Live-Cell Characterization of the JNK Signaling Cascade

Signaling cascades process extracellular information about environmental changes into spatiotemporally regulated signaling events, through a series of signal relays as well as feedback loops and crosstalk mechanisms. The JNK signaling cascade is a major mediator of vital cellular decision making processes, including cellular differentiation and inflammatory response. The cascade transduces a variety of extracellular signals into these functional responses, but its response to TNF, an important cytokine in immune system communications, has not been well characterized, especially at the single mammalian cell level. To characterize JNK quantitatively in single living cells, we used a FRET-based JNK activity reporter to visualize JNK activity in HEK cells. We found that JNK produces a graded response to varying concentrations of TNF, suggesting that JNK relays a graded input signal to its downstream components, which is subsequently converted into a digital response for executing important cellular decisions such as programmed cell death. Using the reporter targeted to the plasma membrane, we also discovered that JNK activation kinetics is slower at the plasma membrane compared to the cytosol, suggesting that JNK activities are regulated spatially. Moreover, JNK in HEK cells exhibited increased sensitivity to TNF when prestimulated with a low dose of the stimulus

Effect of different cytokines on mammaglobin and maspin gene expression in normal leukocytes: possible relevance to the assays for the detection of micrometastatic breast cancer

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-α, IL-1β) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells

Live-Cell Characterization of the JNK Signaling Cascade

Signaling cascades process extracellular information about environmental changes into spatiotemporally regulated signaling events, through a series of signal relays as well as feedback loops and crosstalk mechanisms. The JNK signaling cascade is a major mediator of vital cellular decision making processes, including cellular differentiation and inflammatory response. The cascade transduces a variety of extracellular signals into these functional responses, but its response to TNF, an important cytokine in immune system communications, has not been well characterized, especially at the single mammalian cell level. To characterize JNK quantitatively in single living cells, we used a FRET-based JNK activity reporter to visualize JNK activity in HEK cells. We found that JNK produces a graded response to varying concentrations of TNF, suggesting that JNK relays a graded input signal to its downstream components, which is subsequently converted into a digital response for executing important cellular decisions such as programmed cell death. Using the reporter targeted to the plasma membrane, we also discovered that JNK activation kinetics is slower at the plasma membrane compared to the cytosol, suggesting that JNK activities are regulated spatially. Moreover, JNK in HEK cells exhibited increased sensitivity to TNF when prestimulated with a low dose of the stimulus

Expression of Neurexin, Neuroligin, and Their Cytoplasmic Binding Partners in the Pancreatic β-Cells and the Involvement of Neuroligin in Insulin Secretion

The composition of the β-cell exocytic machinery is very similar to that of neuronal synapses, and the developmental pathway of β-cells and neurons substantially overlap. β-Cells secrete γ-aminobutyric acid and express proteins that, in the brain, are specific markers of inhibitory synapses. Recently, neuronal coculture experiments have identified three families of synaptic cell-surface molecules (neurexins, neuroligins, and SynCAM) that drive synapse formation in vitro and that control the differentiation of nascent synapses into either excitatory or inhibitory fully mature nerve terminals. The inhibitory synapse-like character of the β-cells led us to hypothesize that members of these families of synapse-inducing adhesion molecules would be expressed in β-cells and that the pattern of expression would resemble that associated with neuronal inhibitory synaptogenesis. Here, we describe β-cell expression of the neuroligins, neurexins, and SynCAM, and show that neuroligin expression affects insulin secretion in INS-1 β-cells and rat islet cells. Our findings demonstrate that neuroligins and neurexins are expressed outside the central nervous system and help confer an inhibitory synaptic-like phenotype onto the β-cell surface. Analogous to their role in synaptic neurotransmission, neurexin-neuroligin interactions may play a role in the formation of the submembrane insulin secretory apparatus

Predictive Factors and Risk Model for Positive Circumferential Resection Margin Rate after Transanal Total Mesorectal Excision in 2653 Patients with Rectal Cancer

The aim of this study was to determine the incidence of, and preoperative risk factors for, positive circumferential resection margin (CRM) after transanal total mesorectal excision (TaTME). Background: TaTME has the potential to further reduce the rate of positive CRM for patients with low rectal cancer, thereby improving oncological outcome. Methods: A prospective registry-based study including all cases recorded on the international TaTME registry between July 2014 and January 2018 was performed. Endpoints were the incidence of, and predictive factors for, positive CRM. Univariate and multivariate logistic regressions were performed, and factors for positive CRM were then assessed by formulating a predictive model. Results: In total, 2653 patients undergoing TaTME for rectal cancer were included. The incidence of positive CRM was 107 (4.0%). In multivariate logistic regression analysis, a positive CRM after TaTME was significantly associated with tumors located up to 1 cm from the anorectal junction, anterior tumors, cT4 tumors, extra-mural venous invasion (EMVI), and threatened or involved CRM on baseline MRI (odds ratios 2.09, 1.66, 1.93, 1.94, and 1.72, respectively). The predictive model showed adequate discrimination (area under the receiver-operating characteristic curve >0.70), and predicted a 28% risk of positive CRM if all risk factors were present. Conclusion: Five preoperative tumor-related characteristics had an adverse effect on CRM involvement after TaTME. The predicted risk of positive CRM after TaTME for a specific patient can be calculated preoperatively with the proposed model and may help guide patient selection for optimal treatment and enhance a tailored treatment approach to further optimize oncological outcomes