Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Genome Capture Sequencing Selectively Enriches Bacterial DNA and Enables Genome-Wide Measurement of Intrastrain Genetic Diversity in Human Infections.

Within-host evolution produces genetic diversity in bacterial strains that cause chronic human infections. However, the lack of facile methods to measure bacterial allelic variation in clinical samples has limited understanding of intrastrain diversity's effects on disease. Here, we report a new method termed genome capture sequencing (GenCap-Seq) in which users inexpensively make hybridization probes from genomic DNA or PCR amplicons to selectively enrich and sequence targeted bacterial DNA from clinical samples containing abundant human or nontarget bacterial DNA. GenCap-Seq enables accurate measurement of allele frequencies over targeted regions and is scalable from specific genes to entire genomes, including the strain-specific accessory genome. The method is effective with samples in which target DNA is rare and inhibitory and DNA-degrading substances are abundant, including human sputum and feces. In proof-of-principle experiments, we used GenCap-Seq to investigate the responses of diversified Pseudomonas aeruginosa populations chronically infecting the lungs of people with cystic fibrosis to in vivo antibiotic exposure, and we found that treatment consistently reduced intrastrain genomic diversity. In addition, analysis of gene-level allele frequency changes suggested that some genes without conventional resistance functions may be important for bacterial fitness during in vivo antibiotic exposure. GenCap-Seq's ability to scalably enrich targeted bacterial DNA from complex samples will enable studies on the effects of intrastrain and intraspecies diversity in human infectious disease. IMPORTANCE Genetic diversity evolves in bacterial strains during human infections and could affect disease manifestations and treatment resistance. However, the extent of diversity present in vivo and its changes over time are difficult to measure by conventional methods. We developed a novel approach, GenCap-Seq, to enrich microbial DNA from complex human samples like sputum and feces for genome-wide measurements of bacterial allelic diversity. The approach is inexpensive, scalable to encompass entire targeted genomes, and works in the presence of abundant untargeted nucleic acids and inhibiting substances. We used GenCap-Seq to investigate in vivo responses of diversified bacterial strains to antibiotic treatment. This method will enable new ideas about the effects of intrastrain diversity on human infections to be tested.

Antiviral effects of blueberry proanthocyanidins against Aichi virus.

Blueberry polyphenols are known for their high antioxidant and antimicrobial potential. Aichi virus (AiV) is an emerging human enteric virus that causes gastroenteritis outbreaks worldwide. This study aimed to (1) determine the time- and dose-dependent effects of blueberry proanthocyanidins (B-PAC) against AiV over 24 h at 37 °C; (2) gain insights on their mode of action using pre- and post-treatment of host cells and Transmission Electron Microscopy; and (3) determine their anti-AiV effects in model foods and under simulated gastric conditions. AiV at ∼5 log PFU/ml was incubated with equal volumes of commercial blueberry juice (BJ, pH 2.8), neutralized BJ (pH 7.0), B-PAC (2, 4, and 10 mg/ml) prepared either in 10% ethanol, apple juice (AJ), 2% milk, simulated gastric fluid (SGF, pH 1.5) or simulated intestinal fluid (SIF, pH 7.5), and controls (malic acid (pH 3.0), phosphate buffered saline (pH 7.2), apple juice (pH 3.6) and 2% milk) over 24 h at 37 °C, followed by standard plaque assays. Each experiment was replicated thrice and data were statistically analyzed. Differences in AiV titers with 1 mg/ml B-PAC were 2.13 ±â€¯0.06 log PFU/ml lower after 24 h and ≥3 log PFU/ml (undetectable levels) lower with 2 and 5 mg/ml B-PAC compared to AiV titers in PBS after 24 h and 3 h, respectively. BJ at 37 °C resulted in titer differences (lower titers compared to PBS) of 0.17 ±â€¯0.06, 1.27 ±â€¯0.01, and 1.73 ±â€¯0.23 log PFU/ml after 1, 3, and 6 h and ≥3 log PFU/ml after 24 h. Pre- and post-treatment of host cells with 0.5 mg/ml B-PAC caused titer decreases of 0.62 ±â€¯0.33 and 0.30 ±â€¯0.06 log PFU/ml, respectively suggesting a moderate effect on viral-host cell binding. B-PAC at 2 mg/ml in AJ caused titer differences of ≥3 log PFU/ml after 0.5 h, while differences of 0.84 ±â€¯0.03 log PFU/ml with 5 mg/ml B-PAC in milk, and ≥3 log PFU/ml with B-PAC at 5 mg/ml in SIF after 30 min were obtained. This study shows the ability of BJ and B-PAC to decrease AiV titers to potentially prevent AiV-related illness and outbreaks.

Monomeric catechin and dimeric procyanidin B2 against human norovirus surrogates and their physicochemical interactions.

Plant polyphenols have shown antiviral activity against several human pathogens, but their physicochemical interactions are not well-understood. The objectives of this study were to compare the antiviral activity between monomeric catechin and dimeric procyanidin B2 (PB2) using cultivable human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and to understand their potential antiviral mechanism using virus-like particles (VLPs) and the P domain of human norovirus GII (HNoV GII.4). Surrogate viruses at 5 log PFU/mL were treated with 0.5-5 mg/mL monomeric catechin monohydrate, PB2 or phosphate buffered saline (PBS, pH 7.2; control) at 37 °C over 24 h. Infectivity was determined using plaque assays and data from triplicate experiments were statistically analyzed. PB2 at 0.5 mg/mL and 1 mg/mL reduced FCV-F9 to undetectable levels after 3 h and MNV-1 by 0.21 and 1.23 log PFU after 24 h, respectively. Monomeric catechins at 1 mg/mL reduced FCV-F9 to undetectable levels after 6 h and MNV-1 titers to undetectable levels after 24 h. In addition, PB2 was shown to directly bind the P domain, the main capsid structure of HNoVs in the ratio of 1:1 through spontaneous interactions. Electrostatic interactions played a dominant role between PB2 and the P domain. PB2 significantly altered tertiary but not secondary structures of VLPs. Transmission electron microscopy demonstrated that PB2 aggregated VLPs, further indicating interactions between them. These findings indicate that PB2 causes structural changes of the P domain of VLPs, mainly through direct interaction leading to HNoV inactivation.

Blueberry proanthocyanidins against human norovirus surrogates in model foods and under simulated gastric conditions.

Blueberry proanthocyanidins (B-PAC) are known to decrease titers of human norovirus surrogates in vitro. The application of B-PAC as therapeutic or preventive options against foodborne viral illness needs to be determined using model foods and simulated gastric conditions in vitro. The objective of this study was to evaluate the antiviral effect of B-PAC in model foods (apple juice (AJ) and 2% reduced fat milk) and simulated gastrointestinal fluids against cultivable human norovirus surrogates (feline calicivirus; FCV-F9 and murine norovirus; MNV-1) over 24 h at 37 °C. Equal amounts of each virus (5 log PFU/ml) was mixed with B-PAC (1, 2 and 5 mg/ml) prepared either in AJ, or 2% milk, or simulated gastric fluids and incubated over 24 h at 37 °C. Controls included phosphate buffered saline, malic acid (pH 7.2), AJ, 2% milk or simulated gastric and intestinal fluids incubated with virus over 24 h at 37 °C. The tested viruses were reduced to undetectable levels within 15 min with B-PAC (1, 2 and 5 mg/ml) in AJ (pH 3.6). However, antiviral activity of B-PAC was reduced in milk. FCV-F9 was reduced by 0.4 and 1.09 log PFU/ml with 2 and 5 mg/ml B-PAC in milk, respectively and MNV-1 titers were reduced by 0.81 log PFU/ml with 5 mg/ml B-PAC in milk after 24 h. B-PAC at 5 mg/ml in simulated intestinal fluid reduced titers of the tested viruses to undetectable levels within 30 min. Overall, these results show the potential of B-PAC as preventive and therapeutic options for foodborne viral illnesses.

IL-3 Decreases Cartilage Degeneration by Downregulating Matrix Metalloproteinases and Reduces Joint Destruction in Osteoarthritic Mice.

Osteoarthritis (OA) is a chronic disease of articular joints that leads to degeneration of both cartilage and subchondral bone. These degenerative changes are further aggravated by proinflammatory cytokines including IL-1ß and TNF-α. Previously, we have reported that IL-3, a cytokine secreted by activated T cells, protects cartilage and bone damage in murine models of inflammatory and rheumatoid arthritis. However, how IL-3 protects cartilage degeneration is not yet known. In this study, we investigated the role of IL-3 on cartilage degeneration under both in vitro and in vivo conditions. We found that both mouse and human chondrocytes show strong expression of IL-3R at gene and protein levels. IL-3 increases the expression of mouse chondrocyte-specific genes, Sox9 and collagen type IIa, which were downregulated by IL-1ß. Moreover, IL-3 downregulated IL-1ß- and TNF-α-induced expression of matrix metalloproteinases in both mouse and human chondrocytes. Interestingly, IL-3 reduces the degeneration of articular cartilage and subchondral bone microarchitecture in a mouse model of human OA. Moreover, IL-3 showed the preventive and therapeutic effects on cartilage degeneration induced by IL-1ß in micromass pellet cultures of human mesenchymal stem cells. Thus, to our knowledge, we provide the first evidence that IL-3 has therapeutic potential in amelioration of degeneration of articular cartilage and subchondral bone microarchitecture associated with OA.

Cronobacter sakazakii reduction by blueberry proanthocyanidins.

Blueberry juice and blueberry polyphenols reportedly have antimicrobial properties against foodborne pathogens, without much currently known on their effects against Cronobacter sakazakii. This study evaluated the antimicrobial effects of blueberry proanthocyanidins (PAC) and commercial blueberry juice (BJ) against two strains of C. sakazakii, ATCC 29004 and 29544. BJ (pH 2.8), blueberry PAC (5 mg/ml) and controls (phosphate buffered saline (PBS), pH 7.2, and malic acid pH 3.0) were mixed with equal volumes of washed overnight cultures of C. sakazakii and incubated for 30 min, 1 h, 3 h and 6 h at 37°C. Reductions of ∼1 and 1.50 log CFU/ml were obtained for strains 29004 and 29544, respectively after 30 min with BJ or blueberry PAC. Both C. sakazakii strains 29004 and 29544 were reduced to undetectable levels from 8.25 ± 0.12 log CFU/ml and 8.48 ± 0.03 log CFU/ml, respectively with BJ (pH 2.8) or blueberry PAC after 1 h, while malic acid (pH 3.0) showed ∼1.3 log CFU/ml reduction for both strains. Scanning electron microscopy studies showed differences in cell membrane morphology with clumping and formation of blebs of the treated strains compared to untreated controls. These results warrant further in vivo studies with blueberry bioactives to determine potential for preventing and treating C. sakazakii infections.

IL-3 promotes osteoblast differentiation and bone formation in human mesenchymal stem cells.

IL-3 is an important cytokine that regulates hematopoiesis. We have previously demonstrated that IL-3 is a potent inhibitor of osteoclastogenesis and bone resorption. In the present study, we have investigated the role of IL-3 on human osteoblast differentiation and bone formation. We found that IL-3 in a dose-dependent manner increases osteoblast differentiation and matrix mineralization in human mesenchymal stem cells (MSCs). IL-3 significantly enhances the expression of osteoblast specific genes such as alkaline phosphatase, collagen type-I, osteocalcin and osteopontin; and Runx-2 and osterix transcription factors. Moreover, IL-3 induces the expression of bone morphogenetic protein-2 (BMP-2), and activates smad1/5/8. IL-3 enhances osteoblast differentiation and BMP-2 secretion through JAK/STAT pathway. Interestingly, IL-3 promotes in vivo bone regeneration ability of MSCs. Thus, we reveal for the first time that IL-3 enhances human osteoblast differentiation and bone formation in both in vitro and in vivo conditions, and suggest its therapeutic potential for bone formation in important bone diseases.