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1.
Methods Mol Biol ; 2594: 165-172, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36264495

RESUMO

Single-cell RNA sequencing (scRNA-seq) is gaining popularity as this allows you to profile a large number of individual cells. However, as the volume of the data increases, the need for appropriate computational methods also arises. Here, I will provide an overview of standard computational workflow for scRNA-seq and discuss each step and provide useful tips if applicable.


Assuntos
Análise de Célula Única , Software , Análise de Sequência de RNA/métodos , RNA-Seq , Análise de Célula Única/métodos , Fluxo de Trabalho
2.
Methods Mol Biol ; 2562: 461-469, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36272094

RESUMO

The dramatic increase in accessibility to sequencing technologies has opened new avenues into studying different processes, cells, and animal models. In the amphibian models used for regeneration research, these new datasets have uncovered a variety of information about what genes define the regenerating limb as well as how genes and cells change over the course of regeneration. The accumulation of data from these studies undoubtedly increases our understanding of regeneration. Throughout these studies, it is important to consider how data can be made most useful not only for the primary study but also for reuse within the scientific community. This chapter will focus on best practices for data collection and handling as well as principles to promote access and reuse of big datasets. However, the deposition and thorough description of data of all sizes generated for a publication (e.g., images, fcs files, etc.) can also be done following this generic workflow. The aim is to lower hurdles for reuse, access, and re-evaluation of data which will in turn increase the utility of these datasets and accelerate scientific progress.


Assuntos
Tecnologia , Fluxo de Trabalho , Análise de Sequência , Coleta de Dados
3.
Methods Mol Biol ; 2426: 375-390, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36308698

RESUMO

In this protocol we describe our workflow for analyzing complex, multi-condition quantitative proteomic experiments, with the aim to extract biological insights. The tool we use is an R package, PloGO2, contributed to Bioconductor, which we can optionally precede by running correlation network analysis with WGCNA. We describe the data required and the steps we take, including detailed code examples and outputs explanation. The package was designed to generate gene ontology or pathway summaries for many data subsets at the same time, visualize protein abundance summaries for each biological category examined, help determine enriched protein subsets by comparing them all to a reference set, and suggest key highly correlated hub proteins, if the optional network analysis is employed.


Assuntos
Proteínas , Proteômica , Proteômica/métodos , Ontologia Genética , Fluxo de Trabalho
4.
Chemosphere ; 310: 136865, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36244422

RESUMO

Castor cake is a major by-product generated after castor oil extraction and has been widely used as an organic fertilizer. Once applied to soil, a toxic alkaloid ricinine in castor cake may be released into soils and subsequently taken up by crops, which poses a potential threat to food safety and human health. However, the environmental fate of castor cake derived ricinine in agroecosystems remains unclear. In this study, the release and metabolism of ricinine in soils were conducted using soil pot experiments with different castor cake application rates. The analytical methodology of ricinine quantification in soil pore water was first established using solid phase extraction (SPE) coupled with liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF/MS). A non-target screening workflow associated with LC-QTOF/MS and SIRIUS platform was further developed to identify ricinine metabolites in soil pore water. After castor cake application, the ricinine concentrations in soil pore water significantly increased to 297-7990 µg L-1 at 1 day and then gradually decreased to 62.1-3460 µg L-1 at 7 days and 1.70-279 µg L-1 at 14 days for the selected two tested soils with castor cake application rates of 2, 10, and 20 g castor cake/kg soil. In addition, two ricinine metabolites R-194 and R-180 were tentatively identified and one ricinine metabolite N-demethyl-ricinin was confirmed through authentic reference standard for the first time by the developed non-target screening workflow. This study highlights the release and metabolism of toxic alkaloid ricinine in soils once applied castor cake as an organic fertilizer. Ricinine could be released into soil pore water in a short-term after castor cake application and then undergo demethylation, hydroxylation, and hydroxylation followed by methylation metabolisms over time in agroecosystems.


Assuntos
Alcaloides , Fertilizantes , Humanos , Fertilizantes/análise , Solo , Óleo de Rícino , Fluxo de Trabalho , Cromatografia Líquida , Alcaloides/análise , Espectrometria de Massas , Água/análise
5.
Anal Biochem ; 660: 114969, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36343663

RESUMO

The growing complexity of novel biopharmaceutical formats, such as Fc-fusion proteins, in increasingly competitive environment has highlighted the need of high-throughput analytical platforms. Multi-attribute method (MAM) is an emerging analytical technology that utilizes liquid chromatography coupled with mass spectrometry to monitor critical quality attributes (CQAs) in biopharmaceuticals. MAM is intended to supplement or replace the conventional chromatographic and electrophoretic approaches used for quality control and drug release purpose. In this investigation, we have developed an agile sample preparation approach for deploying MAM workflow for a complex VEGFR-targeted therapeutic Fc-fusion protein. Initially, a systematic time course evaluation of tryptic digestion step was performed to achieve maximum amino acid sequence coverage of >96.5%, in a short duration of 2 h, with minimum assay artifacts. This approach facilitated precise identification of five sites of N-glycosylation with successful monitoring of other CQAs such as deamidation, oxidation, etc. Subsequently, the developed MAM workflow with suitable tryptic digestion time was qualified according to the International council for harmonisation (i.e. ICH) Q2R1 guidelines for method validation. Post-validation, the analytical workflow was also evaluated for its capability to identify unknown moieties, termed as 'New Peak Detection' (i.e. NPD), and assess fold change between the reference and non-reference samples, in a representative investigation of pH stress study. The study, thus, demonstrated the suitability of the MAM workflow for characterization of heavily glycosylated Fc-fusion proteins. Moreover, its NPD feature could offer an all-encompassing view if applied for forced degradation and stability studies.


Assuntos
Produtos Biológicos , Espectrometria de Massas em Tandem , Cromatografia Líquida , Fluxo de Trabalho , Glicosilação
6.
Methods Mol Biol ; 2561: 233-243, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36399273

RESUMO

In recent decades, mass spectrometry-based lipidomics has provided a fertile environment for scientific investigations of biochemical and mechanistic processes in biological systems. Notably, this approach has been used to characterize physiological and pathological processes relevant to the central nervous system by identifying changes in the sphingolipid content in the brain, cerebral spinal fluid, and blood plasma. However, despite a preponderance of studies identifying correlations between specific lipids and disease progression, this powerful resource has not yet substantively translated into clinically useful diagnostic assays. Part of this gap may be explained by insufficient depth of the lipidomic profiles in many studies, by lab-to-lab inconsistencies in methodology, and a lack of absolute quantification. These issues limit the identification of specific molecular species and the harmonization of results across independent studies. In this chapter, we contextualize these issues with recent reports identifying correlations between brain lipids and neurological diseases, and we describe the workflow our group has optimized for analysis of the blood plasma sphingolipidome, adapted to the characterization of the human brain tissue.


Assuntos
Encéfalo , Lipidômica , Humanos , Espectrometria de Massas/métodos , Esfingolipídeos , Fluxo de Trabalho
7.
Methods Mol Biol ; 2603: 151-161, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36370277

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) is a strategic quantitative mass spectrometry method to analyze multiple protein samples in different conditions simultaneously. In recent years, 3D cell growth culture conditions have been developed to establish intestinal organoids from isolated crypts, which mimic the intestine's cell composition and organization. Organoids, isolated from normal or diseased tissues, can be used to compare cell distribution and differentiation, signaling pathways, and cell responses to pharmacological agents, therapeutic drugs, endogenous or exogenous metabolites, and environmental stresses, among others. Here, we describe the process of generating SILAC organoids from the mouse small intestine.


Assuntos
Organoides , Proteômica , Camundongos , Animais , Proteômica/métodos , Marcação por Isótopo/métodos , Organoides/metabolismo , Fluxo de Trabalho , Aminoácidos/química , Intestinos
8.
Magn Reson Med ; 89(1): 322-330, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36120984

RESUMO

PURPOSE: To evaluate the usage of three-dimensional (3D) presaturated TurboFLASH (satTFL) for B 1 + $$ {\mathrm{B}}_1^{+} $$ and B 0 $$ {\mathrm{B}}_0 $$ mapping on single channel and parallel transmission (pTx) systems. METHODS: B 1 + $$ {\mathrm{B}}_1^{+} $$ maps recorded with 3D satTFL were compared to maps from three other 3D B 1 + $$ {\mathrm{B}}_1^{+} $$ mapping sequences in an agar phantom. Furthermore, individual-channel B 1 + $$ {\mathrm{B}}_1^{+} $$ maps of 18 human subjects were recorded with 3D satTFL using B 1 + $$ {\mathrm{B}}_1^{+} $$ interferometry. A neural network was trained for masking of the maps. RESULTS: Out of the sequences compared satTFL was the only one with a mapping range exceeding well over 90°. In regions with lower flip angles there was high correspondence between satTFL and AFI. DREAM and double angle method also showed high qualitative similarity, however the magnitude differed from the other two measurements. The individual-channel B 1 + $$ {\mathrm{B}}_1^{+} $$ maps were successfully used for pTx pulse calculation in a separate study. CONCLUSION: 3D satTFL can record high-quality B 1 + $$ {\mathrm{B}}_1^{+} $$ maps with a high dynamic range in a short time. Correspondence with AFI maps is high, while measurement duration is reduced drastically.


Assuntos
Algoritmos , Imageamento por Ressonância Magnética , Humanos , Imageamento por Ressonância Magnética/métodos , Fluxo de Trabalho , Imagens de Fantasmas , Encéfalo
9.
Nat Commun ; 13(1): 6547, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36319635

RESUMO

Lipids are a structurally diverse class of biomolecules which can undergo a variety of chemical modifications. Among them, lipid (per)oxidation attracts most of the attention due to its significance in the regulation of inflammation, cell proliferation and death programs. Despite their apparent regulatory significance, the molecular repertoire of oxidized lipids remains largely elusive as accurate annotation of lipid modifications is complicated by their low abundance and often unknown, biological context-dependent structural diversity. Here, we provide a workflow based on the combination of bioinformatics and LC-MS/MS technologies to support identification and relative quantification of oxidized complex lipids in a modification type- and position-specific manner. The developed methodology is used to identify epilipidomics signatures of lean and obese individuals with and without type 2 diabetes. The characteristic signature of lipid modifications in lean individuals, dominated by the presence of modified octadecanoid acyl chains in phospho- and neutral lipids, is drastically shifted towards lipid peroxidation-driven accumulation of oxidized eicosanoids, suggesting significant alteration of endocrine signalling by oxidized lipids in metabolic disorders.


Assuntos
Diabetes Mellitus Tipo 2 , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Fluxo de Trabalho , Lipídeos/química , Plasma/química
10.
PLoS One ; 17(11): e0276607, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36350811

RESUMO

High throughput technologies in genomics enable the analysis of small alterations in gene expression levels. Patterns of such deviations are an important starting point for the discovery and verification of new biomarker candidates. Identifying such patterns is a challenging task that requires sophisticated machine learning approaches. Currently, there are a variety of classification models, and a common approach is to compare the performance and select the best one for a given classification problem. Since the association between the features of a data set and the performance of a particular classification method is still not fully understood, the main contribution of this work is to provide a new methodology for predicting the prediction results of different classifiers in the field of biomarker discovery. We propose here a three-steps computational workflow that includes an analysis of the data set characteristics, the calculation of the classification accuracy and, finally, the prediction of the resulting classification error. The experiments were carried out on synthetic and microarray datasets. Using this method, we showed that the predictability strongly depends on the discriminatory ability of the features, e.g., sets of genes, in two or multi-class datasets. If a dataset has a certain discriminatory ability, this method enables prediction of the classification performance before applying a learning model. Thus, our results contribute to a better understanding of the relationship between dataset characteristics and the corresponding performance of a machine learning method, and suggest the optimal classification method for a given dataset based on its discriminatory ability.


Assuntos
Perfilação da Expressão Gênica , Genômica , Perfilação da Expressão Gênica/métodos , Fluxo de Trabalho , Biomarcadores Tumorais , Aprendizado de Máquina
11.
Nat Commun ; 13(1): 6776, 2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36351912

RESUMO

This study compares a deep learning interpretation of 23 echocardiographic parameters-including cardiac volumes, ejection fraction, and Doppler measurements-with three repeated measurements by core lab sonographers. The primary outcome metric, the individual equivalence coefficient (IEC), compares the disagreement between deep learning and human readers relative to the disagreement among human readers. The pre-determined non-inferiority criterion is 0.25 for the upper bound of the 95% confidence interval. Among 602 anonymised echocardiographic studies from 600 people (421 with heart failure, 179 controls, 69% women), the point estimates of IEC are all <0 and the upper bound of the 95% confidence intervals below 0.25, indicating that the disagreement between the deep learning and human measures is lower than the disagreement among three core lab readers. These results highlight the potential of deep learning algorithms to improve efficiency and reduce the costs of echocardiography.


Assuntos
Aprendizado Profundo , Humanos , Feminino , Masculino , Fluxo de Trabalho , Reprodutibilidade dos Testes , Ecocardiografia/métodos , Função Ventricular Esquerda
12.
Microbiology (Reading) ; 168(11)2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36374163

RESUMO

Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick's broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4-5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.


Assuntos
Doenças do Gato , Doenças do Cão , Felis , Animais , Gatos , Cães , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Fluxo de Trabalho , Análise de Sequência de DNA/métodos
13.
Medicina (Kaunas) ; 58(11)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36363569

RESUMO

Dental implants have been placed millions of times worldwide, and the surgical procedure and implant design have steadily improved. The basic prosthetic connection, which makes use of an abutment, has changed little over the past decades. These days, implant placement with immediate provisionalization is an essential stage in implant dentistry and interdisciplinary treatment strategies. Temporary computer-aided design and computer-aided manufacturing (CAD/CAM) of implant-supported crowns restore esthetics and guarantee function during the restoration process and the osseointegration of the dental implant. This case report describes the digital planning; the immediate, static, computer-assisted implant surgery, and the immediate chairside provisionalization of a novel implant system that is directly screw-retained without an abutment.


Assuntos
Coroas , Cirurgia Assistida por Computador , Humanos , Fluxo de Trabalho , Desenho Assistido por Computador , Parafusos Ósseos
14.
Medicina (Kaunas) ; 58(11)2022 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-36363573

RESUMO

Robotic-arm-assisted total hip arthroplasty (RoTHA) offers the opportunity to improve the implant positioning and restoration of native hip mechanics. The concept of individualised, functional implant positioning and how it relates to spinopelvic imbalance is an important yet rather novel consideration in THA. There is mounting evidence that a significant percentage of dislocations occur within the perceived "safe zones"; hence, in the challenging subset of patients with a stiff spinopelvic construct, it is imperative to employ individualised component positioning based on the patients' phenotype. Restoring the native centre of rotation, preserving offset, achieving the desired combined anteversion and avoiding leg length inequality are all very important surgeon-controlled variables that have been shown to be associated with postoperative outcomes. The latest version of the software has a feature of virtual range of motion (VROM), which preoperatively identifies potential dynamic causes of impingement that can cause instability. This review presents the workflow of RoTHA, especially focusing on pragmatic solutions to tackle the challenge of spinopelvic imbalance. Furthermore, it presents an overview of the existing evidence concerning RoTHA and touches upon future direction.


Assuntos
Artroplastia de Quadril , Procedimentos Cirúrgicos Robóticos , Fluxo de Trabalho , Braço , Amplitude de Movimento Articular
15.
J Healthc Qual ; 44(6): 363-372, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36318296

RESUMO

INTRODUCTION: There is often a breakdown in communication between physical therapy and nursing regarding the responsibility of patient mobility. This communication problem contributes to mobility being the most frequently missed clinical intervention. To address this gap, a Mobility Tech (MT) role was implemented at a large academic medical center. The purpose of this article is to describe the implementation of this role. METHODS: Using the Institute for health care improvement (IHI) model for improvement, iterative small tests of change were developed to integrate the MT role on four acute care units. Using process flow mapping, the team developed a MT workflow that was adapted to create MT protocols. RESULTS: Program outcomes included fall rates and the highest level of mobility documented by staff. During the MT pilot from February 2020 through June 2020, MTs provided an average of over 500 interventions each month. There was a clinically and statistically significant increase in mobility noted in two of the four pilot units. CONCLUSION: We have demonstrated that MTs can be safely incorporated into acute care nursing workflow, and comments from staff and patients validate the role is "value added."


Assuntos
Centros Médicos Acadêmicos , Comunicação , Humanos , Cuidados Críticos , Fluxo de Trabalho
16.
J Med Internet Res ; 24(11): e41463, 2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36383427

RESUMO

Digital health interventions are being increasingly incorporated into health care workflows to improve the efficiency of patient care. In turn, sustained patient engagement with digital health interventions can maximize their benefits toward health care outcomes. In this viewpoint, we outline a dynamic patient engagement by using various communication channels and the potential use of omnichannel engagement to integrate these channels. We conceptualize a novel patient care journey where multiple web-based and offline communication channels are integrated through a "digital twin." The principles of implementing omnichannel engagement for digital health interventions and digital twins are also broadly covered. Omnichannel engagement in digital health interventions implies a flexibility for personalization, which can enhance and sustain patient engagement with digital health interventions, and ultimately, patient quality of care and outcomes. We believe that the novel concept of omnichannel engagement in health care can be greatly beneficial to patients and the system once it is successfully realized to its full potential.


Assuntos
Participação do Paciente , Telemedicina , Humanos , Comunicação , Fluxo de Trabalho
17.
Sci Data ; 9(1): 700, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376356

RESUMO

Research can be more transparent and collaborative by using Findable, Accessible, Interoperable, and Reusable (FAIR) principles to publish Earth and environmental science data. Reporting formats-instructions, templates, and tools for consistently formatting data within a discipline-can help make data more accessible and reusable. However, the immense diversity of data types across Earth science disciplines makes development and adoption challenging. Here, we describe 11 community reporting formats for a diverse set of Earth science (meta)data including cross-domain metadata (dataset metadata, location metadata, sample metadata), file-formatting guidelines (file-level metadata, CSV files, terrestrial model data archiving), and domain-specific reporting formats for some biological, geochemical, and hydrological data (amplicon abundance tables, leaf-level gas exchange, soil respiration, water and sediment chemistry, sensor-based hydrologic measurements). More broadly, we provide guidelines that communities can use to create new (meta)data formats that integrate with their scientific workflows. Such reporting formats have the potential to accelerate scientific discovery and predictions by making it easier for data contributors to provide (meta)data that are more interoperable and reusable.


Assuntos
Ciência Ambiental , Projetos de Pesquisa , Metadados , Fluxo de Trabalho
19.
Int J Mol Sci ; 23(21)2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36361669

RESUMO

Quantitative structure-activity relationship (QSAR) and quantitative structure-property relationship (QSPR) are established techniques to relate endpoints to molecular features. We present the Alvascience software suite that takes care of the whole QSAR/QSPR workflow necessary to use models to predict endpoints for untested molecules. The first step, data curation, is covered by alvaMolecule. Features such as molecular descriptors and fingerprints are generated by using alvaDesc. Models are built and validated with alvaModel. The models can then be deployed and used on new molecules by using alvaRunner. We use these software tools on a real case scenario to predict the blood-brain barrier (BBB) permeability. The resulting predictive models have accuracy equal or greater than 0.8. The models are bundled in an alvaRunner project available on the Alvascience website.


Assuntos
Barreira Hematoencefálica , Relação Quantitativa Estrutura-Atividade , Fluxo de Trabalho , Permeabilidade , Software
20.
Int J Implant Dent ; 8(1): 55, 2022 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-36322245

RESUMO

This article describes a case with a full digital procedure to prefabricate an implant-supported interim restoration based on the preoperative digital implant planning. A fully guided surgical template is designed and printed, and then an interim restoration is fabricated based on the planned implant position through a dental computer-aided design (CAD) software. Once the implant was placed at the predetermined position through the fully guided surgical guide, the prefabricated interim restoration could be inserted immediately after the surgery, which can guide the healing of the soft tissue and enhance the esthetic outcomes. This novel technique eliminates the conventional impression making to insert an implant-supported interim restoration immediately after the implant placement surgery, which can guide the healing of the soft tissue, minimize the chairside time and optimize the clinical workflow.


Assuntos
Implantes Dentários , Planejamento de Prótese Dentária , Humanos , Planejamento de Prótese Dentária/métodos , Prótese Dentária Fixada por Implante/métodos , Fluxo de Trabalho , Estética Dentária
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