Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific ...AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.
Regulation of alternative splicing events is an essential step required for the expression of functional cytoskeleton and sarcomere proteins in cardiomyocytes. About 3% of idiopathic dilated ...cardiomyopathy cases present mutations in the RNA binding protein RBM20, a tissue specific regulator of alternative splicing. Transcripts expressed preferentially in skeletal and cardiac muscle, including TTN, CAMK2D, LDB3, LMO7, PDLIM3, RTN4, and RYR2, are RBM20-dependent splice variants. In the present study, we investigated the RBM20 involvement in post-transcriptional regulation of splicing variants expressed by Formin homology 2 domain containing 3 (FHOD3) gene. FHOD3 is a sarcomeric protein highly expressed in the cardiac tissue and required for the assembly of the contractile apparatus. Recently, FHOD3 mutations have been found associated with heart diseases. We identified novel FHOD3 splicing variants differentially expressed in human tissues and provided evidences that FHOD3 transcripts are specific RBM20 and PTBP1 targets. Furthermore, we demonstrated that the expression of RBM20 and PTBP1 promoted the alternative shift, from inclusion to exclusion, of selected FHOD3 exons. These results indicate that RBM20 and PTBP1 play a role in the actin filament functional organization mediated by FHOD3 isoforms and suggest their possible involvement in heart diseases.
Cervical cancer, which main etiologic factor is Human Papillomavirus (HPV) infection, continues to be a burden for public health systems in developing countries. Our laboratory has been working with ...the hypothesis that signals generated in the tumor microenvironment can modulate local and systemic immune responses. In this context, it would be reasonable to think that tumors create pro-tumoral bias in immune cells, even before they are recruited to the tumor microenvironment. To understand if and how signaling started in the tumor microenvironment can influence cells within the tumor and systemically, we investigated the expression of key proteins in signaling pathways important for cell proliferation, viability, immune responses and tolerance. Besides, we used detection of specific phosphorylated residues, which are indicative of activation for Akt, CREB, p65 NFκB, and STAT3. Our findings included the observation of a significant STAT3 expression increase and p65 NFκB decrease in circulating leukocytes in correlation with lesion grade. In light of those observations, we started investigating the result of the inhibition of STAT3 in a tumor experimental model. STAT3 inhibition impaired tumor growth, increased anti-tumor T cell responses and decreased the accumulation of myeloid cells in the spleen. The concomitant inhibition of NFκB partially reversed these effects. This study indicates that STAT3 and NFκB are involved in immunomodulatory tumor effects and STAT3 inhibition could be considered as therapy for patients with cervical cancer.
•Read disturb measurements on HfO2-based RRAMs were performed.•The influence of the Forming conditions and stress entity on the robustness of the low resistance state was investigated.•Electrodes ...material affects the robustness of the cell.•A qualitative picture of the physical mechanism ruling the resistive state transition was given.
An experiment of read disturb is performed on HfO2 based MIM devices designed for resistive memories (RRAM). The experiment elucidates the role of the forming conditions and the electrode materials on the robustness of the low resistance state against electrical disturb. It is performed in three steps: (1) two sets of samples with different electrodes (TiN and Pt) are formed using voltage pulses with different amplitudes; (2) formed samples are subject to constant voltage stress of different entities; (3) the current flowing through the MIM during stress is monitored and processed.
We propose a model of the kinetics of reversible breakdown in metal-insulator-metal structures with afnia based on the growth of fractal patterns of defects when the insulator is subject to an ...external voltage. The probability that a defect is (or is not) generated and the position where it is generated depend on the electric field distribution. The new defect moves accordingly to fractal rules and attach to another defect in a tree branch. When the two electrodes sandwiching the insulating film are connected a conductive filament is formed and the breakdown takes place. The model is calibrated with experiments inducing metastable soft breakdown events in Pt/HfO
2
/Pt capacitors.
We propose a model of the kinetics of reversible breakdown in metal-insulator-metal structures with afnia based on the growth of fractal patterns of defects when the insulator is subject to an ...external voltage. The probability that a defect is (or is not) generated and the position where it is generated depend on the electric field distribution. The new defect moves accordingly to fractal rules and attach to another defect in a tree branch. When the two electrodes sandwiching the insulating film are connected a conductive filament is formed and the breakdown takes place. The model is calibrated with experiments inducing metastable soft breakdown events in Pt/HfO2/Pt capacitors.
Amino acid ester prodrugs of 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB) were synthesized and evaluated for their affinity for hPEPT1, an intestinal oligopeptide transporter. ...Assays of competitive inhibition of 3Hglycylsarcosine (Gly-Sar) uptake in HeLa/hPEPT1 cells by the amino acid ester prodrugs of BDCRB suggested their 2- to 4-fold higher affinity for hPEPT1 compared to BDCRB. Further, promoieties with hydrophobic side chains and l-configuration were preferred by the hPEPT1 transporter.
Background
The development of efficient strategies for managing high‐risk human papillomavirus (HR‐HPV)–positive women is a major challenge when human papillomavirus–based primary screening is being ...performed. The objectives of this study were to evaluate the comparative effectiveness of HR‐HPV testing based on self‐collection (SC) and HR‐HPV testing based on collection by a health professional (HP) and to assess the potential usefulness of HR‐HPV testing combined with testing with the biomarkers p16/Ki‐67, α‐mannosidase, and superoxide dismutase 2 (SOD2).
Methods
This was a cross‐sectional study of 232 women admitted for colposcopy because of an abnormal Papanicolaou smear. The collected material underwent liquid‐based cytology, HR‐HPV detection, and immunocytochemical testing (p16/Ki‐67, α‐mannosidase, and SOD2). The gold standard was the histopathological result; the positive reference was CIN2+.
Results
The overall accuracy of HR‐HPV testing was 76.6%; the results for the SC group (78.1%) and the HP group (75.2%) were similar. The positive predictive values (HP, 76.5%; SC, 80.0%), the negative predictive values (HP, 66.7%; SC, 64.3%), the positive likelihood values (HP, 1.35; SC, 1.36), and the negative likelihood values (HP, 0.21; SC, 0.19) were also similar. p16/Ki‐67 showed higher sensitivity than the other 2 biomarkers: 78.1% versus 45.8% for α‐mannosidase and 44.5% for SOD2. The specificities of the biomarkers were equivalent: 71.4% for p16/Ki‐67, 77.8% for α‐mannosidase, and 71.2% for SOD2. In the HP group, accuracy also leaned more heavily toward the final score (using α‐mannosidase and SOD2) without statistical significance (80.8% vs 77.9%). The contrast with the SC group yielded the same level of accuracy.
Conclusions
SC, when associated with testing with biomarkers, is as accurate as collection by HPs in the detection of women at risk for cervical cancer.
Vaginal self‐collection, when associated with testing with biomarkers (p16/Ki‐67, α‐mannosidase, and superoxide dismutase 2), is as accurate as collection by health professionals in the detection of women at risk for cervical cancer.
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