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1.
Anticancer Res ; 40(10): 5405-5409, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988860

RESUMO

AIM: To investigate the clinical significance of ATP-binding cassette transporter 11 (ABCC11) protein expression in colon cancer. MATERIALS AND METHODS: One hundred thirty nine patients with colon cancer resection between 2009 and 2011 were enrolled. The relationship with immunohistochemical ABCC11 staining and clinicopathological factors was retrospectively analyzed. RESULTS: Median age was 70 years including 67 males and 72 females. The patients with Stage 0, 1, 2, 3a and 4 were 4, 20, 43, 35, 7 and 30, respectively. The patients with curability (Cur) A, B and C were 109, 11 and 19, respectively. Positive expression of ABCC11 was observed in 31 patients (22.3%). There were no significant differences regarding age, gender, location, serum tumor markers, T category, lymphatic invasion and stage in relation to ABCC11 protein expression. Cases with node metastasis and venous invasion as well as unresectable cases were significantly more often found negative for ABCC11 protein (p=0.0246, 0.0285 and 0.0422, respectively). Concerning the 3 year disease free survival (DFS) and the 5 year overall survival (OS) in Stage 2/3 and in Stage 3 with adjuvant chemotherapy, no significant differences were found. However, OS in ABCC11 negative cases was 81.1%, which was significantly lower compared to positive cases, where OS was 96.2%. CONCLUSION: There was significant correlation with ABCC11 expression and lymph node metastasis, venous invasion and curability. The prognosis in ABCC11 negative cases was poor because of increased cases without curative resection.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proliferação de Células/genética , Neoplasias do Colo/genética , Metástase Linfática/genética , Idoso , Neoplasias do Colo/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática/patologia , Masculino , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos
2.
Ecotoxicol Environ Saf ; 202: 110940, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800223

RESUMO

Recent evidence indicates that chronic, low-dose exposure to mixtures of pesticides can cause adverse responses in a variety of cells, tissues and organs, although interactions between pesticides circulating in the blood and cancer cells remain largely unexplored. The aim of this study was to investigate the potential of a mixture of four pesticides to induce multidrug resistance against the chemotherapeutic agents cisplatin, 5-fluorouracil and temozolomide in the human U87 glioblastoma cell line, and to explore the molecular mechanisms underlying this resistance. We found that the repeated administration of the pesticide mixture (containing the insecticides chlorpyrifos-ethyl and deltamethrin, the fungicide metiram, and the herbicide glyphosate) induced a strong drug resistance in U87 cells. The resistance was durable and transferred to subsequent cell generations. In addition, we detected a significant over-expression of the ATP-binding cassette (ABC) membrane transporters P-gp/ABCB1 and BRCP/ABCG2 as well as a glutathione-S-transferase (GST)/M1-type cellular detoxification function, known to have important roles in multidrug resistance, thus providing molecular support for the acquired multidrug resistance phenotype and shedding light on the mechanism of resistance. We further determined that there was lower mortality in the resistant brain tumor cells and that the mitochondrial apoptosis pathway was activated at a lower rate after chemotherapy compared to non-resistant control cells. In addition, multidrug-resistant cells were found to have both higher motility and wound-healing properties, suggesting a greater metastatic potential. Our results suggest that the investigation of P-gp, BRCP and GST/M1 multidrug resistance gene expression and/or protein levels in biopsy specimens of brain tumor patients who were at risk of pesticide exposure could be beneficial in determining chemotherapy dose and prolonging patient survival.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Praguicidas/toxicidade , Testes de Toxicidade Crônica , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia
3.
PLoS Pathog ; 16(8): e1008763, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32834002

RESUMO

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Colite/patologia , Variação Genética , Macrófagos/imunologia , Salmonelose Animal/patologia , Salmonella typhimurium/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem da Célula , Colite/induzido quimicamente , Colite/imunologia , Colite/microbiologia , Análise Mutacional de DNA , Modelos Animais de Doenças , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Replicação Viral
4.
PLoS Pathog ; 16(8): e1008697, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776976

RESUMO

The diamondback moth, Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacterium Bacillus thuringiensis (Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal that P. xylostella requires combined mutations in both PxABCC2 and PxABCC3 to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations of PxABCC2 and PxABCC3 that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) of P. xylostella, with a mutation (RA2) causing the mis-splicing of PxABCC2 and another mutation (RA3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed that RA2 and RA3 were tightly linked to Cry1Ac resistance. Introgression of RA2 and RA3 enabled a susceptible strain (G88) of P. xylostella to obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role of PxABCC2 and PxABCC3 in Cry1Ac resistance, frameshift mutations were introduced into PxABCC2 and PxABCC3 singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion of PxABCC2 or PxABCC3 alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance in P. xylostella by demonstrating mutations within both PxABCC2 and PxABCC3 genes are required for high-level Cry1Ac resistance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Bacillus thuringiensis , Proteínas de Insetos/química , Proteínas de Insetos/genética , Mariposas/química , Mariposas/genética , Mariposas/metabolismo , Mutação , Alinhamento de Sequência
5.
Nucleic Acids Res ; 48(15): 8445-8460, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32644157

RESUMO

DNA lesions or other barriers frequently compromise replisome progress. The SF2 helicase RecG is a key enzyme in the processing of postreplication gaps or regressed forks in Escherichia coli. A deletion of the recG gene renders cells highly sensitive to a range of DNA damaging agents. Here, we demonstrate that RecG function is at least partially complemented by another SF2 helicase, RadD. A ΔrecGΔradD double mutant exhibits an almost complete growth defect, even in the absence of stress. Suppressors appear quickly, primarily mutations that compromise priA helicase function or recA promoter mutations that reduce recA expression. Deletions of uup (encoding the UvrA-like ABC system Uup), recO, or recF also suppress the ΔrecGΔradD growth phenotype. RadD and RecG appear to avoid toxic situations in DNA metabolism, either resolving or preventing the appearance of DNA repair intermediates produced by RecA or RecA-independent template switching at stalled forks or postreplication gaps. Barriers to replisome progress that require intervention by RadD or RecG occur in virtually every replication cycle. The results highlight the importance of the RadD protein for general chromosome maintenance and repair. They also implicate Uup as a new modulator of RecG function.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Adenosina Trifosfatases/genética , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Escherichia coli/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Mutação/genética , Recombinação Genética/genética
6.
Proc Natl Acad Sci U S A ; 117(32): 19228-19236, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32703810

RESUMO

The ATP-binding cassette (ABC) transporter of mitochondria (Atm1) mediates iron homeostasis in eukaryotes, while the prokaryotic homolog from Novosphingobium aromaticivorans (NaAtm1) can export glutathione derivatives and confer protection against heavy-metal toxicity. To establish the structural framework underlying the NaAtm1 transport mechanism, we determined eight structures by X-ray crystallography and single-particle cryo-electron microscopy in distinct conformational states, stabilized by individual disulfide crosslinks and nucleotides. As NaAtm1 progresses through the transport cycle, conformational changes in transmembrane helix 6 (TM6) alter the glutathione-binding site and the associated substrate-binding cavity. Significantly, kinking of TM6 in the post-ATP hydrolysis state stabilized by MgADPVO4 eliminates this cavity, precluding uptake of glutathione derivatives. The presence of this cavity during the transition from the inward-facing to outward-facing conformational states, and its absence in the reverse direction, thereby provide an elegant and conceptually simple mechanism for enforcing the export directionality of transport by NaAtm1. One of the disulfide crosslinked NaAtm1 variants characterized in this work retains significant glutathione transport activity, suggesting that ATP hydrolysis and substrate transport by Atm1 may involve a limited set of conformational states with minimal separation of the nucleotide-binding domains in the inward-facing conformation.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Bactérias/química , Sphingomonadaceae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Glutationa/química , Glutationa/metabolismo , Ferro/metabolismo , Domínios Proteicos , Sphingomonadaceae/química , Sphingomonadaceae/genética
8.
Gene ; 754: 144890, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32534057

RESUMO

PURPOSE: Stargardt disease (STGD) is the most frequent cause of hereditary macular dystrophy in childhood. Variants in the ABCA4, ELOVL4, PROM1, BEST1, and PRPH2 genes have been detected in patients with autosomal recessive or dominant STGD. This study was aimed at identifying the novel disease-associated variants in Chinese patients with STGD. METHODS: Ten Chinese families and two sporadic cases with STGD (n = 32) were enrolled in the study. All subjects underwent genetic analysis with next-generation sequencing (NGS), which was based on a specially customized capture panel targeting exons and untranslated regions (UTRs) of 792 genes related to common hereditary ophthalmopathy. Variants were analyzed to assess possible pathogenicity. RESULTS: Fourteen disease-associated variants of ABCA4 were detected in 9 Chinese families with autosomal recessive STGD, including 11 pathogenic variants and 3 likely pathogenic variants. Variant c.4253 + 4C > T in ABCA4 was identified as one de novo variant. Of the 14 distinct variants in ABCA4, 7 novel variants were found. In addition, one known variant of PROM1, c.1117C > T (p.Arg373Cys), was detected in one family and one sporadic case with autosomal dominant STGD, respectively. One novel missense variant of ELOVL4, c.59A > G (p.Asn20Ser), was found in one sporadic case with autosomal dominant STGD. The potential deleterious effects of these novel variants were confirmed through intensive analysis. CONCLUSION: By panel-based NGS, 8 novel disease-associated variants are identified in two genes responsible for STGD, including ABCA4 and ELOVL4. Our results further extend the mutation spectrum of these two genes in Chinese patients with STGD. One ABCA4 c.4253 + 4C > T variant is identified as a de novo splicing variant.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Marcadores Genéticos , Variação Genética , Mutação , Doença de Stargardt/genética , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Criança , Proteínas do Olho/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Membrana/genética , Linhagem , Adulto Jovem
9.
Int J Infect Dis ; 97: 47-53, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32531432

RESUMO

PURPOSE: To explore the molecular genetic mechanisms underlying different responsiveness to Enterovirus 71 (EV71) vaccine. METHODS: We recruited 10,245 healthy children into a phase 3 clinical trial to evaluate the efficacy of EV71 vaccine in 2012. Fifty subjects from the trial were divided into the potent immune response group (20 subjects) and the ineffective immune response group (30 subjects). Whole-exome sequencing was performed for these 50 samples and we conducted bioinformatics analyses based on online public database. RESULTS: A total of 222,180 germline variants were detected across 50 subjects. Single nucleotide variant (SNV)-based screening of the subjects with potent or ineffective immune response allowed the identification of a potentially detrimental heterozygous missense variant (c.3784C>T) in EEA1. We also retained TRIM59 and ABCA7 genes that contain different loss of function (LoF) variants shared in two cases and involved in the immune response process. Then, we conducted high-resolution typing of 9 classical HLA genes, HLA-DRB1*03:01, HLA-DQA1*05:01 and HLA-DQB1*02:01 alleles were frequently (recurrence ≥5) observed only in ineffective immune responders. CONCLUSIONS: Our study is a meaningful attempt on the comparison of genomic profiles between potent and ineffective immune responders induced by EV71 vaccine, and several candidate potentially detrimental genes were identified.


Assuntos
Enterovirus Humano A/imunologia , Vacinas Virais/imunologia , Transportadores de Cassetes de Ligação de ATP/genética , Alelos , Pré-Escolar , China , Feminino , Variação Genética , Cadeias alfa de HLA-DQ/genética , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas com Motivo Tripartido/genética , Sequenciamento Completo do Exoma
10.
Food Chem ; 331: 127360, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32585548

RESUMO

The influence of food components on nanoparticle (NP) internalization indicates a need to investigate the behaviors of NPs in a complex system. This study measured the changes of TiO2 NP colloidal stability and quenching of anthocyanin fluorescence to indicate NP-anthocyanin interactions, and cytotoxicity, oxidative stress, expression of ABC transporters and intracellular Ti concentrations in 3D Caco-2 spheroids co-exposed to NPs and anthocyanins to indicate the influence of anthocyanins on NP bio-effects. The anthocyanins were observed to have minimal impacts on colloidal properties of TiO2 NPs. Meanwhile, NP-anthocyanin co-exposure did not induce cytotoxicity or oxidative stress. The fluorescence quenching study indicated the binding of anthocyanins onto TiO2 NPs, and the binding affinity was inversely correlated with NP internalization into 3D Caco-2 spheroids. This may be partially related with the up-regulation of ABC transporters. Our results may provide novel insights into understanding the interactions of NPs and anthocyanins with human intestinal cells.


Assuntos
Antocianinas/farmacologia , Nanopartículas Metálicas/química , Titânio/farmacocinética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antocianinas/química , Células CACO-2 , Coloides/farmacocinética , Depuradores de Radicais Livres/farmacologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Titânio/química
12.
PLoS One ; 15(4): e0232476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32353073

RESUMO

P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Biocatálise , Cloreto de Cálcio/metabolismo , Ensaios Enzimáticos , Hidrólise , Mutação , Naftóis , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Triazinas
13.
PLoS One ; 15(4): e0224643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348310

RESUMO

Grey mould is caused by the ascomycetes Botrytis cinerea in a range of crop hosts. As a biological control agent, the nucleoside antibiotic wuyiencin has been industrially produced and widely used as an effective fungicide. To elucidate the effects of wuyiencin on the transcriptional regulation in B. cinerea, we, for the first time, report a genome-wide transcriptomic analysis of B. cinerea treated with wuyiencin. 2067 genes were differentially expressed, of them, 886 and 1181 genes were significantly upregulated and downregulated, respectively. Functional categorization indicated that transcript levels of genes involved in amino acid metabolism and those encoding putative secreted proteins were altered in response to wuyiencin treatment. Moreover, the expression of genes involved in protein synthesis and energy metabolism (oxidative phosphorylation) and of those encoding ATP-binding cassette transporters was markedly upregulated, whereas that of genes participating in DNA replication, cell cycle, and stress response was downregulated. Furthermore, wuyiencin resulted in mycelial malformation and negatively influenced cell growth rate and conidial yield in B. cinerea. Our results suggest that this nucleoside antibiotic regulates all aspects of cell growth and differentiation in B. cinerea. To summarize, some new candidate pathways and target genes that may related to the protective and antagonistic mechanisms in B. cinerea were identified underlying the action of biological control agents.


Assuntos
Antifúngicos/farmacologia , Botrytis/genética , Farmacorresistência Fúngica , Transcriptoma , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Botrytis/efeitos dos fármacos , Botrytis/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação para Cima
14.
PLoS One ; 15(4): e0231893, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298378

RESUMO

BACKGROUND/OBJECTIVES: Vaccination is the most important tool for controlling brucellosis, but currently there is no vaccine available for canine brucellosis, which is a zoonotic disease of worldwide distribution caused by Brucella canis. This study aimed to evaluate protection and immune response induced by Brucella ovis ΔabcBA (BoΔabcBA) encapsulated with alginate against the challenge with Brucella canis in mice and to assess the safety of this strain for dogs. METHODS: Intracellular growth of the vaccine strain BoΔabcBA was assessed in canine and ovine macrophages. Protection induced by BoΔabcBA against virulent Brucella canis was evaluated in the mouse model. Safety of the vaccine strain BoΔabcBA was assessed in experimentally inoculated dogs. RESULTS: Wild type B. ovis and B. canis had similar internalization and intracellular multiplication profiles in both canine and ovine macrophages. The BoΔabcBA strain had an attenuated phenotype in both canine and ovine macrophages. Immunization of BALB/c mice with alginate-encapsulated BoΔabcBA (108 CFU) induced lymphocyte proliferation, production of IL-10 and IFN-γ, and protected against experimental challenge with B. canis. Dogs immunized with alginate-encapsulated BoΔabcBA (109 CFU) seroconverted, and had no hematologic, biochemical or clinical changes. Furthermore, BoΔabcBA was not detected by isolation or PCR performed using blood, semen, urine samples or vaginal swabs at any time point over the course of this study. BoΔabcBA was isolated from lymph nodes near to the site of inoculation in two dogs at 22 weeks post immunization. CONCLUSION: Encapsulated BoΔabcBA protected mice against experimental B. canis infection, and it is safe for dogs. Therefore, B. ovis ΔabcBA has potential as a vaccine candidate for canine brucellosis prevention.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Vacina contra Brucelose/imunologia , Brucella ovis/genética , Brucelose/prevenção & controle , Doenças do Cão/prevenção & controle , Alginatos/química , Animais , Formação de Anticorpos , Brucella canis/patogenicidade , Brucella ovis/imunologia , Brucella ovis/isolamento & purificação , Brucelose/microbiologia , Brucelose/patologia , Doenças do Cão/microbiologia , Doenças do Cão/patologia , Cães , Feminino , Imunização , Fígado/microbiologia , Fígado/fisiologia , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Ovinos
15.
Invest Ophthalmol Vis Sci ; 61(4): 13, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32298433

RESUMO

Purpose: To analyze the progression of choriocapillaris (CC) impairment in recessive Stargardt disease (STGD) and compare it to the progression of retinal pigment epithelium (RPE) atrophy. Methods: Fifty-five patients with a clinical diagnosis of STGD and genetic confirmation of pathogenic biallelic variants in ABCA4 were imaged with short-wavelength fundus autofluorescence (SW-AF) and optical coherence tomography angiography (OCTA) at a single clinic visit, whereas a subset of 12 patients were imaged with the same modalities at two different clinic visits. Results: We observed three stages of CC impairment: an area of bright yet intact macular CC (11 patients), regions of vascular rarefaction and incomplete CC atrophy within an area of bright CC (10 patients), and areas of extensive CC atrophy (26 patients). These changes correlated to the degree of RPE atrophy observed in SW-AF imaging. Furthermore, 8 patients presented with early changes on SW-AF, but healthy CC. Quantitative analyses of the atrophic changes revealed that the area of RPE atrophy is larger (9.6 ± 1.7 mm2 vs. 6.9 ± 1.3 mm2, P < 0.001) and that it progresses at a faster rate (1.1 ± 0.1 mm2/year vs. 0.8 ± 0.2 mm2/year, P = 0.004) than the corresponding area of CC atrophy. Conclusions: CC impairment is progressive and OCTA imaging can be used to demonstrate the stages, which culminate in extensive CC atrophy. Furthermore, CC impairment is secondary to RPE atrophy in STGD. We further advocate the use of SW-AF and OCTA imaging in monitoring the progression of STGD.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Corioide/patologia , Epitélio Pigmentado da Retina/patologia , Doença de Stargardt/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Atrofia , Criança , Progressão da Doença , Feminino , Angiofluoresceinografia , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Óptica , Estudos Retrospectivos , Doença de Stargardt/genética , Tomografia de Coerência Óptica , Adulto Jovem
16.
Nat Commun ; 11(1): 1763, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273501

RESUMO

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients in bacteria and archaea. They consist of an integral membrane unit, the S-component, and a tripartite ECF module. It has been proposed that the S-component mediates the substrate transport by toppling over in the membrane when docking onto an ECF module. Here, we present multi-scale molecular dynamics simulations and in vitro experiments to study the molecular toppling mechanism of the S-component of a folate-specific ECF transporter. Simulations reveal a strong bending of the membrane around the ECF module that provides a driving force for toppling of the S-component. The stability of the toppled state depends on the presence of non-bilayer forming lipids, as confirmed by folate transport activity measurements. Together, our data provide evidence for a lipid-dependent toppling-based mechanism for the folate-specific ECF transporter, a mechanism that might apply to other ECF transporters.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Ácido Fólico/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Membrana Celular/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Proteica
17.
Nature ; 580(7803): 409-412, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296172

RESUMO

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis1-3. Although Mtb can synthesize vitamin B12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis2. Mtb does not encode any characterized cobalamin transporter4-6; however, the gene rv1819c was found to be essential for uptake of cobalamin1. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin7. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold1. Here, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å3, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells8-11.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Bleomicina/metabolismo , Mycobacterium tuberculosis/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Transporte Biológico , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
18.
Ecotoxicol Environ Saf ; 197: 110606, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32304921

RESUMO

ATP binding cassette (ABC) transporters, types C, G, and B were monitored via qPCR in order to investigate the influence of heavy metal (HM) contamination of post-industrial and post-agricultural soils and the effects of its supplementation with sewage sludge, on Sinapis alba plants. Five house-keeping genes were selected and validated to ensure the best reference points. The relative expression of ABC types C and G genes was profoundly affected by experimental conditions and included their upregulation after plants exposure to heavy metals and downregulation after supplementation with sewage sludge. However, ABC type C was more responsive then type G. The experimental conditions altered the expression of ABC type C gene faster than ABC type G and thus, the expression of ABC type C can therefore potentially be used as a bioindicator during assisted phytoremediation of degraded sites. In clean soil, supplementation with sewage sludge with a slight content of heavy metals still caused an upregulation in the expression of ABC types C and G, which showed that proper toxicity assessments are necessary to ensure safe application of sewage sludge into soils. Results showed that the analysed genes take a significant part in plants metal detoxification and that their expression is regulated at transcriptional level after exposure to soil contaminated with heavy metals by both, industrial activities and by sewage sludge supplementation. Thus, their expression can potentially be used as an early-warning biomarker when soil supplementation with sewage sludge is incorporated into the soil-management process.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais Pesados/metabolismo , Esgotos , Sinapis/metabolismo , Poluentes do Solo/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Biodegradação Ambiental , Biomarcadores Ambientais , Metais Pesados/toxicidade , Sinapis/efeitos dos fármacos , Sinapis/genética , Solo/química , Poluentes do Solo/toxicidade
19.
Hum Genet ; 139(10): 1261-1272, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32318854

RESUMO

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect for which only ~ 20% of the underlying genetic variation has been identified. Variants in noncoding regions have been increasingly suggested to contribute to the missing heritability. In this study, we investigated whether variation in craniofacial enhancers contributes to NSCLP. Candidate enhancers were identified using VISTA Enhancer Browser and previous publications. Prioritization was based on patterning defects in knockout mice, deletion/duplication of craniofacial genes in animal models and results of whole exome/whole genome sequencing studies. This resulted in 20 craniofacial enhancers to be investigated. Custom amplicon-based sequencing probes were designed and used for sequencing 380 NSCLP probands (from multiplex and simplex families of non-Hispanic white (NHW) and Hispanic ethnicities) using Illumina MiSeq. The frequencies of identified variants were compared to ethnically matched European (CEU) and Los Angeles Mexican (MXL) control genomes and used for association analyses. Variants in mm427/MSX1 and hs1582/SPRY1 showed genome-wide significant association with NSCLP (p ≤ 6.4 × 10-11). In silico analysis showed that these enhancer variants may disrupt important transcription factor binding sites. Haplotypes involving these enhancers and also mm435/ABCA4 were significantly associated with NSCLP, especially in NHW (p ≤ 6.3 × 10-7). Importantly, groupwise burden analysis showed several enhancer combinations significantly over-represented in NSCLP individuals, revealing novel NSCLP pathways and supporting a polygenic inheritance model. Our findings support the role of craniofacial enhancer sequence variation in the etiology of NSCLP.


Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Variação Genética , Herança Multifatorial , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Doenças Assintomáticas , Fenda Labial/etnologia , Fenda Labial/patologia , Fissura Palatina/etnologia , Fissura Palatina/patologia , Embrião de Mamíferos , Grupo com Ancestrais do Continente Europeu , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Hispano-Americanos , Humanos , Fator de Transcrição MSX1/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Linhagem , Fosfoproteínas/genética , Estados Unidos
20.
Nature ; 580(7803): 413-417, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296173

RESUMO

Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Sideróforos/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
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