Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

Regression-based techniques for statistical decision making in single-case designs

The present study evaluates the performance of four methods for estimating regression coefficients used to make statistical decisions regarding intervention effectiveness in single-case designs. Ordinary least squares estimation is compared to two correction techniques dealing with general trend and one eliminating autocorrelation whenever it is present. Type I error rates and statistical power are studied for experimental conditions defined by the presence or absence of treatment effect (change in level or in slope), general trend, and serial dependence. The results show that empirical Type I error rates do not approximate the nominal ones in presence of autocorrelation or general trend when ordinary and generalized least squares are applied. The techniques controlling trend show lower false alarm rates, but prove to be insufficiently sensitive to existing treatment effects. Consequently, the use of the statistical significance of the regression coefficients for detecting treatment effects is not recommended for short data series.

Correcting surface coil excitation inhomogeneities in single-shot SPEN MRI.

Given their high sensitivity and ability to limit the field of view (FOV), surface coils are often used in magnetic resonance spectroscopy (MRS) and imaging (MRI). A major downside of surface coils is their inherent radiofrequency (RF) B1 heterogeneity across the FOV, decreasing with increasing distance from the coil and giving rise to image distortions due to non-uniform spatial responses. A robust way to compensate for B1 inhomogeneities is to employ adiabatic inversion pulses, yet these are not well adapted to all imaging sequences - including to single-shot approaches like echo planar imaging (EPI). Hybrid spatiotemporal encoding (SPEN) sequences relying on frequency-swept pulses provide another ultrafast MRI alternative, that could help solve this problem thanks to their built-in heterogeneous spatial manipulations. This study explores how this intrinsic SPEN-based spatial discrimination, could be used to compensate for the B1 inhomogeneities inherent to surface coils. Experiments carried out in both phantoms and in vivo rat brains demonstrate that, by suitably modulating the amplitude of a SPEN chirp pulse that progressively excites the spins in a direction normal to the coil, it is possible to compensate for the RF transmit inhomogeneities and thus improve sensitivity and image fidelity.

Elastic properties and secondary structure formation of single-stranded DNA at monovalent and divalent salt conditions

Single-stranded DNA (ssDNA) plays a major role in several biological processes. It is therefore of fundamental interest to understand how the elastic response and the formation of secondary structures are modulated by the interplay between base pairing and electrostatic interactions. Here we measure force-extension curves (FECs) of ssDNA molecules in optical tweezers set up over two orders of magnitude of monovalent and divalent salt conditions, and obtain its elastic parameters by fitting the FECs to semiflexible models of polymers. For both monovalent and divalent salts, we find that the electrostatic contribution to the persistence length is proportional to the Debye screening length, varying as the inverse of the square root of cation concentration. The intrinsic persistence length is equal to 0.7 nm for both types of salts, and the effectivity of divalent cations in screening electrostatic interactions appears to be 100-fold as compared with monovalent salt, in line with what has been recently reported for single-stranded RNA. Finally, we propose an analysis of the FECs using a model that accounts for the effective thickness of the filament at low salt condition and a simple phenomenological description that quantifies the formation of non-specific secondary structure at low forces.

Single centre experience of the application of self navigated 3D whole heart cardiovascular magnetic resonance for the assessment of cardiac anatomy in congenital heart disease.

BACKGROUND: For free-breathing cardiovascular magnetic resonance (CMR), the self-navigation technique recently emerged, which is expected to deliver high-quality data with a high success rate. The purpose of this study was to test the hypothesis that self-navigated 3D-CMR enables the reliable assessment of cardiovascular anatomy in patients with congenital heart disease (CHD) and to define factors that affect image quality. METHODS: CHD patients ≥2 years-old and referred for CMR for initial assessment or for a follow-up study were included to undergo a free-breathing self-navigated 3D CMR at 1.5T. Performance criteria were: correct description of cardiac segmental anatomy, overall image quality, coronary artery visibility, and reproducibility of great vessels diameter measurements. Factors associated with insufficient image quality were identified using multivariate logistic regression. RESULTS: Self-navigated CMR was performed in 105 patients (55% male, 23 ± 12y). Correct segmental description was achieved in 93% and 96% for observer 1 and 2, respectively. Diagnostic quality was obtained in 90% of examinations, and it increased to 94% if contrast-enhanced. Left anterior descending, circumflex, and right coronary arteries were visualized in 93%, 87% and 98%, respectively. Younger age, higher heart rate, lower ejection fraction, and lack of contrast medium were independently associated with reduced image quality. However, a similar rate of diagnostic image quality was obtained in children and adults. CONCLUSION: In patients with CHD, self-navigated free-breathing CMR provides high-resolution 3D visualization of the heart and great vessels with excellent robustness.

CTP in Transient Global Amnesia: A Single-Center Experience of 30 Patients.

BACKGROUND AND PURPOSE: Medial temporal lobe abnormalities on DWI and functional imaging are occasionally observed in patients with transient global amnesia. We used CTP to study these patients during or briefly after resolution of their amnesic syndrome. MATERIALS AND METHODS: From 2002 onward, patients satisfying clinical criteria for transient global amnesia who underwent CTP were included. Patients with additional clinical features suggesting transient ischemic attack or stroke and those with an ischemic lesion on subsequent DWI were excluded. If deemed necessary by the clinician, DWI was performed within 10 days. RESULTS: Thirty patients with transient global amnesia underwent CTP at a median latency of 5.9 hours (interquartile range, 4.3-9.7 hours) after symptom onset. All findings, except for those in 1 patient, were normal, including those in the 14 patients with well-imaged hippocampi. In the patient with abnormal findings, CTP and PWI showed hypoperfusion in both lentiform nuclei extending into the insulae, with normalization on the repeat CTP 6 days later. In 10 patients, DWI was performed at a median latency of 2 days (interquartile range, 0-9 days). Of these, 2 showed punctate hippocampal lesions, often seen in transient global amnesia. In 2 patients excluded because of mildly atypical transient global amnesia and ischemic lesions on subsequent DWI, acute CTP findings were also normal. CONCLUSIONS: Patients with transient global amnesia had normal CTP findings in the acute phase with the exception of 1 patient with transient hypoperfusion in both basal ganglia. If imaging is performed for typical and atypical transient global amnesia, DWI should be the preferred method.

1.84 micron emission of Tm3+ sensitized by Yb3+ ions in monoclinic KGd(WO4)2 single crystals

By exciting at 940 nm, we have characterized the 1.84 m near infrared emission of trivalent thulium ions in Yb3+, Tm3+:KGd WO4 2 single crystals as a function of the dopant concentration and temperature, from 10 K to room temperature. An overall 3H6 Stark splitting of 470 cm−1 for the Tm3+ ions in the Yb3+, Tm3+:KGd WO4 2 was obtained. We also studied the blue emission at 476 nm Tm3+ and the near infrared emissions at 1.48 m Tm3+ and 1 m Yb3+ as a function of the dopant concentration. Experimental decay times of the 1G4, 3H4, and 3F4 Tm3+ and 2F5/2 Yb3+ excited states have been measured as a function of Yb3+ and Tm3+ ion concentrations. For the 3F4 →3H6 transition of Tm3+ ions, we used the reciprocity method to calculate the maximum emission cross section of 3.07 10−20 cm2 at 1.84 m for the polarization parallel to the Nm principal optical direction.

Clinical utility of a molecular test in adjuvant treatment decision making in women with endocrine-sensitive early stage breast cancer: a retrospective, single center one year experience

Background. Molecular tests for breast cancer (BC) risk assessment are reimbursed by health insurances in Switzerland since the beginning of year 2015. The main current role of these tests is to help oncologists to decide about the usefulness of adjuvant chemotherapy in patients with early stage endocrine-sensitive and human epidermal growth factor receptor 2 (HER2)-negative BC. These gene expression signatures aim at predicting the risk of recurrence in this subgroup. One of them (OncotypeDx/OT) also predicts distant metastases rate with or without the addition of cytotoxic chemotherapy to endocrine therapy. The clinical utility of these tests -in addition to existing so-called "clinico-pathological" prognostic and predictive criteria (e.g. stage, grade, biomarkers status)-is still debated. We report a single center one year experience of the use of one molecular test (OT) in clinical decision making. Methods. We extracted from the CHUV Breast Cancer Center data base the total number of BC cases with estrogen-receptor positive (ER+), HER2-negative early breast cancer (node negative (pN0) disease or micrometastases in up to 3 lymph nodes) operated between September 2014 and August 2015. For the cases from this group in which a molecular test had been decided by the tumor board, we collected the clinicopathologic parameters, the initial tumor board decision, and the final adjuvant systemic therapy decision. Results. A molecular test (OT) was done in 12.2% of patients with ER + HER2 negative early BC. The median age was 57.4 years and the median invasive tumor size was 1.7 cm. These patients were classified by ODX testing (Recurrence Score) into low-, intermediate-, and high risk groups, respectively in 27.2%, 63.6% and 9% of cases. Treatment recommendations changed in 18.2%, predominantly from chemotherapyendocrine therapy to endocrine treatment alone. Of 8 patients originally recommended chemotherapy, 25% were recommended endocrine treatment alone after receiving the Recurrence Score result. Conclusions. Though reimbursed by health insurances since January 2015, molecular tests are used moderately in our institution as per the decision of the multidisciplinary tumor board. It's mainly used to obtain a complementary confirmation supporting the decision of no chemotherapy. The OncotypeDx Recurrence Score results were in the intermediate group in 66% of the 9 tested cases but contributed to avoid chemotherapy in 2 patients during the last 12 months.

Single amino acid charge switch defines clinically distinct proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1)-associated inflammatory diseases.

BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.

Outcomes After Liver Resection for Hepatic Alveolar Echinococcosis: A Single-Center Cohort Study.

BACKGROUND: Switzerland is a region in which alveolar echinococcosis (AE) is endemic. Studies evaluating outcomes after liver resection (LR) for AE are scarce. The aim of this study was to assess the short- and long-term outcomes of AE patients after LR in a single tertiary referral center. METHODS: We retrospectively analyzed data pertaining to all patients with liver AE who were treated with LR at our institution between January 1992 and December 2013. Patient demographics, intraoperative data, extent of LR procedures (major vs. minor LR), postoperative outcomes, and negative histological margin (R0) resection rate were recorded in a database. Recurrence rates after LR were analyzed. RESULTS: LR was performed in 59 patients diagnosed with hepatic AE (56 complete surgeries, 3 reduction surgeries). Postoperative morbidity and mortality were observed in 34 % (25 % grade I-II, 9 % grade III-IV) and 2 % of the patients, respectively. R0 (complete) resection rate was 71 % (n = 42), and R1/R2 resection rate was 29 % (n = 17). Extra-hepatic recurrence occurred in 1 case (lung) after R0 resection. In cases of R1/R2 resection, 7 intra-hepatic disease progressions occurred with a median time of 10 months (IQR 6-11 months). Long-term (more than 1 year) benzimidazole treatment stabilized the disease in 64 % (9/14) of patients with R1 status. The overall survival rate was 97 %. CONCLUSIONS: Liver AE can be safely and definitively treated with LR, provided that R0 resection is achieved. In cases of R1 resection, benzimidazole therapy seems to be effective in stabilizing the intra-hepatic disease and preventing extra-hepatic recurrence.

The effects of some variables on CO2 laser-MAG hybrid welding

The CO2-laser-MAG hybrid welding process has been shown to be a productive choice for the welding industry, being used in e.g. the shipbuilding, pipe and beam manufacturing, and automotive industries. It provides an opportunity to increase the productivity of welding of joints containing air gaps compared with autogenous laser beam welding, with associated reductions in distortion and marked increases in welding speeds and penetration in comparison with both arc and autogenous laser welding. The literature study indicated that the phenomena of laser hybrid welding are mostly being studied using bead-on-plate welding or zero air gap configurations. This study shows it very clearly that the CO2 laser-MAG hybrid welding process is completely different, when there is a groove with an air gap. As in case of industrial use it is excepted that welding is performed for non-zero grooves, this study is of great importance for industrial applications. The results of this study indicate that by using a 6 kW CO2 laser-MAG hybrid welding process, the welding speed may also be increased if an air gap is present in the joint. Experimental trials indicated that the welding speed may be increased by 30-82% when compared with bead-on-plate welding, or welding of a joint with no air gap i.e. a joint prepared as optimum for autogenous laser welding. This study demonstrates very clearly, that the separation of the different processes, as well as the relative configurations of the processes (arc leading or trailing) affect welding performance significantly. These matters influence the droplet size and therefore the metal transfer mode, which in turn determined the resulting weld quality and the ability to bridge air gaps. Welding in bead-onplate mode, or of an I butt joint containing no air gap joint is facilitated by using a leading torch. This is due to the preheating effect of the arc, which increases the absorptivity of the work piece to the laser beam, enabling greater penetration and the use of higher welding speeds. With an air gap present, air gap bridging is more effectively achieved by using a trailing torch because of the lower arc power needed, the wider arc, and the movement of droplets predominantly towards the joint edges. The experiments showed, that the mode of metal transfer has a marked effect on gap bridgeability. Transfer of a single droplet per arc pulse may not be desirable if an air gap is present, because most of the droplets are directed towards the middle of the joint where no base material is present. In such cases, undercut is observed. Pulsed globular and rotational metal transfer modes enable molten metal to also be transferred to the joint edges, and are therefore superior metal transfer modes when bridging air gaps. It was also found very obvious, that process separation is an important factor in gap bridgeability. If process separation is too large, the resulting weld often exhibits sagging, or no weld may be formed at all as a result of the reduced interaction between the component processes. In contrast, if the processes are too close to one another, the processing region contains excess molten metal that may create difficulties for the keyhole to remain open. When the distance is optimised - i.e. a separation of 0-4 mm in this study, depending on the welding speed and beam-arc configuration - the processes act together, creating beneficial synergistic effects. The optimum process separation when using a trailing torch was found to be shorter (0-2 mm) than when a leading torch is used (2-4 mm); a result of the facilitation of weld pool motion when the latter configuration is adopted. This study demonstrates, that the MAG process used has a strong effect on the CO2-laser-MAG hybrid welding process. The laser beam welding component is relatively stable and easy to manage, with only two principal processing parameters (power and welding speed) needing to be adjusted. In contrast, the MAG process has a large number of processing parameters to optimise, all of which play an important role in the interaction between the laser beam and the arc. The parameters used for traditional MAG welding are often not optimal in achieving the most appropriate mode of metal transfer, and weld quality in laser hybrid welding, and must be optimised if the full range of benefits provided by hybrid welding are to be realised.

Improving the performance of the single-dish Cherenkov telescope MAGIC through the use of signal timing

The Cherenkov light flashes produced by Extensive Air Showers are very short in time. A high bandwidth and fast digitizing readout, therefore, can minimize the influence of the background from the light of the night sky, and improve the performance in Cherenkov telescopes. The time structure of the Cherenkov image can further be used in single-dish Cherenkov telescopes as an additional parameter to reduce the background from unwanted hadronic showers. A description of an analysis method which makes use of the time information and the subsequent improvement on the performance of the MAGIC telescope (especially after the upgrade with an ultra fast 2 GSamples/s digitization system in February 2007) will be presented. The use of timing information in the analysis of the new MAGIC data reduces the background by a factor two, which in turn results in an enhancement of about a factor 1.4 of the flux sensitivity to point-like sources, as tested on observations of the Crab Nebula.

Future perspectives in melanoma research: meeting report from the "Melanoma Bridge": Napoli, December 3rd-6th 2014.

The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.