RESUMO
The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%) and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%) and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.
Assuntos
Anormalidades do Olho/genética , Olho/embriologia , Fatores de Transcrição Forkhead/genética , Adolescente , Alelos , Catarata/genética , Criança , Opacidade da Córnea/genética , Deficiências do Desenvolvimento/genética , Olho/crescimento & desenvolvimento , Anormalidades do Olho/enzimologia , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Masculino , Mutação , Linhagem , FenótipoRESUMO
The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a-/- single homozygous and foxc1-/- double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/-;foxc1b-/-) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.
Assuntos
Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/genética , Oftalmopatias Hereditárias/genética , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Alelos , Animais , Segmento Anterior do Olho/patologia , Desenvolvimento Embrionário/genética , Anormalidades do Olho/patologia , Oftalmopatias Hereditárias/patologia , Dosagem de Genes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Heterozigoto , Homozigoto , Humanos , Mutação/genética , Escoliose/genética , Escoliose/patologia , Peixe-Zebra/genéticaRESUMO
While genetic causes are known for many syndromes involving developmental anomalies, a large number of individuals with overlapping phenotypes remain undiagnosed. Using exome-sequencing analysis and review of matchmaker databases, we have discovered four de novo missense variants predicted to affect the N-terminal region of WDR37-p.Ser119Phe, p.Thr125Ile, p.Ser129Cys, and p.Thr130Ile-in unrelated individuals with a previously unrecognized syndrome. Features of WDR37 syndrome include the following: ocular anomalies such as corneal opacity/Peters anomaly, coloboma, and microcornea; dysmorphic facial features; significant neurological impairment with structural brain defects and seizures; poor feeding; poor post-natal growth; variable skeletal, cardiac, and genitourinary defects; and death in infancy in one individual. WDR37 encodes a protein of unknown function with seven predicted WD40 domains and no previously reported human pathogenic variants. Immunocytochemistry and western blot studies showed that wild-type WDR37 is localized predominantly in the cytoplasm and mutant proteins demonstrate similar protein levels and localization. CRISPR-Cas9-mediated genome editing generated zebrafish mutants with novel missense and frameshift alleles: p.Ser129Phe, p.Ser129Cys (which replicates one of the human variants), p.Ser129Tyr, p.Lys127Cysfs, and p.Gln95Argfs. Zebrafish carrying heterozygous missense variants demonstrated poor growth and larval lethality, while heterozygotes with frameshift alleles survived to adulthood, suggesting a potential dominant-negative mechanism for the missense variants. RNA-seq analysis of zebrafish embryos carrying a missense variant detected significant upregulation of cholesterol biosynthesis pathways. This study identifies variants in WDR37 associated with human disease and provides insight into its essential role in vertebrate development and possible molecular functions.
Assuntos
Anormalidades Múltiplas/genética , Coloboma/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Repetições WD40/genética , Anormalidades Múltiplas/patologia , Adulto , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Coloboma/patologia , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/patologia , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Homologia de Sequência , Síndrome , Peixe-ZebraRESUMO
Various 4'-R-substituted phenyl azacyclic allenes were synthesized in good yields, and their thermal transformations were studied. For the first time, the obtained rearrangement products-new N-bridged cyclopenta[a]indenes, and the corresponding parent allenes were evaluated as potential inhibitors of acetyl- and butyrylcholinesterase. Among the tested compounds, the allene derivative 2g proved to competitively inhibit human AChE with inhibition constant value (Ki) in the low micromolar range.
Assuntos
Butirilcolinesterase , Inibidores da Colinesterase , Acetilcolinesterase/metabolismo , Alcadienos , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
3-(1H-Indol-3-yl)benzofuran-2(3H)-ones were efficiently accessed via polyphosphoric acid-mediated condensation of 3-(2-nitrovinyl)-1H-indoles with phenols.
Assuntos
Benzofuranos , Indóis , FenóisRESUMO
Missense variants located in the N-terminal region of WDR37 were recently identified to cause a multisystemic syndrome affecting neurological, ocular, gastrointestinal, genitourinary, and cardiac development. WDR37 encodes a WD40 repeat-containing protein of unknown function. We identified three novel WDR37 variants, two likely pathogenic de novo alleles and one inherited variant of uncertain significance, in individuals with phenotypes overlapping those previously reported but clustering in a different region of the protein. The novel alleles are C-terminal to the prior variants and located either within the second WD40 motif (c.659A>G p.(Asp220Gly)) or in a disordered protein region connecting the second and third WD40 motifs (c.778G>A p.(Asp260Asn) and c.770C>A p.(Pro257His)). The three novel mutants showed normal cellular localization but lower expression levels in comparison to wild-type WDR37. To investigate the normal interactions of WDR37, we performed co-immunoprecipitation and yeast two-hybrid assays. This revealed the ability of WDR37 to form homodimers and to strongly bind PACS1 and PACS2 phosphofurin acidic cluster sorting proteins; immunocytochemistry confirmed colocalization of WDR37 with PACS1 and PACS2 in human cells. Next, we analyzed previously reported and novel mutants for their ability to dimerize with wild-type WDR37 and bind PACS proteins. Interaction with wild-type WDR37 was not affected for any variant; however, one novel mutant, p.(Asp220Gly), lost its ability to bind PACS1 and PACS2. In summary, this study presents a novel region of WDR37 involved in human disease, identifies PACS1 and PACS2 as major binding partners of WDR37 and provides insight into the functional effects of various WDR37 variants.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Mutantes/genética , Proteínas Nucleares/genética , Anormalidades Múltiplas/metabolismo , Adolescente , Animais , Células Cultivadas , Criança , Pré-Escolar , Disfunção Cognitiva/genética , Feminino , Humanos , Masculino , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Linhagem , Ligação Proteica , Síndrome , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Transporte Vesicular/metabolismoRESUMO
The recently discovered [4+1]-spirocyclization of nitroalkenes to indoles provided a convenient new approach to 2-(1H-indol-2-yl)acetonitriles. However, this reaction was complicated by the formation of inert 3-(2-nitroethyl)-1H-indole byproducts. Herein, we offer a workaround this problem that allows for effective transformation of the unwanted byproducts into acetonitrile target molecules.
RESUMO
Nitroalkanes activated with polyphosphoric acid could serve as efficient electrophiles in reactions with amines and hydrazines, enabling various cascade transformations toward heterocyclic systems. This strategy was developed for an innovative synthetic protocol employing simultaneous or sequential annulation of two different heterocyclic cores, affording [1,2,4]triazolo[4,3-a]quinolines with 1,3,4-oxadiazole substituents.
RESUMO
The PITX2 (paired-like homeodomain 2) gene encodes a bicoid-like homeodomain transcription factor linked with several human disorders. The main associated congenital phenotype is Axenfeld-Rieger syndrome, type 1, an autosomal dominant condition characterized by variable defects in the anterior segment of the eye, an increased risk of glaucoma, craniofacial dysmorphism and dental and umbilical anomalies; in addition to this, one report implicated PITX2 in ring dermoid of the cornea and a few others described cardiac phenotypes. We report three novel PITX2 mutations-c.271C > T, p.(Arg91Trp); c.259T > C, p.(Phe87Leu); and c.356delA, p.(Gln119Argfs*36)-identified in independent families with typical Axenfeld-Rieger syndrome characteristics and some unusual features such as corneal guttata, Wolf-Parkinson-White syndrome, and hyperextensibility. To gain further insight into the diverse roles of PITX2/pitx2 in vertebrate development, we generated various genetic lesions in the pitx2 gene via TALEN-mediated genome editing. Affected homozygous zebrafish demonstrated congenital defects consistent with the range of PITX2-associated human phenotypes: abnormal development of the cornea, iris and iridocorneal angle; corneal dermoids; and craniofacial dysmorphism. In addition, via comparison of pitx2M64* and wild-type embryonic ocular transcriptomes we defined molecular changes associated with pitx2 deficiency, thereby implicating processes potentially underlying disease pathology. This analysis identified numerous affected factors including several members of the Wnt pathway and collagen types I and V gene families. These data further support the link between PITX2 and the WNT pathway and suggest a new role in regulation of collagen gene expression during development.
Assuntos
Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/genética , Oftalmopatias Hereditárias/genética , Glaucoma/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Síndrome de Wolff-Parkinson-White/genética , Proteínas de Peixe-Zebra/genética , Animais , Segmento Anterior do Olho/fisiopatologia , Colágeno Tipo I/genética , Colágeno Tipo V/genética , Anormalidades do Olho/fisiopatologia , Oftalmopatias Hereditárias/fisiopatologia , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Glaucoma/fisiopatologia , Humanos , Mutação , Linhagem , Fatores de Transcrição/deficiência , Via de Sinalização Wnt , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteína Homeobox PITX2RESUMO
The title compound, C(23)H(23)NO, is the product of a tandem transformation of the double Mannich base bis-(1-oxo-1,2,3,4-tertrahydro-2-naphtho-ylmeth-yl)amine hydro-chloride in HBr solution upon heating. The tetra-hydro-pyridine ring has a non-symmetrical half-chair conformation, whereas the cyclo-hexa-diene and cyclo-hexene rings adopt non-symmetrical half-boat conformations. The dihedral angle between the planes of the terminal benzene rings is 62.85â (6)°. The N atom has a trigonal-pyramidal geometry [sum of the bond angles = 332.4â (3)°]. In the crystal, mol-ecules form [001] chains via weak non-classical C-Hâ¯N hydrogen bonds. The chains are stacked along the b axis.
RESUMO
Laser soldering is a current biophotonic technique for the surgical recovery of the integrity of soft tissues. This technology involves the use of a device providing laser exposure to the cut edges of the wound with a solder applied. The proposed solder consisted of an aqueous dispersion of biopolymer albumin (25 wt.%), single-walled carbon nanotubes (0.1 wt.%) and exogenous indocyanine green chromophore (0.1 wt.%). Under laser exposure, the dispersion transforms into a nanocomposite due to the absorption of radiation and its conversion into heat. The nanocomposite is a frame structure of carbon nanotubes in a biopolymer matrix, which provides adhesion of the wound edges and the formation of a strong laser weld. A new laser device based on a diode laser (808 nm) has been developed to implement the method. The device has a temperature feedback system based on a bolometric infrared matrix sensor. The system determines the hottest area of the laser weld and adjusts the current supplied to the diode laser to maintain the preset laser heating temperature. The laser soldering technology made it possible to heal linear defects (cuts) in the skin of laboratory animals (rabbits) without the formation of a fibrotic scar compared to the control (suture material). The combined use of a biopolymer nanocomposite solder and a laser device made it possible to achieve a tensile strength of the laser welds of 4 ± 0.4 MPa. The results of the experiment demonstrated that the addition of single-walled carbon nanotubes to the solder composition leads to an increase in the ultimate tensile strength of the laser welds by 80%. The analysis of regenerative and morphological features in the early stages (1-3 days) after surgery revealed small wound gaps, a decrease in inflammation, the absence of microcirculatory disorders and an earlier epithelization of laser welds compared to the control. On the 10th day after the surgical operation, the laser weld was characterized by a thin cosmetic scar and a continuous epidermis covering the defect. An immunohistochemical analysis proved the absence of myofibroblasts in the area of the laser welds.
RESUMO
The title compound, C(17)H(18)N(2)O(4), is the methyl ester of the adduct of intra-molecular Diels-Alder reaction between maleic anhydride and 1-(2-fur-yl)-2,3,4,5-tetra-hydro-1H-pyrrolo-[1,2-a][1,4]diazepine. The mol-ecule comprises a fused penta-cyclic system containing four five-membered rings (viz. pyrrole, 2-pyrrolidinone, tetra-hydro-furan and dihydro-furan) and one seven-membered ring (1,4-diazepane). The pyrrole ring is approximately planar (r.m.s. deviation = 0.003â Å) while the 2-pyrrolidinone, tetra-hydro-furan and dihydro-furan five-membered rings have the usual envelope conformations. The central seven-membered diazepane ring adopts a boat conformation. In the crystal, mol-ecules are bound by weak inter-molecular C-Hâ¯O hydrogen-bonding inter-actions into zigzag chains propagating in [010]. In the crystal packing, the chains are stacked along the a axis.
RESUMO
The mol-ecule of the title compound, C15H15Br2NO3, comprises a fused tricyclic system consisting of two five-membered rings (cyclo-pentane and tetra-hydro-furan) and one six-membered ring (tetra-hydro-pyridinone). Both five-membered rings of the tricyclic system have envelope conformations, and the conformation of the six-membered cycle is inter-mediate between chair and half-chair. In the crystal, the mol-ecules are linked by C-Hâ¯O hydrogen bonds and C-Hâ¯π, C-Brâ¯π and Câ¯O inter-actions into double layers. The layers are connected into a three-dimensional network by van der Waals inter-actions.
RESUMO
The title compound, C24H24N2O5S, crystallizes with two independent mol-ecules (A and B) in the asymmetric unit. In the central ring systems of both mol-ecules, the tetra-hydro-furan rings adopt envelope conformations, the pyrrolidine rings adopt a twisted-envelope conformation and the six-membered ring is in a boat conformation. In mol-ecules A and B, the nine-membered groups attached to the central ring system are essentially planar (r.m.s. deviations of 0.002 and 0.003â Å, respectively). They form dihedral angles of 64.97â (9) and 56.06â (10)°, respectively, with the phenyl rings. In the crystal, strong inter-molecular O-Hâ¯O hydrogen bonds and weak inter-molecular C-Hâ¯O contacts link the mol-ecules, forming a three-dimensional network. In addition weak π-π stacking inter-actions [centroid-to centroid distance = 3.7124â (13)â Å] between the pyrrolidine rings of the nine-membered groups of A mol-ecules are observed. Hirshfeld surface analysis and two-dimensional fingerprint plots were used to qu-antify the inter-molecular inter-actions present in the crystal, indicating that the environments of the two mol-ecules are very similar. The most important contributions for the crystal packing are from Hâ¯H (55.8% for mol-ecule A and 53.5% for mol-ecule B), Oâ¯H/Hâ¯O (24.5% for mol-ecule A and 26.3% for mol-ecule B) and Câ¯H/Hâ¯C (12.6% for mol-ecule A and 15.7% for mol-ecule B) inter-actions.
RESUMO
In the title compound, C20H19NO5, the central six-membered ring has a slightly distorted half-chair conformation, with puckering parameters of Q T = 0.3387â (11)â Å, θ = 49.11â (19)° and φ = 167.3â (2)°. The conformation of the fused pyrrolidine ring is that of an envelope. Mol-ecules are connected by inter-molecular C-Hâ¯O hydrogen bonds, C-Hâ¯π inter-actions and π-π stacking inter-actions [centroid-to-centroid distance = 3.9536â (11)â Å, with a slippage of 2.047â Å], forming a three-dimensional network. The most important contributions to the surface contacts are from Hâ¯H (46.3%), Oâ¯H/Hâ¯O (31.5%) and Câ¯H/Hâ¯C (17.3%) inter-actions, as concluded from a Hirshfeld surface analysis.
RESUMO
The mechanistic rationale involving activation of nitroalkanes towards interaction with nucleophilic reagents in the presence of polyphosphoric acid (PPA) was re-evaluated. Could nitrile oxide moieties be formed during this process? This experiment demonstrates that at least in some cases this could happen, as generated nitrile oxides were successfully intercepted as adducts of [3 + 2] cycloadditions.
RESUMO
Attachment of stone crystals to tubular epithelium may initiate kidney stone formation. We previously reported that apical nucleolin related protein (NRP) expression during mitosis enhance attachment of Ca oxalate monohydrate crystals (COM). Some forms of injury may also increase affinity for crystals. We examined changes in subcellular localization of NRP during the course of cisplatin-induced apoptosis in cultured inner medullary collecting duct cells. Caspase-3 activation and chromatin condensation followed by nuclear fragmentation occurred after 20 h exposure to cisplatin, indicating the development of apoptosis. Cells were fixed without permeabilization and stained for surface NRP. Cells with condensed chromatin showed little or no cytoplasmic or apical NRP. Those at an early stage of nuclear fragmentation had cytoplasmic but not apical NRP and cells with advanced nuclear fragmentation were positively stained for apical NRP. Membrane proteins isolated by apical biotinylation and precipitated with avidin were analyzed by Western blot. Apical NRP was markedly increased after cisplatin compared to control, while expression of the apical marker, GP-135, and other putative attachment protein were unchanged. Hyaluronic acid was decreased. Cultures with apoptotic cells demonstrated increased adherence of COM that was inhibited by the polyanion (poly)aspartic acid. We conclude that pre-existing apoptotic injury may promote calcium oxalate crystals attachment to renal tubular epithelium via apical NRP expression.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Oxalato de Cálcio , Cisplatino/farmacologia , Túbulos Renais Coletores/citologia , Adesividade , Células Cultivadas , Cristalização , Humanos , Glicoproteínas de Membrana/biossínteseRESUMO
The title compound, C(27)H(29)NO(11), is a product of the tandem 'domino' Diels-Alder reaction. The mol-ecule comprises a fused hexa-cyclic system containing four five-membered rings (two dihydro-furan and two tetra-hydro-furan) in the usual envelope conformations and two six-membered rings (tetra-hydro-pyridinone and piperidine) adopting slightly flattened boat and chair conformations, respectively. The dispositions of the carboxyl-ate substituents relative to each other are determined by both steric reasons and inter-molecular C-Hâ¯O hydrogen bonding and attractive anti-parallel C=Oâ¯C=O inter-actions [Câ¯O = 2.995â (2)â Å].
RESUMO
The title di-epoxy-phenalene derivative, C17H18O6, comprises a fused cyclic system containing four five-membered rings (two di-hydro-furan and two tetra-hydro-furan) and one six-membered ring (cyclo-hexa-ne). The five-membered di-hydro-furan and tetra-hydro-furan rings adopt envelope conformations, and the six-membered cyclo-hexane ring adopts a distorted chair conformation. Two methyl carboxyl-ate groups occupy adjacent positions (2- and 3-) on a tetra-hydro-furan ring. In the crystal, two pairs of C-Hâ¯O hydrogen bonds link the mol-ecules to form inversion dimers, enclosing two R 2 2(6) ring motifs, that stack along the a-axis direction and are arranged in layers parallel to the bc plane.
RESUMO
Compounds (I), C22H20N2O2, (II), C22H20N2O2 and (III), C20H18N2O3 are the products of three-component reactions between isatoic anhydride, the corresponding amine and 3-(5-methylfuran-2-yl)- or (furan-2-yl)-2-methyl-acryl-aldehyde. Compound (I) crystallizes in the monoclinic space group P21/n, while compounds (II) and (III) are isostructural and crystallize in the ortho-rhom-bic space group Pbca. The tetra-hydro-pyrimidine ring in (I)-(III) adopts a sofa conformation. The NH nitro-gen atom has a trigonal-pyramidal geometry, whereas the N(R) nitro-gen atom is flattened. The furyl-vinyl substituents in (I)-(III) are practically planar and have an E configuration at the C=C double bond. In (I), this bulky fragment occupies the axial position at the quaternary carbon atom of the tetra-hydro-pyrimidine ring, whereas in (II) and (III) it is equatorially disposed. In the crystal of (I), mol-ecules form hydrogen-bonded chains propagating along [001] by strong inter-molecular N-Hâ¯O hydrogen bonds. The chains are packed in stacks along the a-axis direction. In the crystals of (II) and (III), mol-ecules also form hydrogen-bonded chains propagating along [100] by strong inter-molecular N-Hâ¯O hydrogen bonds. However, despite the fact that compounds (II) and (III) are isostructural, steric differences between the phenyl and furyl substituents result in chains with different geometries. Thus in the crystal of (II) the chains have a zigzag-like structure, whereas in the crystal of (III), they are almost linear. In both (II) and (III), the hydrogen-bonded chains are further packed in stacks along the b-axis direction.