Advances In Detection Methods Research Articles

Isolating and Analyzing Protein Containing Granules from Cells

Recent advancements in detection methods have made protein condensates, also called granules, a major area of study, but tools to characterize these assemblies need continued development to keep up with evolving paradigms. We have optimized a protocol to separate condensates from cells using chemical cross-linking followed by size-exclusion chromatography (SEC). After SEC fractionation, the samples can be characterized by a variety of approaches including enzyme-linked immunosorbent assay, dynamic light scattering, electron microscopy, and mass spectrometry. The protocol described here has been optimized for cultured mammalian cells and E. coli expressing recombinant proteins. Since the lysates are fractionated by size, this protocol can be modified to study other large protein assemblies, including the nuclear pore complex, and for other tissues or organisms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: SEC separation of cross-linked mammalian cell lysates Alternate Protocol: Preparation of non-crosslinked mammalian cells Basic Protocol 2: SEC separation of E. coli lysate Support Protocol 1: Detecting protein of interest by ELISA Support Protocol 2: TCA precipitation of SEC fractions.

Recent Advances in In Vivo Neurochemical Monitoring.

The brain is a complex network that accounts for only 5% of human mass but consumes 20% of our energy. Uncovering the mysteries of the brain’s functions in motion, memory, learning, behavior, and mental health remains a hot but challenging topic. Neurochemicals in the brain, such as neurotransmitters, neuromodulators, gliotransmitters, hormones, and metabolism substrates and products, play vital roles in mediating and modulating normal brain function, and their abnormal release or imbalanced concentrations can cause various diseases, such as epilepsy, Alzheimer’s disease, and Parkinson’s disease. A wide range of techniques have been used to probe the concentrations of neurochemicals under normal, stimulated, diseased, and drug-induced conditions in order to understand the neurochemistry of drug mechanisms and develop diagnostic tools or therapies. Recent advancements in detection methods, device fabrication, and new materials have resulted in the development of neurochemical sensors with improved performance. However, direct in vivo measurements require a robust sensor that is highly sensitive and selective with minimal fouling and reduced inflammatory foreign body responses. Here, we review recent advances in neurochemical sensor development for in vivo studies, with a focus on electrochemical and optical probes. Other alternative methods are also compared. We discuss in detail the in vivo challenges for these methods and provide an outlook for future directions.

Effects of mRNA Modifications on Translation: An Overview.

The mRNA epitranscriptome imparts diversity to gene expression by installing chemical modifications. Advances in detection methods have identified chemical modifications in eukaryotic, bacterial, and viral messenger RNAs (mRNAs). The biological functions of modifications in mRNAs still remain to be understood. Chemical modifications are introduced in synthetic mRNAs meant for therapeutic applications to maximize expression from the synthetic mRNAs and to evade the host immune response. This overview provides a background of chemical modifications found in mRNAs, with an emphasis on pseudouridine and its known effects on the mRNA life cycle, its potential applications in synthetic mRNA, and the methods used to assess its effects on mRNA translation.

Sperm DNA Integrity and Male Fertility in Farm Animals: A Review.

The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.

Comparison of Vehicle Detection Techniques applied to IP Camera Video Feeds for use in Intelligent Transport Systems

Vehicle detection is an important area in Transport and Artificial Intelligence. Through vehicle detection techniques, vehicles can be located across different images. Some of these models are robust enough to identify parts of vehicles in images where the vehicle might be partially occluded. Recent advances in detection methods gave rise to a range of different techniques that can be used for recognition and detection of vehicles. Although each technique has its merits, it is not always the case that the adopted model works well for scenarios involving IP Cameras. The motivation for this study is to compare several state-of-the-art techniques, including deep learning models and computer vision approaches. A set of experiments are developed in order to test these models on a number of low quality IP camera footages set in the transport domain in order to measure detection and recognition accuracy. The final evaluation compares detection accuracy using mean average precision, the semantics of the recognised vehicle as well as recognition robustness when applied to a dataset that contains images with different light conditions. The study also looks at persistence in recognition across frames in video data and a detailed description of the dataset used to train the evaluated models. Finally, the paper also goes through some scenarios that applies the results obtained in this study to ITS systems that use IP camera feeds.

Detection of phosphorus species in water: technology and strategies

Phosphorus species are the sum of naturally evolved phosphorus elements with diverse forms of existence and unique properties. The detection and analysis of the optical properties of unknown phosphorus species via direct or indirect strategies offers unique advantages in understanding the growth processes and existence characteristics of various chemicals and microorganisms in water environments. This review highlights recent advances and future trends in methods of detection of total phosphorus in water, including photoelectric strategies, spectroscopy techniques, and modeling algorithms. These methods effectively explore the dynamic changes of total phosphorus content in complex water environments to reveal important signals in water, which is of great guiding significance for achieving accurate detection of water quality and promoting social development. We also discuss some extended strategies for its measurement and prediction via rational design and cross-combination, which may help inspire future design of more accurate and intelligent detection models or systems. The strategies based on these types of total phosphorus detection methods provide a versatile platform for novel sensors and thereby show great potential in the development of future water quality detection applications.

Advances in Detection Methods of β-Amyloid Protein

Abstract Alzheimer's disease (AD) is a neurodegenerative disease with unknown etiology. β-amyloid protein (Aβ) is one of the specific biomarkers of AD, and many clinical studies suggest that abnormal levels of Aβ in blood, cerebrospinal fluid and brain tissue are closely related to the progression of AD. The analysis and evaluation of Aβ are important for early detection, tracking, prevention, and treatment of AD. In this paper, the present situations of the commonly used detection methods of Aβ at home and abroad were summarized and compared. Specifically, the latest application of new electrochemical biosensor in Aβ detection was mainly described, and the summary of its future directions and the potential applications was given.

Analysis of proteins and peptides by electromigration methods in microchips

This review presents the developments and applications of microchip electromigration methods in the separation and analysis of peptides and proteins in the period 2011-mid-2016. The developments in sample preparation and preconcentration, microchannel material, and surface treatment are described. Separations by various microchip electromigration methods (zone electrophoresis in free and sieving media, affinity electrophoresis, isotachophoresis, isoelectric focusing, electrokinetic chromatography, and electrochromatography) are demonstrated. Advances in detection methods are reported and novel applications in the areas of proteomics and peptidomics, quality control of peptide and protein pharmaceuticals, analysis of proteins and peptides in biomatrices, and determination of physicochemical parameters are shown.

Advances in detection methods for pyrethroid insecticides and their metabolites in human biological samples of human

Advances in detection methods for pyrethroid insecticides and their metabolites in human biological samples of human

Advances in detection methods for 1-bromopropane and its metabolites

Advances in detection methods for 1-bromopropane and its metabolites

Significance of IASLC/ATS/ERS classification for early-stage lung adenocarcinoma patients in predicting benefit from adjuvant chemotherapy.

Non-small cell lung cancer (NSCLC) is currently the leading cause of cancer-related deaths worldwide and represents an incredibly challenging problem for clinicians. Despite advances in methods of detection and treatment, there is an expected increase in mortality in both developed and developing countries (1,2). The most common histologic subtype is adenocarcinoma (ADC), which accounts for approximately 50% of NSCLCs (3,4). In developed countries it is detected at an early-stage only in 25% of cases. The current gold standard for treatment of early-stage lung ADC when there are no significant risk factors is lobectomy. Despite recent progress and development of surgical techniques and minimally invasive approaches, approximately 40% of lung ADC patients experience tumor recurrence and death following complete resection (3,4). While meta-analysis has confirmed survival benefits for patients with pathologic stage II or IIIA lung ADC who have undergone adjuvant chemotherapy following surgical resection (5), there is still controversy over the role of chemotherapy in pathologic stage I patients. Additionally, its role in combination with surgery versus radiotherapy has yet to be elucidated.

Glycobiology of the oral pathogen Porphyromonas gingivalis and related species

Until recently, glycoproteins had only been described in eukaryotes. However, advances in detection methods and genome analyses have allowed the discovery of N-linked or O-linked glycoproteins, similar to those found in eukaryotes, in some bacterial species. These prokaryotic glycoproteins play roles in adhesion, solubility, formation of protein complexes, protection from protein degradation, and changes in antigenicity. Periodontal pathogen Porphyromonas gingivalis secretes virulence proteins via the type IX secretion system, some of which localize on the cell surface by binding to lipopolysaccharide (LPS). These virulence proteins have a conserved C-terminal domain (CTD) region, which is used as a secretion signal. However, it is still uncertain how the secreted proteins on the cell surface bind to LPS. In this review, we discuss the synthesis of P. gingivalis O polysaccharide, which plays a role in anchoring the CTD protein on the cell surface, and recent discoveries of glycoproteins in P. gingivalis as well as other species in the phylum Bacteroidetes.

Clinical Significance of Tissue Factor-Exposing Microparticles in Arterial and Venous Thrombosis.

Microparticles (MP) are small extracellular vesicles (30-1,000 nm) that are released from activated cells or platelets. Exposure of negatively charged phospholipids and tissue factor (TF) renders MP procoagulant. Normal plasma levels of intravascular TF-exposing MP (TFMP) are low, but their number may rise in pathological conditions, including cancer and infectious disease. Emerging evidence indicates an important role for these circulating TFMP in the pathogenesis of thrombotic complications such as venous thromboembolism and disseminated intravascular coagulation, whereas their contribution to arterial thrombosis is less studied. Despite serious limitations of the currently available assays for measuring TFMP levels or the procoagulant activity associated with TFMP with respect to sensitivity and specificity, the scientific interest in TFMP is rapidly growing because their application as prognostic biomarkers for thrombotic complications is promising. Future advances in detection methods will likely provide more insight into TFMP and eventually improve their clinical utility.

Diagnosis of viral gastroenteritis: limits and potential of currently available procedures.

The diagnostic approaches to viral gastroenteritis have evolved substantially over the past decades because of the advances in detection methods, the emergence of new pathogens, and the increase in diarrhea hospitalizations attributed to viruses, especially in young children in non-industrialized countries. Overall, these factors have lead to a relevant improvement of types and operating characteristics of diagnostic methods (including sensitivity and specificity), as well as turnaround time. In this review, clinical and laboratory approaches to the diagnosis of viruses causing gastroenteritis are presented; in particular, specimen collection and detection methods are reviewed and discussed, taking into account performance and limitations.

Aberrant methylation patterns in cancer: a clinical view.

Epigenetic mechanisms, such as DNA methylation, DNA hydroxymethylation, post-translational modifications (PTMs) of histone proteins affecting nucleosome remodelling, and regulation by small and large non-coding RNAs (ncRNAs) work in concert with cis and trans acting elements to drive appropriate gene expression. Advances in detection methods and development of dedicated platforms and methylation arrays resulted in an explosion of information on aberrantly methylated sequences linking deviations in epigenetic landscape with the initiation and progression of complex diseases. Here, we consider how DNA methylation changes in malignancies, such as breast, pancreatic, colorectal, and gastric cancer could be exploited for the purpose of developing specific diagnostic tools. DNA methylation changes can be applicable as biomarkers for detection of malignant disease in easily accessible tissues. Methylation signatures are already proving to be an important marker for determination of drug sensitivity. Even more, promoter methylation patterns of some genes, such as MGMT, SHOX2, and SEPT9, have already been translated into commercial clinical assays aiding in patient assessment as adjunct diagnostic tools. In conclusion, the changes in DNA methylation patterns in tumour cells are slowly gaining entrance into routine diagnostic tests as promising biomarkers and as potential therapeutic targets.

Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping

BackgroundEpidermal growth factor receptor (EGFR)-activating mutations are major determinants in predicting the tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). Noninvasive test for the detection of EGFR mutations is required, especially in NSCLC patients from whom tissue is not available. In this study, we assessed the feasibility of detection of EGFR mutations in free DNA circulating in plasma.MethodsPlasma samples of 60 patients with partial response to gefitinib were analyzed to detect EGFR-activating mutations in exons 19 and 21. Forty (66.7%) of patients had tumor EGFR mutation results. EGFR mutations in plasma were detected using the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method. All clinical data and plasma samples were obtained from 11 centers of the Korean Molecular Lung Cancer Group (KMLCG).ResultsOf the 60 patients, 39 were female and the median age was 62.5 years. Forty-three patients never smoked, 53 had adenocarcinomas, and seven had other histologic types. EGFR-activating mutation was detected in plasma of 10 cases (exon 19 deletion in seven and exon 21 L858R point mutation in three). It could not be found in plasma after treatment for 2 months. When only patients with confirmed EGFR mutation in tumor were analyzed, 17% (6 of 35) of them showed positive plasma EGFR mutation and the mutation type was completely matched with that in tumor. There was no statistically significant difference in clinical parameters between patients with EGFR mutations in plasma and those without EGFR mutations.ConclusionsThe detection rate of EGFR mutations from plasma was not so high despite highly sensitive EGFR mutation test suggesting that more advances in detection methods and further exploration of characteristics of circulating free DNA are required.

Environmental Mass Spectrometry in the North American Great Lakes Fish Monitoring and Surveillance Program

The Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) has served to protect the Laurentian Great Lakes of North America for decades. Top predator fish from each lake are employed as bioindicators of chemical stressors within each lake. While a vast database has been created for legacy contaminants, such as polychlorinated biphenyls and organochlorine pesticides, a recent programmatic shift has transformed GLFMSP into a more proactive new chemical discovery/screening endeavour to capture the burden of more contemporary chemicals in the environment. The transition prompted the need for advanced instrumentation and the evaluation of mass spectrometric approaches beyond traditional electron capture detection and unit mass resolution mass spectrometers. Here the advances in detection methods are documented and the current direction of the program in creating a living database of anthropogenic chemicals affecting Great Lakes fish is highlighted.

ChemInform Abstract: Laboratory Studies of Organic Peroxy Radical Chemistry: An Overview with Emphasis on Recent Issues of Atmospheric Significance

AbstractReview: 224 refs.

Infecciones causadas por Clostridium difficile: una visión actualizada

Clostridium difficile is an emerging anaerobic, spore forming pathogen, recognized as the etiological agent of ~ 30% of antibiotic associated diarrheas. Clinical symptoms can fluctuate from mild to moderate diarrhea, pseudomembranous colitis and toxic megacolon. The incidence of C. difficile associated infections (CDAI) is ~ 1% of total hospitalized patients. CDAI has a mortality rate of ~1 to 5%, and a relapse rate of ~ 20%. The appearance of severe outbreaks of CDAI could be attributed to changes in the production of the two major virulence factors, the enterotoxins TcdA and TcdB, which produce massive epithelial damage. C. difficile spores play an essential role in transmission, initiation and persistence of CDAI. Recent advances in detection methods, development of novel therapies and prevention methods could allow a reduction on the frequency of CDAI. The objective of this review is to provide an updated view on the mechanisms of pathogenesis, epidemiology, risk factors, detection methods, treatment and prevention of CDAI.

Laboratory studies of organic peroxy radical chemistry: an overview with emphasis on recent issues of atmospheric significance

Organic peroxy radicals (often abbreviated RO(2)) play a central role in the chemistry of the Earth's lower atmosphere. Formed in the atmospheric oxidation of essentially every organic species emitted, their chemistry is part of the radical cycles that control the oxidative capacity of the atmosphere and lead to the formation of ozone, organic nitrates, organic acids, particulate matter and other so-called secondary pollutants. In this review, laboratory studies of this peroxy radical chemistry are detailed, as they pertain to the chemistry of the atmosphere. First, a brief discussion of methods used to detect the peroxy radicals in the laboratory is presented. Then, the basic reaction pathways - involving RO(2) unimolecular reactions and bimolecular reactions with atmospheric constituents such as NO, NO(2), NO(3), O(3), halogen oxides, HO(2), and other RO(2) species - are discussed. For each of these reaction pathways, basic reaction rates are presented, along with trends in reactivity with radical structure. Focus is placed on recent advances in detection methods and on recent advances in our understanding of radical cycling processes, particularly pertaining to the complex chemistry associated with the atmospheric oxidation of biogenic hydrocarbons.