Protective effect of dexmedetomidine against delayed bone healing caused by morphine via PI3K/Akt mediated Nrf2 antioxidant defense system.

Background: As a class of analgesics, opioids are frequently used to treat both acute and chronic moderate to severe pain. Patients frequently receive opioid painkillers after orthopedic accidents or surgeries. Evidence suggests that opioid drug users have a 55.1% higher risk of fracture and poor bone repair than non-users of opioid drugs. The key pathogenic alterations in the incidence and progression of poor bone repair are over apoptosis and aging of osteoblasts due to the stress caused by oxidation. Dexmedetomidine (Dex) has been proven to protect against a variety of degenerative illnesses by reducing oxidative stress. However, nothing is known about how it affects bone repair. Methods: PI3K/Akt/Nrf2 pathway was detected by immunofluorescence and Western blot. SOD, CAT, JC-1, dihydroethidium and mitosox were used in the Oxidative Stress. Micro-CT, H&E and Masson's staining, immunohistochemically were performed to evaluate the therapeutic effects of DEX on calvarial defects in the morphine-induced rat model. Results: We found that morphine-induced an imbalance in the metabolism and catabolism of primary rat Osteoblasts. However, these conditions could be inhibited by DEX treatment. In the meantime, DEX induced the expression of Nrf2-regulated antioxidant enzymes such as NQO1, HO-1, GCLm, GCLc, and TrxR1. DEX-mediated Nrf2 activation is linked to the PI3K/Akt signaling system. Furthermore, it has been established that intravenous DEX enhanced the growth of bone healing in a model of a surgically produced rat cranial lesion. Conclusion: This is the first description of the unique DEX mechanism acting as a Nrf2 activator against morphine-mediated oxidative harm, raising the possibility that the substance may be used to prevent bone defects.

Cathelicidin-BF regulates the AMPK/SIRT1/NF-κB pathway to ameliorate murine osteoarthritis: In vitro and in vivo studie.

Osteoarthritis (OA) is a chronic degenerative disease with a significant prevalence that causes cartilage damage and can lead to disability. The main factors contributing to the onset and progression of OA include inflammation and degeneration of the extracellular matrix. Cathelicidin-BF (BF-30), a natural peptide derived from Bungarus fasciatus venom, has shown multiple important pharmacological effects. However, the action mechanism of BF-30 in OA treatment remains to be elucidated. In this research, X-ray and Safranin O staining were employed to evaluate the imageology and histomorphology differences in the knee joints of mice in vivo. Techniques such as Western blot analysis, RT-qPCR, ELISA, and immunofluorescence staining were applied to examine gene and protein level changes in in vitro experiments. It was found that BF-30 significantly decreased inflammation and enhanced extracellular matrix metabolism. For the first time, it was demonstrated that the positive effects of BF-30 are mediated through the activation of the AMPK/SIRT1/NF-κB pathway. Moreover, when BF-30 was co-administered with Compound C, an AMPK inhibitor, the therapeutic benefits of BF-30 were reversed in both in vivo and in vitro settings. In conclusion, the findings suggest that BF-30 could be a novel therapeutic agent for OA improvement.

Infant Formulas With Partially or Extensively Hydrolyzed Milk Proteins for the Prevention of Allergic Diseases: A Systematic Review and Meta-Analysis of Clinical Trials.

Despite the widely recommended usage of partially hydrolyzed formula (PHF) or extensively hydrolyzed formula (EHF) of milk protein for preventing allergic diseases (ADs), clinical studies have been inconclusive regarding their efficacy compared with that of cow's milk formula (CMF) or breast milk (BM). We aimed to systematically evaluate the effects of PHF or EHF compared with those of CMF or BM on risk of ADs (cow's milk allergy, allergic rhinitis, eczema, asthma, wheeze, food allergy, and sensitization) in children. We searched PubMed, Embase, Cochrane Library, and Web of Science for clinical trials published from inception to 21 October, 2022. We used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach to grade the strength of evidence. Overall, 24 trials (10,950 infants) were included, 17 of which specifically included high-risk infants. GRADE was low for the evidence that, compared with CMF, infants early fed with EHF had lower risk of cow's milk allergy at age 0-2 y [relative risk (RR): 0.62; 95% CI: 0.39, 0.99]. Moderate evidence supported that PHF and EHF reduced risk of eczema in children aged younger or older than 2 y, respectively (RR: 0.71; 95% CI: 0.52, 0.96; and RR: 0.79; 95% CI: 0.67, 0.94, respectively). We also identified moderate systematic evidence indicating that PHF reduced risk of wheeze at age 0-2 y compared with CMF (RR: 0.50; 95% CI: 0.29, 0.85), but PHF and EHF increased the risk compared with BM (RR: 1.61; 95% CI: 1.11, 2.31; and RR: 1.64; 95% CI: 1.26, 2.14). Neither PHF nor EHF had significant effects on other ADs in children of any age. In conclusion, compared with CMF, PHF, or EHF had different preventive effect on cow's milk allergy, eczema, and wheeze. Compared with BM, both PHF and EHF may increase risk of wheeze but not other ADs. Given that most trials included only high-risk infants, more research on non-high-risk infants is warranted before any generalization is attempted. This protocol was registered at PROSPERO as CRD42022320787.

Comparison of genotypic and phenotypic antimicrobial resistance profiles of Salmonella enterica isolates from poultry diagnostic specimens.

The spread of antimicrobial-resistant bacteria is a significant concern, as it can lead to increased morbidity and mortality in both humans and animals. Whole-genome sequencing (WGS) is a powerful tool that can be used to conduct a comprehensive analysis of the genetic basis of antimicrobial resistance (AMR). We compared the phenotypic and genotypic AMR profiles of 97 Salmonella isolates derived from chicken and turkey diagnostic samples. We focused AMR analysis on 5 antimicrobial classes: aminoglycoside, beta-lactam, phenicol, tetracycline, and trimethoprim. The overall sensitivity and specificity of WGS in predicting phenotypic antimicrobial resistance in the Salmonella isolates were 93.4% and 99.8%, respectively. There were 16 disagreement instances, including 15 that were phenotypically resistant but genotypically susceptible; the other instance involved phenotypic susceptibility but genotypic resistance. Of the isolates examined, 67 of 97 (69%) carried at least 1 resistance gene, with 1 isolate carrying as many as 12 resistance genes. Of the 31 AMR genes analyzed, 16 were identified as aminoglycoside-resistance genes, followed by 4 beta-lactam-resistance, 3 tetracycline-resistance, 2 sulfonamide-resistance, and 1 each of fosfomycin-, quinolone-, phenicol-, trimethoprim-, bleomycin-, and colistin-resistance genes. Most of the resistance genes found were located on plasmids.

Development of a rapid visual detection technology for BmNPV based on CRISPR/Cas13a system.

Pathogenic microorganism of silkworm are important factors that threaten the high-quality development of sericulture. Among them, Bombyx mori nucleopolyhedrovirus (BmNPV) caused diseases often lead to frequent outbreaks and high mortality, resulting in huge losses to sericultural industry. Current molecular detection methods for BmNPV require expensive equipment and sikilled technical personnel. As a result, the most commonly detection method for silkworm egg production enterprises involves observing the presence of polyhedra under a microscope. However, this method has low accuracy and sensitivity. There is an urgent need to develop a new detection technology with high sensitivity, high specificity, and applicability for silkworm farms, silkworm egg production enterprises and quarantine departments. In this study, we successfully established the CRISPR/Cas13a BmNPV visualized detection technology by combining Recombinase Polymerase Amplification (RPA) technology and CRISPR/Cas13a system. This technology is based on microplate lateral, flow test strips and portable fluorescence detector. The detection sensitivity can reach up to 1 copies/µL for positive standard plasmid and 1 fg/µL for BmNPV genome in 30-45 min, demonstrating high sensitivity. By detecting silkworm tissues infected with different pathogens, we determined that CRISPR/Cas13a detection technology has good specificity. In summary, the newly established nucleic acid detection technology for BmNPV is characterized by high sensitivity, high specificity, low cost and convenience for visualization. It can be applied in field detection and silkworm egg quality monitory system.

PPAR γ activation in chondrocytes alleviates glucocorticoid-induced oxidative stress, mitochondrial impairment, and pyroptosis via autophagic flow enhancement.

Osteoarthritis (OA) is a progressive age-related disease characterised by pathological changes in the synovium, articular cartilage, and subchondral bone, significantly reducing the patients' quality of life. This study investigated the role of glucocorticoids, specifically dexamethasone, in OA progression, with a particular focus on their effects on chondrocytes. Although glucocorticoids are commonly used for OA pain relief, our research demonstrated that high concentrations of dexamethasone may accelerate OA progression by enhancing the ability of reactive oxygen species to inhibit chondrocyte autophagy, resulting in cell death and accelerated cartilage degeneration. Despite reports on the acceleration of pathogenesis and cartilage damage in some patients of OA taking corticosteroids, the mechanism behind the same has not been investigated. This necessitates an investigation of the concentration-dependent changes in the cartilage cells upon dexamethasone administration. In addition, the protective effect of PPAR γ on chondrocytes can prevent the decrease in chondrocyte autophagy and delay cartilage degeneration. Therefore, our study suggests that the therapeutic use of glucocorticoids in OA treatment should be more nuanced considering their potential detrimental effects. Future investigations should focus on the mechanisms underlying the glucocorticoid-mediated modulation of cell death processes, which could provide insights into new therapeutic strategies for OA treatment.

Porous organic semiconductor/PET composite fibre for the synergistic removal of hexavalent chromium and organic pollutants under sunlight.

In this study, the porous graphite phase carbon nitride photocatalyst (P-g-C3N4) is prepared by the CaCO3 template method, and then P-g-C3N4/T-polyethylene terephthalate (T-PET) catalytic fibre is prepared by the padding method. P-g-C3N4 can provide more active sites than g-C3N4 as proved by the Brunauer-Emmett-Teller and the UV-Visible diffuse reflectance test. P-g-C3N4 powder catalyst successfully supports PET fibre as proved by scanning electron microscope, Fourier infrared spectroscopy and X-ray diffraction spectroscopy. The photocatalytic performance of P-g-C3N4/T-PET catalytic fibre is tested by constructing a single hexavalent chromium or hexavalent chromium/organic pollutant binary pollution system. The potential application value of P-g-C3N4/T-PET catalytic fibre is further explored by simulating the complex actual water environment. After five recycles, P-g-C3N4/T-PET catalytic fibre shows good catalytic performance. The mechanism of P-g-C3N4/PET photocatalytic degradation of organic pollutants is proposed through the capture agent experiment and electron paramagnetic resonance spectroscopy. Among them, •O2- is the most important active species of P-g-C3N4 catalytic fibre, which is used for the oxidation of organic pollutants. At the same time, photoelectrons generated by the catalytic fibre are used to reduce hexavalent chromium. The efficiency of P-g-C3N4 to remove pollutants is improved by using PET fibre as a carrier, which not only solves the problem of difficult recovery of powder catalysts but also provides more active sites.

Recent Updates on Multifunctional Nanomaterials as Antipathogens in Humans and Livestock: Classification, Application, Mode of Action, and Challenges.

Pathogens cause infections and millions of deaths globally, while antipathogens are drugs or treatments designed to combat them. To date, multifunctional nanomaterials (NMs), such as organic, inorganic, and nanocomposites, have attracted significant attention by transforming antipathogen livelihoods. They are very small in size so can quickly pass through the walls of bacterial, fungal, or parasitic cells and viral particles to perform their antipathogenic activity. They are more reactive and have a high band gap, making them more effective than traditional medications. Moreover, due to some pathogen's resistance to currently available medications, the antipathogen performance of NMs is becoming crucial. Additionally, due to their prospective properties and administration methods, NMs are eventually chosen for cutting-edge applications and therapies, including drug administration and diagnostic tools for antipathogens. Herein, NMs have significant characteristics that can facilitate identifying and eliminating pathogens in real-time. This mini-review analyzes multifunctional NMs as antimicrobial tools and investigates their mode of action. We also discussed the challenges that need to be solved for the utilization of NMs as antipathogens.

Associations of Maternal Serum Iodine Concentration with Obstetric Complications and Birth Outcomes-Longitudinal Analysis Based on the Huizhou Mother-Infant Cohort, South China.

This study aimed to explore the temporal associations between maternal serum iodine concentration (SIC) and common pregnancy outcomes in Chinese women. Eligible singleton pregnant women aged 20-34 years were selected, and their fasting blood samples were collected during early (T1, n = 1101) and mid-pregnancy (T2, n = 403) for SIC testing by inductively coupled plasma mass spectrometry. Multivariable linear regression indicated that log10SIC at T1 (ß = -0.082), T2 (ß = -0.198), and their % change (ß = -0.131) were inversely associated with gestational weight gain (GWG, all p < 0.05). Maternal log10SIC at both T1 (ß = 0.077) and T2 (ß = 0.105) were positively associated with the Apgar score at 1 min (both p < 0.05). Women in the third quartile (Q3) of SIC at T1 had a lower risk of small for gestational age (SGA, OR = 0.405, 95% CI: 0.198-0.829) compared with those in Q4. Restricted cubic spline regression suggested a U-shaped association between SIC and SGA risk, and SIC above 94 µg/L at T1 was the starting point for an increased risk of SGA. The risk of premature rupture of membrane (PROM) increased by 96% (OR = 1.960, 95% CI: 1.010-3.804) in Q4 compared to that in Q1. Our longitudinal data from an iodine-replete region of China indicated that high maternal SIC could restrict GWG and improve Apgar scores at delivery, but might increase the risk of SGA and PROM.

ADAM12 promotes the resistance of lung adenocarcinoma cells to EGFR-TKI and regulates the immune microenvironment by activating PI3K/Akt/mTOR and RAS signaling pathways.

Lung adenocarcinoma (LUAD) is a malignant respiratory disease, resulting in a heavy social burden. Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance and tumor immune microenvironment are important directions in the treatment of LUAD. In this study, we confirmed the role of ADAM metallopeptidase domain 12 (ADAM12) in LUAD development and progression. Our bioinformatic analysis was conducted to screen ADAM12 was correlated with EGFR-TKI and immune infiltration in LUAD patients. Our results showed that the transcription and post-transcription level of ADAM12 is significantly increased in tumor samples compared to normal samples, and ADAM12 correlated with poor prognosis in LUAD patients. High level of ADAM12 accelerated the LUAD progression via promoting proliferation, cell cycle, apoptosis escaping, immune escaping, EGFR-TKI resistance, angiogenesis, invasion and migration based on experiment validation in vitro and in vivo, which could be attenuated by ADAM12 knockdown. Further mechanistic studies suggested that the PI3K/Akt/mTOR and RAS signaling pathways were activated after ADAM12 knockdown. Therefore, ADAM12 might be validated as a possible molecular therapy target and prognostic marker for patients with LUAD.

Metagenomic identification of a novel oomycete cultured from a pyogranulomatous mass on the tail of a domestic cat.

We report here a transiently culturable oomycete pathogen isolated from a pyogranulomatous tail mass in a cat. The organism was morphologically and genetically distinct from Lagenidium and Pythium species. Following next-generation sequencing (NGS) and assembly of contigs, initial phylogenetic analysis using fragments of the cox1 mitochondrial gene identified this specimen as Paralagenidium sp. after nucleotide alignments with sequences obtained from the Barcode of Life Data System (BOLD). However, further analysis of a concatenation of 13 different mitochondrial genes showed that this organism is unique and different from all known oomycetes. A negative PCR result using primers targeting known oomycete pathogens may not be enough to rule out oomycosis in a suspected case. Additionally, the use of a single gene to classify oomycetes may produce misleading results. The advent of metagenomic sequencing and NGS provides a unique opportunity to further explore the diversity of oomycetes as plant and animal pathogens beyond the current capabilities of global barcoding projects that are based on partial genomic sequences.

Isolation and characterization of mammalian orthoreovirus from bats in the United States.

Mammalian orthoreovirus (MRV) infects many mammalian species including humans, bats, and domestic animals. To determine the prevalence of MRV in bats in the United States, we screened more than 900 bats of different species collected during 2015-2019 by a real-time reverse-transcription polymerase chain reaction assay; 4.4% bats tested MRV-positive and 13 MRVs were isolated. Sequence and phylogenetic analysis revealed that these isolates belonged to four different strains/genotypes of viruses in Serotypes 1 or 2, which contain genes similar to those of MRVs detected in humans, bats, bovine, and deer. Further characterization showed that these four MRV strains replicated efficiently on human, canine, monkey, ferret, and swine cell lines. The 40/Bat/USA/2018 strain belonging to the Serotype 1 demonstrated the ability to infect and transmit in pigs without prior adaptation. Taken together, this is evidence for different genotypes and serotypes of MRVs circulating in US bats, which can be a mixing vessel of MRVs that may spread to other species, including humans, resulting in cross-species infections.

Adaptation and uniformity of monolithic zirconia crowns fabricated by additive 3-dimensional gel deposition.

STATEMENT OF PROBLEM: A novel monolithic zirconia restoration fabricated by additive 3-dimensional (3D) gel deposition, named as self-glazed zirconia (SGZ), has recently been developed. SGZ crowns exhibit reliable mechanical properties and esthetic appearance, but their adaptation and uniformity are unclear. PURPOSE: The purpose of this in vitro study was to compare the adaptation and uniformity of monolithic zirconia crowns fabricated by additive 3D gel deposition with that of milled zirconia and lithium disilicate crowns. MATERIAL AND METHODS: Ten identical resin abutments were made based on the scanning data of an extracted and prepared human mandibular first molar. Three types of monolithic crowns were then fabricated by using 2 different processes: 3D gel deposition zirconia (SGZ), milled zirconia (ZZ), and milled lithium disilicate (EMX) (n=10) through a completely digital workflow. The nondestructive direct-view technique and replica technique were used to measure the marginal and internal discrepancy values individually. The uniformity index was calculated to describe the uniformity of the internal space. The results were analyzed by using 1-way ANOVA and Kruskal-Wallis statistical tests (α=.05). RESULTS: The marginal discrepancy of EMX exhibited the highest values among the 3 groups (P=.001). The 2 types of zirconia crowns had comparable marginal discrepancy values (P>.05). The internal discrepancy values of SGZ were significantly lower than those of EMX at the occlusal region and of ZZ at all measured locations (P<.05). All 3 types of monolithic crowns showed comparable good uniformity (P=.056). CONCLUSIONS: The marginal and internal adaptations of novel monolithic zirconia crowns were within clinical requirements. Compared with the zirconia and lithium disilicate crowns fabricated by subtractive milling, monolithic zirconia crowns fabricated by additive 3D gel deposition had comparable uniformity and better internal adaptation.

Optimized conditions for Listeria, Salmonella and Escherichia whole genome sequencing using the Illumina iSeq100 platform with point-and-click bioinformatic analysis.

Whole-genome sequencing (WGS) data have become an integral component of public health investigations and clinical diagnostics. Still, many veterinary diagnostic laboratories cannot afford to implement next generation sequencing (NGS) due to its high cost and the lack of bioinformatic knowledge of the personnel to analyze NGS data. Trying to overcome these problems, and make NGS accessible to every diagnostic laboratory, thirteen veterinary diagnostic laboratories across the United States (US) initiated the assessment of Illumina iSeq100 sequencing platform for whole genome sequencing of important zoonotic foodborne pathogens Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The work presented in this manuscript is a continuation of this multi-laboratory effort. Here, seven AAVLD accredited diagnostic laboratories explored a further reduction in sequencing costs and the usage of user-friendly platforms for genomic data analysis. Our investigation showed that the same genomic library quality could be achieved by using a quarter of the recommended reagent volume and, therefore a fraction of the actual price, and confirmed that Illumina iSeq100 is the most affordable sequencing technology for laboratories with low WGS demand. Furthermore, we prepared step-by-step protocols for genomic data analysis in three popular user-friendly software (BaseSpace, Geneious, and GalaxyTrakr), and we compared the outcomes in terms of genome assembly quality, and species and antimicrobial resistance gene (AMR) identification. No significant differences were found in assembly quality, and the three analysis methods could identify the target bacteria species. However, antimicrobial resistance genes were only identified using BaseSpace and GalaxyTrakr; and GalaxyTrakr was the best tool for this task.

Research on thermoeconomic fault diagnosis for marine low speed two stroke diesel engine.

To satisfy the requirements of low fuel consumption, low emission, and high efficiency of the shipping industry, marine diesel engines are developing in the direction of automation and energy-saving, which increases the possibility and complexity of marine diesel engine failures. A one-dimension thermodynamic model for the marine diesel engine is built with AVL Boost software. The model is applied to a low-speed two-stroke 6S50MC diesel engine, and the error between the main performance parameters obtained by simulation and the test bench data is less than 3% under 100% and 75% load. Based on the model, 6 typical single faults and many typical double faults concomitant phenomena of diesel are reproduced. Based on the second law of thermodynamics, the exergy flow among the components and the external environment is analyzed. The thermoeconomic model of a marine diesel engine is established where the "fuel" and "product" of the components are defined according to their function. The fault diagnosis results show that the effects of faults generally propagate through the diesel engine system and affect the behavior of several components, resulting in induced malfunction in normal components. Therefore the malfunction MFi of each component is the superposition of the intrinsic malfunction and the induced malfunction according to the malfunction and dysfunction analysis. The thermoeconomic fault diagnosis method can be used to narrow the search range of abnormal components though it cannot accurately locate the fault.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

A microfluidic biosensor for rapid simultaneous detection of waterborne pathogens.

A microfluidic based biosensor was investigated for rapid and simultaneous detection of Salmonella, Legionella, and Escherichia coli O157:H7 in tap water and wastewater. The biosensor consisted of two sets of focusing electrodes connected in parallel and three sets of interdigitated electrodes (IDE) arrays. The electrodes enabled the biosensor to concentrate and detect bacteria at both low and high concentrations. The focusing region was designed with vertical metal sidewall pairs and multiple tilted thin-film finger pairs to generate positive dielectrophoresis (p-DEP) to force the bacteria moving toward the microchannel centerline. As a result, the bacterial pathogens were highly concentrated when they reached the detection electrode arrays. The detection IDE arrays were coated with three different antibodies against the target bacterial pathogens and a cross-linker to enhance the binding of antibodies to the detection electrode. As the binding of bacterial pathogen to its specific antibodies took place, the impedance value changed. The results demonstrated that the biosensors were capable of detecting Salmonella, Legionella, and E. coli 0157:H7 simultaneously with a detection limit of 3 bacterial cells/ml in 30 - 40 min.

Detection of virulence and extended spectrum ß-lactamase genes in Salmonella by multiplex high-resolution melt curve real-time PCR assay.

AIMS: Develop and standardize multiplex high-resolution melt curve (HRM) real-time PCR assays for simultaneous detection of Salmonella virulence and extended spectrum ß-lactamase (ESBL) genes in food. METHODS AND RESULTS: Two sets of multiplex real-time PCR assays targeting six virulence and three ESBL genes with internal amplification control were standardized. The first assay detected hilA, fimH, sipA, blaTEM and blaSHV, and the second detected invA, fimA, stn and blaCMY . The PCR assays were validated with DNA samples from 77 different Salmonella strains. The assay specificity was tested with DNA from 47 non-Salmonella strains. Melt curve analyses showed distinct, well-separated melting peaks of each target gene detected by their respective melting temperatures (Tm ). Different food samples were spiked with 10, 102 and 103  CFU/ml of Salmonella. The optimized assays were able to detect all target genes in concentrations of as low as 10 CFU/ml in 25 g foods within 10 h of enrichment. CONCLUSIONS: Multiplex HRM real-time PCR assays can be used as rapid detection methods for detecting Salmonella in foods. SIGNIFICANCE AND IMPACT OF STUDY: The assays developed in this study will allow for accurate detection of virulence and ESBL genes in Salmonella that are present in low concentrations in food samples.

CircPTCH1 Promotes Migration in Lung Cancer by Regulating MYCN Expression Through miR-34c-5p.

BACKGROUND: The incidence rate and mortality rate of lung cancer are the highest in the world. Therefore, further studies are needed to reveal the molecular mechanism of lung cancer progression and development. Previous study demonstrated that the deregulation of circRNAs can regulate cell biological functions in tumorigenesis and development. However, the roles of circPTCH1 in lung cancer have not yet been revealed. MATERIALS AND METHODS: The expression levels of circPTCH1, miR-34c-5p, and MYCN were measured by RT-PCR in lung cancer tissues and cells; dual-luciferase reporter and RIP assay showed that circRNA served as a sponge for miRNA, and miRNA could target mRNA. In vitro, effects of si-circPTCH1 can regulate lung cancer cells' migration, invasion were detected by CCK-8 assay, wound healing assay, and transwell assay. RESULTS: Our research demonstrated that the expression of circPTCH1 was upregulated in lung cancer tissues and cell lines and increased in metastatic tissues compared to that of non-metastatic tissues. circPTCH1 sponging miR-34c-5p to target MYCN was revealed by dual-luciferase reporter and a RIP assay. In addition, the expression level of miR-34c-5p was reduced in lung cancer tumor tissues, and MYCN was significantly increased in lung cancer tumor tissues. Pearson correlation analysis showed that miR-34c-5p with circPTCH1 and MYCN had a moderately negative correlation, and there was a moderately positive correlation between circPTCH1 and MYCN. Further, cytological studies found that circPTCH1 reduced lung cancer cells' migration and invasion by targeting MYCN via miR-34c-5p. CONCLUSION: circPTCH1 plays a tumor enhancement role in lung cancer and that can effectively promote migration, invasion and EMT by targeting the miR-34c-5p/MYCN axis. circPTCH1 may be a novel potential treatment and diagnosis biomarker for lung cancer.

A panel of real-time PCR assays for the detection of Bourbon virus, Heartland virus, West Nile virus, and Trypanosoma cruzi in major disease-transmitting vectors.

Vector-borne pathogens, such as Bourbon virus (BRBV), Heartland virus (HRTV), West Nile virus (WNV), and Trypanosoma cruzi (TCZ) are a great threat to public health and animal health. We developed a panel of TaqMan real-time PCR assays for pathogen surveillance. PCR targets were selected based on nucleic acid sequences deposited in GenBank. Primers and probes were either designed de novo or selected from publications. The coverages and specificities of the primers and probes were extensively evaluated by performing BLAST searches. Synthetic DNA or RNA fragments (gBlocks) were used as PCR templates in initial assay development and PCR positive controls in subsequent assay validation. For operational efficiency, the same thermocycling profile was used in BRBV, HRTV, and WNV reverse-transcription quantitative PCR (RT-qPCR) assays, and a similar thermocycling profile without the initial reverse-transcription step was used in TCZ qPCR. The assays were optimized by titrating primer and probe concentrations. The analytical sensitivities were 100, 100, 10, and 10 copies of gBlock per reaction for BRBV (Cq = 36.0 ± 0.7), HRTV (Cq = 36.6 ± 0.9), WNV (Cq = 35.5 ± 0.4), and TCZ (Cq = 38.8 ± 0.3), respectively. PCR sensitivities for vector genomic DNA or RNA spiked with gBlock reached 100, 100, 10, and 10 copies per reaction for BRBV, HRTV, WNV, and TCZ, respectively. PCR specificity evaluated against a panel of non-target pathogens showed no significant cross-reactivity. Our BRBV, HRTV, WNV, and TCZ PCR panel could support epidemiologic studies and pathogen surveillance.