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The development of Fe-based catalysts for the selective catalytic reduction of NOx by NH3 (NH3-SCR of NOx) has garnered significant attention due to their exceptional SO2 resistance. However, the influence of different sulfur-containing species (e.g., ferric sulfates and ammonium sulfates) on the NH3-SCR activity of Fe-based catalysts as well as its dependence on exposed crystal facets of Fe2O3 has not been revealed. This work disclosed that nanorod-like α-Fe2O3 (Fe2O3-NR) predominantly exposing (110) facet performed better than nanosheet-like α-Fe2O3 (Fe2O3-NS) predominantly exposing (001) facet in NH3-SCR reaction, due to the advantages of Fe2O3-NR in redox properties and surface acidity. Furthermore, the results of the SO2/H2O resistance test at a critical temperature of 250 °C, catalytic performance evaluations on Fe2O3-NR and Fe2O3-NS sulfated by SO2 + O2 or deposited with NH4HSO4 (ABS), and systematic characterization revealed that the reactivity of ammonium sulfates on Fe2O3 catalysts to NO(+O2) contributed to their improved catalytic performance, while ferric sulfates showed enhancing and inhibiting effects on NH3-SCR activity on Fe2O3-NR and Fe2O3-NS, respectively; despite this, Fe2O3-NR showed higher affinity for SO2 + O2. This work set a milestone in understanding the NH3-SCR reaction on Fe2O3 catalysts in the presence of SO2 from the aspect of crystal facet engineering.
Assuntos
Amônia , Catálise , Amônia/química , Dióxido de Enxofre/química , Compostos Férricos/química , OxirreduçãoRESUMO
Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling plays a crucial role in anterior-posterior (A-P) axial patterning of vertebrate embryos by promoting posterior development. In our screens for novel developmental regulators in Xenopus embryos, we identified Fam3b as a secreted factor regulated in ectodermal explants. Family with sequence similarity 3 member B (FAM3B)/PANDER (pancreatic-derived factor) is a cytokine involved in glucose metabolism, type 2 diabetes, and cancer in mammals. However, the molecular mechanism of FAM3B action in these processes remains poorly understood, largely because its receptor is still unidentified. Here we uncover an unexpected role of FAM3B acting as a FGF receptor (FGFR) ligand in Xenopus embryos. fam3b messenger RNA (mRNA) is initially expressed maternally and uniformly in the early Xenopus embryo and then in the epidermis at neurula stages. Overexpression of Xenopus fam3b mRNA inhibited cephalic structures and induced ectopic tail-like structures. Recombinant human FAM3B protein was purified readily from transfected tissue culture cells and, when injected into the blastocoele cavity, also caused outgrowth of tail-like structures at the expense of anterior structures, indicating FGF-like activity. Depletion of fam3b by specific antisense morpholino oligonucleotides in Xenopus resulted in macrocephaly in tailbud tadpoles, rescuable by FAM3B protein. Mechanistically, FAM3B protein bound to FGFR and activated the downstream ERK signaling in an FGFR-dependent manner. In Xenopus embryos, FGFR activity was required epistatically downstream of Fam3b to mediate its promotion of posterior cell fates. Our findings define a FAM3B/FGFR/ERK-signaling pathway that is required for axial patterning in Xenopus embryos and may provide molecular insights into FAM3B-associated human diseases.
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Citocinas/fisiologia , Desenvolvimento Embrionário/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Xenopus/fisiologia , Xenopus laevis/embriologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , HumanosRESUMO
Orofacial clefts (OFCs) are the most common congenital craniofacial disorders and cause serious problems with the appearance, orofacial function and mental health of the patients. The fibroblast growth factor (FGF) signaling pathway is critical for several aspects of craniofacial development and loss-of-function mutations of coding genes for multiple FGFs and FGFRs can lead to OFCs. We recently characterized FAM3B as a novel ligand of FGF signaling, which, through binding to FGFRs and activating downstream ERK, regulates craniofacial development in Xenopus. In this study, we identify two rare variants in FAM3B (p.Q61R and p.D128G) via target region sequencing of FAM3B on 144 unrelated sporadic patients with non-syndromic OFCs (NSOFCs). Bioinformatic analysis predict that these two variants are likely to be damaging and biochemical experiments show that these two variants weaken the FGF ligand activity of FAM3B by decreasing its expression and thus secretion. In summary, our results indicate that FAM3B is a novel candidate gene for NSOFCs in humans.
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Fenda Labial , Fissura Palatina , Humanos , Fenda Labial/genética , Fissura Palatina/genética , Ligantes , Mutação , Fatores de Crescimento de Fibroblastos/genética , Proteínas de Neoplasias/genética , Citocinas/genéticaRESUMO
The Spemann and Mangold Organizer (SMO) is of fundamental importance for dorsal ventral body axis formation during vertebrate embryogenesis. Maternal Huluwa (Hwa) has been identified as the dorsal determinant that is both necessary and sufficient for SMO formation. However, it remains unclear how Hwa is regulated. Here, we report that the E3 ubiquitin ligase zinc and ring finger 3 (ZNRF3) is essential for restricting the spatial activity of Hwa and therefore correct SMO formation in Xenopus laevis. ZNRF3 interacts with and ubiquitinates Hwa, thereby regulating its lysosomal trafficking and protein stability. Perturbation of ZNRF3 leads to the accumulation of Hwa and induction of an ectopic axis in embryos. Ectopic expression of ZNRF3 promotes Hwa degradation and dampens the axis-inducing activity of Hwa. Thus, our findings identify a substrate of ZNRF3, but also highlight the importance of the regulation of Hwa temporospatial activity in body axis formation in vertebrate embryos.
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Organizadores Embrionários , Ubiquitina-Proteína Ligases , Animais , Padronização Corporal , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Lisossomos/metabolismo , Organizadores Embrionários/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
SARS-CoV-2 has infected over 128 million people worldwide, and until a vaccine is developed and widely disseminated, vigilant testing and contact tracing are the most effective ways to slow the spread of COVID-19. Typical clinical testing only confirms the presence or absence of the virus, but rather, a simple and rapid testing procedure that sequences the entire genome would be impactful and allow for tracing the spread of the virus and variants, as well as the appearance of new variants. However, traditional short read sequencing methods are time consuming and expensive. Herein, we describe a tiled genome array that we developed for rapid and inexpensive full viral genome resequencing, and we have applied our SARS-CoV-2-specific genome tiling array to rapidly and accurately resequence the viral genome from eight clinical samples. We have resequenced eight samples acquired from patients in Wyoming that tested positive for SARS-CoV-2. We were ultimately able to sequence over 95% of the genome of each sample with greater than 99.9% average accuracy.
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COVID-19 , SARS-CoV-2 , Genoma Viral , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Neurog2 is a crucial regulator of neuronal fate specification and differentiation in vivo and in vitro However, it remains unclear how Neurog2 transactivates neuronal genes that are silenced by repressive chromatin. Here, we provide evidence that the histone H3 lysine 9 demethylase KDM3A facilitates the Xenopus Neurog2 (formerly known as Xngnr1) chromatin accessibility during neuronal transcription. Loss-of-function analyses reveal that KDM3A is not required for the transition of naive ectoderm to neural progenitor cells but is essential for primary neuron formation. ChIP series followed by qPCR analyses reveal that Neurog2 promotes the removal of the repressive H3K9me2 marks and addition of active histone marks, including H3K27ac and H3K4me3, at the NeuroD1 and Tubb2b promoters; this activity depends on the presence of KDM3A because Neurog2, via its C-terminal domain, interacts with KDM3A. Interestingly, KDM3A is dispensable for the neuronal transcription initiated by Ascl1, a proneural factor related to neurogenin in the bHLH family. In summary, our findings uncover a crucial role for histone H3K9 demethylation during Neurog2-mediated neuronal transcription and help in the understanding of the different activities of Neurog2 and Ascl1 in initiating neuronal development.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Ectoderma/metabolismo , Feminino , Lisina/química , Neurogênese , Neurônios/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ativação Transcricional , Xenopus laevisRESUMO
Maternally expressed proteins function in vertebrates to establish the major body axes of the embryo and to establish a pre-pattern that sets the stage for later-acting zygotic signals. This pre-patterning drives the propensity of Xenopus animal cap cells to adopt neural fates under various experimental conditions. Previous studies found that the maternally expressed transcription factor, encoded by the Xenopus achaete scute-like gene ascl1, is enriched at the animal pole. Asc1l is a bHLH protein involved in neural development, but its maternal function has not been studied. Here, we performed a series of gain- and loss-of-function experiments on maternal ascl1, and present three novel findings. First, Ascl1 is a repressor of mesendoderm induced by VegT, but not of Nodal-induced mesendoderm. Second, a previously uncharacterized N-terminal domain of Ascl1 interacts with HDAC1 to inhibit mesendoderm gene expression. This N-terminal domain is dispensable for its neurogenic function, indicating that Ascl1 acts by different mechanisms at different times. Ascl1-mediated repression of mesendoderm genes was dependent on HDAC activity and accompanied by histone deacetylation in the promoter regions of VegT targets. Finally, maternal Ascl1 is required for animal cap cells to retain their competence to adopt neural fates. These results establish maternal Asc1l as a key factor in establishing pre-patterning of the early embryo, acting in opposition to VegT and biasing the animal pole to adopt neural fates. The data presented here significantly extend our understanding of early embryonic pattern formation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desacetilases/metabolismo , Mesoderma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ectoderma/efeitos dos fármacos , Ectoderma/embriologia , Ectoderma/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Endoderma/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Mesoderma/efeitos dos fármacos , Morfolinos/farmacologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Estrutura Terciária de Proteína , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Ascl1 is a multi-functional regulator of neural development in invertebrates and vertebrates. Ectopic expression of Ascl1 can generate functional neurons from non-neural somatic cells. The abnormal expression of ASCL1 has been reported in several types of carcinomas. We have previously identified Ascl1 as a crucial maternal regulator of the germ layer pattern formation in Xenopus Functional studies have indicated that the maternally-supplied Ascl1 renders embryonic cells a propensity to adopt neural fates on one hand, and represses the mesendoderm formation on the other. However, it remains unclear how Ascl1 achieves its repressor function during the activation of mesendoderm genes by VegT. Here, we performed series of gain- and loss-of-function experiments and found that: (i) VegT, the maternal mesendoderm determinant in Xenopus, is required for the deposition of H3K27ac and H3K9ac at its target gene loci during mesendoderm induction; (ii) Ascl1 and VegT antagonistically modulate the deposition of acetylated histone marks at mesendoderm gene loci; (iii) Ascl1 overexpression reduces the VegT-occupancy at mesendoderm gene loci; (iv) Ascl1 but not Neurog2 possesses a repressive activity during mesendoderm induction. These findings reveal a novel repressive function for Ascl1 in inhibiting non-neural fates during early Xenopus embryogenesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Mesoderma/citologia , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus/embriologia , Acetilação , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Xenopus/genéticaRESUMO
We demonstrate experimentally a chemical codoping approach that would simultaneously narrow the band gap and control the band edge positions of TiO2 semiconductors. It is shown that a sequential doping scheme with nitrogen (N) leading the way, followed by phosphorus (P), is crucial for the incorporation of both N and P into the anion sites. Various characterization techniques confirm the formation of the N-P bonds, and as a consequence of chemical codoping, the band gap of TiO2 is reduced from 3.2 eV to 1.8 eV. The realization of chemical codoping could be an important step forward in improving the general performance of electronic and optoelectronic materials and devices.
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The biosynthetic pathway of actinorhodin in Streptomyces coelicolor A3(2) has been studied for decades as a model system of type II polyketide biosynthesis. The actinorhodin biosynthetic gene cluster includes a gene, actVI-orfA, that encodes a protein that belongs to the nuclear transport factor-2-like (NTF-2-like) superfamily. The function of this ActVI-ORFA protein has been a long-standing question in this field. Several hypothetical functions, including pyran ring cyclase, enzyme complex stability enhancer, and gene transcription regulator, have been proposed for ActVI-ORFA in previous studies. However, although the recent structural analysis of ActVI-ORFA revealed a solvent-accessible cavity, the protein displayed structural differences to the well-characterized cyclase SnoaL and did not possess a DNA-binding domain. The obtained crystal structure facilitates an inspection of the previous hypotheses regarding the function of ActVI-ORFA. In the present study, we investigated the effects of a series of actVI-orfA test plasmids with different mutations in an established vector/host system. Time-course analysis of dynamic metabolism profiles demonstrated that ActVI-ORFA prevented formation of shunt metabolites and may have a metabolic flux directing function, which shepherds the flux of unstable intermediates towards actinorhodin. The expression studies resulted in the isolation and structure elucidation of two new shunt metabolites from the actinorhodin pathway. Next, we utilized computational modeling to probe the active site of ActVI-ORFA and confirmed the importance of residues R76 and H78 in the flux directing functionality by expression studies. This is the first time such a function has been observed for a member of NTF-2-like superfamily in Streptomyces secondary metabolism.
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Antraquinonas , Proteínas de Bactérias , Streptomyces coelicolor , Antraquinonas/metabolismo , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Família Multigênica , Mutação , BenzoisocromanequinonasRESUMO
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and ß-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E(S)] = 1.7 to R enantioselectivity [E(R)] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E(S) > 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.
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Arthrobacter/enzimologia , Hidrolases/genética , Hidrolases/metabolismo , Substituição de Aminoácidos , Arthrobacter/genética , Domínio Catalítico , Evolução Molecular Direcionada , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Calcium leaching using additives is the most critical step in the indirect aqueous carbonation process of CaSO4·2H2O. However, recovery of the soluble additives from the sulfate-rich carbonation filtrate limits the large-scale industrial implementation of current carbonation technologies. To address this issue, we employed aspartic acid (Asp) as a leaching additive. The dissolution capability of CaSO4·2H2O in aqueous ammonia was found to improve significantly owing to the complexation effect between Asp and the Ca2+ ions. The maximum amount of dissolved CaSO4·2H2O was determined according to the competitive relationship between the complexing effect and the inhibitory effect of free ammonia molecules on the dissociation of CaSO4·2H2O, and the solution pH influences such competition. The precipitation of CaCO3 was examined by monitoring the variations in the pH and conductivity of the carbonation reaction system. As a result, the shift in the Asp dissociation equilibrium extended the induction period, and the growth period was divided into three stages according to the relative difference between the consumption and formation rates of CO3 2-. Moreover, it was determined that the carbonation products consisted of stable spherical vaterite particles. The recovery of Asp was also demonstrated at its isoelectric point, with a recovery efficiency of >80% being achieved, and recycling experiments confirmed the stability of the recycled Asp. Finally, the amount of dissolved CaSO4·2H2O and the total carbonation efficiency during cycling were determined as 16.3 ± 0.4 g L-1 and 46.5 ± 1.9%, respectively.
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Despite the fundamental roles of TGF-ß family signaling in cell fate determination in all metazoans, the mechanism by which these signals are spatially and temporally interpreted remains elusive. The cell-context-dependent function of TGF-ß signaling largely relies on transcriptional regulation by SMAD proteins. Here, we discover that the DNA repair-related protein, HMCES, contributes to early development by maintaining nodal/activin- or BMP-signaling-regulated transcriptional network. HMCES binds with R-SMAD proteins, co-localizing at active histone marks. However, HMCES chromatin occupancy is independent on nodal/activin or BMP signaling. Mechanistically, HMCES competitively binds chromatin to limit binding by R-SMAD proteins, thereby forcing their dissociation and resulting in repression of their regulatory effects. In Xenopus laevis embryo, hmces KD causes dramatic development defects with abnormal left-right axis asymmetry along with increasing expression of lefty1. These findings reveal HMCES transcriptional regulatory function in the context of TGF-ß family signaling.
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Ativinas , Proteínas Morfogenéticas Ósseas , Ativinas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Smad Reguladas por Receptor/genética , Proteínas Smad Reguladas por Receptor/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
INTRODUCTION The nuclear pore complex (NPC) resides on the nuclear envelope (NE) and mediates nucleocytoplasmic cargo transport. As one of the largest cellular machineries, a vertebrate NPC consists of cytoplasmic filaments, a cytoplasmic ring (CR), an inner ring, a nuclear ring, a nuclear basket, and a luminal ring. Each NPC has eight repeating subunits. Structure determination of NPC is a prerequisite for understanding its functional mechanism. In the past two decades, integrative modeling, which combines x-ray structures of individual nucleoporins and subcomplexes with cryo-electron tomography reconstructions, has played a crucial role in advancing our knowledge about the NPC. The CR has been a major focus of structural investigation. The CR subunit of human NPC was reconstructed by cryo-electron tomography through subtomogram averaging to an overall resolution of ~20 Å, with local resolution up to ~15 Å. Each CR subunit comprises two Y-shaped multicomponent complexes known as the inner and outer Y complexes. Eight inner and eight outer Y complexes assemble in a head-to-tail fashion to form the proximal and distal rings, respectively, constituting the CR scaffold. To achieve higher resolution of the CR, we used single-particle cryo-electron microscopy (cryo-EM) to image the intact NPC from the NE of Xenopus laevis oocytes. Reconstructions of the core region and the Nup358 region of the X. laevis CR subunit had been achieved at average resolutions of 5 to 8 Å, allowing identification of secondary structural elements. RATIONALE Packing interactions among the components of the CR subunit were poorly defined by all previous EM maps. Additional components of the CR subunit are strongly suggested by the EM maps of 5- to 8-Å resolution but remain to be identified. Addressing these issues requires improved resolution of the cryo-EM reconstruction. Therefore, we may need to enhance sample preparation, optimize image acquisition, and develop an effective data-processing strategy. RESULTS To reduce conformational heterogeneity of the sample, we spread the opened NE onto the grids with minimal force and used the chemical cross-linker glutaraldehyde to stabilize the NPC. To alleviate orientation bias of the NPC, we tilted sample grids and imaged the sample with higher electron dose at higher angles. We improved the image-processing protocol. With these efforts, the average resolutions for the core and the Nup358 regions have been improved to 3.7 and 4.7 Å, respectively. The highest local resolution of the core region reaches 3.3 Å. In addition, a cryo-EM structure of the N-terminal α-helical domain of Nup358 has been resolved at 3.0-Å resolution. These EM maps allow the identification of five copies of Nup358, two copies of Nup93, two copies of Nup205, and two copies of Y complexes in each CR subunit. Relying on the EM maps and facilitated by AlphaFold prediction, we have generated a final model for the CR of the X. laevis NPC. Our model of the CR subunit includes 19,037 amino acids in 30 nucleoporins. A previously unknown C-terminal fragment of Nup160 was found to constitute a key part of the vertex, in which the short arm, long arm, and stem of the Y complex meet. The Nup160 C-terminal fragment directly binds the ß-propeller proteins Seh1 and Sec13. Two Nup205 molecules, which do not contact each other, bind the inner and outer Y complexes through distinct interfaces. Conformational elasticity of the two Nup205 molecules may underlie their versatility in binding to different nucleoporins in the proximal and distal CR rings. Two Nup93 molecules, each comprising an N-terminal extended helix and an ACE1 domain, bridge the Y complexes and Nup205. Nup93 and Nup205 together play a critical role in mediating the contacts between neighboring CR subunits. Five Nup358 molecules, each in the shape of a shrimp tail and named "the clamp," hold the stems of both Y complexes. The innate conformational elasticity allows each Nup358 clamp to adapt to a distinct local environment for optimal interactions with neighboring nucleoporins. In each CR subunit, the α-helical nucleoporins appear to provide the conformational elasticity; the 12 ß-propellers may strengthen the scaffold. CONCLUSION Our EM map-based model of the X. laevis CR subunit substantially expands the molecular mass over the reported composite models of vertebrate CR subunit. In addition to the Y complexes, five Nup358, two Nup205, and two Nup93 molecules constitute the key components of the CR. The improved EM maps reveal insights into the interfaces among the nucleoporins of the CR. [Figure: see text].
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Proteínas de Xenopus , Xenopus laevis , Animais , Microscopia Crioeletrônica , Citoplasma/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , Proteínas de Xenopus/química , Xenopus laevis/metabolismoRESUMO
Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy structure of the inner ring (IR) subunit from the Xenopus laevis NPC at an average resolution of 4.2 Å. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin hetero-trimer of Nup62/58/54 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure reveals the molecular basis for the IR subunit assembly of a vertebrate NPC.
Assuntos
Poro Nuclear , Proteínas de Xenopus , Transporte Ativo do Núcleo Celular , Animais , Microscopia Crioeletrônica , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Nuclear pore complex (NPC) shuttles cargo across the nuclear envelope. Here we present single-particle cryo-EM structure of the nuclear ring (NR) subunit from Xenopus laevis NPC at an average resolution of 5.6 Å. The NR subunit comprises two 10-membered Y complexes, each with the nucleoporin ELYS closely associating with Nup160 and Nup37 of the long arm. Unlike the cytoplasmic ring (CR) or inner ring (IR), the NR subunit contains only one molecule each of Nup205 and Nup93. Nup205 binds both arms of the Y complexes and interacts with the stem of inner Y complex from the neighboring subunit. Nup93 connects the stems of inner and outer Y complexes within the same NR subunit, and places its N-terminal extended helix into the axial groove of Nup205 from the neighboring subunit. Together with other structural information, we have generated a composite atomic model of the central ring scaffold that includes the NR, IR, and CR. The IR is connected to the two outer rings mainly through Nup155. This model facilitates functional understanding of vertebrate NPC.
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Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Animais , Microscopia Crioeletrônica , Citoplasma/metabolismo , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Synthetic biology-based approaches have been employed to generate advanced natural product (NP) pathway intermediates to overcome obstacles in NP drug discovery and production. Type II polyketides (PK-IIs) comprise a major subclass of NPs that provide attractive structures for antimicrobial and anticancer drug development. Herein, we have assembled five biosynthetic pathways using a generalized operon design strategy in Streptomyces coelicolor M1152 to allow comparative analysis of metabolite production in an improved heterologous host. The work resulted in production of four distinct PK-II core structures, namely benzoisochromanequinone, angucycline, tetracenomycin, and pentangular compounds, which serve as precursors to diverse pharmaceutically important NPs. Our bottom-up design strategy provided evidence that the biosynthetic pathway of BE-7585A proceeds via an angucycline core structure, instead of rearrangement of an anthracycline aglycone, and led to the discovery of a novel 26-carbon pentangular polyketide. The synthetic biology platform presented here provides an opportunity for further controlled production of diverse PK-IIs in a heterologous host.
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Produtos Biológicos/metabolismo , Descoberta de Drogas/métodos , Policetídeos/metabolismo , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Genes Bacterianos , Engenharia Metabólica/métodos , Naftacenos/metabolismo , Plasmídeos/genética , Tioaçúcares/metabolismoRESUMO
In Wnt/ß-catenin signaling, the ß-catenin protein level is deliberately controlled by the assembly of the multiprotein ß-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3ß (GSK3ß), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid-liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3ß, CK1α, and ß-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates ß-catenin phosphorylation by GSK3ß and is critical for the regulation of ß-catenin protein stability and thus Wnt/ß-catenin signaling.
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Complexos Multiproteicos/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células HCT116 , Células HEK293 , Humanos , Complexos Multiproteicos/genética , beta Catenina/genéticaRESUMO
Guided bone regeneration (GBR) technique is most commonly used to treat alveolar bone defect. Polylactic acid (PLA) attracts much attention to utilize as a GBR membrane because it has relatively high mechanical strength and biodegradability. However, randomized controlled trials of PLA as a GBR membrane in animals were rare. The aim of this work is to observe the efficacy of polylactic acid membrane in guiding bone regeneration in Beagle canine alveolar bone defect restoration and to compare efficacy with the collagen membrane, providing an experimental basis for further clinical use of the polylactic acid membrane. The tests of physical and chemical properties showed that the PLA membrane has well mechanical strength to maintenance the space for the new bone, and has proper aperture for the attachment of osteoblasts. Through X-ray and histopathological examination of the different time points, the bone grafting material covered with PLA membrane can form similar mature bone compared to collagen membrane ones. Meanwhile, biodegradable speed of the PLA membrane was slower. Thus, this study showed that polylactic acid membrane as synthetic biodegradable polymer was reliably effective in guiding bone regeneration of alveolar bone defects, showed the favorable osteogenic capability and forecasts well applications in bone augmentation.
Assuntos
Regeneração Óssea , Membranas Artificiais , Animais , Cães , Regeneração Tecidual Guiada Periodontal , Osteoblastos , Osteogênese , PolímerosRESUMO
PURPOSE: In order to achieve accurate and minimally invasive root canal treatment and enhance the fracture resistance of tooth tissue after root canal therapy, this study explores digital guided mediated minimally invasive root canal treatment and compares it with conventional root canal treatment to provide a more favorable method for clinical practice. METHODS: Forty freshly extracted first permanent molars were randomly divided into control group and experimental group. Teeth in the control group were treated with conventional root canal treatment, while teeth in the experimental group were treated with precise minimally invasive root canal treatment. The difference between the time of opening of the pulp chamber and the area of the open pores on the total area of the occlusal surface was compared. Loading test was carried out on the subjects using a universal testing machine, and the fracture resistence of the tooth tissues of the two groups were measured. Statistical analysis was performed using SPSS 24.0 software package. RESULTS: In the control group, the time required for opening the pulp chamber was (1.85±0.05) min, the open pore area was (9.18±0.48)% of the total occlusal area, and the load of the tooth tissue was (1.48±0.07) kN. In the experimental group, the time required was (0.72±0.10) min, the open pore area was (3.53±0.13)% of the total occlusal area, and the load of the tooth tissue was (1.81±0.03) kN. The higher the loading value, the stronger the fracture resistance of the tooth tissue. Compared with traditional root canal treatment, digital guided plate mediated minimally invasive root canal treatment had the advantages of short time, small access cavity and strong fracture resistance of tooth tissue. The difference between the two groups was significant (P<0.01). CONCLUSIONS: Digital guided plate-mediated accurate minimally invasive root canal treatment can reduce the occlusal area, shorten the operation time beside the chair, retain more healthy tooth tissue, enhance the fracture resistance of tooth tissue after root canal treatment, and improve the retention rate of affected teeth.