Conserved and Novel Properties of Clathrin-Mediated Endocytosis in Dictyostelium Discoideum

The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. Clathrin functions with a network of interacting accessory proteins, one of which is the adaptor complex AP-2, to co-ordinate vesicle formation. Disruption of genes involved in clathrin-mediated endocytosis causes embryonic lethality in multicellular animals suggesting that clathrin-mediated endocytosis is a fundamental cellular process. However, loss of clathrin-mediated endocytosis genes in single cell eukaryotes, such as S.cerevisiae (yeast), does not cause lethality, suggesting that clathrin may convey specific advantages for multicellularity. Furthermore, the spatiotemporal dynamics and requirements for individual components of the clathrin-mediated endocytic pathway differ between yeast and mammals. I therefore sought to study the components of clathrin-mediated endocytosis in another unicellular system, the organism Dictyostelium, which diverged early from the lineage leading to yeast and mammals. Dictyostelium offers a unique advantage as upon starvation it transitions from a unicellular to multicellular state. In this thesis I studied clathrin-mediated endocytosis in the unicellular growth phase of Dictyostelium and identified a heterotetrameric AP-2 complex in Dictyostelium that is homologous to that present in mammalian cells. Analysis of this pathway at a high spatial and temporal resolution shows the high degree of similarity in the kinetics of internalization of individual clathrin-coated vesicles between Dictyostelium and mammalian cells. These similarities support the conclusion that the formation of clathrin-coated vesicles is homologous between Dictyostelium and mammals and, thus, these features likely evolved early. I also found a role for clathrin-mediated endocytosis in maintenance and biogenesis of the contractile vacuole in Dictyostelium. Contractile vacuoles are specialized organelles found in some single celled organisms that allow cells to osmoregulate by collecting and removing excess water from the cytoplasm; they are not found in yeast or animals. I found that that the contractile vacuole protein, dajumin-GFP, is trafficked via the cell membrane and is a cargo that is internalized by clathrin-mediated endocytosis in Dictyostelium. Internalization of dajumin-GFP is via a clathrin-dependent, AP-2 independent mechanism and is distinct from other endocytic mechanisms. These results suggest the role of clathrin in protein sorting also evolved early, while dependence on specific components of the clathrin-mediated endocytic pathway, may have evolved later, as demonstrated by internalization independent of AP-2 function. In Dictyostelium, clathrin is known to be required for the formation of multicellular structures. Since it is possible to visualize the transition from a unicellular state to multicellularity, Dictyostelium is now uniquely positioned to study the dynamics of clathrin-mediated endocytosis during multicellular development

MEASURING ENERGY EXPENDITURE IN SWIMMING TO ASSESS GROSSMECHANICAL EFFICIENCY

When swimming at submaximal speeds the rate of energy expenditure can be estimated from respiratory analysis. Over the past decades many methods for stationary non-aqueous evaluation of respiratory and ventilatory parameters in swimmers has emerged. Recently there have been advances in portable telemetric systems (Cosmed K4 b2, Italy) that can measure these variables from rest to maximal activity while swimming without the need to stop the exercise. Preliminary data show that the last modified snorkel system can be considered as a valid device for collecting expired gas for BxB analysis, comparable to the standard facemask, with the advantage of being suitable for measurements during swimming

Human neutrophils activated via TLR8 promote Th17 polarization through IL-23

Human neutrophils contribute to the regulation of inflammation via the generation of a range of cytokines that affect all elements of the immune system. Here, we investigated their ability to express some of the members of the IL‐12 family after incubation with TLR8 agonists. Highly pure human neutrophils were thus incubated for up to 48 h with or without R848, or other TLR8 agonists, to then measure the expression levels of transcripts and proteins for IL‐12 family member subunits by RNA‐seq, reverse transcription quantitative PCR, and ELISA. We show a TLR8‐mediated inducible expression of IL‐12B and IL‐23A, but not IL‐12A, mRNA, which occurs via chromatin remodeling (as assessed by ChIP‐seq), and subsequent production of IL‐23 and IL‐12B, but no IL‐12, proteins. Induction of IL‐23 requires endogenous TNF‐α, as both mRNA and protein levels were blocked in TLR8‐activated neutrophils via a TNF‐α‐neutralizing Ab. We also show that supernatants from TLR8‐activated neutrophils, but not autologous monocytes, induce the differentiation of Th17 cells from naïve T cells in an IL‐23‐dependent fashion. This study unequivocally demonstrates that highly pure human neutrophils express and produce IL‐23, further supporting the key roles played by these cells in the important IL‐17/IL‐23 network and Th17 responses

Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through α2β1 integrin

Endorepellin, the COOH-terminal domain of the heparan sulfate proteoglycan perlecan, inhibits several aspects of angiogenesis. We provide evidence for a novel biological axis that links a soluble fragment of perlecan protein core to the major cell surface receptor for collagen I, α2β1 integrin, and provide an initial investigation of the intracellular signaling events that lead to endorepellin antiangiogenic activity. The interaction between endorepellin and α2β1 integrin triggers a unique signaling pathway that causes an increase in the second messenger cAMP; activation of two proximal kinases, protein kinase A and focal adhesion kinase; transient activation of p38 mitogen-activated protein kinase and heat shock protein 27, followed by a rapid down-regulation of the latter two proteins; and ultimately disassembly of actin stress fibers and focal adhesions. The end result is a profound block of endothelial cell migration and angiogenesis. Because perlecan is present in both endothelial and smooth muscle cell basement membranes, proteolytic activity during the initial stages of angiogenesis could liberate antiangiogenic fragments from blood vessels' walls, including endorepellin

Integrated analysis of randomized controlled trials evaluating bortezomib + lenalidomide + dexamethasone or bortezomib + thalidomide + dexamethasone induction in transplant-eligible newly diagnosed multiple myeloma

ObjectiveProviding the most efficacious frontline treatment for newly diagnosed multiple myeloma (NDMM) is critical for patient outcomes. No direct comparisons have been made between bortezomib + lenalidomide + dexamethasone (VRD) and bortezomib + thalidomide + dexamethasone (VTD) induction regimens in transplant-eligible NDMM.MethodsAn integrated analysis was performed using patient data from four trials meeting prespecified eligibility criteria: two using VRD (PETHEMA GEM2012 and IFM 2009) and two using VTD (PETHEMA GEM2005 and IFM 2013-04).ResultsThe primary endpoint was met, with VRD demonstrating a noninferior rate of at least very good partial response (>= VGPR) after induction vs VTD. GEM comparison demonstrated improvement in the >= VGPR rate after induction for VRD vs VTD (66.3% vs 51.2%; P = .00281) that increased after transplant (74.4% vs 53.5%). Undetectable minimal residual disease rates post induction (46.7% vs 34.9%) and post transplant (62.4% vs 47.3%) support the benefit of VRD vs VTD. Treatment-emergent adverse events leading to study and/or treatment discontinuation were less frequent with VRD (3%, GEM2012; 6%, IFM 2009) vs VTD (11%, IFM 2013-04).ConclusionThese results supported the benefit of VRD over VTD for induction in transplant-eligible patients with NDMM. The trials included are registered with ClinicalTrials.gov (NCT01916252, NCT01191060, NCT00461747, and NCT01971658)

Optimizing laser coupling, matter heating, and particle acceleration from solids using multiplexed ultraintense lasers

Realizing the full potential of ultrahigh-intensity lasers for particle and radiation generation will require multi-beam arrangements due to technology limitations. Here, we investigate how to optimize their coupling with solid targets. Experimentally, we show that overlapping two intense lasers in a mirror-like configuration onto a solid with a large preplasma can greatly improve the generation of hot electrons at the target front and ion acceleration at the target backside. The underlying mechanisms are analyzed through multidimensional particle-in-cell simulations, revealing that the self-induced magnetic fields driven by the two laser beams at the target front are susceptible to reconnection, which is one possible mechanism to boost electron energization. In addition, the resistive magnetic field generated during the transport of the hot electrons in the target bulk tends to improve their collimation. Our simulations also indicate that such effects can be further enhanced by overlapping more than two laser beams

Patterns of anti-malarial drug treatment among pregnant women in Uganda

BACKGROUND: Prompt use of an effective anti-malarial drug is essential for controlling malaria and its adverse effects in pregnancy. The World Health Organization recommends an artemisinin-based combination therapy as the first-line treatment of uncomplicated malaria in the second and third trimesters of pregnancy. The study objective was to determine the degree to which presumed episodes of uncomplicated symptomatic malaria in pregnancy were treated with a recommended anti-malarial regimen in a region of Uganda. METHODS: Utilizing a population-based random sample, we interviewed women living in Jinja, Uganda who had been pregnant in the past year. RESULTS: Self-reported malaria during the index pregnancy was reported among 67% (n = 334) of the 500 participants. Among the 637 self-reported episodes of malaria, an anti-malarial drug was used for treatment in 85% of the episodes. Use of a currently recommended treatment in the first trimester was uncommon (5.6%). A contraindicated anti-malarial drug (sulphadoxine-pyrimethamine and/or artemether-lumefantrine) was involved in 70% of first trimester episodes. Recommended anti-malarials were used according to the guidelines in only 30.1% of all second and third trimester episodes. CONCLUSIONS: Self-reported malaria was extremely common in this population and adherence to treatment guidelines for the management of malaria in pregnancy was poor. Use of artemether-lumefantrine combined with non-recommended anti-malarials was common practice. Overuse of anti-malarial drugs, especially ones that are no longer recommended, undermines malaria control efforts by fueling the spread of drug resistance and delaying appropriate treatment of non-malarial febrile illnesses. Improved diagnostic capacity is essential to ultimately improving the management of malaria-like symptoms during pregnancy and appropriate use of currently available anti-malarials

Addressing disparities in maternal health care in Pakistan: gender, class and exclusion

Background: After more than two decades of the Safe Motherhood Initiative and Millennium Development Goals aimed at reducing maternal mortality, women continue to die in childbirth at unacceptably high rates in Pakistan. While an extensive literature describes various programmatic strategies, it neglects the rigorous analysis of the reasons these strategies have been unsuccessful, especially for women living at the economic and social margins of society. A critical gap in current knowledge is a detailed understanding of the root causes of disparities in maternal health care, and in particular, how gender and class influence policy formulation and the design and delivery of maternal health care services. Taking Pakistan as a case study, this research builds upon two distinct yet interlinked conceptual approaches to understanding the phenomenon of inequity in access to maternal health care: social exclusion and health systems as social institutions. Methods/Design: This four year project consists of two interrelated modules that focus on two distinct groups of participants: (1) poor, disadvantaged women and men and (2) policy makers, program managers and health service providers. Module one will employ critical ethnography to understand the key axes of social exclusion as related to gender, class and zaat and how they affect women’s experiences of using maternal health care. Through health care setting observations, interviews and document review, Module two will assess policy design and delivery of maternal health services. Discussion: This research will provide theoretical advances to enhance understanding of the power dynamics of gender and class that may underlie poor women’s marginalization from health care systems in Pakistan. It will also provide empirical evidence to support formulation of maternal health care policies and health care system practices aimed at reducing disparities in maternal health care in Pakistan. Lastly, it will enhance inter-disciplinary research capacity in the emerging field of social exclusion and maternal health and help reduce social inequities and achieve the Millennium Development Goal No. 5