Molecular Modelling and Biochemical Characterization of a DNA Repair Enzyme Binding to DNA

Computational techniques of homology modelling, enzyme–substrate docking, and molecular dynamics were applied to elucidate the structure and substrate binding properties of the DNA repair enzyme polynucleotide kinase/phosphatase from the nematode C. elegans (CePNKP). PNKP is involved in the repair of DNA strand breaks, a form of DNA damage caused by reactive oxygen species, ionizing radiation and certain chemical mutagens. Mutations in human PNKP have been associated with the neurological disorders Microcephaly with Intractable Seizures (MCSZ) and Ataxia Oculomotor Apraxia 4 (AOA4). In addition, human PNKP has been identified as a potential drug target for the development of chemo- and radiosensitizing agents for cancer treatment. CePNKP is a useful model system for studying the human enzyme. The substrate preference for both human and C. elegans PNKP has been investigated, and found to be similar, with higher selectivity for recessed over blunt DNA ends. However, CePNKP exhibits a more exclusive preference for recessed DNA ends than human PNKP. To elucidate the reason for the unique substrate specificity of CePNKP, the structure of CePNKP in complex with its DNA substrate must be determined in atomistic detail by molecular modelling. The generated structural model is compared with our experimental results of kinase activity assays of wild-type CePNKP. Discipline: Biological Sciences/Chemistry Faculty Mentor: Dr. Nina Bernstein, Dr. Jorge Llan

Computational Study of the Structure of Lactoperoxidase and its Active Site

Lactoperoxidase (LPO) is an enzyme that fights in the first line of defense against infection. LPO catalyzes the formation of oxidizing chemicals that indiscriminately kill foreign microbes and viruses caught in the mucous membranes of vulnerable body parts, namely of the eyes and upper airways. Because of its importance for the immune system, the molecular structure and efficacy of native forms of LPO against various pathogens have been studied for potential applications in medical therapies. Despite its frequency in research, the mechanism by which LPO converts common ions, such as chloride, into antimicrobial agents has not been resolved in atomistic detail. Thus, we seek to determine catalytic mechanism of LPO using the methods of computational chemistry, which incorporates classical and quantum mechanics to simulate chemical phenomena. To start, we examined various three-dimensional structures of LPO taken from the Protein Data Bank to estimate the variability and flexibility of the active site and the overall protein. An active-site structural model was then constructed to compute the spatial distribution and strength of intermolecular forces at play in the LPO active site using a force field specially optimized for proteins. The resulting implications to substrate binding and catalysis were analyzed. This allows us to progress to the next stage, where quantum chemical methods will be used to ultimately elucidate the catalytic mechanism of LPO. *Indicates faculty mentor

Plasma endotoxin core antibody concentration and linear growth are unrelated in rural Malawian children aged 2–5 years

BACKGROUND: Environmental enteropathy is subclinical inflammation of the upper gastrointestinal tract associated with reduced linear growth in developing countries. Usually investigators have used biopsy or a dual sugar absorption test to assess environmental enteropathy. Such tests are time and resource intensive, restricting their utility as screening methods. Serum endotoxin core antibody (EndoCab) concentration is a potential indicator of intestinal inflammation and integrity, and thus may be useful to predict environmental enteropathy. We analyzed the association of serum EndoCab levels versus linear growth and lactulose-mannitol assay results in 2–5 year old rural Malawian children. METHODS: This was an observational study of 388 rural, asymptomatic Malawian children who had anthropometric measurements taken at least every 3 months since birth. In June and July 2011, dual sugar permeability tests were performed and serum samples were drawn for EndoCab assays. Pearson correlation, Student’s t test and multivariable linear regression were used to compare ln EndoCab concentrations with height-for-age z scores (HAZ) at time of sampling and 3 months later. Identical analysis was also performed for ln EndoCab versus measurements from dual sugar permeability testing performed in conjunction with serum sampling. In a subgroup of children with anthropometric data in the months prior to serum sampling, Pearson correlation was used to estimate the relationship between ln EndoCab and recent linear growth. RESULTS: Ln EndoCab concentrations were not correlated with HAZ at time of measurement (B = −0.078, P = 0.14) nor change in HAZ over the subsequent 3 months HAZ (B = −0.018, P = 0.27). EndoCab concentration was not associated with %lactulose excretion (B < 0.001, P = 0.98) nor the lactulose:mannitol ratio (B = 0.021, P = 0.62). Subgroup analysis also did not reveal any significant association between EndoCab and recent growth. CONCLUSION: EndoCab titers were not correlated with measurements of growth or intestinal permeability in rural pre-school aged Malawian children

Comparison of Milk and Maize Based Diets in Kwashiorkor

The dual sugar test of intestinal permeability is a reliable non-invasive way of assessing the response of the small intestinal mucosa to nutritional rehabilitation. Our aim was to compare a local mix of maize soya egg to the standard milk diet in the treatment of kwashiorkor. The diets were alternated three monthly in the sequence milk-maize-milk. There' were a total of 533 kwashiorkor admissions of at least' five days during the study who received either milk or maize. Intestinal permeability was assessed at weekly intervals by the lactulose-rhamnose test in 100 kwashiorkor cases, including 55 on milk and 45 on the maize diet. Permeability ratios (95% confidence interval) on the milk diet improved by a mean of 6.4 (1.7 to 11.1) compared with - 6.8 (-16.8 to 5.0) in the maize group. The improved permeability on milk occurred despite more diarrhoea, which constituted 34.8% of hospital days compared to 24.3% in the maize group. Case fatality rates for all 533 kwashiorkor admissions were 13.6% v 20,9%, respectively, giving a relative risk of death in the maize group of 1.54 (1.04 to 2,28). The maize group also had more  clinical sepsis.(60% v 31%) and less weight gain (2.9 v 4.4 g/kg/day) than the milk group. Milk is superior to a local maize based diet in the treatment of kwashiorkor in terms of mortality, weight gain, clinical sepsis, and improvement in intestinal permeability

Droplet digital PCR quantifies host inflammatory transcripts in feces reliably and reproducibly

AbstractThe gut is the most extensive, interactive, and complex interface between the human host and the environment and therefore a critical site of immunological activity. Non-invasive methods to assess the host response in this organ are currently lacking. Feces are the available analyte which have been in proximity to the gut tissue.We applied a method of concentrating host transcripts from fecal specimens using a existing bead-based affinity separation method for nucleic acids and quantified transcripts using droplet digital PCR (ddPCR) to determine the copy numbers of a variety of key transcripts in the gut immune system. ddPCR compartmentalizes the reaction in a small aqueous droplet suspended in oil, and counts droplets as either fluorescent or non-fluorescent. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize transcript concentration.This method was applied to 799 fecal samples from rural Malawian children, and over 20,000 transcript concentrations were quantified. Host mRNA was detected in >99% samples, a threshold for target detection was established at an average expression of 0.02 copies target/GAPDH, above which correlation coefficient between duplicate measurements is >0.95. Quantities of transcript detected using ddPCR were greater than standard qPCR. Fecal sample preservation at the time of collection did not require immediate freezing or the addition of buffers or enzymes. Measurements of transcripts encoding immunoactive proteins correlated with a measure of gut inflammation in the study children, thereby substantiating their relevance. This method allows investigators to interrogate gene expression in the gut

Supplementary feeding with fortified spread among moderately underweight 6-18-month-old rural Malawian children.

We aimed to analyse growth and recovery from undernutrition among moderately underweight ambulatory children receiving micronutrient-fortified maize-soy flour (Likuni Phala, LP) or ready-to-use fortified spread (FS) supplementary diet. One hundred and seventy-six 6-18-month-old individuals were randomized to receive 500 g LP or 350 g FS weekly for 12 weeks. Baseline and end of intervention measurements were used to calculate anthropometric gains and recovery from underweight, wasting and stunting. Mean weight-for-age increased by 0.22 (95% CI 0.07-0.37) and 0.28 (0.18-0.40) Z-score units in the LP and FS groups respectively. Comparable increase for mean weight-for-length was 0.39 (0.20-0.57) and 0.52 (0.38-0.65) Z-score units. Recovery from underweight and wasting was 20% and 93% in LP group and 16% and 75% in FS group. Few individuals recovered from stunting and mean length-for-age was not markedly changed. There were no statistically significant differences between the outcomes in the two intervention groups. In a poor food-security setting, underweight infants and children receiving supplementary feeding for 12 weeks with ready-to-use FS or maize-soy flour porridge show similar recovery from moderate wasting and underweight. Neither intervention, if limited to a 12-week duration, appears to have significant impact on the process of linear growth or stunting

Child Survival in sub Sahara Africa: the role of CAPGAN and regional child health practitioners & scientists

Close to a decade ago the Millennium Development Goals (MDG) were developed and unanimously accepted by the General Assembly of the United Nations. The think tank behind the MDGs, the Earth Institute at ColumbiaUniversity in New York led by the economist Jeffry Sachs , developed 8 overarching goals, which -if achieved- would liberate the world from povert