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.     

Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

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.     

Biochar and alternate partial root-zone irrigation greatly enhance the effectiveness of mulberry in remediating lead-contaminated soils

土壤铅污染日益严重,植物修复是一种环保的污染土壤修复技术。本文旨在研究四种土壤铅污染水平下,添加生物碳和分根区交替 灌溉(Alternative Partial Root-zone Irrigation,APRI)对桑树幼苗的生长、铅适应性和铅积累的影响。我们以生物碳(添加与不添加生物碳)、灌溉方式(APRI 与常规灌溉)和土壤铅水平(0、50、200 和800 mg kg⁻¹ Pb)为三因素实施了温室试验。通过测定桑树幼苗的生长性状、渗透物质代谢、抗氧化酶活性、铅的积累和转运等参数,探讨了不同处理对桑树生长发育的影响。结果表明,桑树对土壤铅污染有较强的适应能力;生物碳和APRI 在不同土壤铅水平上协同提高了生物量和吸收根表面积。桑树通过调节谷胱甘肽 (GSH)、脯氨酸代谢和过氧化物酶(POD)活性,增加了渗透和抗氧化调节能力,进而提高了对重度铅污染土壤(800 mg kg⁻¹)的抗性。桑苗中的铅离子主要集中在根中,与土壤铅浓度具有剂量效应。土壤铅、生物碳和ARPI的交互作用影响了叶片和根系中铅的浓度、转运和生物富集系数。综上所述,在桑树栽培中结合外源生物碳和APRI可有效地用于修复土壤铅污染。     

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.     

Naturally Occurring Bacteria Similar to the Methyl tert-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10³ to 10⁴ cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 μg of MTBE/liter, densities of native PM1 increased to approximately 10⁵ cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.