Diagnosis of Fanconi Anemia: Chromosomal Breakage Analysis

Fanconi anemia (FA) is a rare inherited syndrome with diverse clinical symptoms including developmental defects, short stature, bone marrow failure, and a high risk of malignancies. Fifteen genetic subtypes have been distinguished so far. The mode of inheritance for all subtypes is autosomal recessive, except for FA-B, which is X-linked. Cells derived from FA patients are—by definition—hypersensitive to DNA cross-linking agents, such as mitomycin C, diepoxybutane, or cisplatinum, which becomes manifest as excessive growth inhibition, cell cycle arrest, and chromosomal breakage upon cellular exposure to these drugs. Here we provide a detailed laboratory protocol for the accurate assessment of the FA diagnosis as based on mitomycin C-induced chromosomal breakage analysis in whole-blood cultures. The method also enables a quantitative estimate of the degree of mosaicism in the lymphocyte compartment of the patient

Diagnostic Overlap between Fanconi Anemia and the Cohesinopathies: Roberts Syndrome and Warsaw Breakage Syndrome

Fanconi anemia (FA) is a recessively inherited disease characterized by multiple symptoms including growth retardation, skeletal abnormalities, and bone marrow failure. The FA diagnosis is complicated due to the fact that the clinical manifestations are both diverse and variable. A chromosomal breakage test using a DNA cross-linking agent, in which cells from an FA patient typically exhibit an extraordinarily sensitive response, has been considered the gold standard for the ultimate diagnosis of FA. In the majority of FA patients the test results are unambiguous, although in some cases the presence of hematopoietic mosaicism may complicate interpretation of the data. However, some diagnostic overlap with other syndromes has previously been noted in cases with Nijmegen breakage syndrome. Here we present results showing that misdiagnosis may also occur with patients suffering from two of the three currently known cohesinopathies, that is, Roberts syndrome (RBS) and Warsaw breakage syndrome (WABS). This complication may be avoided by scoring metaphase chromosomes—in addition to chromosomal breakage—for spontaneously occurring premature centromere division, which is characteristic for RBS and WABS, but not for FA

Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor a-chain (IL-7Ra) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Ra extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency

Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates

Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer

Fanconi anemia (FA) is a recessive disorder associated with progressive pancytopenia, multiple developmental defects, and marked predisposition to malignancies. FA is genetically heterogeneous, comprising at least 12 complementation groups (A–M). Activation of one of the FA proteins (FANCD2) by mono-ubiquitination is an essential step in DNA damage response. As FANCD2 interacts with BRCA1, is expressed in proliferating normal breast cells, and FANCD2 knockout mice develop breast tumors, we investigated the expression of FANCD2 in sporadic and hereditary invasive breast cancer patients to evaluate its possible role in breast carcinogenesis. Two tissue microarrays of 129 and 220 sporadic breast cancers and a tissue microarray containing 25 BRCA1 germline mutation-related invasive breast cancers were stained for FANCD2. Expression results were compared with several clinicopathological variables and tested for prognostic value. Eighteen of 96 (19%) sporadic breast cancers and two of 21 (10%) BRCA1-related breast cancers were completely FANCD2-negative, which, however, still showed proliferation. In the remaining cases, the percentage of FANCD2-expressing cells correlated strongly with mitotic index and percentage of cells positive for the proliferation markers Ki-67 and Cyclin A. In immunofluorescence double staining, coexpression of FANCD2 and Ki-67 was apparent. In survival analysis, high FANCD2 expression appeared to be prognostically unfavorable for overall survival (p = 0.03), independent from other major prognosticators (p = 0.026). In conclusion, FANCD2 expression is absent in 10–20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer

Diagnosis of Fanconi Anemia: Mutation Analysis by Next-Generation Sequencing

Fanconi anemia (FA) is a rare genetic instability syndrome characterized by developmental defects, bone marrow failure, and a high cancer risk. Fifteen genetic subtypes have been distinguished. The majority of patients (≈85%) belong to the subtypes A (≈60%), C (≈15%) or G (≈10%), while a minority (≈15%) is distributed over the remaining 12 subtypes. All subtypes seem to fit within the “classical” FA phenotype, except for D1 and N patients, who have more severe clinical symptoms. Since FA patients need special clinical management, the diagnosis should be firmly established, to exclude conditions with overlapping phenotypes. A valid FA diagnosis requires the detection of pathogenic mutations in a FA gene and/or a positive result from a chromosomal breakage test. Identification of the pathogenic mutations is also important for adequate genetic counselling and to facilitate prenatal or preimplantation genetic diagnosis. Here we describe and validate a comprehensive protocol for the molecular diagnosis of FA, based on massively parallel sequencing. We used this approach to identify BRCA2, FANCD2, FANCI and FANCL mutations in novel unclassified FA patients

Bridging the Gap: 3D Real-Space Characterization of Colloidal Assemblies via FIB-SEM Tomography

Insight in the structure of nanoparticle assemblies up to a single particle level is key to understand the collective properties of these assemblies, which critically depend on the individual particle positions and orientations. However, the characterization of large, micron sized assemblies containing small, 10-500 nanometer, sized colloids is highly challenging and cannot easily be done with the conventional light, electron or X-ray microscopy techniques. Here, we demonstrate that focused ion beam-scanning electron microscopy (FIB-SEM) tomography in combination with image processing enables quantitative real-space studies of ordered and disordered particle assemblies too large for conventional transmission electron tomography, containing particles too small for confocal microscopy. First, we demonstrate the high resolution structural analysis of spherical nanoparticle assemblies, containing small anisotropic gold nanoparticles. Herein, FIB-SEM tomography allows the characterization of assembly dimensions which are inaccessible to conventional transmission electron microscopy. Next, we show that FIB-SEM tomography is capable of characterizing much larger ordered and disordered assemblies containing silica colloids with a diameter close to the resolution limit of confocal microscopes. We determined both the position and the orientation of each individual (nano)particle in the assemblies by using recently developed particle tracking routines. Such high precision structural information is essential in the understanding and design of the collective properties of new nanoparticle based materials and processes.Comment: 17 pages, 4 figures, Supplemental Information at articles webpage: https://doi.org/10.1039/C8NR09753

Modelación molecular de cinco flavonoides como antagonistas del receptor de hidrocarburos de arilo. Potencialidades para la salud y producción animal

Background: The Aryl hydrocarbon receptor (AHR) plays a significant role in the development of the mammary gland, as it is related to the transforming growth factor β1 (TGF-β1), which regulates several cellular processes. Hence, its overexpression may lead to pathological processes in the animals, and affect their health and production. Materials and methods: The 3-methyl luteoline, kaempferol, resveratrol, myricetin, and quercetin flavonoid molecules were studied. Modelling relied on the AHR:ARNT structures obtained from Swiss Model software, for coupling program MOE 2019.01, and to determine the protein-protein interactions (PPI). The Cocomaps (bioComplexes Contact MAPS) servers, and Robetta and Rosetta Backruband were used for determining the mutations of alanine. Results: The flavonoids studied associated with contact interfaces at the bHLH, PAS-A domain level and the bHLH/PAS-A and PAS-A/PAS-B of AHR interfaces, and they can undergo an antagonistic behavior due to the interactions at the contact surface level to block or modulate the protein-protein interactions between AHR and ARNT. Conclusions: The five flavonoids can interact at different AHR superficial interfaces to modulate the formation of the functional heterodimer, acting as antagonist agents. The order of occurrence probability of these actions is higher with 3-methyl luteoline, kaempferol, resveratrol, and lower with myricetin, and quercetin. Feed supplementation using foliage rich in these flavonoids might improve animal health and production. Keywords: Animal nutrition, Aryl hydrocarbon receptor, flavonoids, breeding, transcription factors (Source: DeCS)Antecedentes: El receptor de arilo de hidrocarburos (AHR) juega un papel importante en el desarrollo de la glándula mamaria, se relaciona con el factor de crecimiento transformante β1 (TGF-β1) que regula diversos procesos celulares, por lo que su sobreexpresión puede provocar procesos patológicos en los animales y afectar su salud y producción. Materiales y Métodos: Se investigaron las moléculas de flavonoides 3-Metilluteolina, Kaempferol, Resveratrol, Miricetina y Quercetina. Para el modelado se utilizaron las estructuras de AHR:ARNT obtenidas del programa Swiss Model, para el acoplamiento el programa MOE 2019.01., y para la determinación de interacciones proteína-proteína (IPP) y mutaciones de alanina, los servidores Cocomaps (bioCOmplexes Contact MAPS)  y Robetta y Rosetta Backrub, respectivamente.   Resultados: Los flavonoides estudiados se unen a interfaces de contacto a nivel de los dominios bHLH, PAS-A y las interfaces bHLH/PAS-A y PAS-A/PAS-B de la AHR y pueden exhibir un comportamiento antagónico debido a las interacciones a nivel de las superficies de contacto para bloquear o modular las interacciones proteína-proteína entre AHR y ARNT. Conclusiones: Los cinco flavonoides pueden interactuar a nivel de diferentes interfaces superficiales del AHR para modular la formación del heterodímero funcional, actuando como agentes antagonistas. El orden de probabilidad de estas acciones es mayor con 3-Metilluteolina, Kaempferol, Resveratrol y menor con Miricetina y Quercetina. La suplementación del pienso con follaje rico en estos flavonoides podría mejorar la salud y la producción animal. Palabras clave: Nutrición animal, Receptor de Hidrocarburos de Arilo, Flavonoides, Reproducción, Factores de Transcripción. (Fuente: DeCS

Social networks and political participation in a Sicilian community context

AbstractThis study shows the linkage between political and social participation, underlining the relevance of the motivational sphere. The aim is to evaluate politically relevant social capital by adopting a relational perspective and ego-network measures, so that we can understand the interdependence between cognitive maps, motivational factors and relational dimension, both in qualitative and quantitative dimensions

Modelación molecular de cinco flavonoides como antagonistas del receptor de hidrocarburos de arilo. Potencialidades para la salud y producción animal

Antecedentes: El receptor de arilo de hidrocarburos (AHR) juega un papel importante en el desarrollo de la glándula mamaria, se relaciona con el factor de crecimiento transformante β1 (TGF-β1) que regula diversos procesos celulares, por lo que su sobreexpresión puede provocar procesos patológicos en los animales y afectar su salud y producción. Materiales y Métodos: Se investigaron las moléculas de flavonoides 3-Metilluteolina, Kaempferol, Resveratrol, Miricetina y Quercetina. Para el modelado se utilizaron las estructuras de AHR:ARNT obtenidas del programa Swiss Model, para el acoplamiento el programa MOE 2019.01., y para la determinación de interacciones proteína-proteína (IPP) y mutaciones de alanina, los servidores Cocomaps (bioCOmplexes Contact MAPS) y Robetta y Rosetta Backrub, respectivamente. Resultados: Los flavonoides estudiados se unen a interfaces de contacto a nivel de los dominios bHLH, PAS-A y las interfaces bHLH/PAS-A y PAS-A/PAS-B de la AHR y pueden exhibir un comportamiento antagónico debido a las interacciones a nivel de las superficies de contacto para bloquear o modular las interacciones proteína-proteína entre AHR y ARNT. Conclusiones: Los cinco flavonoides pueden interactuar a nivel de diferentes interfaces superficiales del AHR para modular la formación del heterodímero funcional, actuando como agentes antagonistas. El orden de probabilidad de estas acciones es mayor con 3-Metilluteolina, Kaempferol, Resveratrol y menor con Miricetina y Quercetina. La suplementación del pienso con follaje rico en estos flavonoides podría mejorar la salud y la producción animal