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1.
Adv Sci (Weinh) ; 11(14): e2305998, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38298098

RESUMO

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.


Assuntos
Células Matadoras Naturais , Neoplasias , Humanos , Receptores de Células Matadoras Naturais/metabolismo , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Neoplasias/metabolismo
2.
Development ; 150(17)2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-37519269

RESUMO

Changes in gene expression represent an important source of phenotypic innovation. Yet how such changes emerge and impact the evolution of traits remains elusive. Here, we explore the molecular mechanisms associated with the development of masculinizing ovotestes in female moles. By performing integrative analyses of epigenetic and transcriptional data in mole and mouse, we identified the co-option of SALL1 expression in mole ovotestes formation. Chromosome conformation capture analyses highlight a striking conservation of the 3D organization at the SALL1 locus, but an evolutionary divergence of enhancer activity. Interspecies reporter assays support the capability of mole-specific enhancers to activate transcription in urogenital tissues. Through overexpression experiments in transgenic mice, we further demonstrate the capability of SALL1 to induce kidney-related gene programs, which are a signature of mole ovotestes. Our results highlight the co-option of gene expression, through changes in enhancer activity, as a plausible mechanism for the evolution of traits.


Assuntos
Rim , Toupeiras , Animais , Feminino , Camundongos , Rim/metabolismo , Camundongos Transgênicos , Toupeiras/genética
3.
Angew Chem Int Ed Engl ; 62(32): e202218850, 2023 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-36637348

RESUMO

Hydrogen (H2 ) produced from renewables will have a growing impact on the global energy dynamics towards sustainable and carbon-neutral standards. The share of green H2 is still too low to meet the net-zero target, while the demand for high-quality hydrogen continues to rise. These factors amplify the need for economically viable H2 generation technologies. The present article aims at evaluating the existing technologies for high-quality H2 production based on solar energy. Technologies such as water electrolysis, photoelectrochemical and solar thermochemical water splitting, liquid metal reactors and plasma conversion utilize solar power directly or indirectly (as carbon-neutral electrons) and are reviewed from the perspective of their current development level, technical limitations and future potential.

4.
Cell ; 185(20): 3689-3704.e21, 2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36179666

RESUMO

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.


Assuntos
Cromatina , Placenta , Animais , Fator de Ligação a CCCTC/metabolismo , Montagem e Desmontagem da Cromatina , Elementos Facilitadores Genéticos , Evolução Molecular , Feminino , Genoma , Mamíferos/metabolismo , Placenta/metabolismo , Gravidez , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Science ; 370(6513): 208-214, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033216

RESUMO

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.


Assuntos
Adaptação Fisiológica/genética , Fator 9 de Crescimento de Fibroblastos/genética , Toupeiras/genética , Elementos Reguladores de Transcrição , Diferenciação Sexual/genética , Esteroide 17-alfa-Hidroxilase/genética , Animais , Inversão Cromossômica , Conjuntos de Dados como Assunto , Feminino , Regulação da Expressão Gênica , Genoma , Camundongos , Camundongos Transgênicos , Sequências de Repetição em Tandem , Testosterona/sangue , Testosterona/genética
6.
PLoS Comput Biol ; 16(5): e1007843, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32469863

RESUMO

Reconstructing haplotypes from sequencing data is one of the major challenges in genetics. Haplotypes play a crucial role in many analyses, including genome-wide association studies and population genetics. Haplotype reconstruction becomes more difficult for higher numbers of homologous chromosomes, as it is often the case for polyploid plants. This complexity is compounded further by higher heterozygosity, which denotes the frequent presence of variants between haplotypes. We have designed Ranbow, a new tool for haplotype reconstruction of polyploid genome from short read sequencing data. Ranbow integrates all types of small variants in bi- and multi-allelic sites to reconstruct haplotypes. To evaluate Ranbow and currently available competing methods on real data, we have created and released a real gold standard dataset from sweet potato sequencing data. Our evaluations on real and simulated data clearly show Ranbow's superior performance in terms of accuracy, haplotype length, memory usage, and running time. Specifically, Ranbow is one order of magnitude faster than the next best method. The efficiency and accuracy of Ranbow makes whole genome haplotype reconstruction of complex genome with higher ploidy feasible.


Assuntos
Haplótipos , Poliploidia , Algoritmos , Conjuntos de Dados como Assunto , Heterozigoto , Humanos
7.
Viruses ; 12(1)2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31936115

RESUMO

Virus replication displays a large cell-to-cell heterogeneity; yet, not all sources of this variability are known. Here, we study the effect of defective interfering (DI) particle (DIP) co-infection on cell-to-cell variability in influenza A virus (IAV) replication. DIPs contain a large internal deletion in one of their eight viral RNAs (vRNA) and are, thus, defective in virus replication. Moreover, they interfere with virus replication. Using single-cell isolation and reverse transcription polymerase chain reaction, we uncovered a large between-cell heterogeneity in the DI vRNA content of infected cells, which was confirmed for DI mRNAs by single-cell RNA sequencing. A high load of intracellular DI vRNAs and DI mRNAs was found in low-productive cells, indicating their contribution to the large cell-to-cell variability in virus release. Furthermore, we show that the magnitude of host cell mRNA expression (some factors may inhibit virus replication), but not the ribosome content, may further affect the strength of single-cell virus replication. Finally, we show that the load of viral mRNAs (facilitating viral protein production) and the DI mRNA content are, independently from one another, connected with single-cell virus production. Together, these insights advance single-cell virology research toward the elucidation of the complex multi-parametric origin of the large cell-to-cell heterogeneity in virus infections.


Assuntos
Vírus Defeituosos/genética , Variação Genética , Vírus da Influenza A/genética , RNA Viral/genética , Análise de Célula Única , Replicação Viral , Animais , Cães , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino
8.
Proc Natl Acad Sci U S A ; 116(25): 12390-12399, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31147463

RESUMO

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in the loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele, resulting in severe Shh loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.


Assuntos
Cromatina/metabolismo , Extremidades/embriologia , Proteínas Hedgehog/genética , Transcrição Gênica , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação para Baixo , Elementos Facilitadores Genéticos , Proteínas de Membrana/genética , Camundongos , Regiões Promotoras Genéticas , Coesinas
9.
Front Mol Neurosci ; 12: 60, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30914922

RESUMO

The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3- R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3- R356Q and CB3SH3- R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.

10.
Mol Psychiatry ; 24(11): 1748-1768, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-29728705

RESUMO

RLIM, also known as RNF12, is an X-linked E3 ubiquitin ligase acting as a negative regulator of LIM-domain containing transcription factors and participates in X-chromosome inactivation (XCI) in mice. We report the genetic and clinical findings of 84 individuals from nine unrelated families, eight of whom who have pathogenic variants in RLIM (RING finger LIM domain-interacting protein). A total of 40 affected males have X-linked intellectual disability (XLID) and variable behavioral anomalies with or without congenital malformations. In contrast, 44 heterozygous female carriers have normal cognition and behavior, but eight showed mild physical features. All RLIM variants identified are missense changes co-segregating with the phenotype and predicted to affect protein function. Eight of the nine altered amino acids are conserved and lie either within a domain essential for binding interacting proteins or in the C-terminal RING finger catalytic domain. In vitro experiments revealed that these amino acid changes in the RLIM RING finger impaired RLIM ubiquitin ligase activity. In vivo experiments in rlim mutant zebrafish showed that wild type RLIM rescued the zebrafish rlim phenotype, whereas the patient-specific missense RLIM variants failed to rescue the phenotype and thus represent likely severe loss-of-function mutations. In summary, we identified a spectrum of RLIM missense variants causing syndromic XLID and affecting the ubiquitin ligase activity of RLIM, suggesting that enzymatic activity of RLIM is required for normal development, cognition and behavior.


Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Transtorno da Conduta/genética , Feminino , Genes Ligados ao Cromossomo X , Células HEK293 , Humanos , Recém-Nascido , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Camundongos , Pessoa de Meia-Idade , Mutação , Linhagem , Fatores de Transcrição/genética , Ubiquitinação , Inativação do Cromossomo X , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
11.
Cancer Discov ; 8(5): 600-615, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29483136

RESUMO

Small cell lung cancer (SCLC) patient-derived xenografts (PDX) can be generated from biopsies or circulating tumor cells (CTC), though scarcity of tissue and low efficiency of tumor growth have previously limited these approaches. Applying an established clinical-translational pipeline for tissue collection and an automated microfluidic platform for CTC enrichment, we generated 17 biopsy-derived PDXs and 17 CTC-derived PDXs in a 2-year timeframe, at 89% and 38% efficiency, respectively. Whole-exome sequencing showed that somatic alterations are stably maintained between patient tumors and PDXs. Early-passage PDXs maintain the genomic and transcriptional profiles of the founder PDX. In vivo treatment with etoposide and platinum (EP) in 30 PDX models demonstrated greater sensitivity in PDXs from EP-naïve patients, and resistance to EP corresponded to increased expression of a MYC gene signature. Finally, serial CTC-derived PDXs generated from an individual patient at multiple time points accurately recapitulated the evolving drug sensitivities of that patient's disease. Collectively, this work highlights the translational potential of this strategy.Significance: Effective translational research utilizing SCLC PDX models requires both efficient generation of models from patients and fidelity of those models in representing patient tumor characteristics. We present approaches for efficient generation of PDXs from both biopsies and CTCs, and demonstrate that these models capture the mutational landscape and functional features of the donor tumors. Cancer Discov; 8(5); 600-15. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.


Assuntos
Genômica , Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Animais , Biópsia , Modelos Animais de Doenças , Genômica/métodos , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/terapia , Tomografia Computadorizada por Raios X , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Mol Neurobiol ; 55(5): 4240-4252, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28616718

RESUMO

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor and non-motor symptoms. The underlying pathology of non-motor symptoms is poorly understood. Discussed are pathological changes of extrastriatal brain structures. In this study, we characterized histopathological alterations of extrastriatal brain structures in the 6-hydroxydopamine (6-OHDA) PD animal model. Lesions were induced by unilateral stereotactic injections of 6-OHDA into the striatum or medial forebrain bundle of adult male mice. Loss of tyrosine hydroxylase positive (TH+) fibers as well as glia activation was quantified following stereological principles. Loss of dopaminergic innervation was further investigated by western-blotting. As expected, 6-OHDA injection into the nigrostriatal route induced retrograde degeneration of dopaminergic neurons within the substantia nigra pars compacta (SNpc), less so within the ventral tegmental area. Furthermore, we observed a region-specific drop of TH+ projection fiber density in distinct cortical regions. This pathology was most pronounced in the cingulate- and motor cortex, whereas the piriform cortex was just modestly affected. Loss of cortical TH+ fibers was not paralleled by microglia or astrocyte activation. Our results demonstrate that the loss of dopaminergic neurons within the substantia nigra pars compacta is paralleled by a cortical dopaminergic denervation in the 6-OHDA model. This model serves as a valuable tool to investigate mechanisms operant during cortical pathology in PD patients. Further studies are needed to understand why cortical dopaminergic innervation is lost in this model, and what functional consequence is associated with the observed denervation.


Assuntos
Corpo Estriado/patologia , Animais , Astrócitos/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Injeções , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologia , Degeneração Neural/patologia , Oxidopamina , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/patologia
13.
Brain ; 140(11): 2879-2894, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29053855

RESUMO

Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.


Assuntos
Encefalopatias/genética , Fissura Palatina/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Fácies , Deficiência Intelectual/genética , Nistagmo Patológico/genética , Receptores de GABA-A/genética , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Feminino , Variação Genética , Humanos , Masculino , Microcefalia/genética , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Técnicas de Patch-Clamp , Linhagem , Receptores de GABA-A/metabolismo , Síndrome , Xenopus laevis , Adulto Jovem , Ácido gama-Aminobutírico/metabolismo
14.
Nat Plants ; 3(9): 696-703, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28827752

RESUMO

Here we present the 15 pseudochromosomes of sweet potato, Ipomoea batatas, the seventh most important crop in the world and the fourth most significant in China. By using a novel haplotyping method based on genome assembly, we have produced a half haplotype-resolved genome from ~296 Gb of paired-end sequence reads amounting to roughly 67-fold coverage. By phylogenetic tree analysis of homologous chromosomes, it was possible to estimate the time of two recent whole-genome duplication events as occurring about 0.8 and 0.5 million years ago. This half haplotype-resolved hexaploid genome represents the first successful attempt to investigate the complexity of chromosome sequence composition directly in a polyploid genome, using sequencing of the polyploid organism itself rather than any of its simplified proxy relatives. Adaptation and application of our approach should provide higher resolution in future genomic structure investigations, especially for similarly complex genomes.


Assuntos
Cromossomos de Plantas/genética , Genoma de Planta/genética , Ipomoea batatas/genética , China , Produtos Agrícolas , Haplótipos , Filogenia , Poliploidia
15.
J Neurosci Methods ; 285: 49-57, 2017 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-28478074

RESUMO

BACKGROUND: The nigrostriatal pathway is of great importance for the execution of movements, especially in the context of Parkinson's disease. In research, analysis of this pathway often requires the application of severe animal experiments. Organotypic nigrostriatal slice cultures offer a resource-saving alternative to animal experiments for research on the nigrostriatal system. NEW METHOD: We have established a time-saving protocol for the preparation of murine sagittal nigrostriatal slice cultures by using a tissue chopper and agarose embedding instead of a vibratome. Furthermore, we developed the first murine co-culture model and the first co-culture utilising sagittal slices for modelling the nigrostriatal pathway. RESULTS: Sagittal nigrostriatal slice cultures show good overall tissue preservation and a high number of morphologically unimpaired dopaminergic neurons in the substantia nigra. Sagittal-frontal co-culture demonstrates massive outgrowth of dopaminergic fibres from the substantia nigra into co-cultured tissue. COMPARISON WITH EXISTING METHODS: The use of a tissue chopper instead of a vibratome allows notable time-saving during culture preparation, therefore allowing optimisation of the preparation time. Sagittal co-cultures offer the opportunity to study dopaminergic fibres in their physiological environment and in co-cultured tissue from a different animal in the same culture system. CONCLUSION: We here present a possibility to optimise the slice culture preparation process with the simple means of using a tissue chopper and fast agarose embedding. Furthermore, our sagittal-frontal co-culture system is suitable for the observation of dopaminergic outgrowth in both co-cultured tissues.


Assuntos
Técnicas de Cocultura , Técnicas In Vitro/métodos , Neurônios/metabolismo , Técnicas de Cultura de Órgãos , Animais , Animais Recém-Nascidos , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos/instrumentação , Técnicas de Cultura de Órgãos/métodos , Substância Negra/citologia , Substância Negra/fisiologia , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismo
16.
PLoS Genet ; 13(1): e1006567, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28103242

RESUMO

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes (HOXA9-13, and HOXD9-13) in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups (Group 1: HOXA/D9, HOXA/D10, HOXD12, and HOXA13 and Group 2: HOXA/D11 and HOXD13), which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Genoma , Proteínas de Homeodomínio/metabolismo , Proteínas Repressoras/metabolismo , Ativação Transcricional , Animais , Fator de Ligação a CCCTC , Galinhas , Condrogênese , Cromatina/metabolismo , Mesoderma/metabolismo , Ligação Proteica
17.
Hum Mutat ; 38(4): 409-425, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28055140

RESUMO

Impairment of translation initiation and its regulation within the integrated stress response (ISR) and related unfolded-protein response has been identified as a cause of several multisystemic syndromes. Here, we link MEHMO syndrome, whose genetic etiology was unknown, to this group of disorders. MEHMO is a rare X-linked syndrome characterized by profound intellectual disability, epilepsy, hypogonadism and hypogenitalism, microcephaly, and obesity. We have identified a C-terminal frameshift mutation (Ile465Serfs) in the EIF2S3 gene in three families with MEHMO syndrome and a novel maternally inherited missense EIF2S3 variant (c.324T>A; p.Ser108Arg) in another male patient with less severe clinical symptoms. The EIF2S3 gene encodes the γ subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ISR. Studies in patient fibroblasts confirm increased ISR activation due to the Ile465Serfs mutation and functional assays in yeast demonstrate that the Ile465Serfs mutation impairs eIF2γ function to a greater extent than tested missense mutations, consistent with the more severe clinical phenotype of the Ile465Serfs male mutation carriers. Thus, we propose that more severe EIF2S3 mutations cause the full MEHMO phenotype, while less deleterious mutations cause a milder form of the syndrome with only a subset of the symptoms.


Assuntos
Epilepsia , Fator de Iniciação 2 em Eucariotos/genética , Hipogonadismo , Deficiência Intelectual/genética , Deficiência Intelectual Ligada ao Cromossomo X/genética , Microcefalia , Mutação , Sequência de Aminoácidos , Saúde da Família , Feminino , Genitália/anormalidades , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Obesidade , Linhagem , Análise de Sequência de DNA/métodos , Homologia de Sequência de Aminoácidos , Síndrome
18.
Genome Res ; 27(2): 223-233, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27923844

RESUMO

Complex regulatory landscapes control the pleiotropic transcriptional activities of developmental genes. For most genes, the number, location, and dynamics of their associated regulatory elements are unknown. In this work, we characterized the three-dimensional chromatin microarchitecture and regulatory landscape of 446 limb-associated gene loci in mouse using Capture-C, ChIP-seq, and RNA-seq in forelimb, hindlimb at three developmental stages, and midbrain. The fine mapping of chromatin interactions revealed a strong preference for functional genomic regions such as repressed or active domains. By combining chromatin marks and interaction peaks, we annotated more than 1000 putative limb enhancers and their associated genes. Moreover, the analysis of chromatin interactions revealed two regimes of chromatin folding, one producing interactions stable across tissues and stages and another one associated with tissue and/or stage-specific interactions. Whereas stable interactions associate strongly with CTCF/RAD21 binding, the intensity of variable interactions correlates with changes in underlying chromatin modifications, specifically at the viewpoint and at the interaction site. In conclusion, this comprehensive data set provides a resource for the characterization of hundreds of limb-associated regulatory landscapes and a framework to interpret the chromatin folding dynamics observed during embryogenesis.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos , Fatores de Transcrição/genética , Ativação Transcricional/genética , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Histonas/genética , Camundongos , Regiões Promotoras Genéticas
19.
Nat Commun ; 7: 12681, 2016 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-27601181

RESUMO

Photoelectrochemical water splitting promises both sustainable energy generation and energy storage in the form of hydrogen. However, the realization of this vision requires laboratory experiments to be engineered into a large-scale technology. Up to now only few concepts for scalable devices have been proposed or realized. Here we introduce and realize a concept which, by design, is scalable to large areas and is compatible with multiple thin-film photovoltaic technologies. The scalability is achieved by continuous repetition of a base unit created by laser processing. The concept allows for independent optimization of photovoltaic and electrochemical part. We demonstrate a fully integrated, wireless device with stable and bias-free operation for 40 h. Furthermore, the concept is scaled to a device area of 64 cm(2) comprising 13 base units exhibiting a solar-to-hydrogen efficiency of 3.9%. The concept and its successful realization may be an important contribution towards the large-scale application of artificial photosynthesis.

20.
PLoS One ; 11(2): e0147904, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26828654

RESUMO

INTRODUCTION: Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing. METHODS: We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99th percentile and 176 lean adults (BMI ≤ 15th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults. RESULTS AND CONCLUSION: We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (pTDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.


Assuntos
Peso Corporal/genética , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Criança , Simulação por Computador , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Heterozigoto , Humanos , Desequilíbrio de Ligação/genética , Masculino , Obesidade/genética , Controle de Qualidade , Reprodutibilidade dos Testes , Magreza/genética
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