Evolutionary patterning: a novel approach to the identification of potential drug target sites in Plasmodium falciparum

Malaria continues to be the most lethal protozoan disease of humans. Drug development programs exhibit a high attrition rate and parasite resistance to chemotherapeutic drugs exacerbates the problem. Strategies that limit the development of resistance and minimize host side-effects are therefore of major importance. In this study, a novel approach, termed evolutionary patterning (EP), was used to identify suitable drug target sites that would minimize the emergence of parasite resistance. EP uses the ratio of non-synonymous to synonymous substitutions (omega) to assess the patterns of evolutionary change at individual codons in a gene and to identify codons under the most intense purifying selection (omega < or = 0.1). The extreme evolutionary pressure to maintain these residues implies that resistance mutations are highly unlikely to develop, which makes them attractive chemotherapeutic targets. Method validation included a demonstration that none of the residues providing pyrimethamine resistance in the Plasmodium falciparum dihydrofolate reductase enzyme were under extreme purifying selection. To illustrate the EP approach, the putative P. falciparum glycerol kinase (PfGK) was used as an example. The gene was cloned and the recombinant protein was active in vitro, verifying the database annotation. Parasite and human GK gene sequences were analyzed separately as part of protozoan and metazoan clades, respectively, and key differences in the evolutionary patterns of the two molecules were identified. Potential drug target sites containing residues under extreme evolutionary constraints were selected. Structural modeling was used to evaluate the functional importance and drug accessibility of these sites, which narrowed down the number of candidates. The strategy of evolutionary patterning and refinement with structural modeling addresses the problem of targeting sites to minimize the development of drug resistance. This represents a significant advance for drug discovery programs in malaria and other infectious diseases.     

Bacterial pili,with emphasis on Mycobacterium tuberculosis curli pili: potential biomarkers for point-of care tests and therapeutics

Context: Novel biomarkers are essential for developing rapid diagnostics and therapeutic interventions

Objective: This review aimed to highlight biomarker characterisation and assessment of unique bacterial pili.

Methods: A PubMed search for bacterial pili, diagnostics, vaccine and therapeutics was performed, with emphasis on the well characterised pili.

Results: In total, 46 papers were identified and reviewed.

Conclusion: Extensive analyses of pili enabled by advanced nanotechnology and whole genome sequencing provide evidence that they are strong biomarker candidates. Mycobacterium tuberculosis curli pili are emphasised as important epitopes for the development of much needed point-of-care diagnostics and therapeutics.     

Relationship between morphological and physiological responses to waterlogging and salinity in Sporobolus virginicus (L.) Kunth

The effects of waterlogging and salinity on morphological and physiological responses in the marsh grass Sporobolus virginicus (L.) Kunth were investigated in a 4×2 factorial experiment. Plants were subjected to four salinity levels (0, 100, 200 and 400 mol m^–3 NaCl) and two soil inundation conditions (drained and flooded) for 42 days. Flooding at 0 mol m^–3 NaCl caused initiation of adventitious surface roots, increased internal acration and plant height, induced alcohol dehydrogenase activity (ADH), and decreased belowground biomass and the number of culms per plant. Salinity increase from 0 to 400 mol m^–3 NaCl under drained conditions increased leaf and root proline concentrations and decreased photosynthesis, aboveground biomass, number of culms per plant and number of internodes per culm. Concurrent waterlogging and salinity induced ADH activity and adventitious surface roots but decreased plant height and aboveground biomass. Internal air space increased with waterlogging from 0 to 100 mol m^–3 NaCl but further increases in salinity to 400 mol m^–3 reduced air space. Combined waterlogging and salinity stresses, however, had no effect on photosynthesis or on the concentrations of proline in leaves or roots. These results are discussed in relation to the widespread colonization by S. virginicus of a wide range of coastal environments varying in soil salinity and in the frequency and intensity of waterlogging.     

Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D.

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry. Truncated forms of gD lacking the transmembrane and cytoplasmic tail regions have been shown to bind to cells and block plaque formation. Using complementation analysis and a panel of gD mutants, we previously identified four regions of gD (regions I to IV) which are important for virus entry. Here, we used baculovirus vectors to overexpress truncated forms of wild-type gD from HSV type 1 (HSV-1) [gD-1(306t)] and HSV-2 [gD-2(306t)] and four mutants, gD-1(inverted delta 34t), gD-1(inverted delta 126t), gD-1(inverted delta 243t), and gD-1(delta 290-299t), each having a mutation in one of the four functional regions. We used an enzyme-linked immunosorbent assay and circular dichroism to analyze the structure of these proteins, and we used functional assays to study the role of gD in binding, penetration, and cell-to-cell spread. gD-1 and gD-2 are similar in antigenic structure and thermal stability but vary in secondary structure. Mutant proteins with insertions in region I or II were most altered in structure and stability, while mutants with insertions in region III or IV were less altered. gD-1(306t) and gD-2(306t) inhibited both plaque formation and cell-to-cell transmission of HSV-1. In spite of obvious structural differences, all of the mutant proteins bound to cells, confirming that binding is not the only function of gD. The region I mutant did not inhibit HSV plaque formation or cell-to-cell spread, suggesting that this region is necessary for the function of gD in these processes. Surprisingly, the other three mutant proteins functioned in all of the in vitro assays, indicating that the ability of gD to bind to cells and inhibit infection does not correlate with its ability to initiate infection as measured by the complementation assay. The region IV mutant, gD-1(delta 290-299t), had an unexpected enhanced inhibitory effect on HSV infection. Taken together, the results argue against a single functional domain in gD. It is likely that different gD structural elements are involved in successive steps of infection.     

Metabolic profiles and lipoprotein lipid concentrations in non-obese and obese patients with polycystic ovarian disease

Clinical parameters, androgen status and lipoprotein lipid profiles were assessed in 10 non-obese and 10 obese patients with polycystic ovarian disease (PCOD) and reference subjects matched for age, height and weight. Both obese and non-obese women with PCOD had significantly higher androgen levels when compared to the reference groups. When comparison of lipoprotein lipid profiles were made between groups, non-obese women with PCOD had significantly higher total cholesterol, triglycerides and LDL-cholesterol levels than non-obese reference subjects. Obese PCOD women manifested significantly higher total cholesterol, LDL-cholesterol, cholesterol/HDL, and LDL/HDL values than did obese reference subjects. Correlations between serum androgens and lipoprotein lipid concentrations in PCOD and normal women were unhelpful. Both non-obese and obese patients with PCOD had significantly higher systolic and diastolic blood pressures (BPs) than the reference groups. Thus, both non-obese and obese women with PCOD manifest hyperandrogenaemia which may result in a male pattern of lipoprotein lipid concentrations.     

Tumour metabolites regulate tissue kallikrein in human umbilical vein endothelial cells

Angiogenesis, the sprouting of new blood vessels, is tightly mediated via a myriad of endogenous factors. A pro-angiogenic alteration facilitates the formation of neovascular tumour networks, thereby providing mechanisms for uncontrolled growth. The kallikrein-kinin system is postulated to be pro-angiogenic since its components have been detected in both endothelial cells and tumour tissue. No studies have, however, focussed on the role of tissue kallikrein (TK) in human angiogenic endothelial cell-tumour interactions. This study has optimised a challenge model whereby endothelial cells are presented with neuroblastoma metabolites, and vice versa. Image analysis of immunoreactive TK revealed a dose-dependant, significant reduction of TK localisation within endothelial cells, while gene expression remained unchanged, the latter determined by in situ RT-PCR. Neuroblastoma cells, when challenged with endothelial cell metabolites, displayed no change in TK synthesis or localisation. Alterations in TK synthesis and/or storage by angiogenic endothelial cells may be mediated by tumour-released signals and possibly indicate a shift from a proteolytic to a mitogenic function of TK. The challenge model provides a relatively simple experimental system to study angiogenic factors in tumour-endothelial cell interaction, and is the first to localise both TK and its mRNA within angiogenic endothelial and tumour cells.     

Sleep deprivation induces the unfolded protein response in mouse cerebral cortex

Little is known about the molecular mechanisms underlying sleep. We show the induction of key regulatory proteins in a cellular protective pathway, the unfolded protein response (UPR), following 6 h of induced wakefulness. Using C57/B6 male mice maintained on a 12:12 light/dark cycle, we examined, in cerebral cortex, the effect of different durations of prolonged wakefulness (0, 3, 6, 9 and 12 h) from the beginning of the lights-on inactivity period, on the protein expression of BiP/GRP78, a chaperone and classical UPR marker. BiP/GRP78 expression is increased with increasing durations of sleep deprivation (6, 9 and 12 h). There is no change in BiP/GRP78 levels in handling control experiments carried out during the lights-off period. PERK, the transmembrane kinase responsible for attenuating protein synthesis, which is negatively regulated by binding to BiP/GRP78, is activated by dissociation from BiP/GRP78 and by autophosphorylation. There is phosphorylation of the elongation initiation factor 2alpha and alteration in ribosomal function. These changes are first observed after 6 h of induced wakefulness. Thus, prolonging wakefulness beyond a certain duration induces the UPR indicating a physiological limit to wakefulness.     

Quassinoids from the leaves of the Madagascan Simaroubaceae Samadera madagascariensis

An investigation of the leaves of the Madagascan Simaroubaceae Samadera madagascariensis has yielded three C18 quassinoids, 5beta,6-dihydrosamaderine A, 2-chlorosamaderine A, and samaderolactone A, and a C19 quassinoid, 3,4beta-dihydrosamaderine C, together with the known quassinoids samaderine A, samaderine B, and cedronin. The compounds isolated displayed little or no anti-tumour activity.     

EM investigation of myoblast origin in regenerating hamster skeletal muscle explants.

This study attempted to dispel the confusion that exists in the understanding of the origin of myoblasts during muscle regeneration. Regenerating hamster muscle explants from cultures were studied under the EM on 4 consecutive days, after incubation. Preincubation specimens served as controls. Revelations were that euchromatic myonuclei underwent dense granulation and activation after incubation. Presumptive myoblasts (PM) lying clearly within the myofibre increased in numbers with incubation time. Some myonuclei showed partial transformation towards a PM. This study concluded that myonuclei transformed into myoblasts during the process of muscle regeneration and that the PM, produced from a myonucleus, was a stage in the development of the satellite cell (SC) in regenerating muscle. These SC, myoblasts from myonuclear origin, proliferated, fused, and formed multinucleate myotubes that matured into myofibres which replaced damaged muscle. Findings of this study may have new implications for the proposed myoblast transplant or gene transfer therapy, both of which, whilst being possible answers for muscular dystrophy, depend on a sound knowledge of muscle regeneration mechanisms.