Identification and characterization of secreted proteins of Theileria lestoquardi schizonts

T. lestoquardi is a tick-borne parasite that infects leukocytes of sheep and goats. It causes the death of millions of sheep and goats every year and continues to spread in the world. The disease is mainly caused by the schizont stage, which induces transformation of the infected cell to a state of uncontrolled proliferation. In response to the proliferation of infected cells, there is evidence that the host deploys its immune system to eliminate the pathogen by cytotoxic T lymphocyte (CTL) responses. The purpose of this study was to identify schizont secreted proteins, which are likely to include molecules targeted by the immune response as well as those associated with the transformation process. An efficient method for the lysis and purification of schizonts from infected cells was established. This involved use of complement treatment to release schizonts from infected cells and an optimal density of Nycoprep gradient medium to separate schizonts from host cell components. An optimised method for metabolic labelling of schizont-infected cells was also established. Putative secreted proteins were detected in metabolically labelled schizont culture supernatants by SDS-PAGE and autoradiographic analysis. Using two-dimensional electrophoresis, mass spectrometry, including MALDI-TOF and Q-TOF, and N-terminal sequencing analysis, three putative secreted proteins of parasite origin were identified, namely Theileria mhsp70, hsp70 and inorganic pyrophosphatase. Seven host-derived proteins were also identified, namely mhsp70, hsp60, mitochondrial ribosomal protein LI2 (MRPL12, TFS4), ATP synthase-5, ATP synthase 6, PDH-Eip and ATP-dependent proteinase SP-22. Further investigation of the relationship of the host-derived proteins with the schizont using dual-labelling and confocal microscopy analysis provided evidence that the hostderived protein, MRPL12, is associated with intracellular schizonts with some colocalising with schizont mitochondria. The analysis also revealed that some host mitochondria are closely associated with the schizonts of T. lestoquardi

Repressive Mutations Restore Function-Loss Caused by the Disruption of Trimerization in \u3cem\u3eEscherichia coli\u3c/em\u3e Multidrug Transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding

Repressive Mutations Restore Function-Loss Caused by the Disruption of Trimerization in \u3cem\u3eEscherichia coli\u3c/em\u3e Multidrug Transporter AcrB

AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding

Functional Relevance of AcrB Trimerization in Pump Assembly and Substrate Binding

AcrB is a multidrug transporter in the inner membrane of Escherichia coli. It is an obligate homotrimer and forms a tripartite efflux complex with AcrA and TolC. AcrB is the engine of the efflux machinery and determines substrate specificity. Active efflux depends on several functional features including proton translocation across the inner membrane through a proton relay pathway in the transmembrane domain of AcrB; substrate binding and migration through the substrate translocation pathway; the interaction of AcrB with AcrA and TolC; and the formation of AcrB homotrimer. Here we investigated two aspects of the inter-correlation between these functional features, the dependence of AcrA-AcrB interaction on AcrB trimerization, and the reliance of substrate binding and penetration on protein-protein interaction. Interaction between AcrA and AcrB was investigated through chemical crosslinking, and a previously established in vivo fluorescent labeling method was used to probe substrate binding. Our data suggested that dissociation of the AcrB trimer drastically decreased its interaction with AcrA. In addition, while substrate binding with AcrB seemed to be irrelevant to the presence or absence of AcrA and TolC, the capability of trimerization and conduction of proton influx did affect substrate binding at selected sites along the substrate translocation pathway in AcrB

FSD-C10: A more promising novel ROCK inhibitor than Fasudil for treatment of CNS autoimmunity.

Rho-Rho kinase (Rho-ROCK) triggers an intracellular signalling cascade that regulates cell survival, death, adhesion, migration, neurite outgrowth and retraction and influences the generation and development of several neurological disorders. Although Fasudil, a ROCK inhibitor, effectively suppressed encephalomyelitis (EAE), certain side effects may limit its clinical use. A novel and efficient ROCK inhibitor, FSD-C10, has been explored. In the present study, we present chemical synthesis and structure of FSD-C10, as well as the relationship between compound concentration and ROCK inhibition. We compared the inhibitory efficiency of ROCKI and ROCK II, the cell cytotoxicity, neurite outgrowth and dendritic formation, neurotrophic factors and vasodilation between Fasudil and FSD-C10. The results demonstrated that FSD-C10, like Fasudil, induced neurite outgrowth of neurons and dendritic formation of BV-2 microglia and enhanced the production of neurotrophic factor brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3). However, the cell cytotoxicity and vasodilation of FSD-C10 were relatively small compared with Fasudil. Although Fasudil inhibited both ROCK I and ROCK II, FSD-C10 more selectively suppressed ROCK II, but not ROCK I, which may be related to vasodilation insensitivity and animal mortality. Thus, FSD-C10 may be a safer and more promising novel ROCK inhibitor than Fasudil for the treatment of several neurological disorders

Measurement of proton electromagnetic form factors in e+e−→ppˉe^+e^- \to p\bar{p} in the energy region 2.00-3.08 GeV

The process of $e^+e^- \rightarrow p\bar{p}$ is studied at 22 center-of-mass energy points ($\sqrt{s}$) from 2.00 to 3.08 GeV, exploiting 688.5~pb$^{-1}$ of data collected with the BESIII detector operating at the BEPCII collider. The Born cross section~($\sigma_{p\bar{p}}$) of $e^+e^- \rightarrow p\bar{p}$ is measured with the energy-scan technique and it is found to be consistent with previously published data, but with much improved accuracy. In addition, the electromagnetic form-factor ratio ($|G_{E}/G_{M}|$) and the value of the effective ($|G_{\rm{eff}}|$), electric ($|G_E|$) and magnetic ($|G_M|$) form factors are measured by studying the helicity angle of the proton at 16 center-of-mass energy points. $|G_{E}/G_{M}|$ and $|G_M|$ are determined with high accuracy, providing uncertainties comparable to data in the space-like region, and $|G_E|$ is measured for the first time. We reach unprecedented accuracy, and precision results in the time-like region provide information to improve our understanding of the proton inner structure and to test theoretical models which depend on non-perturbative Quantum Chromodynamics

Observation of ηc→ωω\eta_c\to\omega\omega in J/ψ→γωωJ/\psi\to\gamma\omega\omega

Using a sample of $(1310.6\pm7.0)\times10^6$ $J/\psi$ events recorded with the BESIII detector at the symmetric electron positron collider BEPCII, we report the observation of the decay of the $(1^1 S_0)$ charmonium state $\eta_c$ into a pair of $\omega$ mesons in the process $J/\psi\to\gamma\omega\omega$. The branching fraction is measured for the first time to be $\mathcal{B}(\eta_c\to\omega\omega)= (2.88\pm0.10\pm0.46\pm0.68)\times10^{-3}$, where the first uncertainty is statistical, the second systematic and the third is from the uncertainty of $\mathcal{B}(J/\psi\to\gamma\eta_c)$. The mass and width of the $\eta_c$ are determined as $M=(2985.9\pm0.7\pm2.1)\,$MeV/$c^2$ and $\Gamma=(33.8\pm1.6\pm4.1)\,$MeV.Comment: 13 pages, 6 figure

Observation of the WW-Annihilation Decay Ds+→ωπ+D^{+}_{s} \rightarrow \omega \pi^{+} and Evidence for Ds+→ωK+D^{+}_{s} \rightarrow \omega K^{+}

We report on the observation of the $W$-annihilation decay $D^{+}_{s} \rightarrow \omega \pi^{+}$ and the evidence for $D_{s}^{+} \rightarrow \omega K^{+}$ with a data sample corresponding to an integrated luminosity of 3.19 fb$^{-1}$ collected with the BESIII detector at the center-of-mass energy $\sqrt{s} = 4.178$ GeV. We obtain the branching fractions $\mathcal{B}(D^{+}_{s} \rightarrow \omega \pi^{+}) = (1.77\pm0.32_{{\rm stat.}}\pm0.11_{{\rm sys.}}) \times 10^{-3}$ and $\mathcal{B}(D^{+}_{s} \rightarrow \omega K^{+}) = (0.87\pm0.24_{{\rm stat.}}\pm0.07_{{\rm sys.}}) \times 10^{-3}$, respectively

Mercury's three‐dimensional asymmetric magnetopause

Mercury's magnetopause is unique in the solar system due to its relatively small size and its close proximity to the Sun. Based on 3 years of MErcury Surface, Space ENvironment, GEochemistry, and Ranging orbital Magnetometer and the Fast Imaging Plasma Spectrometer data, the mean magnetopause location was determined for a total of 5694 passes. We fit these magnetopause locations to a three‐dimensional nonaxially symmetric magnetopause which includes an indentation for the cusp region that has been successfully applied to the Earth. Our model predicts that Mercury's magnetopause is highly indented surrounding the cusp with central depth ~0.64 RM and large dayside extension. The dayside polar magnetopause dimension is, thus, smaller than the equatorial magnetopause dimension. Cross sections of the dayside magnetopause in planes perpendicular to the Mercury‐Sun line are prolate and elongated along the dawn‐dusk direction. In contrast, the magnetopause downstream of the terminator plane is larger in the north‐south than the east‐west directions by a ratio of 2.6 RM to 2.2 RM at a distance of 1.5 RM downstream of Mercury. Due to the northward offset of the internal dipole, the model predicts that solar wind has direct access to the surface of Mercury at middle magnetic latitudes in the southern hemisphere. During extremely high solar wind pressure conditions, the northern hemisphere middle magnetic latitudes may also be subject to direct solar wind impact.Key PointsMercury's magnetopause near‐cusp indentationMagnetotail cross sections elongated in north‐south directionExtreme solar wind pressure events were analyzedPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/115924/1/jgra52083_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/115924/2/jgra52083.pd