Minor and trace sterols in marine invertebrates 55. The isolation, structure elucidation and synthesis of ergosta-5, 24(28), 25-trien-3 beta-ol

The isolation, structure determination and synthesis of ergosta-5, 24(28), 25-trien-3 beta-ol, as well as the synthesis of its 28-14C analog--a possible biosynthetic precursor of several marine sterols--is described.     

Characterization of the genetic system of the xylose-fermenting yeast Pichia stipitis

High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads. Received: 29 February 1996 / Accepted: 29 March 1996     

Selective protection of restriction endonuclease sites

A difficulty that is encountered when attempting to insert a PCR-amplified product or DNA fragment of interest into a particular vector is the presence within the insert of one or more internal restriction endonuclease (RE) sites identical to those selected for the flanks of the insert. Our method circumvents this problem by partially protecting internal RE sites while flanking sites for the same RE are cleaved. The amplified product is first heat denatured in the presence of excess amounts of perfectly complementary oligonucleotides that can anneal to the flanks of the insert. The mixture is allowed to anneal and is subsequently digested with the appropriate endonucleases. This results in the cleavage of the flanking RE sites while digestion at the internal RE site is not efficient. The mixture is subsequently heat denatured and column purified to remove the oligonucleotides. The product is then allowed to anneal and can be used directly in a ligation reaction with the plasmid vector. This method facilitates the construction of recombinant molecules by creating desired flanks while preserving internal RE sites.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR 15N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Time Series Analysis of Trends in Malaria Cases and Deaths at Hospitals and the Effect of Antimalarial Interventions, 2001–2011, Ethiopia

Background

The Government of Ethiopia and its partners have deployed artemisinin-based combination therapies (ACT) since 2004 and long-lasting insecticidal nets (LLINs) since 2005. Malaria interventions and trends in malaria cases and deaths were assessed at hospitals in malaria transmission areas during 2001–2011.

Methods

Regional LLINs distribution records were used to estimate the proportion of the population-at-risk protected by LLINs. Hospital records were reviewed to estimate ACT availability. Time-series analysis was applied to data from 41 hospitals in malaria risk areas to assess trends of malaria cases and deaths during pre-intervention (2001–2005) and post-interventions (2006–2011) periods.

Findings

The proportion of the population-at-risk potentially protected by LLINs increased to 51% in 2011. The proportion of facilities with ACTs in stock exceeded 87% during 2006–2011. Among all ages, confirmed malaria cases in 2011 declined by 66% (95% confidence interval [CI], 44–79%) and SPR by 37% (CI, 20%–51%) compared to the level predicted by pre-intervention trends. In children under 5 years of age, malaria admissions and deaths fell by 81% (CI, 47%–94%) and 73% (CI, 48%–86%) respectively. Optimal breakpoint of the trendlines occurred between January and June 2006, consistent with the timing of malaria interventions. Over the same period, non-malaria cases and deaths either increased or remained unchanged, the number of malaria diagnostic tests performed reflected the decline in malaria cases, and rainfall remained at levels supportive of malaria transmission.

Conclusions

Malaria cases and deaths in Ethiopian hospitals decreased substantially during 2006–2011 in conjunction with scale-up of malaria interventions. The decrease could not be accounted for by changes in hospital visits, malaria diagnostic testing or rainfall. However, given the history of variable malaria transmission in Ethiopia, more data would be required to exclude the possibility that the decrease is due to other factors.     

A single 24 h recall overestimates exclusive breastfeeding practices among infants aged less than six months in rural Ethiopia

Background

Exclusive breastfeeding (EBF) to six months is one of the World Health Organization’s (WHOs) infant and young child feeding (IYCF) core indicators. Single 24 h recall method is currently in use to measure exclusive breastfeeding practice among children of age less than six months. This approach overestimates the prevalence of EBF, especially among small population groups. This justifies the need to look for alternative measurement techniques to have a valid estimate regardless of population characteristics.

Method

The study involved 422 infants of age less than six months, living in Gurage zone, Southern Ethiopia. The study was conducted from January to February 2016. Child feeding practices were measured for seven consecutive days using 24 h recall method. Recall since birth, was used to measure breastfeeding practices from birth to the day of data collection. Data on EBF obtained by using single 24 h recall were compared with seven days repeated 24 h recall method. McNemar’s test was done to assess if a significant difference existed in rates of EBF between measurement methods.

Result

The mean age of infants in months was 3 (SD ?1.43). Exclusive breastfeeding prevalence was highest (76.7%; 95% CI 72.6, 80.8) when EBF was estimated using single 24 h recall. The prevalence of EBF based on seven repeated 24 h recall was 53.2% (95% CI: 48.3, 58.0). The estimated prevalence of EBF since birth based on retrospective data (recall since birth) was 50.2% (95% CI 45.4, 55.1). Compared to the EBF estimates obtained from seven repeated 24 h recall, single 24 h recall overestimated EBF magnitude by 23 percentage points (95% CI 19.2, 27.8). As the number of days of 24 h recall increased, a significant decrease in overestimation of EBF was observed.

Conclusion

A significant overestimation was observed when single 24 h recall was used to estimate prevalence of EBF compared to seven days of 24 h recall. By increasing the observation days we can significantly decrease the degree of overestimation. Recall since birth presented estimates of EBF that is close to seven repeated 24 h recall. This suggests that a week recall could be an alternative indicator to single 24 h recall.

     

Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10⁵ cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH₄)₂SO₄ application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10⁶ cells/g of dry soil to peak values (day 7) of 35 × 10⁶ and 66 × 10⁶ cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10⁹ and 2.2 × 10⁹ cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10⁶ to 38.0 × 10⁶ cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH₄⁺ h⁻¹ cell⁻¹ and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10⁶, 17.5 × 10⁶, and 1.7 × 10⁶ cells/mol of NH₄⁺ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.     

Marine 4-methyl sterols: synthesis of C-24 epimers of 4 alpha, 24-dimethyl-5 alpha-cholestan-3 beta-ol and 360 MHz 1HNMR comparisons to the natural product from Plexaura homomalla

Comparison of the highfield 1HNMR spectrum of 4 alpha, 24-dimethyl-5 alpha-cholestan-3 beta-o1 isolated by open column adsorptive chromatography and reversed-phase HPLC from P. homomalla with those of the corresponding synthetic 24 alpha and 24 beta compounds demonstrate that the gorgonian natural product is purely 24 beta, the same C-24 configuration found in sterols related to dinosterol and gorgosterol. 360 MHz 1HNMR data are also reported for synthetic 4 alpha, 24 beta-dimethyl-5 alpha-cholest-22E-en-3 beta-o1 (another P. homomalla natural product). The use of 1HNMR correlations in assigning C-24 configurations of 24-methyl marine sterols possessing various nuclei is examined and discussed. Analyses of the methyl sterol components of P. homomalla are tabulated and discussed with regard to origin and plausible biosynthetic interrelationships in light of the C-24 configurational findings.     

A Qualitative Study Exploring Barriers Related to Use of Footwear in Rural Highland Ethiopia: Implications for Neglected Tropical Disease Control

Background

The role of footwear in protection against a range of Neglected Tropical Diseases (NTDs) is gaining increasing attention. Better understanding of the behaviors that influence use of footwear will lead to improved ability to measure shoe use and will be important for those implementing footwear programs.

Methodology/Principal Findings

Using the PRECEDE-PROCEED model we assessed social, behavioral, environmental, educational and ecological needs influencing whether and when children wear shoes in a rural highland Ethiopian community endemic for podoconiosis. Information was gathered from 242 respondents using focus groups, semi-structured interviews and extended case studies. Shoe-wearing norms were said to be changing, with going barefoot increasingly seen as ‘shameful’. Shoes were thought to confer dignity as well as protection against injury and cold. However, many practical and social barriers prevented the desire to wear shoes from being translated into practice. Limited financial resources meant that people were neither able to purchase more than one pair of shoes to ensure their longevity nor afford shoes of the preferred quality. As a result of this limited access, shoes were typically preserved for special occasions and might not be provided for children until they reached a certain age. While some barriers (for example fit of shoe and fear of labeling through use of a certain type of shoe) may be applicable only to certain diseases, underlying structural level barriers related to poverty (for example price, quality, unsuitability for daily activities and low risk perception) are likely to be relevant to a range of NTDs.

Conclusions/Significance

Using well established conceptual models of health behavior adoption, we identified several barriers to shoe wearing that are amenable to intervention and which we anticipate will be of benefit to those considering NTD prevention through shoe distribution.     

High risk of early sub-therapeutic penicillin concentrations after intramuscular benzathine penicillin G injections in Ethiopian children and adults with rheumatic heart disease

IntroductionIntramuscular benzathine penicillin G (BPG) injections are a cornerstone of secondary prophylaxis to prevent acute rheumatic fever (ARF) and rheumatic heart disease (RHD). Uncertainties regarding inter-ethnic and preparation variability, and target exposure profiles of BPG injection are key knowledge gaps for RHD control.MethodsTo evaluate BPG pharmacokinetics (PK) in patients receiving 4-weekly doses in Ethiopia, we conducted a prospective cohort study of ARF/RHD patients attending cardiology outpatient clinics. Serum samples were collected weekly for one month after injection and assayed with a liquid chromatography-mass spectroscopy assay. Concentration-time datasets for BPG were analyzed by nonlinear mixed effects modelling using NONMEM.ResultsA total of 190 penicillin concentration samples from 74 patients were included in the final PK model. The median age, weight, BMI was 21 years, 47 kg and 18 kg/m², respectively. When compared with estimates derived from Indigenous Australian patients, the estimate for median (95% confidence interval) volume of distribution (V/F) was lower (54.8 [43.9–66.3] l.70kg^-1) whilst the absorption half-life (t_1/2-abs2) was longer (12.0 [8.75–17.7] days). The median (IQR) percentage of time where the concentrations remained above 20 ng/mL and 10 ng/mL within the 28-day treatment cycle was 42.5% (27.5–60) and 73% (58.5–99), respectively.ConclusionsThe majority of Ethiopian patients receiving BPG as secondary prophylaxis to prevent RHD do not attain target concentrations for more than two weeks during each 4-weekly injection cycle, highlighting the limitations of current BPG strategies. Between-population variation, together with PK differences between different preparations may be important considerations for ARF/RHD control programs.