Identification of Highly Expressed, Soluble Proteins Using an Improved, High-Throughput Pooled ORF Expression Technology

This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into three separate E. coli cultures. After bulk expression and purification, the proteins from the three separate pools were digested into tryptic peptides. Each of these samples was subsequently analyzed in triplicate using reversed-phase high-performance liquid chromatography (LC) coupled directly online with MS/MS. The abundance of each protein was determined by calculating the average exponentially modified protein abundance index (emPAI) of each protein across the three protein pools. Human proteins that consistently gave high emPAI values were subjected to small-scale expression and purification. These clones showed high levels of expression of soluble protein. Conversely, proteins that were not observed by LC-MS/MS did not show any detectable soluble expression in small-scale validation studies. Using this improved POET method allows the expression characteristics of hundreds of proteins to be quickly determined in a single experiment

Feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors

Neurofibromatosis type 1, including the highly aggressive malignant peripheral nerve sheath tumors (MPNSTs), is featured by the loss of functional neurofibromin 1 (NF1) protein resulting from genetic alterations. A major function of NF1 is suppressing Ras activities, which is conveyed by an intrinsic GTPase-activating protein-related domain (GRD). In this study, we explored the feasibility of restoring Ras GTPase via exogenous expression of various GRD constructs, via gene delivery using a panel of adeno-associated virus (AAV) vectors in MPNST and human Schwann cells (HSCs). We demonstrated that several AAV serotypes achieved favorable transduction efficacies in those cells and a membrane-targeting GRD fused with an H-Ras C-terminal motif (C10) dramatically inhibited the Ras pathway and MPNST cells in a NF1-specific manner. Our results opened up a venue of gene replacement therapy in NF1-related tumors

The Orbit of NGC 5907 ULX-1

© 2024. The Author(s). Published by the American Astronomical Society. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/Wereport on the orbit of the binary system powering the most extreme ultraluminous X-ray pulsar known to date: NGC5907ULX-1(hereafter ULX1). ULX1 has been the target of a substantial multi-instrument campaign, mainly in the X-ray band, but no clear counterparts are known in other bands. Although ULX1 is highly variable and pulsations can be transient (regardless of the source flux), the timing data collected so far allow us to investigate the orbit of this system. We find an orbital period P orb =+ 5.7 0.1 0.6 days and a projected semi-axis 0.8 1 A 3.1 lt s =+. The most likely ephemeris is Porb = 5.6585(6) days, A1 =3.1(4) lt-s, and the epoch of ascending nodes passage is Tasc = 57751.37(5) MJD. However, there are six similar solutions acceptable within 3σ.Wefind further indications that ULX1 is a high-mass X-ray binary. This implies that we are observing its orbit face on, with an inclination <5°.Peer reviewe

Approaches to monitor and evaluate OER policies in higher education : tracing developments in Germany, Austria, and Switzerland

The 2019 UNESCO recommendation on Open Educational Resources (OER) encourages member states to monitor policies and mechanisms in OER across the world. In higher education, there are many initiatives and policies around OER. This contribution gives insights into the current situation concerning OER policy documents that are of national or institutional relevance for public higher education institutions in Germany, Switzerland, and Austria. For each country, a different approach for identifying OER policy documents was chosen, dependent on the availability of documents and different dominant forms of documentation. Whereas digital documents available on the web were found as helpful sources for Germany, and performance agreements between the national ministry and individual universities were used for analysis in Austria, a survey amongst all universities was the chosen research approach in Switzerland to give an overview about potentially OER related policy documents. All these documents are now made available via the OER World Map. With this contribution, the authors also highlight the possibility of using the OER World Map as a powerful tool to collect and evaluate OER policy documents

Approaches to monitor and evaluate OER policies in higher education : tracing developments in Germany, Austria, and Switzerland

The 2019 UNESCO recommendation on Open Educational Resources (OER) encourages member states to monitor policies and mechanisms in OER across the world. In higher education, there are many initiatives and policies around OER. This contribution gives insights into the current situation concerning OER policy documents that are of national or institutional relevance for public higher education institutions in Germany, Switzerland, and Austria. For each country, a different approach for identifying OER policy documents was chosen, dependent on the availability of documents and different dominant forms of documentation. Whereas digital documents available on the web were found as helpful sources for Germany, and performance agreements between the national ministry and individual universities were used for analysis in Austria, a survey amongst all universities was the chosen research approach in Switzerland to give an overview about potentially OER related policy documents. All these documents are now made available via the OER World Map. With this contribution, the authors also highlight the possibility of using the OER World Map as a powerful tool to collect and evaluate OER policy documents

Deconvoluting Post-Transplant Immunity: Cell Subset-Specific Mapping Reveals Pathways for Activation and Expansion of Memory T, Monocytes and B Cells

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO+CD62L− effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant

MicroRNA-1 is a candidate tumor suppressor and prognostic marker in human prostate cancer

We previously reported that miR-1 is among the most consistently down-regulated miRs in primary human prostate tumors. In this follow-up study, we further corroborated this finding in an independent data set and made the novel observation that miR-1 expression is further reduced in distant metastasis and is a candidate predictor of disease recurrence. Moreover, we performed in vitro experiments to explore the tumor suppressor function of miR-1. Cell-based assays showed that miR-1 is epigenetically silenced in human prostate cancer. Overexpression of miR-1 in these cells led to growth inhibition and down-regulation of genes in pathways regulating cell cycle progression, mitosis, DNA replication/repair and actin dynamics. This observation was further corroborated with protein expression analysis and 3′-UTR-based reporter assays, indicating that genes in these pathways are either direct or indirect targets of miR-1. A gene set enrichment analysis revealed that the miR-1-mediated tumor suppressor effects are globally similar to those of histone deacetylase inhibitors. Lastly, we obtained preliminary evidence that miR-1 alters the cellular organization of F-actin and inhibits tumor cell invasion and filipodia formation. In conclusion, our findings indicate that miR-1 acts as a tumor suppressor in prostate cancer by influencing multiple cancer-related processes and by inhibiting cell proliferation and motility

Optimasi Portofolio Resiko Menggunakan Model Markowitz MVO Dikaitkan dengan Keterbatasan Manusia dalam Memprediksi Masa Depan dalam Perspektif Al-Qur`an

Risk portfolio on modern finance has become increasingly technical, requiring the use of sophisticated mathematical tools in both research and practice. Since companies cannot insure themselves completely against risk, as human incompetence in predicting the future precisely that written in Al-Quran surah Luqman verse 34, they have to manage it to yield an optimal portfolio. The objective here is to minimize the variance among all portfolios, or alternatively, to maximize expected return among all portfolios that has at least a certain expected return. Furthermore, this study focuses on optimizing risk portfolio so called Markowitz MVO (Mean-Variance Optimization). Some theoretical frameworks for analysis are arithmetic mean, geometric mean, variance, covariance, linear programming, and quadratic programming. Moreover, finding a minimum variance portfolio produces a convex quadratic programming, that is minimizing the objective function ðð¥with constraintsð ð 𥠥 ðandð´ð¥ = ð. The outcome of this research is the solution of optimal risk portofolio in some investments that could be finished smoothly using MATLAB R2007b software together with its graphic analysis