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https://www.readbyqxmd.com/read/28812671/sulfate-radicals-enable-a-non-enzymatic-krebs-cycle-precursor
#1
Markus A Keller, Domen Kampjut, Stuart A Harrison, Markus Ralser
The evolutionary origins of the Krebs cycle (tricarboxylic acid cycle) are not currently clear. Despite the existence of a simple non-enzymatic Krebs cycle catalyst being dismissed only a few years ago as 'an appeal to magic', citrate and other intermediates have since been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify a metabolism-like non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate compounds in a reaction mixture that orients on the typical components of Archaean sediment...
March 13, 2017: Nature ecology & evolution
https://www.readbyqxmd.com/read/28811111/cell-cultures-in-drug-discovery-and-development-the-need-of-reliable-in-vitro-in-vivo-extrapolation-for-pharmacodynamics-and-pharmacokinetics-assessment
#2
REVIEW
Karol Jaroch, Alina Jaroch, Barbara Bojko
For ethical and cost-related reasons, use of animals for the assessment of mode of action, metabolism and/or toxicity of new drug candidates has been increasingly scrutinized in research and industrial applications. Implementation of the 3 "Rs"(1); rule (Reduction, Replacement, Refinement) through development of in silico or in vitro assays has become an essential element of risk assessment. Physiologically based pharmacokinetic (PBPK(2)) modeling is the most potent in silico tool used for extrapolation of pharmacokinetic parameters to animal or human models from results obtained in vitro...
July 27, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28809961/biochemical-characterization-of-recombinant-influenza-a-polymerase-heterotrimer-complex-endonuclease-activity-and-evaluation-of-inhibitors
#3
Weimei Xing, Ona Barauskas, Thorsten Kirschberg, Anita Niedziela-Majka, Michael Clarke, Gabriel Birkus, Perry Weissburg, Xiaohong Liu, Brian E Schultz, Roman Sakowicz, HyockJoo Kwon, Joy Y Feng
Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PAN) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8...
2017: PloS One
https://www.readbyqxmd.com/read/28809826/high-throughput-screening-for-protein-based-inheritance-in-s-cerevisiae
#4
James S Byers, Daniel F Jarosz
The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold...
August 8, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28809115/an-intuitive-approach-for-predicting-potential-human-health-risk-with-the-tox21-10k-library
#5
Nisha S Sipes, John F Wambaugh, Robert Pearce, Scott S Auerbach, Barbara Anne Wetmore, Jui-Hua Hsieh, Andrew J Shapiro, Daniel Svoboda, Michael J DeVito, Stephen S Ferguson
In vitro-in vivo extrapolation (IVIVE) analyses translating high-throughput screening (HTS) data to human relevance have been limited. This study represents the first report applying IVIVE approaches and exposure comparisons using the entirety of the Tox21 federal collaboration chemical screening data, incorporating assay response efficacy and quality of concentration-response fits, and providing quantitative anchoring to first address the likelihood of human in vivo interactions with Tox21 compounds. This likelihood was assessed using a maximum blood concentration to in vitro response ratio approach (Cmax/AC50), analogous to decision-making methods for clinical drug-drug interactions...
August 15, 2017: Environmental Science & Technology
https://www.readbyqxmd.com/read/28809009/docking-and-virtual-screening-in-drug-discovery
#6
Maria Kontoyianni
Stages in a typical drug discovery organization include target selection, hit identification, lead optimization, preclinical and clinical studies. Hit identification and lead optimization are very much intertwined with computational modeling. Structure-based virtual screening (VS) has been a staple for more than a decade now in drug discovery with its underlying computational technique, docking, extensively studied. Depending on the objective, the parameters for VS may change, but the overall protocol is very straightforward...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28809005/resazurin-live-cell-assay-setup-and-fine-tuning-for-reliable-cytotoxicity-results
#7
José Á Rodríguez-Corrales, Jatinder S Josan
In vitro cytotoxicity tests allow for fast and inexpensive screening of drug efficacy prior to in vivo studies. The resazurin assay (commercialized as Alamar Blue(®)) has been extensively utilized for this purpose in 2D and 3D cell cultures, and high-throughput screening. However, improper or lack of assay validation can generate unreliable results and limit reproducibility. Herein, we report a detailed protocol for the optimization of the resazurin assay to determine relevant analytical (limits of detection, quantification, and linear range) and biological (growth kinetics) parameters, and, thus, provide accurate cytotoxicity results...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28809004/identification-of-lipid-binding-modulators-using-the-protein-lipid-overlay-assay
#8
Tuo-Xian Tang, Wen Xiong, Carla V Finkielstein, Daniel G S Capelluto
The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808998/microfluidics-mass-spectrometry-of-protein-carbohydrate-interactions-applications-to-the-development-of-therapeutics-and-biomarker-discovery
#9
Alina D Zamfir
The functional interactions of carbohydrates and their protein receptors are the basis of biological events critical to the evolution of pathological states. Hence, for the past years, such interactions have become the focus of research for the development of therapeutics and discovery of novel glycan biomarkers based on their binding affinity. Due to the high sensitivity, throughput, reproducibility, and capability to ionize minor species in heterogeneous mixtures, microfluidics-mass spectrometry (MS) has recently emerged as a method of choice in protein-glycan interactomics...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808971/high-throughput-screening-for-identification-of-novel-innate-immune-activators
#10
Bryan J Gall, Victor R DeFilippis
Modern drug discovery has embraced in vitro platforms that enable investigation of large numbers of compounds within tractable timeframes and for feasible costs. These endeavors have been greatly aided in recent years by advances in molecular and cell-based methods such as gene delivery and editing technology, advanced imaging, robotics, and quantitative analysis. As such, the examination of phenotypic impacts of novel molecules may only be limited by the size of the compound collection. Innate immune signaling processes in mammalian cells are especially amenable to high-throughput screening platforms since the cellular responses elicited by their activation often result in high level transcription that can be harnessed in the form of bioluminescent or fluorescent signal...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808970/genome-wide-crispr-cas9-screening-for-high-throughput-functional-genomics-in-human-cells
#11
Shiyou Zhu, Yuexin Zhou, Wensheng Wei
It is highly desirable to identify gene's function in a high-throughput fashion, and the CRISPR/Cas9 system has been harnessed to meet such a need. Here, we describe a general method to generate genome-scale lentiviral single-guide RNA (sgRNA) library and conduct a pooled function-based screening in human cells. This protocol would be of interest to researchers to rapidly identify genes in a variety of biological processes.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808963/extraction-and-qpcr-based-detection-of-mirnas-from-cultured-pbmcs-of-bubaline-origin
#12
Chandra S Mukhopadhyay, Ramneek Verma, Jasdeep Singh
MicroRNAs are small noncoding but functionally important RNA molecules that are involved in regulating diverse cellular, metabolic, and immune processes. Their small size necessitates modification in traditional acid phenol-chloroform based RNA isolation procedures to get highly enriched fraction of small RNA that includes miRNAs and siRNAs . Further, of the different methods available, real-time PCR is a powerful tool for precise and specific detection and quantification of miRNA. Moreover, real-time PCR is used to validate the screening or expression of miRNAs that are discovered during high-throughput sequencing, or microarray analysis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28808272/a-combinatorial-approach-for-the-discovery-of-cytochrome-p450-2d6-inhibitors-from-nature
#13
Johannes Hochleitner, Muhammad Akram, Martina Ueberall, Rohan A Davis, Birgit Waltenberger, Hermann Stuppner, Sonja Sturm, Florian Ueberall, Johanna M Gostner, Daniela Schuster
The human cytochrome P450 2D6 (CYP2D6) enzyme is part of phase-I metabolism and metabolizes at least 20% of all clinically relevant drugs. Therefore, it is an important target for drug-drug interaction (DDI) studies. High-throughput screening (HTS) assays are commonly used tools to examine DDI, but show certain drawbacks with regard to their applicability to natural products. We propose an in silico - in vitro workflow for the reliable identification of natural products with CYP2D6 inhibitory potential. In order to identify candidates from natural product-based databases that share similar structural features with established inhibitors, a pharmacophore model was applied...
August 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28807932/reducing-inflammatory-cytokine-production-from-renal-collecting-duct-cells-by-inhibiting-gata2-ameliorates-acute-kidney-injury
#14
Lei Yu, Takashi Moriguchi, Hiroshi Kaneko, Makiko Hayashi, Atsushi Hasegawa, Masahiro Nezu, Hideyuki Saya, Masayuki Yamamoto, Ritsuko Shimizu
Acute kidney injury (AKI) is a leading cause of chronic kidney disease. Proximal tubules are considered to be the primary origin of pathogenic inflammatory cytokines in AKI. However, it remains unclear whether other cell types, including collecting duct (CD) cells, participate in inflammatory processes. The transcription factor GATA2 is specifically expressed in CD cells and maintains their cellular identity. To explore the pathophysiological function of GATA2 in AKI, we generated renal tubular cell-specific Gata2 deletion (G2CKO) mice and examined their susceptibility to ischemia-reperfusion injury (IRI)...
August 14, 2017: Molecular and Cellular Biology
https://www.readbyqxmd.com/read/28807232/ovarian-cancer-novel-molecular-aspects-for-clinical-assessment
#15
REVIEW
Raffaele Palmirotta, Erica Silvestris, Stella D'Oronzo, Angela Cardascia, Franco Silvestris
Ovarian cancer is a very heterogeneous tumor which has been traditionally characterized according to the different histological subtypes and differentiation degree. In recent years, innovative molecular screening biotechnologies have allowed to identify further subtypes of this cancer based on gene expression profiles, mutational features, and epigenetic factors. These novel classification systems emphasizing the molecular signatures within the broad spectrum of ovarian cancer have not only allowed a more precise prognostic prediction, but also proper therapeutic strategies for specific subgroups of patients...
September 2017: Critical Reviews in Oncology/hematology
https://www.readbyqxmd.com/read/28807090/mass-spectral-characterization-and-uplc-quantitation-of-3-deoxyanthocyanidins-in-sorghum-bicolor-varietals
#16
Nathan P Stern, Jatinder Rana, Amitabh Chandra, John Balles
A quantitative ultra-performance LC (UPLC) method was developed and validated to successfully separate, identify, and quantitate the major polyphenolic compounds present in different varieties of sorghum (Sorghum bicolor) feedstock. The method was linear from 3.2 to 320 ppm, with an r² of 0.99999 when using luteolinidin chloride as the external standard. Method accuracy was determined to be 99.5%, and precision of replicate preparations was less than 1% RSD. Characterization by UPLC-MS determined that the predominant polyphenolic components of the sorghum varietals were 3-deoxyanthocyanidins (3-DXAs)...
August 10, 2017: Journal of AOAC International
https://www.readbyqxmd.com/read/28806050/linking-high-throughput-screens-to-identify-moas-and-novel-inhibitors-of-mycobacterium-tuberculosis-dihydrofolate-reductase
#17
John P Santa Maria, Yumi Park, Lihu Yang, Nicholas Murgolo, Michael D Altman, Paul Zuck, Greg Adam, Chad Chamberlin, Peter Saradjian, Peter Dandliker, Helena I M Boshoff, Clifton E Barry, Charles Garlisi, David B Olsen, Katherine Young, Meir Glick, Elliott Nickbarg, Peter S Kutchukian
Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for mechanism of action (MoA) and reveal actionable chemical matter. Here we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning is employed to identify chemical features with predicted efficacy targets for a given phenotypic screen...
August 14, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28805610/validation-of-a-high-throughput-and-robust-technique-bacs-on-beads-assay-karyolite-bobs-for-pre-implantation-aneuploidy-screening
#18
Grace Wing Shan Kong, Yanlin Ma, Jian Ou, Yvonne Ka Yin Kwok, Wei Wang, Queenie Sum Yee Yeung, Cherry Kit Man Wong, Qi Li, Wen Xu, Weiying Lu, Hong Li, Tin Chiu Li, Kwong Wai Choy
OBJECTIVE: This study aims to validate the BACs-on-Beads (BoB) technology as a robust and high throughput method for pre-implantation genetic screening (PGS) for aneuploidy. MATERIAL AND METHODS: The performances with respect to the sensitivity, specificity, success rate and detection rate of this technique from new BoBs technology and traditional array chromosomal genomic hybridization (aCGH) were compared. And the use of BoBs as a screening tool for euploid embryos in PGS was evaluated...
August 2017: Taiwanese Journal of Obstetrics & Gynecology
https://www.readbyqxmd.com/read/28805314/evolved-%C3%AE-factor-prepro-leaders-for-directed-laccase-evolution-in-saccharomyces-cerevisiae
#19
Ivan Mateljak, Thierry Tron, Miguel Alcalde
Although the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution experiments. In this study, twelve constructs were prepared by fusing native and evolved α-factor prepro-leaders from S. cerevisiae to four different laccases with low-, medium- and high-redox potential (PM1L from basidiomycete PM1; PcL from Pycnoporus cinnabarinus; TspC30L from Trametes sp...
August 14, 2017: Microbial Biotechnology
https://www.readbyqxmd.com/read/28804628/from-saccharomyces-cerevisiae-to-human-the-important-gene-co-expression-modules
#20
Wei Liu, Li Li, Hua Ye, Haiwei Chen, Weibiao Shen, Yuexian Zhong, Tian Tian, Huaqin He
Network-based systems biology has become an important method for analyzing high-throughput gene expression data and gene function mining. Yeast has long been a popular model organism for biomedical research. In the current study, a weighted gene co-expression network analysis algorithm was applied to construct a gene co-expression network in Saccharomyces cerevisiae. Seventeen stable gene co-expression modules were detected from 2,814 S. cerevisiae microarray data. Further characterization of these modules with the Database for Annotation, Visualization and Integrated Discovery tool indicated that these modules were associated with certain biological processes, such as heat response, cell cycle, translational regulation, mitochondrion oxidative phosphorylation, amino acid metabolism and autophagy...
August 2017: Biomedical Reports
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