"Virtual patch clamp analysis" for predicting the functional significance of pathogenic variants in sodium channels.

OBJECTIVE: Opening of voltage-gated sodium channels is crucial for neuronal depolarization. Proper channel opening and influx of Na+ through the ion pore, is dependent upon binding of Na+ ion to a specific amino-acid motif (DEKA) within the pore. In this study we used molecular dynamic simulations, an advanced bioinformatic tool, to research the dysfunction caused by pathogenic variants in SCN1a, SCN2a and SCN8a genes. METHOD: Molecular dynamic simulations were performed in six patients: three patients with Dravet syndrome (p.Gly177Ala,p.Ser259Arg and p.Met1267Ile, SCN1a), two patients with early onset drug resistant epilepsy(p.Ala263Val, SCN2a and p.Ile251Arg, SCN8a), and a patient with autism (p.Thr155Ala, SCN2a). After predicting the 3D-structure of mutated proteins by homology modeling, time dependent molecular dynamic simulations were performed, using the Schrödinger algorithm. The opening of the sodium channel, including the detachment of the sodium ion to the DEKA motif and pore diameter were assessed. Results were compared to the existent patch clamp analysis in four patients, and consistency with clinical phenotype was noted. RESULTS: The Na+ ion remained attached to DEKA filter longer when compared to wild type in the p.Gly177Ala, p.Ser259Arg,SCN1a, and p.Thr155Ala, SCN2a variants, consistent with loss-of-function. In contrast, it detached quicker from DEKA than wild type in the p.Ala263Val,SCN2a variant, consistent with gain-of-function. In the p.Met1267Ile,SCN1a variant, detachment from DEKA was quicker, but pore diameter decreased, suggesting partial loss-of-function. In the p.Leu251Arg,SCN8a variant, the pore remained opened longer when compared to wild type, consistent with a gain-of-function. The molecular dynamic simulation results were consistent with the existing patch-clamp analysis studies, as well as the clinical phenotype. SIGNIFICANCE: Molecular dynamic simulation can be useful in predicting pathogenicity of variants and the disease phenotype, and selecting targeted treatment based on channel dysfunction. Further development of these bioinformatic tools may lead to "virtual patch-clamp analysis".

Deciphering radiological stable disease to immune checkpoint inhibitors.

BACKGROUND: 'Stable disease (SD)' as per RECIST is a common but ambiguous outcome in patients receiving immune checkpoint inhibitors (ICIs). This study aimed to characterize SD and identify the subset of patients with SD who are benefiting from treatment. Understanding SD would facilitate drug development and improve precision in correlative research. PATIENTS AND METHODS: A systematic review was carried out to characterize SD in ICI trials. SD and objective response were compared to proliferation index using The Cancer Genome Atlas gene expression data. To identify a subgroup of SD with outcomes mirroring responders, we examined a discovery cohort of non-small-cell lung cancer (NSCLC). Serial cutpoints of two variables, % best overall response and progression-free survival (PFS), were tested to define a subgroup of patients with SD with similar survival as responders. Results were then tested in external validation cohorts. RESULTS: Among trials of ICIs (59 studies, 14 280 patients), SD ranged from 16% to 42% in different tumor types and was associated with disease-specific proliferation index (ρ = -0.75, P = 0.03), a proxy of tumor kinetics, rather than relative response to ICIs. In a discovery cohort of NSCLC [1220 patients, 313 (26%) with SD to ICIs], PFS ranged widely in SD (0.2-49 months, median 4.9 months). The subset with PFS >6 months and no tumor growth mirrored partial response (PR) minor (overall survival hazard ratio 1.0) and was proposed as the definition of SD responder. This definition was confirmed in two validation cohorts from trials of NSCLC treated with durvalumab and found to apply in tumor types treated with immunotherapy in which depth and duration of benefit were correlated. CONCLUSIONS: RECIST-defined SD to immunotherapy is common, heterogeneous, and may largely reflect tumor growth rate rather than ICI response. In patients with NSCLC and SD to ICIs, PFS >6 months and no tumor growth may be considered 'SD responders'. This definition may improve the efficiency of and insight derivable from clinical and translational research.

The associations of CD4 count, CD4/CD8 ratio, and HIV viral load with survival from non-small cell lung cancer in persons living with HIV.

HIV status may influence survival from non-small cell lung cancer (NSCLC). Among NSCLC patients in the Bronx, NY, we assessed (1) associations of CD4 count, CD4/CD8 ratio and HIV viral load (VL) with survival and (2) prognostic factors among persons living with HIV (PLWH). We compared survival from NSCLC diagnosis (2004-2017) between HIV-negative persons (HIV-, n=2,881) and PLWH (n=88) accounting for clinical and sociodemographic factors. HIV-survival was also compared with PLWH, dichotomized by CD4 (<200 vs. ≥200cells/µL), CD4/CD8 (median, <0.43 vs. ≥0.43) and VL (<75 vs. ≥75copies/mL) at NSCLC diagnosis. Among PLWH, we assessed the relationships of CD4, CD4/CD8, and VL with survival, adjusting for age, sex, and cancer stage. PLWH with CD4< 200cells/µL had lower survival than HIV- [hazard ratio, 95% confidence interval [HR(95%CI)]=1.86(0.98-3.55)]. Survival was similar between PLWH with CD4≥ 200cells/µL and HIV- [HR(95%CI) = 0.90(0.61-1.33)]. Results were similar when categorizing PLWH by CD4/CD8 [vs. HIV-: low CD4/CD8: HR(95%CI) = 1.74(1.07-3.89); high CD4/CD8: HR(95%CI) = 0.63(0.37-1.07)] and VL [vs. HIV-: <75copies/mL: HR(95%CI) = 0.74(0.46-1.21), ≥75copies/mL: HR(95%CI) = 1.41(0.88-2.27)]. Among PLWH, CD4< 200cells/µL was associated with worse survival [vs. CD4≥ 200cells/µL: HR(95%CI) = 2.37(1.14-4.92)]. CD4, CD4/CD8, and VL may be prognostic markers for PLWH with NSCLC, suggesting immune status may be important in NSCLC survival among PLWH.

Imaging following thermal ablation of early lung cancers: expected post-treatment findings and tumour recurrence.

Thermal ablation is a minimally invasive technique that is growing in acceptance and popularity in the management of early lung cancers. Although curative resection remains the optimal treatment strategy for stage I pulmonary malignancies, percutaneous ablative treatments may also be considered for selected patients. These techniques can additionally be used in the treatment of oligometastatic disease. Thermal ablation of early lung tumours can be achieved using several different techniques. For example, microwave ablation (MWA) and radiofrequency ablation (RFA) utilise extreme heat, whereas cryoablation uses extremely cold temperatures to cause necrosis and ultimately cell death. Typically, post-ablation imaging studies are performed within the first 1-3 months with subsequent imaging performed at regular intervals to ensure treatment response and to evaluate for signs of recurrent disease. Surveillance imaging is usually undertaken with computed tomography (CT) and integrated positron-emission tomography (PET)/CT. Typical imaging findings are usually seen on CT and PET/CT following thermal ablation of lung tumours, and it is vital that radiologists are familiar with these appearances. In addition, radiologists should be aware of the imaging findings that indicate local recurrence following ablation. The objective of this review is to provide an overview of the expected post-treatment findings on CT and PET/CT following thermal ablation of early primary lung malignancies, as well as describing the imaging appearances of local recurrence.

Investigation of patterns of nodal metastases in BRAF mutant lung cancer.

Axillary lymph nodes (axLN) are a rare site of nodal metastases in patients with lung cancer. BRAF mutated lung cancer is a genetically distinct subtype that occurs in 2-5% of non-small cell lung carcinomas (NSCLC). A recent study identified a highly unusual pattern of metastatic spread to axLN in patients with BRAF mutated colorectal cancer (CRC). The purpose of the study is to assess the incidence of axLN metastases in BRAF mutated NSCLC. Baseline computed tomography (CT) imaging at diagnosis and all follow up CTs of patients with BRAF mutated NSCLC treated at our institution were retrospectively reviewed by two radiologists for evidence of axLN metastases. Positron emission tomography (PET)/CT was reviewed when available. A control group of patients with non-BRAF mutated NSCLC was assessed. Three criteria were used for the diagnosis of a metastatic node; pathologic confirmation, radiologic size greater ≥1.5cm in short axis diameter or fluorodeoxyglucose avidity on PET/CT and radiologic size ≥1.0cm in short axis diameter. Forty-six patients with BRAF mutated NSCLC and CT images on the institutional PACS were identified. 7 (15%) patients with BRAF mutated NSCLC had axLN metastases using the proposed diagnostic criteria. One patient had a pathologic proven axLN metastasis, 3 had axLNs measuring ≥1.5cm in short axis, and 3 had nodes which were FDG avid on PET/CT and measured ≥1.0cm in short axis. By comparison, 1 of 46 (2%) control patients with non-BRAF mutated NSCLC had axLN metastases. Previous series have reported the prevalence of axLN metastases in patients with NSCLC as 0.61-0.75%. We have found a higher incidence of axLN metastases in BRAF mutated NSCLC patients than described in non-BRAF mutated NSCLC patients. Examination of the axilla should be a routine part of physical examination in this genetically distinct subgroup of lung cancer patients.

Comparison of CT volumetric measurement with RECIST response in patients with lung cancer.

PURPOSE: To examine the correlations between uni-dimensional RECIST and volumetric measurements in patients with lung adenocarcinoma and to assess their association with overall survival (OS) and progression-free survival (PFS). MATERIALS AND METHODS: In this study of patients receiving chemotherapy for lung cancer in the setting of a clinical trial, response was prospectively evaluated using RECIST 1.0. Retrospectively, volumetric measurements were recorded and response was assessed by two different volumetric methods at each followup CT scan using a semi-automated segmentation algorithm. We subsequently evaluated the correlation between the uni-dimensional RECIST measurements and the volumetric measurements and performed landmark analyses for OS and PFS at the completion of the first and second follow-ups. Kaplan-Meier curves together with log-rank tests were used to evaluate the association between the different response criteria and patient outcome. RESULTS: Forty-two patients had CT scans at baseline, after the first follow up scan and second followup scan, and then every 8 weeks. The uni-dimensional RECIST measurements and volumetric measurements were strongly correlated, with a Spearman correlation coefficient (ρ) of 0.853 at baseline, ρ=0.861 at the first followup, ρ=0.843 at the 2nd followup, and ρ=0.887 overall between-subject. On first follow-up CT, partial responders and non responders as assessed by an "ellipsoid" volumetric criteria showed a significant difference in OS (p=0.008, 1-year OS of 70% for partial responders and 46% for non responders). There was no difference between the groups when assessed by RECIST criteria on first follow-up CT (p=0.841, 1-year OS rate of 64% for partial responders and 64% for non responders). CONCLUSION: Volumetric response on first follow-up CT may better predict OS than RECIST response. CLINICAL RELEVANCE STATEMENT: Assessment of tumor size and response is of utmost importance in clinical trials. Volumetric measurements may help to better predict OS than uni-dimensional RECIST criteria.

The California Pertussis Epidemic 2010: A Review of 986 Pediatric Case Reports From San Diego County.

BACKGROUND: The California Department of Public Health (CDPH) declared a pertussis epidemic on 23 June 2010. More cases were reported in 2010 (9146) than in any year since 1947. We describe the characteristics of pertussis epidemiology and disease from 986 reported cases in children in San Diego County (population 3.2 million). METHODS: Descriptive statistics were abstracted from CDPH pertussis case report forms that were completed by public health nurses investigating reports of positive laboratory results for pertussis and reports of illnesses compatible with pertussis. RESULTS: Of 1144 reported adult and pediatric cases, 753 (66%) were confirmed and 391 were probable/suspect. Children aged <19 years comprised 86% of all reported cases in San Diego County; of these, 22% were aged 11-18 years, 29% were aged 6-10 years, 27% were aged 1-5 years, and 22% were aged <1 year (with 70% aged <6 months). Case rates were highest in infants aged <6 months (651 per 100 000 population). Of those aged >1 year, the highest attack rates were in preschool children aged 1-5 years (114 per 100 000) and elementary school children aged 6-10 years (141 per 100 000). Of 51 children hospitalized, 82% were aged <6 months; 2 deaths occurred in these young infants. Paroxysmal cough was noted in over 70% of children in all age groups; post-tussive vomiting occurred in 36% (aged 11-18 years) to 57% (aged <6 months) of children. CONCLUSIONS: Pertussis vaccine efficacy may decrease more rapidly than previously believed, facilitating spread of pertussis in elementary school-aged children. The highest case rates and the only mortality occurred in infants aged <6 months.

A defect in the thymidine kinase 2 gene causing isolated mitochondrial myopathy without mtDNA depletion.

Isolated mitochondrial myopathies (IMM) are either due to primary defects in mtDNA, in nuclear genes that control mtDNA abundance and structure such as thymidine kinase 2 (TK2), or due to CoQ deficiency. Defects in the TK2 gene have been found to be associated with mtDNA depletion attributed to a depleted mitochondrial dNTP pool in non-dividing cells. We report an unusual case of IMM, homozygous for the H90N mutation in the TK2 gene but unlike other cases with the same mutation, does not demonstrate mtDNA depletion. The patient's clinical course is relatively mild and a muscle biopsy showed ragged red muscle fibers with a mild decrease in complexes I and an increase in complexes IV and II activities. This report extends the phenotypic expression of TK2 defects and suggests that all patients who present with an IMM even with normal quantities of mtDNA should be screened for TK2 mutations.

Stilbazulenyl nitrone decreases oxidative stress and reduces lung injury after hemorrhagic shock/resuscitation and LPS.

Multiorgan failure is a major cause of late morbidity and mortality after trauma. Reactive oxygen species generated during shock/resuscitation contribute to tissue injury by priming the immune system for an exaggerated response to subsequent inflammatory stimuli such as LPS. Stilbazulenyl nitrone (STAZN) is a novel second-generation azulenyl nitrone that has been shown to have potent antioxidant properties in a rat model of brain ischemia. We hypothesized that STAZN may confer protection against lung injury after shock/resuscitation and LPS by reducing oxidative stress and lowering the production of NF-kappaB-dependent pro-inflammatory cytokines. Sprague-Dawley rats were submitted to a two-hit model of lung injury involving hemorrhagic shock/resuscitation and subsequent intratracheal LPS injection, with and without intraperitoneal injections of STAZN. STAZN reduced overall lung injury in response to LPS alone and also after shock/resuscitation plus LPS. STAZN also reduced plasma levels of 8-isoprostane, a proxy measure of oxidative stress, indicating its antioxidant activity in vivo. The effect of STAZN was, at least in part, related to its effect on nuclear translocation of NF-kappaB and generation of the pro-inflammatory cytokine TNF-alpha. Azulenyl nitrones such as STAZN represent a promising novel class of antioxidants for treating organ injury.

The ALIAS (ALbumin In Acute Stroke) Phase III randomized multicentre clinical trial: design and progress report.

High-dose human ALB (albumin) therapy is highly neuroprotective in animal models of ischaemic stroke. A recently completed 82-subject pilot-phase dose-escalation trial has shown that ALB is safe, with strong preliminary suggestions of possible efficacy. We are now proceeding to a large randomized, double-blinded, placebo-controlled multicentre trial funded by the NIH (National Institutes of Health), the ALIAS (Albumin In Acute Stroke) Phase III Trial, which is designed to ascertain definitively whether high-dose ALB therapy confers neuroprotection in subjects with acute ischaemic stroke treated within 5 h of stroke onset. The primary efficacy outcome measure is a favourable outcome, defined as an NIHSS (NIH Stroke Scale) score of 0-1 or a modified Rankin Scale score of 0-1 at 3 months post-randomization. Separate randomization (1:1) to ALB or placebo therapy will be carried out in two cohorts of 900 subjects each, one that receives standard-of-care thrombolytic therapy and the other that does not. Approx. 60 North American clinical sites will participate. Subject enrollment is expected to commence in July 2006.

Measuring tumor response and shape change on CT: esophageal cancer as a paradigm.

BACKGROUND: Accurate response assessment is essential for evaluating new cancer treatments. We evaluated the impact of Response Evaluation Criteria in Solid Tumors (RECIST), World Health Organization (WHO) criteria and tumor shape on response assessment in patients with metastatic esophageal cancer. PATIENTS AND METHODS: In 19 patients with metastatic esophageal cancer in a phase II trial of bryostatin-1 and paclitaxel, response was retrospectively assessed for 89 lesions with RECIST and WHO criteria on baseline and serial follow-up CT scans. The eccentricity factor (EF) was introduced for measuring the degree to which tumor shape diverges from a perfect sphere [EF = radical1-(LPD/MD)(2), where LPD is the largest perpendicular diameter and MD is the maximal diameter]. RESULTS: The disagreement rate in best overall response categorization between RECIST (unidimensional) and WHO (bidimensional) criteria was 26.3%. Change in eccentricity was significantly greater (P < 0.01) for patients with disagreement (mean 0.31, range 0-0.91). When the short axis was used for unidimensional lymph node measurement, disagreement between WHO and RECIST lessened. CONCLUSIONS: Response assessment by WHO and RECIST differs substantially. Greater change in eccentricity is associated with greater discordance between WHO and RECIST. The discordance between WHO and RECIST may impact on how effective a therapy is judged to be.

Antitumor effect of E1A in ovarian cancer by cytoplasmic sequestration of activated ERK by PEA15.

The adenovirus type 5 gene E1A is known to suppress tumorigenicity by transcriptionally downregulating HER-2/neu (HER2) or by inducing apoptosis. We show here that E1A also suppressed the tumorigenicity of the low-HER2-expressing ovarian cancer cell line OVCAR-3 by decreasing cell proliferation. We further found that the mechanism responsible for this reduced proliferation is the presence of PEA15 (phosphoprotein enriched in astrocytes), which is upregulated by E1A in ovarian cancer; PEA15 promotes translocation of ERK from the nucleus to the cytoplasm, leading to inhibition of ERK-dependent transcription and proliferation. Indeed, siRNA-mediated knockdown of PEA15 expression in OVCAR-3 stable E1A transfectants resulted in a nuclear accumulation of the active form of ERK, followed by an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. Finally, PEA15 by itself suppressed colony formation in breast and ovarian cancer cell lines, in which E1A is known to have antitumor activity. We conclude that part of the antitumor effect of E1A in ovarian cancer results from cytoplasmic sequestration of the activated form of ERK by PEA15.

Integrin activation by talin.

The development and integrity of the cardiovascular system depends on integrins, a family of adhesion receptors, vitally important for homeostasis of animal species from fruit fly to man. Integrins are critical players in cell migration, cell adhesion, cell cycle progression, differentiation, and apoptosis. Consequently, integrins have a major impact on the patterning and functions of the blood and cardiovascular system. Integrins undergo conformational changes, which alter their affinity for ligands through a process operationally defined as integrin activation. Integrin activation is important for platelet aggregation, leukocyte extravasation, and cell adhesion and migration, thus influencing such processes as hemostasis, inflammation and angiogenesis. Recently, a series of studies have begun to define the mechanism of integrin activation by demonstrating that binding of a cytoskeletal protein, talin, to integrin beta subunit cytoplasmic tail is a last common step in integrin activation. These findings indicate that talin is likely to be at the center of converging signaling pathways regulating integrin activation.

Phase II trial of branched peginterferon-alpha 2a (40 kDa) for patients with advanced renal cell carcinoma.

BACKGROUND: Peginterferon-alpha 2a (40 kDa), PEGASYS(TM) (PEG-IFN), is a modified form of recombinant human interferon (IFN)-alpha 2a with sustained absorption and prolonged half-life after subcutaneous administration. A phase II trial was conducted in previously untreated patients with advanced renal cell carcinoma (RCC) to assess efficacy, toxicity and pharmacokinetic profile. PATIENTS AND METHODS: Forty previously untreated patients with advanced RCC were enrolled on this multicenter trial. The median age was 60 years and 63% had prior nephrectomy. PEG-IFN was administered at a dose of 450 micro g/week on a weekly basis by subcutaneous injection. Serial venous blood samples were drawn to assess concentrations of PEG-IFN. RESULTS: Five (13%) patients achieved a major response (four partial and one complete). The median time to progression was 3.8 months, and 63% of patients were alive at 1 year. The toxicity profile was mostly mild to moderate in intensity. Toxicity higher than grade 2 included neutropenia (six patients), fatigue/asthenia (four patients), nausea/vomiting (three patients) and elevated hepatic transaminase concentrations (four patients). Serum drug levels were studied in all patients; mean C(max) at week 1 was 19 ng/ml, and levels were sustained at close to peak over 1 week. With chronic dosing, drug concentration was increased 3-fold, and steady state was achieved in 5-9 weeks. CONCLUSIONS: The sustained maintenance of serum levels of PEG-IFN allows once-weekly dosing. The efficacy and tolerability profile was qualitatively similar to standard IFN-alpha, and adverse events were mostly mild to moderate in nature.

Integrin activation takes shape.

Integrins are cell surface adhesion receptors that are essential for the development and function of multicellular animals. Here we summarize recent findings on the regulation of integrin affinity for ligand (activation), one mechanism by which cells modulate integrin function. The focus is on the structural basis of integrin activation, the role of the cytoplasmic domain in integrin affinity regulation, and potential mechanisms by which activation signals are propagated from integrin cytoplasmic domains to the extracellular ligand-binding domain.

Ischemic preconditioning preserves mitochondrial function after global cerebral ischemia in rat hippocampus.

Ischemic tolerance in brain develops when sublethal ischemic insults occur before "lethal" cerebral ischemia. Two windows for the induction of tolerance by ischemic preconditioning (IPC) have been proposed: one that occurs within 1 hour after IPC, and another that occurs 1 or 2 days after IPC. The authors tested the hypotheses that IPC would reduce or prevent ischemia-induced mitochondrial dysfunction. IPC and ischemia were produced by bilateral carotid occlusions and systemic hypotension (50 mm Hg) for 2 and 10 minutes, respectively. Nonsynaptosomal mitochondria were harvested 24 hours after the 10-minute "test" ischemic insult. No significant changes were observed in the oxygen consumption rates and activities for hippocampal mitochondrial complexes I to IV between the IPC and sham groups. Twenty-four hours of reperfusion after 10 minutes of global ischemia (without IPC) promoted significant decreases in the oxygen consumption rates in presence of substrates for complexes I and II compared with the IPC and sham groups. These data suggest that IPC protects the integrity of mitochondrial oxidative phosphorylation after cerebral ischemia.

Talampanel, a novel noncompetitive AMPA antagonist, is neuroprotective after traumatic brain injury in rats.

Talampanel [(R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo[4,5-h][2,3] benzodiazepine] is an orally active noncompetitive antagonist of the AMPA subtype of glutamate excitatory amino acid receptors. The purpose of this study was to determine whether treatment with talampanel would protect in a rat model of traumatic brain injury (TBI). Twenty-four hours prior to TBI, a fluid-percussion interface was positioned parasagittally over the right cerebral cortex. On the following day, fasted rats were anesthetized with 3% halothane, 70% nitrous oxide, and a balance of oxygen; mechanically ventilated and physiologically regulated; and subjected to right parieto-occipital parasagittal fluid-percussion injury (1.5-2.0 atm). The agent (talampanel, bolus infusion of 4 mg/kg followed by infusion of 4 mg/kg/h over 72 h) or vehicle was administered i.v. starting at either 30 min or 3 h after trauma. Seven days after TBI, brains were perfusion-fixed, coronal sections at various levels were digitized, and contusion areas were measured. Treatment with talampanel, when instituted 30 min after trauma, significantly reduced total contusion area compared to vehicle-treated rats (0.54 +/- 0.25 vs. 1.79 +/- 0.42 mm2, respectively). When talampanel treatment was begun at 3 h, the neuroprotective effect of the drug was lost. In addition, treatment with talampanel starting at 30 min significantly attenuated neuronal damage in all three subsectors of the hippocampal CA1 sector compared to vehicle-treated rats (normal-neuron counts, right (ipsilateral) medial CA1: 80.3 +/- 2.0 [talampanel] vs. 66.3 +/- 2.1 [vehicle] (mean +/- SEM); middle CA1: 71.5 +/- 2.0 vs. 60.3 +/- 2.2; lateral CA1: 74.5 +/- 3.0 vs. 63.0 +/- 3.2, respectively). By contrast, when talampanel treatment was begun at 3 h, normal pyramidal-neuron counts were almost identical in both groups. Our findings document that talampanel therapy instituted 30 min after trauma significantly reduces histological damage.

PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains.

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.

Computerized weight loss intervention optimizes staff time: the clinical and cost results of a controlled clinical trial conducted in a managed care setting.

OBJECTIVE: To evaluate the costs and effects of incremental components of a weight-loss program. DESIGN: A 3-arm, 12-month randomized controlled clinical trial to evaluate 3 incremental levels of intervention intensity. SUBJECTS/SETTING: The study included 588 individuals (BMI > 25 kg/m2) in a freestanding health maintenance organizalion and achieved an 81% completion rate. INTERVENTION: Using a cognitive behavioral approach for tailoring lifestyle modification goals, the incremental levels of intervention included a) a workbook alone, b) the addition of computerized tailoring using onsite computer kiosks with touch screen monitors, and c) the addition of both computers and staff consultation. MAIN OUTCOME MEASURES: Endpoints included weight parameters, lipid profile, plasma glucose, blood pressure, intervention costs, dietary intake, and physical activity. STATISTICAL ANALYSIS PERFORMED: Study endpoints were analyzed using analysis of variance for normally distributed variables and analysis of covariance to control for any baseline differences. Regression and correlation analysis assessed the relationship between weight loss and other variables. RESULTS: For the increasing levels of intervention intensity, the mean 12-month weight losses were 2.2, 4.7, and 7.4 pounds, with the respective cost per participant being $12.33, $41.99, and $133.74. The decreases in mean BMIs for these respective intervelation levels were 0.4, 0.9 and 1.2. All groups reported a decrease in energy and fat intake and an increase in blocks walked (P<.01). Intervention variables that correlated with weight loss included more computer log-ons, achieving computer-selected goals, more self-monitoring, increased walking, and decreased energy and fat intake, as well as higher attendance in staff consultation group sessions for that treatment condition. Weight loss correlated with decreases in fasting glucose and blood pressure. APPLICATIONS/CONCLUSIONS: In a weight-loss program, computers can facilitate selecting behavioral change goals. More frequent usage resulted in greater weight loss. Staff counseling to augment the computer intervention achieved the most weight loss.