Digital and Absolute Assays for Low Abundance Molecular Biomarkers.

There has been a recent surge of advances in biomolecular assays based on the measurement of discrete molecular targets as opposed to signals averaged across molecular ensembles. Many of these "digital" assay designs derive from now-mature technologies involving single-molecule imaging and microfluidics and provide an assortment of new modalities to quantify nucleic acids and proteins in biospecimens such as blood and tissue homogenates. A primary new benefit is the robust detection of trace analytes at attomolar to femtomolar concentrations for which many ensemble assays cannot distinguish signals above noise levels. In addition, multiple biomolecules can be differentiated within a mixture using optical barcodes, with much faster and simpler readouts compared with sequencing methods. In ideal digital assays, signals should, in theory, further represent absolute molecular counts, rather than relative levels, eliminating the need for calibration standards that are the mainstay of typical assays. Several digital assay platforms have now been commercialized but challenges hinder the adoption and diversification of these new formats, as there are broad needs to balance sensitivity and dynamic range of detection, increase analyte multiplexing, improve sample throughput, and reduce cost. Our lab and others have developed technologies to address these challenges by redesigning molecular probes and labels, improving molecular transport within detection focal volumes, and applying solution-based readout methods in flow.This Account describes the principles, formats, and design constraints of digital biomolecular assays that apply optical labels toward the goal of simple and routine target counting that may ultimately approach absolute readout standards. The primary challenges can be understood from fundamental concepts in thermodynamics and kinetics of association reactions, mass transport, and discrete statistics. Major advances include (1) new inorganic nanocrystal probes for more robust counting compared with dyes, (2) diverse molecular amplification tools that endow attachment of numerous labels to single targets, (3) specialized surfaces with patterned features for electromagnetic coupling to labels for signal amplification, (4) surface capture enhancement methods to concentrate targets through disruption of diffusion depletion zones, and (5) flow counting in which analytes are rapidly counted in solution without pull-down to a surface. Further progress and integration of these tools for biomolecular counting could improve the precision of laboratory measurements in life sciences research and benefit clinical diagnostic assays for low abundance biomarkers in limiting biospecimen volumes that are out of reach of traditional ensemble-level bioassays.

An optimization model for reducing thrombectomy center rotations while maintaining medical accessibility.

BACKGROUND/PURPOSE: This study addresses the delicate balance between healthcare personnel burnout and medical accessibility in the context of endovascular thrombectomy (EVT) services in urban areas. We aimed to determine the minimum number of hospitals providing EVT on rotation each day without compromising patient access. METHODS: Employing an optimization model, we developed shift schedules based on patient coverage rates and volumes during the pre-pandemic (2016-2018) and pandemic (2019-2021) periods. Starting with a minimum of two hospitals on duty per day, we gradually increased to a maximum of eight. Patient coverage rates, defined as the proportion of patients meeting bypass criteria and transported to rotating hospitals capable of EVT, were the primary outcomes. Sensitivity analyses explored the impact of varying patient transport intervals and accumulating patients over multiple years. RESULTS: Results from 7024 patient records revealed patient coverage rates of 92.5% (standard deviation [SD] 2.8%) during the pre-pandemic and 91.4% (SD 2.8%) during the pandemic, with at least two rotating hospitals daily. No significant differences were observed between schedules based on the highest patient volume and coverage rate months. A patient coverage rate of 98.99% was achieved with four rotating hospitals per day during the pre-pandemic period, with limited improvement beyond this threshold. Changing patient transport intervals and accumulating patients over six years (p = 0.83) had no significant impact on coverage rates. CONCLUSION: Our optimization model supports reducing the number of daily rotating hospitals by half while preserving a balance between patient accessibility and alleviating strain on medical teams.

Effect of the Functional VP1 Unique Region of Human Parvovirus B19 in Causing Skin Fibrosis of Systemic Sclerosis.

Human parvovirus B19 (B19V) is a single-stranded non-enveloped DNA virus of the family Parvoviridae that has been associated with various autoimmune disorders. Systemic sclerosis (SSc) is an autoimmune connective tissue disorder with high mortality and has been linked to B19V infection. However, the precise mechanism underlying the B19V contribution to the development of SSc remains uncertain. This study investigated the impacts of the functional B19V-VP1 unique region (VP1u) in macrophages and bleomycin (BLE)-induced SSc mice. Cell experimental data showed that significantly decreased viability and migration of both B19V-VP1u-treated U937 and THP-1 macrophages are detected in the presence of celastrol. Significantly increased MMP9 activity and elevated NF-kB, MMP9, IL-6, TNF-α, and IL-1ß expressions were detected in both B19V-VP1u-treated U937 and THP-1 macrophages. Conversely, celastrol revealed an inhibitory effect on these molecules. Notably, celastrol intervened in this pathogenic process by suppressing the sPLA2 activity of B19V-VP1u and subsequently reducing the inflammatory response. Notably, the administration of B19V-VP1u exacerbated BLE-induced skin fibrosis in mice, with augmented expressions of TGF-ß, IL-6, IL-17A, IL-18, and TNF-α, ultimately leading to α-SMA and collagen I deposits in the dermal regions of BLE-induced SSc mice. Altogether, this study sheds light on parvovirus B19 VP1u linked to scleroderma and aggravated dermal fibrosis.

Rapid quantification of microRNA-375 through one-pot primer-generating rolling circle amplification.

A recent surge of interest in microRNA has been driven by its discovery as a circulating biomarker of disease, with many diagnostic test platforms currently under development. Alternatives to widely used microRNA quantification methods such as quantitative reverse transcriptase PCR (qRT-PCR) are needed for use in portable and point-of-care devices which are incompatible with complex sample processing workflows and thermal cycling. Rolling circle amplification (RCA) is a one-pot assay technique which directly amplifies nucleic acids using sequence-specific microRNA priming to initiate a single-step isothermal reaction that is compatible with simple devices. Sensitivity remains a limitation of RCA methods, however, and detection limits do not typically reach the femtomolar level in which microRNA targets are present in blood. RCA assays have previously been improved by digestion of the amplification products using a nicking endonuclease to exponentially generate new reaction primers. Here we describe how a ligation-free version of this technique performed in a single tube can be used to improve the limit of detection for microRNA-375, an important blood biomarker for prostate cancer. Endonuclease addition changes a linear process into an exponential amplification reaction which results in a 61-fold improvement of the limit of detection (5.9 fM), a dynamic range wider than 5-log(10), and a shorter reaction time. By eliminating the need for microRNA reverse transcription and thermal cycling, this single-step one-pot method provides a more rapid and simplified alternative to qRT-PCR for ultrasensitive microRNA quantification in blood extracts.

Antioxidative effect of DJ-1 is enhanced in NG108-15 cells by DPMQ-induced copper influx.

Disruption of copper homeostasis is closely involved in neurodegenerative disorders. This study examined whether a hybrid copper-binding compound, (E)-2-(4-(dimethylamino)phenylimino)methyl)quinolin-8-ol (DPMQ), is able to protect NG108-15 cells against oxidative stress. We found that treatment of cells with rotenone or hydrogen peroxide increased cellular oxidative stress and resulted in mitochondrial dysfunction and apoptosis. The cellular levels of Nrf2 and the Cu2+ chaperone DJ-1 were also decreased. These oxidative detrimental effects were all inhibited when cells were cotreated with DPMQ. DPMQ increased cellular Cu2+ content, DJ-1 protein level, superoxide dismutase (SOD) activity, and Nrf2 nuclear translocation under basal state. The activity of SOD decreased under redox imbalance and this decrease was blocked by DPMQ treatment, while the protein level of SOD1 remained unaltered regardless of the oxidative stress and DPMQ treatment. Using endogenous proteins, coimmunoprecipitation showed that DJ-1 bound with SOD1 and Nrf2 individually. The amount of Nrf2, bound to DJ-1, consistently reflected its cellular level, while the amount of SOD1, bound to DJ-1, was potentiated by DPMQ, being greater in the basal state than under redox imbalance. Simultaneous inclusion of nonpermeable Cu2+ chelator tetrathiomolybdate or triethylenetetramine during DPMQ treatment blocked all aforementioned effects of DPMQ, showing that the dependency of the effect of DPMQ on extracellular Cu2+. In addition, silencing of DJ-1 blocked the protection of DPMQ against oxidative stress. Taken all together, our results suggest that DPMQ stabilizes DJ-1 in a Cu2+-dependent manner, which then brings about SOD1 activation and Nrf2 nuclear translocation; these together alleviate cellular oxidative stress.

Murray secretion scale and fiberoptic endoscopic evaluation of swallowing in predicting aspiration in dysphagic patients.

The objective of this retrospective review is to evaluate the ability of the Murray secretion scale to predict aspiration as determined by fiberoptic endoscopic evaluation of swallowing. Patients with dysphagia undergoing a fiberoptic endoscopic evaluation of swallowing study between January 2013 and November 2015 from a single, tertiary care institution were retrospectively reviewed. The Murray secretion scale and penetration aspiration scale on fiberoptic endoscopic evaluation of swallowing examination were determined. Spearman's correlation analysis, sensitivity, specificity, predictive values, and relative risk evaluating the relationship between the Murray secretion scale and aspiration on fiberoptic endoscopic evaluation of swallowing were calculated. Subgroups of head and neck cancer patients, penetration group, and aspiration group were also analyzed. The mean age of the cases (N = 212) was 62.4 years. Eighty percent were male. There was a strong correlation between Murray secretion scale grade and penetration aspiration scale score (r = 0.785, p < 0.001). The sensitivity and specificity of a Murray secretion scale grade 2 or higher in predicting aspiration were 74 and 90%, respectively. Individuals with a Murray secretion scale grade of 2 or higher were 13.6 times more likely to aspirate than patients with a lower Murray secretion scale grade. All subgroups showed similar trend. Determination of a Murray secretion scale grade, determined by flexible nasopharyngoscopy, may predict patients at high risk for aspiration. In clinical scenarios where more complete assessments of aspiration risk are immediately impossible or impractical, the Murray secretion scale grade may add valuable information to assist in clinical decision-making in patients with dysphagia.

Quantum Dot-Fab' Conjugates as Compact Immunolabels for Microtubule Imaging and Cell Classification.

Antibodies and their conjugates of fluorescent labels are widely applied in life sciences research and clinical pathology. Among diverse label types, compact quantum dots (QDs) provide advantages of multispectral multiplexing, bright signals in the deep red and infrared, and low steric hindrance. However, QD-antibody conjugates have random orientation of the antigen-binding domain which may interfere with labeling and are large (20-30 nm) and heterogeneous, which limits penetration into biospecimens. Here, we develop conjugates of compact QDs and Fab' antibody fragments as primary immunolabels. Fab' fragments are conjugated site-specifically through sulfhydryl groups distal to antigen-binding domains, and the multivalent conjugates have small and homogeneous sizes (∼12 nm) near those of full-sized antibodies. Their performance as immunolabels for intracellular antigens is evaluated quantitatively by metrics of microtubule labeling density and connectivity in fixed cells and for cytological identification in fixed brain specimens, comparing results with probes based on spectrally-matched dyes. QD-Fab' conjugates outperformed QD conjugates of full-sized antibodies and could be imaged with bright signals with 1-photon and 2-photon excitation. The results demonstrate a requirement for smaller bioaffinity agents and site-specific orientation for the success of nanomaterial-based labels to enhance penetration in biospecimens and minimize nonspecific staining.

Structural Design of Multidentate Copolymers as Compact Quantum Dot Coatings for Live-Cell Single-Particle Imaging.

Quantum dots (QDs) are a class of semiconductor nanocrystal used broadly as fluorescent emitters for analytical studies in the life sciences. These nanomaterials are particularly valuable for single-particle imaging and tracking applications in cells and tissues. An ongoing technological goal is to reduce the hydrodynamic size of QDs to enhance access to sterically hindered biological targets. Multidentate polymer coatings are a focus of these efforts and have resulted in compact and stable QDs with hydrodynamic diameters near 10 nm. New developments are needed to reach smaller sizes to further enhance transport through pores in cells and tissues. Here, we describe how structural characteristics of linear multidentate copolymers determine hydrodynamic size, colloidal stability, and biomolecular interactions of coated QDs. We tune copolymer composition, degree of polymerization, and hydrophilic group length, and coat polymers on CdSe and (core)shell (HgCdSe)CdZnS QDs. We find that a broad range of polymer structures and compositions yield stable colloidal dispersions; however, hydrodynamic size minimization and nonspecific binding resistance can only be simultaneously achieved within a narrow range of properties, requiring short polymers, balanced compositions, and small nanocrystals. In quantitative single-molecule imaging assays in synapses of live neurons, size reduction progressively increases labeling specificity of neurotransmitter receptors. Our findings provide a design roadmap to next-generation QDs with sizes approaching fluorescent protein labels that are the standard of many live-cell biomolecular studies.

Significance of the glycolytic pathway and glycolysis related-genes in tumorigenesis of human colorectal cancers.

We investigated gene expressions involved in the glycolytic pathways in colorectal cancer. The study was designed to use gene ontology and its relevant bioinformatics tools to analyze the microarray data obtained from CRC tissues and their corresponding normal tissues, in order to explore the correlation between the glycolytic metabolic pathway and possible pathogenesis of this disease. The overexpression of glycolysis-related genes was observed in over 76% of CRC tissues. In addition, we stimulated the SW480 and SW620 CRC cell lines with 15 mM D-(+)-glucose and 10 mM 2-deoxy-D-glucose respectively. The results indicate that the proliferation response of both the SW480 and SW620 cell lines increased remarkably with a time-dependent effect by D-(+)-glucose administration. In contrast, the proliferation response of both the SW480 and SW620 cell lines was significantly inhibited by 2-DG administration. Likewise, further analyses of the expression of related genes triggered by the D-(+)-glucose in vivo show that the activation process of these eight genes - GLUT1, HK1, GPI, GAPD, PGK1, PGK2, ENO2, PKM2 - prominently increased with a time-dependent effect. In conclusion, this study demonstrates that the glycolytic pathway and glycolysis-related genes may play an important role in the tumorigenesis of CRC, but their molecular mechanisms need further investigation to verify this.

Fully endoscopic laser stapedotomy: is it comparable with microscopic surgery?

BACKGROUND: Microscopic stapedotomy is very successful and has long history, but it still has some constraints. Thus, otoendoscopy is increasingly popular nowadays. AIMS/OBJECTIVES: The retrospective review study was to investigate the role of endoscopic laser stapedotomy in treating patients with otosclerosis. MATERIALS AND METHODS: Seventeen patients who received endoscopic laser stapedotomy from April 2014 to July 2017 were enrolled and compared to 13 patients who had microscopic stapedotomy from February 2009 to March 2012. The anatomical structures, operative time, and postoperative hearing outcomes were assessed in two groups. Relation between external acoustic canal and operative time was also analyzed. RESULTS: Using an endoscope, the operative field was clear, with easily identified anatomy, without need to sacrifice bony structures. The operative time was significantly longer in the endoscopic group in 2014 and decreased in the following years. There was no significant difference of hearing improvements between the two groups. There was a weak correlation between the width of the external auditory canal and the operative time. CONCLUSIONS AND SIGNIFICANCE: Fully endoscopic stapes surgery is a feasible and safe surgical technique and results in satisfactory hearing outcomes. However, surgeons take longer to master the technique and to achieve shorter endoscopic operative times.

Resolution of transmission electron backscatter diffraction in aluminum and silver: Effect of the atomic number.

This work aims to investigate the influence of intrinsic and extrinsic factors on the physical resolution of the transmission electron backscattered diffraction technique (t-EBSD) in aluminum and silver. Here, we focus on the intrinsic factors, namely, atomic number and thickness of the specimen, and extrinsic set-up factors, which include the electron beam voltage, working distance, and specimen tilt. The working distance and tilt angle, which are selected as 12 mm and 60° for Al and 12 mm and 50° for Ag, respectively, reveal a sharp pattern with high contrast. The physical resolutions at the lateral and longitudinal directions depend on the depth resolution. The depth and lateral and longitudinal resolutions increase in Al but decrease in Ag with increased accelerating voltage. The decrease in specimen thickness for Al and Ag from 400 nm to 100 nm reduces the lateral and longitudinal resolutions. The most ideal depth and lateral and longitudinal resolutions obtained under a thickness of 100 nm are 22.7, 18.9, and 33.7 nm at 30 kV for Ag and 34.7, 22.8, and 36.6 nm at 15 kV for Al, respectively.

Honokiol rescues sepsis-associated acute lung injury and lethality via the inhibition of oxidative stress and inflammation.

PURPOSE: Sepsis has a high mortality rate despite the recent advances in intensive care medicine and antibiotics. Honokiol, a low molecular weight natural product, is known to possess anti-inflammatory activity. Here, we investigate whether honokiol can ameliorate acute lung injury and lethal response in murine models of sepsis. METHODS: Mice were intraperitoneally given vehicle or honokiol 30 min after the induction of sepsis by cecal ligation and puncture (CLP) and endotoxemia by administration of E. coli lipopolysaccharide (LPS). RESULTS: The productions of serum tumor necrosis factor-α (TNF-α), nitric oxide (NO), and high mobility group box 1 (HMGB 1) were increased in mice during sepsis, which could be reversed by honokiol. Honokiol could also effectively reduce the increased blood lipid peroxidation and nitrotyrosine in septic mice. Honokiol significantly reversed the inductions of inducible NO synthase and nuclear factor-κB (NF-κB) activation in the lungs of mice during sepsis. Honokiol also effectively rescued the lung edema, lung pathological changes, and lethality in septic mice. CONCLUSIONS: These findings suggest that honokiol is capable of suppressing the lethal response and acute lung injury associated with sepsis, and support the potential use of honokiol as a therapeutic agent for the conditions associated with septic shock.

CDC25A, VAV1, TP73, BRCA1 and ZAP70 gene overexpression correlates with radiation response in colorectal cancer.

Radiotherapy is increasingly used in adjuvant approaches for colorectal cancer (CRC) to reduce local recurrence and improve survival. However, the principal limitation is the large variability in response among different individuals due to tumor heterogeneity. In the present study, we compared gene expression profiles between radiosensitive and radioresistant colorectal cancer cell lines to identify radiation-related molecules that can be used to evaluate the effects of radiation. The CRC cell line SW620 was irradiated with a high-energy photo beam. Following radiation treatment, RNA was extracted from non-irradiated and irradiated cells, respectively, and gene expression analysis was performed by oligonucleotide microarray and the DAVID bioinformatics method. To further confirm the results, an additional 4 CRC cell lines, COLO205, T84, HCT116, SW480 and SW403 were purchased from ATCC. The radiosensitivities of each were determined by the survival fraction at 2 Gray (SF2) of the surviving cells using the ATPLite assay, and the gene expression profiles after irradiation among the radiosensitive and radioresistant cell lines were analyzed by membrane arrays. The relationships between gene expression and patient clinicopathological features were also analyzed using membrane arrays and RT-PCR. The results from oligonucleotide microarray analysis show that 1601 genes were up-regulated (gene expression ratio of post- to pre-radiation treatment>2). By bioinformatic database analysis, 30 up-regulated genes were identified as involved in DNA damage response pathways, immune response pathways and the complement and coagulation cascades pathway. Fifteen genes showed differential gene expression profiles between radiosensitive (HCT116 and SW620) and radioresistant CRC cell lines (SW403 and SW480). In 110 CRC tissues, we detected five genes CDC25A, VAV1, TP73, BRCA1 and ZAP70 from 15 overexpressed genes that significantly related to prognostic factors (tumor size, advanced stage, invasive depth, lymph node metastasis and differentiation). These findings suggest that CDC25A, VAV1, TP73, BRCA1 and ZAP70 may be novel markers for predicting the effectiveness of radiotherapy in CRC patients.

Nanoparticles-doped guest-host liquid crystal displays.

The technique, nanoparticles-induced vertical alignment (NIVA), was applied to fabricate a guest-host liquid crystal display (GH-LCD) without conventional alignment layers. The GH-LCD produced by NIVA exhibited a high reflectance of approximately 59% in the voltage-off state and a low threshold voltage of approximately 2.1 V(rms). NIVA is very suitable to be applied for fabricating a plastic LCD requiring a low temperature process, and a flexible timepiece by using NIVA was demonstrated.