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1.
J Nanosci Nanotechnol ; 20(2): 1288-1295, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31383130

RESUMO

A DFT investigation was performed to evaluate the structure, electronic properties of the doped graphene, and the adsorption behavior of C2H2 and H2 on the graphene sheet. Two kinds of doping scenarios are considered, namely dopants into pristine graphene and vacancy graphene. It is observed that the doping energy is negative for dopant (N, P, S) into vacancy graphene at pyridinictype site, yet it is positive at graphitic-type site for pristine graphene. It could be inferred that the introducing process is exothermic reaction for defective graphene. Meanwhile, for the adsorption of C2H2 on the surface of defective graphene, we notice that C2H2 would prefer to be adsorbed at the top site of the doping atom and the adsorption energy increases with the introducing of dopants, indicating that the dopant would enhance the interaction between C2H2 and graphene. Regarding hydrogen molecule, the dopant has less promotion effect on the adsorption. Moreover, the graphene plays a role of electron donor while the gas molecule (C2H2/H2) is the electron acceptor when it is adsorbed. For the co-adsorption, the C2H2 is privileged to interact with the graphene and the pre-adsorbed C2H2 on doped graphene would weaken the opportunity of the uptake of H2 molecule. We anticipate that our results would provide information to design the optimized catalysts.

2.
Rev Environ Contam Toxicol ; 251: 131-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31129734

RESUMO

Maternal exposure to endocrine-disrupting chemicals (EDCs) is associated with long-term hormone-dependent effects that are sometimes not revealed until maturity, middle age, or adulthood. The aim of this study was to conduct descriptive reviews on animal experimental and human epidemiological evidence of the adverse health effects of in utero and lactational exposure to selected EDCs on the first generation and subsequent generation of the exposed offspring. PubMed, Web of Science, and Toxline databases were searched for relevant human and experimental animal studies on 29 October 29 2018. Search results were screened for relevance, and studies that met the inclusion criteria were evaluated and qualitative data extracted for analysis. The search yielded 73 relevant human and 113 animal studies. Results from studies show that in utero and lactational exposure to EDCs is associated with impairment of reproductive, immunologic, metabolic, neurobehavioral, and growth physiology of the exposed offspring up to the fourth generation without additional exposure. Little convergence is seen between animal experiments and human studies in terms of the reported adverse health effects which might be associated with methodologic challenges across the studies. Based on the available animal and human evidence, in utero and lactational exposure to EDCs is detrimental to the offspring. However, more human studies are necessary to clarify the toxicological and pathophysiological mechanisms underlying these effects.


Assuntos
Disruptores Endócrinos , Exposição Materna/estatística & dados numéricos , Animais , Feminino , Humanos , Gravidez
3.
Einstein (Sao Paulo) ; 18: eAO4739, 2020.
Artigo em Inglês, Português | MEDLINE | ID: mdl-31553355

RESUMO

OBJECTIVE: To use magnetic resonance imaging to assess the prevalence of foot and ankle ligament injuries and fractures associated with ankle sprain and not diagnosed by x-ray. METHODS: We included 180 consecutive patients with a history of ankle sprain, assessed at a primary care service in a 12-month period. Magnetic resonance imaging findings were recorded and described. RESULTS: Approximately 92% of patients had some type of injury shown on the magnetic resonance imaging. We found 379 ligament injuries, 9 osteochondral injuries, 19 tendinous injuries and 51 fractures. Only 14 magnetic resonance imaging tests (7.8%) did not show any sort of injury. We observed a positive relation between injuries of the lateral complex, syndesmosis and medial ligaments. However, there was a negative correlation between ankle ligament injuries and midfoot injuries. CONCLUSION: There was a high rate of injuries secondary to ankle sprains. We found correlation between lateral ligament injuries and syndesmosis and deltoid injuries. We did not observe a relation between deltoid and syndesmosis injuries or between lateral ligamentous and subtalar injuries. Similarly, no relation was found between ankle and midfoot injuries.


Assuntos
Traumatismos do Tornozelo/epidemiologia , Serviço Hospitalar de Emergência/estatística & dados numéricos , Adolescente , Adulto , Idoso , Traumatismos do Tornozelo/diagnóstico por imagem , Brasil/epidemiologia , Cartilagem Articular/lesões , Criança , Feminino , Humanos , Ligamentos Laterais do Tornozelo/lesões , Imagem por Ressonância Magnética/métodos , Masculino , Pessoa de Meia-Idade , Prevalência , Adulto Jovem
4.
Methods Mol Biol ; 2065: 1-4, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578683

RESUMO

Quantitative polymerase chain reaction (PCR) is the basis of a variety of scientific applications and publications in a broad range of interests. It also plays a fundamental role in nucleic acid sequencing applications, including Next Generation Sequencing (NGS)-based ones. The potential of PCR diagnostics is enormous, particularly for the early diagnosis of life-threatening infections. Some other fields of applications that use PCR on a regular basis include oncology, genetics, microbiology, biochemistry, immunogenetics, NGS, ecology, comparative genome evolution, ancestry DNA, pharmacogenomics, personalized medicine, and even general medicine.

5.
Methods Mol Biol ; 2065: 5-22, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578684

RESUMO

Primers are critical components of any PCR assay, as they are the main determinants of its specificity, sensitivity, and robustness. Despite the publication of numerous guidelines, the actual design of many published assays is often unsound: primers lack the claimed specificity, they may have to compete with secondary structures at their binding sites, primer dimer formation may affect the assay's sensitivity or they may bind only within a narrow temperature range. This chapter provides simple guidance to avoid these most common issues.

6.
Methods Mol Biol ; 2065: 23-38, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578685

RESUMO

MicroRNAs (miRNAs), a class of small non-coding RNAs that modulate gene expression at the post-transcriptional level, are attractive targets in many academic and diagnostic applications. Among them, assessing miRNA biomarkers in minimally invasive liquid biopsies was shown to be a promising tool for managing diseases, particularly cancer. The initial screening of disease-relevant transcripts is often performed by high-throughput next-generation sequencing (NGS), in here RNA sequencing (RNA-Seq). After complex processing of small RNA-Seq data, differential gene expression analysis is performed to evaluate miRNA biomarker signatures. To ensure experimental validity, biomarker candidates are commonly validated by an orthogonal technology such as reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). This chapter outlines in detail the material and methods one can apply to reproducibly identify miRNA biomarker signatures from blood total RNA. After screening miRNA profiles by small RNA-Seq, resulting data is validated in compliance with the "Minimum Information for Publication of Quantitative Real-Time PCR Experiments" (MIQE) guidelines.

7.
Methods Mol Biol ; 2065: 39-54, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578686

RESUMO

Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.

8.
Methods Mol Biol ; 2065: 55-64, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578687

RESUMO

The levels of expression of the HLA-class I molecules are critical for modulating T/NK lymphocytes effector functions. Among HLA molecules, HLA-C, the most recent developed form of class I antigens, is subjected to multiple post transcriptional level of regulation that affect its cell surface expression.We describe a new method of allele-specific real-time PCR that monitor the integrity/disruption of the binding site of the microRNA Hsa-miR-148a, a key factor associated to the levels of HLA-C expression in the Caucasian populations.

9.
Methods Mol Biol ; 2065: 65-77, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578688

RESUMO

The recent resurgence of yellow fever virus (YFV) activity in the tropical regions of Africa and South America has sparked renewed interest in this infamous arboviral disease. Also, the development and production of viral vaccines involve several steps that need the monitoring of viral load throughout the process (antigen production, purification, and inactivation). Currently, these steps are followed by plaque lysis titration assay, whose results take about 7-10 days to come out and thus resulting in a laborious and time-consuming approach. With the advent of quantitative real-time PCR (qPCR), we have a faster method to be applied during vaccine production and also to be effectively used for the diagnosis of YFV infection. The technique herein standardized proved to be effective for determining YF viral load both in vivo and in vitro, thus becoming a very important tool for laboratory analysis to verify the vaccination status of individuals, beyond acting as a quality control for vaccine production and diagnosis.

10.
Methods Mol Biol ; 2065: 79-94, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578689

RESUMO

Assessment of the abundance of fungi in environmental samples by quantitative PCR (qPCR) of community DNA is often a difficult task due to biases introduced during PCR amplification, resulting from the differences associated with length polymorphism and the varying number of copies of the rRNA operon among fungal species, the lack of specificity of the primers targeting the different regions of the rRNA operon, or their insufficient coverage of the fungal lineages. To overcome those limitations, it is crucial to test and select the specific primers sets which provide the more accurate approximation to the quantification of the targeted fungal populations in a given set of samples. Fungi are a significant fraction of the microbiota in wastewater treatment plants (WWTPs), but the activated sludge microbial communities comprise many other eukaryotic microorganisms whose molecular markers are often coamplified by primers initially designed as fungal-specific. Here, the use of the FungiQuant primer set is recommended for the quantification of fungal molecular markers (18S rRNA genes) by qPCR in activated sludge samples and the full protocol is described.

11.
Methods Mol Biol ; 2065: 95-104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578690

RESUMO

This chapter reports the use of real-time quantitative PCR to detect Diplodia sapinea, a fungal plant pathogen that causes shoot tip dieback and tree mortality on pine trees. This molecular approach represents a reliable and sensitive tool to detect fungal pathogens in DNA extracted from plant tissues and its use can be also recommended to study fungal behavior in host tissues by quantifying fungal growth in the latent phase, when symptoms in the host are not present yet.

12.
Methods Mol Biol ; 2065: 105-118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578691

RESUMO

Gene expression analysis by means of RT-qPCR is a highly sensitive technique. However, this requires an accurate protocol for the whole procedure from sampling to data analysis. We have optimized this protocol specifically for the analysis of plant tissues. Special attention is paid to RNA quality and integrity and to the appropriate setup of the assays in order to be compliant with the MIQE guidelines. This protocol was already successfully applied in ten different plant species.

13.
Methods Mol Biol ; 2065: 119-137, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578692

RESUMO

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.

14.
Methods Mol Biol ; 2065: 139-151, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578693

RESUMO

We propose two different approaches involving the use of quantitative real-time PCR for the detection or analysis of circulating tumor cells. In one case cells are indirectly identified through the expression of a marker mRNA, while in the other one cells are enriched by size prior to be submitted to mutational analysis for a specific target. Both methods have been successfully applied to the study of circulating melanoma cells.

15.
Methods Mol Biol ; 2065: 153-173, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578694

RESUMO

Molecular diagnosis and measurement of minimal residual disease (MRD) in patients with chronic myeloid leukemia (CML) is essential for clinical management. In the era of tyrosine kinase inhibitor therapy molecular tests including BCR-ABL1 transcript monitoring and kinase domain mutation analysis are the main tools used to inform choice of treatment, appropriate dosage and even whether therapy can be safely withdrawn. Quantitation of BCR-ABL1 oncogene transcript by real-time quantitative PCR (qPCR) is currently the gold-standard method for monitoring as it provides superior sensitivity over karyotyping and fluorescent in situ hybridization (FISH). Here we describe step-by-step methods of RNA conversion to cDNA along with the qPCR protocol which is used in one of the main reference laboratories for this test.

16.
Methods Mol Biol ; 2065: 175-190, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578695

RESUMO

For tissues obtained from glioma samples with/without nonneoplastic brain there is no consensus for universal reference gene but there are some potential genes that might have good stability, under certain conditions. Considering all points described in this work, the care with tissue collection, until gene amplification, directly impacts on the reliable characterization of its mRNA levels. Moreover, it is clear the importance of selecting the most appropriate reference genes for each experimental situation, to allow the accurate normalization of target genes, especially for genes that are subtly regulated.

17.
Methods Mol Biol ; 2065: 191-197, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578696

RESUMO

Individual age is a phenotypic trait that provides useful information not only in forensic investigations but also in the aging research which is becoming an urgent call due to the dramatic growth of the aging population worldwide.TaqMan quantification PCR (qPCR) can be successfully applied to biological age estimation, using method defined in Zubakov et al. (Curr Biol 20:R970-R971, 2010). Since levels of signal joint T-cell receptor rearrangement excision circle (sjTREC) in human lymphocytes are known to decrease with age increasing, the qPCR of sjTREC represents a simple and relatively reproducible technique which offers highly accurate age estimation results.

18.
Methods Mol Biol ; 2065: 199-208, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578697

RESUMO

Real time technology provides great advancements over PCR-based methods for a broad range of applications. With the increased availability of sequencing information, there is a need for the development and application of high-throughput real time PCR genotyping and gene expression methods that significantly broaden the current screening capabilities. Thermo Fisher Scientific (USA) has released a platform (QuantStudio™ 12K Flex system coupled with OpenArray® technology) with key elements required for high-throughput SNP genotyping and gene expression analysis. This allows for a rapid screening of large numbers of TaqMan® assays (up to 256) in many samples (up to 480) per run. This advanced real-time method involves the use of an array composed of 3,000 through-holes running on the QuantStudio™ 12K with OpenArray® block. The aim of this chapter is to outline the OpenArray® approach while providing a comprehensive in-depth review of the scientific literature on this topic. In agreement with a large number of independent studies, we conclude that the use of OpenArray® technology is a rapid and accurate method for high-throughput and large-scale systems biology studies with high specificity and sensitivity.

19.
Methods Mol Biol ; 2065: 209-231, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31578698

RESUMO

The great promise of digital PCR is the potential for unparalleled precision enabling accurate measurements for detection and quantification of genetic material. This chapter walks the reader through the fundamentals of digital PCR technology including digital PCR modeling using Poisson statistics. It describes a highly successful implementation of digital PCR technology using the chip-based nanofluidic Applied Biosystems™ QuantStudio™ 3D digital PCR system. It reviews the large number of applications where digital PCR is poised to make significant impacts. These include applications where detection of rare genetic targets is prioritized such as liquid biopsy, rare mutation detection, confirmation of NGS variant detection, detection of fusion transcripts, detection of chimerism and GMO detection and monitoring. These further include applications where accurate quantification of genetic targets is prioritized such as generation of references and standards, copy number variation, and NGS Library quantification.

20.
Dev Comp Immunol ; 102: 103453, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31326564

RESUMO

Small organisms, like the nematode C. elegans, are emerging as insightful models in which to study host/pathogen interactions and the evolving interplay between host defenses and microbial offenses. In C. elegans the innate immune response has been shown to be connected to the DAF-2 insulin/insulin-like growth factor 1 (IGF-1) signal pathway, a critical transduction pathway that mediates stress response in the worms via the DAF-16 FOXO/forkhead transcription factor. Our studies of the C. elegans' phenotypes that are associated with behavioral innate immune response (avoidance behavior) and IGF-1 signaling perturbations (lifespan effects) led us to question the cause of the avoidance behavior observed when C. elegans are challenged with B. anthracis. While worms indeed avoid B. anthracis, and this behavior seems to be partly tied to IGF-1 signaling, the bacteria have neither nematocidal nor visible pathogenic effects on the worms. In fact, worms fed B. anthracis alone exhibit extended lifespans. We demonstrate that the extended lifespan phenotype seen in worms fed B. anthracis is likely the result of calorie restriction, and that worms do not eat B. anthracis even when avoidance behaviors have been suppressed. We further demonstrate a large time lag between the onset of avoidance behavior (which occurs upon contact with B. anthracis), and the induction of IGF-1 signaling (which occurs much later) in worms fed B. anthracis. Taken together, our data demonstrate behavioral avoidance that does not appear to be linked to a measurable immune response. We propose that, in some situations, avoidance behaviors categorized as immunological might be more accurately described as broad foraging behaviors induced in worms presented with a non-preferred food choice, or with a food choice that is either difficult or impossible for the worms to ingest.

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