7196 research outputs found
Sort by
Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo
No full textThe phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals
Mammalian cell culture density determination using a laser thru-beam sensor
No full textHigh-throughput protein expression platforms are increasingly used to produce proteins for many applications: to support studies in structure/function, regulation, and proteomics, as well as for direct use as potential biotherapeutic agents for medical applications. Here we describe a modification to the automated protein expression platform used at GNF in which a spectrophotometric sensor and a customized culture flask-receiving nest have been combined in a device that we refer to as the flask density reader (FDR). The FDR enables rapid, non-invasive, automated spectrophotometric determination of cell densities. The FDR reduces the risk of contamination with frequent flask sampling and greatly reduces the time and effort needed to count cells using off-line methods
FDA Public Meeting Report on “Drug Interactions with Hormonal Contraceptives: Public Health and Drug Development Implication”
No full textPotential drug interactions with hormonal contraceptives (HCs) are an important public health concern. A public meeting on “Drug Interaction with Hormonal Contraceptives: Public Health and Drug Development Implication”1 was hosted by the United States (US) Food and Drug Administration (FDA). The meeting endeavored to provide an opportunity for the FDA to seek input from experts on the public health concerns associated with the use of HCs and interacting drugs that might affect efficacy and safety, including pharmacokinetic (PK) / pharmacodynamic (PD) considerations in designing drug interaction studies with HCs during drug development and approaches to translate the results of drug interaction information into informative labeling and communication. The input received could be used to refine FDA's thinking on HC drug interaction study design and interpretation, and labeling communication on drug interaction risk. This meeting benefited from strong and diverse participation from the Center for Drug Evaluation and Research (CDER) at the FDA, Center for Disease Control and Prevention (CDC), National Institute of Health (NIH), Medical Product Agency (MPA) of Sweden, pharmaceutical industry, and academia representatives. This report provides a summary of the key discussion based on the presentations and panel discussion
Aqueous soft dispersed interface-rich systems that promote and guide chemical reactions
No full textAlthough aqueous solutions are considered as sustainable, environmentally friendly reaction media, their use often is limited due to incompatibilities in terms of the reactants’ solubilities. This apparent limitation can be overcome by converting aqueous solutions into soft dispersed interface-rich systems, for example into polyelectrolyte solutions, micellar solutions, oil-in-water microemulsions, or vesicle dispersions. All consist of homogenously distributed dynamic structures which provide local environments which are different from the bulk solution, in analogy to one of the key features of enzymes. The presence of soft dispersed interface-rich structures may not only lead to selective reaction accelerations, but also to changes in reaction pathways, whereby chemical reactions are guided by the structures towards desired products, again in analogy to enzyme-catalysed transformations. General concepts are illustrated with selected examples, ranging from enzyme mimics, the preparation of conductive polymers, and transfer metal-catalysed organic syntheses on industrial scale, to prebiotic systems chemistry considerations
Impact of inducible blaDHA-1 on susceptibility of Klebsiella pneumoniae clinical isolates to LYS228 and identification of chromosomal mpl mutations mediating upregulation of plasmid borne DHA-1 expression
No full textA panel of twenty three K. pneumoniae clinical isolates harboring plasmid-borne inducible β-lactamase DHA-1 (blaDHA-1) exhibited a wide range of susceptibilities to the novel monobactam LYS228 (MIC range 0.125 - >64 µg/mL). This panel was considerably less susceptible (MIC ≥ 8 µg/mL for 9/23 of the isolates) than was a previously reported Enterobacteriaceae strain panel comprised of 88 isolates expressing ESBLs, KPCs and MBL (MIC90 of 2 µg/mL), suggesting that blaDHA-1 can impact LYS228 susceptibility in clinical isolates. Mutants with decreased in vitro susceptibility to LYS228 and upregulated expression of blaDHA-1 were selected in vitro from K. pneumoniae blaDHA-1 strains. These had mutations in the chromosomal peptidoglycan recycling gene mpl. Pre-existing mpl mutations were identified among our clinical strains and these had strongly upregulated expression of blaDHA-1 and reduced susceptibility to LYS228. Therefore we have identified a novel mechanism of blaDHA upregulation in K. pneumoniae clinical isolates, furthering our understanding of the factors underlying β-lactam resistance and the variability in β-lactam susceptibility among these clinical strains
Guide Swap enables genome-scale pooled CRISPR–Cas9 screening in human primary cells
No full textCRISPR–Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells—an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR–Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems
Historical Data: Histopathology Lesions Observed in the Eyes of Control Rabbits in Topical Ocular Administration and Contact Lens Studies
No full textInformation on background changes in the ocular tissues of rabbits (Oryctolagus cuniculus), a common species employed in ophthalmic toxicology studies, is sparse. This complicates interpretation of changes in light of small sample sizes on any single study. The purpose of this publication is to document the interstudy incidence of spontaneous or iatrogenic changes occurring in eyes of control rabbits. Photomicrographs of select lesions are provided. The data set was derived from a total of 54 studies conducted over an eleven-year period at Alcon Research Ltd., a Novartis Division, which featured topical ocular and contact lens routes of administration. It includes a total of 1,222 pigmented and albino New Zealand rabbits and a total of 2,084 eyes which were either untreated or treated with innocuous control articles. There were no noteworthy differences across routes of administration. Changes in anterior segment ocular and adnexal tissues were more common than in posterior segment ocular tissues. Overall, mononuclear cell infiltration was the most common finding. The retina was the posterior tissue most commonly observed with spontaneous changes, with folds and rosettes being the most common retinal finding. Retinal changes were more common in albino as compared to pigmented rabbits. Understanding the incidence and characteristics of spontaneous ocular lesions facilitates accurate and consistent diagnosis and data interpretation
MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma
No full textCombination anti–cytotoxic T lymphocyte antigen 4 (CTLA-4) and anti–programmed cell death protein 1 (PD-1) therapy promotes antitumor immunity and provides superior benefit to patients with advanced-stage melanoma compared with either therapy alone. T cell immunity requires recognition of antigens in the context of major histocompatibility complex (MHC) class I and class II proteins by CD8+ and CD4+ T cells, respectively. We examined MHC class I and class II protein expression on tumor cells from previously untreated melanoma patients and correlated the results with transcriptional and genomic analyses and with clinical response to anti–CTLA-4, anti–PD-1, or combination therapy. Most (>50% of cells) or complete loss of melanoma MHC class I membrane expression was observed in 78 of 181 cases (43%), was associated with transcriptional repression of HLA-A, HLA-B, HLA-C, and B2M, and predicted primary resistance to anti–CTLA-4, but not anti–PD-1, therapy. Melanoma MHC class II membrane expression on >1% cells was observed in 55 of 181 cases (30%), was associated with interferon-γ (IFN-γ) and IFN-γ–mediated gene signatures, and predicted response to anti–PD-1, but not anti–CTLA-4, therapy. We conclude that primary response to anti–CTLA-4 requires robust melanoma MHC class I expression. In contrast, primary response to anti–PD-1 is associated with preexisting IFN-γ–mediated immune activation that includes tumor-specific MHC class II expression and components of innate immunity when MHC class I is compromised. The benefits of combined checkpoint blockade may be attributable, in part, to distinct requirements for melanoma-specific antigen presentation to initiate antitumor immunity
Practical Aspects of Machine Learning for the Design-Synthesis-Purify-Assay Workflow
No full textChimia column: no abstrac
Drug Analogs from Fragment Based Long Short-Term Memory Generative Neural Networks
No full textSeveral recent reports have shown that long short-term memory generative neural networks (LSTM) of the type used for grammar learning efficiently learn to write SMILES of drug-like compounds when trained with SMILES from a database of bioactive compounds such as ChEMBL and can later produce focused sets upon transfer learning with compounds of specific bioactivity profiles. Here we trained an LSTM using molecules taken either from ChEMBL, DrugBank, commercially available fragments, or from FDB-17 (a database of fragments up to 17 atoms) and performed transfer learning to a single known drug to obtain new analogs of this drug. We found that this approach readily generates hundreds of relevant and diverse new drug analogs and works best with training sets of around 40,000 compounds as simple as commercial fragments. These data suggest that fragment-based LSTM offer a promising method for new molecule generation