A yeast-based assay for protein tyrosine kinase substrate specificity and inhibitor resistance

Phosphorylation of tyrosines by protein kinases is a fundamental mode of signal transduction in all eukaryotic cells, leading to a wide variety of cellular outcomes, including proliferation, differentiation, transcriptional activation, and programmed cell death. Perturbations to tyrosine kinase signaling networks by activation, overexpression, or mutation is the driving factor in many diseases, most notably cancers. The development of tyrosine kinase inhibitors, 37 of which are currently FDA-approved, has led to a revolution in cancer treatment. Imatinib, the first FDA-approved kinase inhibitor, has drastically improved prognosis for patients with Bcr-abl-positive leukemias. Despite this unprecedented success, however, up to one-third of patients lose response to imatinib due to mutations within the tyrosine kinase domain of Bcr-abl. Subsequent generations of Bcr-abl inhibitors, including dasatinib and ponatinib, have been developed to overcome these resistance mutations, but in each case, novel resistance mutations have arisen. We present a high-throughput yeast-based assay for the prediction of dasatinib- and ponatinib-resistant mutations in the ABL1 kinase domain. Our results not only recapitulate all known dasatinib-resistant mutations, but confirm recent patient data emphasizing the importance of compound mutations in ponatinib resistance. Furthermore, with hundreds of kinase inhibitors in development for the treatment of a wide range of diseases, understanding the cellular pathway of each kinase is critically important to the selection of ideal drug targets and avoiding potentially toxic side effects. Discovery of novel tyrosine kinase substrates is hindered by the presence of 90 human tyrosine kinases, which are often active in the same pathways. Phosphoproteomics, chemical genetics, and in vitro assays have been used to great success, yet only 30% of phosphorylated tyrosines in the human proteome have been assigned to a specific kinase. Recent advances in predicting tyrosine kinase substrates have been made by combining large data sets on kinase domain specificity, cellular localization, and protein-protein interactions in probabilistic algorithms. However, the high-quality data sets required for accurate predictions are often lacking. In chapter 2, we present a high-throughput yeastbased assay for screening millions of putative kinase substrates, which we then use to build a probabilistic model to accurately predict the in vitro phosphorylation of candidate substratesBiochemistr

Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy (vol 47, pg 73, 2015)

"Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy" published in Nature Genetics (2015), volume 47, pp.73-77

Subsequent Surgery After Revision Anterior Cruciate Ligament Reconstruction: Rates and Risk Factors From a Multicenter Cohort

BACKGROUND: While revision anterior cruciate ligament reconstruction (ACLR) can be performed to restore knee stability and improve patient activity levels, outcomes after this surgery are reported to be inferior to those after primary ACLR. Further reoperations after revision ACLR can have an even more profound effect on patient satisfaction and outcomes. However, there is a current lack of information regarding the rate and risk factors for subsequent surgery after revision ACLR. PURPOSE: To report the rate of reoperations, procedures performed, and risk factors for a reoperation 2 years after revision ACLR. STUDY DESIGN: Case-control study; Level of evidence, 3. METHODS: A total of 1205 patients who underwent revision ACLR were enrolled in the Multicenter ACL Revision Study (MARS) between 2006 and 2011, composing the prospective cohort. Two-year questionnaire follow-up was obtained for 989 patients (82%), while telephone follow-up was obtained for 1112 patients (92%). If a patient reported having undergone subsequent surgery, operative reports detailing the subsequent procedure(s) were obtained and categorized. Multivariate regression analysis was performed to determine independent risk factors for a reoperation. RESULTS: Of the 1112 patients included in the analysis, 122 patients (11%) underwent a total of 172 subsequent procedures on the ipsilateral knee at 2-year follow-up. Of the reoperations, 27% were meniscal procedures (69% meniscectomy, 26% repair), 19% were subsequent revision ACLR, 17% were cartilage procedures (61% chondroplasty, 17% microfracture, 13% mosaicplasty), 11% were hardware removal, and 9% were procedures for arthrofibrosis. Multivariate analysis revealed that patients aged <20 years had twice the odds of patients aged 20 to 29 years to undergo a reoperation. The use of an allograft at the time of revision ACLR (odds ratio [OR], 1.79; P = .007) was a significant predictor for reoperations at 2 years, while staged revision (bone grafting of tunnels before revision ACLR) (OR, 1.93; P = .052) did not reach significance. Patients with grade 4 cartilage damage seen during revision ACLR were 78% less likely to undergo subsequent operations within 2 years. Sex, body mass index, smoking history, Marx activity score, technique for femoral tunnel placement, and meniscal tearing or meniscal treatment at the time of revision ACLR showed no significant effect on the reoperation rate. CONCLUSION: There was a significant reoperation rate after revision ACLR at 2 years (11%), with meniscal procedures most commonly involved. Independent risk factors for subsequent surgery on the ipsilateral knee included age <20 years and the use of allograft tissue at the time of revision ACLR

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Development and Validation of a Symptom-Based Activity Index for Adults With Eosinophilic Esophagitis

Standardized instruments are needed to assess the activity of eosinophilic esophagitis (EoE), to provide endpoints for clinical trials and observational studies. We aimed to develop and validate a patient-reported outcome (PRO) instrument and score, based on items that could account for variations in patients’ assessments of disease severity. We also evaluated relationships between patients’ assessment of disease severity and EoE-associated endoscopic, histologic, and laboratory findings

Distributed PCA and network anomaly detection

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In-network PCA and anomaly detection

Copyright © 2007, by the author(s)

Minos Garofalakis

We consider the problem of network anomaly detection in large distributed systems. In this setting, Principal Component Analysis (PCA) has been proposed as a method for discovering anomalies by continuously tracking the projection of the data onto a residual subspace. While successful empirically in small networks, this approach has serious scalability limitations. To overcome these limitations, we develop a PCA-based anomaly detector in which adaptive local data filters send to a coordinator just enough data to enable accurate global detection. Our method is based on a stochastic matrix perturbation analysis that characterizes the tradeoff between the accuracy of anomaly detection and the amount of data communicated over the network.