This paper reviews methods for detecting proteins based on molecular digitization,
, the isolation and detection of single protein molecules or singulated ensembles of protein molecules. The single ...molecule resolution of these methods has resulted in significant improvements in the sensitivity of immunoassays beyond what was possible using traditional "analog" methods: the sensitivity of some digital immunoassays approach those of methods for measuring nucleic acids, such as the polymerase chain reaction (PCR). The greater sensitivity of digital protein detection has resulted in immuno-diagnostics with high potential societal impact,
, the early diagnosis and therapeutic intervention of Alzheimer's Disease. In this review, we will first provide the motivation for developing digital protein detection methods given the limitations in the sensitivity of analog methods. We will describe the paradigm shift catalyzed by single molecule detection, and will describe in detail one digital approach - which we call digital bead assays (DBA) - based on the capture and labeling of proteins on beads, identifying "on" and "off" beads, and quantification using Poisson statistics. DBA based on the single molecule array (Simoa) technology have sensitivities down to attomolar concentrations, equating to ∼10 proteins in a 200 μL sample. We will describe the concept behind DBA, the different single molecule labels used, the ways of analyzing beads (imaging of arrays and flow), the binding reagents and substrates used, and integration of these technologies into fully automated and miniaturized systems. We provide an overview of emerging approaches to digital protein detection, including those based on digital detection of nucleic acids labels, single nanoparticle detection, measurements using nanopores, and methods that exploit the kinetics of single molecule binding. We outline the initial impact of digital protein detection on clinical measurements, highlighting the importance of customized assay development and translational clinical research. We highlight the use of DBA in the measurement of neurological protein biomarkers in blood, and how these higher sensitivity methods are changing the diagnosis and treatment of neurological diseases. We conclude by summarizing the status of digital protein detection and suggest how the lab-on-a-chip community might drive future innovations in this field.
The 'precision medicine (systems medicine)' concept promises to achieve a shift to future healthcare systems with a more proactive and predictive approach to medicine, where the emphasis is on ...disease prevention rather than the treatment of symptoms. The individualization of treatment for each patient will be at the centre of this approach, with all of a patient's medical data being computationally integrated and accessible. Precision medicine is being rapidly embraced by biomedical researchers, pioneering clinicians and scientific funding programmes in both the European Union (EU) and USA. Precision medicine is a key component of both Horizon 2020 (the EU Framework Programme for Research and Innovation) and the White House's Precision Medicine Initiative. Precision medicine promises to revolutionize patient care and treatment decisions. However, the participants in precision medicine are faced with a considerable central challenge. Greater volumes of data from a wider variety of sources are being generated and analysed than ever before; yet, this heterogeneous information must be integrated and incorporated into personalized predictive models, the output of which must be intelligible to non-computationally trained clinicians. Drawing primarily from the field of 'oncology', this article will introduce key concepts and challenges of precision medicine and some of the approaches currently being implemented to overcome these challenges. Finally, this article also covers the criticisms of precision medicine overpromising on its potential to transform patient care.
This paper describes the need for technologies that improve analytical sensitivity to proteins to better define and monitor the progression from heath to disease over the course of an individual's ...life. These technologies have the potential to allow the early diagnosis of disease, and trigger treatments at the time when they have the greatest opportunity to be effective. We will describe a technology that we have developed for high sensitivity protein detection, namely, single molecule arrays (Simoa). Simoa is based on the capture of protein molecules on magnetic beads, labeling each protein with an enzyme, and counting of single enzyme labels on beads that are isolated in arrays of femtoliter wells. Simoa has enabled the detection of proteins at subfemtomolar concentrations in a variety of biological fluids. We describe the impact of higher sensitivity of proteins using Simoa on: less invasive testing; earlier detection of disease; providing biomarker baseline profiles for healthy individuals; testing of small sample volumes; monitoring of therapeutic efficacy; faster tests; and detection of proteins in complex samples. We also provide a perspective of how new technologies that allow the low-cost manufacture and miniaturization of Simoa could drive the next wave of analytical devices, including wearables.
Abstract
Novel forensics-inspired molecular approaches have revolutionized species detection in the wild and are particularly useful for tracing endangered or invasive species. These new ...environmental DNA or RNA (eDNA or eRNA)–based techniques are now being applied to human and animal pathogen surveillance, particularly in aquatic environments. They allow better disease monitoring (presence or absence and geographical spread) and understanding of pathogen occurrence and transmission, benefitting species conservation and, more recently, our understanding of the COVID-19 global human pandemic. In the present article, we summarize the benefits of eDNA-based monitoring, highlighted by two case studies: The first is a fibropapillomatosis tumor-associated herpesvirus (chelonid herpesvirus 5) driving a sea turtle panzootic, and the second relates to eRNA-based detection of the SARS-CoV-2 coronavirus driving the COVID-19 human pandemic. The limitations of eDNA- or eRNA-based approaches are also summarized, and future directions and recommendations of the field are discussed. Continuous eDNA- or eRNA-based monitoring programs can potentially improve human and animal health by predicting disease outbreaks in advance, facilitating proactive rather than reactive responses.
Sclerosants: A Comparative Review DUFFY, DAVID M.
Dermatologic surgery,
June 2010, 2010-Jun, 2010-06-00, 20100601, Letnik:
36, Številka:
Sup 2
Journal Article
Recenzirano
BACKGROUND
Sclerotherapy is popular for the treatment of lower extremity telangiectasias and varicose and reticular veins. Although a large number of sclerosants are commonly employed, there are few ...data that directly compare their advantages and drawbacks.
OBJECTIVES
To analyze and present the differences between sclerosants that make them more or less suitable agents in specific clinical applications.
MATERIALS AND METHODS
A systemic review of published medical literature that compares and contrasts different classes of sclerosants is presented.
RESULTS
There is no perfect sclerosant that is complication free and 100% effective. The ability to match the sclerosant to the clinical problem being approached makes the availability of more Food and Drug Administration–approved sclerosants appealing.
CONCLUSION
Modern sclerosants that have been subjected to rigorous experimental and clinical trials will provide even more efficacious and safer patient treatments.
David M. Duffy, MD, received a grant from Bioform for support of this article.
Asthma is a heterogeneous disease that results from the complex interaction between genetic factors and environmental exposures that occur at critical periods throughout life. It seems plausible to ...regard childhood-onset and adult-onset asthma as different entities, each with a different pathophysiology, trajectory, and outcome. This review provides an overview about the role of genetics and gene-environment interactions in these two conditions. Looking at the genetic overlap between childhood and adult onset disease gives one window into whether there is a correlation, as well as to mechanism. A second window is offered by the genetics of the relationship between each type of asthma and other phenotypes e.g., obesity, chronic obstructive pulmonary disease (COPD), atopy, vitamin D levels, and inflammatory and immune status; and third, the genetic-specific responses to the many environmental exposures that influence risk throughout life, and particularly those that occur during early-life development. These represent a large number of possible combinations of genetic and environmental factors, at least 150 known genetic loci
. tobacco smoke, outdoor air pollutants, indoor exposures, farming environment, and microbial exposures. Considering time of asthma onset extends the two-dimensional problem of gene-environment interactions to a three-dimensional problem, since identified gene-environment interactions seldom replicate for childhood and adult asthma, which suggests that asthma susceptibility to environmental exposures may biologically differ from early life to adulthood as a result of different pathways and mechanisms of the disease.
We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by ...polymerases. An abasic peptide nucleic acid (PNA) probe-containing a reactive amine instead of a nucleotide at a specific position in the sequence-for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase-containing an aldehyde group-that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)-an established biomarker of liver toxicity-extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use.
Abstract Objective To conduct a pilot study to evaluate the prognostic potential of serum tau protein measurements to predict neurological outcome 6 months following resuscitation from cardiac ...arrest. Methods In this retrospective observational study, we employed a new ultra sensitive digital immunoassay technology to examine serial serum samples from 25 cardiac arrest patients to examine tau release into serum as a result of brain hypoxia, and probe for its significance predicting six-month neurological outcome. Serial blood samples were obtained from resuscitated cardiac arrest survivors during their first five days in an intensive care unit, and serum total tau was measured. Cerebral function assessments were made using Cerebral Performance Categorization (CPC) at discharge from the ICU and six months later. Tau data were analyzed in the context of 6-month CPC scores. Results Tau elevations ranged from modest (<10 pg/mL) to very high (hundreds of pg/mL), and exhibited unexpected bi-modal kinetics in some patients. Early tau elevations appeared within 24 h of cardiac arrest, and delayed elevations appeared after 24–48 h. In patients with delayed elevations, areas under the curves of tau concentration vs. hours since cardiac arrest were highly predictive of 6-month outcome ( P < 0.0005). Conclusion High-sensitivity serum tau measurements combined with an understanding of tau release kinetics could have utility for hypoxic brain injury assessment and prediction of cerebral function outcome.
Previous studies have successfully identified genetic variants in several genes associated with human iris (eye) color; however, they all used simplified categorical trait information. Here, we ...quantified continuous eye color variation into hue and saturation values using high-resolution digital full-eye photographs and conducted a genome-wide association study on 5,951 Dutch Europeans from the Rotterdam Study. Three new regions, 1q42.3, 17q25.3, and 21q22.13, were highlighted meeting the criterion for genome-wide statistically significant association. The latter two loci were replicated in 2,261 individuals from the UK and in 1,282 from Australia. The LYST gene at 1q42.3 and the DSCR9 gene at 21q22.13 serve as promising functional candidates. A model for predicting quantitative eye colors explained over 50% of trait variance in the Rotterdam Study. Over all our data exemplify that fine phenotyping is a useful strategy for finding genes involved in human complex traits.
Amyloid β (Aβ) peptides are proteolytic products from amyloid precursor protein (APP) and are thought to play a role in Alzheimer disease (AD) pathogenesis. While much is known about molecular ...mechanisms underlying cerebral Aβ accumulation in familial AD, less is known about the cause(s) of brain amyloidosis in sporadic disease. Animal and postmortem studies suggest that Aβ secretion can be up-regulated in response to hypoxia. We employed a new technology (Single Molecule Arrays, SiMoA) capable of ultrasensitive protein measurements and developed a novel assay to look for changes in serum Aβ42 concentration in 25 resuscitated patients with severe hypoxia due to cardiac arrest. After a lag period of 10 or more hours, very clear serum Aβ42 elevations were observed in all patients. Elevations ranged from approximately 80% to over 70-fold, with most elevations in the range of 3-10-fold (average approximately 7-fold). The magnitude of the increase correlated with clinical outcome. These data provide the first direct evidence in living humans that ischemia acutely increases Aβ levels in blood. The results point to the possibility that hypoxia may play a role in the amyloidogenic process of AD.
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