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1.
Food Chem ; 306: 125628, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31629297

RESUMO

Microencapsulation of Zingiber officinale essential oil (EO) in polysaccharide, chitosan (CH) and sodium carboxymethyl cellulose (CMC) based on the electrostatic interaction between charged polysaccharides at pH 3.0 in dual delivery system. Ratio variations of CH and CMC in microencapsulation were studied at 1:2, 2:1 and 1:1. This study aimed to evaluate the influence of the encapsulating materials combination on freeze-dried EO powders and to present the mechanisms for loading and releasing EO involved in the preparation of CH/CMC microcapsules. The spectroscopy analysis, physical properties, microstructural, encapsulation efficiency and EO release behavior in obtained EO microparticles were evaluated by using the analysis of fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and gas chromatography mass spectrometry (GC-MS), respectively. Afterwards, the above prepared microcapsules were applied on winter jujube fruit (Ziziphus jujuba Mill.) preservation. Results demonstrated that both the microstructure and stability of microencapsulation were improved in delivery system loading with CH and CMC (1:1) with the encapsulation efficiency of 88.50%, compared to other ratios of CH and CMC (1:2 and 2:1). Furthermore, the microencapsulation had a capacity to control and reduce the EO release, therefore the morphological and sensory quality of jujube fruits in EO delivery system during storage was enhanced significantly (P < 0.05), in comparison to control. Results revealed that the microparticles produced with CH and CMC (1:1) was considered to present better characteristics of microstructure, encapsulation efficiency, as well as to maintain higher nutritional quality for jujube fruit. Thus, EO microencapsulation loaded in CH/CMC-based dual delivery system has potential application and developmental value prospects in food industries.


Assuntos
Gengibre/química , Óleos Voláteis/química , Ziziphus/química , Cápsulas/análise , Quitosana/análogos & derivados , Quitosana/química , Frutas/química
2.
Eur J Hum Genet ; 26(10): 1441-1450, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29899370

RESUMO

Orofacial development is a multifaceted process involving tightly regulated genetic signaling networks, that when perturbed, lead to orofacial abnormalities including cleft lip and/or cleft palate. We and others have shown an association between the cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2) gene and nonsyndromic cleft lip with or without cleft palate (NSCLP). Further, we demonstrated that knockdown of Crispld2 in zebrafish alters neural crest cell migration patterns resulting in abnormal jaw and palate development. In this study, we performed RNA profiling in zebrafish embryos and identified 249 differentially expressed genes following knockdown of Crispld2. In silico pathway analysis identified a network of seven genes previously implicated in orofacial development for which differential expression was validated in three of the seven genes (CASP8, FOS, and MMP2). Single nucleotide variant (SNV) genotyping of these three genes revealed significant associations between NSCLP and FOS/rs1046117 (GRCh38 chr14:g.75746690 T > C, p = 0.0005) in our nonHispanic white (NHW) families and MMP2/rs243836 (GRCh38 chr16:g.55534236 G > A; p = 0.002) in our Hispanic families. Nominal association was found between NSCLP and CASP8/rs3769825 (GRCh38 chr2:g.202111380 C > A; p < 0.007). Overtransmission of MMP2 haplotypes were identified in the Hispanic families (p < 0.002). Significant gene-gene interactions were identified for FOS-MMP2 in the NHW families and for CASP8-FOS in the NHW simplex family subgroup (p < 0.004). Additional in silico analysis revealed a novel gene regulatory network including five of these newly identified and 23 previously reported NSCLP genes. Our results demonstrate that animal models of orofacial clefting can be powerful tools to identify novel candidate genes and gene regulatory networks underlying NSCLP.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Predisposição Genética para Doença , Glicoproteínas/genética , Proteínas de Peixe-Zebra/genética , Animais , Fenda Labial/patologia , Fissura Palatina/patologia , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Genótipo , Haplótipos , Humanos , Polimorfismo de Nucleotídeo Único , Peixe-Zebra
3.
Mol Genet Genomic Med ; 5(6): 730-741, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29178643

RESUMO

BACKGROUND: Oligodontia is a severe form of tooth agenesis characterized by the absence of six or more permanent teeth. Oligodontia has complex etiology and variations in numerous genes have been suggested as causal for the condition. METHODS: We applied whole-exome sequencing (WES) to identify the cause of oligodontia in a 9-year-old girl missing 11 permanent teeth. Protein modeling and functional analysis in zebrafish were also performed to understand the impact of identified variants on the phenotype. RESULTS: We identified a novel compound heterozygous missense mutation in WNT10A (c.637G>A:p.Gly213Ser and c.1070C>T:p.Thr357Ile) as the likely cause of autosomal recessive oligodontia in the child. Affected residues are located in conserved regions and variants are predicted to be highly deleterious for potentially destabilizing the protein fold and inhibiting normal protein function. Functional studies in zebrafish embryos showed that wnt10a is expressed in the craniofacies at critical time points for tooth development, and that perturbations of wnt10a expression impaired normal tooth development and arrested tooth development at 5 days postfertilization (dpf). Furthermore, mRNA expression levels of additional tooth development genes were directly correlated with wnt10a expression; expression of msx1, dlx2b, eda, and axin2 was decreased upon wnt10a knockdown, and increased upon wnt10a overexpression. CONCLUSIONS: Our results reveal a novel compound heterozygous variant in WNT10A as pathogenic for oligodontia, and demonstrate that perturbations of wnt10a expression in zebrafish may directly and/or indirectly affect tooth development recapitulating the agenesis phenotype observed in humans.


Assuntos
Anodontia/genética , Dente/crescimento & desenvolvimento , Proteínas Wnt/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/genética , Anodontia/diagnóstico , Sequência de Bases , Criança , Dentição Permanente , Embrião não Mamífero/metabolismo , Feminino , Heterozigoto , Humanos , Modelos Animais , Morfolinos/genética , Morfolinos/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Dente/patologia , Sequenciamento Completo do Exoma , Proteínas Wnt/química , Proteínas Wnt/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
4.
Mol Genet Genomic Med ; 3(5): 440-51, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26436110

RESUMO

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect affecting 135,000 newborns worldwide each year. While a multifactorial etiology has been suggested as the cause, despite decades of research, the genetic underpinnings of NSCLP remain largely unexplained. In our previous genome-wide linkage study of a large NSCLP African-American family, we identified a candidate locus at 8q21.3-24.12 (LOD = 2.98). This region contained four genes, Frizzled-6 (FZD6), Matrilin-2 (MATN2), Odd-skipped related 2 (OSR2) and Solute Carrier Family 25, Member 32 (SLC25A32). FZD6 was located under the maximum linkage peak. In this study, we sequenced the coding and noncoding regions of these genes in two affected family members, and identified a rare variant in intron 1 of FZD6 (rs138557689; c.-153 + 432A>C). The variant C allele segregated with NSCLP in this family, through affected and unaffected individuals, and was found in one other NSCLP African-American family. Functional assays showed that this allele creates an allele-specific protein-binding site and decreases promoter activity. We also observed that loss and gain of fzd6 in zebrafish contributes to craniofacial anomalies. FZD6 regulates the WNT signaling pathway, which is involved in craniofacial development, including midfacial formation and upper labial fusion. We hypothesize, therefore, that alteration in FZD6 expression contributes to NSCLP in this family by perturbing the WNT signaling pathway.

5.
Genesis ; 53(10): 660-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26297922

RESUMO

The CAP superfamily member, CRISPLD2, has previously been shown to be associated with nonsyndromic cleft lip and palate (NSCLP) in human populations and to be essential for normal craniofacial development in the zebrafish. Additionally, in rodent models, CRISPLD2 has been shown to play a role in normal lung and kidney development. However, the specific role of CRISPLD2 during these developmental processes has yet to be determined. In this study, it was demonstrated that Crispld2 protein localizes to the orofacial region of the zebrafish embryo and knockdown of crispld2 resulted in abnormal migration of neural crest cells (NCCs) during both early and late time points. An increase in cell death after crispld2 knockdown as well as an increase in apoptotic marker genes was also shown. This data suggests that Crispld2 modulates the migration, differentiation, and/or survival of NCCs during early craniofacial development. These results indicate an important role for Crispld2 in NCC migration during craniofacial development and suggests involvement of Crispld2 in cell viability during formation of the orofacies.


Assuntos
Movimento Celular/genética , Glicoproteínas/genética , Crista Neural/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Apoptose/genética , Padronização Corporal/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Cabeça/embriologia , Humanos , Microscopia Confocal , Crista Neural/citologia , Crista Neural/embriologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Crânio/embriologia , Crânio/metabolismo , Fatores de Tempo , Imagem com Lapso de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
6.
Genesis ; 50(12): 871-81, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22887593

RESUMO

Nonsyndromic cleft lip and palate (NSCLP), a common birth defect, affects 4,000 newborns in the US each year. Previously, we described an association between CRISPLD2 and NSCLP and showed Crispld2 expression in the murine palate. These results suggested that a perturbation in CRISPLD2 activity affects craniofacial development. Here, we describe crispld2 expression and the phenotypic consequence of its loss of function in zebrafish. crispld2 was expressed at all stages of zebrafish morphogenesis examined and localized to the rostral end by 1-day postfertilization. Morpholino knockdown of crispld2 resulted in significant jaw and palatal abnormalities in a dose-dependent manner. Loss of crispld2 caused aberrant patterning of neural crest cells (NCC) suggesting that crispld2 is necessary for normal NCC formation. Altogether, we show that crispld2 plays a significant role in the development of the zebrafish craniofacies and alteration of normal protein levels disturbs palate and jaw formation. These data provide support for a role of CRISPLD2 in NSCLP.


Assuntos
Moléculas de Adesão Celular/genética , Anormalidades Craniofaciais/genética , Glicoproteínas/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Encéfalo/anormalidades , Moléculas de Adesão Celular/metabolismo , Fenda Labial/genética , Fissura Palatina/genética , Técnicas de Silenciamento de Genes , Glicoproteínas/metabolismo , Arcada Osseodentária/embriologia , Anormalidades Maxilomandibulares/genética , Dados de Sequência Molecular , Morfolinos , Crista Neural/anormalidades , Crista Neural/embriologia , Palato/anormalidades , Palato/embriologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
8.
Adv Otorhinolaryngol ; 70: 107-113, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21358192

RESUMO

Nonsyndromic cleft lip with or without an associated cleft palate (NSCLP) is one of the most common birth defects affecting 135,000 babies worldwide each year. It causes severe facial dysmorphism and treatment requires a multifaceted team approach. While treatment modalities have improved, the costs to families and the health care resources are still enormous. The causes of NSCLP are multifactorial with both genetic and environmental factors. Progress is being made towards defining the genetic variation underpinning NSCLP by utilizing gene discovery techniques including genome wide linkage, association mapping and a candidate gene approaches. To date, approximately 20% of the genetic contributions to NSCLP have been assigned to a small number of genes. This chapter provides a review of recent progress in defining the genetic causes of NSCLP.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Estudo de Associação Genômica Ampla , Humanos , Fenótipo
9.
J Am Chem Soc ; 132(19): 6663-71, 2010 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-20426415

RESUMO

The biosynthetic gene cluster for tautomycetin (TTN), a highly potent and selective protein phosphatase (PP) inhibitor isolated from Streptomyces griseochromogenes, has recently been cloned and sequenced. To better understand the transformations responsible for converting the post-polyketide synthase product into the exciting anticancer and immunosuppressive chemotherapeutic candidate TTN, we produced and characterized new analogues resulting from inactivation of two genes, ttnD and ttnF, in S. griseochromogenes. Inactivation of ttnD and ttnF, which encode for putative decarboxylase and dehydratase enzymes, respectively, afforded mutant strains SB13013 and SB13014. The DeltattnD mutant SB13013 accumulated four new TTN analogues, TTN D-1, TTN D-2, TTN D-3, and TTN D-4, whereas the DeltattnF mutant accumulated only one new TTN analogue, TTN F-1. The accumulation of these new TTN analogues defines the function of TtnD and TtnF and the timing of their chemistries in relation to installation of the C5 ketone moiety within TTN. Notably, all new analogues possess a structurally distinguishing carboxylic acid moiety, revealing that TtnD apparently cannot catalyze decarboxylation in the absence of TtnF. Additionally, cytotoxicity and PP inhibition assays reveal the importance of the functional groups installed by TtnDF and, consistent with earlier proposals, the C2''-C5 fragment of TTN to be a critical structural determinant behind the important and unique PP-1 selectivity displayed by TTN.


Assuntos
Proteínas de Bactérias/genética , Lipídeos/biossíntese , Alcenos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Carboxiliases/química , Carboxiliases/genética , Carboxiliases/isolamento & purificação , Carboxiliases/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Inativação Gênica , Humanos , Hidroliases/química , Hidroliases/genética , Hidroliases/isolamento & purificação , Hidroliases/metabolismo , Concentração Inibidora 50 , Lipídeos/farmacologia , Família Multigênica , Mutação , Fosfoproteínas Fosfatases/antagonistas & inibidores , Streptomyces/enzimologia , Streptomyces/genética , Streptomyces/metabolismo
10.
Org Lett ; 11(7): 1639-42, 2009 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-19281218

RESUMO

The biosynthetic gene cluster for tautomycin (TTM), a potent protein phosphatase (PP) inhibitor has recently been characterized. Inactivation of ttmM, which encodes a putative C3' hydroxylase, afforded mutant SB6005 which accumulated three new 3'-deshydroxy TTM analogs, supporting the function of TtmM and the previously proposed linear pathway for TTM biosynthesis. Bioassays reveal the importance of the C3' OH moiety in PP inhibition and that PP inhibition is not the exclusive mechanism driving TTM-induced cell death.


Assuntos
Fosfoproteínas Fosfatases/antagonistas & inibidores , Piranos/síntese química , Compostos de Espiro/síntese química , Streptomyces/genética , Furanos/síntese química , Furanos/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Lipídeos/síntese química , Lipídeos/química , Estrutura Molecular , Família Multigênica/efeitos dos fármacos , Piranos/química , Piranos/farmacologia , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Streptomyces/metabolismo , Relação Estrutura-Atividade
11.
BMC Med Genet ; 9: 6, 2008 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-18254962

RESUMO

BACKGROUND: Mental Retardation is a common heterogeneous neurodevelopment condition, which causes are still largely elusive. It has been suggested that half of the phenotypic variation of intelligence is explained by genetic variation. And genetic or inherited factors indeed account for most of the cases of mental retardation with an identifiable cause. However, only a few autosomal genes have been mapped and identified to date. In this report, the genetic causes for an apparently recessive form of mental retardation, in a large nordern swedish pedigree, are investigated. METHODS: After extensive evaluation of the patients, which ruled out recognizable patterns of malformation and excluded known causes of MR, a comprehensive genome-wide linkage analysis, with 500 microsatellite markers, was performed in 24 members of this family. Additionally, a genome-wide copy number analysis, using an affimetrix 250 K SNP chip, was performed in this pedigree. RESULTS: No significant LOD score was found with either parametric and non-parametric linkage analysis. The highest scores are located at chromosomes 13, 15 and 17. Genome-wide copy number analysis identified no clear cause for the disorder; but rather, several variants were present in the family members, irrespective of their affected status. CONCLUSION: These results suggest that mental retardation in this family, unlikely what was expected, has a heterogeneous aetiology; and that several lower effect genes variants might be involved. To demonstrate such effects, our family may be too small. This study also indicates that the ascertainment of the cause of MR may be challenging, and that a complex aetiology may be present even within a pedigree, constituting an additional obstacle for genetic counselling. Variants in genes involved in molecular mechanisms of cellular plasticity, in genes involved in the development of underlying neural architectures, and in genes involved in neurodevelopment and in the ongoing function of terminally differentiated neurons may underlie the phenotypic variation of intelligence and explain instances of intellectual impairment.


Assuntos
Ligação Genética , Padrões de Herança , Deficiência Intelectual/genética , Feminino , Dosagem de Genes , Marcadores Genéticos , Genoma Humano , Humanos , Escore Lod , Masculino , Repetições de Microssatélites/genética , Linhagem , Polimorfismo de Nucleotídeo Único
12.
Biochemistry ; 44(9): 3615-25, 2005 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-15736971

RESUMO

Therapeutic heparin concentrations selectively inhibit the intrinsic tenase complex in an antithrombin-independent manner. To define the molecular target and mechanism for this inhibition, recombinant human factor IXa with alanine substituted for solvent-exposed basic residues (H92, R170, R233, K241) in the protease domain was characterized with regard to enzymatic activity, heparin affinity, and inhibition by low molecular weight heparin (LMWH). These mutations only had modest effects on chromogenic substrate hydrolysis and the kinetics of factor X activation by factor IXa. Likewise, factor IXa H92A and K241A showed factor IXa-factor VIIIa affinity similar to factor IXa wild type (WT). In contrast, factor IXa R170A demonstrated a 4-fold increase in apparent factor IXa-factor VIIIa affinity and dramatically increased coagulant activity relative to factor IXa WT. Factor IXa R233A demonstrated a 2.5-fold decrease in cofactor affinity and reduced ability to stabilize cofactor half-life relative to wild type, suggesting that interaction with the factor VIIIa A2 domain was disrupted. Markedly (R233A) or moderately (H92A, R170A, K241A) reduced binding to immobilized LMWH was observed for the mutant proteases. Solution competition demonstrated that the EC(50) for LMWH was increased less than 2-fold for factor IXa H92A and K241A but over 3.5-fold for factor IXa R170A, indicating that relative heparin affinity was WT > H92A/K241A > R170A >> R233A. Kinetic analysis of intrinsic tenase inhibition demonstrated that relative affinity for LMWH was WT > K241A > H92A > R170A >> R233A, correlating with heparin affinity. Thus, LMWH inhibits intrinsic tenase by interacting with the heparin-binding exosite in the factor IXa protease domain, which disrupts interaction with the factor VIIIa A2 domain.


Assuntos
Antitrombinas/fisiologia , Coenzimas/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Dalteparina/metabolismo , Fator IXa/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Alanina/genética , Sítios de Ligação , Testes de Coagulação Sanguínea , Linhagem Celular , Compostos Cromogênicos/metabolismo , Inibidores de Cisteína Proteinase/química , Dalteparina/química , Fator IXa/química , Fator IXa/genética , Fator VIIIa/metabolismo , Fator X/antagonistas & inibidores , Fator X/metabolismo , Meia-Vida , Humanos , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Ligação Proteica/genética
13.
Methods Mol Biol ; 277: 47-59, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15201448

RESUMO

To facilitate identification of disease genes containing an expanded trinucleotide repeat, a repeat expansion detection (RED) and gene cloning system was established. The RED method was developed to enable detection of expanded trinucleotide repeat sequences in any DNA sample from any species without prior knowledge of the DNA sequences flanking the repeat. The DNA to be tested is used as a template for a repeat oligonucleotide to anneal and ligate in a two-step cycling procedure. After hundreds of annealing/ligation cycles, a large amount of oligonucleotide multimers is accumulated. The longest multimer represents the largest repeat expansion in the genome tested. The gene isolation strategy is based on size separation of genomic fragments, followed by subcloning and library hybridization with an oligonucleotide probe. The expanded trinucleotide repeat is identified throughout the procedure using the RED method. Using this approach, two disease genes, the Huntington's disease gene and the MJD/SCA3 gene, were cloned. This cloning strategy should be applicable to isolation of any DNA fragment containing large trinucleotide repeat expansions in any species.


Assuntos
Técnicas Genéticas , Repetições de Trinucleotídeos/genética , Autorradiografia , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida
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