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1.
Nat Commun ; 11(1): 4917, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004802

RESUMO

Maternal mRNA clearance is an essential process that occurs during maternal-to-zygotic transition (MZT). However, the dynamics, functional importance, and pathological relevance of maternal mRNA decay in human preimplantation embryos have not yet been analyzed. Here we report the zygotic genome activation (ZGA)-dependent and -independent maternal mRNA clearance processes during human MZT and demonstrate that subgroups of human maternal transcripts are sequentially removed by maternal (M)- and zygotic (Z)-decay pathways before and after ZGA. Key factors regulating M-decay and Z-decay pathways in mouse have similar expression pattern during human MZT, suggesting that YAP1-TEAD4 transcription activators, TUT4/7-mediated mRNA 3'-oligouridylation, and BTG4/CCR4-NOT-induced mRNA deadenylation may also be involved in the regulation of human maternal mRNA stability. Decreased expression of these factors and abnormal accumulation of maternal transcripts are observed in the development-arrested embryos of patients who seek assisted reproduction. Defects of M-decay and Z-decay are detected with high incidence in embryos that are arrested at the zygote and 8-cell stages, respectively. In addition, M-decay is not found to be affected by maternal TUBB8 mutations, although these mutations cause meiotic cell division defects and zygotic arrest, which indicates that mRNA decay is regulated independent of meiotic spindle assembly. Considering the correlations between maternal mRNA decay defects and early developmental arrest of in vitro fertilized human embryos, M-decay and Z-decay pathway activities may contribute to the developmental potential of human preimplantation embryos.


Assuntos
Blastocisto/metabolismo , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Animais , Técnicas de Cultura Embrionária , Feminino , Fertilização In Vitro/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Meiose/genética , Camundongos , Mutação , Oócitos/metabolismo , Cultura Primária de Células , RNA-Seq , Tubulina (Proteína)/genética , Zigoto/metabolismo
2.
Int J Food Microbiol ; 334: 108833, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32911159

RESUMO

This work aims to provide the first study on the mycobiota present in Chilean pepper Capsicum annuum L. cv. "Cacho de Cabra" throughout the early production stages. Two hundred and forty berry fruits were sampled: 1) at the ripe fruits harvest day; 2) during drying; and 3) smoking processes. A total of 192 strains, encompassing 11 genera and 44 species, were identified through analysis of ß-tubulin (benA) gene and internal transcribed spacer of ribosomal DNA (ITS) region. All collection points showed samples with high fungal contamination, but the mycobiota composition varied as a result of different environmental conditions. Alternaria spp. and Fusarium spp. were predominantly isolated from fresh fruits of C. annuum. Penicillium spp. was the most frequent genus in all analysed points. Penicillium brevicompactum and P. crustosum were the most abundant species. Among Aspergillus, A. niger and A. flavus were dominant after the drying phase. In our study, none of the analysed strains of Penicillium (113) and Aspergillus (35) produced Ochratoxin A at detectable levels. The broad characterization of the fungal community of C. annuum carried out in this study, could be a guideline for future mycotoxin analyses performed directly on the pod. Understanding the role and dynamics of mycobiota and its relationship with the toxins present in this substrate, will be useful to establish and improve control measures considering the specificities of each point in the C. annuum production chain.


Assuntos
Capsicum/microbiologia , Microbiologia de Alimentos/métodos , Alimentos em Conserva/microbiologia , Micobioma , Chile , DNA Espaçador Ribossômico/genética , Frutas/microbiologia , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Micotoxinas/análise , Tubulina (Proteína)/genética
3.
PLoS One ; 15(8): e0237111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750100

RESUMO

Animal Tubulin-Based-Polymorphism (aTBP), an intron length polymorphism method recently developed for vertebrate genotyping, has been successfully applied to the identification of several fish species. Here, we report data that demonstrate the ability of the aTBP method to assign a specific profile to fish species, each characterized by the presence of commonly shared amplicons together with additional intraspecific polymorphisms. Within each aTBP profile, some fragments are also recognized that can be attributed to taxonomic ranks higher than species, e.g. genus and family. Versatility of application across different taxonomic ranks combined with the presence of a significant number of DNA polymorphisms, makes the aTBP method an additional and useful tool for fish genotyping, suitable for different purposes such as species authentication, parental recognition and detection of allele variations in response to environmental changes.


Assuntos
Proteínas de Peixes/genética , Peixes/genética , Técnicas de Genotipagem/métodos , Polimorfismo Genético , Tubulina (Proteína)/genética , Animais
4.
PLoS Pathog ; 16(8): e1008710, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32817722

RESUMO

Rice stripe virus (RSV, genus Tenuivirus, family Phenuiviridae) is the causal agent of rice stripe disease transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent propagative manner. The midgut and salivary glands of SBPH are the first and last barriers to the viral circulation and transmission processes, respectively; however, the precise mechanisms used by RSV to cross these organs and transmit to rice plants have not been fully elucidated. We obtained the full-length cDNA sequence of L. striatellus α-tubulin 2 (LsTUB) and found that RSV infection increased the level of LsTUB in vivo. Furthermore, LsTUB was shown to co-localize with RSV nonstructural protein 3 (NS3) in vivo and bound NS3 at positions 74-76 and 80-82 in vitro. Transient gene silencing of LsTUB expression caused a significant reduction in detectable RSV loads and viral NS3 expression levels, but had no effect on NS3 silencing suppressor activity and viral replication in insect cells. However, suppression of LsTUB attenuated viral spread in the bodies of SBPHs and decreased RSV transmission rates to rice plants. Electrical penetration graphs (EPG) showed that LsTUB knockdown by RNAi did not impact SBPH feeding; therefore, the reduction in RSV transmission rates was likely caused by a decrease in viral loads inside the planthopper. These findings suggest that LsTUB mediates the passage of RSV through midgut and salivary glands and leads to successful horizontal transmission.


Assuntos
Hemípteros/metabolismo , Proteínas de Insetos/metabolismo , Insetos Vetores/metabolismo , Oryza/virologia , Doenças das Plantas/virologia , Tenuivirus/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Sistema Digestório/metabolismo , Sistema Digestório/virologia , Hemípteros/genética , Hemípteros/virologia , Proteínas de Insetos/genética , Insetos Vetores/genética , Insetos Vetores/virologia , Glândulas Salivares/metabolismo , Glândulas Salivares/virologia , Tubulina (Proteína)/genética
5.
Int J Syst Evol Microbiol ; 70(8): 4798-4807, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32783804

RESUMO

Leptographium panxianense and L. puerense are proposed as new taxa based on sequence data and morphological characters. The phylogenetic analyses based on ITS2-partial LSU rDNA region, ß-tubulin and elongation factor 1-α genes showed that L. panxianense and L. puerense formed well-supported clades and were closely related to L. yunnanense, L. wushanense and L. conjunctum, and then nested within the L. lundbergii complex. The two species differ in their conidial size and shape. The conidia of L. panxianense are larger than those of L. puerense while the conidial shape of L. puerense is more ovovoid. The optimal growth temperature of both L. panxianense and L. puerense is at 20 °C, which is different from those of L. yunnanense, L. wushanense and L. conjunctum. Comparison of sequence data and morphological characters confirmed the placement of the two undescribed taxa in the genus of Leptographium.


Assuntos
Besouros/microbiologia , Ophiostomatales/classificação , Filogenia , Pinus , Animais , China , DNA Fúngico/genética , DNA Ribossômico/genética , Técnicas de Tipagem Micológica , Ophiostomatales/isolamento & purificação , Fator 1 de Elongação de Peptídeos/genética , Análise de Sequência de DNA , Esporos Fúngicos , Tubulina (Proteína)/genética
6.
Exp Parasitol ; 217: 107957, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32687847

RESUMO

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.


Assuntos
Anti-Helmínticos/farmacologia , Haemonchus/genética , Taxa de Mutação , Tiabendazol/farmacologia , Tubulina (Proteína)/genética , Animais , DNA de Helmintos/isolamento & purificação , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Frequência do Gene , Marcadores Genéticos , Genótipo , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/classificação , Haemonchus/efeitos dos fármacos , Dose Letal Mediana , Óvulo/efeitos dos fármacos , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/parasitologia
7.
Mol Cell ; 79(1): 191-198.e3, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619469

RESUMO

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Glicina/análogos & derivados , Microtúbulos/efeitos dos fármacos , Neoplasias/patologia , Preparações Farmacêuticas/metabolismo , Sulfonas/farmacologia , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografia por Raios X , Contaminação de Medicamentos , Glicina/farmacologia , Humanos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Preparações Farmacêuticas/química , Conformação Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genética
8.
Mol Cell ; 79(1): 180-190.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619468

RESUMO

Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Glicina/análogos & derivados , Neoplasias Pulmonares/patologia , Sulfonas/farmacologia , Tubulina (Proteína)/metabolismo , Contaminação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glicina/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mutação , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Células Tumorais Cultivadas
9.
Parasitol Res ; 119(9): 2851-2862, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32651637

RESUMO

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 ß-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.


Assuntos
Albendazol/farmacologia , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos/genética , Haemonchus/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Cromatografia Líquida , Glutamato Desidrogenase/metabolismo , Hemoncose/tratamento farmacológico , Haemonchus/genética , Reação em Cadeia da Polimerase , Proteômica , Ovinos , Doenças dos Ovinos/parasitologia , Espectrometria de Massas em Tandem , Tubulina (Proteína)/genética
10.
Int J Syst Evol Microbiol ; 70(7): 4321-4328, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32579096

RESUMO

A collection of fungal isolates obtained from crop plants, specifically grapevine and blueberry, in Peru were characterised through morphological and DNA sequence analyses of the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (tub2) and translation elongation factor 1-alpha (tef-1α) regions. Isolates produced monomorphic and dimorphic conidiophores typical of members of the genus Clonostachys. Single- and multi-locus gene phylogenies confirmed the isolates as representing members of the genus Clonostachys, more closely related to species in the subgenus Bionectria. In phylogenetic analyses the isolates grouped in two separate clades, one corresponding to the species Clonostachys pseudochroleuca and the other one distinct from all known species of the genus Clonostachys. These isolates are recognized as representing a novel species species for which the name Clonostachys viticola is proposed.


Assuntos
Hypocreales/classificação , Filogenia , Vitis/microbiologia , DNA Espaçador Ribossômico/genética , Hypocreales/isolamento & purificação , Técnicas de Tipagem Micológica , Fator 1 de Elongação de Peptídeos/genética , Peru , Análise de Sequência de DNA , Tubulina (Proteína)/genética
11.
Oncology ; 98(10): 689-698, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32585672

RESUMO

BACKGROUND: ßIII-Tubulin, encoded by the TUBB3 gene, is a microtubule protein. Several studies have shown that overexpression of TUBB3 is linked to poor prognosis and is involved in taxane resistance in some cancers. OBJECTIVE: The aim of this study was to analyze the expression and function of TUBB3 in clear cell renal cell carcinoma (ccRCC). METHODS: The expression of TUBB3 was determined using immuno-histochemistry in ccRCC specimens. The effects of TUBB3 knockdown on cell growth and invasion were evaluated in RCC cell lines. We analyzed the interaction between TUBB3, p53, cancer stem cell markers, and PD-L1. RESULTS: In 137 cases of ccRCC, immunohistochemistry showed that 28 (20%) of the ccRCC cases were positive for TUBB3. High TUBB3 expression was significantly correlated with high nuclear grade, high T stage, and N stage. A Kaplan-Meier analysis showed that high expression of TUBB3 was associated with poor overall survival after nephrectomy. In silico analysis also showed that high TUBB3 expression was correlated with overall survival. Knockdown of TUBB3 suppressed cell growth and invasion in 786-O and Caki-1 cells. High TUBB3 expression was associated with CD44, CD133, PD-L1, and p53 in ccRCC. We generated p53 knockout cells using the CRISPR-Cas9 system. Western blotting revealed that p53 knockout upregulated the expression of TUBB3. CONCLUSION: These results suggest that TUBB3 may play an oncogenic role and could be a potential therapeutic target in ccRCC.


Assuntos
Tubulina (Proteína)/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Idoso , Antígeno B7-H1/biossíntese , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Imuno-Histoquímica , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Prognóstico , Tubulina (Proteína)/genética , Proteína Supressora de Tumor p53/genética
12.
Phytopathology ; 110(9): 1522-1529, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32352861

RESUMO

Fusarium graminearum causes Fusarium head blight (FHB), a destructive disease of cereal crops worldwide. Carbendazim (methylbenzimidazol-2-ylcarbamate [MBC]) is widely used for controlling FHB. A previous study showed that the F240L mutation in the ß2-tubulin of F. graminearum (Fgß2-tubulin) confers hypersensitivity to MBC. Whether the substitution of phenylalanine by other amino acids in position 240 of the Fgß2-tubulin gene also confers hypersensitivity to MBC is unknown. Moreover, the biological fitness of these mutants is poorly understood. In this study, we substituted position 240 of Fgß2-tubulin with other amino acids. We found that the F240A, F240E, F240I, and F240Y mutations in Fgß2-tubulin could also confer F. graminearum hypersensitivity to MBC, although the effective concentration resulting in 50% inhibition (EC50) differed among the mutations. The F240G mutation, in contrast, decreased the sensitivity to MBC. In addition, a molecular docking assay indicated that the binding affinity between Fgß2-tubulin and MBC were increased by the F240A, F240E, F240I, and F240Y mutations but decreased by the F240G mutation. All mutants had normal conidial morphology, but the growth rates and pathogenicity of the F240A, F240E, F240G, F240I, and F240Y mutants were significantly decreased. Moreover, the F240A and F240G mutants produced twisted hyphae. In addition, microtubules were sparse and rarely observed in ß2F240A-EGFP, ß2F240E-EGFP, and ß2F240G-EGFP. These results indicate that position 240 (phenylalanine) is not only vital to the function of Fgß2-tubulin but also plays an important role in regulating the sensitivity of F. graminearum to MBC. Any mutation in this site would be detrimental to survival.


Assuntos
Fungicidas Industriais , Fusarium , Simulação de Acoplamento Molecular , Mutação , Doenças das Plantas , Tubulina (Proteína)/genética
13.
PLoS Negl Trop Dis ; 14(3): e0008151, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32226021

RESUMO

Sporothrix chilensis is a mild-pathogenical specie of Sporothrix pallida complex, until now, known as restrict to Chile. Herein, we describe the first clinical isolates identified as S. chilensis in Brazil, preserved in the URM Culture Collection, by polyphasic taxonomy, and their respective antifungal profile of this emergent fungus.


Assuntos
Sporothrix/classificação , Sporothrix/isolamento & purificação , Esporotricose/microbiologia , Antifúngicos/farmacologia , Brasil , Humanos , Testes de Sensibilidade Microbiana , Técnicas de Tipagem Micológica , Análise de Sequência de DNA , Sporothrix/genética , Sporothrix/fisiologia , Tubulina (Proteína)/genética
14.
J Ovarian Res ; 13(1): 42, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32316999

RESUMO

PURPOSE: Variations in many genes may lead to the occurrence of oocyte maturation defects. To investigate the genetic basis of oocyte maturation defects, we performed clinical and genetic analysis of a pedigree. METHODS: The proband with oocyte maturation defect-2 receiving ovulation induction therapy and her parents were selected for clinical detection, whole exome sequencing and Sanger sequencing. One unrelated healthy woman received ovulation induction therapy as control. Mutations were assessed after frequency screening of public exome databases. Then homozygous variants shared by the proband and her parents were selected. RESULTS: Arrest of oocytes maturation was observed. A new missense mutation in TUBB8 (TUBB8: NM_177,987: exon 2: c. C161T: p. A54V) was identified, which was shown to be rare compared with public databases. The variant was highly conserved among primates, and was suggested to be deleterious by online software prediction. CONCLUSIONS: The homozygote of this variant (TUBB8: NM_ 177,987: exon 2:c.C161T: p.A54V) might affect spindle assembly, cause arrest of oocyte maturation and lead to oocyte maturation defect-2.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Infertilidade Feminina/genética , Oócitos/fisiologia , Tubulina (Proteína)/genética , Adulto , Consanguinidade , Feminino , Homozigoto , Humanos , Infertilidade Feminina/terapia , Mutação de Sentido Incorreto , Indução da Ovulação
15.
PLoS Biol ; 18(3): e3000647, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163403

RESUMO

Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.


Assuntos
Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas Wnt/metabolismo , Animais , Complexo de Sinalização da Axina/genética , Complexo de Sinalização da Axina/metabolismo , Axônios/metabolismo , Polaridade Celular , Dendritos/genética , Drosophila , Proteínas de Drosophila/genética , Endossomos/genética , Microtúbulos/genética , Mutação , Receptores Wnt/genética , Receptores Wnt/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Wnt/genética , Via de Sinalização Wnt/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
16.
Parasit Vectors ; 13(1): 114, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122383

RESUMO

BACKGROUND: Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. METHODS: Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 ß-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. RESULTS: Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. CONCLUSIONS: To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.


Assuntos
Benzimidazóis/farmacologia , Códon , Resistência a Medicamentos/genética , Doenças das Cabras/parasitologia , Haemonchus/genética , Polimorfismo de Nucleotídeo Único , Tubulina (Proteína)/genética , Albendazol/farmacologia , Animais , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Feminino , Frequência do Gene , Genótipo , Doenças das Cabras/tratamento farmacológico , Cabras , Haemonchus/efeitos dos fármacos , Masculino , Análise de Sequência de DNA , Sudão , Trichostrongyloidea/genética , Trichostrongylus/genética
17.
Proc Natl Acad Sci U S A ; 117(14): 7799-7802, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32205434

RESUMO

Cytoskeletons are self-organized networks based on polymerized proteins: actin, tubulin, and driven by motor proteins, such as myosin, kinesin, and dynein. Their positive Darwinian evolution enables them to approach optimized functionality (self-organized criticality). Dynein has three distinct titled subunits, but how these units connect to function as a molecular motor is mysterious. Dynein binds to tubulin through two coiled coil stalks and a stalk head. The energy used to alter the head binding and propel cargo along tubulin is supplied by ATP at a ring 1,500 amino acids away. Here, we show how many details of this extremely distant interaction are explained by water waves quantified by thermodynamic scaling. Water waves have shaped all proteins throughout positive Darwinian evolution, and many aspects of long-range water-protein interactions are universal (described by self-organized criticality). Dynein water waves resembling tsunami produce nearly optimal energy transport over 1,500 amino acids along dynein's one-dimensional peptide backbone. More specifically, this paper identifies many similarities in the function and evolution of dynein compared to other cytoskeleton proteins such as actin, myosin, and tubulin.


Assuntos
Trifosfato de Adenosina/genética , Citoesqueleto/genética , Dineínas/genética , Evolução Molecular , Actinas , Sequência de Aminoácidos/genética , Animais , Fenômenos Biofísicos , Aptidão Genética/genética , Cinesina/genética , Microtúbulos/genética , Miosinas/genética , Conformação Proteica , Tubulina (Proteína)/genética
18.
Eur J Paediatr Neurol ; 26: 46-60, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32169460

RESUMO

OBJECTIVE: To describe fetal, clinical, radiological, morphological features of TUBB3 related syndrome. METHODS: We report two families each of two generations harboring a novel and a previously described heterozygous TUBB3 pathogenic variants. We compared these patients with other published TUBB3-related cases. We describe the pathological features of dysgyria in the two aborted fetuses. RESULTS: The mother and son from family 1 had a history of mild developmental delay in motor and language skills and demonstrated mild cerebellar signs and mirror movements. Neuroimaging findings included: hypoplastic corpus callosum (CC), asymmetric ventriculomegaly and cerebellar vermis hypoplasia in all patients and frontal dysgyria in three. Autopsy of the fetal brain showed an unusual shape and orientation of the frontal sulci and gyri with normal cortical layering and no abnormal cell types. The mother of family 2 had congenital strabismus, mild muscle weakness on the right and a past history of developmental delay. Fetal brain MRI showed abnormal cerebral sulcation, hemispheric asymmetry, asymmetric ventriculomegaly, dysmorphic short CC and frontal cortical interdigitation. Autopsy demonstrated fronto-parietal predominant dysgyria, bilateral ventriculomegaly, hippocampal and CC hypoplasia, abnormal Sylvian fissure. Lamination and neuron morphology in the areas of dysgyria were normal. CONCLUSIONS: TUBB3 related cortical malformations can be mild, consistent with dysgyria rather than typical pachygyria or polymicrogyria. The autopsy findings in fetal TUBB3 related dysgyria are abnormal orientation of sulci and gyri, but normal neuron morphology and layering. We suggest that TUBB3 - associated brain malformations can be suspected in-utero which in turn can aid in prognostic counselling and interpretation of genetic testing.


Assuntos
Feto/anormalidades , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Tubulina (Proteína)/genética , Adulto , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Lactente , Recém-Nascido , Imagem por Ressonância Magnética/métodos , Masculino , Mutação , Gravidez , Síndrome
19.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118673, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32057919

RESUMO

Microtubules are polymers of α/ß-tubulin, with microtubule organization being regulated by microtubule-associated proteins (MAPs). Herein, we describe a novel role for the epithelial gene repressor, zinc finger E-box-binding homeobox 1 (ZEB1), that "switches" from a chromatin-associated protein during interphase, to a MAP that associates with α-, ß- and γ-tubulin during mitosis. Additionally, ZEB1 was also demonstrated to associate with γ-tubulin at the microtubule organizing center (MTOC). Using confocal microscopy, ZEB1 localization was predominantly nuclear during interphase, with α/ß-tubulin being primarily cytoplasmic and the association between these proteins being minimal. However, during the stages of mitosis, ZEB1 co-localization with α-, ß-, and γ-tubulin was significantly increased, with the association commonly peaking during metaphase in multiple tumor cell-types. ZEB1 was also observed to accumulate in the cleavage furrow during cytokinesis. The increased interaction between ZEB1 and α-tubulin during mitosis was also confirmed using the proximity ligation assay. In contrast to ZEB1, its paralog ZEB2, was mainly perinuclear and cytoplasmic during interphase, showing some co-localization with α-tubulin during mitosis. Considering the association between ZEB1 with α/ß/γ-tubulin during mitosis, studies investigated ZEB1's role in the cell cycle. Silencing ZEB1 resulted in a G2-M arrest, which could be mediated by the up-regulation of p21Waf1/Cip1 and p27Kip1 that are known downstream targets repressed by ZEB1. However, it cannot be excluded the G2/M arrest observed after ZEB1 silencing is not due to its roles as a MAP. Collectively, ZEB1 plays a role as a MAP during mitosis and could be functionally involved in this process.


Assuntos
Cromatina/genética , Proteínas Associadas aos Microtúbulos/genética , Mitose/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Pontos de Checagem do Ciclo Celular/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Citocinese/genética , Humanos , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica/genética , Fuso Acromático/genética , Tubulina (Proteína)/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/química
20.
Nat Commun ; 11(1): 695, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019927

RESUMO

Cellular processes are influenced by liquid phase separation, but its role in DNA repair is unclear. Here, we show that in Saccharomyces cerevisiae, liquid droplets made up of DNA repair proteins cooperate with different types of DNA damage-inducible intranuclear microtubule filaments (DIMs) to promote the clustering of DNA damage sites and maintain genome stability. Rad52 DNA repair proteins at different DNA damage sites assemble in liquid droplets that fuse into a repair centre droplet via the action of petite DIMs (pti-DIMs). This larger droplet concentrates tubulin and projects short aster-like DIMs (aster-DIMs), which tether the repair centre to longer DIMs mediating the mobilization of damaged DNA to the nuclear periphery for repair. Our findings indicate that cooperation between Rad52 liquid droplets and various types of nuclear filaments promotes the assembly and function of the DNA repair centre.


Assuntos
Reparo do DNA , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA , DNA Fúngico/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
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