A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells

Background: Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. Results: In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. Conclusion: By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides

Clinical application of a cancer genomic profiling assay to guide precision medicine decisions

AIM: Develop and apply a comprehensive and accurate next-generation sequencing based assay to help clinicians to match oncology patients to therapies. MATERIALS and METHODS: The performance of the CANCERPLEX(R) assay was assessed using DNA from well-characterized routine clinical formalin-fixed paraffin-embedded (FFPE) specimens and cell lines. RESULTS: The maximum sensitivity of the assay is 99.5% and its accuracy is virtually 100% for detecting somatic alterations with an allele fraction of as low as 10%. Clinically actionable variants were identified in 93% of patients (930 of 1000) who underwent testing. CONCLUSION: The test\u27s capacity to determine all of the critical genetic changes, tumor mutation burden, microsatellite instability status and viral associations has important ramifications on clinical decision support strategies, including identification of patients who are likely to benefit from immune checkpoint blockage therapies

YAP/TAZ Activation Drives Uveal Melanoma Initiation and Progression

Uveal melanoma (UM), the most common ocular malignancy, is characterized by GNAQ/11 mutations. Hippo/YAP and Ras/mitogen-activated protein kinase (MAPK) emerge as two important signaling pathways downstream of G protein alpha subunits of the Q class (GalphaQ/11)-mediated transformation, although whether and how they contribute to UM genesis in vivo remain unclear. Here, we adapt an adeno-associated virus (AAV)-based ocular injection method to directly deliver Cre recombinase into the mouse uveal tract and demonstrate that Lats1/2 kinases suppress UM formation specifically in uveal melanocytes. We find that genetic activation of YAP, but not Kras, is sufficient to initiate UM. We show that YAP/TAZ activation induced by Lats1/2 deletion cooperates with Kras to promote UM progression via downstream transcriptional reinforcement. Furthermore, dual inhibition of YAP/TAZ and Ras/MAPK synergizes to suppress oncogenic growth of human UM cells. Our data highlight the functional significance of Lats-YAP/TAZ in UM initiation and progression in vivo and suggest combination inhibition of YAP/TAZ and Ras/MAPK as a new therapeutic strategy for UM

Structural correlations in heterogeneous electron transfer at monolayer and multilayer graphene electrodes

As a new form of carbon, graphene is attracting intense interest as an electrode material with widespread applications. In the present study, the heterogeneous electron transfer (ET) activity of graphene is investigated using scanning electrochemical cell microscopy (SECCM), which allows electrochemical currents to be mapped at high spatial resolution across a surface for correlation with the corresponding structure and properties of the graphene surface. We establish that the rate of heterogeneous ET at graphene increases systematically with the number of graphene layers, and show that the stacking in multilayers also has a subtle influence on ET kinetics. © 2012 American Chemical Society

Pleiotropic functions of the tumor- and metastasis-suppressing Matrix Metalloproteinase-8 in mammary cancer in MMTV-PyMT transgenic mice

Matrix metalloproteinase-8 (MMP-8; neutrophil collagenase) is an important regulator of innate immunity which has onco-suppressive actions in numerous tumor types

Visualization and validation of twin nucleation and early-stage growth in magnesium

The abrupt occurrence of twinning when Mg is deformed leads to a highly anisotropic response, making it too unreliable for structural use and too unpredictable for observation. Here, we describe an in-situ transmission electron microscopy experiment on Mg crystals with strategically designed geometries for visualization of a long-proposed but unverified twinning mechanism. Combining with atomistic simulations and topological analysis, we conclude that twin nucleation occurs through a pure-shuffle mechanism that requires prismatic-basal transformations. Also, we verified a crystal geometry dependent twin growth mechanism, that is the early-stage growth associated with instability of plasticity flow, which can be dominated either by slower movement of prismatic-basal boundary steps, or by faster glide-shuffle along the twinning plane. The fundamental understanding of twinning provides a pathway to understand deformation from a scientific standpoint and the microstructure design principles to engineer metals with enhanced behavior from a technological standpoint

A new view of electrochemistry at highly oriented pyrolytic graphite

Major new insights on electrochemical processes at graphite electrodes are reported, following extensive investigations of two of the most studied redox couples, Fe(CN)64–/3– and Ru(NH3)63+/2+. Experiments have been carried out on five different grades of highly oriented pyrolytic graphite (HOPG) that vary in step-edge height and surface coverage. Significantly, the same electrochemical characteristic is observed on all surfaces, independent of surface quality: initial cyclic voltammetry (CV) is close to reversible on freshly cleaved surfaces (>400 measurements for Fe(CN)64–/3– and >100 for Ru(NH3)63+/2+), in marked contrast to previous studies that have found very slow electron transfer (ET) kinetics, with an interpretation that ET only occurs at step edges. Significantly, high spatial resolution electrochemical imaging with scanning electrochemical cell microscopy, on the highest quality mechanically cleaved HOPG, demonstrates definitively that the pristine basal surface supports fast ET, and that ET is not confined to step edges. However, the history of the HOPG surface strongly influences the electrochemical behavior. Thus, Fe(CN)64–/3– shows markedly diminished ET kinetics with either extended exposure of the HOPG surface to the ambient environment or repeated CV measurements. In situ atomic force microscopy (AFM) reveals that the deterioration in apparent ET kinetics is coupled with the deposition of material on the HOPG electrode, while conducting-AFM highlights that, after cleaving, the local surface conductivity of HOPG deteriorates significantly with time. These observations and new insights are not only important for graphite, but have significant implications for electrochemistry at related carbon materials such as graphene and carbon nanotubes