Nanoparticle-chaperoned urinary 'synthetic biomarkers' for profiling proteases in cancer

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 61-63).Many biomarker-based diagnostics have poor predictive value because of their dependence on naturally occurring endogenous biomolecules to indicate disease. This work presents a diagnostic platform that uses nanoparticles to profile underlying proteolytic signatures of diseases. In this thesis, work is presented on long circulating peptide-nanoparticle probes that can survey, sense, and remotely report on dysregulated protease activities in cancer. In this strategy, iron oxide nanoparticles are utilized as chaperons to deliver protease-specific peptide libraries to tumors whereupon selective cleavage by active proteases releases peptide fragments that are cleared by the renal system into the urine. These peptide fragments are pre-designed with internal photolabile triggers that un-cage isobaric peptide mass tags optimized for multiplexed LC MS/MS quantification. Results demonstrate that such peptide 'synthetic biomarker' panels uncover unique proteolytic signatures that can be correlated with disease states, allowing for the detection of cancer and potential long-term monitoring of disease using an implantable form. This concept of administering prodiagnostic reagents and analyzing remote reporters is amenable to a broad range of protease-dependent complex diseases, such as liver fibrosis and coagulopathies, and infectious disease.by Omar O. Abudayyeh.S.B

RNA targeting with CRISPR–Cas13

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference1-3 can efficiently knockdown RNAs, but it is prone to off-target effects4, and visualizing RNAs typically relies on the introduction of exogenous tags5. Here we demonstrate that the class 2 type VI6,7 RNA-guided RNA-targeting CRISPR-Cas effector Cas13a8(previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049

Structure and Engineering of Francisella novicida Cas9

Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAM, thus expanding the target space of the CRISPR-Cas9 toolbox

Avaliação do cumprimento da NR-18 em função do porte de obra residencial e proposta de lista de verificação da NR-18

O presente trabalho investiga as reais condições dos ambientes de trabalho da indústria da construção, tendo em vista os problemas relacionados à segurança do trabalho nos canteiros de obras. Formulou-se a hipótese de haver discrepância no grau de cumprimento da NR-18 em obras de diferentes portes (pequena, média e grande). Baseado em uma lista de verificação do grau de cumprimento dos requisitos da NR-18, foram avaliados 115 canteiros de obra na cidade de Chapecó - SC. Para cada item da lista de verificação foi atribuída uma nota e, após compilar as informações, estas foram avaliadas conforme os objetivos da pesquisa. Fez-se uma análise crítica para os cinco piores e os cinco melhores itens observados para as obras pequenas, médias e grandes. Analisando-se os resultados é possível afirmar que há uma diferença significativa de aplicação da NR-18 nos diferentes portes de obras, sendo que as obras de grande porte obtiveram uma média final (6,47) significativamente superior em relação às obras pequenas (1,97). Desta forma, foi corroborada a hipótese formulada, podendo se atribuir este resultado a diversos fatores, entre os quais a falta de habilidades gerenciais de segurança e saúde do trabalho nas pequenas empresas. Outra contribuição deste artigo foi a proposta de uma nova lista de verificação para avaliar o grau de cumprimento da NR-18 nos canteiros de obra

Acute kidney injury in patients treated with immune checkpoint inhibitors

BACKGROUND: Immune checkpoint inhibitor-associated acute kidney injury (ICPi-AKI) has emerged as an important toxicity among patients with cancer. METHODS: We collected data on 429 patients with ICPi-AKI and 429 control patients who received ICPis contemporaneously but who did not develop ICPi-AKI from 30 sites in 10 countries. Multivariable logistic regression was used to identify predictors of ICPi-AKI and its recovery. A multivariable Cox model was used to estimate the effect of ICPi rechallenge versus no rechallenge on survival following ICPi-AKI. RESULTS: ICPi-AKI occurred at a median of 16 weeks (IQR 8-32) following ICPi initiation. Lower baseline estimated glomerular filtration rate, proton pump inhibitor (PPI) use, and extrarenal immune-related adverse events (irAEs) were each associated with a higher risk of ICPi-AKI. Acute tubulointerstitial nephritis was the most common lesion on kidney biopsy (125/151 biopsied patients [82.7%]). Renal recovery occurred in 276 patients (64.3%) at a median of 7 weeks (IQR 3-10) following ICPi-AKI. Treatment with corticosteroids within 14 days following ICPi-AKI diagnosis was associated with higher odds of renal recovery (adjusted OR 2.64; 95% CI 1.58 to 4.41). Among patients treated with corticosteroids, early initiation of corticosteroids (within 3 days of ICPi-AKI) was associated with a higher odds of renal recovery compared with later initiation (more than 3 days following ICPi-AKI) (adjusted OR 2.09; 95% CI 1.16 to 3.79). Of 121 patients rechallenged, 20 (16.5%) developed recurrent ICPi-AKI. There was no difference in survival among patients rechallenged versus those not rechallenged following ICPi-AKI. CONCLUSIONS: Patients who developed ICPi-AKI were more likely to have impaired renal function at baseline, use a PPI, and have extrarenal irAEs. Two-thirds of patients had renal recovery following ICPi-AKI. Treatment with corticosteroids was associated with improved renal recovery

Discovery of novel clustered regularly interspaced short palindromic repeat enzymes for transcriptome engineering and human health

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, September 2018.Page 399 blank. Cataloged from PDF version of thesis.Includes bibliographical references (pages 210-229).RNA plays important and diverse roles in biology, yet molecular tools to measure and manipulate RNA are limited. Recently, the bacterial adaptive immune system, CRISPR, has revolutionized our ability to manipulate DNA, but no known RNA-targeting versions exist. To discover parallel bacterial RNA-targeting systems that could be used for transcriptome engineering, we developed a computational pipeline to mine for novel Class 2 CRISPR systems across more than 25,000 bacterial genomes. Among the many novel CRISPR systems, we found a programmable RNA-targeting CRISPR system, CRISPR-Cas 13, that could provide immunity to E. coli against the ssRNA MS2 phage and biochemically characterized the enzyme. We adapted CRISPR-Casl3 for modulating the transcriptome in mammalian and plant cells by heterologously expressing Casl 3 and engineering the enzyme to precisely knockdown, bind, and edit RNA. Cas 13 knockdown was as efficient as RNA interference, but much more specific, across many transcripts tested. RNA editing with Cas 13 was also highly efficient, with up to 90% base editing rates, and as low as 20 off-targets with engineered specificity versions. Lastly, we combined Cas13 with isothermal amplification to develop a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with single-molecule sensitivity and singlebase mismatch specificity. We used this Casl3a-based molecular detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to specifically detect pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Our results establish CRISPR-Cas13 as a flexible platform for RNA targeting with wide applications in RNA biology, diagnostics, and therapeutics.by Omar O. Abudayyeh.Ph. D. in Medical Engineering and Medical Physic

Modeling and analysis of concrete production plant using petri nets

Computer modeling and analysis of construction processes have gained importance in recent years because of an increase in the complexity of construction processes. Petri nets provide a modeling and analysis approach that can be used to effectively study, understand, analyze, and improve construction processes. This paper highlights the advanced features of petri nets and describes their utilization as a process modeling and analysis tool for the study of a ready-mix concrete plant. Usually process modeling and analysis are performed by initially developing a graphical portrayal of the process and then dynamically studying the response of the process to external and internal factors. A petri net is a formal graphical modeling tool that can be efficiently utilized as a process modeling and analysis tool because it can graphically portray and dynamically analyze a process in an integrated manner. The paper contributes significantly in the area of computer-based decision making and provides value to practicing schedulers, estimators, and project managers who deal with complex construction processes

Modeling and analysis of a mail processing plant using Petri nets

This article outlines the utilization of Petri nets as a computerized process modeling and analysis tool for the study of the Edmonton Mail Processing Plant (MPP). Development of new systems or designing improvement in existing system-such as the MPP-are some of the challenges faced by decision makers and managers on a regular basis in the industry. In order to make prudent decisions, modeling and analysis of the underlying processes that constitute new or existing systems has become essential. Usually process modeling and analysis is performed by initially developing a graphical portrayal of the process and then by dynamically studying the response of the process to external and internal factors. In this article an overview of Petri net based process modeling and analysis is provided. Petri net is a formal graphical modeling tool that can be efficiently utilized as a process modeling and analysis tool because it can graphically portray and dynamically simulate a process in an integrated manner. The objective of this article is to highlight the symbolic graphic format and dynamic simulation capabilities of Petri nets. The article contributes significantly in the area of computer based decision making and provides value to practicing engineers and project managers who manage complex tasks. (C) 1999 Elsevier Science Ltd. All rights reserved