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1.
Cell ; 165(3): 643-55, 2016 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-27104980

RESUMO

Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.


Assuntos
Glicina/análogos & derivados , Proteínas de Ligação a RNA/química , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Cristalografia por Raios X , Dimerização , Glicina/administração & dosagem , Glicina/química , Glicina/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Neoplasias Pancreáticas/tratamento farmacológico , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência , Sulfonas/administração & dosagem , Sulfonas/química , Proteínas ras/metabolismo , Quinase 1 Polo-Like
2.
Mol Cell ; 83(23): 4239-4254.e10, 2023 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-38065062

RESUMO

A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver.


Assuntos
Leucemia , Síndromes Mielodisplásicas , Neoplasias , Metilação de RNA , Fatores de Processamento de Serina-Arginina , Humanos , Leucemia/genética , Síndromes Mielodisplásicas/genética , Neoplasias/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Metilação de RNA/genética
3.
Plant Cell ; 36(2): 447-470, 2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-37820736

RESUMO

Plant nucleotide-binding leucine-rich repeat (NLRs) immune receptors directly or indirectly recognize pathogen-secreted effector molecules to initiate plant defense. Recognition of multiple pathogens by a single NLR is rare and usually occurs via monitoring for changes to host proteins; few characterized NLRs have been shown to recognize multiple effectors. The barley (Hordeum vulgare) NLR gene Mildew locus a (Mla) has undergone functional diversification, and the proteins encoded by different Mla alleles recognize host-adapted isolates of barley powdery mildew (Blumeria graminis f. sp. hordei [Bgh]). Here, we show that Mla3 also confers resistance to the rice blast fungus Magnaporthe oryzae in a dosage-dependent manner. Using a forward genetic screen, we discovered that the recognized effector from M. oryzae is Pathogenicity toward Weeping Lovegrass 2 (Pwl2), a host range determinant factor that prevents M. oryzae from infecting weeping lovegrass (Eragrostis curvula). Mla3 has therefore convergently evolved the capacity to recognize effectors from diverse pathogens.


Assuntos
Ascomicetos , Eragrostis , Hordeum , Magnaporthe , Virulência/genética , Hordeum/genética , Eragrostis/metabolismo , Plantas/metabolismo , Especificidade de Hospedeiro , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
4.
Plant Cell ; 34(9): 3214-3232, 2022 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-35689625

RESUMO

Fungal interactions with plant roots, either beneficial or detrimental, have a crucial impact on agriculture and ecosystems. The cosmopolitan plant pathogen Fusarium oxysporum (Fo) provokes vascular wilts in more than a hundred different crops. Isolates of this fungus exhibit host-specific pathogenicity, which is conferred by lineage-specific Secreted In Xylem (SIX) effectors encoded on accessory genomic regions. However, such isolates also can colonize the roots of other plants asymptomatically as endophytes or even protect them against pathogenic strains. The molecular determinants of endophytic multihost compatibility are largely unknown. Here, we characterized a set of Fo candidate effectors from tomato (Solanum lycopersicum) root apoplastic fluid; these early root colonization (ERC) effectors are secreted during early biotrophic growth on main and alternative plant hosts. In contrast to SIX effectors, ERCs have homologs across the entire Fo species complex as well as in other plant-interacting fungi, suggesting a conserved role in fungus-plant associations. Targeted deletion of ERC genes in a pathogenic Fo isolate resulted in reduced virulence and rapid activation of plant immune responses, while ERC deletion in a nonpathogenic isolate led to impaired root colonization and biocontrol ability. Strikingly, some ERCs contribute to Fo infection on the nonvascular land plant Marchantia polymorpha, revealing an evolutionarily conserved mechanism for multihost colonization by root infecting fungi.


Assuntos
Fusarium , Solanum lycopersicum , Ecossistema , Doenças das Plantas
5.
Nucleic Acids Res ; 51(22): 12224-12241, 2023 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-37953292

RESUMO

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family. We discovered that human TATDN2 has RNA 3' exonuclease and endonuclease activity on double-stranded hairpin RNA structures. Given the cleavage of hairpin RNA by TATDN2, and that BRCA1-deficient cells have difficulty resolving R-loops, we tested whether TATDN2 could resolve R-loops. Using in vitro biochemical reconstitution assays, we found TATDN2 bound to R-loops and degraded the RNA strand but not DNA of multiple forms of R-loops in vitro in a Mg2+-dependent manner. Mutations in amino acids E593 and E705 predicted by Alphafold-2 to chelate an essential Mg2+ cation completely abrogated this R-loop resolution activity. Depleting TATDN2 increased cellular R-loops, DNA damage and chromosomal instability. Loss of TATDN2 resulted in poor replication fork progression in the presence of increased R-loops. Significantly, we found that TATDN2 is essential for survival of BRCA1-deficient cancer cells, but much less so for cognate BRCA1-repleted cancer cells. Thus, we propose that TATDN2 is a novel target for therapy of BRCA1-deficient cancers.


Assuntos
Neoplasias , Humanos , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Replicação do DNA , Instabilidade Genômica , Magnésio , MicroRNAs/genética , Neoplasias/genética , Estruturas R-Loop
6.
Am J Gastroenterol ; 119(7): 1365-1372, 2024 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-38131615

RESUMO

INTRODUCTION: Intravenous corticosteroids are the mainstay of treatment of patients hospitalized with acute severe ulcerative colitis (ASUC). However, 30%-40% of the patients are refractory to corticosteroids. We investigated whether addition of tofacitinib to corticosteroids improved the treatment responsiveness in patients with ASUC. METHODS: This single-center, double-blind, placebo-controlled trial randomized adult patients with ASUC (defined by the Truelove Witts severity criteria) to receive either tofacitinib (10 mg thrice daily) or a matching placebo for 7 days while continuing intravenous corticosteroids (hydrocortisone 100 mg every 6 hours). The primary end point was response to treatment (decline in the Lichtiger index by >3 points and an absolute score <10 for 2 consecutive days without the need for rescue therapy) by day 7. The key secondary outcome was the cumulative probability of requiring initiation of infliximab or undergoing colectomy within 90 days following randomization. All analyses were performed in the intention-to-treat population. RESULTS: A total of 104 patients were randomly assigned to a treatment group (53 to tofacitinib and 51 to placebo). At day 7, response to treatment was achieved in 44/53 (83.01%) patients receiving tofacitinib vs 30/51 (58.82%) patients receiving placebo (odds ratio 3.42, 95% confidence interval 1.37-8.48, P = 0.007). The need for rescue therapy by day 7 was lower in the tofacitinib arm (odds ratio 0.27, 95% confidence interval 0.09-0.78, P = 0.01). The cumulative probability of need for rescue therapy at day 90 was 0.13 in patients who received tofacitinib vs 0.38 in patients receiving placebo (log-rank P = 0.003). Most of the treatment-related adverse effects were mild. One patient, receiving tofacitinib, developed dural venous sinus thrombosis. DISCUSSION: In patients with ASUC, combination of tofacitinib and corticosteroids improved treatment responsiveness and decreased the need for rescue therapy.


Assuntos
Colite Ulcerativa , Piperidinas , Pirimidinas , Pirróis , Humanos , Piperidinas/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Pirimidinas/uso terapêutico , Masculino , Feminino , Método Duplo-Cego , Adulto , Pirróis/uso terapêutico , Pirróis/administração & dosagem , Pessoa de Meia-Idade , Doença Aguda , Resultado do Tratamento , Índice de Gravidade de Doença , Quimioterapia Combinada , Inibidores de Proteínas Quinases/uso terapêutico , Colectomia , Infliximab/uso terapêutico
7.
Dement Geriatr Cogn Disord ; : 1-11, 2024 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-39068924

RESUMO

INTRODUCTION: The safety and therapeutic effects of Gingko biloba extract EGb 761® to treat cognitive decline have been demonstrated in numerous clinical trials. However, trials in Indian populations have been lacking. METHODS: This open-label, multicenter, single-arm, phase IV trial enrolled 150 patients aged ≥50 years with major neurocognitive disorder due to Alzheimer's disease, major vascular neurocognitive disorder, or mixed forms of both according to the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5) criteria and a Mini-Mental State Examination (MMSE) score of 12-24. Patients took 120 mg EGb 761® twice daily for 18 weeks. Therapeutic effects were assessed by CERAD constructional praxis and recall of constructional praxis (CERAD CP, CERAD recall of CP), Trail-Making Test (TMT), Behavioral Pathology in Alzheimer's Disease (BEHAVE-AD), Clinical Global Impressions (CGI) scale, and 11-point box scales for tinnitus and vertigo. Safety assessment was based on the occurrence of adverse events as well as changes in clinical, laboratory, and functional parameters. RESULTS: After 18 weeks, significant improvements compared to baseline were found in constructional praxis (CERAD CP, p < 0.0001), memory (CERAD recall of CP, p < 0.0001), speed and executive functioning (TMT A, p < 0.0001; TMT B, p < 0.0001), and behavioral symptoms (BEHAVE-AD, p < 0.0001). Forty-five adverse events were reported in 33 (22.0%) patients in total, including ten presumed adverse drug reactions in 9 (6.0%) patients. Headache and diarrhea of mild-to-moderate severity were the most frequent events. Two serious adverse events, both considered unrelated to the study drug, occurred in 2 (1.3%) patients. CONCLUSION: This study confirmed the favorable safety profile and suggested therapeutic benefits of EGb 761® in Indian patients with major neurocognitive disorder.

8.
CNS Spectr ; 29(2): 109-118, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38053347

RESUMO

BACKGROUND: Repetitive transcranial magnetic stimulation (rTMS) has been increasingly used for treating obsessive-compulsive disorder (OCD). Although several meta-analyses have explored its effectiveness and safety, there is no umbrella review specifically focused on rTMS for OCD. This umbrella review followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and analyzed relevant meta-analyses on rTMS for OCD. METHODS: Twenty-three articles were identified from PubMed, and after screening, 12 meta-analyses were included in the review. The studies analyzed in the meta-analyses ranged from 10 to 27, with total participants ranging from 282 to 791. The most commonly studied regions were the dorsolateral prefrontal cortex (DLPFC), supplementary motor area (SMA), and orbito-frontal cortex (OFC). RESULT: The majority of the meta-analyses consistently supported the effectiveness of rTMS in reducing OCD symptoms when applied to the DLPFC and SMA. Encouraging results were also observed when targeting the medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC) through deep transcranial magnetic stimulation (dTMS). However, there was a high level of heterogeneity in the findings of nine out of 12 meta-analyses. CONCLUSION: In conclusion, existing evidence suggests that rTMS targeting the DLPFC and SMA consistently reduces OCD symptoms, but targeting the mPFC and ACC through dTMS shows variable results. However, the high heterogeneity in the study findings indicates a need for further research and standardization in the field.


Assuntos
Córtex Motor , Transtorno Obsessivo-Compulsivo , Humanos , Transtorno Obsessivo-Compulsivo/terapia , Córtex Pré-Frontal , Estimulação Magnética Transcraniana/métodos , Resultado do Tratamento , Metanálise como Assunto
9.
Mol Cell ; 58(5): 767-79, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25936804

RESUMO

The PIDDosome-PIDD-RAIDD-caspase-2 complex-is a proapoptotic caspase-activation platform of elusive significance. DNA damage can initiate complex assembly via ATM phosphorylation of the PIDD death domain (DD), which enables RAIDD recruitment to PIDD. In contrast, the mechanisms limiting PIDDosome formation have remained unclear. We identify the mitotic checkpoint factor BubR1 as a direct PIDDosome inhibitor, acting in a noncanonical role independent of Mad2. Following its phosphorylation by ATM at DNA breaks, "primed" PIDD relocates to kinetochores via a direct interaction with BubR1. BubR1 binds the PIDD DD, competes with RAIDD recruitment, and negates PIDDosome-mediated apoptosis after ionizing radiation. The PIDDosome thus sequentially integrates DNA damage and mitotic checkpoint signals to decide cell fate in response to genotoxic stress. We further show that by sequestering PIDD at the kinetochore, BubR1 acts to delay PIDDosome formation until the next cycle, defining a new mechanism by which cells evade apoptosis during mitosis.


Assuntos
Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Caspase 2/metabolismo , Cisteína Endopeptidases/metabolismo , Dano ao DNA , Células HCT116 , Células HeLa , Humanos , Cinetocoros/enzimologia , Proteínas Mad2/metabolismo , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais
10.
Arch Pharm (Weinheim) ; 355(12): e2200299, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36058643

RESUMO

Quantum dots (QDs) are semiconducting nanoparticles having different optical and electrical properties when compared to larger particles. They exhibit photoluminescence when irradiated with ultraviolet light, which is due to the transition of an excited electron from the valence band to the conductance band followed by the return of the exciting electron back into the valence band. The size and material of QDs can affect their optical and other properties too. The QDs possess special attributes like high brightness, protection from photobleaching, photostability, color tunability, low toxicity, low production cost, a multiplexing limit, and a high surface-to-volume proportion, which make them a promising tool for biomedical applications. Here, in this study, we summarize the utilization of QDs in different applications including bioimaging, diagnostics, immunostaining, single-cell analysis, drug delivery, and protein detection. Moreover, we discuss the advantages and challenges of using QDs in biomedical applications when compared with other conventional tools.


Assuntos
Nanopartículas , Pontos Quânticos , Pontos Quânticos/metabolismo , Relação Estrutura-Atividade , Sistemas de Liberação de Medicamentos/métodos
11.
Radiology ; 297(1): E197-E206, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32407255

RESUMO

Background Chest radiography has not been validated for its prognostic utility in evaluating patients with coronavirus disease 2019 (COVID-19). Purpose To analyze the prognostic value of a chest radiograph severity scoring system for younger (nonelderly) patients with COVID-19 at initial presentation to the emergency department (ED); outcomes of interest included hospitalization, intubation, prolonged stay, sepsis, and death. Materials and Methods In this retrospective study, patients between the ages of 21 and 50 years who presented to the ED of an urban multicenter health system from March 10 to March 26, 2020, with COVID-19 confirmation on real-time reverse transcriptase polymerase chain reaction were identified. Each patient's ED chest radiograph was divided into six zones and examined for opacities by two cardiothoracic radiologists, and scores were collated into a total concordant lung zone severity score. Clinical and laboratory variables were collected. Multivariable logistic regression was used to evaluate the relationship between clinical parameters, chest radiograph scores, and patient outcomes. Results The study included 338 patients: 210 men (62%), with median age of 39 years (interquartile range, 31-45 years). After adjustment for demographics and comorbidities, independent predictors of hospital admission (n = 145, 43%) were chest radiograph severity score of 2 or more (odds ratio, 6.2; 95% confidence interval [CI]: 3.5, 11; P < .001) and obesity (odds ratio, 2.4 [95% CI: 1.1, 5.4] or morbid obesity). Among patients who were admitted, a chest radiograph score of 3 or more was an independent predictor of intubation (n = 28) (odds ratio, 4.7; 95% CI: 1.8, 13; P = .002) as was hospital site. No significant difference was found in primary outcomes across race and ethnicity or those with a history of tobacco use, asthma, or diabetes mellitus type II. Conclusion For patients aged 21-50 years with coronavirus disease 2019 presenting to the emergency department, a chest radiograph severity score was predictive of risk for hospital admission and intubation. © RSNA, 2020 Online supplemental material is available for this article.


Assuntos
Infecções por Coronavirus , Pulmão/diagnóstico por imagem , Pandemias , Pneumonia Viral , Adulto , Betacoronavirus , COVID-19 , Infecções por Coronavirus/diagnóstico por imagem , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Intubação Intratraqueal/estatística & dados numéricos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/diagnóstico por imagem , Pneumonia Viral/epidemiologia , Pneumonia Viral/patologia , Valor Preditivo dos Testes , Prognóstico , Radiografia Torácica , Estudos Retrospectivos , SARS-CoV-2 , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Adulto Jovem
12.
J Gastroenterol Hepatol ; 35(3): 418-424, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31408913

RESUMO

BACKGROUND AND AIM: Fecal microbiota transplantation (FMT) targets gut microbiome dysbiosis and is an emerging therapy for ulcerative colitis (UC). Although initial results with FMT in patients with active UC are encouraging, data regarding its acceptability, tolerability, and safety are scant. METHODS: A retrospective analysis of patients with active UC (Mayo clinic score ≥ 4), who received multisession FMT (at weeks 0, 2, 6, 10, 14, 18, and 22) via colonoscopy between June 2016 and June 2018, was performed. Patient acceptability, tolerability, and immediate and long-term safety of the therapy were assessed. RESULTS: Of the 129 patients with active UC who were offered FMT, 101 patients consented, giving acceptability of 78.3%. Fecal slurry retention time improved with each session (3.27 ± 1.06 h for the first session vs 5.12 ± 0.5 h for the seventh session). Abdominal discomfort, flatulence, abdominal distension, borborygmi, and low-grade fever (30.8%, 15.9%, 9.8%, 7.9%, and 7.6%, respectively) were the most common post-procedural short-term adverse events. Long-term adverse events included new-onset urticaria (n = 2, 4.3%), arthritis/arthralgia (n = 3, 6.5%), depression (n = 1, 2.2%), partial sensorineural hearing loss (n = 1, 2.2%), and allergic bronchitis (n = 1, 2.2%). Thirteen (12.9%) patients dropped out because of adverse events. CONCLUSION: Fecal microbiota transplantation appears to be a safe and well-tolerated procedure, with good acceptability in patients with active UC.


Assuntos
Colite Ulcerativa/terapia , Transplante de Microbiota Fecal , Adulto , Transplante de Microbiota Fecal/efeitos adversos , Feminino , Humanos , Masculino , Estudos Retrospectivos , Segurança , Resultado do Tratamento , Adulto Jovem
13.
New Phytol ; 222(3): 1474-1492, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30663769

RESUMO

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long time scales without macroscopic symptoms. However, how plant-pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown. Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi. The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of these, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection. Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long-lived infection of its perennial hosts.


Assuntos
Basidiomycota/genética , Brassicaceae/microbiologia , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Transcriptoma/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiologia , Basidiomycota/patogenicidade , Brassicaceae/imunologia , Sequência Conservada , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Ontologia Genética , Anotação de Sequência Molecular , Filogenia , Plantas Geneticamente Modificadas , Ácido Salicílico/metabolismo , Especificidade da Espécie , Sintenia/genética , Zea mays/microbiologia
14.
PLoS Biol ; 14(4): e1002442, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27082731

RESUMO

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5'-TCCRAC-3'; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5'-TCCRAC-3'). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5'-TCCRAC-3'). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.


Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Sequência de Bases , Metilação de DNA , Hidrólise , Dados de Sequência Molecular
15.
Xenobiotica ; 49(6): 646-654, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29898636

RESUMO

1-Aminobenzotriazole (ABT) is a mechanism-based inactivator of major cytochrome P450 (CYP) enzymes, which is used in multiple mechanistic studies. The purpose was to evaluate the effect of 2 and 16-h pretreatment regimens of ABT on the exposures of triazolam in rat. Another objective was to evaluate the effect of ABT on gastric emptying of acetaminophen. Plasma area under the curve (AUC) of triazolam was increased by 101-fold and 81-fold for the rats pretreated with ABT at 2 and 16 h, respectively, compared to control rats. Time to reach maximum concentration was 0.3, 4.8 and 3.7 h in control, 2 and 16-h pretreatment animals, respectively. In the case of acetaminophen, where Tmax was not delayed, the mean absorption time (MAT) in control, 2 and 16 h ABT pretreatment groups were 0.3, 4.6 and 2.9 h, respectively, suggesting delayed absorption. This hypothesis was further supported by GastroPlusTM simulation. In summary, extent of triazolam absorption was increased to a similar extent with both 2 and 16 h ABT pretreatment regimens, suggesting that either of the regimen can be used to increase parent exposures in rat. With ABT pretreatment, delayed absorption of triazolam and acetaminophen was observed, as suggested by delay in Tmax and MAT, respectively.


Assuntos
Inibidores das Enzimas do Citocromo P-450/farmacologia , Esvaziamento Gástrico/efeitos dos fármacos , Triazolam/farmacocinética , Triazóis/farmacologia , Acetaminofen/farmacocinética , Administração Oral , Animais , Área Sob a Curva , Inibidores das Enzimas do Citocromo P-450/administração & dosagem , Sistema Enzimático do Citocromo P-450/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Triazolam/administração & dosagem , Triazóis/administração & dosagem
16.
Emerg Radiol ; 26(3): 257-262, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30613930

RESUMO

PURPOSE: The main purpose of the study is to determine if the presence of a particular computed tomography (CT) imaging finding, a bursal lipohematoma, portends the presence of a concomitant rotator cuff tear (RCT) in patients with proximal humerus fractures by reviewing previous CTs. METHODS: Three hundred eighty-six CT scans were retrospectively reviewed by two board-certified radiologists to determine the presence of a proximal humerus fracture and to assess for the presence of a subacromial/subdeltoid or subcoracoid bursal hematoma. The medical record including operative documentation was then examined in the patients with proximal humerus fractures, with or without a concomitant bursal lipohematoma. RESULTS: Of the surgically managed patients, four had an intraoperative diagnosis of RCT. The preoperative CT scans of all of these patients demonstrated a bursal lipohematoma. Additionally, a non-surgically managed patient with a subacromial/subdeltoid bursal lipohematoma on CT scan was found to have a RCT on subsequent MRI. Of note, a rotator cuff tear was not documented in operative reports of patients with CT scans that were not found to contain a bursal lipohematoma. CONCLUSIONS: Bursal lipohematoma is a potentially useful preoperative CT sign of full thickness rotator cuff tear in patients with proximal humerus fractures, providing the clinician with more information in the optimization of the management approach.


Assuntos
Bolsa Sinovial/diagnóstico por imagem , Bolsa Sinovial/lesões , Hematoma/diagnóstico por imagem , Lesões do Manguito Rotador/diagnóstico por imagem , Fraturas do Ombro/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Adulto , Bolsa Sinovial/cirurgia , Diagnóstico Diferencial , Feminino , Hematoma/cirurgia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Lesões do Manguito Rotador/cirurgia , Fraturas do Ombro/cirurgia
17.
Plant Cell ; 27(11): 3277-89, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26566920

RESUMO

Magnaporthe oryzae is the causal agent of rice blast disease, the most devastating disease of cultivated rice (Oryza sativa) and a continuing threat to global food security. To cause disease, the fungus elaborates a specialized infection cell called an appressorium, which breaches the cuticle of the rice leaf, allowing the fungus entry to plant tissue. Here, we show that the exocyst complex localizes to the tips of growing hyphae during vegetative growth, ahead of the Spitzenkörper, and is required for polarized exocytosis. However, during infection-related development, the exocyst specifically assembles in the appressorium at the point of plant infection. The exocyst components Sec3, Sec5, Sec6, Sec8, and Sec15, and exocyst complex proteins Exo70 and Exo84 localize specifically in a ring formation at the appressorium pore. Targeted gene deletion, or conditional mutation, of genes encoding exocyst components leads to impaired plant infection. We demonstrate that organization of the exocyst complex at the appressorium pore is a septin-dependent process, which also requires regulated synthesis of reactive oxygen species by the NoxR-dependent Nox2 NADPH oxidase complex. We conclude that septin-mediated assembly of the exocyst is necessary for appressorium repolarization and host cell invasion.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/fisiologia , Doenças das Plantas/microbiologia , Septinas/metabolismo , Hifas/metabolismo , Imunoprecipitação , Subunidades Proteicas/metabolismo , Transporte Proteico , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/metabolismo , Frações Subcelulares/metabolismo
18.
RNA Biol ; 15(11): 1420-1432, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30362859

RESUMO

RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.


Assuntos
Glioblastoma/tratamento farmacológico , Luteolina/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Terapia Combinada , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Luteolina/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fenótipo , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , RNA/química , RNA/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Radiação Ionizante , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
19.
Proc Natl Acad Sci U S A ; 111(50): 17995-8000, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25453078

RESUMO

A variety of human cancers, including nonsmall cell lung (NSCLC), breast, and colon cancers, are driven by the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases. Having shown that bisphosphonates, a class of drugs used widely for the therapy of osteoporosis and metastatic bone disease, reduce cancer cell viability by targeting HER1, we explored their potential utility in the prevention and therapy of HER-driven cancers. We show that bisphosphonates inhibit colony formation by HER1(ΔE746-A750)-driven HCC827 NSCLCs and HER1(wt)-expressing MB231 triple negative breast cancers, but not by HER(low)-SW620 colon cancers. In parallel, oral gavage with bisphosphonates of mice xenografted with HCC827 or MB231 cells led to a significant reduction in tumor volume in both treatment and prevention protocols. This result was not seen with mice harboring HER(low) SW620 xenografts. We next explored whether bisphosphonates can serve as adjunctive therapies to tyrosine kinase inhibitors (TKIs), namely gefitinib and erlotinib, and whether the drugs can target TKI-resistant NSCLCs. In silico docking, together with molecular dynamics and anisotropic network modeling, showed that bisphosphonates bind to TKIs within the HER1 kinase domain. As predicted from this combinatorial binding, bisphosphonates enhanced the effects of TKIs in reducing cell viability and driving tumor regression in mice. Impressively, the drugs also overcame erlotinib resistance acquired through the gatekeeper mutation T790M, thus offering an option for TKI-resistant NSCLCs. We suggest that bisphosphonates can potentially be repurposed for the prevention and adjunctive therapy of HER1-driven cancers.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/prevenção & controle , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/prevenção & controle , Difosfonatos/farmacologia , Receptores ErbB/antagonistas & inibidores , Animais , Western Blotting , Difosfonatos/uso terapêutico , Reposicionamento de Medicamentos/métodos , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Dinâmica Molecular , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Ensaio Tumoral de Célula-Tronco
20.
Proc Natl Acad Sci U S A ; 111(50): 17989-94, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25453081

RESUMO

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Difosfonatos/farmacologia , Receptores ErbB/antagonistas & inibidores , Modelos Moleculares , Anisotropia , Western Blotting , Linhagem Celular Tumoral , Cristalografia , Difosfonatos/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Fluorescência , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Sais de Tetrazólio , Tiazóis
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