7196 research outputs found
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Runtime Maximization of Continuous Precipitation in an Ultrasonic Process Chamber
No full textThe goal of this investigation was to develop a continuous process in order to produce as much material on dry basis as possible, at stable conditions, in a harsh, precipitating environment. Therefore a novel approach, the direct application of ultrasound (US) in the process chamber, was used to prolong the process time. The main focus was to avoid fouling and build-up, which is an undesired effect in continuous manufacturing. Fouling can occur for many reasons and has several mechanisms (precipitation fouling, particulate fouling, biofouling, corrosion fouling, chemical reaction fouling etc.). In the worst case, fouling can lead to a complete shutdown due to equipment blocking. In this work, two model substance combinations were used (lactose/water/isopropanol and ibuprofen/ethanol/water). A feed suspension was mixed with an anti-solvent in an ultrasonic process chamber, which transduces the power directly into the mixture. The feed suspension is composed of solids distributed in a saturated liquid, which was saturated with the respective solid. The solids are precipitated during mixing, and blockage of the system can take place due to introduced fouling and accumulation in the system. Critical process parameters (the product temperature, the US input and the solid loading) were analyzed in terms of their influence on the process stability and duration. Since this process could also be applied to produce or purify particles, the particle size distribution (PSD) of the two substances was evaluated with regard to agglomeration and attrition
Acoustic Droplet Ejection and Open Port Interface for Rapid Analysis of Metabolic Stability Assays
No full textIn vitro absorption, distribution, metabolism and elimination (ADME) assays are widely used for profiling compounds in pharmaceutical drug discovery programs. Many compounds are screened in metabolic stability assays, using liver microsomes as a model of intrinsic hepatic clearance. Analysis of metabolic stability assays has relied on high throughput LC-MS/MS techniques to keep up with automated assays and compound profiling needs. An experimental alternative to sample analysis via fast chromatography employs an open port interface (OPI) which dilutes and directs acoustically-ejected droplets from microtiter plates to a conventional electrospray ion source for ionization and introduction into a mass spectrometer. Metabolic stability assays of 37 commercial drug compounds using in human, dog, rat and mouse liver microsomes (LMs), were analyzed by LC-MS/MS and an experimental breadboard version of an ADE-OPI-MS/MS system. Results from the experiments comparing intrinsic clearance (CLint) generated with ADE-OPI-MS/MS vs fast LC-MS/MS for all compounds showed ≥86% of CLint values were within a factor of two with R2 ≥ 0.86 using 25 nL and 5 nL sample ejection volumes on the ADE-OPI-MS/MS instrument. Throughput with the experimental ADE-OPI-MS/MS system used in this study was more than ten-fold faster than analysis by the fast LC-MS/MS at 1.3 s/sample versus 17.2 s/sample, respectively
Functional atlas of primary miRNA maturation by the Microprocessor
No full textPrimary microRNAs are the precursors of microRNAs which modulate the expression of most mRNAs in humans. They fold up into a hairpin structure that is cleaved at its base by an enzyme complex known as the Microprocessor (Drosha/DGCR8). While many of the molecular details are known, distinguishing features of primary microRNA compared to other hairpin forming transcripts be further explored. We developed a massively parallel functional assay termed Dro-seq that enabled testing of hundreds of known primary microRNA substrates and thousands of single nucleotide variants of well-known microRNAs. We found an additional feature of primary microRNAs, called Shannon entropy, describing the structural ensemble important for processing. In a deep mutagenesis experiment, we find particular apical loop U bases are likely recognized by DGCR8 and are important for efficient processing. These findings build on existing knowledge about primary miRNA maturation by Microprocessor and further explore the substrate RNA sequence-structure relationship
High-dimensional immune monitoring for chimeric antigen receptor T cell therapies
No full textPurpose of Review: high-dimensional flow cytometry experiments have become a method of
choice for high-throughput integration and characterization of cell populations. Here, we
present a summary of state-of-the-art R-based pipelines used for differential analyses of
cytometry data, largely based on Chimeric Antigen Receptor (CAR) T cell therapies.
Recent Findings: in recent years, existing tools tailored to analyze complex high-dimensional
data such as single cell RNA sequencing (scRNAseq) have been successfully ported to
cytometry studies due to the similar nature of flow cytometry and scRNAseq platforms.
Existing environments like Cytobank24, FlowJo27 and FCS Express 28 already offer a variety of
these ported tools, but they either come at a premium or are fairly complicated to manage by an
inexperienced user. To mitigate these limitations, experienced cytometrists and
bioinformaticians usually incorporate these functions into an R-Shiny 17 application that
ultimately offers a user-friendly, intuitive environment that can be used to analyze flow
cytometry data.
Summary: computational tools and Shiny based tools are the perfect answer to the ever-growing
dimensionality and complexity of flow cytometry data, by offering a dynamic, yet user friendly
exploratory space, tailored to bridge the space between the lab experimental world and the
computational, machine learning space
Semiparametric estimation of the cure fraction in population‐based cancer survival analysis
No full textWith rapid development in medical research, the treatment of diseases including cancer has progressed dramatically and those survivors may die from causes other than the one under study, especially among elderly patients. Motivated by the Surveillance, Epidemiology, and End Results (SEER) female breast cancer study, background mortality is incorporated into the mixture cure proportional hazards (MCPH) model to improve the cure fraction estimation in population‐based cancer studies. Here, that patients are “cured” is defined as when the mortality rate of the individuals in diseased group returns to the same level as that expected in the general population, where the population level mortality is presented by the mortality table of the United States. The semiparametric estimation method based on the EM algorithm for the MCPH model with background mortality (MCPH+BM) is further developed and validated via comprehensive simulation studies. Real data analysis shows that the proposed semiparametric MCPH+BM model may provide more accurate estimation in population‐level cancer study
LGR5 controls extracellular matrix production by stem cells in the developing intestine
No full textThe Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2-Wnt-mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs
Mission Accomplished: The ACVP/STP Coalition for Veterinary Pathology Fellows Completes Its Objectives, but Its Legacy and Spirit Live On
No full textSummary of outcome of Coalition for Society of Tox Path and ACVP for which I am on the Board of Governors. NO NOVARTIS DATA or INFORMATION.
The Coalition was formed in late 2004 to provide a unified mechanism for the American College of Veterinary Pathologists (ACVP) and Society of Toxicologic Pathology (STP) to solicit and allocate funds to establish new training positions for veterinary anatomic and clinical pathologists. Oversight was provided by ACVP Council and STP Executive Committee. Strategic leadership was provided by a ten member Board of Governors, with representatives from academia and industry, appointed by Council and Executive Committee to serve 3 year terms. In addition, current Presidents of the ACVP and STP served as ad hoc members. A Director and Administrative Manager were responsible for implementing and administering Coalition activities; Gary Cockerell and The Rees Group, respectively, have served in these capacities since inception
Systematic Chemogenetic Library Assembly
No full textAt the dawn of the 21st century, human genetic sequencing in combination with computationally-enabled data analysis was heralded to streamline the understanding and curing of diseases. Despite great strides in the use of human genetics and large-scale informatics, a long path remains to parlay candidate genes into successful drug candidates. As such, pharmacological modulators, both chemical and biological probes, assembled into chemogenetic libraries continue to be a mainstay of biomedical research and have a tremendous impact in helping to interrogate complex biological systems. Over the course of 6 years, we assembled and grew a dynamic chemogenetic library of chemical probes (a Mechanism-of-Action Box, or MoA Box) using a mixture of data mining and crowdsourcing institutional expertise across a range of cross-functional scientists. While a chemogenetic library is not conceptually unique, we feel that the lessons learned in construction, annotation, application, and sharing of a MoA Box may help spur discussions to advance the field of chemical biology. Additionally, we herein are disclosing, 4518 compounds with primary annotated gene targets; much of our library (~57% of which are commercially available), should readily enable the larger chemical probe community. We hope our efforts will continue to expand through pre-competitive collaboration across biotech, academia, and chemical vendors. Alliances, such as ChemicalProbes.org, will be instrumental in enabling the rapid discovery and enhanced the reproducibility of chemical probes in biomedical research
BRAF-mutant Transcriptional Subtypes Predict Outcome of Combined BRAF, MEK, and EGFR Blockade With Dabrafenib, Trametinib, and Panitumumab in Patients With Colorectal Cancer
No full textPurpose: The influence of the transcriptional and immunologic context of mutations on therapeutic outcomes with targeted therapy in cancer has not been well defined. BRAF V600E-mutant (BM) colorectal cancer comprises two main transcriptional subtypes, BM1 and BM2. We sought to determine the impact of BM subtype, as well as distinct biological features of those subtypes, on response to BRAF/MEK/EGFR inhibition in patients with CRC. Experimental Design: Paired fresh tumour biopsies were acquired at baseline and on day 15 of treatment from all consenting patients with BM CRC enrolled in a Phase II clinical trial of dabrafenib, trametinib, and panitumumab. For each sample, BM subtype, cell cycle, and immune gene signature expression were determined using RNA-sequencing (RNA-seq), and a Cox proportional hazards model was applied to determine association with progression-free survival (PFS). Results: Confirmed response rates, median PFS, and overall survival (OS) were higher in BM1 subtype patients compared to BM2 subtype patients. Evaluation of immune contexture identified greater immune reactivity in BM1, while cell cycle signatures were more highly expressed in BM2. A multivariate model of PFS incorporating BM subtype plus immune and cell cycle signatures revealed that BM subtype encompasses the majority of the effect. Conclusions: BM subtype is significantly associated with the outcome of combination dabrafenib, trametinib, and panitumumab therapy and may serve as a standalone predictive biomarker beyond mutational status. Our findings support a more nuanced approach to targeted therapeutic decisions that incorporates assessment of transcriptional context
Inhibition of wnt/β-catenin signaling in neuroendocrine tumors in vitro: Antitumoral effects
No full textBackground and aims: Inhibition of Wnt/β-catenin signaling by specific inhibitors is currently being investigated as an antitumoral strategy for various cancers. The role of Wnt/β-catenin signaling in neuroendocrine tumors still needs to be further investigated. Methods: This study investigated the antitumor activity of the porcupine (PORCN) inhibitor WNT974 and the β-catenin inhibitor PRI-724 in human neuroendocrine tumor (NET) cell lines BON1, QGP-1, and NCI-H727 in vitro. NET cells were treated with WNT974, PRI-724, or small interfering ribonucleic acids against β-catenin, and subsequent analyses included cell viability assays, flow cy-tometric cell cycle analysis, caspase3/7 assays and Western blot analysis. Results: Treatment of NET cells with WNT974 significantly reduced NET cell viability in a dose-and time-dependent manner by inducing NET cell cycle arrest at the G1 and G2/M phases without inducing apoptosis. WNT974 primarily blocked Wnt/β-catenin signaling by the dose-and time-dependent downregulation of low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation and non-phosphorylated β-catenin and total β-catenin, as well as the genes targeting the latter (c-Myc and cyclinD1). Furthermore, the WNT974-induced reduction of NET cell viability occurred through the inhibition of GSK-3-dependent or independent signaling (including pAKT/mTOR, pEGFR and pIGFR signaling). Similarly, treatment of NET cells with the β-catenin inhibitor PRI-724 caused significant growth inhibition, while the knockdown of β-catenin expression by siRNA reduced NET tumor cell viability of BON1 cells but not of NCI-H727 cells. Conclusions: The PORCN inhibitor WNT974 possesses antitumor properties in NET cell lines by inhibiting Wnt and related signaling. In addition, the β-catenin inhibitor PRI-724 possesses antitumor properties in NET cell lines. Future studies are needed to determine the role of Wnt/β-catenin signaling in NET as a potential therapeutic target