Design, synthesis, nuclear localization, and biological activity of a fluorescent duocarmycin analog, HxTfA

HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318 nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis

A semi-automated method for counting fluorescent malaria oocysts increases the throughput of transmission blocking studies

<p>Abstract</p> <p>Background</p> <p>Malaria transmission is now recognized as a key target for intervention. Evaluation of the <it>Plasmodium </it>oocyst burden in the midguts of <it>Anopheles spp</it>. is important for many of assays investigating transmission. However, current assays are very time-consuming, manually demanding and patently subject to observer-observer variation.</p> <p>Methods</p> <p>This report presents the development of a method to rapidly, accurately and consistently determine oocyst burdens on mosquito midguts using GFP-expressing <it>Plasmodium berghei </it>and a custom-written macro for ImageJ. The counting macro was optimized and found to be fit-for-purpose by performing gametocyte membrane feeds with parasite infected blood. Dissected midguts were counted both manually and using the automated macro, then compared. The optimized settings for the macro were then validated by using it to determine the transmission blocking efficacies of two anti-malarial compounds - dehydroepiandrosterone sulphate and lumefantrine, in comparison to manually determined analysis of the same experiment.</p> <p>Results</p> <p>Concurrence of manual and macro counts was very high (R²= 0.973) and reproducible. Estimated transmission blocking efficacies between manual and automated analysis were highly concordant, indicating that dehydroepiandrosterone sulphate has little or no transmission blocking potential, whilst lumefantrine strongly inhibits sporogony.</p> <p>Conclusion</p> <p>Recognizing a potential five-fold increase in throughput, the resulting reduction in personnel costs, and the absence of inter-operator/laboratory variation possible with this approach, this counting macro may be a benefit to the malaria community.</p

Ubiquitous molecular substrates for associative learning and activity-dependent neuronal facilitation.

Recent evidence suggests that many of the molecular cascades and substrates that contribute to learning-related forms of neuronal plasticity may be conserved across ostensibly disparate model systems. Notably, the facilitation of neuronal excitability and synaptic transmission that contribute to associative learning in Aplysia and Hermissenda, as well as associative LTP in hippocampal CA1 cells, all require (or are enhanced by) the convergence of a transient elevation in intracellular Ca2+ with transmitter binding to metabotropic cell-surface receptors. This temporal convergence of Ca2+ and G-protein-stimulated second-messenger cascades synergistically stimulates several classes of serine/threonine protein kinases, which in turn modulate receptor function or cell excitability through the phosphorylation of ion channels. We present a summary of the biophysical and molecular constituents of neuronal and synaptic facilitation in each of these three model systems. Although specific components of the underlying molecular cascades differ across these three systems, fundamental aspects of these cascades are widely conserved, leading to the conclusion that the conceptual semblance of these superficially disparate systems is far greater than is generally acknowledged. We suggest that the elucidation of mechanistic similarities between different systems will ultimately fulfill the goal of the model systems approach, that is, the description of critical and ubiquitous features of neuronal and synaptic events that contribute to memory induction

Assay for Evaluating the Abundance of Vibrio cholerae and Its O1 Serogroup Subpopulation from Water without DNA Extraction

10.3390/pathogens11030363Pathogens113363-36

Genetic analysis of primaquine tolerance in a patient with relapsing vivax malaria.

Patients with Plasmodium vivax malaria are treated with primaquine to prevent relapse infections. We report primaquine failure in a patient with 3 relapses without any possibility of re-infection. Using whole genome sequencing of the relapsing parasite isolates, we identified single nucleotide variants as candidate molecular markers of resistance

Co-evolution of transcriptional and posttranslational cell-cycle regulation

DNA microarray studies have shown that hundreds of genes are transcribed periodically during the mitotic cell cycle of humans, budding yeast, fission yeast and the plant Arabidopsis thaliana. Here we show that despite the fact the protein complexes involved in this process are largely the same among all eukaryotes, their regulation has evolved considerably. Our comparative analysis of several large-scale data sets reveals that although the regulated subunits of each protein complex are expressed just before its time of action, the identity of the periodically expressed proteins differs significantly between organisms. Moreover, we show that these changes in transcriptional regulation have co-evolved with post-translational control independently in several lineages; loss or gain of cell-cycle-regulated transcription of specific genes is often mirrored by changes in phosphorylation of the proteins that they encode. Our results indicate that many different solutions have evolved for assembling the same molecular machines at the right time during the cell cycle, involving both transcriptional and post-translational layers that jointly control the dynamics of biological systems