Thirty-four years of climatic selection in the land snail Theba pisana

Few continuous, long-term studies have measured the intensity and variability of natural selection within a framework of clear adaptive hypotheses. In the snail Theba pisana, the proportion of effectively unbanded shells is higher in exposed habitats than in adjacent acacia thickets, which has been explained by microclimatic selection. Comparisons across an ecotone for 34 consecutive years determined the combined effects on morph frequencies of habitat and changes in weather conditions during summer. The long-term average (±s.e.) frequency of effectively unbanded shells was 0.577±0.011 in the open habitat when compared with 0.353±0.005 in the acacia. The persistent association of shell banding with habitat accounted for 34% of the variation in morph frequencies. Differences among years were also large, representing 23% of the variation. Higher proportions of effectively unbanded snails were associated with hotter, sunnier summers. Thus, temporal variation supports the hypothesis of microclimatic selection, consistent with the spatial association with habitat. Based on observed rates of change, the mean annual selection on this polymorphism was about 0.13, but with a large variance: s was as high as 0.5, but ⩽0.05 in about 40% of the years. The large variance and frequent reversals in direction of selection indicate a potential for rapid genetic change, but with little net change in morph frequencies over three decades, highlighting the value of long-term continuous studies of populations facing natural environmental variation.     

Cost-Effectiveness of an Intervention to Reduce Emergency Re-Admissions to Hospital among Older Patients

Background

The objective is to estimate the cost-effectiveness of an intervention that reduces hospital re-admission among older people at high risk. A cost-effectiveness model to estimate the costs and health benefits of the intervention was implemented.

Methodology/Principal Findings

The model used data from a randomised controlled trial conducted in an Australian tertiary metropolitan hospital. Participants were acute medical admissions aged >65 years with at least one risk factor for re-admission: multiple comorbidities, impaired functionality, aged >75 years, recent multiple admissions, poor social support, history of depression. The intervention was a comprehensive nursing and physiotherapy assessment and an individually tailored program of exercise strategies and nurse home visits with telephone follow-up; commencing in hospital and continuing following discharge for 24 weeks. The change to cost outcomes, including the costs of implementing the intervention and all subsequent use of health care services, and, the change to health benefits, represented by quality adjusted life years, were estimated for the intervention as compared to existing practice. The mean change to total costs and quality adjusted life years for an average individual over 24 weeks participating in the intervention were: cost savings of $333 (95% Bayesian credible interval $ -1,932∶1,282) and 0.118 extra quality adjusted life years (95% Bayesian credible interval 0.1∶0.136). The mean net-monetary-benefit per individual for the intervention group compared to the usual care condition was $7,907 (95% Bayesian credible interval $5,959∶$9,995) for the 24 week period.

Conclusions/Significance

The estimation model that describes this intervention predicts cost savings and improved health outcomes. A decision to remain with existing practices causes unnecessary costs and reduced health. Decision makers should consider adopting this program for elderly hospitalised patients.     

Does food shortage delay development of homeothermy in European shag nestlings (<Emphasis Type="Italic">Phalacrocorax aristotelis</Emphasis>)?

Nestlings seem to face a trade-off between reducing the basal level of energy metabolism, as an energy-saving response, and maintaining thermogenic capacity during temporal food shortage. In the present study we examined developmental responses to short-term diet restriction of 12–16 day old nestling European shags kept under laboratory conditions and tested whether temporal food shortage delay the development of homeothermy. During food shortage the European shag nestlings substantially reduced basal level of energy metabolism, resulting in significant energy savings. The reduction in basal level of energy metabolism corresponded with a reduction in peak metabolic rate. At the same time, the low peak metabolic rate of diet-restricted nestlings was offset by a lower mass-specific minimal thermal conductance, and an increased mass-specific absolute scope. Consequently, the insulation and the portion of peak metabolic rate available for regulatory thermogenesis seemed to develop normally, as expected from age, during the period of food shortage. Further, the degree of homeothermy, measured as the index of homeothermy, was not significantly lower in diet-restricted nestlings compared to controls at the same age. We conclude that temporal food shortage did not significantly delay the development of homeothermy in the European shag nestlings despite substantial reductions in basal level of energy metabolism and peak metabolic rate.Communicated by: G. Heldmaier     

The Long-Term Effects of a Peer-Led Sex Education Programme (RIPPLE): A Cluster Randomised Trial in Schools in England

Background

Peer-led sex education is widely believed to be an effective approach to reducing unsafe sex among young people, but reliable evidence from long-term studies is lacking. To assess the effectiveness of one form of school-based peer-led sex education in reducing unintended teenage pregnancy, we did a cluster (school) randomised trial with 7 y of follow-up.

Methods and Findings

Twenty-seven representative schools in England, with over 9,000 pupils aged 13–14 y at baseline, took part in the trial. Schools were randomised to either peer-led sex education (intervention) or to continue their usual teacher-led sex education (control). Peer educators, aged 16–17 y, were trained to deliver three 1-h classroom sessions of sex education to 13- to 14-y-old pupils from the same schools. The sessions used participatory learning methods designed to improve the younger pupils'' skills in sexual communication and condom use and their knowledge about pregnancy, sexually transmitted infections (STIs), contraception, and local sexual health services. Main outcome measures were abortion and live births by age 20 y, determined by anonymised linkage of girls to routine (statutory) data. Assessment of these outcomes was blind to sex education allocation. The proportion of girls who had one or more abortions before age 20 y was the same in each arm (intervention, 5.0% [95% confidence interval (CI) 4.0%–6.3%]; control, 5.0% [95% CI 4.0%–6.4%]). The odds ratio (OR) adjusted for randomisation strata was 1.07 (95% CI 0.80–1.42, p = 0.64, intervention versus control). The proportion of girls with one or more live births by 20.5 y was 7.5% (95% CI 5.9%–9.6%) in the intervention arm and 10.6% (95% CI 6.8%–16.1%) in the control arm, adjusted OR 0.77 (0.51–1.15). Fewer girls in the peer-led arm self-reported a pregnancy by age 18 y (7.2% intervention versus 11.2% control, adjusted OR 0.62 [95% CI 0.42–0.91], weighted for non-response; response rate 61% intervention, 45% control). There were no significant differences for girls or boys in self-reported unprotected first sex, regretted or pressured sex, quality of current sexual relationship, diagnosed sexually transmitted diseases, or ability to identify local sexual health services.

Conclusion

Compared with conventional school sex education at age 13–14 y, this form of peer-led sex education was not associated with change in teenage abortions, but may have led to fewer teenage births and was popular with pupils. It merits consideration within broader teenage pregnancy prevention strategies.

Trial registration:

ISRCTN (ISRCTN94255362).     

The Grey Mouse Lemur Uses Season-Dependent Fat or Protein Sparing Strategies to Face Chronic Food Restriction

During moderate calorie restriction (CR) the heterotherm Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days (winter; SD40 and SD80, respectively) and long-days (summer; LD40 and LD80, respectively) acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.     

Cost Effectiveness of Seasonal Intermittent Preventive Treatment Using Amodiaquine & Artesunate or Sulphadoxine-Pyrimethamine in Ghanaian Children

Background

Intermittent preventive treatment for malaria in children (IPTc) involves the administration of a full course of an anti-malarial treatment to children under 5 years old at specified time points regardless of whether or not they are known to be infected, in areas where malaria transmission is seasonal. It is important to determine the costs associated with IPTc delivery via community based volunteers and also the potential savings to health care providers and caretakers due to malaria episodes averted as a consequence of IPTc.

Methods

Two thousand four hundred and fifty-one children aged 3–59 months were randomly allocated to four groups to receive: three days of artesunate plus amodiaquine (AS+AQ) monthly, three days of AS+AQ bimonthly, one dose of sulphadoxine-pyrimethamine (SP) bi-monthly or placebo. This paper focuses on incremental cost effectiveness ratios (ICERs) of the three IPTc drug regimens as delivered by community based volunteers (CBV) in Hohoe, Ghana compared to current practice, i.e. case management in the absence of IPTc. Financial and economic costs from the publicly funded health system perspective are presented. Treatment costs borne by patients and their caretakers are also estimated to present societal costs. The costs and effects of IPTc during the intervention period were considered with and without a one year follow up. Probabilistic sensitivity analysis was undertaken to account for uncertainty.

Results

Economic costs per child receiving at least the first dose of each course of IPTc show SP bimonthly, at US$8.19, is the cheapest to deliver, followed by AS+AQ bimonthly at US$10.67 and then by AS+AQ monthly at US$14.79. Training, drug delivery and supervision accounted for approximately 20–30% each of total unit costs. During the intervention period AS & AQ monthly was the most cost effective IPTc drug regimen at US$67.77 (61.71–74.75, CI 95%) per malaria case averted based on intervention costs only, US$64.93 (58.92–71.92, CI 95%) per malaria case averted once the provider cost savings are included and US$61.00 (54.98, 67.99, CI 95%) when direct household cost savings are also taken into account. SP bimonthly was US$105.35 (75.01–157.31, CI 95%) and AS & AQ bimonthly US$211.80 (127.05–399.14, CI 95%) per malaria case averted based on intervention costs only. The incidence of malaria in the post intervention period was higher in children who were <1 year old when they received AS+AQ monthly compared to the placebo group leading to higher cost effectiveness ratios when one year follow up is included. The cost per child enrolled fell considerably when modelled to district level as compared to those encountered under trial conditions.

Conclusions

We demonstrate how cost-effective IPTc is using three different drug regimens and the possibilities for reducing costs further if the intervention was to be scaled up to the district level. The need for effective training, drug delivery channels and supervision to support a strong network of community based volunteers is emphasised.     

Systematic review of dietary intervention trials to lower blood total cholesterol in free-living subjects

Objectives: To estimate the efficacy of dietary advice to lower blood total cholesterol concentration in free-living subjects and to investigate the efficacy of different dietary recommendations. Design: Systematic overview of 19 randomised controlled trials including 28 comparisons. Subjects: Free-living subjects. Interventions: Individualised dietary advice to modify fat intake. Main outcome measure: Percentage difference in blood total cholesterol concentration between the intervention and control groups. Results: The percentage reduction in blood total cholesterol attributable to dietary advice after at least six months of intervention was 5.3% (95% confidence interval 4.7% to 5.9%). Including both short and long duration studies, the effect was 8.5% at 3 months and 5.5% at 12 months. Diets equivalent to the step 2 diet of the American Heart Association were of similar efficacy to diets that aimed to lower total fat intake or to raise the polyunsaturated to saturated fatty acid ratio. These diets were moderately more effective than the step 1 diet of the American Heart Association (6.1% v 3.0% reduction in blood total cholesterol concentration; P<0.0001). On the basis of reported food intake, the targets for dietary change were seldom achieved. The observed reductions in blood total cholesterol concentrations in the individual trials were consistent with those predicted from dietary intake on the basis of the Keys equation. Conclusions: Individualised dietary advice for reducing cholesterol concentration is modestly effective in free-living subjects. More intensive diets achieve a greater reduction in serum cholesterol concentration. Failure to comply fully with dietary recommendations is the likely explanation for this limited efficacy.

Key messages

• Results from metabolic ward studies suggest that dietary change can reduce blood cholesterol concentrations by up to 15%
• In free-living subjects the standard step 1 diet of the American Heart Association lowers cholesterol concentration by about 3%, and about another 3% can be achieved with more intensive diets
• Difficulties in complying with the prescribed dietary change explain the failure to achieve the expected reductions in cholesterol concentrations
• It is important to be realistic about the reductions in cardiovascular risk that can be achieved by individual dietary counselling

     

Spatial and Genetic Epidemiology of Hookworm in a Rural Community in Uganda

There are remarkably few contemporary, population-based studies of intestinal nematode infection for sub-Saharan Africa. This paper presents a comprehensive epidemiological analysis of hookworm infection intensity in a rural Ugandan community. Demographic, kinship, socioeconomic and environmental data were collected for 1,803 individuals aged six months to 85 years in 341 households in a cross-sectional community survey. Hookworm infection was assessed by faecal egg count. Spatial variation in the intensity of infection was assessed using a Bayesian negative binomial spatial regression model and the proportion of variation explained by host additive genetics (heritability) and common domestic environment was estimated using genetic variance component analysis. Overall, the prevalence of hookworm was 39.3%, with the majority of infections (87.7%) of light intensity (≤1000 eggs per gram faeces). Intensity was higher among older individuals and was associated with treatment history with anthelmintics, walking barefoot outside the home, living in a household with a mud floor and education level of the household head. Infection intensity also exhibited significant household and spatial clustering: the range of spatial correlation was estimated to be 82 m and was reduced by a half over a distance of 19 m. Heritability of hookworm egg count was 11.2%, whilst the percentage of variance explained by unidentified domestic effects was 17.8%. In conclusion, we suggest that host genetic relatedness is not a major determinant of infection intensity in this community, with exposure-related factors playing a greater role.     

Temporal variation overshadows the response of leaf litter microbial communities to simulated global change

Bacteria and fungi drive the decomposition of dead plant biomass (litter), an important step in the terrestrial carbon cycle. Here we investigate the sensitivity of litter microbial communities to simulated global change (drought and nitrogen addition) in a California annual grassland. Using 16S and 28S rDNA amplicon pyrosequencing, we quantify the response of the bacterial and fungal communities to the treatments and compare these results to background, temporal (seasonal and interannual) variability of the communities. We found that the drought and nitrogen treatments both had significant effects on microbial community composition, explaining 2–6% of total compositional variation. However, microbial composition was even more strongly influenced by seasonal and annual variation (explaining 14–39%). The response of microbial composition to drought varied by season, while the effect of the nitrogen addition treatment was constant through time. These compositional responses were similar in magnitude to those seen in microbial enzyme activities and the surrounding plant community, but did not correspond to a consistent effect on leaf litter decomposition rate. Overall, these patterns indicate that, in this ecosystem, temporal variability in the composition of leaf litter microorganisms largely surpasses that expected in a short-term global change experiment. Thus, as for plant communities, future microbial communities will likely be determined by the interplay between rapid, local background variability and slower, global changes.     

Seasonal Intermittent Preventive Treatment for the Prevention of Anaemia and Malaria in Ghanaian Children: A Randomized,Placebo Controlled Trial

Background

Malaria and anaemia are the leading causes of morbidity and mortality in children in sub-Saharan Africa. We have investigated the effect of intermittent preventive treatment with sulphadoxine-pyrimethamine or artesunate plus amodiaquine on anaemia and malaria in children in an area of intense, prolonged, seasonal malaria transmission in Ghana.

Methods

2451 children aged 3–59 months from 30 villages were individually randomised to receive placebo or artesunate plus amodiaquine (AS+AQ) monthly or bimonthly, or sulphadoxine-pyrimethamine (SP) bimonthly over a period of six months. The primary outcome measures were episodes of anaemia (Hb<8.0 g/dl) or malaria detected through passive surveillance.

Findings

Monthly artesunate plus amodiaquine reduced the incidence of malaria by 69% (95% CI: 63%, 74%) and anaemia by 45% (95% CI: 25%,60%), bimonthly sulphadoxine-pyrimethamine reduced the incidence of malaria by 24% (95% CI: 14%,33%) and anaemia by 30% (95% CI: 6%, 49%) and bimonthly artesunate plus amodiaquine reduced the incidence of malaria by 17% (95% CI: 6%, 27%) and anaemia by 32% (95% CI: 7%, 50%) compared to placebo. There were no statistically significant reductions in the episodes of all cause or malaria specific hospital admissions in any of the intervention groups compared to the placebo group. There was no significant increase in the incidence of clinical malaria in the post intervention period in children who were >1 year old when they received IPTc compared to the placebo group. However the incidence of malaria in the post intervention period was higher in children who were <1 year old when they received AS+AQ monthly compared to the placebo group.

Interpretation

IPTc is safe and efficacious in reducing the burden of malaria in an area of Ghana with a prolonged, intense malaria transmission season.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00119132","term_id":"NCT00119132"}}NCT00119132     

A technique for conditioning and calibrating force-sensing resistors for repeatable and reliable measurement of compressive force

Miniature sensors that could measure forces applied by the fingers and hand without interfering with manual dexterity or range of motion would have considerable practical value in ergonomics and rehabilitation. In this study, techniques have been developed to use inexpensive pressure-sensing resistors (FSRs) to accurately measure compression force. The FSRs are converted from pressure-sensing to force-sensing devices. The effects of nonlinear response properties and dependence on loading history are compensated by signal conditioning and calibration. A fourth-order polynomial relating the applied force to the current voltage output and a linearly weighted sum of prior outputs corrects for sensor hysteresis and drift. It was found that prolonged (>20 h) shear force loading caused sensor gain to change by approximately 100%. Shear loading also had the effect of eliminating shear force effects on sensor output, albeit only in the direction of shear loading. By applying prolonged shear loading in two orthogonal directions, the sensors were converted into pure compression sensors. Such preloading of the sensor is, therefore, required prior to calibration. The error in compression force after prolonged shear loading and calibration was consistently <5% from 0 to 30 N and <10% from 30 to 40 N. This novel method of calibrating FSRs for measuring compression force provides an inexpensive tool for biomedical and industrial design applications where measurements of finger and hand force are needed.     

Triage of Women with Minor Cervical Lesions: Data Suggesting a “Test and Treat” Approach for HPV E6/E7 mRNA Testing

Background

Human papillomavirus (HPV) testing is included in the cervical cancer screening program in the triage of women with equivocal (ASC-US) or low-grade (LSIL) cytological lesions. These women have an increased risk for developing high grade dysplasia and cancer (CIN2+) compared to women with normal cytology. However, in order to avoid unnecessary follow-up, as well as overtreatment, a high positive predictive value (PPV) of the triage test is important.

Methodology/Principal Findings

The HPV test PreTect HPV-Proofer, detecting E6/E7 mRNA from the HPV types 16, 18, 31, 33 and 45, is used as triage test together with repeat cytology. PPV data for HPV E6/E7 mRNA testing during the period from January 2006 up to June 2009 are reported. In total, 406 of 2099 women (19.3%) had a positive HPV test result. Of the women with a positive test result and with a histological diagnosis (n = 347), 243 women had histological high-grade dysplasia or cancer (CIN2+), giving a PPV of 70.0% (95% confidence interval [CI], 65.2%–74.8%). For HPV 16 or HPV 33 positive women above 40 years of age, the PPV was 83.7% (95% CI, 73.3%–94.0%) and 84.6% (95% CI, 65.0%–100.0%) respectively. The PPV of test positive women with HSIL cytology was 94.2% (95% CI, 88.7%–99.7%).

Conclusions

When the result in triage is HPV mRNA positive, our data suggest direct treatment for women above 40 years of age or for women with a concurrent cytological HSIL diagnosis, contributing to better clinical safety for these women. In addition, by decreasing the time to treatment, thereby reducing the number of recalls, the patient management algorithm will be considerably improved, in turn reducing follow-up costs as well as unnecessary psychological stress among patients.     

Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling

The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott-Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. In vitro, the Cdc42 sensors produce up to 3.2-fold FRET emission ratio changes upon binding active Cdc42. The GEF sensors yield up to 1.7-fold changes in FRET upon exchange of GDP for GTP. The GEF-catalyzed rate of nucleotide exchange for the GEF sensor is indistinguishable from that of wild-type Cdc42, but GAP-catalyzed nucleotide hydrolysis is slowed approximately 16-fold. In vivo, both sensors faithfully report on Cdc42 and/or Cdc42-GEF activity. These results establish the successful creation of rationally designed and genetically encoded tools that can be used to image the activity of biologically and medically important molecules in living systems.     

The Potential Involvement of E-cadherin and β-catenins in Meningioma

Objective

To investigate the potential involvements of E-cadherin and β-catenin in meningioma.

Methods

Immunohistochemistry staining was performed on samples from patients with meningioma. The results were graded according to the positive ratio and intensity of tissue immunoreactivity. The expression of E-cadherin and β-catenin in meningioma was analyzed by its relationship with WHO2007 grading, invasion, peritumoral edema and postoperative recurrence.

Results

The positive rates of E-cadherin in meningioma WHO I, II, III were 92.69%, 33.33% and 0, respectively, (P<0.05); while the positive rates of β-catenin in meningioma WHO I, II, III were 82.93%, 33.33% and 20.00%, respectively, (P<0.05). The positive rate of E-cadherin in meningioma without invasion (94.12%) was higher than that with invasion (46.67%) (P<0.05). The difference in the positive rate of β-catenin between meningioma without invasion (88.24%) and meningioma with invasion (33.33%, P<0.05) was also statically significant. The positive rates of E-cadherin in meningioma with peritumoral edema 0, 1, 2, 3 were 93.75%, 85.71%, 60.00% and 0 respectively, (P<0.05); the positive rates of β-catenin in meningioma with peritumoral edema 0, 1, 2, 3 were 87.50%, 85.71%, 30.00% and 0 respectively, (P<0.01). The positive rates of E- cadherin in meningioma with postoperative recurrence were 33.33%, and the positive rate with postoperative non-recurrence was 90.00% (P<0.01). The positive rates of β-catenin in meningioma with postoperative recurrence and non-recurrence were 11.11%, 85.00%, respectively (P<0.01).

Conclusion

The expression levels of E- cadherin and β-catenin correlated closely to the WHO 2007 grading criteria for meningioma. In atypical or malignant meningioma, the expression levels of E-cadherin and β-catenin were significantly lower. The expression levels of E- cadherin and β-catenin were also closely correlated with the invasion status of meningioma, the size of the peritumoral edema and the recurrent probabilities of the meningioma, all in an inverse correlationship. Taken together, the present study provided novel molecular targets in clinical treatments to meningioma.     

Modeling HIV Vaccines in Brazil: Assessing the Impact of a Future HIV Vaccine on Reducing New Infections,Mortality and Number of People Receiving ARV

Background

The AIDS epidemic in Brazil remains concentrated in populations with high vulnerability to HIV infection, and the development of an HIV vaccine could make an important contribution to prevention. This study modeled the HIV epidemic and estimated the potential impact of an HIV vaccine on the number of new infections, deaths due to AIDS and the number of people receiving ARV treatment, under various scenarios.

Methods and Findings

The historical HIV prevalence was modeled using Spectrum and projections were made from 2010 to 2050 to study the impact of an HIV vaccine with 40% to 70% efficacy, and 80% coverage of adult population, specific groups such as MSM, IDU, commercial sex workers and their partners, and 15 year olds. The possibility of disinhibition after vaccination, neglecting medium- and high-risk groups, and a disease-modifying vaccine were also considered. The number of new infections and deaths were reduced by 73% and 30%, respectively, by 2050, when 80% of adult population aged 15–49 was vaccinated with a 40% efficacy vaccine. Vaccinating medium- and high-risk groups reduced new infections by 52% and deaths by 21%. A vaccine with 70% efficacy produced a great decline in new infections and deaths. Neglecting medium- and high-risk population groups as well as disinhibition of vaccinated population reduced the impact or even increased the number of new infections. Disease-modifying vaccine also contributed to reducing AIDS deaths, the need for ART and new HIV infections.

Conclusions

Even in a country with a concentrated epidemic and high levels of ARV coverage, such as Brazil, moderate efficacy vaccines as part of a comprehensive package of treatment and prevention could have a major impact on preventing new HIV infections and AIDS deaths, as well as reducing the number of people on ARV. Targeted vaccination strategies may be highly effective and cost-beneficial.     

Comparison of Free-Living Amoebae in Hot Water Systems of Hospitals with Isolates from Moist Sanitary Areas by Identifying Genera and Determining Temperature Tolerance

Legionella-contaminated hot water systems and moist sanitary areas in six hospitals were sampled for amoebae by following a standardized collection protocol. Genus identifications and temperature tolerance determinations were made. Amoebae identified as Hartmannella vermiformis (65%), Echinamoebae spp. (15%), Saccamoebae spp. (12%), and Vahlkampfia spp. (9%) were detected in 29 of 56 (52%) hot water samples. Twenty-three of 49 (47%) swabs obtained from moist areas were amoeba positive. The following genera were identified: Acanthamoeba (22%), Naegleria (22%), Vahlkampfia (20%), Hartmannella (15%), and Vanella (7%). The temperature tolerance of amoebae from hot water systems was strikingly different from that of amoebae from moist areas. At 44°C on agar, 59% of amoebic isolates sampled from hot water systems showed growth. The corresponding value for isolates from moist areas was only 17%. Six Acanthamoeba isolates from the moist areas were considered potential pathogens. Four Hartmannella and two Saccamoeba isolates from hot water could be cultured at 53°C.     

Short-term observations of marine bacterial and viral communities: patterns,connections and resilience

Observation of short-term temporal variation in bacterial and viral communities is important for understanding patterns of aquatic microbial diversity. We collected surface seawater once daily for 38 consecutive days with seven more samples interspersed over 40 more days at one location ∼2 km from Santa Catalina Island, California. Bacterial communities were analyzed by automated ribosomal intergenic spacer analysis (ARISA) and viral communities were analyzed by terminal restriction fragment length polymorphism (TRFLP) of the conserved T4-like myoviral gene encoding the major capsid protein (g23). Common bacterial and viral taxa were consistently dominant, and relatively few displayed dramatic increases/decreases or ‘boom/bust'' patterns that might be expected from dynamic predator-prey interactions. Association network analysis showed most significant covariations (associations) occurred among bacterial taxa or among viral taxa and there were several modular (highly-interconnected) associations (P⩽0.005). Associations observed between bacteria and viruses (P⩽0.005) occurred with a median time lag of 2 days. Regression of all pairwise Bray-Curtis similarities between samples indicated a rate of bacterial community change that slows from 2.1%–0.18% per day over a week to 2 months; the rate stays around 0.4% per day for viruses. Our interpretation is that, over the scale of days, individual bacterial and viral OTUs can be dynamic and patterned; resulting in statistical associations regarded as potential ecological interactions. However, over the scale of weeks, average bacterial community variation is slower, suggesting that there is strong community-level ecological resilience, that is, a tendency to converge towards a ‘mean'' microbial community set by longer-term controlling factors.     

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background

Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination.

Methodology/Principal Findings

We applied BRET to develop an autoluminescent Ca²⁺ indicator, BRAC, which is composed of Ca²⁺-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein''s chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca²⁺ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca²⁺-independent signal drifts due to change in cell shape, focus shift, etc.

Conclusions/Significance

The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca²⁺ imaging not only in single live cells but also in small living subjects.     

Unique Flexibility in Energy Metabolism Allows Mycobacteria to Combat Starvation and Hypoxia

Mycobacteria are a group of obligate aerobes that require oxygen for growth, but paradoxically have the ability to survive and metabolize under hypoxia. The mechanisms responsible for this metabolic plasticity are unknown. Here, we report on the adaptation of Mycobacterium smegmatis to slow growth rate and hypoxia using carbon-limited continuous culture. When M. smegmatis is switched from a 4.6 h to a 69 h doubling time at a constant oxygen saturation of 50%, the cells respond through the down regulation of respiratory chain components and the F₁F_o-ATP synthase, consistent with the cells lower demand for energy at a reduced growth rate. This was paralleled by an up regulation of molecular machinery that allowed more efficient energy generation (i.e. Complex I) and the use of alternative electron donors (e.g. hydrogenases and primary dehydrogenases) to maintain the flow of reducing equivalents to the electron transport chain during conditions of severe energy limitation. A hydrogenase mutant showed a 40% reduction in growth yield highlighting the importance of this enzyme in adaptation to low energy supply. Slow growing cells at 50% oxygen saturation subjected to hypoxia (0.6% oxygen saturation) responded by switching on oxygen scavenging cytochrome bd, proton-translocating cytochrome bc₁-aa₃ supercomplex, another putative hydrogenase, and by substituting NAD⁺-dependent enzymes with ferredoxin-dependent enzymes thus highlighting a new pattern of mycobacterial adaptation to hypoxia. The expression of ferredoxins and a hydrogenase provides a potential conduit for disposing of and transferring electrons in the absence of exogenous electron acceptors. The use of ferredoxin-dependent enzymes would allow the cell to maintain a high carbon flux through its central carbon metabolism independent of the NAD⁺/NADH ratio. These data demonstrate the remarkable metabolic plasticity of the mycobacterial cell and provide a new framework for understanding their ability to survive under low energy conditions and hypoxia.     

Rapid Scaling Up of Insecticide-Treated Bed Net Coverage in Africa and Its Relationship with Development Assistance for Health: A Systematic Synthesis of Supply,Distribution, and Household Survey Data

Background

Development assistance for health (DAH) targeted at malaria has risen exponentially over the last 10 years, with a large fraction of these resources directed toward the distribution of insecticide-treated bed nets (ITNs). Identifying countries that have been successful in scaling up ITN coverage and understanding the role of DAH is critical for making progress in countries where coverage remains low. Sparse and inconsistent sources of data have prevented robust estimates of the coverage of ITNs over time.

Methods and Principal Findings

We combined data from manufacturer reports of ITN deliveries to countries, National Malaria Control Program (NMCP) reports of ITNs distributed to health facilities and operational partners, and household survey data using Bayesian inference on a deterministic compartmental model of ITN distribution. For 44 countries in Africa, we calculated (1) ITN ownership coverage, defined as the proportion of households that own at least one ITN, and (2) ITN use in children under 5 coverage, defined as the proportion of children under the age of 5 years who slept under an ITN. Using regression, we examined the relationship between cumulative DAH targeted at malaria between 2000 and 2008 and the change in national-level ITN coverage over the same time period. In 1999, assuming that all ITNs are owned and used in populations at risk of malaria, mean coverage of ITN ownership and use in children under 5 among populations at risk of malaria were 2.2% and 1.5%, respectively, and were uniformly low across all 44 countries. In 2003, coverage of ITN ownership and use in children under 5 was 5.1% (95% uncertainty interval 4.6% to 5.7%) and 3.7% (2.9% to 4.9%); in 2006 it was 17.5% (16.4% to 18.8%) and 12.9% (10.8% to 15.4%); and by 2008 it was 32.8% (31.4% to 34.4%) and 26.6% (22.3% to 30.9%), respectively. In 2008, four countries had ITN ownership coverage of 80% or greater; six countries were between 60% and 80%; nine countries were between 40% and 60%; 12 countries were between 20% and 40%; and 13 countries had coverage below 20%. Excluding four outlier countries, each US$1 per capita in malaria DAH was associated with a significant increase in ITN household coverage and ITN use in children under 5 coverage of 5.3 percentage points (3.7 to 6.9) and 4.6 percentage points (2.5 to 6.7), respectively.

Conclusions

Rapid increases in ITN coverage have occurred in some of the poorest countries, but coverage remains low in large populations at risk. DAH targeted at malaria can lead to improvements in ITN coverage; inadequate financing may be a reason for lack of progress in some countries. Please see later in the article for the Editors'' Summary