Effects of amoebae on the growth of microbes isolated from moisture-damaged buildings

Dampness, moisture, and mold in buildings are associated with adverse health outcomes. In addition to fungi and bacteria, amoebae have been found in moisture-damaged building materials. Amoebae and a growing list of bacteria have been shown to have mutual effects on each other's growth, but the interactions between amoebae and microbes common in moisture-damaged buildings have not been reported. We co-cultivated the amoeba Acanthamoeba polyphaga with bacteria and fungi isolated from moisture-damaged buildings in laboratory conditions for up to 28 days. The microbes selected were the bacteria Streptomyces californicus, Bacillus cereus, and Pseudomonas fluorescens, and the fungi Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum. Fungi and bacteria generally benefited from the presence of the amoebae, whereas the growth of amoebae was hindered by Streptomyces californicus, Stachybotrys chartarum, and Bacillus cereus. Pseudomonas fluorescens slightly enhanced amoebae viability. Amoebae were indifferent to the presence of Aspergillus versicolor and Penicillium spinulosum. Thus, our results show that amoebae can alter the survival and growth of some microbes in moisture-damaged buildings.     

Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.     

Effect of dietary protein on embryo recovery rate and quality in superovulated heifers

For almost 3 decades, superovulation and embryo transfer have been used in cattle breeding to increase the number of offspring from genetically superior female animals. Several factors including nutrition affect the number of transferable embryos recovered. We compared the effects of two different dietary protein levels easily achieved in practical conditions on embryo number and quality in superovulated heifers. Finnish Ayrshire heifers (n = 37) were allocated to isoenergic diets containing either 14% (D14) or 18% (D18) crude protein (CP). Estruses were synchronized, and the heifers were subsequently superovulated and inseminated using a standard FSH-protocol. Embryos were collected 7 days after inseminations (71-72 days after the beginning of the treatment period) by uterine flushing. The number of corpora lutea, and the number and quality of embryos were determined. Protein feeding did not affect superovulatory response, the number of embryos or the number of transferable embryos recovered. Proportionally more poor-quality embryos were found in group D14 than in group D18 (20.2% versus 13.2%, respectively, P = 0.053). It is concluded that a long-term moderate increase in the content of crude protein fed to energy-adequate heifers does not seem to affect superovulatory response and the number of embryos recovered, but it may be advantageous to the quality of embryos.     

An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes

An enzymatic assay adapted to photometric analysis with 96-well microplates was evaluated for the measurement of 2-deoxyglucose (2DG) uptake in insulin-responsive tissues and differentiated 3T3-L1 adipocytes. For in vivo measurements, a small amount of nonradiolabeled 2DG was injected into mice without affecting glucose metabolism. For photometric quantification of the small amount of 2-deoxyglucose 6-phosphate (2DG6P) that accumulates in cells, we introduced glucose-6-phosphate dehydrogenase, glutathione reductase, and 5,5′-dithiobis(2-nitrobenzoic acid) to the recycling amplification reaction of NADPH. We optimized the enzyme reaction for complete oxidation of endogenous glucose 6-phosphate (G6P) and glucose in mouse tissues in vivo and serum as well as in 3T3-L1 adipocytes in vitro. All reactions are performed in one 96-well microplate by consecutive addition of reagents, and the assay is able to quantify 2DG and 2DG6P in the range of 5–80 pmol. The results obtained with the assay for 2DG uptake in vitro and in vivo in the absence or presence of insulin stimulation was similar to those obtained with the standard radioisotopic method. Thus, the enzymatic assay should prove to be useful for measurement of 2DG uptake in insulin-responsive tissues in vivo as well as in cultured cells.     

Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides

Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.     

Impact of early growth on postprandial responses in later life

Background

Low birth weight and slow growth during infancy are associated with increased rates of chronic diseases in adulthood. Associations with risk factors such as fasting glucose and lipids concentrations are weaker than expected based on associations with disease. This could be explained by differences in postprandial responses, which, however, have been little studied. Our aim was to examine the impact of growth during infancy on postprandial responses to a fast-food meal (FF-meal) and a meal, which followed the macro-nutrient composition of the dietary guidelines (REC-meal).

Methodology/Principal Findings

We recruited 24 overweight 65–75 year-old subjects, 12 with slow growth during infancy (SGI-group) and 12 with normal early growth. All the subjects were born at term. The study meals were isocaloric and both meals were consumed once. Plasma glucose, insulin, triglycerides (TG) and free fatty acids (FFA) were measured in fasting state and over a 4-h period after both meals. Subjects who grew slowly during infancy were also smaller at birth. Fasting glucose, insulin or lipid concentrations did not differ significantly between the groups. The TG responses were higher for the SGI-group both during the FF-meal (P = 0.047) and the REC-meal (P = 0.058). The insulin responses were significantly higher for the SGI-group after the FF-meal (P = 0.036). Glucose and FFA responses did not differ significantly between the groups.

Conclusions

Small birth size and slow early growth predict postprandial TG and insulin responses. Elevated responses might be one explanation why subjects who were small at birth and experiencing slow growth in infancy are at an increased risk of developing cardiovascular diseases in later life.     

Obesity and Occupational Injury: A Prospective Cohort Study of 69,515 Public Sector Employees

Background

Obesity and overweight are suggested to increase the risk of occupational injury but longitudinal evidence to confirm this is rare. We sought to evaluate obesity and overweight as risk factors for occupational injuries.

Methodology/Principal Findings

A total of 69,515 public sector employees (80% women) responded to a survey in 2000–2002, 2004 or 2008. Body mass index (kg/m²) was derived from self-reported height and weight and was linked to records of subsequent occupational injuries obtained from national registers. Different injury types, locations and events or exposures (the manner in which the injury was produced or inflicted) were analyzed by body mass index category adjusting for baseline socio-demographic characteristics, work characteristics, health-risk behaviors, physical and mental health, insomnia symptoms, and sleep duration. During the mean follow-up of 7.8 years (SD = 3.2), 18% of the employees (N = 12,204) recorded at least one occupational injury. Obesity was associated with a higher overall risk of occupational injury; multivariable adjusted hazard ratio (HR) 1.21 (95% CI 1.14–1.27). A relationship was observed for bone fractures (HR = 1.37; 95% CI: 1.10–1.70), dislocations, sprains and strains (HR = 1.36; 95% CI: 1.25–1.49), concussions and internal injuries (HR = 1.26; 95% CI: 1.11–1.44), injuries to lower extremities (HR = 1.62; 95%: 1.46–1.79) and injuries to whole body or multiple sites (HR = 1.37; 95%: 1.10–1.70). Furthermore, obesity was associated with a higher risk of injuries caused by slipping, tripping, stumbling and falling (HR = 1.55; 95% CI: 1.40–1.73), sudden body movement with or without physical stress (HR = 1.24; 95% CI: 1.10–1.41) and shock, fright, violence, aggression, threat or unexpected presence (HR = 1.33; 95% CI: 1.03–1.72). The magnitude of the associations between overweight and injuries was smaller, but the associations were generally in the same direction as those of obesity.

Conclusions/Significance

Obese employees record more occupational injuries than those with recommended healthy weight.     

Chronic HIV Infection Enhances the Responsiveness of Antigen Presenting Cells to Commensal Lactobacillus

Chronic immune activation despite long-term therapy poses an obstacle to immune recovery in HIV infection. The role of antigen presenting cells (APCs) in chronic immune activation during HIV infection remains to be fully determined. APCs, the frontline of immune defense against pathogens, are capable of distinguishing between pathogens and non-pathogenic, commensal bacteria. We hypothesized that HIV infection induces dysfunction in APC immune recognition and response to some commensal bacteria and that this may promote chronic immune activation. Therefore we examined APC inflammatory cytokine responses to commensal lactobacilli. We found that APCs from HIV-infected patients produced an enhanced inflammatory response to Lactobacillus plantarum WCFS1 as compared to APCs from healthy, HIV-negative controls. Increased APC expression of TLR2 and CD36, signaling through p38-MAPK, and decreased expression of MAP kinase phosphatase-1 (MKP-1) in HIV infection was associated with this heightened immune response. Our findings suggest that chronic HIV infection enhances the responsiveness of APCs to commensal lactobacilli, a mechanism that may partly contribute to chronic immune activation.