Engineering ePTEN,an enhanced PTEN with increased tumor suppressor activities

The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a key regulator of cell proliferation, survival, and migration and the enzyme that dephosphorylates it, phosphatase and tensin homolog (PTEN), is an important tumor suppressor. As excess PIP3 signaling is a hallmark of many cancers, its suppression through activation of PTEN is a potential cancer intervention. Using a heterologous expression system in which human PTEN-GFP is expressed in Dictyostelium cells, we identified mutations in the membrane-binding regulatory interface that increase the recruitment of PTEN to the plasma membrane due to enhanced association with PI(4,5)P2. We engineered these into an enhanced PTEN (ePTEN) with approximately eightfold increased ability to suppress PIP3 signaling. Upon expression in human cells, ePTEN decreases PIP3 levels in the plasma membrane; phosphorylation of AKT, a major downstream event in PIP3 signaling; and cell proliferation and migration. Thus, the activation of PTEN can readjust PIP3 signaling and may serve as a feasible target for anticancer therapies.Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a potent second messenger that drives many biological processes, such as cell growth, survival, and migration (1, 2). In many cancers, PIP3 levels are elevated due to mutations that either elevate the activity of phosphoinositide 3-kinases (PI3Ks) or decrease that of tumor suppressor phosphatase and tensin homolog (PTEN) (3–5). Although inhibition of PI3Ks has been extensively tried as a cancer drug target, activation of PTEN has been rarely studied (6). As PTEN is mainly located in the cytosol and its PIP3 phosphatase activity is suppressed at this location (7, 8), recruiting more PTEN to the plasma membrane and thereby stimulating its lipid phosphatase activity would seem to be an effective method to repress abnormal PIP3 levels in cancer cells.PTEN comprises an N-terminal “PIP2-binding” motif, globular catalytic and C2 domains, and a C-terminal tail (8–10). Positively charged residues in the PIP2-binding and C2 domains have been proposed to recruit PTEN to the plasma membrane through associations with negatively charged head groups of membrane lipids (11–13). The C-terminal tail is thought to fold back and bind to the membrane-binding regions, maintaining the majority of PTEN in the cytoplasm (11, 14, 15). This intramolecular inhibition is controlled by phosphorylation of four serine/threonine residues in the tail domain. A PTEN mutant that carries an alanine substitution in the phosphorylation sites of the C-terminal tail (termed PTEN_A4) increases the membrane association of PTEN. However, most PTEN_A4 is still present in the cytoplasm, suggesting that the A4 mutations may not completely liberate the membrane-binding sites from inhibition by the tail.To decipher the mechanisms underlying the membrane association of PTEN, we developed a visual screen for the localization of human PTEN expressed in Dictyostelium cells. PTEN is evolutionarily conserved, and human PTEN can functionally replace Dictyostelium PTEN (16–19). Using this heterologous expression system, we identified a membrane-binding regulatory interface in PTEN, consisting of regions of the catalytic domain and the CBR3 and Cα2 loops of the C2 domain (20). In the current study, we introduce multiple mutations in the membrane-binding regulatory interface that completely release the inhibitory effects of the tail, generating a synthetic enzyme, referred to as enhanced PTEN (ePTEN), with greatly increased membrane localization and PIP3 phosphatase activity. Our findings demonstrate that activation of PTEN is a feasible therapeutic strategy for cancers with increased PIP3 signaling.     

Factors affecting long-term survival of patients with bronchogenic carcinoma.

From January 1963 to December 1968, 148 patients underwent thoracotomy for bronchogenic carcinoma. In 123 patients either lobectomy or pneumonectomy was performed (resectability rate of 84 per cent). The over-all operative mortality was 3.4 per cent. Forty of the patients undergoing resection (34 per cent) are alive and free of cancer five years after surgery. Tumor size, nodal involvement, cell type, location, symptoms, and extent of surgery were studied in relation to the long-term results. Five year survival was directly related to the size of the tumor and the extent of nodal involvement. No patients with mediastinal nodal involvement or with lesions larger than 7 cm in diameter were among the long-term survivors.     

Acute graft-versus-host disease after pediatric solid organ transplant

Acute graft-versus-host disease (GVHD) is a rare and life threatening complication after solid organ transplantation. The diagnosis can be made with clinical and laboratory evidence of skin, liver, or intestinal involvement. The role of skin biopsy in confirming acute GVHD is debatable. However, it is proposed that the skin biopsy is a valuable tool in confirming the diagnosis in low prior probability settings. An atypical case of acute GVHD following orthotopic liver and small bowel transplantation in a 2-year-old male is presented. Seven weeks posttransplantation, the patient developed a bullous eruption limited to the buttocks and upper thighs. A skin biopsy was performed which showed interface dermatitis and epidermal necrosis consistent with acute GVHD. Prompt treatment with daclizumab and intravenous corticosteroids was given and the patient survived without evidence of systemic GVHD. This case highlights the importance of skin biopsy in establishing the prompt diagnosis of GVHD in low prior probability settings.     

An arteriolar compliance model of the cerebral blood flow response to neural stimulus

Although functional magnetic resonance imaging (fMRI) is a widely used and powerful tool for studying brain function, the quantitative interpretation of fMRI measurements for basic neuroscience and clinical studies can be complicated by inter-subject and inter-session variability arising from modulation of the baseline vascular state by disease, aging, diet, and pharmacological agents. In particular, recent studies have shown that the temporal dynamics of the cerebral blood flow (CBF) and the blood oxygenation level dependent (BOLD) responses to stimulus are modulated by changes in baseline CBF induced by various vasoactive agents and by decreases in vascular compliance associated with aging. These effects are not readily explained using current models of the CBF and BOLD responses. We present here a second-order nonlinear feedback model of the evoked CBF response in which neural activity modulates the compliance of arteriolar smooth muscle. Within this model framework, the baseline vascular state affects the dynamic response by changing the relative contributions of an active smooth muscle component and a passive connective tissue component to the overall vessel compliance. Baseline dependencies of the BOLD signal are studied by coupling the arteriolar compliance model with a previously described balloon model of the venous compartment. Numerical simulations show that the proposed model describes to first order the observed dependence of CBF and BOLD responses on the baseline vascular state.     

Individualized Dosing of Therapeutic Monoclonal Antibodies—a Changing Treatment Paradigm?

The introduction of monoclonal antibodies (mAbs) to the treatment of inflammatory bowel disease (IBD) was an important medical milestone. MAbs have been demonstrated as safe and efficacious treatments of IBD. However, a large percentage of patients either fail to respond initially or lose response to therapy after a period of treatment. Although there are factors associated with poor treatment outcomes in IBD, one cause for treatment failure may be low mAb exposure. Consequently, gastroenterologists have begun using therapeutic drug monitoring (TDM) to guide dose adjustment. However, while beneficial, TDM does not provide sufficient information to effectively adjust doses. The pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs are complex, with numerous factors impacting on mAb PK and PD. The concept of dashboard-guided dosing based on Bayesian PK models allows physicians to combine TDM with factors influencing mAb PK to individualize therapy more effectively. One issue with TDM has been the slow turnaround of assay results, either necessitating an additional clinic visit for a sample or reacting to TDM results at a subsequent, rather than the current, dose. New point-of-care (POC) assays for mAbs are being developed that would potentially allow physicians to determine drug concentration quickly. However, work remains to understand how to determine what target exposure is needed for an individual patient, and whether the combination of POC assays and dashboards presents a safe approach with substantial outcome benefit over the current standard of care.     

Intradiploic orbital roof meningioma with pneumosinus dilatans in a child: a case report and review of the literature

Intradiploic meningioma of the orbital roof is a type of intraosseous meningioma. It is very rare in children, and only 7 cases have been reported in the pediatric age group. We report a case of a 14-year-old female who presented with progressive exophthalmos, diplopia and pain involving the right eye. Computed tomography (CT) scan and magnetic resonance imaging (MRI) revealed an intradiploic orbital roof tumor with diploe expansion and pneumosinus dilatans. The tumor was completely removed surgically. The histopathological diagnosis was transitional meningioma. The clinical and radiological findings of intadiploic meningioma are discussed with a review of the relevant literature.