RESUMO
In Drosophila, a group of zinc finger architectural proteins recruits the CP190 protein to the chromatin, an interaction that is essential for the functional activity of promoters and insulators. In this study, we describe a new architectural C2H2 protein called Madf and Zinc-Finger Protein 1 (Mzfp1) that interacts with CP190. Mzfp1 has an unusual structure that includes six C2H2 domains organized in a C-terminal cluster and two tandem MADF domains. Mzfp1 predominantly binds to housekeeping gene promoters located in both euchromatin and heterochromatin genome regions. In vivo mutagenesis studies showed that Mzfp1 is an essential protein, and both MADF domains and the CP190 interaction region are required for its functional activity. The C2H2 cluster is sufficient for the specific binding of Mzfp1 to regulatory elements, while the second MADF domain is required for Mzfp1 recruitment to heterochromatin. Mzfp1 binds to the proximal part of the Fub boundary that separates regulatory domains of the Ubx and abd-A genes in the Bithorax complex. Mzfp1 participates in Fub functions in cooperation with the architectural proteins Pita and Su(Hw). Thus, Mzfp1 is a new architectural C2H2 protein involved in the organization of active promoters and insulators in Drosophila.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Elementos Isolantes , Proteínas Nucleares , Regiões Promotoras Genéticas , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Elementos Isolantes/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Genes Essenciais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ligação Proteica , Regulação da Expressão Gênica , Eucromatina/metabolismo , Eucromatina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas Associadas aos MicrotúbulosRESUMO
In arthropods, zinc finger-associated domains (ZADs) are found at the N-termini of many DNA-binding proteins with tandem arrays of Cys2-His2 zinc fingers (ZAD-C2H2 proteins). ZAD-C2H2 proteins undergo fast evolutionary lineage-specific expansion and functional diversification. Here, we show that all ZADs from Drosophila melanogaster form homodimers, but only certain ZADs with high homology can also heterodimerize. CG2712, for example, is unable to heterodimerize with its paralog, the previously characterized insulator protein Zw5, with which it shares 46% homology. We obtained a crystal structure of CG2712 protein's ZAD domain that, in spite of a low sequence homology, has similar spatial organization with the only known ZAD structure (from Grauzone protein). Steric clashes prevented the formation of heterodimers between Grauzone and CG2712 ZADs. Using detailed structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that rapid evolutionary acquisition of interaction specificity was mediated by the more energy-favorable formation of homodimers in comparison to heterodimers, and that this specificity was achieved by multiple amino acid substitutions resulting in the formation or breaking of stabilizing interactions. We speculate that specific homodimerization of ZAD-C2H2 proteins is important for their architectural role in genome organization.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Dedos de Zinco , Animais , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Dimerização , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster , Modelos Moleculares , Mutagênese , Multimerização Proteica , Fatores de Transcrição/químicaRESUMO
The binding of the Drosophila male-specific lethal dosage compensation complex (DCC) exclusively to the male X chromosome provides an excellent model system to understand mechanisms of selective recruitment of protein complexes to chromatin. Previous studies showed that the male-specific organizer of the complex, MSL2, and the ubiquitous DNA-binding protein CLAMP are key players in the specificity of X chromosome binding. The CXC domain of MSL2 binds to genomic sites of DCC recruitment in vitro Another conserved domain of MSL2, named Clamp-binding domain (CBD) directly interacts with the N-terminal zinc-finger domain of CLAMP. Here, we found that inactivation of CBD or CXC individually only modestly affected recruitment of the DCC to the X chromosome in males. However, combination of these two genetic lesions within the same MSL2 mutant resulted in an increased loss of DCC recruitment to the X chromosome. Thus, proper MSL2 positioning requires an interaction with either CLAMP or DNA to initiate dosage compensation in Drosophila males.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Feminino , Masculino , Modelos Genéticos , Mutação/genética , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Cromossomo X/genéticaRESUMO
All-dielectric optical metasurfaces are a workhorse in nano-optics, because of both their ability to manipulate light in different degrees of freedom and their excellent performance at light frequency conversion. Here, we demonstrate first-time generation of photon pairs via spontaneous parametric-down conversion in lithium niobate quantum optical metasurfaces with electric and magnetic Mie-like resonances at various wavelengths. By engineering the quantum optical metasurface, we tailor the photon-pair spectrum in a controlled way. Within a narrow bandwidth around the resonance, the rate of pair production is enhanced up to 2 orders of magnitude, compared to an unpatterned film of the same thickness and material. These results enable flat-optics sources of entangled photons-a new promising platform for quantum optics experiments.
RESUMO
The basidiomycete Schizophyllum commune has the highest level of genetic polymorphism known among living organisms. In a previous study, it was also found to have a moderately high per-generation mutation rate of 2×10-8, likely contributing to its high polymorphism. However, this rate has been measured only in an experiment on Petri dishes, and it is unclear how it translates to natural populations. Here, we used an experimental design that measures the rate of accumulation of de novo mutations in a linearly growing mycelium. We show that S. commune accumulates mutations at a rate of 1.24×10-7 substitutions per nucleotide per meter of growth, or â¼2.04×10-11 per nucleotide per cell division. In contrast to what has been observed in a number of species with extensive vegetative growth, this rate does not decline in the course of propagation of a mycelium. As a result, even a moderate per-cell-division mutation rate in S. commune can translate into a very high per-generation mutation rate when the number of cell divisions between consecutive meiosis is large.
Assuntos
Taxa de Mutação , Schizophyllum/genética , Acúmulo de Mutações , Micorrizas/genética , Micorrizas/crescimento & desenvolvimento , Polimorfismo Genético , Schizophyllum/crescimento & desenvolvimentoRESUMO
Lithium niobate is an excellent and widely used material for nonlinear frequency conversion due to its strong optical nonlinearity and broad transparency region. Here, we report the fabrication and experimental investigation of resonant nonlinear metasurfaces for second-harmonic generation based on thin-film lithium niobate. In the fabricated metasurfaces, we observe pronounced Mie-type resonances leading to enhanced second-harmonic generation in the direction normal to the metasurface. We find the largest second-harmonic generation efficiency for the resonance dominated by the electric contributions because its specific field distribution enables the most efficient usage of the largest element of the lithium niobate nonlinear susceptibility tensor. This is confirmed by polarization-resolved second-harmonic measurements, where we study contributions from different elements of the nonlinear susceptibility tensor to the total second-harmonic signal. Our work facilitates establishing lithium niobate as a material for resonant nanophotonics.
RESUMO
Polyploidization and subsequent sub- and neofunctionalization of duplicated genes represent a major mechanism of plant genome evolution. Capsella bursa-pastoris, a widespread ruderal plant, is a recent allotetraploid and, thus, is an ideal model organism for studying early changes following polyploidization. We constructed a high-quality assembly of C. bursa-pastoris genome and a transcriptome atlas covering a broad sample of organs and developmental stages (available online at http://travadb.org/browse/Species=Cbp). We demonstrate that expression of homeologs is mostly symmetric between subgenomes, and identify a set of homeolog pairs with discordant expression. Comparison of promoters within such pairs revealed emerging asymmetry of regulatory elements. Among them there are multiple binding sites for transcription factors controlling the regulation of photosynthesis and plant development by light (PIF3, HY5) and cold stress response (CBF). These results suggest that polyploidization in C. bursa-pastoris enhanced its plasticity of response to light and temperature, and allowed substantial expansion of its distribution range.
Assuntos
Capsella/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Poliploidia , Sequências Reguladoras de Ácido Nucleico , Anotação de Sequência MolecularRESUMO
Insulators are multiprotein-DNA complexes that regulate the nuclear architecture. The Drosophila CP190 protein is a cofactor for the DNA-binding insulator proteins Su(Hw), CTCF, and BEAF-32. The fact that CP190 has been found at genomic sites devoid of either of the known insulator factors has until now been unexplained. We have identified two DNA-binding zinc-finger proteins, Pita, and a new factor named ZIPIC, that interact with CP190 in vivo and in vitro at specific interaction domains. Genomic binding sites for these proteins are clustered with CP190 as well as with CTCF and BEAF-32. Model binding sites for Pita or ZIPIC demonstrate a partial enhancer-blocking activity and protect gene expression from PRE-mediated silencing. The function of the CTCF-bound MCP insulator sequence requires binding of Pita. These results identify two new insulator proteins and emphasize the unifying function of CP190, which can be recruited by many DNA-binding insulator proteins.
Assuntos
Cromatina/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Sítios de Ligação , Cromatina/metabolismo , Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/genética , Expressão Gênica , Estudos de Associação Genética , Genômica , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
According to recent models, as yet poorly studied architectural proteins appear to be required for local regulation of enhancer-promoter interactions, as well as for global chromosome organization. Transcription factors ZIPIC, Pita and Zw5 belong to the class of chromatin insulator proteins and preferentially bind to promoters near the TSS and extensively colocalize with cohesin and condensin complexes. ZIPIC, Pita and Zw5 are structurally similar in containing the N-terminal zinc finger-associated domain (ZAD) and different numbers of C2H2-type zinc fingers at the C-terminus. Here we have shown that the ZAD domains of ZIPIC, Pita and Zw5 form homodimers. In Drosophila transgenic lines, these proteins are able to support long-distance interaction between GAL4 activator and the reporter gene promoter. However, no functional interaction between binding sites for different proteins has been revealed, suggesting that such interactions are highly specific. ZIPIC facilitates long-distance stimulation of the reporter gene by GAL4 activator in yeast model system. Many of the genomic binding sites of ZIPIC, Pita and Zw5 are located at the boundaries of topologically associated domains (TADs). Thus, ZAD-containing zinc-finger proteins can be attributed to the class of architectural proteins.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Multimerização Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Feminino , Genes Reporter/genética , Masculino , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Fatores de Transcrição/genética , Transgenes/genética , Dedos de ZincoRESUMO
The objective of the current review is to summarize the current state of optical methods in redox biology. It consists of two parts, the first is dedicated to genetically encoded fluorescent indicators and the second to Raman spectroscopy. In the first part, we provide a detailed classification of the currently available redox biosensors based on their target analytes. We thoroughly discuss the main architecture types of these proteins, the underlying engineering strategies for their development, the biochemical properties of existing tools and their advantages and disadvantages from a practical point of view. Particular attention is paid to fluorescence lifetime imaging microscopy as a possible readout technique, since it is less prone to certain artifacts than traditional intensiometric measurements. In the second part, the characteristic Raman peaks of the most important redox intermediates are listed, and examples of how this knowledge can be implemented in biological studies are given. This part covers such fields as estimation of the redox states and concentrations of Fe-S clusters, cytochromes, other heme-containing proteins, oxidative derivatives of thiols, lipids, and nucleotides. Finally, we touch on the issue of multiparameter imaging, in which biosensors are combined with other visualization methods for simultaneous assessment of several cellular parameters.
Assuntos
Técnicas Biossensoriais , Análise Espectral Raman , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Técnicas Biossensoriais/métodos , Oxirredução , BiologiaRESUMO
Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.
Assuntos
Cromatina , Proteínas de Drosophila , Interfase , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , Cromatina/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/embriologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
Locomotion triggers a coordinated response of both neurons and astrocytes in the brain. Here we performed calcium (Ca2+) imaging of these two cell types in the somatosensory cortex in head-fixed mice moving on the airlifted platform. Ca2+ activity in astrocytes significantly increased during locomotion from a low quiescence level. Ca2+ signals first appeared in the distal processes and then propagated to astrocytic somata, where it became significantly larger and exhibited oscillatory behaviour. Thus, astrocytic soma operates as both integrator and amplifier of Ca2+ signal. In neurons, Ca2+ activity was pronounced in quiescent periods and further increased during locomotion. Neuronal Ca2+ concentration ([Ca2+]i) rose almost immediately following the onset of locomotion, whereas astrocytic Ca2+ signals lagged by several seconds. Such a long lag suggests that astrocytic [Ca2+]i elevations are unlikely to be triggered by the activity of synapses among local neurons. Ca2+ responses to pairs of consecutive episodes of locomotion did not significantly differ in neurons, while were significantly diminished in response to the second locomotion in astrocytes. Such astrocytic refractoriness may arise from distinct mechanisms underlying Ca2+ signal generation. In neurons, the bulk of Ca2+ enters through the Ca2+ channels in the plasma membrane allowing for steady-level Ca2+ elevations in repetitive runs. Astrocytic Ca2+ responses originate from the intracellular stores, the depletion of which affects subsequent Ca2+ signals. Functionally, neuronal Ca2+ response reflects sensory input processed by neurons. Astrocytic Ca2+ dynamics is likely to provide metabolic and homeostatic support within the brain active milieu.
Assuntos
Astrócitos , Cálcio , Camundongos , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Neurônios/metabolismo , Cálcio da Dieta/metabolismoRESUMO
Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of H2O2 in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of H2O2 in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.
Assuntos
Isquemia Encefálica , Diabetes Mellitus , Hiperglicemia , AVC Isquêmico , Acidente Vascular Cerebral , Ratos , Camundongos , Animais , Peróxido de Hidrogênio , Acidente Vascular Cerebral/patologia , Hiperglicemia/patologia , Isquemia Encefálica/patologiaRESUMO
It is natural to assume that patterns of genetic variation in hyperpolymorphic species can reveal large-scale properties of the fitness landscape that are hard to detect by studying species with ordinary levels of genetic variation. Here, we study such patterns in a fungus Schizophyllum commune, the most polymorphic species known. Throughout the genome, short-range linkage disequilibrium (LD) caused by attraction of minor alleles is higher between pairs of nonsynonymous than of synonymous variants. This effect is especially pronounced for pairs of sites that are located within the same gene, especially if a large fraction of the gene is covered by haploblocks, genome segments where the gene pool consists of two highly divergent haplotypes, which is a signature of balancing selection. Haploblocks are usually shorter than 1000 nucleotides, and collectively cover about 10% of the S. commune genome. LD tends to be substantially higher for pairs of nonsynonymous variants encoding amino acids that interact within the protein. There is a substantial correlation between LDs at the same pairs of nonsynonymous mutations in the USA and the Russian populations. These patterns indicate that selection in S. commune involves positive epistasis due to compensatory interactions between nonsynonymous alleles. When less polymorphic species are studied, analogous patterns can be detected only through interspecific comparisons.
Changes to DNA known as mutations may alter how the proteins and other components of a cell work, and thus play an important role in allowing living things to evolve new traits and abilities over many generations. Whether a mutation is beneficial or harmful may differ depending on the genetic background of the individual that is, depending on other mutations present in other positions within the same gene due to a phenomenon called epistasis. Epistasis is known to affect how various species accumulate differences in their DNA compared to each other over time. For example, a mutation that is rare in humans and known to cause disease may be widespread in other primates because its negative effect is canceled out by another mutation that is standard for these species but absent in humans. However, it remains unclear whether epistasis plays a significant part in shaping genetic differences between individuals of the same species. A type of fungus known as Schizophyllum commune lives on rotting wood and is found across the world. It is one of the most genetically diverse species currently known, so there is a higher chance of pairs of compensatory mutations occurring and persisting for a long time in S. commune than in most other species, providing a unique opportunity to study epistasis. Here, Stolyarova et al. studied two distinct populations of S. commune, one from the USA and one from Russia. The team found that unlike in humans, flies and other less genetically diverse species epistasis maintains combinations of mutations in S. commune that individually would be harmful to the fungus but together compensate for each other. For example, pairs of mutations affecting specific molecules known as amino acids the building blocks of proteins that physically interact with each other tended to be found together in the same individuals. One potential downside of having pairs of compensatory mutations in the genome is that when the organism reproduces, the process of making sex cells may split up these pairs so that harmful mutations are inherited without their partner mutations. Thus, epistasis may have helped shape the way S. commune and other genetically diverse species have evolved.
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Epistasia Genética , Aptidão Genética , Alelos , Haplótipos , Desequilíbrio de Ligação , Mutação , Federação RussaRESUMO
AIM: A high-fat diet (HFD) is generally considered to negatively influence the body, the brain, and cognition. Nonetheless, fat and fatty acids are essential for nourishing and constructing brain tissue. Astrocytes are central for lipolysis and fatty acids metabolism. We tested how HFD affects astrocyte metabolism, morphology, and physiology. METHODS: We used Raman microspectroscopy to assess the redox state of mitochondria and lipid content in astrocytes and neurons in hippocampal slices of mice subjected to HFD. Astrocytes were loaded with fluorescent dye through patch pipette for morphological analysis. Whole-cell voltage-clamp recordings were performed to measure transporter and potassium currents. Western blot analysis quantified the expression of astrocyte-specific proteins. Field potential recordings measured the magnitude of long-term potentiation (LTP). Open filed test was performed to evaluate the effect of HFD on animal behavior. RESULTS: We found that exposure of young mice to 1 month of HFD increases lipid content and relative amount of reduced cytochromes in astrocytes but not in neurons. Metabolic changes were paralleled with an enlargement of astrocytic territorial domains due to an increased outgrowth of branches and leaflets. Astrocyte remodeling was associated with an increase in expression of ezrin and with no changes in glial fibrillary acidic protein (GFAP), glutamate transporter-1 (GLT-1), and glutamine synthetase (GS). Such physiological (non-reactive) enlargement of astrocytes in the brain active milieu promoted glutamate clearance and LTP and translated into behavioral changes. CONCLUSION: Dietary fat intake is not invariably harmful and might exert beneficial effects depending on the biological context.
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Astrócitos , Dieta Hiperlipídica , Animais , Astrócitos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Lipídeos , Camundongos , Plasticidade NeuronalRESUMO
High-throughput sequencing of fetal DNA is a promising and increasingly common method for the discovery of all (or all coding) genetic variants in the fetus, either as part of prenatal screening or diagnosis, or for genetic diagnosis of spontaneous abortions. In many cases, the fetal DNA (from chorionic villi, amniotic fluid, or abortive tissue) can be contaminated with maternal cells, resulting in the mixture of fetal and maternal DNA. This maternal cell contamination (MCC) undermines the assumption, made by traditional variant callers, that each allele in a heterozygous site is covered, on average, by 50% of the reads, and therefore can lead to erroneous genotype calls. We present a panel of methods for reducing the genotyping error in the presence of MCC. All methods start with the output of GATK HaplotypeCaller on the sequencing data for the (contaminated) fetal sample and both of its parents, and additionally rely on information about the MCC fraction (which itself is readily estimated from the high-throughput sequencing data). The first of these methods uses a Bayesian probabilistic model to correct the fetal genotype calls produced by MCC-unaware HaplotypeCaller. The other two methods "learn" the genotype-correction model from examples. We use simulated contaminated fetal data to train and test the models. Using the test sets, we show that all three methods lead to substantially improved accuracy when compared with the original MCC-unaware HaplotypeCaller calls. We then apply the best-performing method to three chorionic villus samples from spontaneously terminated pregnancies.
Assuntos
Amostra da Vilosidade Coriônica/métodos , Contaminação por DNA , Testes Genéticos/métodos , Análise de Sequência de DNA/métodos , Adulto , Teorema de Bayes , Amostra da Vilosidade Coriônica/normas , Feminino , Testes Genéticos/normas , Humanos , Aprendizado de Máquina , Mutação , Gravidez , Análise de Sequência de DNA/normas , Razão Sinal-RuídoRESUMO
BACKGROUND: Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. RESULTS: Here, we have used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7. CONCLUSIONS: The subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.
Assuntos
Proteínas Correpressoras/química , Proteínas de Drosophila/química , Multimerização Proteica , Animais , Sítios de Ligação , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos Isolantes , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Intramedullary nailing is considered a "gold standard" for treatment of tibial shaft fractures. However, some types of fractures are typically considered as "difficult for nailing". This group includes the periarticular fractures, fractures of both bones at the same level, comminuted and segmental fractures of the tibia. Fixator-assisted nailing (FAN) is an effective method treatment of these types of fractures. The main requirements for the ideal reduction device are an ease of its installation and an ability of multiplanar fracture reduction. Fixator-assisted nailing (FAN) with the use of two perpendicular to each other monolateral tubular frames perfectly meets these requirements. In this study we present this new surgical technique and the analysis of first 30 cases. METHODS: A prospective analysis was conducted for 30 patients with "difficult for nailing" tibial fractures treated with fixator-assisted nailing in our institution between September 1st, 2017, and March 1st, 2018. The duration of surgery and its different stages, the time of fluoroscopy, difficulties encountered during surgery, were analyzed. Clinical and radiological methods were used to evaluated reduction quality. RESULTS: In all 30 cases the acceptable reduction was achieved. The mean duration of the surgical procedure was 73.7 ± 3 min. The mean duration of fluoroscopy 85.9 ± 4.8 s. In 7 cases we faced with technical difficulties, which were successfully addressed. CONCLUSION: The described technique of FAN is an effective method for the treatment of "difficult for nailing" tibial fractures. Future multi-centered studies with a larger number of patients are needed to validate our results.
Assuntos
Fixação Intramedular de Fraturas/métodos , Consolidação da Fratura/fisiologia , Fraturas Intra-Articulares/cirurgia , Fraturas da Tíbia/cirurgia , Adulto , Idoso , Feminino , Fluoroscopia , Fixação Intramedular de Fraturas/instrumentação , Fixação Intramedular de Fraturas/tendências , Humanos , Fraturas Intra-Articulares/diagnóstico por imagem , Fraturas Intra-Articulares/fisiopatologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/fisiopatologia , Resultado do TratamentoRESUMO
Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.