Depletion of the mini-chromosome maintenance complex binding protein allows the progression of cytokinesis despite abnormal karyokinesis during the asexual development of Plasmodium falciparum

The eukaryotic cell cycle is typically divided into distinct phases with cytokinesis immediately following mitosis. To ensure proper cell division, each phase is tightly coordinated via feedback controls named checkpoints. During its asexual replication cycle, the malaria parasite Plasmodium falciparum undergoes multiple asynchronous rounds of mitosis with segregation of uncondensed chromosomes followed by nuclear division with intact nuclear envelope. The multi-nucleated schizont is then subjected to a single round of cytokinesis that produces dozens of daughter cells called merozoites. To date, no cell cycle checkpoints have been identified that regulate the Plasmodium spp. mode of division. Here, we identify the Plasmodium homologue of the Mini-Chromosome Maintenance Complex Binding Protein (PfMCMBP), which co-purified with the Mini-Chromosome Maintenance (MCM) complex, a replicative helicase required for genomic DNA replication. By conditionally depleting PfMCMBP, we disrupt nuclear morphology and parasite proliferation without causing a block in DNA replication. By immunofluorescence microscopy, we show that PfMCMBP depletion promotes the formation of mitotic spindle microtubules with extensions to more than one DNA focus and abnormal centrin distribution. Strikingly, PfMCMBP-deficient parasites complete cytokinesis and form aneuploid merozoites with variable cellular and nuclear sizes. Our study demonstrates that the parasite lacks a robust checkpoint response to prevent cytokinesis following aberrant karyokinesis

Chronic GLP1 Therapy Reduces Postprandial IL6 in Obese Humans With Prediabetes

Single-dose glucagon-like peptide 1 (GLP1) therapy increases postprandial plasma IL6 levels in prediabetic, obese humans. GLP1-IL6 interactions underly multiple antidiabetic effects, but these may differ after acute versus chronic therapy. This study examines postprandial effects of GLP1 after chronic therapy. Seven humans (six Black) with prediabetes and obesity completed 6 weeks of exenatide extended release therapy. Then subjects returned for pre- and post-meal measurements of plasma IL6, GLP1, glucagon, and related inflammatory markers. Weight, which was measured before and after therapy, did not change. Plasma IL6 decreased from baseline to postmeal state ( = 0.016), with decreases in free fatty acids

Comparative gender analysis of the efficacy and safety of atazanavir/ritonavir and lopinavir/ritonavir at 96 weeks in the CASTLE study.

OBJECTIVES: To examine whether the overall results of the CASTLE study pertain to both genders, we analysed the efficacy and safety of atazanavir/ritonavir and lopinavir/ritonavir in 277 female and 606 male patients in the open-label, multinational trial over 96 weeks. The trial is registered with ClinicalTrials.gov, number NCT00272779. METHODS: Treatment-naive patients aged ≥ 18 years with HIV-1 RNA ≥ 5000 copies/mL were randomized to receive either atazanavir/ritonavir 300/100 mg once daily or lopinavir/ritonavir 400/100 mg twice daily, with fixed-dose tenofovir/emtricitabine 300/200 mg once daily. RESULTS: At week 96, confirmed virological response rates (HIV RNA \u3c50 copies\u3e/mL; intent-to-treat analysis) were higher in women and men receiving atazanavir/ritonavir than those receiving lopinavir/ritonavir and lower in women than men in both treatment arms (67% of women and 77% of men on atazanavir/ritonavir and 63% of women and 71% of men on lopinavir/ritonavir). These differences were not observed in the on-treatment analysis. Mean change in CD4 cell count from baseline to week 96 was 265 cells/mm(3) for women and 269 cells/mm(3) for men on atazanavir/ritonavir and 298 cells/mm(3) for women and 286 cells/mm(3) for men on lopinavir/ritonavir. Discontinuation rates were higher in women than men in each treatment arm (22% of women and 15% of men on atazanavir/ritonavir and 29% of women and 18% of men on lopinavir/ritonavir). In women and men, grade 2-4 nausea and diarrhoea were more frequent in the lopinavir/ritonavir group; jaundice and hyperbilirubinaemia occurred more frequently in the atazanavir/ritonavir group. CONCLUSIONS: Once-daily atazanavir/ritonavir is an effective and well-tolerated therapeutic option for women and men with HIV-1 infection. The sex-based differences in response may be due to higher discontinuation rates in women

Calcium-Dependent Protein Kinase 5 Is Required for Release of Egress-Specific Organelles in Plasmodium falciparum

ABSTRACT The human malaria parasite Plasmodium falciparum requires efficient egress out of an infected red blood cell for pathogenesis. This egress event is highly coordinated and is mediated by several signaling proteins, including the plant-like P. falciparum calcium-dependent protein kinase 5 (PfCDPK5). Knockdown of PfCDPK5 results in an egress block where parasites are trapped inside their host cells. The mechanism of this PfCDPK5-dependent block, however, remains unknown. Here, we show that PfCDPK5 colocalizes with a specialized set of parasite organelles known as micronemes and is required for their discharge, implicating failure of this step as the cause of the egress defect in PfCDPK5-deficient parasites. Furthermore, we show that PfCDPK5 cooperates with the P. falciparum cGMP-dependent kinase (PfPKG) to fully activate the protease cascade critical for parasite egress. The PfCDPK5-dependent arrest can be overcome by hyperactivation of PfPKG or by physical disruption of the arrested parasite, and we show that both treatments facilitate the release of the micronemes required for egress. Our results define the molecular mechanism of PfCDPK5 function and elucidate the complex signaling pathway of parasite egress

A Bivalent Meningococcal B Vaccine in Adolescents and Young Adults.

BACKGROUND: MenB-FHbp is a licensed meningococcal B vaccine targeting factor H-binding protein. Two phase 3 studies assessed the safety of the vaccine and its immunogenicity against diverse strains of group B meningococcus. METHODS: We randomly assigned 3596 adolescents (10 to 18 years of age) to receive MenB-FHbp or hepatitis A virus vaccine and saline and assigned 3304 young adults (18 to 25 years of age) to receive MenB-FHbp or saline at baseline, 2 months, and 6 months. Immunogenicity was assessed in serum bactericidal assays that included human complement (hSBAs). We used 14 meningococcal B test strains that expressed vaccine-heterologous factor H-binding proteins representative of meningococcal B epidemiologic diversity; an hSBA titer of at least 1:4 is the accepted correlate of protection. The five primary end points were the proportion of participants who had an increase in their hSBA titer for each of 4 primary strains by a factor of 4 or more and the proportion of those who had an hSBA titer at least as high as the lower limit of quantitation (1:8 or 1:16) for all 4 strains combined after dose 3. We also assessed the hSBA responses to the primary strains after dose 2; hSBA responses to the 10 additional strains after doses 2 and 3 were assessed in a subgroup of participants only. Safety was assessed in participants who received at least one dose. RESULTS: In the modified intention-to-treat population, the percentage of adolescents who had an increase in the hSBA titer by a factor of 4 or more against each primary strain ranged from 56.0 to 85.3% after dose 2 and from 78.8 to 90.2% after dose 3; the percentages of young adults ranged from 54.6 to 85.6% and 78.9 to 89.7%, after doses 2 and 3, respectively. Composite responses after doses 2 and 3 in adolescents were 53.7% and 82.7%, respectively, and those in young adults were 63.3% and 84.5%, respectively. Responses to the 4 primary strains were predictive of responses to the 10 additional strains. Most of those who received MenB-FHbp reported mild or moderate pain at the vaccination site. CONCLUSIONS: MenB-FHbp elicited bactericidal responses against diverse meningococcal B strains after doses 2 and 3 and was associated with more reactions at the injection site than the hepatitis A virus vaccine and saline. (Funded by Pfizer; ClinicalTrials.gov numbers, NCT01830855 and NCT01352845 )

An essential malaria protein defines the architecture of blood-stage and transmission-stage parasites

Blood-stage replication of the human malaria parasite Plasmodium falciparum occurs via schizogony, wherein daughter parasites are formed by a specialized cytokinesis known as segmentation. Here we identify a parasite protein, which we name P. falciparum Merozoite Organizing Protein (PfMOP), as essential for cytokinesis of blood-stage parasites. We show that, following PfMOP knockdown, parasites undergo incomplete segmentation resulting in a residual agglomerate of partially divided cells. While organelles develop normally, the structural scaffold of daughter parasites, the inner membrane complex (IMC), fails to form in this agglomerate causing flawed segmentation. In PfMOP-deficient gametocytes, the IMC formation defect causes maturation arrest with aberrant morphology and death. Our results provide insight into the mechanisms of replication and maturation of malaria parasites

The Malaria Parasite Cyclin H Homolog PfCyc1 Is Required for Efficient Cytokinesis in Blood-Stage Plasmodium falciparum

ABSTRACT All well-studied eukaryotic cell cycles are driven by cyclins, which activate cyclin-dependent kinases (CDKs), and these protein kinase complexes are viable drug targets. The regulatory control of the Plasmodium falciparum cell division cycle remains poorly understood, and the roles of the various CDKs and cyclins remain unclear. The P. falciparum genome contains multiple CDKs, but surprisingly, it does not contain any sequence-identifiable G1-, S-, or M-phase cyclins. We demonstrate that P. falciparum Cyc1 (PfCyc1) complements a G1 cyclin-depleted Saccharomyces cerevisiae strain and confirm that other identified malaria parasite cyclins do not complement this strain. PfCyc1, which has the highest sequence similarity to the conserved cyclin H, cannot complement a temperature-sensitive yeast cyclin H mutant. Coimmunoprecipitation of PfCyc1 from P. falciparum parasites identifies PfMAT1 and PfMRK as specific interaction partners and does not identify PfPK5 or other CDKs. We then generate an endogenous conditional allele of PfCyc1 in blood-stage P. falciparum using a destabilization domain (DD) approach and find that PfCyc1 is essential for blood-stage proliferation. PfCyc1 knockdown does not impede nuclear division, but it prevents proper cytokinesis. Thus, we demonstrate that PfCyc1 has a functional divergence from bioinformatic predictions, suggesting that the malaria parasite cell division cycle has evolved to use evolutionarily conserved proteins in functionally novel ways

Atlas of Plasmodium falciparum intraerythrocytic development using expansion microscopy

Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample by ~4.5×. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have cataloged 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the outer centriolar plaque and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the outer centriolar plaque until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an association with the outer centriolar plaque during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology