A novel method for liquid-phase extraction of cell-free DNA for detection of circulating tumor DNA.

Low yields of extracted cell-free DNA (cfDNA) from plasma limit continued development of liquid biopsy in cancer, especially in early-stage cancer diagnostics and cancer screening applications. We investigate a novel liquid-phase-based DNA isolation method that utilizes aqueous two-phase systems to purify and concentrate circulating cfDNA. The PHASIFY MAX and PHASIFY ENRICH kits were compared to a commonly employed solid-phase extraction method on their ability to extract cfDNA from a set of 91 frozen plasma samples from cancer patients. Droplet digital PCR (ddPCR) was used as the downstream diagnostic to detect mutant copies. Compared to the QIAamp Circulating Nucleic Acid (QCNA) kit, the PHASIFY MAX method demonstrated 60% increase in DNA yield and 171% increase in mutant copy recovery, and the PHASIFY ENRICH kit demonstrated a 35% decrease in DNA yield with a 153% increase in mutant copy recovery. A follow-up study with PHASIFY ENRICH resulted in the positive conversion of 9 out of 47 plasma samples previously determined negative with QCNA extraction (all with known positive tissue genotyping). Our results indicate that this novel extraction technique offers higher cfDNA recovery resulting in better sensitivity for detection of cfDNA mutations compared to a commonly used solid-phase extraction method.

Evaluation of the INDICAID COVID-19 Rapid Antigen Test in Symptomatic Populations and Asymptomatic Community Testing.

As the COVID-19 pandemic progresses, there is an increasing need for rapid, accessible assays for SARS-CoV-2 detection. We present a clinical evaluation and real-world implementation of the INDICAID COVID-19 rapid antigen test (INDICAID rapid test). A multisite clinical evaluation of the INDICAID rapid test using prospectively collected nasal (bilateral anterior) swab samples from symptomatic subjects was performed. The INDICAID rapid test demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 85.3% (95% confidence interval [95% CI], 75.6% to 91.6%) and 94.9% (95% CI, 91.6% to 96.9%), respectively, compared to laboratory-based reverse transcriptase PCR (RT-PCR) using nasal specimens. The INDICAID rapid test was then implemented at COVID-19 outbreak screening centers in Hong Kong as part of a testing algorithm (termed "dual-track") to screen asymptomatic individuals for prioritization for confirmatory RT-PCR testing. In one approach, preliminary positive INDICAID rapid test results triggered expedited processing for laboratory-based RT-PCR, reducing the average time to confirmatory result from 10.85 h to 7.0 h. In a second approach, preliminary positive results triggered subsequent testing with an onsite rapid RT-PCR, reducing the average time to confirmatory result to 0.84 h. In 22,994 asymptomatic patients, the INDICAID rapid test demonstrated a PPA of 84.2% (95% CI, 69.6% to 92.6%) and an NPA of 99.9% (95% CI, 99.9% to 100%) compared to laboratory-based RT-PCR using combined nasal/oropharyngeal specimens. The INDICAID rapid test has excellent performance compared to laboratory-based RT-PCR testing and, when used in tandem with RT-PCR, reduces the time to confirmatory positive result. IMPORTANCE Laboratory-based RT-PCR, the current gold standard for COVID-19 testing, can require a turnaround time of 24 to 48 h from sample collection to result. The delayed time to result limits the effectiveness of centralized RT-PCR testing to reduce transmission and stem potential outbreaks. To address this, we conducted a thorough evaluation of the INDICAID COVID-19 rapid antigen test, a 20-minute rapid antigen test, in both symptomatic and asymptomatic populations. The INDICAID rapid test demonstrated high sensitivity and specificity with RT-PCR as the comparator method. A dual-track testing algorithm was also evaluated utilizing the INDICAID rapid test to screen for preliminary positive patients, whose samples were then prioritized for RT-PCR testing. The dual-track method demonstrated significant improvements in expediting the reporting of positive RT-PCR test results compared to standard RT-PCR testing without prioritization, offering an improved strategy for community testing and controlling SARS-CoV-2 outbreaks.

Histamine receptors of cones and horizontal cells in Old World monkey retinas.

In primates the retina receives input from histaminergic neurons in the posterior hypothalamus that are active during the day. In order to understand how this input contributes to information processing in Old World monkey retinas, we have been localizing histamine receptors (HR) and studying the effects of histamine on the neurons that express them. Previously, we localized HR3 to the tips of ON bipolar cell dendrites and showed that histamine hyperpolarizes the cells via this receptor. We raised antisera against synthetic peptides corresponding to an extracellular domain of HR1 between the 4th and 5th transmembrane domains and to an intracellular domain near the carboxyl terminus of HR2. Using these, we localized HR1 to horizontal cells and a small number of amacrine cells and localized HR2 to puncta closely associated with synaptic ribbons inside cone pedicles. Consistent with this, HR1 mRNA was detected in horizontal cell perikarya and primary dendrites and HR2 mRNA was found in cone inner segments. We studied the effect of 5 µM exogenous histamine on primate cones in macaque retinal slices. Histamine reduced I(h) at moderately hyperpolarized potentials, but not the maximal current. This would be expected to increase the operating range of cones and conserve ATP in bright, ambient light. Thus, all three major targets of histamine are in the outer plexiform layer, but the retinopetal axons containing histamine terminate in the inner plexiform layer. Taken together, the findings in these three studies suggest that histamine acts primarily via volume transmission in primate retina.

BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism.

The GGCC-specific restriction endonuclease BspRI is one of the few Type IIP restriction endonucleases, which were suggested to be a monomer. Amino acid sequence information obtained by Edman sequencing and mass spectrometry analysis was used to clone the gene encoding BspRI. The bspRIR gene is located adjacently to the gene of the cognate modification methyltransferase and encodes a 304 aa protein. Expression of the bspRIR gene in Escherichia coli was dependent on the replacement of the native TTG initiation codon with an ATG codon, explaining previous failures in cloning the gene using functional selection. A plasmid containing a single BspRI recognition site was used to analyze kinetically nicking and second-strand cleavage under steady-state conditions. Cleavage of the supercoiled plasmid went through a relaxed intermediate indicating sequential hydrolysis of the two strands. Results of the kinetic analysis of the first- and second-strand cleavage are consistent with cutting the double-stranded substrate site in two independent binding events. A database search identified eight putative restriction-modification systems in which the predicted endonucleases as well as the methyltransferases share high sequence similarity with the corresponding protein of the BspRI system. BspRI and the related putative restriction endonucleases belong to the PD-(D/E)XK nuclease superfamily.

Isolation and production of cells suitable for human therapy: challenges ahead.

Considerable practical hurdles must be overcome prior to the broad application of stem cell therapies. We outline challenges that may vary across different models of cell therapy, including the following broad concepts: issues related to the sourcing of material, and issues related to product manufacturing, shipping, storage and tracking, and standardization.

New ISSCR guidelines underscore major principles for responsible translational stem cell research.

The International Society for Stem Cell Research (ISSCR) task force that developed new Guidelines for the Clinical Translation of Stem Cells discusses core principles that should guide the responsible transition of basic stem cell research into appropriate clinical applications.

Differentiating primary human cells in rapid-throughput discovery applications.

Primary cultures of human cells provide an increasingly important alternative to using virally transformed or otherwise immortalized cell lines or to using cloned cell lines derived from human or animal tumors. Advances in primary cell culture techniques, media formulations, and other reagents have enabled routine culture of primary cells derived from human tissues for biomedical research and drug discovery approaches such as high content screening. That primary cells retain the phenotypic characteristics of the original tissue is one main advantage over immortalized cell lines. However, securing reliable supplies of primary cells on a large scale has been problematic. Here, two primary differentiating cell types, preadipocytes and preosteoclasts, are described to illustrate the utility of commercially produced cell systems in discovery research and rapid-throughput applications.

Effect of water-soluble metal working fluid aerosols on respiratory system after 13 weeks of repeated inhalation exposure in F344 rats.

Three groups of male F344 rats were exposed to a water-soluble metal working fluid (MWF) aerosol at concentrations of 20, 60 or 180 mg/m3 for 6 h/day, five days a week, for 13 weeks in inhalation chambers. The aerosol particles were normally distributed and the mass median aerodynamic diameter was 1.56 microm. Despite the absence of clinical findings or significant changes in body weight during the 13-week exposure period, the numbers of white blood cells and lung weights were significantly higher at the end of the 13-week exposure period. Exposure to 20 mg/m3 of the MWF aerosol was found to have an effect on the respiratory system, including an accumulation of foamy macrophages in the bronchoalveolar lavage (BAL) fluid and thickening of the alveolar walls in the histopathology. The level of histamine and number of polymorphonuclear (PMN) cells were also higher in the BAL fluid from the rats exposed to 60 mg/m3 of the MWF aerosol, while the respiratory inflammation was most pronounced in the rats exposed to 180 mg/m3 of the MWF aerosol, including the accumulation of PMNs and foamy macrophages in the BAL cells, lung weight increase and thickening of the alveolar walls. Immunoglobulin IgG2a level was also lower in the sera from the rats exposed to 180 mg/m3 of the MWF aerosol. Therefore, even though no clinical symptoms were observed in the rats exposed to the high MWF concentration, respiratory inflammation was still induced by a relatively low concentration of the MWF, while the immune system was affected by the high MWF concentration.

Inflammatory and immunological responses to subchronic exposure to endotoxin-contaminated metalworking fluid aerosols in F344 rats.

Rats were exposed for 6 h per day in inhalation chambers to a 10 mg/m(3) concentration of metalworking fluid (MWF) contaminated with endotoxin at concentrations of 1813 (low dose) and 20,250 eu/m(3) (high dose) 5 days per week for 8 weeks. It was found that 94.7% of the MWF aerosol particles had diameters in the range of 0.42-4.6 microm, with geometric mean diameter of 1.56 microm. The body weight and pulmonary function parameters were measured every week during the 8 weeks of exposure, whereas bronchoalveolar lavage (BAL) fluid was prepared to measure the inflammatory markers and cytokines after the 8 weeks of exposure. There were no changes in body weight and respiratory function (tidal volume and respiratory frequency) during the 8 weeks of exposure to the MWF containing endotoxins, yet lung weight increased significantly (P < 0.05) in the rats exposed to the MWF both with and without endotoxins. The number of polymorphonuclear (PMN) cells in the BAL fluid increased significantly (P < 0.05) in the rats exposed to MWF with endotoxins, and the levels of cytokines such as IL-4, INF-gamma, IL-1beta, and TNF-alpha also were significantly increased (P < 0.05) compared to the control. The NOx production activity of the BAL cells increased significantly (P < 0.05) in the rats exposed to the MWF with and without endotoxins. Increases in lung weight, number of PMN cells, and levels of extracellular cytokines and NOx were all more significant in the rats exposed to the MWF with endotoxins rather than in those exposed to MWF without endotoxins. In spleen cell cultures, T-cell proliferation activity was decreased, yet cytokine levels (INF-gamma, IL-1beta, IL-4, and TNF-alpha) remained unchanged after repeated exposure to MWF with and without endotoxins. Although the levels of total IgG(1), IgG(2a), and IgE antibodies in the serum were not changed, the levels of endotoxin-specific antibodies, including IgG(2a) and IgE, were increased significantly (P < 0.05) in the rats exposed to endotoxins, but there was not a significant increase in endotoxin-specific IgG(1). When taken together, the results indicate that lung inflammatory responses can be induced without changing pulmonary function after repeated exposure to MWFs contaminated with endotoxins. In addition, endotoxin-specific IgG(2a) and IgE may be effective biomarkers for workers exposed to MWFs contaminated with endotoxins in the workplace.

Respiratory effect of acute and subacute exposure to endotoxin-contaminated metal working fluid (MWF) aerosols on Sprague-Dawley rats.

Male Sprague-Dawley rats were exposed to a water-soluble metal working fluid (MWF) (5% v/v) contaminated with endotoxins (10,000 eu/ml or 100,000 eu/ml) at 10 mg/m3 for six hours per day for three days (acute exposure) or two weeks (subacute exposure). The geometric mean diameter of the MWF aerosols was 1.56 microm, and the airborne endotoxin concentrations ranged from 1,231 to 2,173 eu/m3 (10,000 eu/ml in the bulk MWF) for the low dose and 19,263-27,386 eu/m3 (100,000 eu/ml in the bulk MWF) for the high dose. Minimal effects were observed after exposure to 10 mg/m3 of the MWF without endotoxins for three days or two weeks. However, an increase in the number of polymorphonuclear cells (PMNs) and the level of protein was noted in the bronchoalveolar lavage (BAL) fluid from the rats acutely exposed to the MWF with endotoxins. The acute exposure produced a greater increase in the number of PMNs and total cell number in the BAL fluid than the subacute exposure. The number of white blood cells in the peripheral blood and the weight of the lungs both increased after the subacute exposure to the MWF aerosol with endotoxins, indicating increased vascular permeability in response to the endotoxin exposure. The levels of cyotokines such as IL-4, INF-gamma, and IL-1beta in the BAL fluid from the rats exposed to the MWF with or without endotoxins remained unchanged. Although the level of nitric oxide (NO(x)) in the BAL supernatant did not show any change, the induction of NO(x) from the alveolar macrophages increased in the rats acutely or subacutely exposed to the MWF contaminated with endotoxins. The ConA-induced proliferation response showed no change, yet the LPS-induced proliferation response was significantly increased in the splenocytes from the rats subacutely exposed to the MWF with and without endotoxins. The level of TNF-alpha in the spleen cell culture obtained from the rats exposed to the MWF with or without endotoxins increased without changing the levels of IL-1beta, IL-4, and INF-gamma. The level of endotoxin-specific IgE in the serum obtained from the rats exposed to the MWF with endotoxins increased dose-dependently, while the levels of total immunoglobulins (IgG(1), IgG(2a) and IgE) and endotoxin-specific IgG(1) and IgG(2a) remained unchanged. Accordingly, the current results indicate that lung inflammation can be immediately induced by acute or subacute exposure to an MWF contaminated with endotoxins, and macrophages would appear to play a role in the induction of inflammation along with B-cell functions rather than T-cell functions, after subacute exposure to an MWF with endotoxins. In addition, endotoxin-specific IgE is an early marker for endotoxin exposure in the workplace.