Interference of aging media on the assessment of yeast chronological life span by propidium iodide staining

An increasing number of researchers are using the Saccharomyces cerevisiae chronological aging model to gain insight into the post-mitotic cellular aging. Recently, an alternative approach to the traditional cellular viability assay by colony-forming unit (CFU) counts, based on the propidium iodide (PI) staining combined with flow cytometry (PI-FCM), was proposed for the assessment of yeast chronological aging. Since the chronological aging assessment shows variations particularly concerning the aging media, in this work, the influence of the most common aging media (exhausted media or water) on the assessment of chronological aging by PI staining was studied. Our results show that this methodology is highly affected by the aging media. Indeed, a correlation between CFU counts and the percentage of PI-stained cells is only achieved with the exhausted media. As such, the assessment of yeast chronological aging by PI-FCM water should not be used.     

Pyrolytic Methylation-Gas Chromatography of Whole Bacterial Cells for Rapid Profiling of Cellular Fatty Acids

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.     

Pioglitazone Improves Fat Distribution,the Adipokine Profile and Hepatic Insulin Sensitivity in Non-Diabetic End-Stage Renal Disease Subjects on Maintenance Dialysis: A Randomized Cross-Over Pilot Study

Background

Fat redistribution, increased inflammation and insulin resistance are prevalent in non-diabetic subjects treated with maintenance dialysis. The aim of this study was to test whether pioglitazone, a powerful insulin sensitizer, alters body fat distribution and adipokine secretion in these subjects and whether it is associated with improved insulin sensitivity.

Trial Design

This was a double blind cross-over study with 16 weeks of pioglitazone 45 mg vs placebo involving 12 subjects.

Methods

At the end of each phase, body composition (anthropometric measurements, dual energy X-ray absorptometry (DEXA), abdominal CT), hepatic and muscle insulin sensitivity (2-step hyperinsulinemic euglycemic clamp with 2H2-glucose) were measured and fasting blood adipokines and cardiometabolic risk markers were monitored.

Results

Four months treatment with pioglitazone had no effect on total body weight or total fat but decreased the visceral/sub-cutaneous adipose tissue ratio by 16% and decreased the leptin/adiponectin (L/A) ratio from 3.63×10⁻³ to 0.76×10⁻³. This was associated with a 20% increase in hepatic insulin sensitivity without changes in muscle insulin sensitivity, a 12% increase in HDL cholesterol and a 50% decrease in CRP.

Conclusions/Limitations

Pioglitazone significantly changes the visceral-subcutaneous fat distribution and plasma L/A ratio in non diabetic subjects on maintenance dialysis. This was associated with improved hepatic insulin sensitivity and a reduction of cardio-metabolic risk markers. Whether these effects may improve the outcome of non diabetic end-stage renal disease subjects on maintenance dialysis still needs further evaluation.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT01253928","term_id":"NCT01253928"}}NCT01253928     

Anthropophilic Anopheles species composition and malaria in Tierradentro,Córdoba,Colombia

Malaria is still a primary health problem in Colombia. The locality of Tierradentro is situated in the municipality of Montelíbano, Córdoba, in the northwest of Colombia, and has one of the highest annual parasite index of malaria nationwide. However, the vectors involved in malaria transmission in this locality have not yet been identified. In this study, the local anthropophilic Anopheles composition and natural infectivity with Plasmodium were investigated. In August 2009, 927 female Anopheles mosquitoes were collected in eight localities using the human landing catch method and identified based on their morphology. Cryptic species were determined by restriction fragment length polymorphism-internal transcribed spacer (ITS)2 molecular analysis. Eight species [Anopheles nuneztovari s.l. (92.8%), Anopheles darlingi (5.1%), Anopheles triannulatus s.l. (1.8%), Anopheles pseudopunctipennis s.l. (0.2%), Anopheles punctimacula s.l. (0.2%), Anopheles apicimacula (0.1%), Anopheles albimanus (0.1%) and Anopheles rangeli (0.1%)] were identified and species identity was confirmed by ITS2 sequencing. This is the first report of An. albimanus, An. rangeli and An. apicimacula in Tierradentro. Natural infectivity with Plasmodium was determined by ELISA. None of the mosquitoes was infectious for Plasmodium. An. nuneztovari s.l. was the predominant species and is considered the primary malaria vector; An. darlingi and An. triannulatus s.l. could serve as secondary vectors.     

Interaction of <i>Acaena elongata</i> L. with Arbuscular Mycorrhizal Fungi under Phosphorus Limitation Conditions in a Temperate Forest

The aim of this study was to analyze the performance of Acaena elongata colonized by arbuscular mycorrhizal fungi (AMF) to different phosphorus (P) concentrations, as a measure of AMF dependency. A. elongata, is a species from soils where P availability is limited, such as temperate forests. Our research questions were: 1) How do different P concentrations affect the AMF association in Acaena elongata, and 2) How does the AMF association influence A. elongata’s growth under different P concentrations? A. elongata’s growth, P content in plant tissue, AMF colonization and dependency were measured under four P concentrations: control (0 g P kg⁻¹ ), low (0.05 g P kg⁻¹ ), intermediate (0.2 g P kg⁻¹ ) and high (2 g P kg⁻¹ ) in different harvests. A complete randomized block design was applied. A. elongata’s growth was higher under -AMF in intermediate and high P concentrations, and the lowest growth corresponded to +AMF in the low and intermediate P concentration. We observed a negative effect on the root biomass under +AMF in intermediate P concentration, while the P concentration had a positive effect on the leaf area ratio. The AMF colonization in A. elongata decreased in the highest P concentration and it was favored under intermediate P concentration; while the low and the high concentrations generated a cost-benefit imbalance. Our results suggest that the performance of some plant species in soils with low P availability may not be favored by their association with AMF, but a synergy between AMF and intermediate P concentrations might drive A. elongata’s growth.     

Sugars fermented byBifidobacterium infantis ATCC 27920 in relation to growth and α-galactosidase activity

Summary The ability of Bifidobacterium infantis ATCC 27 920 to ferment glucose, galactose, lactose, melibiose and raffinose was investigated with respect to -galactosidase (-d-galactoside galactohydrolase, E.C. 3.2.1.22). The sugars were tested at three concentrations: 0.5, 1.0 and 2.0%. The growth of B. infantis was slower on glucose compared with the other sugars. The highest specific growth rate was observed on melibiose followed by lactose. High cell numbers could be rapidly obtained on galactose-containing sugars. For each carbohydrate, enzyme activity was maximal at the end of the exponential phase and the highest specific -galactosidase activities were recorded on the two -1,6 galactosaccharides (melibiose and raffinose: 3.0 and 4.5 nkat · 10⁹ colony-forming units, respectively).Contribution no. 186 from the Food Research and Development Centre Offprint requests to: D. Roy     

Simultaneous Saccharification and Fermentation of Hydrothermal Pretreated Lignocellulosic Biomass: Evaluation of Process Performance Under Multiple Stress Conditions

Industrial lignocellulosic bioethanol processes are exposed to different environmental stresses (such as inhibitor compounds, high temperature, and high solid loadings). In this study, a systematic approach was followed where the liquid and solid fractions were mixed to evaluate the influence of varied solid loadings, and different percentages of liquor were used as liquid fraction to determine inhibitor effect. Ethanol production by simultaneous saccharification and fermentation (SSF) of hydrothermally pretreated Eucalyptus globulus wood (EGW) was studied under combined diverse stress operating conditions (30–38 °C, 60–80 g of liquor from hydrothermal treatment or autohydrolysis (containing inhibitor compounds)/100 g of liquid and liquid to solid ratio between 4 and 6.4 g liquid in SSF/g unwashed pretreated EGW) using an industrial Saccharomyces cerevisiae strain supplemented with low-cost byproducts derived from agro-food industry. Evaluation of these variables revealed that the combination of temperature and higher solid loadings was the most significant variable affecting final ethanol concentration and cellulose to ethanol conversion, whereas solid and autohydrolysis liquor loadings had the most significant impact on ethanol productivity. After optimization, an ethanol concentration of 54 g/L (corresponding to 85 % of conversion and 0.51 g/Lh of productivity at 96 h) was obtained at 37 °C using 60 % of autohydrolysis liquor and 16 % solid loading (liquid to solid ratio of 6.4 g/g). The selection of a suitable strain along with nutritional supplementation enabled to produce noticeable ethanol titers in quite restrictive SSF operating conditions, which can reduce operating cost and boost the economic feasibility of lignocellulose-to-ethanol processes.