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1.
Nat Commun ; 15(1): 3324, 2024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38637512

RESUMEN

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.


Asunto(s)
Proteínas Asociadas a CRISPR , ARN Catalítico , ARN/metabolismo , ARN Catalítico/metabolismo , Sistemas CRISPR-Cas/genética , ADN/metabolismo , Dominio Catalítico , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , División del ARN
2.
J Bronchology Interv Pulmonol ; 31(1): 63-69, 2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-37246305

RESUMEN

BACKGROUND: Benign airway stenosis (BAS) represents a significant burden on patients, providers, and healthcare systems. Spray cryotherapy (SCT) has been proposed as an adjunctive treatment to reduce BAS recurrence. We sought to examine safety and practice variations of the latest SCT system when used for BAS. METHODS: We conducted a retrospective multicenter cohort study in seven academic institutions within the Interventional Pulmonary Outcomes Group. All patients who underwent at least one SCT session with a diagnosis of BAS at the time of procedure at these institutions were included. Demographics, procedure characteristics, and adverse events were captured through each center's procedural database and electronic health record. RESULTS: A total of 102 patients underwent 165 procedures involving SCT from 2013 to 2022. The most frequent etiology of BAS was iatrogenic (n = 36, 35%). In most cases, SCT was used prior to other standard BAS interventions (n = 125; 75%). The most frequent SCT actuation time per cycle was five seconds. Pneumothorax complicated four procedures, requiring tube thoracostomy in two. Significant post-SCT hypoxemia was noted in one case, with recovery by case conclusion and no long-term effects. There were no instances of air embolism, hemodynamic compromise, or procedural or in-hospital mortality. CONCLUSION: SCT as an adjunctive treatment for BAS was associated with a low rate of complications in this retrospective multicenter cohort study. SCT-related procedural aspects varied widely in examined cases, including actuation duration, number of actuations, and timing of actuations relative to other interventions.


Asunto(s)
Criocirugía , Crioterapia , Humanos , Estudios Retrospectivos , Estudios de Cohortes , Constricción Patológica/etiología , Crioterapia/efectos adversos , Criocirugía/efectos adversos
3.
PLoS One ; 18(10): e0290393, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37878622

RESUMEN

OBJECTIVES: To evaluate the reliability of a novel segmentation-based volume rendering approach for quantification of benign central airway obstruction (BCAO). DESIGN: A retrospective single-center cohort study. SETTING: Data were ascertained using electronic health records at a tertiary academic medical center in the United States. PARTICIPANTS AND INCLUSION: Patients with airway stenosis located within the trachea on two-dimensional (2D) computed tomography (CT) imaging and documentation of suspected benign etiology were included. Four readers with varying expertise in quantifying tracheal stenosis severity were selected to manually segment each CT using a volume rendering approach with the available free tools in the medical imaging viewing software OsiriX (Bernex, Switzerland). Three expert thoracic radiologists were recruited to quantify the same CTs using traditional subjective methods on a continuous and categorical scale. OUTCOME MEASURES: The interrater reliability for continuous variables was calculated by the intraclass correlation coefficient (ICC) using a two-way mixed model with 95% confidence intervals (CI). RESULTS: Thirty-eight patients met the inclusion criteria, and fifty CT scans were selected for measurement. The most common etiology of BCAO was iatrogenic in 22 patients (58%). There was an even distribution of chest and neck CT imaging within our cohort. The average ICC across all four readers for the volume rendering approach was 0.88 (95% CI, 0.84 to 0.93), suggesting good to excellent agreement. The average ICC for thoracic radiologists for subjective methods on the continuous scale was 0.38 (95% CI, 0.20 to 0.55), suggesting poor to fair agreement. The kappa for the categorical approach was 0.26, suggesting a slight to fair agreement amongst the raters. CONCLUSION: In this retrospective cohort study, agreement was good to excellent for raters with varying expertise in airway cross-sectional imaging using a novel segmentation-based volume rendering approach to quantify BCAO. This proposed measurement outperformed our expert thoracic radiologists using conventional subjective grading methods.


Asunto(s)
Tomografía Computarizada por Rayos X , Humanos , Estudios Retrospectivos , Reproducibilidad de los Resultados , Estudios de Cohortes , Constricción Patológica , Tomografía Computarizada por Rayos X/métodos , Variaciones Dependientes del Observador
4.
Xenotransplantation ; 30(6): e12828, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37767640

RESUMEN

Thrombomodulin is important for the production of activated protein C (APC), a molecule with significant regulatory roles in coagulation and inflammation. To address known molecular incompatibilities between pig thrombomodulin and human thrombin that affect the conversion of protein C into APC, GalTKO.hCD46 pigs have been genetically modified to express human thrombomodulin (hTBM). The aim of this study was to evaluate the impact of transgenic hTBM expression on the coagulation dysregulation that is observed in association with lung xenograft injury in an established lung perfusion model, with and without additional blockade of nonphysiologic interactions between pig vWF and human GPIb axis. Expression of hTBM was variable between pigs at the transcriptional and protein level. hTBM increased the activation of human protein C and inhibited thrombosis in an in vitro flow perfusion assay, confirming that the expressed protein was functional. Decreased platelet activation was observed during ex vivo perfusion of GalTKO.hCD46 lungs expressing hTBM and, in conjunction with transgenic hTBM, blockade of the platelet GPIb receptor further inhibited platelets and increased survival time. Altogether, our data indicate that expression of transgenic hTBM partially addresses coagulation pathway dysregulation associated with pig lung xenograft injury and, in combination with vWF-GP1b-directed strategies, is a promising approach to improve the outcomes of lung xenotransplantation.


Asunto(s)
Proteína C , Factor de von Willebrand , Animales , Porcinos , Humanos , Trasplante Heterólogo , Proteína C/metabolismo , Factor de von Willebrand/metabolismo , Células Endoteliales/metabolismo , Trombomodulina/genética , Animales Modificados Genéticamente/metabolismo , Pulmón/metabolismo , Perfusión
5.
Res Sq ; 2023 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-37163044

RESUMEN

CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic6,7. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors8,9, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.

6.
Nat Commun ; 13(1): 2829, 2022 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-35595728

RESUMEN

CRISPR-Cas systems are adaptive immune systems that protect prokaryotes from foreign nucleic acids, such as bacteriophages. Two of the most prevalent CRISPR-Cas systems include type I and type III. Interestingly, the type I-D interference proteins contain characteristic features of both type I and type III systems. Here, we present the structures of type I-D Cascade bound to both a double-stranded (ds)DNA and a single-stranded (ss)RNA target at 2.9 and 3.1 Å, respectively. We show that type I-D Cascade is capable of specifically binding ssRNA and reveal how PAM recognition of dsDNA targets initiates long-range structural rearrangements that likely primes Cas10d for Cas3' binding and subsequent non-target strand DNA cleavage. These structures allow us to model how binding of the anti-CRISPR protein AcrID1 likely blocks target dsDNA binding via competitive inhibition of the DNA substrate engagement with the Cas10d active site. This work elucidates the unique mechanisms used by type I-D Cascade for discrimination of single-stranded and double stranded targets. Thus, our data supports a model for the hybrid nature of this complex with features of type III and type I systems.


Asunto(s)
Proteínas Asociadas a CRISPR , Ácidos Nucleicos , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismo , División del ADN , ARN
7.
Nat Commun ; 11(1): 5931, 2020 11 23.
Artículo en Inglés | MEDLINE | ID: mdl-33230133

RESUMEN

Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.


Asunto(s)
Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Microscopía por Crioelectrón , ADN/química , ADN/metabolismo , Desulfovibrio vulgaris/química , Desulfovibrio vulgaris/genética , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Motivos de Nucleótidos , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo
8.
Mol Cell ; 80(6): 971-979.e7, 2020 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-33248026

RESUMEN

CRISPR-Cas adaptive immune systems provide prokaryotes with defense against viruses by degradation of specific invading nucleic acids. Despite advances in the biotechnological exploitation of select systems, multiple CRISPR-Cas types remain uncharacterized. Here, we investigated the previously uncharacterized type I-D interference complex and revealed that it is a genetic and structural hybrid with similarity to both type I and type III systems. Surprisingly, formation of the functional complex required internal in-frame translation of small subunits from within the large subunit gene. We further show that internal translation to generate small subunits is widespread across diverse type I-D, I-B, and I-C systems, which account for roughly one quarter of CRISPR-Cas systems. Our work reveals the unexpected expansion of protein coding potential from within single cas genes, which has important implications for understanding CRISPR-Cas function and evolution.


Asunto(s)
Inmunidad Adaptativa/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Evolución Molecular , Proteínas Asociadas a CRISPR/inmunología , Células Procariotas/inmunología , Células Procariotas/virología , Biosíntesis de Proteínas , Virus/inmunología
9.
Artículo en Inglés | MEDLINE | ID: mdl-31534978

RESUMEN

As sources of reactive halogens, snowpacks in sea ice regions control the oxidative capacity of the Arctic atmosphere. However, measurements of snowpack halide concentrations remain sparse, particularly in the high Arctic, limiting our understanding of and ability to parameterize snowpack participation in tropospheric halogen chemistry. To address this gap, we measured concentrations of chloride, bromide, and sodium in snow samples collected during polar spring above remote multi-year sea ice (MYI) and first-year sea ice (FYI) north of Greenland and Alaska, as well as in the central Arctic, and compared these measurements to a larger dataset collected in the Alaskan coastal Arctic by Krnavek et al. (2012). Regardless of sea ice region, these surface snow samples generally featured lower salinities, compared to coastal snow. Surface snow in FYI regions was typically enriched in bromide and chloride compared to seawater, indicating snowpack deposition of bromine and chlorine-containing trace gases and an ability of the snowpack to participate further in bromine and chlorine activation processes. In contrast, surface snow in MYI regions was more often depleted in bromide, indicating it served as a source of bromine-containing trace gases to the atmosphere prior to sampling. Measurements at various snow depths indicate that the deposition of sea salt aerosols and halogen-containing trace gases to the snowpack surface played a larger role in determining surface snow halide concentrations compared to upward brine migration from sea ice. Calculated enrichment factors for bromide and chloride, relative to sodium, in the MYI snow samples suggests that MYI regions, in addition to FYI regions, have the potential to play an active role in Arctic boundary layer bromine and chlorine chemistry. The ability of MYI regions to participate in springtime atmospheric halogen chemistry should be considered in regional modeling of halogen activation and interpretation of satellite-based tropospheric bromine monoxide column measurements.

10.
Xenotransplantation ; 26(2): e12458, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30175863

RESUMEN

BACKGROUND: Elevated pulmonary vascular resistance (PVR), platelet adhesion, coagulation activation, and inflammation are prominent features of xenolung rejection. Here, we evaluate the role of thromboxane and histamine on PVR, and their contribution to other lung xenograft injury mechanisms. METHODS: GalTKO.hCD46 single pig lungs were perfused ex vivo with fresh heparinized human blood: lungs were either treated with 1-Benzylimidazole (1-BIA) combined with histamine receptor blocker famotidine (n = 4) or diphenhydramine (n = 6), 1-BIA alone (n = 6) or were left untreated (n = 9). RESULTS: Six of the nine control experiments (GalTKO.hCD46 untreated), "survived" until elective termination at 4 hours. Without treatment, initial PVR elevation within the first 30 minutes resolved partially over the following hour, and increased progressively during the final 2 hours of perfusion. In contrast, 1-BIA, alone or in addition to either antihistamine treatment, was associated with low stable PVR. Combined treatments significantly lowered the airway pressure when compared to untreated reference. Although platelet and neutrophil sequestration and coagulation cascade activation were not consistently altered by any intervention, increased terminal wet/dry weight ratio in untreated lungs was significantly blunted by combined treatments. CONCLUSION: Combined thromboxane and histamine pathway blockade prevents PVR elevation and significantly inhibits loss of vascular barrier function when GalTKO.hCD46 lungs are perfused with human blood. Platelet activation and platelet and neutrophil sequestration persist in all groups despite efficient complement regulation, and appear to occur independent of thromboxane and histamine antagonism. Our work identifies thromboxane and histamine as key mediators of xenolung injury and defines those pathways as therapeutic targets to achieve successful xenolung transplantation.


Asunto(s)
Supervivencia de Injerto/fisiología , Xenoinjertos/inmunología , Histamina/farmacología , Resistencia Vascular , Animales , Animales Modificados Genéticamente , Plaquetas/inmunología , Humanos , Pulmón/metabolismo , Trasplante de Pulmón/métodos , Porcinos , Trasplante Heterólogo/métodos , Resistencia Vascular/fisiología
11.
FASEB J ; 30(3): 1171-86, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26631480

RESUMEN

In sickle cell disease (SCD), treatment of recurrent vasoocclusive episodes, leading to pain crises and organ damage, is still a therapeutic challenge. Vasoocclusion is caused primarily by adherence of homozygous for hemoglobin S (SS) red blood cells (SSRBCs) and leukocytes to the endothelium. We tested the therapeutic benefits of MEK1/2 inhibitors in reversing vasoocclusion in nude and humanized SCD mouse models of acute vasoocclusive episodes using intravital microscopy. Administration of 0.2, 0.3, 1, or 2 mg/kg MEK1/2 inhibitor to TNF-α-pretreated nude mice before human SSRBC infusion inhibited SSRBC adhesion in inflamed vessels, prevented the progression of vasoocclusion, and reduced SSRBC organ sequestration. By use of a more clinically relevant protocol, 0.3 or 1 mg/kg MEK1/2 inhibitor given to TNF-α-pretreated nude mice after human SSRBC infusion and onset of vasoocclusion reversed SSRBC adhesion and vasoocclusion and restored blood flow. In SCD mice, 0.025, 0.05, or 0.1 mg/kg MEK1/2 inhibitor also reversed leukocyte and erythrocyte adhesion after the inflammatory trigger of vasoocclusion and improved microcirculatory blood flow. Cell adhesion was reversed by shedding of endothelial E-selectin, P-selectin, and αvß3 integrin, and leukocyte CD44 and ß2 integrin. Thus, MEK1/2 inhibitors, by targeting the adhesive function of SSRBCs and leukocytes, could represent a valuable therapeutic intervention for acute sickle cell vasoocclusive crises.


Asunto(s)
Anemia de Células Falciformes/tratamiento farmacológico , Anemia de Células Falciformes/enzimología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Selectina E/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Femenino , Hemoglobina Falciforme/metabolismo , Humanos , Receptores de Hialuranos/metabolismo , Integrina alfaVbeta3/metabolismo , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Selectina-P/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedades Vasculares/tratamiento farmacológico , Enfermedades Vasculares/metabolismo
12.
Xenotransplantation ; 22(2): 102-11, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25470239

RESUMEN

BACKGROUND: Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series. METHODS: Data from 157 porcine lung xenoperfusion experiments using otherwise unmodified heparinized human blood were aggregated as either continuous or dichotomous variables. Lungs were wild type in 17 perfusions (11% of the study group), while 31 lungs (20% of the study group) had one genetic modification, 40 lungs (39%) had 2, and 47 lungs (30%) had 3 or more modifications. The primary endpoint was functional lung survival to 4 h of perfusion. Secondary analyses evaluated previously identified markers associated with known lung xenograft injury mechanisms. In addition to comparison among all xenografts grouped by survival status, a subgroup analysis was performed of lungs incorporating the GalTKO.hCD46 genotype. RESULTS: Each increase in the number of genetic modifications was associated with additional prolongation of lung xenograft survival. Lungs that exhibited survival to 4 h generally had reduced platelet activation and thrombin generation. GalTKO and the expression of hCD46, HO-1, hCD55, or hEPCR were associated with improved survival. hTBM, HLA-E, and hCD39 were associated with no significant effect on the primary outcome. CONCLUSION: This meta-analysis of an extensive lung xenotransplantation series demonstrates that increasing the number of genetic modifications targeting known xenogeneic lung injury mechanisms is associated with incremental improvements in lung survival. While more detailed mechanistic studies are needed to explore the relationship between gene expression and pathway-specific injury and explore why some genes apparently exhibit neutral (hTBM, HLA-E) or inconclusive (CD39) effects, GalTKO, hCD46, HO-1, hCD55, and hEPCR modifications were associated with significant lung xenograft protection. This analysis supports the hypothesis that multiple genetic modifications targeting different known mechanisms of xenograft injury will be required to optimize lung xenograft survival.


Asunto(s)
Animales Modificados Genéticamente/inmunología , Xenoinjertos/inmunología , Trasplante de Pulmón/métodos , Sus scrofa/genética , Sus scrofa/inmunología , Trasplante Heterólogo/métodos , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos Heterófilos/genética , Sangre/inmunología , Receptor de Proteína C Endotelial , Galactosiltransferasas/deficiencia , Galactosiltransferasas/genética , Galactosiltransferasas/inmunología , Técnicas de Inactivación de Genes , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/inmunología , Humanos , Técnicas In Vitro , Trasplante de Pulmón/efectos adversos , Proteína Cofactora de Membrana/genética , Proteína Cofactora de Membrana/inmunología , Perfusión , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Trasplante Heterólogo/efectos adversos
13.
Clin Proteomics ; 10(1): 1, 2013 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-23286773

RESUMEN

BACKGROUND: In sickle cell disease (SCD), the mitogen-activated protein kinase (MAPK) ERK1/2 is constitutively active and can be inducible by agonist-stimulation only in sickle but not in normal human red blood cells (RBCs). ERK1/2 is involved in activation of ICAM-4-mediated sickle RBC adhesion to the endothelium. However, other effects of the ERK1/2 activation in sickle RBCs leading to the complex SCD pathophysiology, such as alteration of RBC hemorheology are unknown. RESULTS: To further characterize global ERK1/2-induced changes in membrane protein phosphorylation within human RBCs, a label-free quantitative phosphoproteomic analysis was applied to sickle and normal RBC membrane ghosts pre-treated with U0126, a specific inhibitor of MEK1/2, the upstream kinase of ERK1/2, in the presence or absence of recombinant active ERK2. Across eight unique treatment groups, 375 phosphopeptides from 155 phosphoproteins were quantified with an average technical coefficient of variation in peak intensity of 19.8%. Sickle RBC treatment with U0126 decreased thirty-six phosphopeptides from twenty-one phosphoproteins involved in regulation of not only RBC shape, flexibility, cell morphology maintenance and adhesion, but also glucose and glutamate transport, cAMP production, degradation of misfolded proteins and receptor ubiquitination. Glycophorin A was the most affected protein in sickle RBCs by this ERK1/2 pathway, which contained 12 unique phosphorylated peptides, suggesting that in addition to its effect on sickle RBC adhesion, increased glycophorin A phosphorylation via the ERK1/2 pathway may also affect glycophorin A interactions with band 3, which could result in decreases in both anion transport by band 3 and band 3 trafficking. The abundance of twelve of the thirty-six phosphopeptides were subsequently increased in normal RBCs co-incubated with recombinant ERK2 and therefore represent specific MEK1/2 phospho-inhibitory targets mediated via ERK2. CONCLUSIONS: These findings expand upon the current model for the involvement of ERK1/2 signaling in RBCs. These findings also identify additional protein targets of this pathway other than the RBC adhesion molecule ICAM-4 and enhance the understanding of the mechanism of small molecule inhibitors of MEK/1/2/ERK1/2, which could be effective in ameliorating RBC hemorheology and adhesion, the hallmarks of SCD.

14.
Nano Lett ; 11(3): 1153-60, 2011 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-21280663

RESUMEN

Photoinduced ordering of disordered block copolymers (BCPs) would provide an on-demand, nonintrusive route for formation of well-ordered nanostructures in arbitrarily defined regions of an otherwise disordered material. Here we achieve this objective using a rapid and simple approach in which photoconversion of an additive blended with the BCP introduces strong interactions between the additive and one of the chain segments and induces strong order in the BCP blend. The strategy is generally applicable to block copolymers containing chain segments capable of hydrogen bonding with the additive.

15.
Macromolecules ; 43(9): 4253-4260, 2010 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-21116459

RESUMEN

The real time changes occurring within films of cylinder-forming poly(α-methylstyrene-block-4-hydroxystyrene) (PαMS-b-PHOST) were monitored as they were swollen in tetrahydrofuran (THF) and acetone solvent vapors. In situ information was obtained by combining grazing incidence small angle X-ray scattering (GISAXS) with film thickness monitoring of the solvent vapor swollen films. We show that for self assembly to occur, the polymer thin film must surpass a swollen thickness ratio of 212% of its original thickness when swollen in THF vapors and a ratio of 268% for acetone vapor annealing. As the polymer becomes plasticized by solvent vapor uptake, the polymer chains must become sufficiently mobile to self assemble, or reorganize, at room temperature. Using vapors of a solvent selective to one of the blocks, in our case PHOST-selective acetone, an order-order transition occured driven by the shift in volume fraction. The BCC spherical phase assumed in the highly swollen state can be quenched by rapid drying. Upon treatment with vapor of a non-selective solvent, THF, the film maintained the cylindrical morphology suggested by its dry-state volume fraction. In situ studies indicate that self-assembly occurs spontaneously upon attaining the threshold swelling ratios.

16.
ACS Appl Mater Interfaces ; 1(10): 2363-70, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20355874

RESUMEN

We report a new approach to solution-processable low-dielectric-constant (low-k) materials including photolithographic patterning of these materials in chemically benign and environmentally friendly solvents. A series of semiperfluorinated molecular glasses with styrenic substituents were successfully synthesized. These small molecular materials were thermally stable up to 400 degrees C and also exhibited an amorphous nature, which is essential to forming uniform films. Differential scanning calorimetry studies revealed that a cross-linking reaction occurred in the presence of acid, resulting in the formation of robust polymeric films. Atomic force microscopy images of the cross-linked films showed uniform and pinhole-free surface properties. Dielectric constants determined by a capacitance measurement were 2.6-2.8 (100 kHz) at ambient conditions, which are comparable to other polymeric low-k materials. The incorporation of semiperfluorinated substituents was effective in decreasing the dielectric constant; in particular, the fluorinated alkyl ether structure proved best. In addition, the fluorinated substituents contributed to good solubility in hydrofluoroether (HFE) solvents, which enabled the successful photolithographic patterning of those materials in HFEs down to a submicrometer scale.

17.
ACS Nano ; 2(7): 1396-402, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19206307

RESUMEN

Poly(alpha-methylstyrene)-block-poly(4-hydroxystyrene) acts as both a lithographic deep UV photoresist and a self-assembling material, making it ideal for patterning simultaneously by both top-down and bottom-up fabrication methods. Solvent vapor annealing improves the quality of the self-assembled patterns in this material without compromising its ability to function as a photoresist. The choice of solvent used for annealing allows for control of the self-assembled pattern morphology. Annealing in a nonselective solvent (tetrahydrofuran) results in parallel orientation of cylindrical domains, while a selective solvent (acetone) leads to formation of a trapped spherical morphology. Finally, we have self-assembled both cylindrical and spherical phases within lithographically patterned features, demonstrating the ability to precisely control ordering. Observing the time evolution of switching from cylindrical to spherical morphology within these features provides clues to the mechanism of ordering by selective solvent.


Asunto(s)
Cristalización/métodos , Membranas Artificiales , Nanoestructuras/química , Nanoestructuras/ultraestructura , Nanotecnología/métodos , Poliestirenos/química , Sustancias Macromoleculares/química , Ensayo de Materiales , Conformación Molecular , Tamaño de la Partícula , Propiedades de Superficie
18.
Am J Orthop (Belle Mead NJ) ; 36(2): 68-70, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17405634

RESUMEN

Distal radius fractures are common injuries in children. Displaced fractures have traditionally been treated with closed reduction followed by immobilization in a long arm cast. Because of variable success rates with this technique, a trend in the literature is toward operative fixation of these fractures. A popular alternative practice involves temporary immobilization in a sugar-tong splint, though we are unaware of any studies demonstrating the efficacy of this technique in children. We present our experience in treating these injuries initially with a sugar-tong splint and then with a short arm cast. We retrospectively reviewed the cases of 53 patients (age range, 2-12 years) treated with closed reduction and a sugar-tong splint followed by conversion to a short arm cast after 2 to 3 weeks. In 51 (96%) of 53 fractures, reduction was maintained without more aggressive intervention. The sugar-tong splint is effective in maintaining reductions in pediatric distal radius fractures and has none of the added risks associated with current alternative methods.


Asunto(s)
Fracturas del Radio/terapia , Férulas (Fijadores) , Vendajes , Moldes Quirúrgicos , Niño , Preescolar , Protocolos Clínicos , Humanos , Radiografía , Fracturas del Radio/diagnóstico por imagen , Fracturas del Radio/cirugía , Estudios Retrospectivos
19.
Langmuir ; 22(21): 8684-9, 2006 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-17014105

RESUMEN

Oil-filled silica nanocapsules consisting of a hydrophobic liquid core and a silicate shell have been shown to efficiently extract hydrophobic compounds from aqueous media. With a view toward quantifying the selectivity of these systems, a series of electrochemical and spectroscopic measurements was performed. Uptake and kinetics experiments were carried out through electrochemical measurements by using solutions of lipophilic electroactive molecules of different sizes and with different affinities for silica. Other solutions with fluorescent probes were used for spectrophotometry measurements. In this work we report the environment where the lipophilic compounds studied end up after absorption and the kinetics of their uptake by the oil-filled silica nanocapsules with different shell thicknesses.

20.
J Am Chem Soc ; 127(15): 5528-39, 2005 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-15826191

RESUMEN

Sc(OTf)(3) efficiently catalyzes the self-sufficient transimination reaction between various types of C=N bonds in organic solvents, with turnover frequencies up to 3600 h(-)(1) and rate accelerations up to 6 x 10(5). The mechanism of the crossover reaction in mixtures of amines and imines is studied, comparing parallel individual reactions with coupled equilibria. The intrinsic kinetic parameters for isolated reactions cannot simply be added up when several components are mixed, and the behavior of the system agrees with the presence of a unique mediator that constitutes the core of a network of competing reactions. In mixed systems, every single amine or imine competes for the same central hub, in accordance with their binding affinity for the catalyst metal ion center. More generally, the study extends the basic principles of constitutional dynamic chemistry to interconnected chemical transformations and provides a step toward dynamic systems of increasing complexity.

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