759 research outputs found
Vaccinia virus as a tool to study novel mechanisms of host cell contraction and blebbing
Vaccinia is a widely studied member of the poxvirus family. Throughout its life cycle, vaccinia manipulates cellular processes of the host to aid its replication and spread. Early in infection vaccinia stimulates transient host cell contraction, associated with dynamic plasma membrane blebbing and migration, reminiscent of the RhoA-ROCK regulated amoeboid migration of cancer cells. Cell contraction required the expression of early vaccinia genes whilst subsequent re-spreading required late vaccinia genes. Using a virus that does not express the vaccinia protein F11, I showed that F11 was required for virus infection to stimulate contraction. However, using a highly attenuated vaccinia strain rescued with wild type F11, MVA-F11, I found that cell contraction additionally required other vaccinia proteins. Live cell imaging of F11-GFP revealed that F11 is recruited to the plasma membrane during blebbing, further supporting a role for F11 in regulating these membrane events. Despite previous evidence from this laboratory showing F11 interacts with RhoA, I found that cell contraction was independent of RhoA. Instead vaccinia induced contraction required RhoC to ROCK signalling. Furthermore, using a small siRNA screen of several Rho GTPases, I discovered that RhoD negatively regulates vaccinia-induced contraction. I suggest that RhoD can supress cell contraction in uninfected and ∆F11L-infected cells by inhibiting RhoC activation, and suggest that RhoD signalling is inhibited during infection to allow for efficient cell contraction
The Salmonella effector SteD mediates MARCH8-1 dependent ubiquitination of MHC II molecules and inhibits T cell activation
The SPI-2 type III secretion system (T3SS) of intracellular Salmonella enterica translocates effector proteins into mammalian cells. Infection of antigen-presenting cells results in SPI-2 T3SS-dependent ubiquitination and reduction of surface-localized mature MHC class II (mMHCII). We identify the effector SteD as required and sufficient for this process. In Mel Juso cells, SteD localized to the Golgi network and vesicles containing the E3 ubiquitin ligase MARCH8 and mMHCII. SteD caused MARCH8-dependent ubiquitination and depletion of surface mMHCII. One of two transmembrane domains and the C-terminal cytoplasmic region of SteD mediated binding to MARCH8 and mMHCII, respectively. Infection of dendritic cells resulted in SteD-dependent depletion of surface MHCII, the co-stimulatory molecule B7.2, and suppression of T cell activation. SteD also accounted for suppression of T cell activation during Salmonella infection of mice. We propose that SteD is an adaptor, forcing inappropriate ubiquitination of mMHCII by MARCH8 and thereby suppressing T cell activation
The molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases
Pathogenic bacteria rely on secreted effector proteins to manipulate host signaling pathways, often in creative ways. CE clan proteases, specific hydrolases for ubiquitin-like modifications (SUMO and NEDD8) in eukaryotes, reportedly serve as bacterial effector proteins with deSUMOylase, deubiquitinase, or, even, acetyltransferase activities. Here, we characterize bacterial CE protease activities, revealing K63-linkage-specific deubiquitinases in human pathogens, such as Salmonella, Escherichia, and Shigella, as well as ubiquitin/ubiquitin-like cross-reactive enzymes in Chlamydia, Rickettsia, and Xanthomonas. Five crystal structures, including ubiquitin/ubiquitin-like complexes, explain substrate specificities and redefine relationships across the CE clan. Importantly, this work identifies novel family members and provides key discoveries among previously reported effectors, such as the unexpected deubiquitinase activity in Xanthomonas XopD, contributed by an unstructured ubiquitin binding region. Furthermore, accessory domains regulate properties such as subcellular localization, as exemplified by a ubiquitin-binding domain in Salmonella Typhimurium SseL. Our work both highlights and explains the functional adaptations observed among diverse CE clan proteins
The Physiotherapy eSkills Training Online resource improves performance of practical skills: A controlled trial
Background: E-learning is a common and popular mode of educational delivery, but little is known about its effectiveness in teaching practical skills. The aim of this study was to determine whether the Physiotherapy eSkills Training Online resource in addition to usual teaching improved the performance of practical skills in physiotherapy students. Method: This study was a non-randomised controlled trial. The participants were graduate entry physiotherapy students enrolled in consecutive semesters of a neurological physiotherapy unit of study. The experimental group received the Physiotherapy eSkills Training Online resource as well as usual teaching. The Physiotherapy eSkills Training Online resource is an online resource incorporating (i) video-clips of patient-therapist simulations; (ii) supportive text describing the aim, rationale, equipment, key points, common errors and methods of progression; and (iii) a downloadable PDF document incorporating the online text information and a still image of the video-clip for each practical skill. The control group received usual teaching only. The primary outcomes were the overall performance of practical skills as well as their individual components, measured using a practical examination. Results: The implementation of the Physiotherapy eSkills Training Online resource resulted in an increase of 1.6 out of 25 (95% CI -0.1 to 3.3) in the experimental group compared with the control group. In addition, the experimental group scored 0.5 points out of 4 (95% CI 0 to 1.1) higher than the control group for 'effectiveness of the practical skill' and 0.6 points out of 4 (95% CI 0.1 to 1.1) higher for 'rationale for the practical skill'. Conclusion: There was improvement in performance of practical skills in students who had access to the Physiotherapy eSkills Training Online resource in addition to usual teaching. Students considered the resource to be very useful for learning.7 page(s
DNA topoisomerases participate in fragility of the oncogene RET
Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APHinduced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication
D* Production in Deep Inelastic Scattering at HERA
This paper presents measurements of D^{*\pm} production in deep inelastic
scattering from collisions between 27.5 GeV positrons and 820 GeV protons. The
data have been taken with the ZEUS detector at HERA. The decay channel
(+ c.c.) has been used in the study. The
cross section for inclusive D^{*\pm} production with
and is 5.3 \pms 1.0 \pms 0.8 nb in the kinematic region
{ GeV and }. Differential cross
sections as functions of p_T(D^{*\pm}), and are
compared with next-to-leading order QCD calculations based on the photon-gluon
fusion production mechanism. After an extrapolation of the cross section to the
full kinematic region in p_T(D^{*\pm}) and (D^{*\pm}), the charm
contribution to the proton structure function is
determined for Bjorken between 2 10 and 5 10.Comment: 17 pages including 4 figure
Observation of Scaling Violations in Scaled Momentum Distributions at HERA
Charged particle production has been measured in deep inelastic scattering
(DIS) events over a large range of and using the ZEUS detector. The
evolution of the scaled momentum, , with in the range 10 to 1280
, has been investigated in the current fragmentation region of the Breit
frame. The results show clear evidence, in a single experiment, for scaling
violations in scaled momenta as a function of .Comment: 21 pages including 4 figures, to be published in Physics Letters B.
Two references adde
Observation of hard scattering in photoproduction events with a large rapidity gap at HERA
Events with a large rapidity gap and total transverse energy greater than 5
GeV have been observed in quasi-real photoproduction at HERA with the ZEUS
detector. The distribution of these events as a function of the
centre of mass energy is consistent with diffractive scattering. For total
transverse energies above 12 GeV, the hadronic final states show predominantly
a two-jet structure with each jet having a transverse energy greater than 4
GeV. For the two-jet events, little energy flow is found outside the jets. This
observation is consistent with the hard scattering of a quasi-real photon with
a colourless object in the proton.Comment: 19 pages, latex, 4 figures appended as uuencoded fil
Utilisation of an operative difficulty grading scale for laparoscopic cholecystectomy
Background
A reliable system for grading operative difficulty of laparoscopic cholecystectomy would standardise description of findings and reporting of outcomes. The aim of this study was to validate a difficulty grading system (Nassar scale), testing its applicability and consistency in two large prospective datasets.
Methods
Patient and disease-related variables and 30-day outcomes were identified in two prospective cholecystectomy databases: the multi-centre prospective cohort of 8820 patients from the recent CholeS Study and the single-surgeon series containing 4089 patients. Operative data and patient outcomes were correlated with Nassar operative difficultly scale, using Kendall’s tau for dichotomous variables, or Jonckheere–Terpstra tests for continuous variables. A ROC curve analysis was performed, to quantify the predictive accuracy of the scale for each outcome, with continuous outcomes dichotomised, prior to analysis.
Results
A higher operative difficulty grade was consistently associated with worse outcomes for the patients in both the reference and CholeS cohorts. The median length of stay increased from 0 to 4 days, and the 30-day complication rate from 7.6 to 24.4% as the difficulty grade increased from 1 to 4/5 (both p < 0.001). In the CholeS cohort, a higher difficulty grade was found to be most strongly associated with conversion to open and 30-day mortality (AUROC = 0.903, 0.822, respectively). On multivariable analysis, the Nassar operative difficultly scale was found to be a significant independent predictor of operative duration, conversion to open surgery, 30-day complications and 30-day reintervention (all p < 0.001).
Conclusion
We have shown that an operative difficulty scale can standardise the description of operative findings by multiple grades of surgeons to facilitate audit, training assessment and research. It provides a tool for reporting operative findings, disease severity and technical difficulty and can be utilised in future research to reliably compare outcomes according to case mix and intra-operative difficulty
Measurement of the diffractive structure function in deep inelastic scattering at HERA
This paper presents an analysis of the inclusive properties of diffractive
deep inelastic scattering events produced in interactions at HERA. The
events are characterised by a rapidity gap between the outgoing proton system
and the remaining hadronic system. Inclusive distributions are presented and
compared with Monte Carlo models for diffractive processes. The data are
consistent with models where the pomeron structure function has a hard and a
soft contribution. The diffractive structure function is measured as a function
of \xpom, the momentum fraction lost by the proton, of , the momentum
fraction of the struck quark with respect to \xpom, and of . The \xpom
dependence is consistent with the form \xpoma where
in all bins of and
. In the measured range, the diffractive structure function
approximately scales with at fixed . In an Ingelman-Schlein type
model, where commonly used pomeron flux factor normalisations are assumed, it
is found that the quarks within the pomeron do not saturate the momentum sum
rule.Comment: 36 pages, latex, 11 figures appended as uuencoded fil
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