In Situ Measurement of Electron Energy Evolution in a Laser-Plasma Accelerator.

We report on a novel, noninvasive method applying Thomson scattering to measure the evolution of the electron beam energy inside a laser-plasma accelerator with high spatial resolution. The determination of the local electron energy enabled the in-situ detection of the acting acceleration fields without altering the final beam state. In this Letter we demonstrate that the accelerating fields evolve from (265±119) GV/m to (9±4) GV/m in a plasma density ramp. The presented data show excellent agreement with particle-in-cell simulations. This method provides new possibilities for detecting the dynamics of plasma-based accelerators and their optimization.

Compact all-optical precision-tunable narrowband hard Compton X-ray source.

Readily available bright X-ray beams with narrow bandwidth and tunable energy promise to unlock novel developments in a wide range of applications. Among emerging alternatives to large-scale and costly present-day radiation sources which severely restrict the availability of such beams, compact laser-plasma-accelerator-driven inverse Compton scattering sources show great potential. However, these sources are currently limited to tens of percent bandwidths, unacceptably large for many applications. Here, we show conceptually that using active plasma lenses to tailor the electron bunch-photon interaction, tunable X-ray and gamma beams with percent-level bandwidths can be produced. The central X-ray energy is tunable by varying the focusing strength of the lens, without changing electron bunch properties, allowing for precision-tuning the X-ray beam energy. This method is a key development towards laser-plasma-accelerator-driven narrowband, precision tunable femtosecond photon sources, enabling a paradigm shift and proliferation of compact X-ray applications.

Stress urinary incontinence after hysterectomy: a 10-year national follow-up study.

PURPOSE: Hysterectomy has been associated with increased risk for developing stress urinary incontinence (SUI) and having a SUI operation. We examined the long-term rate of SUI operations after hysterectomy and associated risk factors. METHODS: We followed up 5000 women without prior urinary incontinence (UI) who had a hysterectomy in a prospective FINHYST 2006 cohort study until the end of 2016 through a national health register. The main outcome was SUI operations, and secondary outcomes were outpatient visits for UI, and their association of preoperative patient and operation factors. RESULTS: During the median follow-up time of 10.6 years (IQR 10.3-10.8), 111 (2.2%) women had a SUI operation and 241 (4.8%) had an outpatient visit for UI. The SUI operation rate was higher after vaginal hysterectomy and laparoscopic hysterectomy (n = 71 and 28, 3.3% and 1.8%, respectively) compared to abdominal hysterectomy (n = 11, 0.8%). In a multivariate risk analysis by Cox regression, the association with vaginal hysterectomy and SUI operation remained significant when adjusted for vaginal deliveries, preceding pelvic organ prolapse (POP), uterus size, age and BMI (HR 2.4, 95% CI 1.1-5.3). Preceding POP, three or more deliveries and laparoscopic hysterectomy were significantly associated with UI visits but not with SUI operations. CONCLUSION: After hysterectomy, 2.2% of women underwent operative treatment for SUI. The number of SUI operations was more than double after vaginal hysterectomy compared to abdominal hysterectomy, but preceding POP explained this added risk partially. Preceding POP and three or more vaginal deliveries were independently associated with UI visits after hysterectomy.

FLASHForward: plasma wakefield accelerator science for high-average-power applications.

The FLASHForward experimental facility is a high-performance test-bed for precision plasma wakefield research, aiming to accelerate high-quality electron beams to GeV-levels in a few centimetres of ionized gas. The plasma is created by ionizing gas in a gas cell either by a high-voltage discharge or a high-intensity laser pulse. The electrons to be accelerated will either be injected internally from the plasma background or externally from the FLASH superconducting RF front end. In both cases, the wakefield will be driven by electron beams provided by the FLASH gun and linac modules operating with a 10 Hz macro-pulse structure, generating 1.25 GeV, 1 nC electron bunches at up to 3 MHz micro-pulse repetition rates. At full capacity, this FLASH bunch-train structure corresponds to 30 kW of average power, orders of magnitude higher than drivers available to other state-of-the-art LWFA and PWFA experiments. This high-power functionality means FLASHForward is the only plasma wakefield facility in the world with the immediate capability to develop, explore and benchmark high-average-power plasma wakefield research essential for next-generation facilities. The operational parameters and technical highlights of the experiment are discussed, as well as the scientific goals and high-average-power outlook. This article is part of the Theo Murphy meeting issue 'Directions in particle beam-driven plasma wakefield acceleration'.

MicroRNA-146a reduces MHC-II expression via targeting JAK/STAT signaling in dendritic cells after stem cell transplantation.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Cooperation of BRAF(F595L) and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling.

Activating BRAF mutations, in particular V600E/K, drive many cancers and are considered mutually exclusive with mutant RAS, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif cooperate with RAS via paradoxical MEK/ERK activation. Due to the increasing use of comprehensive tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We investigated an atypical BRAF mutation, F595L, which was identified along with mutant HRAS in histiocytic sarcoma and also occurs in epithelial cancers, melanoma and neuroblastoma, and determined its interaction with mutant RAS. Unlike other DFG motif mutants, BRAF(F595L) is a gain-of-function variant with intermediate activity that does not act paradoxically, but nevertheless cooperates with mutant RAS to promote oncogenic signaling, which is efficiently blocked by pan-RAF and MEK inhibitors. Mutation data from patients and cell lines show that BRAF(F595L), as well as other intermediate-activity BRAF mutations, frequently coincide with mutant RAS in various cancers. These data define a distinct class of activating BRAF mutations, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway and underscore the value of comprehensive genomic testing for uncovering the vulnerabilities of individual tumors.

Depletion of STAT5 blocks TEL-SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice.

The spleen tyrosine kinase (SYK) was identified as an oncogenic driver in a broad spectrum of hematologic malignancies. The in vivo comparison of three SYK containing oncogenes, SYK(wt), TEL-SYK and IL-2-inducible T-cell kinase (ITK)-SYK revealed a general myeloexpansion and the establishment of three different hematologic (pre)diseases. SYK(wt) enhanced the myeloid and T-cell compartment, without leukemia/lymphoma development. ITK-SYK caused lethal T-cell lymphomas and the cytoplasmic TEL-SYK fusion induced an acute panmyelosis with myelofibrosis-type acute myeloid leukemia (AML) with up to 50% immature megakaryoblasts infiltrating bone marrow, spleen and liver, additional MPN features (myelofibrosis and granulocyte expansion) and MDS stigmata with megakaryocytic and erythroid dysplasia. LKS cells were reduced and all subsets (LT/ST/MPP) showed reduced proliferation rates. SYK inhibitor treatment (R788) of diseased TEL-SYK mice reduced leukocytosis, spleen and liver infiltration, enhanced the hematocrit and prolonged survival time, but could not significantly reduce myelofibrosis. Stat5 was identified as a major downstream mediator of TEL-SYK in vitro as well as in vivo. Consequently, targeted deletion of Stat5 in vivo completely abrogated TEL-SYK-induced AML and myelofibrosis development, proving Stat5 as a major driver of SYK-induced transformation. Our experiments highlight the important role of SYK in AML and myelofibrosis and prove SYK and STAT5 inhibitors as potent treatment options for those diseases.

Antibiotic prophylaxis for hysterectomy, a prospective cohort study: cefuroxime, metronidazole, or both?

OBJECTIVE: To evaluate cefuroxime and metronidazole antibiotic prophylaxis. DESIGN: Observational nonrandomised 1-year prospective cohort study. SETTING: Fifty-three hospitals in Finland. POPULATION: A total of 5279 women undergoing hysterectomy for benign indications, with cefuroxime given to 4301 and metronidazole given to 2855. Excluding other antibiotics, cefuroxime alone was given to 2019, metronidazole alone was given to 518, and they were administered in combination to 2252 women. METHODS: Data on 1115 abdominal hysterectomies (AHs), 1541 laparoscopic hysterectomies (LHs), and 2133 vaginal hysterectomies (VHs) were analysed using logistic regression adjusted for confounding factors. MAIN OUTCOME MEASURES: Postoperative infections. RESULTS: Cefuroxime had a risk-reductive effect for total infections (adjusted odds ratio, OR, 0.29; 95% confidence interval, 95% CI, 0.22-0.39), but the independent effect of metronidazole and the interaction effect of cefuroxime and metronidazole were nonsignificant. In subgroup analyses of AHs, LHs, and VHs involving those receiving the two main antibiotics only, the effect of cefuroxime alone nonsignificantly differed from that of cefuroxime and metronidazole in combination for all types of infection. The absence of cefuroxime, assessed by comparing metronidazole alone with cefuroxime and metronidazole in combination, led to an increased risk for total infections in AHs (adjusted OR 3.63; 95% CI 1.99-6.65), in LHs (OR 3.53; 95% CI 1.74-7.18), and in VHs (OR 4.05; 95% CI 2.30-7.13), and also increased risks for febrile events in all categories (AHs, OR 2.86; 95% CI 1.09-7.46; LHs, OR 13.19; 95% CI 3.66-47.49; VHs, OR 12.74; 95% CI 3.01-53.95), wound infections in AHs (OR 6.88; 95% CI 1.09-7.49), and pelvic infections in VHs (OR 4.26; 95% CI 1.76-10.31). CONCLUSIONS: In this study, cefuroxime appeared to be effective in prophylaxis against infections. Metronidazole appeared to be ineffective, with no additional risk-reductive effect when combined with cefuroxime.

Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors.

Grb2-associated binder 2 (Gab2) serves as a critical amplifier in the signaling network of Bcr-Abl, the driver of chronic myeloid leukemia (CML). Despite the success of tyrosine kinase inhibitors (TKIs) in CML treatment, TKI resistance, caused by mutations in Bcr-Abl or aberrant activity of its network partners, remains a clinical problem. Using inducible expression and knockdown systems, we analyzed the role of Gab2 in Bcr-Abl signaling in human CML cells, especially with respect to TKI sensitivity. We show for the first time that Gab2 signaling protects CML cells from various Bcr-Abl inhibitors (imatinib, nilotinib, dasatinib and GNF-2), whereas Gab2 knockdown or haploinsufficiency leads to increased TKI sensitivity. We dissected the underlying molecular mechanism using various Gab2 mutants and kinase inhibitors and identified the Shp2/Ras/ERK and the PI3K/AKT/mTOR axes as the two critical signaling pathways. Gab2-mediated TKI resistance was associated with persistent phosphorylation of Gab2 Y452, a PI3K recruitment site, and consistent with this finding, the protective effect of Gab2 was completely abolished by the combination of dasatinib with the dual PI3K/mTOR inhibitor NVP-BEZ235. The identification of Gab2 as a novel modulator of TKI sensitivity in CML suggests that Gab2 could be exploited as a biomarker and therapeutic target in TKI-resistant disease.

Functional characterization of cancer-associated Gab1 mutations.

Grb2-associated binder 1 (Gab1) is a docking protein that transduces signals from a variety of tyrosine kinases, including Met and the epidermal growth factor receptor (EGFR). Although the related protein Gab2 is strongly implicated in human cancer, a role for Gab1 has been less clear. However, a screen for gene mutations in breast cancer identified two somatic mutations in Gab1, Y83C and T387N. In this paper we describe the functional characterization of these Gab1 mutants. MCF-10A immortalized mammary epithelial cells overexpressing Gab1 Y83C and T387N exhibited a more elongated, fibroblastic phenotype compared with wild-type Gab1 controls. Expression of Gab1 or the mutants promoted epidermal growth factor (EGF)-independent proliferation in monolayer culture to a similar degree. However, in Matrigel culture, both mutants enhanced the formation of acini exhibiting an aberrant, branched morphology. In addition, expression of the mutants modestly increased Erk activation. The two mutants also enhanced branching morphogenesis in a different mammary epithelial cell line, HC11. To gain further insights into the mechanism of action of these mutations, we mapped Gab1 phosphorylation sites by mass spectrometry. This detected phosphorylation of T387 but ;not Y83. Cellular stimulation with EGF or hepatocyte growth factor (HGF) led to a transient, or sustained, induction of T387 phosphorylation, respectively. As T387 corresponds in position to Gab2 T391, which suppresses Gab2 signaling in a phosphorylation-dependent manner, these data support a model in which the T387N mutation abrogates negative-feedback regulation of Gab1. Interrogation of publically-available databases revealed additional cancer-associated mutations at, or in close proximity to, identified serine/threonine phosphorylation sites in other docking proteins. These data indicate that aberrant Gab1 signaling can directly contribute to breast cancer progression, and that negative feedback sites in docking proteins can be targeted by oncogenic mutations.

Pharmaceutical thrombosis prophylaxis, bleeding complications and thromboembolism in a national cohort of hysterectomy for benign disease.

BACKGROUND: Pharmaceutical thrombosis prophylaxis (PTP) with low-molecular-weight heparin (LMWH) is highly effective in preventing venous thromboembolic events (VTEs) and fatal pulmonary embolism. Important risk factors for VTEs are surgery and immobilization, along with malignancy. Many studies involving gynaecological malignancies show no increased risk for bleeding complications with PTP. Little is known about the PTP-associated risk for bleeding complications with hysterectomy for benign disease, or about current VTE incidence in the less-invasive hysterectomy methods. METHODS: Our observational prospective national 1-year cohort from 1 January to 31 December 2006 in 53 hospitals represented 79.4% (5297 of 6645) of hysterectomies performed for benign cause in Finland in 2006. We evaluated PTP use and VTE incidence. Operative and post-operative bleeding complications were analysed with logistic regression adjusted for confounders: age, BMI, experience of the gynaecological surgeon, hospital type, indication for hysterectomy, uterine weight, operative haemorrhage, concomitant surgery, adhesiolysis and antibiotic prophylaxis. RESULTS: Hysterectomies were performed by three main approaches: 2345 vaginal hysterectomies (VHs, 44%), of which 1433 were for uterine prolapse and 912 for other indications, 1679 laparoscopic hysterectomies (LHs, 32%) and 1255 abdominal hysterectomies (AHs, 24%). PTP was given to 64.8% of patients (3420 of 5279) and was identified as LMWH in 3313 patients (97%); 107 left unidentified. By type of hysterectomy, PTP was given in VH for uterine prolapse to 73.2% of patients, VH for other indication to 51.6%, in LH to 59.4% and in AH to 71.9%. For all hysterectomies analysed together, PTP doubled the odds for post-operative haemorrhage or haematoma. By type of hysterectomy, PTP associated with post-operative haemorrhage or haematoma in VH for prolapse [2.7% of PTP given, versus 0.8% of no PTP; odds ratio (OR): 4.82, 95% confidence interval (CI): 1.38-16.83]; and in AH (3.1% versus 1.4%; OR: 2.87, 95% CI: 1.03-7.98), and in AH also with post-operative transfusion (3.1% versus 1.4%; OR: 3.34, 95% CI: 1.41-7.88). For LH and VH for indications other than prolapse, the effect of PTP on post-operative haemorrhage was non-significant. For VH, the risk for post-operative haemorrhage fell with age. Operative mean haemorrhage with all hysterectomy types, and operative bleeding complications in AH and VH also fell with age. Obesity increased haemorrhage and operative bleeding complications for LH and VH, whereas post-operative bleeding complications were less for the obese in AH. VTEs were 6 of 5279 (0.1%): two PEs each occurred after AH and VH, and two deep venous thromboses after LH. CONCLUSIONS: With a relatively wide PTP coverage (64.8%), VTEs were rare (0.1%). All affected had received PTP. Analysis of efficacy, meaning interpretation of how many VTEs or deaths were prevented, cannot be done from our observational study but related to safety in hysterectomy for benign disease, PTP associated with post-operative bleeding complications with AH and with VH for prolapse. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov protocol (NCT00744172).

The RhoGAP protein Deleted in Liver Cancer 3 (DLC3) is essential for adherens junctions integrity.

Epithelial cell-cell contacts are mediated by E-cadherin interactions, which are regulated by the balanced local activity of Rho GTPases. Despite the known function of Rho at adherens junctions (AJs), little is known about the spatial control of Rho activity at these sites. Here we provide evidence that in breast epithelial cells the Deleted in Liver Cancer 3 (DLC3) protein localizes to AJs and is essential for E-cadherin function. DLC3 is a still poorly characterized RhoA-specific GTPase-activating protein that is frequently downregulated in various types of cancer. We demonstrate that DLC3 depletion leads to mislocalization of E-cadherin and catenins, which was associated with impaired cell aggregation and increased migration. This is explained by aberrant local Rho signaling because ROCK inhibition restored cell-cell contacts in DLC3 knockdown cells. We thus identify DLC3 as a novel negative regulator of junctional Rho and propose that DLC3 loss contributes to carcinogenesis by compromising epithelial integrity.

Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis.

The Gab2 docking protein is a target of several oncogenic protein tyrosine kinases and potentiates activation of the Ras/extracellular signal regulated kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways. Since Gab2 is phosphorylated by c-Src, and both proteins are overexpressed in breast cancers, we have determined the biological consequences of their co-expression in the immortalized human mammary epithelial cell line MCF-10A. While overexpression of c-Src did not affect acinar morphogenesis or growth factor dependence in three-dimensional culture, c-Src co-operated with Gab2 to promote epidermal growth factor (EGF)-independent acinar growth. In contrast, expression of v-Src or the activated mutant c-SrcY527F led to a spectrum of aberrant phenotypes ranging from spheroids with incomplete luminal clearance to highly disrupted, dispersed structures. Gab2 co-expression shifted the phenotypic distribution towards the dispersed phenotype, an effect not observed with a Gab2 mutant unable to bind the p85 subunit of PI3-kinase (Gab2Deltap85). In v-Src-expressing cells, Gab2, but not Gab2Deltap85, significantly decreased E-cadherin adhesive strength without altering its surface expression. Gab2 associated with E-cadherin in the presence and absence of v-Src, indicating that the ability of Gab2 to weaken the strength of cell-cell contacts may reflect enhanced activation of PI3-kinase at adherens junctions. Gab2 also increased migration and invasion of these cells in transwell assays, but these effects were p85-independent. Overall, these findings demonstrate a novel mechanism whereby Gab2 may promote metastatic spread and indicate that Gab2 may play several roles during breast cancer progression.

Functional analysis of the regulatory requirements of B-Raf and the B-Raf(V600E) oncoprotein.

The BRAF(V600E) mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf(wt)), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5'-triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf(V600E), although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf(V600E) was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf(wt) and B-Raf(V600E) and suggests that B-Raf(V600E) cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation.

Prognostic factors of advanced colorectal cancer patients.

The cooperative oncology group for Chemotherapy of Gastrointestinal Tumors (CGT) retrospectively examined 139 patients with metastatic colorectal cancer for prognostic factors. Clinical characteristics, tumor parameters, and blood parameters were investigated for prognostic explanation of survival from the start of chemotherapy for the advanced disease. A combination of a univariate regression and a multivariate step down procedure with Cox's regression model led to the identification of performance status, sex, white blood count and, to a lesser degree, blood sedimentation rate and albumin as important prognostic factors. Based on these variables an individual risk score was calculated for each patient.