Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors

BUENO: Biotechnology Unified Education Network of Opportunities Project

In this video, published by InnovATEBIO, Deborah R. Overath explores the Biotechnology Unified Education Network of Opportunities (BUENO) project at Texas Southmost College, a Hispanic-Serving Institution. The project aims to create curriculum and educational materials, facilitate professional development, and develop programs for biotechnology students. The video recording runs 25:28 minutes in length

Appendix C. The control matrices, the values of sij, aij, and sijaij for each matrix, results of tests of density effects on individual matrix elements, additional results of the alternative analysis of density effects in the manipulated quadrats, results of the check of the linearity assumption of the LTRE, and a discussion of the sampling errors of and the separate matrix elements.

The control matrices, the values of sij, aij, and sijaij for each matrix, results of tests of density effects on individual matrix elements, additional results of the alternative analysis of density effects in the manipulated quadrats, results of the check of the linearity assumption of the LTRE, and a discussion of the sampling errors of and the separate matrix elements

Appendix B. A generalization to stage-specific population projection matrices of Pollard et al.'s (1987) bootstrap method for time series data and a description of the comparable method used as a supplementary analysis of the experimental data.

A generalization to stage-specific population projection matrices of Pollard et al.'s (1987) bootstrap method for time series data and a description of the comparable method used as a supplementary analysis of the experimental data

Appendix A. Methods of population projection matrix construction, including "false match" correction, and direct effects of the density manipulations.

Methods of population projection matrix construction, including "false match" correction, and direct effects of the density manipulations

Index Files

Index File

Genetic diversity across the mitochondrial genome of eastern oysters (Crassostrea virginica) in the northern Gulf of Mexico

The eastern oyster, Crassostrea virginica, is divided into four populations along the western North Atlantic, however, the only published mitochondrial genome sequence was assembled using one individual in Delaware. This study aimed to 1) assemble C. virginica mitochondrial genomes from Texas with pooled restriction-site-associated DNA sequence (ezRAD), 2) evaluate the validity of the mitochondrial genome assemblies including comparison with Sanger sequencing data, and 3) evaluate genetic differentiation both between the Delaware and Texas genomes, as well as among three bays in Texas. The pooled-genome-assembled-genomes (PAGs) from Texas exhibited several characteristics indicating that they were valid, including elevated nucleotide diversity in non-coding and the third position of codons, placement as the sister haplotype of the genome from Delaware in a phylogenetic reconstruction of Crassostrea mitochondrial genomes, and a lack of genetic structure in the ND4 gene among the three Texas bays as was found with Sanger amplicons in samples from the same bays several years prior. In the comparison between the Delaware and Texas genome, 27 of 38 coding regions exhibited variability between the two populations, which were differentiated by 273 mutations, versus 1-13 mutations among the Texas samples. Using the full PAGs, there was no additional evidence for population structure among the three Texas bays. While population genetics is rapidly moving towards larger high-density datasets, studies of mitochondrial DNA (and genomes) can be particularly useful for comparing historic data prior to the modern era of genomics. As such, being able to reliably compile mitochondrial genomes from genomic data can improve the ability to compare results across studies

N-myristoyltransferase inhibitors as new leads to treat sleeping sickness

African sleeping sickness or human African trypanosomiasis (HAT), caused by Trypanosoma brucei spp., is responsible for ~30,000 deaths each year. Available treatments for this neglected disease are poor, with unacceptable efficacy and safety profiles, particularly in the late stage of the disease, when the parasite has infected the central nervous system. Here, we report the validation of a molecular target and discovery of associated lead compounds with potential to address this unmet need. Inhibition of this target, T. brucei N-myristoyltransferase (TbNMT), leads to rapid killing of trypanosomes both in vitro and in vivo and cures trypanosomiasis in mice. These high affinity inhibitors bind into the peptide substrate pocket of the enzyme and inhibit protein N-myristoylation in trypanosomes. The compounds identified have very promising pharmaceutical properties and represent an exciting opportunity to develop oral drugs to treat this devastating disease. Our studies validate TbNMT as a promising therapeutic target for HAT