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The 16th GCC Closed Forum was held in Orlando, FL, USA, on 23 June 2023. Representatives from international bioanalytical Contract Research Organizations were in attendance in order to discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at the meeting included: IS response, flow cytometry, changes to the bioanalytical industry, NGS assays, biomarker assay for tissues, dPCR validation, immunogenicity harmonization and ICH M10 implementation. Conclusions and consensus from discussions of these topics are included in this article.
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Biomarcadores , Citometria de Fluxo , Citometria de Fluxo/normas , Citometria de Fluxo/métodos , Biomarcadores/análise , Humanos , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Camonsertib is a novel ATR kinase inhibitor in clinical development for advanced cancers targeting sensitizing mutations. This article describes the identification and biosynthesis of an N-glucuronide metabolite of camonsertib. This metabolite was first observed in human hepatocyte incubations and was subsequently isolated to determine the structure, evaluate its stability as part of bioanalytical method development and for use as a standard for estimating its concentration in Phase I samples. The N-glucuronide was scaled-up using a purified bacterial culture preparation and was subsequently isolated using preparative chromatography. The bacterial culture generated sufficient material of the glucuronide to allow for one- and two-dimensional 1H and 13C NMR structural elucidation and further bioanalytical characterization. The NOE data combined with the gradient HMBC experiment and molecular modeling, strongly suggests that the point of attachment of the glucuronide results in the formation of (2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(5-(4-((1R,3r,5S)-3-hydroxy-8-oxabicyclo[3.2.1]octan-3-yl)-6-((R)-3-methylmorpholino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-1H-pyrazol-1-yl)tetrahydro-2H-pyran-2-carboxylic acid. Significance Statement This is the first report of a glucuronide metabolite of camonsertib formed by human hepatocyte incubations. This study reveals the structure of an N-glucuronide metabolite of camonsertib using detailed elucidation by one- and two-dimensional NMR after scale-up using a novel bacterial culture approach yielding significant quantities of a purified metabolite.
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INTRODUCTION: Medical operations are vulnerable to global supply chain fluctuations. The ability to locally produce and reliably sterilize medical equipment may mitigate this risk. This project developed a reliable high-level disinfection process for 3D printed surgical tools. METHODS: Surgical instruments and consumables were designed and printed from various materials. Devices contaminated with known and unknown bacteria underwent one of three cleaning methods followed by high-level disinfection using submersion in a Cidex OPA Solution. Devices were then cultured on blood agar plates and incubated for 48 h. Positive and negative controls were performed. RESULTS: The results of control experiments showed no growth on negative controls and significant growth on all positive control plates. Of the three cleaning methods tested, one showed no growth: cleaning with isopropyl alcohol and chlorhexidine followed by Cidex bath. DISCUSSION: This project successfully developed a rapid high-level disinfection process for 3D printed surgical instruments made from two different types of 3D printing material.
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Desinfecção , Esterilização , Humanos , Glutaral , Esterilização/métodos , Desinfecção/métodos , Clorexidina , Impressão Tridimensional , Instrumentos Cirúrgicos , Contaminação de Equipamentos/prevenção & controleRESUMO
In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein assembly polypeptide-1 (AP-1) complex operates as part of the secretory pathway at the trans-Golgi network (TGN), while the AP-2 complex and the TPLATE complex jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched TGN/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis (Arabidopsis thaliana) cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.
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Arabidopsis , Vesículas Revestidas por Clatrina , Arabidopsis/genética , Arabidopsis/metabolismo , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Proteoma/metabolismo , Proteômica , Fator de Transcrição AP-1/análise , Fator de Transcrição AP-1/metabolismoRESUMO
BACKGROUND: Minimally invasive resuscitative endovascular balloon occlusion of the aorta (REBOA) following noncompressible hemorrhage results in significant ischemia reperfusion injury (IRI). Adverse outcomes from IRI include organ dysfunction and can result in profound hemodynamic and molecular compromise. We hypothesized that adenosine, lidocaine, and magnesium (ALM) attenuates organ injury and inflammation responses following REBOA IRI in a porcine model of hemorrhage. METHODS: Animals underwent a 20% controlled hemorrhage followed by 45 minutes of supraceliac balloon occlusion. They were randomized into two groups: control (n = 9) and ALM intervention (n = 9) to include a posthemorrhage, pre-REBOA bolus (200 mL of 3% NaCl ALM) followed by a continuous drip (2 mL/kg per hour of 0.9% NaCl ALM) during the 4-hour resuscitative period. Primary outcomes included hemodynamic parameters, gene expression of inflammatory signaling molecules, and plasma concentrations of select cytokines and chemokines. RESULTS: The ALM cohort demonstrated a significant reduction in cardiac output and cardiac index. Plasma concentrations of interleukin 2 and interleukin 10 were significantly lower 3 hours post-REBOA in animals treated with ALM versus vehicle. Interleukin 4 levels in plasma were also lower with ALM at 3 and 4 hours post-REBOA (p < 0.05). Liver expression of IL1RN, MTOR, and LAMP3 messenger RNA was significantly lower with ALM as compared with the vehicle. No significant difference in large bowel gene expression was observed between treatments. CONCLUSION: In a porcine model of hemorrhage, ALM treatment mitigated inflammatory responses early during post-REBOA resuscitation. Our findings suggest that ALM use with trauma may reduce inflammatory injury and improve outcomes related to REBOA utilization.
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Oclusão com Balão , Procedimentos Endovasculares , Traumatismo por Reperfusão , Choque Hemorrágico , Animais , Adenosina/uso terapêutico , Aorta/cirurgia , Oclusão com Balão/métodos , Modelos Animais de Doenças , Procedimentos Endovasculares/métodos , Hemorragia/terapia , Lidocaína/farmacologia , Lidocaína/uso terapêutico , Magnésio/farmacologia , Magnésio/uso terapêutico , Traumatismo por Reperfusão/terapia , Choque Hemorrágico/etiologia , Choque Hemorrágico/terapia , SuínosRESUMO
Systemic acquired resistance (SAR) is a mechanism through which plants may respond to initial challenge by a pathogen through activation of inducible defense responses, thereby increasing resistance to subsequent infection attempts. Fitness costs are assumed to be incurred by plants induced for SAR, and several studies have attempted to quantify these costs. We developed a mathematical model, motivated by game-theoretic concepts, to simulate competition between hypothetical plant populations with and without SAR to examine conditions under which the phenomenon of SAR may have evolved. Data were gathered from various studies on fitness costs of induced resistance on life history traits in different plant hosts and scaled as a proportion of the values in control cohorts in each study (i.e., healthy plants unprimed for SAR). With unprimed healthy control plants set to a fitness value of 1, primed healthy plants incurred a fitness cost of about 10.4% (0.896, n = 157), primed diseased plants incurred a fitness cost of about 15.5% (0.845, n = 54), and unprimed diseased plants incurred a fitness cost of about 28.9% (0.711, n = 69). Starting from a small proportion of the population (0.5%) and competing against a population with constitutive defenses alone in stochastic simulations, the SAR phenotype almost always dominated the population after 1000 generations when the probability of disease was greater than or equal to 0.5 regardless of the probability for priming errors.
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Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta, indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Proteases Específicas de Ubiquitina/genética , UbiquitinaçãoRESUMO
We present TrackMate, an open source Fiji plugin for the automated, semi-automated, and manual tracking of single-particles. It offers a versatile and modular solution that works out of the box for end users, through a simple and intuitive user interface. It is also easily scriptable and adaptable, operating equally well on 1D over time, 2D over time, 3D over time, or other single and multi-channel image variants. TrackMate provides several visualization and analysis tools that aid in assessing the relevance of results. The utility of TrackMate is further enhanced through its ability to be readily customized to meet specific tracking problems. TrackMate is an extensible platform where developers can easily write their own detection, particle linking, visualization or analysis algorithms within the TrackMate environment. This evolving framework provides researchers with the opportunity to quickly develop and optimize new algorithms based on existing TrackMate modules without the need of having to write de novo user interfaces, including visualization, analysis and exporting tools. The current capabilities of TrackMate are presented in the context of three different biological problems. First, we perform Caenorhabditis-elegans lineage analysis to assess how light-induced damage during imaging impairs its early development. Our TrackMate-based lineage analysis indicates the lack of a cell-specific light-sensitive mechanism. Second, we investigate the recruitment of NEMO (NF-κB essential modulator) clusters in fibroblasts after stimulation by the cytokine IL-1 and show that photodamage can generate artifacts in the shape of TrackMate characterized movements that confuse motility analysis. Finally, we validate the use of TrackMate for quantitative lifetime analysis of clathrin-mediated endocytosis in plant cells.
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Rastreamento de Células/métodos , Embrião não Mamífero/ultraestrutura , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Análise de Célula Única/métodos , Software , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Algoritmos , Animais , Arabidopsis/metabolismo , Arabidopsis/ultraestrutura , Caenorhabditis elegans , Rastreamento de Células/estatística & dados numéricos , Clatrina/genética , Clatrina/metabolismo , Embrião não Mamífero/metabolismo , Endocitose , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica de Plantas , Transdução de Sinal Luminoso , Células Vegetais/metabolismo , Células Vegetais/ultraestrutura , Análise de Célula Única/estatística & dados numéricosRESUMO
Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological barriers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the characterization of three exciting new gyramide analogs-from a library of 183 derivatives-that are potent inhibitors of DNA gyrase and are active against clinical strains of gram-negative bacteria (Escherichia coli, Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and gram-positive bacteria (Bacillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent inhibitors of the DNA supercoiling activity of DNA gyrase (IC50s of 47-170 nM) but do not alter the enzyme's ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations support the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other known inhibitors.
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Methods for the subcellular fractionation and enrichment of specific intracellular compartments are essential tools in the analysis of compartment composition and function. In vitro characterization of isolated cell organelles and other endomembrane intermediates, including exploration of the compartment protein ensemble, offers strong clues of in vivo function identity. Here, we describe methodology for the isolation of clathrin-coated vesicles from Arabidopsis thaliana suspension-cultured cells on the basis of differential and density centrifugation.
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Arabidopsis/ultraestrutura , Vesículas Revestidas por Clatrina/metabolismo , Biologia Molecular/métodos , Arabidopsis/metabolismo , Fracionamento Celular , Centrifugação com Gradiente de Concentração/métodos , Vesículas Revestidas por Clatrina/químicaRESUMO
Stomatal cytokinesis defective1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Clatrina/metabolismo , Citocinese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Dados de Sequência Molecular , Mutação , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genéticaRESUMO
Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an epsin N-terminal homology protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein nevershed/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.
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Proteínas de Arabidopsis/metabolismo , Clatrina/metabolismo , Vacúolos/metabolismo , Rede trans-Golgi/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Proteínas Ativadoras de GTPase , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Meristema/genética , Meristema/metabolismo , Meristema/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Dados de Sequência Molecular , Mutação , Filogenia , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico/genética , Homologia de Sequência de Aminoácidos , Vacúolos/ultraestruturaRESUMO
Rhizoctonia crown and root rot (RCRR), caused by Rhizoctonia solani AG-2-2, is an increasingly important disease of sugar beet in Minnesota and North Dakota. Disease ratings are based on subjective, visual estimates of root rot severity (0-to-7 scale, where 0 = healthy and 7 = 100% rotted, foliage dead). Remote sensing was evaluated as an alternative method to assess RCRR. Field plots of sugar beet were inoculated with R. solani AG 2-2 IIIB at different inoculum densities at the 10-leaf stage in 2008 and 2009. Data were collected for (i) hyperspectral reflectance from the sugar beet canopy and (ii) visual ratings of RCRR in 2008 at 2, 4, 6, and 8 weeks after inoculation (WAI) and in 2009 at 2, 3, 5, and 9 WAI. Green, red, and near-infrared reflectance and several calculated narrowband and wideband vegetation indices (VIs) were correlated with visual RCRR ratings, and all resulted in strong nonlinear regressions. Values of VIs were constant until at least 26 to 50% of the root surface was rotted (RCRR = 4, wilting of foliage starting to develop) and then decreased significantly as RCRR ratings increased and plants began dying. RCRR also was detected using airborne, color-infrared imagery at 0.25- and 1-m resolution. Remote sensing can detect RCRR but not before initial appearance of foliar symptoms.
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Propionibacterium species are members of the normal flora of skin and the mouth but their pathogenic potential is often overlooked. Three fatal cases of endocarditis caused by Propionibacterium species over an 8-year period are reported, and a review is presented of a further 33 cases from the world literature. In most cases, infection was protracted, with minimal signs in the early stages. Fourteen cases (42.4%) involved native valves, 16 (48.5%) involved prosthetic valves and three (9.1%) were associated with other intracardiac prosthetic material. Intracardiac abscesses were commonly encountered, with Propionibacterium endocarditis occurring in 28.6% of native valve infections and 52.9% of prosthetic valve infections. A very high proportion of all of the cases (70.6%) required surgical intervention. Several factors appeared to delay institution of appropriate therapy and may have contributed to abscess formation, including an indolent clinical course, negative or delayed culture results, and the tendency to consider this organism as a blood-culture contaminant. The authors recommend careful clinical evaluation before disregarding a blood-culture isolate of Propionibacterium spp. as a skin contaminant, and consideration of this bacterium as a potential cause of apparently culture-negative endocarditis.