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1.
Biosens Bioelectron ; 253: 116166, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38428069

RESUMO

Eccrine sweat can serve as a source of biomarkers for assessing physiological health and nutritional balance, for tracking loss of essential species from the body and for evaluating exposure to hazardous substances. The growing interest in this relatively underexplored class of biofluid arises in part from its non-invasive ability for capture and analysis. The simplest devices, and the only ones that are commercially available, exploit soft microfluidic constructs and colorimetric assays with purely passive modes of operation. The most sophisticated platforms exploit batteries, electronic components and radio hardware for inducing sweat, for electrochemical evaluation of its content and for wireless transmission of this information. The work reported here introduces a technology that combines the advantages of these two different approaches, in the form of a cost-effective, easy-to-use device that supports on-demand evaluation of multiple biomarkers in sweat. This flexible, skin-interfaced, miniaturized system incorporates a hydrogel that contains an approved drug to activate eccrine sweat glands, electrodes and a simple circuit and battery to delivery this drug by iontophoresis through the surface of the skin, microfluidic channels and microreservoirs to capture the induced sweat, and multiple colorimetric assays to evaluate the concentrations of chloride, zinc, and iron. As demonstrated in healthy human participants monitored before and after a meal, such devices yield results that match those of traditional laboratory analysis techniques. Clinical studies that involve cystic fibrosis pediatric patients illustrate the use of this technology as a simple, painless, and reliable alternative to traditional hospital systems for measurements of sweat chloride.


Assuntos
Técnicas Biossensoriais , Suor , Humanos , Criança , Cloretos , Colorimetria , Biomarcadores
2.
Cell ; 187(2): 446-463.e16, 2024 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-38242087

RESUMO

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Modelos Biológicos , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Epigenômica , Genômica , Glioblastoma/genética , Glioblastoma/patologia , Análise de Célula Única , Microambiente Tumoral , Heterogeneidade Genética
3.
J Pediatr Surg ; 59(3): 515-521, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38092651

RESUMO

BACKGROUND: Clostridioides Difficile Infection (CDI) is a serious antibiotic related complication that has been reported among children undergoing treatment of appendicitis. CDI likelihood amongst different empiric antibiotic regimens for appendicitis remains unclear but likely has important implications for antibiotic stewardship. METHODS: A retrospective cohort study of the Pediatric Health Information System was used to examine patients ages 1 through 18 who received operative management of acute appendicitis. Common empiric antibiotic regimens 1) Ceftriaxone & Metronidazole (CM) 2) Piperacillin & Tazobactam (PT) and 3) Cefoxitin were compared. Study outcomes were CDI within 28 days post-appendectomy and 30-day post-appendectomy percutaneous drainage procedures. Subset analyses were repeated to only include hospitals that standardized empiric antibiotic choice. RESULTS: Of 105,911 patients, 220 (0.21 %) developed CDI. CDI was more common in patients that received CM (CM 0.29 % vs PT 0.15 % vs Cefoxitin 0.18 %; P < 0.01). On adjusted analysis, PT was associated with a lower likelihood of CDI (OR, 0.48; 95%CI, 0.31-0.74) compared to CM which was consistent in hospitals with standardized antibiotic choice. Exposure to more unique antibiotic regimens (OR, 1.70; 95 % CI, 1.50-1.93) and higher total antibiotic days (OR, 1.17; 95 % CI 1.13-1.21) were associated with an increased likelihood of CDI. There was no significant difference in the likelihood of post-appendectomy percutaneous drainage between antibiotic regimens. CONCLUSIONS: CDI is rare following appendectomy for pediatric appendicitis. While PT was associated with statistically lower rates of CDI compared to CM, antibiotic stewardship efforts to avoid mixed regimens and decrease overall antibiotic exposure warrant exploration. LEVEL OF EVIDENCE: Level III.


Assuntos
Apendicite , Infecções por Clostridium , Humanos , Criança , Antibacterianos/uso terapêutico , Cefoxitina , Estudos Retrospectivos , Apendicite/tratamento farmacológico , Apendicite/cirurgia , Resultado do Tratamento , Metronidazol/uso terapêutico , Ceftriaxona/efeitos adversos , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/etiologia , Combinação Piperacilina e Tazobactam
4.
Neuro Oncol ; 26(4): 640-652, 2024 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-38141254

RESUMO

BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (R = 0.85), IDH1 mutation in OD (R = 0.87), and with tumor purity (R = 0.91 for GBM; R = 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (R = 0.52 for GBM; R = 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Oligodendroglioma , Telomerase , Humanos , Neoplasias Encefálicas/patologia , Glioma/patologia , Glioblastoma/genética , Glioblastoma/patologia , Oligodendroglioma/genética , Mutação , Biomarcadores Tumorais/genética , Isocitrato Desidrogenase/genética , Telomerase/genética , Proteínas Proto-Oncogênicas/genética , Proteínas de Ciclo Celular/genética
5.
Cell Rep ; 40(12): 111344, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36130485

RESUMO

Telomerase activation counteracts senescence and telomere erosion caused by uncontrolled proliferation. Epidermal growth factor receptor (EGFR) amplification drives proliferation while telomerase reverse transcriptase promoter (TERTp) mutations underlie telomerase reactivation through recruitment of GA-binding protein (GABP). EGFR amplification and TERTp mutations typically co-occur in glioblastoma, the most common and aggressive primary brain tumor. To determine if these two frequent alterations driving proliferation and immortality are functionally connected, we combine analyses of copy number, mRNA, and protein data from tumor tissue with pharmacologic and genetic perturbations. We demonstrate that proliferation arrest decreases TERT expression in a GABP-dependent manner and elucidate a critical proliferation-to-immortality pathway from EGFR to TERT expression selectively from the mutant TERTp through activation of AMP-mediated kinase (AMPK) and GABP upregulation. EGFR-AMPK signaling promotes telomerase activity and maintains telomere length. These results define how the tumor cell immortality mechanism keeps pace with persistent oncogene signaling and cell cycling.


Assuntos
Glioblastoma , Telomerase , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Glioblastoma/genética , Humanos , Mutação/genética , Oncogenes , RNA Mensageiro , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo
6.
J Surg Res ; 279: 511-517, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35863100

RESUMO

INTRODUCTION: Pediatric appendicitis clinical practice guidelines (CPGs) do not typically address postdischarge healthcare encounters. This study aims to examine common indications for returns to the health system to identify novel quality improvement targets. METHODS: This retrospective cohort study analyzed patients aged 3 to 18 y undergoing appendectomy at a single institution from July 1, 2019, to July 31, 2020. The primary outcome was physical postdischarge encounters comprising emergency department (ED) visits and hospital readmissions. Indications for each encounter were categorized and stratified by appendicitis type (i.e., simple, gangrenous, or perforated). Multivariable logistic regression models were used to estimate association between appendicitis category and postdischarge encounters. RESULTS: Of 434 patients, 240 (55.3%) had simple appendicitis, 77 (17.7%) gangrenous, and 117 (29.9%) perforated appendicitis. Overall, 48 patients had at least one instance of an unplanned postdischarge encounter with a total of 56 unplanned ED presentations and 24 readmissions. Perforated patients were significantly more likely to experience postdischarge ED (odds ratio 2.55; 95% confidence interval 1.29-5.02) and readmission encounters (odds ratio 6.63; 95% confidence interval 2.28-19.28). Common indications for ED encounters included abdominal pain (n = 20) with 25.0% readmitted, abdominal pain and gastrointestinal symptoms (e.g., diarrhea, vomiting, distention) (n = 16) with 87.5% readmitted, and incision concerns (n = 6) with 16.7% readmitted. Common indications for readmissions included intraabdominal abscesses (n = 8) and small bowel obstruction (n = 4). CONCLUSIONS: Assessing indications for postdischarge healthcare encounters enables identification of novel quality improvement targets, including proactively addressing incision concerns and abdominal pain.


Assuntos
Apendicectomia , Apendicite , Dor Abdominal/etiologia , Dor Abdominal/cirurgia , Assistência ao Convalescente , Apendicite/cirurgia , Criança , Atenção à Saúde , Gangrena , Humanos , Alta do Paciente , Readmissão do Paciente , Melhoria de Qualidade , Estudos Retrospectivos
9.
Indian Pediatr ; 58(4): 358-362, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452767

RESUMO

OBJECTIVE: To study clinical characteristics and outcome of children with admitted to a paediatric hospital in Mumbai, India. METHODS: Review of medical records of 969 children admitted between 19 March and 7 August, 2020, to assess the clinico-demographic characteristics, disease severity and factors predicting outcome in COVID-19 children. Variables were compared between children who were previously healthy (Group I) and those with co-morbidity (Group II). RESULTS: 123 (71 boys) children with median (IQR) age of 3 (0.7- 6) years were admitted, of which 47 (38%) had co-morbidities. 39 (32 %) children required intensive care and 14 (11.4%) died. Male sex, respiratory manifestation, oxygen saturation <94%; at admission, mechanical ventilation, inotrope, hospital stay of <10 days were independent predictors of mortality. Oxygen saturation <94% at admission (OR 35.9, 95% CI 1.5-856) and hospital stay <10 days (OR 9.1, 95% CI 1.04-99.1) were significant. CONCLUSION: COVID-19 in children with co-morbidities causes severe disease. Association of mortality with oxygen saturation by pulse oximeter <94% on admission, and hospital stay <10 days, needs further evaluation.


Assuntos
COVID-19 , Cardiotônicos/uso terapêutico , Hospitalização/estatística & dados numéricos , Hipóxia , Unidades de Terapia Intensiva Pediátrica/estatística & dados numéricos , Respiração Artificial , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/terapia , Pré-Escolar , Comorbidade , Feminino , Humanos , Hipóxia/diagnóstico , Hipóxia/etiologia , Índia/epidemiologia , Masculino , Mortalidade , Avaliação de Processos e Resultados em Cuidados de Saúde , Prognóstico , Respiração Artificial/métodos , Respiração Artificial/estatística & dados numéricos , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença
10.
Proc Natl Acad Sci U S A ; 117(35): 21319-21327, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817489

RESUMO

Organisms can adapt to a broad spectrum of sudden and dramatic changes in their environment. These abrupt changes are often perceived as stress and trigger responses that facilitate survival and eventual adaptation. The ubiquitin-proteasome system (UPS) is involved in most cellular processes. Unsurprisingly, components of the UPS also play crucial roles during various stress response programs. The budding yeast SCFMet30 complex is an essential cullin-RING ubiquitin ligase that connects metabolic and heavy metal stress to cell cycle regulation. Cadmium exposure results in the active dissociation of the F-box protein Met30 from the core ligase, leading to SCFMet30 inactivation. Consequently, SCFMet30 substrate ubiquitylation is blocked and triggers a downstream cascade to activate a specific transcriptional stress response program. Signal-induced dissociation is initiated by autoubiquitylation of Met30 and serves as a recruitment signal for the AAA-ATPase Cdc48/p97, which actively disassembles the complex. Here we show that the UBX cofactor Shp1/p47 is an additional key element for SCFMet30 disassembly during heavy metal stress. Although the cofactor can directly interact with the ATPase, Cdc48 and Shp1 are recruited independently to SCFMet30 during cadmium stress. An intact UBX domain is crucial for effective SCFMet30 disassembly, and a concentration threshold of Shp1 recruited to SCFMet30 needs to be exceeded to initiate Met30 dissociation. The latter is likely related to Shp1-mediated control of Cdc48 ATPase activity. This study identifies Shp1 as the crucial Cdc48 cofactor for signal-induced selective disassembly of a multisubunit protein complex to modulate activity.


Assuntos
Proteínas F-Box/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteína com Valosina/metabolismo , Cádmio , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Domínios Proteicos , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales , Estresse Fisiológico
11.
Neuro Oncol ; 22(11): 1580-1590, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-32166314

RESUMO

BACKGROUND: Emerging data suggest that a subset of patients with diffuse isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG) who receive adjuvant temozolomide (TMZ) recur with hypermutation in association with malignant progression to higher-grade tumors. It is currently unclear why some TMZ-treated LGG patients recur with hypermutation while others do not. MGMT encodes O6-methylguanine-DNA methyltransferase, a DNA repair protein that removes cytotoxic and potentially mutagenic lesions induced by TMZ. Here, we hypothesize that epigenetic silencing of MGMT by promoter methylation facilitates TMZ-induced mutagenesis in LGG patients and contributes to development of hypermutation at recurrence. METHODS: We utilize a quantitative deep sequencing assay to characterize MGMT promoter methylation in 109 surgical tissue specimens from initial tumors and post-treatment recurrences of 37 TMZ-treated LGG patients. We utilize methylation arrays to validate our sequencing assay, RNA sequencing to assess the relationship between methylation and gene expression, and exome sequencing to determine hypermutation status. RESULTS: Methylation level at the MGMT promoter is significantly higher in initial tumors of patients that develop hypermutation at recurrence relative to initial tumors of patients that do not (45.7% vs 34.8%, P = 0.027). Methylation level in initial tumors can predict hypermutation at recurrence in univariate models and multivariate models that incorporate patient age and molecular subtype. CONCLUSIONS: These findings reveal a mechanistic basis for observed differences in patient susceptibility to TMZ-driven hypermutation. Furthermore, they establish MGMT promoter methylation level as a potential biomarker to inform clinical management of LGG patients, including monitoring and treatment decisions, by predicting risk of hypermutation at recurrence.


Assuntos
Neoplasias Encefálicas , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glioma , Proteínas Supressoras de Tumor/genética , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Metilação de DNA , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Temozolomida/uso terapêutico
12.
Blood Adv ; 2(23): 3428-3442, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30504235

RESUMO

To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.


Assuntos
Quinase I-kappa B/metabolismo , Leucemia Mieloide Aguda/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteômica , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de Sinais
13.
Pharmacol Ther ; 190: 15-23, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29730444

RESUMO

Genes encoding subunits of SWI/SNF chromatin remodeling complexes are collectively mutated in 20% of all human cancers. ARID1A is the SWI/SNF subunit gene that is most frequently mutated, at variable frequencies across molecular and histological subtypes of cancer. Mouse modeling has revealed that the role of ARID1A in tumor suppression is highly dependent upon context. Recent mechanistic studies have identified a crucial role for ARID1A in targeting SWI/SNF complexes to tissue-specific enhancers and in maintaining their chromatin accessibility. In the absence of ARID1A, defects in control of enhancer activity impair developmental programs and cause extensive dysregulation of gene expression, thus driving tumor formation. Roles for ARID1A have also been described in other processes linked to tumor suppression including control of the cell cycle/DNA damage checkpoint, regulation of P53 targets, and telomerase activation. Here, we synthesize a mechanistic understanding of the role of ARID1A in tumor suppression and discuss the implications of these new discoveries for therapy.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Dano ao DNA/genética , Proteínas de Ligação a DNA , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Mutação , Neoplasias/patologia
15.
Nat Genet ; 49(2): 296-302, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27941798

RESUMO

Genes encoding subunits of SWI/SNF (BAF) chromatin-remodeling complexes are collectively mutated in ∼20% of all human cancers. Although ARID1A is the most frequent target of mutations, the mechanism by which its inactivation promotes tumorigenesis is unclear. Here we demonstrate that Arid1a functions as a tumor suppressor in the mouse colon, but not the small intestine, and that invasive ARID1A-deficient adenocarcinomas resemble human colorectal cancer (CRC). These tumors lack deregulation of APC/ß-catenin signaling components, which are crucial gatekeepers in common forms of intestinal cancer. We find that ARID1A normally targets SWI/SNF complexes to enhancers, where they function in coordination with transcription factors to facilitate gene activation. ARID1B preserves SWI/SNF function in ARID1A-deficient cells, but defects in SWI/SNF targeting and control of enhancer activity cause extensive dysregulation of gene expression. These findings represent an advance in colon cancer modeling and implicate enhancer-mediated gene regulation as a principal tumor-suppressor function of ARID1A.


Assuntos
Neoplasias do Colo/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Células HCT116 , Humanos , Camundongos , Mutação/genética , beta Catenina/genética
16.
PLoS Genet ; 11(12): e1005727, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26656496

RESUMO

Abundance of substrate receptor subunits of Cullin-RING ubiquitin ligases (CRLs) is tightly controlled to maintain the full repertoire of CRLs. Unbalanced levels can lead to sequestration of CRL core components by a few overabundant substrate receptors. Numerous diseases, including cancer, have been associated with misregulation of substrate receptor components, particularly for the largest class of CRLs, the SCF ligases. One relevant mechanism that controls abundance of their substrate receptors, the F-box proteins, is autocatalytic ubiquitylation by intact SCF complex followed by proteasome-mediated degradation. Here we describe an additional pathway for regulation of F-box proteins on the example of yeast Met30. This ubiquitylation and degradation pathway acts on Met30 that is dissociated from Skp1. Unexpectedly, this pathway required the cullin component Cdc53/Cul1 but was independent of the other central SCF component Skp1. We demonstrated that this non-canonical degradation pathway is critical for chromosome stability and effective defense against heavy metal stress. More importantly, our results assign important biological functions to a sub-complex of cullin-RING ligases that comprises Cdc53/Rbx1/Cdc34, but is independent of Skp1.


Assuntos
Proteínas Culina/genética , Proteínas F-Box/genética , Proteínas Ligases SKP Culina F-Box/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitinação/genética , Sequência de Aminoácidos , Cádmio/toxicidade , Ciclo Celular/genética , Divisão Celular/genética , Homeostase/efeitos dos fármacos , Redes e Vias Metabólicas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Saccharomyces cerevisiae , Ubiquitinas/genética
17.
FASEB J ; 29(10): 4201-13, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26116701

RESUMO

Parasite lipids can serve as signaling molecules, important membrane components, energy suppliers, and pathogenesis factors critical for survival. Functional roles of lipid changes in response to drug-generated stress in parasite survival remains unclear. To investigate this, Leishmania donovani parasites, the causative agents of kala-azar, were exposed to the antileishmanial agent potassium antimony tartrate (PAT) (half-maximal inhibitory concentration ∼ 284 µg/ml). Analysis of cell extracts using gas chromatography-mass spectrometry showed significant increases in very long-chain fatty acids (VLCFAs) prior to an increase in ergosterol in PAT-treated parasites as compared with vehicle-treated controls. Ergosterol biosynthesis inhibition during PAT treatment decreased cell viability. VLCFA inhibition with specific inhibitors completely abrogated ergosterol upsurge followed by a reduction in cell viability. Following PAT-induced VLCFA increase, an upsurge in reactive oxygen species (ROS) occurred and inhibition of this ROS with antioxidants abrogated ergosterol increase. Genetically engineered parasites expressing low constitutive ergosterol levels showed more susceptibility to PAT as compared with wild-type control cells but ergosterol supplementation during PAT treatment increased cell viability. In conclusion, we propose that during antimony treatment, the susceptibility of parasites is determined by the levels of cellular ergosterol that are regulated by oxidative stress generated by VLCFAs.


Assuntos
Antimônio/farmacologia , Ergosterol/biossíntese , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/metabolismo , Acetamidas/farmacologia , Antiprotozoários/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fragmentação do DNA/efeitos dos fármacos , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Engenharia Genética , Hemólise/efeitos dos fármacos , Humanos , Leishmania donovani/genética , Leishmaniose Visceral/parasitologia , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/metabolismo , Espécies Reativas de Oxigênio/metabolismo
18.
Methods Mol Biol ; 1205: 37-44, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25213238

RESUMO

Epitope tagging of genes is a powerful technique facilitating assays for gene function, determination of subcellular distribution of proteins, affinity purification, study of protein interaction with other proteins, DNA or RNA, and any other antibody-based approach in the absence of protein-specific antibodies. Here, we describe a one-step PCR-based strategy for insertion of epitope tags at the chromosomal locus. This method takes advantage of efficient homologous recombination in yeast. PCR amplified tags are directed to desired chromosomal loci with the help of primer-encoded flanking homologous sequences enabling selective epitope tagging of genes of interest.


Assuntos
Epitopos/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Reação em Cadeia da Polimerase/métodos , Leveduras/genética , Western Blotting/métodos , Cromossomos Fúngicos , Primers do DNA/genética , Mapeamento de Epitopos , Loci Gênicos , Recombinação Homóloga , Transformação Genética
19.
Antimicrob Agents Chemother ; 58(8): 4682-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24890590

RESUMO

Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 half-knockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.


Assuntos
Amidinas/farmacologia , Antiprotozoários/farmacologia , Furanos/farmacologia , Cetoconazol/farmacologia , Leishmania donovani/efeitos dos fármacos , Estágios do Ciclo de Vida/efeitos dos fármacos , Proteínas de Protozoários/genética , Esterol 14-Desmetilase/genética , Triazóis/farmacologia , Animais , Cultura Axênica , Western Blotting , Cricetinae , Combinação de Medicamentos , Resistência a Medicamentos , Sinergismo Farmacológico , Regulação da Expressão Gênica , Leishmania donovani/genética , Leishmania donovani/crescimento & desenvolvimento , Leishmania donovani/metabolismo , Estágios do Ciclo de Vida/genética , Camundongos , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Proteínas de Protozoários/metabolismo , Esterol 14-Desmetilase/metabolismo
20.
Mol Cell ; 48(2): 288-97, 2012 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-23000173

RESUMO

A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA(+) ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCF(Met30) ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.


Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Proteínas Ligases SKP Culina F-Box , Saccharomyces cerevisiae/enzimologia , Ubiquitinação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Cádmio/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Hidrólise/efeitos dos fármacos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Especificidade por Substrato , Proteína com Valosina
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