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The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org .
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Gerenciamento de Dados , Metadados , Reprodutibilidade dos Testes , Bases de Dados Factuais , CinéticaRESUMO
Renal autoregulation is critical in maintaining stable renal blood flow (RBF) and glomerular filtration rate (GFR). Renal ischemia-reperfusion (IR)-induced kidney injury is characterized by reduced RBF and GFR. The mechanisms contributing to renal microvascular dysfunction in IR have not been fully determined. We hypothesized that increased reactive oxygen species (ROS) contributed to impaired renal autoregulatory capability in IR rats. Afferent arteriolar autoregulatory behavior was assessed using the blood-perfused juxtamedullary nephron preparation. IR was induced by 60 min of bilateral renal artery occlusion followed by 24 h of reperfusion. Afferent arterioles from sham rats exhibited normal autoregulatory behavior. Stepwise increases in perfusion pressure caused pressure-dependent vasoconstriction to 65 ± 3% of baseline diameter (13.2 ± 0.4 µm) at 170 mmHg. In contrast, pressure-mediated vasoconstriction was markedly attenuated in IR rats. Baseline diameter averaged 11.7 ± 0.5 µm and remained between 90% and 101% of baseline over 65-170 mmHg, indicating impaired autoregulatory function. Acute antioxidant administration (tempol or apocynin) to IR kidneys for 20 min increased baseline diameter and improved autoregulatory capability, such that the pressure-diameter profiles were indistinguishable from those of sham kidneys. Furthermore, the addition of polyethylene glycol superoxide dismutase or polyethylene glycol-catalase to the perfusate blood also restored afferent arteriolar autoregulatory responsiveness in IR rats, indicating the involvement of superoxide and/or hydrogen peroxide. IR elevated mRNA expression of NADPH oxidase subunits and monocyte chemoattractant protein-1 in renal tissue homogenates, and this was prevented by tempol pretreatment. These results suggest that ROS accumulation, likely involving superoxide and/or hydrogen peroxide, impairs renal autoregulation in IR rats in a reversible fashion.NEW & NOTEWORTHY Renal ischemia-reperfusion (IR) leads to renal microvascular dysfunction manifested by impaired afferent arteriolar autoregulatory efficiency. Acute administration of scavengers of reactive oxygen species, polyethylene glycol-superoxide dismutase, or polyethylene glycol-catalase following renal IR restored afferent arteriolar autoregulatory capability in IR rats, indicating that renal IR led to reversible impairment of afferent arteriolar autoregulatory capability. Intervention with antioxidant treatment following IR may improve outcomes in patients by preserving renovascular autoregulatory function and potentially preventing the progression to chronic kidney disease after acute kidney injury.
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Arteríolas/metabolismo , Taxa de Filtração Glomerular/fisiologia , Homeostase/fisiologia , Insuficiência Renal Crônica/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Pressão Sanguínea/fisiologia , NADPH Oxidases/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Circulação Renal/fisiologiaRESUMO
Prior studies reported that haploinsufficiency of the transcription factor ETS-1 is renoprotective in Dahl salt-sensitive rats, but the mechanism is unclear. Here, we tested whether ETS-1 is involved in hypertension-induced renal microvascular pathology and autoregulatory impairment. Hypertension was induced in salt-sensitive rats and salt-sensitive rats that are heterozygous with 1 wild-type or reference allele of Ets1 (SSEts1+/-) by feeding a diet containing 4% sodium chloride for 1 week. Increases in blood pressure did not differ. However, phosphorylated ETS-1 increased in afferent arterioles of hypertensive salt-sensitive rats, but not in hypertensive SSEts1+/- rats. Afferent arterioles of hypertensive salt-sensitive rats showed increased monocyte chemotactic protein-1 expression and infiltration of CD68 positive monocytes/macrophages. Isolated kidney microvessels showed increased mRNA expression of vascular cell adhesion molecule, intercellular adhesion molecule, P-selectin, fibronectin, transforming growth factor-ß, and collagen I in hypertensive salt-sensitive rats compared with hypertensive SSEts1+/- rats. Using the in vitro blood-perfused juxtamedullary nephron preparation, pressure-mediated afferent arteriolar responses were significantly blunted in hypertensive salt-sensitive rats compared to hypertensive SSEts1+/- rats. Over a 65-170 mm Hg pressure range tested baseline arteriolar diameters averaged 15.1 µm and remained between 107% and 89% of baseline diameter in hypertensive salt-sensitive rats vs. 114% and 73% in hypertensive SSEts1+/- rats (significantly different). Thus, ETS-1 participates in renal arteriolar pathology and autoregulation and thereby is involved in hypertension-mediated kidney injury in salt-sensitive rats.
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Alpharetrovirus , Hipertensão , Proteína Proto-Oncogênica c-ets-1/genética , Animais , Pressão Sanguínea , Hipertensão/genética , Rim , Oncogenes , Ratos , Ratos Endogâmicos DahlRESUMO
Voltage-dependent L-type Ca2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 (P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction (P < 0.05). Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca2+]i in A7r5 cells was suppressed by 1-10 µM Y-27632 (P < 0.05), but 10 µM Y-27632 was required to suppress Ca2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca2+]i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca2+]i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.
Assuntos
Aorta/citologia , Canais de Cálcio/metabolismo , Rim/irrigação sanguínea , Miócitos de Músculo Liso/efeitos dos fármacos , Transdução de Sinais/fisiologia , Quinases Associadas a rho/antagonistas & inibidores , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Amidas/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Proteínas de Bactérias , Linhagem Celular , Masculino , Miócitos de Músculo Liso/metabolismo , Cloreto de Potássio/farmacologia , Piridinas/farmacologia , Ratos , Proteínas Repressoras , Tiazóis/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia , Vasoconstrição/efeitos dos fármacosRESUMO
Access to dual-enrollment courses, which allow high school students to earn college credit, is stratified by race/ethnicity, class, and geography. States and colleges have begun using multiple measures of readiness, including non-cognitive measures of student preparedness, in lieu of strict reliance on test scores in an attempt to expand and equalize access. This practice was accelerated by COVID-19 due to disruptions in standardized testing. However, limited research has examined how non-cognitive beliefs shape students' experiences and outcomes in dual-enrollment courses. We study a large dual-enrollment program created by a university in the Southwest to examine these patterns. We find that mathematics self-efficacy and educational expectations predict performance in dual-enrollment courses, even when controlling for students' academic preparedness, while factors such as high school belonging, college belonging, and self-efficacy in other academic domains are unrelated to academic performance. However, we find that students of color and first-generation students have lower self-efficacy and educational expectations before enrolling in dual-enrollment courses, in addition to having lower levels of academic preparation. These findings suggest that using non-cognitive measures to determine student eligibility for dual-enrollment courses could exacerbate, rather than ameliorate, inequitable patterns of participation. Students from historically marginalized populations may benefit from social-psychological as well as academic supports in order to receive maximum benefits from early postsecondary opportunities such as dual-enrollment. Our findings have implications for how states and dual-enrollment programs determine eligibility for dual-enrollment as well as how dual-enrollment programs should be designed and delivered in order to promote equity in college preparedness. Supplementary Information: The online version contains supplementary material available at 10.1007/s11162-023-09740-z.
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A palladium-catalyzed spirocyclization reaction is reported, which is proposed to arise via insertion of an oxabicycle into a palladacycle, formed from carbocyclization and a C-H functionalization sequence. Mechanistic studies suggest the insertion is diastereoselective and a post-catalytic retro-Diels-Alder step furnishes an alkene, wherein the oxibicycle has served as an acetylene surrogate. Aryl iodides and carbamoyl chlorides were compatible as starting materials under the same reaction conditions, enabling the convergent and complementary synthesis of spirooxindoles, as well as other azacycles. These spirooxindoles allowed further transformations that were previously unaccessible.
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Amino ester hydrolases (AEHs) are capable of rapid synthesis of cephalexin but suffer from rapid deactivation even at low temperatures. Previous efforts to engineer AEH have generated several improved variants but have been limited in scope in part due to limitations in activity assay throughput for ß-lactam synthesis reactions. Rational design of 'whole variants' was explored to rapidly improve AEH thermostability by mutating between 3-15% of residues. Most variants were found to be inactive due to a mutated calcium binding site, the function of which has not previously been described. Four active variants, all with improved melting temperatures, were characterized in terms of synthesis and hydrolysis activity, melting temperature, and deactivation at 25°C. Two variants were found to have improved total turnover numbers relative to the initial AEH variant; however, a clear tradeoff exists between improved stability and overall activity of each variant.
Assuntos
Hidrolases de Éster Carboxílico , beta-Lactamas , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Sítios de Ligação , Temperatura , Hidrólise , Estabilidade EnzimáticaRESUMO
A widely appreciated principle is that all reactions are fundamentally reversible. Observing reversible transition metal-catalysed reactions, particularly those that include the cleavage of C-C bonds, is more challenging. The development of palladium- and nickel-catalysed carboiodination reactions afforded access to the cis and trans diastereomers of the iodo-dihydroisoquinolone products. Using these substrates, an extensive study investigating the reversibility of C-C bond formation using a simple palladium catalyst was undertaken. Herein we report a comprehensive investigation of reversible C-C bond formation using palladium catalysis employing diastereomeric neopentyl iodides as the starting point. It was shown that both diastereomers could be converted to a common product under identical catalytic conditions. A combination of experimental and computational studies were used to probe the operative mechanism. A variety of concepts key to understanding the process of reversible C-C bond formations were investigated, including the effect of electronic and steric parameters on the C-C bond-cleavage step.
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Pharmaceutical production quality has recently been a focus for improvement through incorporation of end-to-end continuous processing. Enzymatic ß-lactam antibiotic synthesis has been one focus for continuous manufacturing, and α-amino ester hydrolases (AEHs) are currently being explored for use in the synthesis of cephalexin due to their high reactivity and selectivity. In this study, several reactors were simulated to determine how reactor type and configuration impacts reactant conversion, fractional yield toward cephalexin, and volumetric productivity for AEH-catalyzed cephalexin synthesis. The primary reactor configurations studied are single reactors including a continuous stirred-tank reactor (CSTR) and plug flow reactor (PFR) as well as two CSTRS and a CSTR + PFR in series. Substrate concentrations fed to the reactors as well as enzyme concentration in the reactor were varied. The presence of substrate inhibition was found to have a negative impact on all reactor configurations studied. No reactor configuration simultaneously allowed high substrate conversion, high fractional yield, and high productivity; however, a single PFR was found to enable the highest substrate conversion with higher fractional yields than all other reactor configurations, by minimizing substrate inhibition. Finally, to further demonstrate the impact of substrate inhibition, an AEH engineered to improve substrate inhibition was simulated and Pareto optimal fronts for a CSTR catalyzed with the current AEH were compared to Pareto fronts for the improved AEH. Overall, reduced substrate inhibition would allow for high substrate conversion, fractional yield, and productivity with only a single CSTR.
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BACKGROUND: Nucleosome-mediated chromatin compaction has a direct effect on the accessibility of trans-acting activators and repressors to DNA targets and serves as a primary regulatory agent of genetic expression. Understanding the nature and dynamics of chromatin is fundamental to elucidating the mechanisms and factors that epigenetically regulate gene expression. Previous work has shown that there are three types of canonical sequences that strongly regulate nucleosome positioning and thus chromatin accessibility: putative nucleosome-positioning elements, putative nucleosome-repelling sequences, and homopolymeric runs of A/T. It is postulated that these elements can be used to remodel chromatin in C. elegans. Here we show the utility of such elements in vivo, and the extreme efficacy of a newly discovered repelling sequence, PRS-322. RESULTS: In this work, we show that it is possible to manipulate nucleosome positioning in C. elegans solely using canonical and putative positioning sequences. We have not only tested previously described sequences such as the Widom 601, but also have tested additional nucleosome-positioning sequences: the Trifonov sequence, putative repelling sequence-322 (PRS-322), and various homopolymeric runs of A and T nucleotides. CONCLUSIONS: Using each of these types of putative nucleosome-positioning sequences, we demonstrate their ability to alter the nucleosome profile in C. elegans as evidenced by altered nucleosome occupancy and positioning in vivo. Additionally, we show the effect that PRS-322 has on nucleosome-repelling and chromatin remodeling.
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Caenorhabditis elegans , Cromatina , Animais , Caenorhabditis elegans/genética , Nucleossomos , Montagem e Desmontagem da Cromatina , Fatores de Transcrição/genéticaRESUMO
Protecting the future of forests in the United States and other countries depends in part on our ability to monitor and map forest health conditions in a timely fashion to facilitate management of emerging threats and disturbances over a multitude of spatial scales. Remote sensing data and technologies have contributed to our ability to meet these needs, but existing methods relying on supervised classification are often limited to specific areas by the availability of imagery or training data, as well as model transferability. Scaling up and operationalizing these methods for general broadscale monitoring and mapping may be promoted by using simple models that are easily trained and projected across space and time with widely available imagery. Here, we describe a new model that classifies high resolution (~1 m2) 3-band red, green, blue (RGB) imagery from a single point in time into one of four color classes corresponding to tree crown condition or health: green healthy crowns, red damaged or dying crowns, gray damaged or dead crowns, and shadowed crowns where the condition status is unknown. These Tree Crown Health (TCH) models trained on data from the United States (US) Department of Agriculture, National Agriculture Imagery Program (NAIP), for all 48 States in the contiguous US and spanning years 2012 to 2019, exhibited high measures of model performance and transferability when evaluated using randomly withheld testing data (n = 122 NAIP state x year combinations; median overall accuracy 0.89-0.90; median Kappa 0.85-0.86). We present examples of how TCH models can detect and map individual tree mortality resulting from a variety of nationally significant native and invasive forest insects and diseases in the US. We conclude with discussion of opportunities and challenges for extending and implementing TCH models in support of broadscale monitoring and mapping of forest health.
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Monitoramento Ambiental , Árvores , Cor , Monitoramento Ambiental/métodos , Florestas , Simulação de Ambiente Espacial , Estados UnidosRESUMO
Global aluminum demand is anticipated to triple by 2050, by which time global greenhouse gas (GHG) emissions are advised to be cut 50-85% to avoid catastrophic climate impacts. To explore mitigation strategies systematically, a dynamic material flow model was developed to simulate the stocks and flows of the U.S. aluminum cycle and analyze the corresponding GHG emissions. Theoretical and realistic reduction potentials were identified and quantified. The total GHG emissions for the U.S. aluminum cycle in 2006 amount to 38 Mt CO(2)-equivalence. However, the U.S. has increasingly relied on imports of aluminum embodied in various products. The in-use stock is still growing fast in most product categories, which limits current scrap availability for recycling and emissions saving. Nevertheless, there is still large emission mitigation potential through recycling. The potentials from "100% old scrap collection" and "low emission energy" were each calculated to be higher than all process technology potential. Total emissions will decrease dramatically and mitigation priorities will change significantly under a stock saturation situation as much more old scrap becomes available for recycling. The nature of in-use stock development over the coming decades will be decisive for the aluminum industry to reach deeper emission cuts.
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Alumínio/análise , Efeito Estufa , Dióxido de Carbono/análise , Indústria Química , Marketing , Modelos Teóricos , Reciclagem , Estados UnidosRESUMO
Emulsion-based techniques have dramatically advanced our understanding of single-cell biology and complex single-cell features over the past two decades. Most approaches for precise single cell isolation rely on microfluidics, which has proven highly effective but requires substantial investment in equipment and expertise that can be difficult to access for researchers that specialize in other areas of bioengineering and molecular biotechnology. Inspired by the robust droplet generation technologies in modern flow cytometry instrumentation, here we established a new platform for high-throughput isolation of single cells within droplets of tunable sizes by combining flow focusing with ultrasonic vibration for rapid and effective droplet formation. Application of ultrasonic pressure waves to the flowing jet provided enhanced control of emulsion droplet size, permitting capture of 25,000 to 50,000 single cells per minute. As an example application, we applied this new droplet generation platform to sequence the antibody variable region heavy and light chain pairings (VH:VL) from large repertoires of single B cells. We demonstrated the recovery of > 40,000 paired CDRH3:CDRL3 antibody clusters from a single individual, validating that these droplet systems can enable the genetic analysis of very large single-cell populations. These accessible new technologies will allow rapid, large-scale, and precise single-cell analyses for a broad range of bioengineering and molecular biotechnology applications.
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Análise de Célula Única/métodos , Ondas Ultrassônicas , Linfócitos B/imunologia , Emulsões , Citometria de Fluxo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologiaRESUMO
During the inflammatory response to an implanted biomaterial, monocytes undergo a striking phenotypic progression of differentiation into macrophages, which may subsequently fuse to form foreign body giant cells (FBGCs). Taking advantage of an in vitro system of cytokine-induced FBGC formation together with the optical slicing capabilities of a confocal microscope, we investigated the cytoskeletal reorganization and adhesive structure development during this dramatic morphological progression. Human monocytes demonstrated diffuse cytoplasmic staining of adhesive structural proteins. Punctate filamentous (F)-actin structures appeared along the ventral cell membrane of macrophages and were identified as the core of podosome adhesive structures by the distinctive ring staining of vinculin, talin, and paxillin around the F-actin. Cytokine-induced FBGCs were characterized by a restriction of podosomes to the extreme periphery of the ventral cell surface. Although macrophages and FBGC contained equivalent amounts of F-actin, significantly more F-actin was located within 1 micron of the ventral plasma membrane in FBGCs compared to macrophages. Taken together, these results provide new information on the dynamic cytoskeletal reorganization and adhesive structure development that occur during phenotypic progression from human monocytes to macrophages to FBGC. Furthermore, they suggest the acquisition of functional specializations on FBGC formation, which may enhance our understanding of chronic inflammatory processes.
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Polaridade Celular/efeitos dos fármacos , Proteínas do Citoesqueleto/metabolismo , Interleucina-13/farmacologia , Macrófagos/metabolismo , Fusão de Membrana , Actinas/metabolismo , Adesão Celular , Diferenciação Celular , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Citocalasina D/farmacologia , Células Gigantes de Corpo Estranho/efeitos dos fármacos , Células Gigantes de Corpo Estranho/metabolismo , Histocitoquímica , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Microscopia Confocal , Monócitos/efeitos dos fármacos , Monócitos/metabolismoRESUMO
BACKGROUND: The twin reversed-arterial-perfusion sequence is a complication of monochorionic twin pregnancies characterized by the hemodynamic dependence of a "recipient" twin from a "pump" twin. The recipient twin exhibits lethal abnormalities including acardia and acephaly. The pump twin has a mortality rate of 50% as a result of high-output heart failure. CASE: The blood supply to an acardiac-acephalus twin was interrupted at 24 weeks' gestation using endoscopic laser coagulation. The co-twin was delivered at 35 weeks and had an uneventful neonatal course following correction of a persistent patent ductus arteriosus. Review of the literature reveals 22 cases of acardiac twinning treated with invasive procedures, seven of them using endoscopic laser coagulation. Pump twin mortality with fetal surgery was 13.6% in comparison with 50% mortality with expectant management (P < .001). CONCLUSION: Fetal surgery is the best available treatment for acardiac twinning. Endoscopic laser coagulation at or before 24 weeks and endoscopic or sonographic guided umbilical cord ligation after this gestational age seem to be the best treatments for this condition.
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Anormalidades Teratoides Graves , Doenças em Gêmeos , Doenças Fetais , Anormalidades Teratoides Graves/diagnóstico por imagem , Adulto , Feminino , Humanos , Recém-Nascido , Fotocoagulação a Laser , Gravidez , Ultrassonografia Pré-Natal , Artérias Umbilicais/cirurgiaRESUMO
The first phase of this study involves injecting a different fluorescent retrograde axonal tracer into the subthalamic nucleus, the substantia nigra, and the mesopontine tegmentum. Multiple labeled cells are found within the caudal third of the globus pallidus. The entopeduncular nucleus and adjacent basal forebrain structures such as the substantia innominata, lateral hypothalamus, bed nucleus of the stria terminalis, and central nucleus of the amygdala all exhibit some dye containing cell, although multiple labeled cells are rare. The second phase of this study involves injecting a different fluorescent retrograde tracer into either the substantia nigra, or the mesopontine tegmentum, and subsequent processing of the tissue for glutamic acid decarboxylase (GAD) immunocytochemistry. Many dye and antibody double-labeled cells could be found within the entopeduncular nucleus and the caudal third of the globus pallidus. This is in contrast to the surrounding basal forebrain regions with brainstem efferents which were rarely GAD-positive. This study indicates that the collateral pattern and immunocytochemistry of globus pallidus neurons with descending efferents are distinct from other basal forebrain neurons having similar efferents. These results also extend previous findings and suggest that the neuron of the pallidal complex are heterogeneous with respect to their patterns of projections. In particular, the present findings question previous assumptions concerning the homology of pallidal segments between primate and rodent species.
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Globo Pálido/metabolismo , Glutamato Descarboxilase/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Mapeamento Encefálico , Feminino , Globo Pálido/citologia , Imuno-Histoquímica , Vias Neurais , Ratos , Ratos EndogâmicosRESUMO
Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems.
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Montagem e Desmontagem da Cromatina , DNA/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Análise de Sequência de DNA , Animais , Fracionamento Celular/métodos , DNA/análise , Técnicas In Vitro , Plasmídeos/química , Plasmídeos/genética , Reprodutibilidade dos Testes , XenopusRESUMO
The studies presented in this paper evaluate the modulatory role of protein pre-adsorbed polydimethylsiloxane (PDMS) and Biomer on the secretion of fibroblast stimulating growth factors from human monocytes/macrophages. The results of these studies show that Biomer and PDMS selectively activate human monocytes to produce fibroblast "progression-like" and to a lesser extent "competence-like" stimulating growth factors. Polydimethylsiloxane stimulated the monocytes/macrophages to produce more "progression-like" fibro-blast stimulating growth factors than Biomer. The induction of "competence-like" fibroblast stimulating activity from the monocytes was enhanced by preadsorption of PDMS with human derived fibrinogen, fibronectin, IgG, hemoglobin, or albumin. This phenomenon was not observed with protein pre-adsorbed Biomer. These studies support the hypothesis that protein pre-adsorbed polymers will selectively modulate monocyte/macrophage activation and induction of growth factors which have the potential to participate in tissue-implant interactions in vivo.