Mechanisms of cellular invasion by intracellular parasites.

Numerous disease-causing parasites must invade host cells in order to prosper. Collectively, such pathogens are responsible for a staggering amount of human sickness and death throughout the world. Leishmaniasis, Chagas disease, toxoplasmosis, and malaria are neglected diseases and therefore are linked to socio-economical and geographical factors, affecting well-over half the world's population. Such obligate intracellular parasites have co-evolved with humans to establish a complexity of specific molecular parasite-host cell interactions, forming the basis of the parasite's cellular tropism. They make use of such interactions to invade host cells as a means to migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality.

Interrogating a hexokinase-selected small-molecule library for inhibitors of Plasmodium falciparum hexokinase.

Parasites in the genus Plasmodium cause disease throughout the tropic and subtropical regions of the world. P. falciparum, one of the deadliest species of the parasite, relies on glycolysis for the generation of ATP while it inhabits the mammalian red blood cell. The first step in glycolysis is catalyzed by hexokinase (HK). While the 55.3-kDa P. falciparum HK (PfHK) shares several biochemical characteristics with mammalian HKs, including being inhibited by its products, it has limited amino acid identity (~26%) to the human HKs, suggesting that enzyme-specific therapeutics could be generated. To that end, interrogation of a selected small-molecule library of HK inhibitors has identified a class of PfHK inhibitors, isobenzothiazolinones, some of which have 50% inhibitory concentrations (IC50s) of <1 µM. Inhibition was reversible by dilution but not by treatment with a reducing agent, suggesting that the basis for enzyme inactivation was not covalent association with the inhibitor. Lastly, six of these compounds and the related molecule ebselen inhibited P. falciparum growth in vitro (50% effective concentration [EC50] of ≥ 0.6 and <6.8 µM). These findings suggest that the chemotypes identified here could represent leads for future development of therapeutics against P. falciparum.

Prevalence of herbal and dietary supplement usage in Thai outpatients with chronic kidney disease: a cross-sectional survey.

BACKGROUND: There are few studies of the prevalence and patterns of herbal and dietary supplement (HDS) use in patients with chronic kidney disease (CKD), although many researchers and health professionals worldwide have raised concern about the potential effects of HDS on patients with renal insufficiency. A survey was conducted to determine: the prevalence and patterns of HDS use in Thai patients with CKD; the demographic factors related to HDS use; the reasons why Thai patients with CKD use HDS; respondent experiences of benefits and adverse effects from HDS; and the association between conventional medication adherence and HDS use. METHODS: This cross-sectional survey recruited patients with CKD attending two teaching hospitals in Thailand. Data were collected via an interview using a semi-structured interview schedule regarding demographics, HDS usage, reasons for HDS use, and respondent experiences of effects from HDS. Conventional medication adherence was measured using the Thai version of 8-Item Morisky Medication Adherence Scale. Descriptive statistics were used to analyse the prevalence and the patterns of HDS use. Chi-square tests and multiple logistic regression were used to determine any associations between HDS use, demographics and conventional medication adherence. RESULTS: Four hundred and twenty-one eligible patients were recruited. The prevalence of HDS use in the previous 12 months was 45%. There were no demographic differences between HDS users and non-users, except former drinkers were less likely to use HDS, compared with non-drinkers (OR 0.43, 95% CI 0.25-0.75). Those with a medium level of adherence to conventional medication were less likely to use HDS compared with those with a low level of adherence (OR 0.53, 95% CI 0.32-0.87). Maintaining well-being was most common purpose for using HDS (36%). Nearly 18% used HDS, such as holy mushroom, river spiderwort and boesenbergia, to treat kidney disease. The top three most often reported reasons why respondents used HDS were family and friend's recommendation, followed by expecting to gain benefit from HDS and wanting to try them. Perceived beneficial effects on renal function from HDS were reported by around 10% of HDS users. Among HDS users, seven patients perceived worsening CKD from HDS, such as river spiderwort, kariyat and wheatgrass. Additionally, 72% of respondents did not inform their doctor about their HDS use mainly because their doctor did not ask (46%) or would disapprove of their HDS use (15%). CONCLUSIONS: Around half of the Thai patients with CKD used HDS. Health professionals should be aware of HDS use amongst such patients and enquire about HDS use as a part of standard practice in order to prevent any detrimental effects on kidney function.

Expression and characterization of penicillin-binding proteins in Burkholderia cenocepacia.

Putative penicillin-binding proteins (PBPs) were identified in the genome of the Burkholderia cenocepacia strain J2315 based on homology to E. coli PBPs. The three sequences identified as homologs of E. coli PBP1a, BCAL2021, BCAL0274, and BCAM2632, were cloned and expressed as His(6)-tagged fusion proteins in E. coli. The fusion proteins were isolated and shown to bind beta-lactams, indicating these putative PBPs have penicillin-binding activity.

Plasmodium falciparum erythrocytic stage parasites require the putative autophagy protein PfAtg7 for normal growth.

Analysis of the Plasmodium falciparum genome reveals a limited number of putative autophagy genes, specifically the four genes involved in ATG8 lipidation, an essential step in formation of autophagosomes. In yeast, Atg8 lipidation requires the E1-type ligase Atg7, an E2-type ligase Atg3, and a cysteine protease Atg4. These four putative P. falciparum ATG (PfATG) genes are transcribed during the parasite's erythrocytic stages. PfAtg7 has relatively low identity and similarity to yeast Atg7 (14.7% and 32.2%, respectively), due primarily to long insertions typical of P. falciparum. Excluding the insertions the identity and similarity are higher (38.0% and 70.8%, respectively). This and the fact that key residues are conserved, including the catalytic cysteine and ATP binding domain, we hypothesize that PfAtg7 is the activating enzyme of PfAtg8. To assess the role of PfAtg7 we have generated two transgenic parasite lines. In one, the PfATG7 locus was modified to introduce a C-terminal hemagglutinin tag. Western blotting reveals two distinct protein species, one migrating near the predicted 150 kDa and one at approximately 65 kDa. The second transgenic line introduces an inducible degradation domain into the PfATG7 locus, allowing us to rapidly attenuate PfAtg7 protein levels. Corresponding species are also observed in this parasite line at approximately 200 kDa and 100 kDa. Upon PfATG7 attenuation parasites exhibit a slow growth phenotype indicating the essentiality of this putative enzyme for normal growth.

Manic fringe is not required for embryonic development, and fringe family members do not exhibit redundant functions in the axial skeleton, limb, or hindbrain.

Tight regulation of Notch pathway signaling is important in many aspects of embryonic development. Notch signaling can be modulated by expression of fringe genes, encoding glycosyltransferases that modify EGF repeats in the Notch receptor. Although Lunatic fringe (Lfng) has been shown to play important roles in vertebrate segmentation, comparatively little is known regarding the developmental functions of the other vertebrate fringe genes, Radical fringe (Rfng) and Manic fringe (Mfng). Here we report that Mfng expression is not required for embryonic development. Further, we find that despite significant overlap in expression patterns, we detect no obvious synergistic defects in mice in the absence of two, or all three, fringe genes during development of the axial skeleton, limbs, hindbrain, and cranial nerves.