gulf of Mexico. Most research has focused on culture isolates from the eastern Gulf of Mexico. In this investigation, we examine the effects of light, temperature and salinity on the growth rate of K. brevis from the western Gulf of Mexico. Growth rates of K. brevis were determined under various combinations of irradiance (19, 31, 52, 67, and 123 μmol m⁻² s⁻¹), salinity (25, 30, 35, 40 and 45), and temperature (15, 20, 25, and 30 °C). Maximum growth rates varied from 0.17 to 0.36 div day⁻¹ with exponential growth rates increasing with increasing irradiance. Little or no growth was supported at 19 μmol photons m⁻² s⁻¹ for any experiment. Maximum growth rates at 15 °C were much lower than at other temperatures. Maximum growth rates of the Texas clone (SP3) fell within the range of Florida clones reported in the literature (0.17–0.36 div day⁻¹ versus 0.2–1.0 div day⁻¹). The Texas clone SP3 had a very similar light saturation point compared to that of a Florida isolate (Wilson's clone) (67 μmol m⁻² s⁻¹ versus 65 μmol m⁻² s⁻¹), and light compensation (20–30 μmol m⁻² s⁻¹1). The upper and lower salinity tolerance of the Texas clone was similar than that of some Florida clones (45 versus 46 and 25 versus 22.5, respectively). In our study, the Texas clone had the same temperature tolerance reported for Florida clones (15–30 °C). While individual clones can vary considerably in maximum growth rates, our results indicate only minor differences exist between the Texas and Florida strains of K. brevis in their temperature and salinity tolerance for growth. While the literature notes lower salinity occurrences of K. brevis in nearby Louisiana, our isolate from the southern Texas coast has the higher salinity requirements typical of K. brevis in the eastern Gulf of Mexico.     

Sustained Reduction of the Dengue Vector Population Resulting from an Integrated Control Strategy Applied in Two Brazilian Cities

Aedes aegypti has developed evolution-driven adaptations for surviving in the domestic human habitat. Several trap models have been designed considering these strategies and tested for monitoring this efficient vector of Dengue. Here, we report a real-scale evaluation of a system for monitoring and controlling mosquito populations based on egg sampling coupled with geographic information systems technology. The SMCP-Aedes, a system based on open technology and open data standards, was set up from March/2008 to October/2011 as a pilot trial in two sites of Pernambuco -Brazil: Ipojuca (10,000 residents) and Santa Cruz (83,000), in a joint effort of health authorities and staff, and a network of scientists providing scientific support. A widespread infestation by Aedes was found in both sites in 2008–2009, with 96.8%–100% trap positivity. Egg densities were markedly higher in SCC than in Ipojuca. A 90% decrease in egg density was recorded in SCC after two years of sustained control pressure imposed by suppression of >7,500,000 eggs and >3,200 adults, plus larval control by adding fishes to cisterns. In Ipojuca, 1.1 million mosquito eggs were suppressed and a 77% reduction in egg density was achieved. This study aimed at assessing the applicability of a system using GIS and spatial statistic analysis tools for quantitative assessment of mosquito populations. It also provided useful information on the requirements for reducing well-established mosquito populations. Results from two cities led us to conclude that the success in markedly reducing an Aedes population required the appropriate choice of control measures for sustained mass elimination guided by a user-friendly mosquito surveillance system. The system was able to support interventional decisions and to assess the program’s success. Additionally, it created a stimulating environment for health staff and residents, which had a positive impact on their commitment to the dengue control program.     

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.     

Lactobacillus reuteri beta-galactosidase activity and low milk acidification ability

Beta-galactosidase activity was studied as a possible cause of the low milk acidification ability observed in Lactobacillus reuteri NRRL 14171. Enzymatic activity was determined in MRS broth supplemented with either glucose or lactose and milk at the middle and final stage of the exponential phase, as well as at the stationary phase. Results were compared with beta-galactosidase activity in Lactobacillus casei NRRL-B1922, a strain that shows the milk acidification ability. The effects of the types of carbon and nitrogen sources were established by comparison of growth parameters (higher maximum cell concentration and specific growth rate) in broth culture and skim milk supplemented with 2% glucose or 1% casein peptone. In milk, L. reuteri showed higher beta-galactosidase activity in all growth phases compared with L. casei. Greater cell concentration maxima, specific growth rates, and acidification abilities were observed in L. reuteri when it was cultured in milk supplemented with 1% casein peptone compared with non-supplemented milk cultures. Results suggest that the poor milk acidification ability observed in L. reuteri may be more related to a weak proteolytic system than to deficient beta-galactosidase activity.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Drug action against intracellularly growingMycobacterium xenopi

The intracellular growth kinetics ofMycobacterium xenopi was studied in the murine J-774 macrophage cell line model. During the initial 4 days of infection, the bacilli divided about every 33 h. Electron microscopy of infected macrophages showed that bacteria inside phagosomes were surrounded by a protective electron-transparent zone (ETZ). This model was used for comparing the extracellular and intracellular activities of the following drugs: pristinamycin (PRISTINA), isoniazid (INH), clofazimine (CLOFA), rifabutin (=ansamycin; ANSA), rifampicine (RIFA), streptomycin (SM), ethambutol (EMB), and five fluoroquinolones, namely, ciprofloxacin (CIPRO), ofloxacin (OFLO), pefloxacin (PEFLO), enoxacin (ENOX) and norfloxacin (NORFLO). All the drugs were tested within their obtainable serum level concentrations in man. Under these conditions, CLOFA, SM, CIPRO, and OFLO were highly active against intracellularly growingM. xenopi, INH and RIFA were moderately active, whereas ANSA, PRISTINA, EMB, PEFLO, ENOX, and NORFLO were only growth inhibiting. The comparison of these data with extracellular activities of the same drugs underlined the discrepancies observed in test-tube drug activity evaluation and its correlation with results of chemotherapy in patients in whom the drug has essentially an intracellular bacterial killing role.     

Mu-calpain is functionally required for alpha-processing of Alzheimer's beta-amyloid precursor protein

Alzheimer's beta-amyloid precursor protein (APP) is normally processed by an unidentified alpha-secretase. A unique feature of this protease is its high sensitivity to phorbol esters, yet the mechanism involved is unclear. We have previously reported that phorbol 12,13-dibutyrate (PDBu) activates calpain, a Ca2+-dependent protease, and PDBu-induced release of APPs (secreted APP) is sensitive to calpain inhibitors, suggesting that calpain is involved in APP alpha-processing. In the present study, we found that PDBu markedly promoted the expression of both mu- and m-calpains in cultured fibroblasts. Dose-response and time course studies revealed that mu-calpain was more sensitive to PDBu than m-calpain and the temporal course of the mu-calpain change coincides better with that of APPs release. Moreover, the stimulatory effect of PDBu on mu-calpain was selectively blocked by mu-calpain-specific siRNA (small interference RNA) and the blockage was accompanied by a concomitant decrease in APPs release. In contrast, m-calpain siRNA did not affect APPs release significantly. Measurement of amyloid beta protein (Abeta) release in the mu-calpain siRNA-treated cells indicated that Abeta40 and Abeta42 levels inversely changed in relation to APPs, and the changes in Abeta42 were more prominent than in Abeta40. Together, these data suggest that calpain, particularly mu-calpain, is a potential candidate for alpha-secretase in the regulated APP alpha-processing, and that changes in this protease can affect the outcome of the overall APP processing.