RESUMO
Candida auris is an emerging multidrug-resistant yeast with high mortality. We report the sentinel C. auris case on the United States West Coast in a patient who relocated from India. We identified close phylogenetic relatedness to the South Asia clade and ERG11 Y132F and FKS1 S639Y mutations potentially explaining antifungal resistance.
Assuntos
Antifúngicos/farmacologia , Candida/genética , Candidíase/microbiologia , Farmacorresistência Fúngica Múltipla/genética , Neoplasias Retais/microbiologia , Idoso , California , Candida/classificação , Candida/isolamento & purificação , Candida/patogenicidade , Candidíase/complicações , Candidíase/tratamento farmacológico , Candidíase/patologia , Equinocandinas/farmacologia , Evolução Fatal , Fluconazol/farmacologia , Humanos , Índia , Masculino , Testes de Sensibilidade Microbiana , Filogenia , Neoplasias Retais/complicações , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/patologia , Vigilância de Evento Sentinela , ViagemRESUMO
Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6-NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6-NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6-NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6-NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6-NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Carcinoma Secretor Análogo ao Mamário/genética , Adolescente , Adulto , Idoso , Neoplasias da Mama/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Carcinoma Secretor Análogo ao Mamário/patologia , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Adulto JovemRESUMO
Genetic manipulation of the deadly malaria parasite Plasmodium falciparum remains challenging, but the rise of CRISPR/Cas9-based genome editing tools is increasing the feasibility of altering this parasite's genome in order to study its biology. Of particular interest is the investigation of drug targets and drug resistance mechanisms, which have major implications for fighting malaria. We present a new method for introducing drug resistance mutations in P. falciparum without the use of plasmids or the need for cloning homologous recombination templates. We demonstrate this method by introducing edits into the sodium efflux channel PfATP4 by transfection of a purified CRISPR/Cas9-guide RNA ribonucleoprotein complex and a 200-nucleotide single-stranded oligodeoxynucleotide (ssODN) repair template. Analysis of whole genome sequencing data with the variant-finding program MinorityReport confirmed that only the intended edits were made, and growth inhibition assays confirmed that these mutations confer resistance to the antimalarial SJ733. The method described here is ideally suited for the introduction of mutations that confer a fitness advantage under selection conditions, and the novel finding that an ssODN can function as a repair template in P. falciparum could greatly simplify future editing attempts regardless of the nuclease used or the delivery method.
Assuntos
Sistemas CRISPR-Cas/genética , Resistência a Medicamentos/genética , Genoma de Protozoário , ATPase Trocadora de Hidrogênio-Potássio/genética , Isoquinolinas/farmacologia , Mutação/genética , Plasmodium falciparum/genética , Sequência de Aminoácidos , Sequência de Bases , Vetores Genéticos , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , Plasmídeos , Edição de RNA/genéticaRESUMO
Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the α-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique α-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (â¼10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (â¼25% of total), and finally, other endoproteolytic cuts (â¼65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells.
Assuntos
Apoptose , Bases de Dados de Proteínas , Proteólise , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Caspases/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Humanos , Internet , Células Jurkat , Peptídeo Sintases/metabolismo , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Proteoma/química , Proteoma/metabolismo , Subtilisinas/metabolismoRESUMO
We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401-1407). We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.
Assuntos
Dissulfetos/metabolismo , Peptídeos/química , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas/química , Naftalenossulfonato de Anilina , Dicroísmo Circular , Corantes Fluorescentes , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Lisina/metabolismo , Modelos Moleculares , Peptídeos/genética , Peptídeos/metabolismo , Desnaturação Proteica , Engenharia de Proteínas , Dobramento de Proteína , Estabilidade Proteica , Proteínas/genética , Proteínas/metabolismo , Cloreto de Sódio , Eletricidade Estática , Termodinâmica , UltracentrifugaçãoRESUMO
To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this "Connectivity Map" resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.
Assuntos
Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos/métodos , Perfilação da Expressão Gênica , Expressão Gênica/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Linhagem Celular , Linhagem Celular Tumoral , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Estrogênios/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Inibidores de Histona Desacetilases , Humanos , Limoninas/farmacologia , Obesidade/genética , Obesidade/fisiopatologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenotiazinas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Sirolimo/farmacologia , Sirolimo/uso terapêutico , SoftwareRESUMO
Protein design studies using coiled coils have illustrated the potential of engineering simple peptides to self-associate into polymers and networks. Although basic aspects of self-assembly in protein systems have been demonstrated, it remains a major challenge to create materials whose large-scale structures are well determined from design of local protein-protein interactions. Here, we show the design and characterization of a helical peptide, which uses phased hydrophobic interactions to drive assembly into nanofilaments and fibrils ("nanoropes"). Using the hydrophobic effect to drive self-assembly circumvents problems of uncontrolled self-assembly seen in previous approaches that used electrostatics as a mode for self-assembly. The nanostructures designed here are characterized by biophysical methods including analytical ultracentrifugation, dynamic light scattering, and circular dichroism to measure their solution properties, and atomic force microscopy to study their behavior on surfaces. Additionally, the assembly of such structures can be predictably regulated by using various environmental factors, such as pH, salt, other molecular crowding reagents, and specifically designed "capping" peptides. This ability to regulate self-assembly is a critical feature in creating smart peptide biomaterials.