Structural organization of erythrocyte membrane microdomains and their relation with malaria susceptibility.

Cholesterol-rich microdomains are membrane compartments characterized by specific lipid and protein composition. These dynamic assemblies are involved in several biological processes, including infection by intracellular pathogens. This work provides a comprehensive analysis of the composition of human erythrocyte membrane microdomains. Based on their floating properties, we also categorized the microdomain-associated proteins into clusters. Interestingly, erythrocyte microdomains include the vast majority of the proteins known to be involved in invasion by the malaria parasite Plasmodium falciparum. We show here that the Ecto-ADP-ribosyltransferase 4 (ART4) and Aquaporin 1 (AQP1), found within one specific cluster, containing the essential host determinant CD55, are recruited to the site of parasite entry and then internalized to the newly formed parasitophorous vacuole membrane. By generating null erythroid cell lines, we showed that one of these proteins, ART4, plays a role in P. falciparum invasion. We also found that genetic variants in both ART4 and AQP1 are associated with susceptibility to the disease in a malaria-endemic population.

INSPIRED but Tired: How Medical Faculty's Job Demands and Resources Lead to Engagement, Work-Life Conflict, and Burnout.

BACKGROUND: Past research shows that physicians experience high ill-being (i.e., work-life conflict, stress, burnout) but also high well-being (i.e., job satisfaction, engagement). OBJECTIVE: To shed light on how medical faculty's experiences of their job demands and job resources might differentially affect their ill-being and their well-being with special attention to the role that the work-life interface plays in these processes. METHODS: Qualitative thematic analysis was used to analyze interviews from 30 medical faculty (19 women, 11 men, average tenure 13.36 years) at a top research hospital in Canada. FINDINGS: Medical faculty's experiences of work-life conflict were severe. Faculty's job demands had coalescing (i.e., interactive) effects on their stress, work-life conflict, and exhaustion. Although supportive job resources (e.g., coworker support) helped to mitigate the negative effects of job demands, stimulating job resources (e.g., challenging work) contributed to greater work-life conflict, stress, and exhaustion. Thus, for these medical faculty job resources play a dual-role for work-life conflict. Moreover, although faculty experienced high emotional exhaustion, they did not experience the other components of burnout (i.e., reduced self-efficacy, and depersonalization). Some faculty engaged in cognitive reappraisal strategies to mitigate their experiences of work-life conflict and its harmful consequences. CONCLUSION: This study suggests that the precise nature and effects of job demands and job resources may be more complex than current research suggests. Hospital leadership should work to lessen unnecessary job demands, increase supportive job resources, recognize all aspects of job performance, and, given faculty's high levels of work engagement, encourage a climate that fosters work-life balance.

A cryptic cycle in haematopoietic niches promotes initiation of malaria transmission and evasion of chemotherapy.

Blood stage human malaria parasites may exploit erythropoietic tissue niches and colonise erythroid progenitors; however, the precise influence of the erythropoietic environment on fundamental parasite biology remains unknown. Here we use quantitative approaches to enumerate Plasmodium infected erythropoietic precursor cells using an in vivo rodent model of Plasmodium berghei. We show that parasitised early reticulocytes (ER) in the major sites of haematopoiesis establish a cryptic asexual cycle. Moreover, this cycle is characterised by early preferential commitment to gametocytogenesis, which occurs in sufficient numbers to generate almost all of the initial population of circulating, mature gametocytes. In addition, we show that P. berghei is less sensitive to artemisinin in splenic ER than in blood, which suggests that haematopoietic tissues may enable origins of recrudescent infection and emerging resistance to antimalarials. Continuous propagation in these sites may also provide a mechanism for continuous transmission and infection in malaria endemic regions.

Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs.

OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.

Characterization of glutathione uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea.

PURPOSE: To compare and contrast glutathione (GSH) uptake, synthesis, and efflux pathways in the epithelium and endothelium of the rat cornea. METHODS: Whole eyes were cryosectioned in an equatorial orientation and sections fixed in either 0.75% paraformaldehyde alone or 0.75% paraformaldehyde plus 0.01% glutaraldehyde. Sections were then labeled with GSH, γ-glutamylcysteine synthetase (γ-GCS), cysteine, xCT, or multidrug resistance-associated proteins (MRP1, 2, 4, and 5 isoforms) antibodies and then with secondary antibodies to visualize labeling patterns. Cornea morphology was visualized using propidium iodide. Reverse transcriptase-polymerase chain reaction was used to determine which of the 3 putative GSH transporters, NaDC3, C-terminal organic anion transporter 1 (OAT1), and/or N-terminal organic anion transporter 3 (OAT3), were present at the transcript level in the cornea. Colocalization of OAT3 and sodium dependent dicarboxylate transporter 3 (NaDC3) was performed by labeling with OAT3 and NaDC3 primary antibodies that were visualized with secondary antibodies and then mounted in 4'6-diamidino-2-phenylindole to visualize cell morphology. RESULTS: Although immunohistochemical labeling showed GSH to be localized throughout the cornea, labeling for cysteine, γ-GCS, xCT, MRP4, and MRP5 was strongest in the epithelium. In contrast, although GSH labeling was strong in the endothelium, xCT and MRP labeling was absent and cysteine and γ-GCS labeling was weak relative to the epithelium. Reverse transcriptase-polymerase chain reaction revealed OAT3 and NaDC3, but not OAT1, to be present at the transcript level. Immunohistochemical labeling showed OAT3 and NaDC3 to be localized to the endothelium but absent from the epithelium. CONCLUSIONS: The corneal epithelium and endothelium exhibit differences in GSH uptake, synthesis, and efflux pathways. The corneal epithelium seems to be the region where GSH synthesis and GSH efflux occurs. However, in the endothelium, GSH accumulation is likely to be predominantly by direct uptake of GSH from the aqueous humor.