Enantiomeric Cross-Inhibition in the Synthesis of Oligonucleotides on a Nonchiral Template

Prebiotic syntheses of chiral monomers always yield racemic mixtures. Living systems, however, utilize L-amino acids and D-nucleotides in their biopolymers. The generation of optical asymmetry by selection and amplification in an autocatalytic process is, therefore, an important element in many theories of the origin of life. Replication of polynucleotides in template-directed syntheses is an obvious candidate for such an amplification step in a pre-'RNA world'. A serious objection to this suggestion is the observation that the efficiency of template-directed syntheses of RNA is limited by enantiomeric cross-inhibition. Peptide Nucleic Acids (PNAs), amide-linked, nonchiral analogues of RNA, have been 'copied' into RNA and constitute an alternative to chiral polynucleotides as an informational replicating system. Here, we use PNA as model for a hypothetical, nonchiral precursor of RNA in experiments re-examining enantiomeric cross-inhibition. We find that enantiomeric cross-inhibition is as serious in the polymerization of nucleotides on a PNA template as it is on a conventional RNA or DNA template

Separation of 'Uncharged' Oligodeoxynucleotide Analogs by Anion-Exchange Chromatography at High pH

Ion-exchange chromatography is a well-established method for the analysis and purification of phosphodiester-linked oligonucleotides. If elution is carried out under alkaline conditions, the secondary structure of G- and C-rich oligomers is disrupted. Furthermore, elution times become more sensitive to the G and T content of the oligomer, because G and T are deprotonated at pH 10. In recent work on peptide-nucleic acids (PNAs) we noted that mixtures of PNA oligomers G(sub 4), G(sub 6), G(sub 8), and G9(sub 10) are readily separated by elution at pH 12 on an RPC-5 column. Here we show that this separation method is more generally applicable

Unraveling heme detoxification in the malaria parasite by in situ correlative X-ray fluorescence microscopy and soft X-ray tomography

A key drug target for malaria has been the detoxification pathway of the iron containing molecule heme, which is the toxic byproduct of hemoglobin digestion. The cornerstone of heme detoxification is its sequestration into hemozoin crystals, but how this occurs remains uncertain. We report new results of in vivo rate of heme crystallization in the malaria parasite, based on a new technique to measure element specific concentrations at defined locations in cell ultrastructure. Specifically, a high resolution correlative combination of cryo soft X ray tomography has been developed to obtain 3D parasite ultrastructure with cryo X ray fluorescence microscopy to measure heme concentrations. Our results are consistent with a model for crystallization via the heme detoxification protein. Our measurements also demonstrate the presence of considerable amounts of non crystalline heme in the digestive vacuole, which we show is most likely contained in hemoglobin. These results suggest a tight coupling between hemoglobin digestion and heme crystallization, highlighting a new link in the crystallization pathway for drug developmen

Evaluation of Anthelmintic Resistance and Exhaust Air Dust PCR as a Diagnostic Tool in Mice Enzootically Infected with Aspiculuris tetraptera

The entry of infectious agents in rodent colonies occurs despite robust sentinel monitoring programs, strict quarantine measures, and stringent biosecurity practices. In light of several outbreaks with Aspiculuris tetraptera in our facilities, we investigated the presence of anthelmintic resistance and the use of exhaust air dust (EAD) PCR for early detection of A. tetraptera infection. To determine anthelmintic resistance, C57BL/6, DBA/2, and NCr nude mice were experimentally inoculated with embryonated A. tetraptera ova harvested from enzootically infected mice, followed by treatment with 150 ppm fenbendazole in feed, 150 ppm fenbendazole plus 5 ppm piperazine in feed, or 2.1 mg/mL piperazine in water for 4 or 8 wk. Regardless of the mouse strain or treatment, no A. tetraptera were recovered at necropsy, indicating the lack of resistance in the worms to anthelmintic treatment. In addition, 10 of 12 DBA/2 positive-control mice cleared the A. tetraptera infection without treatment. To evaluate the feasibility of EAD PCR for A. tetraptera, 69 cages of breeder mice enzootically infected with A. tetraptera were housed on a Tecniplast IVC rack as a field study. On day 0, 56% to 58% of the cages on this rack tested positive for A. tetraptera by PCR and fecal centrifugation flotation (FCF). PCR from EAD swabs became positive for A. tetraptera DNA within 1 wk of placing the above cages on the rack. When these mice were treated with 150 ppm fenbendazole in feed, EAD PCR reverted to pinworm-negative after 1 mo of treatment and remained negative for an additional 8 wk. The ability of EAD PCR to detect few A. tetraptera positive mice was investigated by housing only 6 infected mice on another IVC rack as a field study. The EAD PCR from this rack was positive for A. tetraptera DNA within 1 wk of placing the positive mice on it. These findings demonstrate that fenbendazole is still an effective anthelmintic and that EAD PCR is a rapid, noninvasive assay that may be a useful diagnostic tool for antemortem detection of A. tetraptera infection, in conjunction with fecal PCR and FCF

Self-aggregation of a,co-Alkanediols into Two and Three Dimensional Crystallites at the Air-Water Interface. Relevance to Ice Nucleation

A correlation is presented between the crystalline structure of monolayers and multilayers of a,co-alkanediols HO-(CH2)„-OH (n = 16, 18, 19, 21, 22, 23, 24, 30) at the air-water interface and their function as ice nucleators. Structural elucidation was carried out by the following methods; grazing incidence X-ray diffraction, scanning force microscopy, cryo-transmission electron microscopy and external reflection Fourier transform-infrared spectroscopy

Self-aggregation of a,co-Alkanediols into Two and Three Dimensional Crystallites at the Air-Water Interface. Relevance to Ice Nucleation

A correlation is presented between the crystalline structure of monolayers and multilayers of a,co-alkanediols HO-(CH2)„-OH (n = 16, 18, 19, 21, 22, 23, 24, 30) at the air-water interface and their function as ice nucleators. Structural elucidation was carried out by the following methods; grazing incidence X-ray diffraction, scanning force microscopy, cryo-transmission electron microscopy and external reflection Fourier transform-infrared spectroscopy

Recruitment and retention of women in fishing communities in HIV prevention research.

INTRODUCTION: Women in fishing communities in Uganda are more at risk and have higher rates of HIV infection. Socio-cultural gender norms, limited access to health information and services, economic disempowerment, sexual abuse and their biological susceptibility make women more at risk of infection. There is need to design interventions that cater for women's vulnerability. We explore factors affecting recruitment and retention of women from fishing communities in HIV prevention research. METHODS: An HIV incidence cohort screened 2074 volunteers (1057 men and 1017 women) aged 13-49 years from 5 fishing communities along Lake Victoria using demographic, medical history, risk behaviour assessment questionnaires.1000 HIV negative high risk volunteers were enrolled and followed every 6 months for 18 months. Factors associated with completion of study visits among women were analyzed using multivariable logistic regression. RESULTS: Women constituted 1,017(49%) of those screened, and 449(45%) of those enrolled with a median (IQR) age of 27 (22-33) years. Main reasons for non-enrolment were HIV infection (33.9%) and reported low risk behaviour (37.5%). A total of 382 (74%) women and 332 (69%) men completed all follow up visits. Older women (>24 yrs) and those unemployed, who had lived in the community for 5 years or more, were more likely to complete all study visits. CONCLUSION: Women had better retention rates than men at 18 months. Strategies for recruiting and retaining younger women and those who have stayed for less than 5 years need to be developed for improved retention of women in fishing communities in HIV prevention and research Programs

Factors associated with dropout in a long term observational cohort of fishing communities around lake Victoria, Uganda.

BACKGROUND: Fishing communities are potentially suitable for Human immunodeficiency virus (HIV) efficacy trials due to their high risk profile. However, high mobility and attrition could decrease statistical power to detect the impact of a given intervention. We report dropout and associated factors in a fisher-folk observational cohort in Uganda. METHODS: Human immunodeficiency virus-uninfected high-risk volunteers aged 13-49 years living in five fishing communities around Lake Victoria were enrolled and followed every 6 months for 18 months at clinics located within each community. Volunteers from two of the five communities had their follow-up periods extended to 30 months and were invited to attend clinics 10-40 km (km) away from their communities. Human immunodeficiency virus counseling and testing was provided, and data on sexual behaviour collected at all study visits. Study completion was defined as completion of 18 or 30 months or visits up to the date of sero-conversion and dropout as missing one or more visits. Discrete time survival models were fitted to find factors independently associated with dropout. RESULTS: A total of 1000 volunteers (55% men) were enrolled. Of these, 91.9% completed 6 months, 85.2% completed 12 months and 76.0% completed 18 months of follow-up. In the two communities with additional follow-up, 76.9% completed 30 months. In total 299 (29.9%) volunteers missed at least one visit (dropped out). Dropout was independently associated with age (volunteers aged 13-24 being most likely to dropout), gender [men being more likely to dropout than women [adjusted hazard ratio (aHR) 1.4; 95% confidence interval (CI) 1.1-1.8)], time spent in the fishing community (those who stayed <1 year being most likely to dropout), History of marijuana use (users being more likely to dropout than non-users [1.7; (1.2-2.5)], ethnicity (non-Baganda being more likely to dropout than Baganda [1.5; (1.2-1.9)], dropout varied between the five fishing communities, having a new sexual partner in the previous 3 months [1.3 (1.0-1.7)] and being away from home for ≥2 nights in the month preceding the interview [1.4 (1.1-1.8)]. CONCLUSION: Despite a substantial proportion dropping out, retention was sufficient to suggest that by incorporating retention strategies it will be possible to conduct HIV prevention efficacy trials in this community