Chlamydial Infection-Dependent Synthesis of Sphingomyelin as a Novel Anti-Chlamydial Target of Ceramide Mimetic Compounds

The obligate intracellular bacterium Chlamydia trachomatis is the major causative agent of bacterial sexually transmitted diseases worldwide. In infected cells, the ceramide transport protein (CERT) is recruited to inclusions, where C. trachomatis replicates using host-synthesized ceramide. The ceramide is converted to sphingomyelin (SM) by a chlamydial infection-dependent SM synthesis (cidSM-synthesis) pathway, which occurs even in the absence of the SM synthases (SMS)-1 and -2 of host cells. The ceramide mimetic compound (1R,3S)-HPA-12 and the nonmimetic compound E16A, both of which are potent inhibitors of CERT, repressed the proliferation of C. trachomatis in HeLa cells. Unexpectedly, (1R,3R)-HPA-12, a ceramide mimetic compound that lacks CERT inhibitory activity, also exhibited potent anti-chlamydial activity. Using endogenous SMS-knockout mutant HeLa cells, we revealed that (1R,3R)-HPA-12 mildly inhibited cidSM-synthesis. In addition, LC-MS analysis revealed that (1R,3R)-HPA-12 is converted to a phosphocholine-conjugated metabolite in an infection-dependent manner. Imaging analysis with a fluorescent analog of ceramide suggested that cidSM-synthesis occurs in the bacterial bodies and/or inclusions. Collectively, these results suggested that (1R,3R)-HPA-12 exerts its anti-chlamydia activity not only as an inhibitor of cidSM-synthesis, but also via putative toxic effects of its phosphocholine adduct, which is most likely produced by the cidSM-synthesis route

Comprehensive analysis of resistance-nodulation-cell division superfamily (RND) efflux pumps from Serratia marcescens, Db10

We investigated the role of the resistance-nodulation-cell division superfamily (RND) efflux system on intrinsic multidrug resistance in Serratia marcescens. We identified eight putative RND efflux system genes in the S. marcescens Db10 genome that included the previously characterized systems, sdeXY, sdeAB, and sdeCDE. Six out of the eight genes conferred multidrug resistance on KAM32, a drug hypersensitive strain of Escherichia coil. Five out of the eight genes conferred resistance to benzalkonium, suggesting the importance of RND efflux systems in biocide resistance in S. marcescens. The energy-dependent efflux activities of five of the pumps were examined using a rhodamine 6G efflux assay. When expressed in the toiC-deficient strain of E. coil, KAM43, none of the genes conferred resistance on E. coil. When hasF, encoding the S. marcescens ToIC ortholog, was expressed in KAM43, all of the genes conferred resistance on E. coil, suggesting that HasF is a major outer membrane protein that is used by all RND efflux systems in this organism. We constructed a sdeXY deletion mutant from a derivative strain of the clinically isolated multidrug-resistant S. marcescens strain and found that the sdeXY deletion mutant was sensitive to a broad spectrum of antimicrobial agents

Natural ligand-nonmimetic inhibitors of the lipid-transfer protein CERT

Lipid transfer proteins mediate inter-organelle transport of membrane lipids at organelle contact sites in cells, playing fundamental roles in the lipidome and membrane biogenesis in eukaryotes. We previously developed a ceramide-mimetic compound as a potent inhibitor of the ceramide transport protein CERT. Here we develop CERT inhibitors with structures unrelated to ceramide. To this aim, we identify a seed compound with no ceramide-like structure but with the capability of forming a hydrogen-bonding network in the ceramide-binding START domain, by virtual screening of ~3 × 106 compounds. We also establish a surface plasmon resonance-based system to directly determine the affinity of compounds for the START domain. Then, we subject the seed compound to a series of in silico docking simulations, efficient chemical synthesis, affinity analysis, protein-ligand co-crystallography, and various in vivo assays. This strategy allows us to obtain ceramide-unrelated compounds that potently inhibited the function of CERT in human cultured cells

Dynamics of thermochemical plumes: 2. Complexity of plume structures and its implications for mapping mantle plumes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94911/1/ggge780.pd

Relation between Inner Structural Dynamics and Ion Dynamics of Laser-Heated Nanoparticles

When a nanoparticle is irradiated by an intense laser pulse, it turns into a nanoplasma, a transition that is accompanied by many interesting nonequilibrium dynamics. So far, most experiments on nanoplasmas use ion measurements, reflecting the outside dynamics in the nanoparticle. Recently, the direct observation of the ultrafast structural dynamics on the inside of the nanoparticle also became possible with the advent of x-ray free electron lasers (XFELs). Here, we report on combined measurements of structural dynamics and speeds of ions ejected from nanoplasmas produced by intense near-infrared laser irradiations, with the control of the initial plasma conditions accomplished by widely varying the laser intensity (9 x 10(14) W/cm(2) to 3 x 10(16) W/cm(2)). The structural change of nanoplasmas is examined by time-resolved x-ray diffraction using an XFEL, while the kinetic energies of ejected ions are measured by an ion time-of-fight method under the same experimental conditions. We find that the timescale of crystalline disordering in nanoplasmas strongly depends on the laser intensity and scales with the inverse of the average speed of ions ejected from the nanoplasma. The observations support a recently suggested scenario for nanoplasma dynamics in the wide intensity range, in which crystalline disorder in nanoplasmas is caused by a rarefaction wave propagating at a speed comparable with the average ion speed from the surface toward the inner crystalline core. We demonstrate that the scenario is also applicable to nanoplasma dynamics in the hard x-ray regime. Our results connect the outside nanoplasma dynamics to the loss of structure inside the sample on the femtosecond timescale

Zds2p Regulates Swe1p-dependent Polarized Cell Growth in Saccharomyces cerevisiae via a Novel Cdc55p Interaction Domain

A C-terminal region in Zds2p (ZH4) is required for regulation of Swe1p-dependent polarized cell growth and this region is necessary and sufficient for interaction with protein phosphatase 2A regulatory subunit, Cdc55p. Our results indicate that the Zds proteins regulate the Swe1p-dependent G2/M checkpoint in a CDC55-dependent manner

Molecular biology of the blood-brain and the blood-cerebrospinal fluid barriers: similarities and differences

Efficient processing of information by the central nervous system (CNS) represents an important evolutionary advantage. Thus, homeostatic mechanisms have developed that provide appropriate circumstances for neuronal signaling, including a highly controlled and stable microenvironment. To provide such a milieu for neurons, extracellular fluids of the CNS are separated from the changeable environment of blood at three major interfaces: at the brain capillaries by the blood-brain barrier (BBB), which is localized at the level of the endothelial cells and separates brain interstitial fluid (ISF) from blood; at the epithelial layer of four choroid plexuses, the blood-cerebrospinal fluid (CSF) barrier (BCSFB), which separates CSF from the CP ISF, and at the arachnoid barrier. The two barriers that represent the largest interface between blood and brain extracellular fluids, the BBB and the BCSFB, prevent the free paracellular diffusion of polar molecules by complex morphological features, including tight junctions (TJs) that interconnect the endothelial and epithelial cells, respectively. The first part of this review focuses on the molecular biology of TJs and adherens junctions in the brain capillary endothelial cells and in the CP epithelial cells. However, normal function of the CNS depends on a constant supply of essential molecules, like glucose and amino acids from the blood, exchange of electrolytes between brain extracellular fluids and blood, as well as on efficient removal of metabolic waste products and excess neurotransmitters from the brain ISF. Therefore, a number of specific transport proteins are expressed in brain capillary endothelial cells and CP epithelial cells that provide transport of nutrients and ions into the CNS and removal of waste products and ions from the CSF. The second part of this review concentrates on the molecular biology of various solute carrier (SLC) transport proteins at those two barriers and underlines differences in their expression between the two barriers. Also, many blood-borne molecules and xenobiotics can diffuse into brain ISF and then into neuronal membranes due to their physicochemical properties. Entry of these compounds could be detrimental for neural transmission and signalling. Thus, BBB and BCSFB express transport proteins that actively restrict entry of lipophilic and amphipathic substances from blood and/or remove those molecules from the brain extracellular fluids. The third part of this review concentrates on the molecular biology of ATP-binding cassette (ABC)-transporters and those SLC transporters that are involved in efflux transport of xenobiotics, their expression at the BBB and BCSFB and differences in expression in the two major blood-brain interfaces. In addition, transport and diffusion of ions by the BBB and CP epithelium are involved in the formation of fluid, the ISF and CSF, respectively, so the last part of this review discusses molecular biology of ion transporters/exchangers and ion channels in the brain endothelial and CP epithelial cells

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362