RESUMEN
Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Modelos Biológicos , Técnicas de Cultivo de Órganos , Organoides/patología , Neoplasias Pancreáticas/patología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Páncreas/metabolismo , Páncreas/patologíaRESUMEN
Spatiotemporally precise and robust cell fate transitions, which depend on specific signaling cues, are fundamental to the development of appropriately patterned tissues. The fidelity and precision with which photoreceptor fates are recruited in the Drosophila eye exemplifies these principles. The fly eye consists of a highly ordered array of ~750 ommatidia, each of which contains eight distinct photoreceptors, R1-R8, specified sequentially in a precise spatial pattern. Recruitment of R1-R7 fates requires reiterative receptor tyrosine kinase / mitogen activated protein kinase (MAPK) signaling mediated by the transcriptional effector Pointed (Pnt). However the overall signaling levels experienced by R2-R5 cells are distinct from those experienced by R1, R6 and R7. A relay mechanism between two Pnt isoforms initiated by MAPK activation directs the universal transcriptional response. Here we ask how the generic Pnt response is tailored to these two rounds of photoreceptor fate transitions. We find that during R2-R5 specification PntP2 is coexpressed with a closely related but previously uncharacterized isoform, PntP3. Using CRISPR/Cas9-generated isoform specific null alleles we show that under otherwise wild type conditions, R2-R5 fate specification is robust to loss of either PntP2 or PntP3, and that the two activate pntP1 redundantly; however under conditions of reduced MAPK activity, both are required. Mechanistically, our data suggest that intrinsic activity differences between PntP2 and PntP3, combined with positive and unexpected negative transcriptional auto- and cross-regulation, buffer first-round fates against conditions of compromised RTK signaling. In contrast, in a mechanism that may be adaptive to the stronger signaling environment used to specify R1, R6 and R7 fates, the Pnt network resets to a simpler topology in which PntP2 uniquely activates pntP1 and auto-activates its own transcription. We propose that differences in expression patterns, transcriptional activities and regulatory interactions between Pnt isoforms together facilitate context-appropriate cell fate specification in different signaling environments.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sistema de Señalización de MAP Quinasas/genética , Proteínas del Tejido Nervioso/metabolismo , Células Fotorreceptoras de Invertebrados/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Redes Reguladoras de Genes , Modelos Animales , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Análisis Espacio-Temporal , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: A thorough knowledge of sonography is essential in clinical practice. Therefore, sonography training is increasingly incorporated into the medical school curriculum, entailing different course models. The question arises which model is most effective to convey sustained sonographic skills. METHODS: Two different peer-assisted learning (PAL) sonography course models were developed as part of a clinical prospective study. The course content was based on the national resident curriculum of the German Society for Ultrasound in Medicine (DEGUM). Model A consists of a 10-week course and model B of a two-day compact course. Each model entailed 20 teaching units (TU). A script was used to prepare for each unit. Two modified OSCE exams of the ultrasound skills (max = 50 points per exam) were performed during the last teaching unit to assess the competence level. For subjective self-assessment and model evaluation, a questionnaire with a 7-point Likert scale was employed. RESULTS: A total of 888 students of the 3rd year participated as part of a voluntary elective in the study (744 in model A and 144 in model B). In the exams, participants in model A (median 43 points) scored significantly higher than those in model B (median 39; p < 0.01). Participants in model A (mean 1.71 points) obtained significantly higher mean competency gain scores in subject knowledge than model B (mean 1.43 points; p < 0.01) participants. All participants were satisfied with the course concept (A: mean 1.68 vs. B: mean 1.78 points; p = 0.05), the teaching materials (A: mean 1.81 vs. B: mean 1.69 points; p = 0.52), and the tutor's didactic skills (A: mean 1.24 vs. B: mean 1.15 points; p < 0.05). CONCLUSION: These results suggest that sonography-specific competency may be obtained through different course models, with a model stretching over several weeks leading to a higher competence level. Further research should assess the long-term retention of the skills obtained in different models.
Asunto(s)
Educación de Pregrado en Medicina , Estudiantes de Medicina , Humanos , Evaluación Educacional , Estudios Prospectivos , Educación de Pregrado en Medicina/métodos , Competencia Clínica , Curriculum , EnseñanzaRESUMEN
Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.
Asunto(s)
Aberraciones Cromosómicas , Proteínas de Unión al ADN/genética , Enfermedades Fetales/genética , Mutación , Obstrucción del Cuello de la Vejiga Urinaria/congénito , Obstrucción del Cuello de la Vejiga Urinaria/genética , Adulto , Animales , Niño , Femenino , Enfermedades Fetales/patología , Genes Dominantes , Edad Gestacional , Humanos , Masculino , Ratones , Persona de Mediana Edad , Linaje , Embarazo , Obstrucción del Cuello de la Vejiga Urinaria/patología , Pez CebraRESUMEN
Mineral nanoparticle suspensions with consolidating properties have been successfully applied in the restoration of weathered architectural surfaces. However, the design of these consolidants is usually stone-specific and based on trial and error, which prevents their robust operation for a wide range of highly heterogeneous monumental stone materials. In this work, we develop a facile and versatile method to systematically study the consolidating mechanisms in action using a surface forces apparatus (SFA) with real-time force sensing and an X-ray surface forces apparatus (X-SFA). We directly assess the mechanical tensile strength of nanosilica-treated single mineral contacts and show a sharp increase in their cohesion. The smallest used nanoparticles provide an order of magnitude stronger contacts. We further resolve the microstructures and forces acting during evaporation-driven, capillary-force-induced nanoparticle aggregation processes, highlighting the importance of the interactions between the nanoparticles and the confining mineral walls. Our novel SFA-based approach offers insight into nano- and microscale mechanisms of consolidating silica treatments, and it can aid the design of nanomaterials used in stone consolidation.
RESUMEN
PURPOSE: This retrospective study aimed to describe the B-mode lung ultrasound (B-LUS) and contrast-enhanced ultrasound (CEUS) follow-up patterns of peripheral pulmonary lesions (PPLs) in patients with confirmed pulmonary embolism (PE). PATIENTS AND METHODS: Data from 27 patients with a confirmed diagnosis of PE and PPLs over 5 mm from October 2009 to November 2018 were included retrospectively in the study. The inclusion criteria were performance of a baseline CEUS examination, a short-term B-LUS and CEUS follow-up, and a long-term B-LUS follow-up of PPLs. The homogeneity of enhancement of PPLs (homogeneous/inhomogeneous/absent) on CEUS and the presence and size of PPLs on B-LUS were evaluated. RESULTS: A total of n = 25/27 (92.6%) lesions showed absent or inhomogeneous enhancement during baseline examination or short-term follow-up, indicating impaired perfusion. On short-term CEUS follow-up, 9/27 cases (33.3%) showed a pattern shift. On B-LUS long-term follow-up, 26/27 lesions (96.3%) were detectable for an average of 10 weeks (range 3-32 weeks). The size of reference lesions was significantly reduced at the time of the final follow-up examination (P < .05). CONCLUSION: B-LUS follow-up showed that, in patients with confirmed PE, PPLs had a delayed regression. On CEUS follow-up examination, various perfusion patterns of PPLs were observed, indicating the different ages and the variable reparative processes of pulmonary infarction. In PPLs independent of the underlying signs and symptoms, follow-up B-LUS and CEUS examinations may be helpful for a possible retrospective diagnosis of peripheral pulmonary infarction suggestive of PE.
Asunto(s)
Hipertensión Pulmonar , Embolia Pulmonar , Infarto Pulmonar , Medios de Contraste , Estudios de Seguimiento , Humanos , Pulmón/diagnóstico por imagen , Perfusión , Embolia Pulmonar/diagnóstico por imagen , Estudios Retrospectivos , UltrasonografíaRESUMEN
In this work, two vintages (2019 and 2020) of red-fleshed 'Weirouge' apples were processed with the innovative spiral filter press technology to investigate juice production in an oxygen-reduced atmosphere. After pressing, a more brilliant red color and appreciably higher amounts of oxidation-sensitive constituents (ascorbic acid, anthocyanins, and colorless (poly)phenols) were seen in spiral filter pressed juices compared to those produced with conventional systems (horizontal filter press and decanter). In a subsequent stability study (24 weeks storage at 4, 20, and 37 °C), the color and phenolic compounds were monitored and differences in the juices produced with the different pressing-systems were widely maintained during the storage period. The analyses of the anthocyanins and colorless (poly)phenols were conducted by UHPLC-DAD-ESI-QTOF-HR-MS/MS and UHPLC-DAD. The spiral filter press emerged as a promising technology for the production of juices with a more attractive color and a better retention of oxidation-sensitive constituents during processing and storage compared to conventional juices.
Asunto(s)
Malus , Antocianinas , Frutas/química , Fenoles/análisis , Espectrometría de Masas en TándemRESUMEN
Fibrinogen nanofibers are very attractive biomaterials to mimic the native blood clot architecture. Previously, we reported the self-assembly of fibrinogen nanofibers in the presence of monovalent salts and have now studied how divalent salts influence fibrinogen precipitation. Although the secondary fibrinogen structure was significantly altered with divalent metal ions, morphological analysis revealed exclusively smooth fibrinogen precipitates. In situ monitoring of the surface roughness facilitated predicting the tendency of various salts to form fibrinogen fibers or smooth films. Analysis of the chemical composition revealed that divalent salts were removed from smooth fibrinogen films upon rinsing while monovalent Na+ species were still present in fibrinogen fibers. Therefore, we assume that the decisive factor controlling the morphology of fibrinogen precipitates is direct ion-protein contact, which requires disruption of the ion-surrounding hydration shells. We conclude that in fibrinogen aggregates, this mechanism is effective only for monovalent ions, whereas divalent ions are limited to indirect fibrinogen adsorption.
Asunto(s)
Fibrinógeno , Nanofibras , Adsorción , Cationes Bivalentes , IonesRESUMEN
The description of forces across confined complex fluids still holds many challenges due to the possible overlap of different contributions. Here, an attempt is made to untangle the interaction between charged surfaces across nanoparticle suspensions. Interaction forces are measured using colloidal-probe atomic force microscopy. The experimental force profiles are considered as a superposition of double layer and structural forces. In order to independently describe the decay of the double layer force, the ionic strength of the suspension is determined by electrolytic conductivity measurements. Jellium approximation is used to define the impact of the fluid on screening the surface potential. There, the nanoparticles are considered homogeneously distributed across the fluid and screening is only carried out via the particles counterions and added salt. The structural force follows a damped oscillatory profile due to the layer-wise expulsion of the nanoparticles upon approach of both surfaces. The description of the oscillatory structural force is extended by a depletion layer next to the confining surfaces, with no nanoparticles present. The thickness of the depletion layer is related to the electrostatic repulsion of the charged nanoparticles from the like-charged surfaces. The results show that the total force profile is a superposition of independent force contributions without any mutual effects. Using this rather simple model describes the complete experimentally determined interaction force profiles very well from surface separations of a few hundred nanometres down to the surfaces being almost in contact.
RESUMEN
Aqueous solutions of a nonionic surfactant (either Tween20 or BrijL23) and an anionic surfactant (sodium dodecyl sulfate, SDS) are investigated, using small-angle neutron scattering (SANS). SANS spectra are analysed by using a core-shell model to describe the form factor of self-assembled surfactant micelles; the intermicellar interactions are modelled by using a hard-sphere Percus-Yevick (HS-PY) or a rescaled mean spherical approximation (RMSA) structure factor. Choosing these specific nonionic surfactants allows for comparison of the effect of branched (Tween20) and linear (BrijL23) surfactant headgroups, both constituted of poly-ethylene oxide (PEO) groups. The nonionic-anionic surfactant mixtures are studied at various concentrations up to highly concentrated samples (Ï â² 0.45) and various mixing ratios, from pure nonionic to pure anionic surfactant solutions. The scattering data reveal the formation of mixed micelles already at concentrations below the critical micelle concentration of SDS. At higher volume fractions, excluded volume effects dominate the intermicellar structuring, even for charged micelles. In consequence, at high volume fractions, the intermicellar structuring is the same for charged and uncharged micelles. At all mixing ratios, almost spherical mixed micelles form. This offers the opportunity to create a system of colloidal particles with a variable surface charge. This excludes only roughly equimolar mixing ratios (X≈ 0.4-0.6) at which the micelles significantly increase in size and ellipticity due to specific sulfate-EO interactions.
RESUMEN
RESEARCH QUESTION: Are there differences in the cardiometabolic health of ICSI-conceived adolescents compared with a control group, taking parental risk factors into account? DESIGN: ICSI-conceived adolescents (nâ¯=â¯272), their mothers (nâ¯=â¯273) and naturally conceived control adolescents (nâ¯=â¯273) and their mothers (nâ¯=â¯273) provided a blood test and answered a health-related questionnaire. The adolescents also attended a physical examination. RESULTS: ICSI-conceived males showed significantly higher mean weight (72.6 ± 15.1 versus 67.7 ± 12.6 kg, Pâ¯=â¯0.005), body mass index (BMI) (22.2 ± 3.7 versus 21.0 ± 3.2 kg/m2, Pâ¯=â¯0.007) and waist circumference (79.1 ± 11.6 versus 74.5 ± 8.7 cm, P < 0.001). The mean values for weight and BMI were also significantly higher in the ICSI parents. In the ICSI-conceived females significant differences in high-density lipoprotein cholesterol (1.5 ± 0.3 versus 1.6 ± 0.3 mmol/l, Pâ¯=â¯0.033) and triglyceride values (1.1 ± 0.5 versus 1.0 ± 0.4 mmol/l, Pâ¯=â¯0.013) were observed. ICSI mothers also had significantly higher triglycerides (Pâ¯=â¯0.002), higher glutamate pyruvate transaminase/alanine aminotransferase (P < 0.001) and higher alkaline phosphatase values (P < 0.001). CONCLUSIONS: Increased values for weight were found in the male and differences in lipid parameters in the female ICSI-conceived adolescents, which were reflected in the values of their parents. Adjustment for parental risk factors generally attenuated the differences between the ICSI and the control groups, but did not completely remove them. Whether these observed differences are clinically relevant for the future health of the participants requires further study. To increase knowledge in this area, future studies should also include parental data.
Asunto(s)
Índice de Masa Corporal , Colesterol/sangre , Obesidad/sangre , Inyecciones de Esperma Intracitoplasmáticas , Triglicéridos/sangre , Adolescente , Presión Sanguínea/fisiología , Femenino , Humanos , Masculino , Encuestas y CuestionariosRESUMEN
Emerging evidence suggests an inverse association between cancer and neurodegenerative diseases (NDD). Although phenotypically different, both diseases display a significant imbalance in the ubiquitination/deubiquitination processes. Therefore, we particularly investigated the expression of ubiquitin C-terminal hydrolases (UCHs: UCH-L1, UCH-L3, UCH-L5 and BAP1), a subfamily of deubiquitinating enzymes (DUBs), using publically available datasets (GTEx, TCGA) and observed altered expression of UCH-L1, UCH-L3, UCH-L5 in 17 cancer types. Interestingly, UCH-L1 (known to be enriched in neurons and interacting with the Parkinson's disease-associated protein α-synuclein) appeared to be a prognostic indicator of unfavorable outcome in endometrial and urothelial cancer, while increased expression of UCH-L3 and UCH-L5 was associated with poor survival in liver and thyroid cancer, respectively. In normal tissues, UCH-L1 was found to be strongly expressed in the cerebral cortex and hypothalamus, while UCH-L3 expression was somewhat higher in the testis. The occurrence of mutation rates in UCHs also suggests that BAP1 and UCH-L5 may play a more dominant role in cancers than UCH-L1 and UCH-L3. We also characterized the functional context and configuration of the repeat elements in the promoter of DUBs genes and found that UCHs are highly discriminatory for catabolic function and are mainly enriched with LINE/CR1 repeats. Regarding the thesis of an inverse association between cancer and NDD, we observed that among all DUBs, UCHs are the one most involved in both entities. Considering a putative therapeutic potential based on presumed common mechanisms, it will be useful to determine whether other DUBs can compensate for the loss of UCH activity under physiological conditions. However, experimental evidence is required to substantiate this argument.
Asunto(s)
Neoplasias/enzimología , Enfermedades Neurodegenerativas/enzimología , Ubiquitina Tiolesterasa/metabolismo , Encéfalo/metabolismo , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Fenotipo , Pronóstico , Dominios Proteicos , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
Dynamic regulation of the interactions between specific molecules on functional surfaces and biomolecules, for example, proteins or cells, is critical for biosensor and biomedical devices. Herein, we present a spiropyran (SP)-based light-responsive surface coating, hPG (hyperbranched polyglycerol)-SP, to control the adsorption of proteins and adhesion of cells. In the normal state, the SP groups on the coating surface were in hydrophobic ring-closed form, which promotes the nonspecific protein adsorption and cell adhesion. Under UV irradiation, the grafted SP groups were dynamically isomerized into hydrophilic/zwitterionic merocyanine. Both hydrophilicity and zwitterions support the formation of a hydrated layer and hence the resulting hPG-MC coatings highly resist protein adsorption and cell adhesion. Moreover, the presented hPG also provided a robust bioinert background to suppress the nonspecific protein adsorption and cells adhesion. Therefore, this functionalized coating exhibited a good photoregulated antifouling behavior. Moreover, the detachment of adsorbed proteins and adhered cells from the coating surface was also realized.
Asunto(s)
Benzopiranos/química , Materiales Biocompatibles Revestidos/química , Glicerol/química , Indoles/química , Nitrocompuestos/química , Polímeros/química , Proteínas/química , Adsorción , Adhesión Celular , Interacciones Hidrofóbicas e Hidrofílicas , Propiedades de SuperficieRESUMEN
Background: Lowe syndrome (LS) and Dent-2 disease (DD2) are disorders associated with mutations in the OCRL gene and characterized by progressive chronic kidney disease (CKD). Here, we aimed to investigate the long-term renal outcome and identify potential determinants of CKD and its progression in children with these tubulopathies. Methods: Retrospective analyses were conducted of clinical and genetic data in a cohort of 106 boys (LS: 88 and DD2: 18). For genotype-phenotype analysis, we grouped mutations according to their type and localization. To investigate progression of CKD we used survival analysis by Kaplan-Meier method using stage 3 CKD as the end-point. Results: Median estimated glomerular filtration rate (eGFR) was lower in the LS group compared with DD2 (58.8 versus 87.4 mL/min/1.73 m2, P < 0.01). CKD stage II-V was found in 82% of patients, of these 58% and 28% had moderate-to-severe CKD in LS and DD2, respectively. Three patients (3%), all with LS, developed stage 5 of CKD. Survival analysis showed that LS was also associated with a faster CKD progression than DD2 (P < 0.01). On multivariate analysis, eGFR was dependent only on age (b = -0.46, P < 0.001). Localization, but not type of mutations, tended to correlate with eGFR. There was also no significant association between presence of nephrocalcinosis, hypercalciuria, proteinuria and number of adverse clinical events and CKD. Conclusions: CKD is commonly found in children with OCRL mutations. CKD progression was strongly related to the underlying diagnosis but did not associate with clinical parameters, such as nephrocalcinosis or proteinuria.
Asunto(s)
Hipercalciuria/epidemiología , Mutación , Nefrocalcinosis/epidemiología , Monoéster Fosfórico Hidrolasas/genética , Proteinuria/epidemiología , Insuficiencia Renal Crónica/genética , Adolescente , Niño , Preescolar , Progresión de la Enfermedad , Femenino , Genotipo , Tasa de Filtración Glomerular , Humanos , Hipercalciuria/genética , Masculino , Nefrocalcinosis/genética , Fenotipo , Proteinuria/genética , Insuficiencia Renal Crónica/fisiopatología , Insuficiencia Renal Crónica/terapia , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10-12. No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region.
Asunto(s)
Extrofia de la Vejiga/genética , Estudio de Asociación del Genoma Completo , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/genética , Animales , Estudios de Casos y Controles , Humanos , Proteínas con Homeodominio LIM/metabolismo , Ratones , Factores de Transcripción/metabolismoRESUMEN
Bladder exstrophy, a severe congenital urological malformation when a child is born with an open urinary bladder, is the most common form of bladder exstrophy-epispadias complex (BEEC) with an incidence of 1:30,000 children of Caucasian descent. Recent studies suggest that WNT genes may contribute to the etiology of bladder exstrophy. Here, we evaluated WNT-pathway genes in 20 bladder exstrophy patients using massively parallel sequencing. In total 13 variants were identified in WNT3, WNT6, WNT7A, WNT8B, WNT10A, WNT11, WNT16, FZD5, LRP1 and LRP10 genes and predicted as potentially disease causing, of which seven variants were novel. One variant, identified in a patient with a de novo nonsynonymous substitution in WNT3 (p.Cys91Arg), was further evaluated in zebrafish. Knock down of wnt3 in zebrafish showed cloaca malformations, including disorganization of the cloaca epithelium and expansion of the cloaca lumen. Our study suggests that the function of the WNT3 p.Cys91Arg variant was altered, since RNA overexpression of mutant Wnt3 RNA does not result in embryonic lethality as seen with wild-type WNT3 mRNA. Finally, we also mutation screened the WNT3 gene further in 410 DNA samples from BEEC cases and identified one additional mutation c.638G>A (p.Gly213Asp), which was paternally inherited. In aggregate our data support the involvement of WNT-pathway genes in BEEC and suggest that WNT3 in itself is a rare cause of BEEC.
Asunto(s)
Extrofia de la Vejiga/genética , Cloaca/embriología , Cloaca/metabolismo , Proteína Wnt3/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Expresión Génica , Técnicas de Silenciamiento del Gen , Estudios de Asociación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Modelos Moleculares , Mutación , Células 3T3 NIH , Sistemas de Lectura Abierta , Penetrancia , Fenotipo , Polimorfismo de Nucleótido Simple , Conformación Proteica , Transporte de Proteínas , ARN Mensajero/genética , Proteína Wnt3/química , Proteína Wnt3/metabolismoRESUMEN
Deep eutectic solvents (DESs) are a mixture of a salt and a molecular hydrogen bond donor, which form a eutectic liquid with a depressed melting point. Quantum mechanical molecular dynamics (QM/MD) simulations have been used to probe the 1 : 2 choline chloride-urea (ChCl : U), choline chloride-ethylene glycol (ChCl : EG) and choline chloride-glycerol (ChCl : Gly) DESs. DES nanostructure and interactions between the ions is used to rationalise differences in DES eutectic point temperatures and viscosity. Simulations show that the structure of the bulk hydrogen bond donor is largely preserved for hydroxyl based hydrogen bond donors (ChCl:Gly and ChCl:EG), resulting in a smaller melting point depression. By contrast, ChCl:U exhibits a well-established hydrogen bond network between the salt and hydrogen bond donor, leading to a larger melting point depression. This extensive hydrogen bond network in ChCl:U also leads to substantially higher viscosity, compared to ChCl:EG and ChCl:Gly. Of the two hydroxyl based DESs, ChCl:Gly also exhibits a higher viscosity than ChCl:EG. This is attributed to the over-saturation of hydrogen bond donor groups in the ChCl:Gly bulk, which leads to more extensive hydrogen bond donor self-interaction and hence higher cohesive forces within the bulk liquid.
RESUMEN
BACKGROUND: Dent disease is a rare X-linked recessive proximal tubulopathy caused by mutations in CLCN5 (Dent-1) or OCRL (Dent-2). As a rule, total protein excretion (TPE) is low in tubular proteinuria compared with glomerular disease. Several authors have reported nephrotic-range proteinuria (NP) and glomerulosclerosis in Dent disease. Therefore, we aimed to analyze protein excretion in patients with documented CLCN5 or OCRL mutations in a systematic literature review. DESIGN: PubMed and Embase were searched for cases with documented CLCN5 or OCRL mutations and (semi-)quantitative data on protein excretion. The most reliable data (i.e., TPE > protein-creatinine ratio > Albustix) was used for NP classification. RESULTS: Data were available on 148 patients from 47 reports: 126 had a CLCN5 and 22 an OCRLmutation. TPE was not significantly different between both forms (p = 0.11). Fifty-five of 126 (43.7 %) Dent-1 vs 13/22 (59.1 %) Dent-2 patients met the definition of NP (p = 0.25). Serum albumin was normal in all reported cases (24/148). Glomerulosclerosis was noted in 20/32 kidney biopsies and was strongly related to tubulointerstitial fibrosis, but not to kidney function or proteinuria. CONCLUSION: More than half of the patients with both forms of Dent disease have NP, and the presence of low molecular weight proteinuria in a patient with NP in the absence of edema and hypoalbuminemia should prompt genetic testing. Even with normal renal function, glomerulosclerosis and tubulointerstitial fibrosis are present in Dent disease. The role of proteinuria in the course of the disease needs to be examined further in longitudinal studies.
Asunto(s)
Enfermedad de Dent/orina , Nefritis Intersticial/orina , Proteinuria/genética , Eliminación Renal/genética , Biopsia , Canales de Cloruro/genética , Enfermedad de Dent/sangre , Enfermedad de Dent/diagnóstico , Enfermedad de Dent/genética , Pruebas Genéticas , Humanos , Riñón/patología , Riñón/fisiopatología , Mutación , Nefritis Intersticial/sangre , Nefritis Intersticial/diagnóstico , Nefritis Intersticial/genética , Monoéster Fosfórico Hidrolasas/genética , Proteinuria/sangre , Proteinuria/diagnóstico , Proteinuria/orina , Albúmina Sérica/análisisRESUMEN
We have applied a serologic proteomic workflow involving three complementary MS approaches to a tissue-specific Kras(G12D) -knockin mouse model of pancreatic cancer that consistently forms precancerous lesions by 4 months of age. The three proteomics applications were highly complementary and allowed us to survey the entire range of low to high molecular weight serologic proteins. Combined, we identified 121 (49↓, 72↑) unique and statistically relevant serologic biomarkers with 88% previously reported to be associated with cancer and 38% specifically correlated with pancreatic cancer. Four markers, lysozyme C2, cytokeratin 19, Serpina1A and Pcf11, were further verified by Western blotting. When applying systems analysis, the top-associated gene ontology functions were tied to wound healing, RXR signaling, growth, differentiation and innate immune activation through the JAK/STAT pathway. Upon further investigation of the apparent immune response using a multiplex cytokine screen, we found that IFN-γ, VEGF and GM-CSF were significantly increased in serum from the Kras(G12D) animals compared to littermate controls. By combining three complementary MS applications, we were able to survey the native intact peptidome and the global proteome in parallel, unveiling pathways that may be biologically relevant to promotion of pancreatic cancer progression and serologic markers of noninvasive early-stage neoplasia.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/genética , Proteoma/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Biomarcadores de Tumor/sangre , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnicas de Sustitución del Gen , Factor Estimulante de Colonias de Granulocitos y Macrófagos/sangre , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Interferón gamma/sangre , Interferón gamma/genética , Queratina-19/sangre , Queratina-19/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muramidasa/sangre , Muramidasa/genética , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Proteoma/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/sangre , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/genética , alfa 1-Antitripsina/sangre , alfa 1-Antitripsina/genética , Factores de Escisión y Poliadenilación de ARNm/sangre , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The evolution of canalized traits is a central question in evolutionary biology. Natural variation in highly conserved traits can provide clues about their evolutionary potential. Here we investigate natural variation in a conserved trait-even-skipped (eve) expression at the cellular blastoderm stage of embryonic development in Drosophila melanogaster. Expression of the pair-rule gene eve was quantitatively measured in three inbred lines derived from a natural population of D. melanogaster. One line showed marked differences in the spacing, amplitude and timing of formation of the characteristic seven-striped pattern over a 50-min period prior to the onset of gastrulation. Stripe 5 amplitude and the width of the interstripe between stripes 4 and 5 were both reduced in this line, while the interstripe distance between stripes 3 and 4 was increased. Engrailed expression in stage 10 embryos revealed a statistically significant increase in the length of parasegment 6 and a decrease in the length of parasegments 8 and 9. These changes are larger than those previously reported between D. melanogaster and D. pseudoobscura, two species that are thought to have diverged from a common ancestor over 25 million years ago. This line harbors a rare 448 bp deletion in the first intron of knirps (kni). This finding suggested that reduced Kni levels caused the deviant eve expression, and indeed we observed lower levels of Kni protein at early cycle 14A in L2 compared to the other two lines. A second of the three lines displayed an approximately 20% greater level of expression for all seven eve stripes. The three lines are each viable and fertile, and none display a segmentation defect as adults, suggesting that early-acting variation in eve expression is ameliorated by developmental buffering mechanisms acting later in development. Canalization of the segmentation pathway may reduce the fitness consequences of genetic variation, thus allowing the persistence of mutations with unexpectedly strong gene expression phenotypes.