Regulation of Rab5 Function during Phagocytosis of Live Pseudomonas aeruginosa in Macrophages

Pseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment of P. aeruginosa infection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages of P. aeruginosa invasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated, P. aeruginosa inhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of live P. aeruginosa. These studies provide evidence that live P. aeruginosa cells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5

Magnetic Resonance Diffusion Tensor Imaging and Diffusion Compartmental Modeling in an Animal Model of Chronic Kidney Disease

poster abstractPurpose: According to National Health and Nutrition Examination Survey (NHANES), Chronic Kidney Disease (CKD) affects 25% of the US population over age 601. Renal fibrosis, a common pathological consequence of CKD, is a progressive process that ultimately leads to end-stage renal failure that requires dialysis or kidney transplantation2. There is a compelling need for non-invasive biomarkers that track changes in the tissue microenvironment associated with CKD. Several studies using magnetic resonance diffusion tensor imaging (DTI) have been proposed as imaging biomarkers for CKD3. In this study, in addition to DTI, we explored a diffusion-compartmental modeling technique4 to study the microstructures of hypoxia induced animal models of CKD. Method: Preparation of the animal CKD model: Experiments were performed in 4 Wistar Rats using protocols approved by the Institutional Animal Care and Use Committee (IACUC). Two days prior to the first magnetic resonance imaging (MRI) scan; surgical intervention in right renal artery was performed in all the animals to create hypoxia induced renal fibrosis. The MRI scans were repeated at an interval of approximately one month. During the imaging session, the rats were sedated and kept in head-first supine position. MRI imaging: The MRI diffusion pulse sequence was a single-shot spin-echo echo-planar imaging (SS-SE-EPI) sequence with multiple diffusion-weighting b-values (i.e. 3 shells with b-values of 150, 300 and 450 s/mm2) and multiple diffusion-weighting directions at each shell (i.e., 10, 19 and 30, respectively). Diffusion directions in each shell and in the projected sphere with all directions (i.e., total 59) were optimized for uniform diffusion sampling in the spherical space5. The repetition time (TR) is 2200 ms and echo time (TE) is 73.6 ms. A total of four signal averages was performed. The imaging parameters were field-of-view (FOV) = 128 x 64 mm, matrix size = 128 x 64, isotropic voxel size of 1 mm3, and 20 oblique coronal slices. Image data processing: DTI derived parameters including axial diffusivity (Da), radial diffusivity (Dr), mean diffusivity (MD), and fractional anisotropy (FA) were computed6. The diffusion compartmental model originally proposed for the brain called neutrite orientation dispersion and density imaging (NODDI)4 was modified to fit the water diffusivities of kidneys. The NODDI model with Watson stick framework produces the volume fraction of stick like diffusion compartment that may explain the active diffusion (transport) of water in the interstitial space between renal tubules, ellipsoid like diffusion compartment that may explain diffusion inside renal tubule, and a fast isotropic diffusion to account for the pseudo-diffusion term relating to bulk vascular flow. The normalized diffusion intensity was fit with a non-linear mathematical model given by A = (1-Viso) (VicAic+(1-Vic) Aec) + VisoAiso ; where Vic and Viso are the volume fraction of active water transport and free diffusion compartments in the kidney, respectively. Aic, Aec and Aiso are the normalized diffusion signal contribution from stick, tubule and free diffusion compartments, respectively. In the raw DW data, the b-value=0 volume clearly shows three distinct layers in the rat kidney representing the inner medulla, outer medulla and cortex (Figure1). Non-overlapping ROI's were constructed from the b-value =0 images. Figure 1: The DTI and Diffusion compartmental modeling parameter for RAT Kidney 2 days after surgical intervention. The Cortex (C), the Outer Medulla (OM) and Inner Medulla (IM) are shown in raw b0 maps. The orientation of the images follows radiology convention. Results: On post-surgical day 2, the overall water diffusivity (i.e., mean diffusivity (MD)) decreased significantly in the outer medullae and inner medullae of the surgical kidneys (Figure 2 B green bars). In the compartmental model, the volume fraction of the stick (interstitial) diffusion compartment (Vic) in right outer and inner medulla was significantly increased compared to the left (Figure 2A blue bars), whereas the volume fraction of water diffusion inside the tubules (Vec = (1-Vic)) decreased significantly. In addition, isotropic free diffusion compartment (Viso) was significantly lower in the inner medullae of the right kidneys. The axial diffusivity (Da) that may describe the diffusion parallel to the tubules decreased significantly in outer and inter medullae of the right surgical kidneys (Figure 2 B blue bars). The radial diffusivity (Dr) that may describe the water diffusion perpendicularly to the renal tubules decreased significantly in only the outer medullae of the right kidneys (Figure 2B gray bars). While FA shows high value in the inner medullae for both left and right kidneys, no significant results were found between left and right kidneys and between two time points. Over the one-month period of time, right inner medullae continued the significant changes in the diffusivity measurements (Figure 2C and D, right groups), but the diffusivities remained similar in the outer medullae (Figure 2 C and D, middle groups). No significant findings were found in the renal cortices between the right and left kidneys on post-surgical day 2 (Figure 2 A and B). Interestingly, the right renal cortices did have significant increase in Vic and decreases in Da, Dr, and MD over the one-month time period (Figure 2 C and D). Figure 2: Diffusion Compartmental (Figure 2A) and DTI (Figure 2B) parameters for Right Cortex (RC) and Left Cortex (LC), Right Outer Medulla (ROM) and Left Outer Medulla (LOM) and Right Inner Medulla (RIM) and Left Inner Medulla (LIM) on post-surgical day 2. (Figure 2C) Is the time series study of diffusion compartmental parameters and (Figure 2D) for DTI parameters for the right kidneys at post-surgical day 2 and 30, respectively. The bars represent diffusion measurements of all four rats. The overhead connecting lines represent significant statistical student t-test with p-value < 0.01. Discussions and Conclusion: The DTI and NODDI analogous diffusion compartment derived parameters are sensitive to the micro-structural changes in kidneys after surgical hypoxia intervention. The outer and inner medullae appear most sensitive to the surgical hypoxia intervention as early as post-surgical day 2. The preliminary result suggests that water diffusion decreases due to renal fibrosis, and more so inside the Henle tubules. In post-surgical day 30, renal cortices start to show changes in water diffusivities while inner medullae continue pathological changes. The NODDI compartmental model shows promising preliminary results in revealing renal microenvironments under the influences of hypoxia induced renal fibrosis. Further study is required to optimize and validate the model

Fate of charge order in overdoped La-based cuprates

In high-temperature cuprate superconductors, stripe order refers broadly to a coupled spin and charge modulation with a commensuration of eight and four lattice units, respectively. How this stripe order evolves across optimal doping remains a controversial question. Here we present a systematic resonant inelastic x-ray scattering study of weak charge correlations in La$_{2−x}$Sr$_{x}$CuO$_{4}$ and La$_{1.8−x}$Eu$_{0.2}$Sr$_{x}$CuO$_{4}$. Ultra high energy resolution experiments demonstrate the importance of the separation of inelastic and elastic scattering processes. Long-range temperature-dependent stripe order is only found below optimal doping. At higher doping, short-range temperature-independent correlations are present up to the highest doping measured. This transformation is distinct from and preempts the pseudogap critical doping. We argue that the doping and temperature-independent short-range correlations originate from unresolved electron–phonon coupling that broadly peaks at the stripe ordering vector. In La$_{2−x}$Sr$_{x}$CuO$_{4}$, long-range static stripe order vanishes around optimal doping and we discuss both quantum critical and crossover scenarios

Rpgrip1 is required for rod outer segment development and ciliary protein trafficking in zebrafish

The authors would like to thank the Royal Society of London, the National Eye Research Centre, the Visual Research Trust, Fight for Sight, the W.H. Ross Foundation, the Rosetrees Trust, and the Glasgow Children’s Hospital Charity for supporting this work. This work was also supported by the Deanship of Scientific Research at King Saud University for funding this research (Research Project) grant number ‘RGP – VPP – 219’.Mutations in the RPGR-interacting protein 1 (RPGRIP1) gene cause recessive Leber congenital amaurosis (LCA), juvenile retinitis pigmentosa (RP) and cone-rod dystrophy. RPGRIP1 interacts with other retinal disease-causing proteins and has been proposed to have a role in ciliary protein transport; however, its function remains elusive. Here, we describe a new zebrafish model carrying a nonsense mutation in the rpgrip1 gene. Rpgrip1homozygous mutants do not form rod outer segments and display mislocalization of rhodopsin, suggesting a role for RPGRIP1 in rhodopsin-bearing vesicle trafficking. Furthermore, Rab8, the key regulator of rhodopsin ciliary trafficking, was mislocalized in photoreceptor cells of rpgrip1 mutants. The degeneration of rod cells is early onset, followed by the death of cone cells. These phenotypes are similar to that observed in LCA and juvenile RP patients. Our data indicate RPGRIP1 is necessary for rod outer segment development through regulating ciliary protein trafficking. The rpgrip1 mutant zebrafish may provide a platform for developing therapeutic treatments for RP patients.Publisher PDFPeer reviewe

Effi cacy of a Russian-backbone live attenuated infl uenza vaccine among young children in Bangladesh: a randomised, double-blind, placebo-controlled trial

Background The rates of infl uenza illness and associated complications are high among children in Bangladesh. We assessed the clinical effi cacy and safety of a Russian-backbone live attenuated infl uenza vaccine (LAIV) at two fi eld sites in Bangladesh. Methods Between Feb 27 and April 9, 2013, children aged 2–4 years in urban Kamalapur and rural Matlab, Bangladesh, were randomly assigned in a 2:1 ratio, according to a computer-generated schedule, to receive one intranasal dose of LAIV or placebo. After vaccination, we monitored children in weekly home visits until Dec 31, 2013, with study clinic surveillance for infl uenza illness. The primary outcome was symptomatic, laboratory-confi rmed infl uenza illness due to vaccine-matched strains. Analysis was per protocol. The trial is registered with ClinicalTrials.gov, number NCT01797029. Findings Of 1761 children enrolled, 1174 received LAIV and 587 received placebo. Laboratory-confi rmed infl uenza illness due to vaccine-matched strains was seen in 93 (15·8%) children in the placebo group and 79 (6·7%) in the LAIV group. Vaccine effi cacy of LAIV for vaccine-matched strains was 57·5% (95% CI 43·6–68·0). The vaccine was well tolerated, and adverse events were balanced between the groups. The most frequent adverse events were tachypnoea (n=86 in the LAIV group and n=54 in the placebo group), cough (n=73 and n=43), and runny nose (n=68 and n=39), most of which were mild. Interpretation This single-dose Russian-backbone LAIV was safe and effi cacious at preventing symptomatic laboratory-confi rmed infl uenza illness due to vaccine-matched strains. LAIV programmes might reduce the burden of infl uenza illness in Bangladesh

Quantum Fluctuations in a Weakly Correlated Mott Insulator

Quantum fluctuations in low-dimensional systems and near quantum phase transitions have significant influences on material properties. Yet, it is difficult to experimentally gauge the strength and importance of quantum fluctuations. Here we provide a resonant inelastic x-ray scattering study of magnon excitations in Mott insulating cuprates. From the thin film of SrCuO$_2$, single- and bi-magnon dispersions are derived. Using an effective Heisenberg Hamiltonian generated from the Hubbard model, we show that the single magnon dispersion is only described satisfactorily when including significant renormalization stemming from quantum fluctuations. Comparative results on La$_2$CuO$_4$ indicate that quantum fluctuations are much stronger in SrCuO$_2$ suggesting closer proximity to a magnetic quantum critical point. Monte Carlo calculations suggest an exotic incommensurate magnetic order as the ground state that competes with the antiferromagnetic N\'eel order. Our results indicate that SrCuO$_2$ -- due to strong quantum fluctuations -- is a unique starting point for the exploration of novel magnetic ground states.Comment: Supplementary Information available upon reques

Uniaxial pressure induced stripe order rotation in La1.88Sr0.12CuO4

Static stripe order is detrimental to superconductivity. Yet, it has been proposed that transverse stripe fluctuations may enhance the inter-stripe Josephson coupling and thus promote superconductivity. Direct experimental studies of stripe dynamics, however, remain difficult. From a strong-coupling perspective, transverse stripe fluctuations are realized in the form of dynamic “kinks”—sideways shifting stripe sections. Here, we show how modest uniaxial pressure tuning reorganizes directional kink alignment. Our starting point is La1.88Sr0.12CuO4 where transverse kink ordering results in a rotation of stripe order away from the crystal axis. Application of mild uniaxial pressure changes the ordering pattern and pins the stripe order to the crystal axis. This reordering occurs at a much weaker pressure than that to detwin the stripe domains and suggests a rather weak transverse stripe stiffness. Weak spatial stiffness and transverse quantum fluctuations are likely key prerequisites for stripes to coexist with superconductivity

RRM3 Fluid Management Device

The current development progress of the fluid management device (FMD) for the Robotic Resupply Mission 3 (RRM3) cryogen source Dewar is described. RRM3 is an on-orbit cryogenic transfer experiment payload for the International Space Station. The fluid management device is a key component of the source Dewar to ensure the ullage bubble is located away from the outlet during transfer. The FMD also facilitates demonstration of radio frequency mass gauging within the source Dewar. The preliminary design of the RRM3 FMD is a number of concentric cones of Mylar which maximizes the volume of liquid in contact with the FMD in the source Dewar. This paper describes the design of the fluid management device and progress of hardware developmen

Human cardiac tissue in a microperfusion chamber simulating extracorporeal circulation - ischemia and apoptosis studies

<p>Abstract</p> <p>Background</p> <p>After coronary artery bypass grafting ischemia/reperfusion injury inducing cardiomyocyte apoptosis may occur. This surgery-related inflammatory reaction appears to be of extreme complexity with regard to its molecular, cellular and tissue mechanisms and many studies have been performed on animal models. However, finding retrieved from animal studies were only partially confirmed in humans. To investigate this phenomenon and to evaluate possible therapies in vitro, adequate human cardiomyocyte models are required. We established a tissue model of human cardiomyocytes preserving the complex tissue environment. To our knowledge human cardiac tissue has not been investigated in an experimental setup mimicking extracorporeal circulation just in accordance to clinical routine, yet.</p> <p>Methods</p> <p>Cardiac biopsies were retrieved from the right auricle of patients undergoing elective coronary artery bypass grafting before cardiopulmonary bypass. The extracorporeal circulation was simulated by submitting the biopsies to varied conditions simulating cardioplegia (cp) and reperfusion (rep) in a microperfusion chamber. Cp/rep time sets were 20/7, 40/13 and 60/20 min. For analyses of the calcium homoeostasis the fluorescent calcium ion indicator FURA-2 and for apoptosis detection PARP-1 cleavage immunostaining were employed. Further the anti-apoptotic effect of carvedilol [10 μM] was investigated by adding into the perfusate.</p> <p>Results</p> <p>Viable cardiomyocytes presented an intact calcium homoeostasis under physiologic conditions. Following cardioplegia and reperfusion a time-dependent elevation of cytosolic calcium as a sign of disarrangement of the calcium homoeostasis occurred. PARP-1 cleavage also showed a time-dependence whereas reperfusion had the highest impact on apoptosis. Cardioplegia and carvedilol could reduce apoptosis significantly, lowering it between 60-70% (p < 0.05).</p> <p>Conclusions</p> <p>Our human cardiac preparation served as a reliable cellular model tool to study apoptosis in vitro. Decisively cardiac tissue from the right auricle can be easily obtained at nearly every cardiac operation avoiding biopsying of the myocardium or even experiments on animals.</p> <p>The apoptotic damage induced by the ischemia/reperfusion stimulus could be significantly reduced by the cold crystalloid cardioplegia. The additional treatment of cardiomyocytes with a non-selective β-blocker, carvedilol had even a significantly higher reduction of apoptotis.</p

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca(2+)-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca(2+)-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca(2+)-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues (4)SE(5); (7)TP(8); and (31)LS(32)). The cleavage between the (31)LS(32) peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Results argue for a mechanism where Ca(2+)-calpain may regulate nuclear availability and degradation of dUTPase