Rapid and Visual Detection of Barley Yellow Dwarf Virus by Reverse Transcription Recombinase Polymerase Amplification with Lateral Flow Strips.

Barley yellow dwarf virus (BYDV) has been a major viral pathogen causing significant losses of cereal crops including oats worldwide. It spreads naturally through aphids, and a rapid, specific, and reliable diagnostic method is imperative for disease monitoring and management. Here, we established a rapid and reliable method for isothermal reverse transcription recombinase polymerase amplification (RT-RPA) combined with a lateral flow strips (LFS) assay for the detection of BYDV-infected oat samples based on the conserved sequences of the BYDV coat protein gene. Specific primers and a probe for RT-RPA reacted and optimally incubated at 42 o C for 10 min, and the end-labeled amplification products were visualized on LFS within 10 min. The RT-RPA-LFS assay showed no cross-reactivity with other major cereal viruses, including barley mild mosaic virus, barley yellow mosaic virus, and rice black streaked dwarf virus, indicating high specificity of the assay. The sensitivity of the RT-RPA-LFS assay was similar to that of reverse transcription polymerase chain reaction, and it was successfully validated to detect BYDV in oat samples from six different regions and in individual aphids. These results confirm the out-standing potential of the RT-RPA-LFS assay for rapid detection of BYDV.

Identification of Viruses Infecting Oats in Korea by Metatranscriptomics.

Controlling infectious plant viruses presents a constant challenge in agriculture. As a source of valuable nutrients for human health, the cultivation of oats (Avena sativa L.) has recently been increased in Korea. To date, however, few studies have been undertaken to identify the viruses infecting oats in this country. In this study, we carried out RNA-sequencing followed by bioinformatics analyses to understand the virosphere in six different geographical locations in Korea where oats are cultivated. We identified three different virus species, namely, barley yellow dwarf virus (BYDV) (BYDV-PAV and BYDV-PAS), cereal yellow dwarf virus (CYDV) (CYDV-RPS and CYDV-RPV), and rice black-streaked dwarf virus (RBSDV). Based on the number of virus-associated reads and contigs, BYDV-PAV was a dominant virus infecting winter oats in Korea. Interestingly, RBSDV was identified in only a single region, and this is the first report of this virus infecting oats in Korea. Single nucleotide polymorphisms analyses indicated that most BYDV, CYDV, and RBSDV isolates show considerable genetic variations. Phylogenetic analyses indicated that BYDVs and CYDVs were largely grouped in isolates from Asia and USA, whereas RBSDV was genetically similar to isolates from China. Overall, the findings of this study provide a preliminary characterization of the types of plant viruses infecting oats in six geographical regions of Korea.

Detection of Plant Pathogenic Viruses in Commercial Gochujang (Fermented Red Pepper Paste) from Korea.

The potential transmission of plant pathogenic viruses through processed foods could be a source of concern for global crop production; however, there is a lack of supporting evidence. The present study was conducted to investigate the presence of plant pathogenic viruses in five samples of gochujang (fermented red pepper paste) manufactured in Korea. Several viruses infecting pepper were detected by reverse transcriptionpolymerase chain reaction, among which the pepper mild mottle virus (PMMoV) was detected in all five samples, at concentrations ranging from 2.8 to 7.0 (log10 copies/ml). In addition, PMMoV was observed by transmission electron microscopy in all five samples. The samples exhibited viral pathogenicity to Nicotiana benthamiana plants, indicating that global trade of processed products could be a possible source of the transmission of plant viruses.

Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid and Sensitive Detection of Barley Yellow Dwarf Virus in Oat.

Barley yellow dwarf virus (BYDV) is an economically important plant pathogen that causes stunted growth, delayed heading, leaf yellowing, and purple leaf tip, thereby reducing the yields of cereal crops worldwide. In the present study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed for the detection of BYDV in oat leaf samples. The RT-RPA assay involved incubation at an isothermal temperature (42°C) and could be performed rapidly in 5 min. In addition, no cross-reactivity was observed to occur with other cereal-infecting viruses, and the method was 100 times more sensitive than conventional reverse transcription polymerase chain reaction. Furthermore, the assay was validated for the detection of BYDV in both field-collected oat leaves and viruliferous aphids. Thus, the RT-RPA assay developed in the present study represents a simple, rapid, sensitive, and reliable method for detecting BYDV in oats.

GM-CSF Enhances Mobilization of Bone Marrow Mesenchymal Stem Cells via a CXCR4-Medicated Mechanism.

BACKGROUND: This study was conducted to investigate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on the mobilization of mesenchymal stem cells (MSCs) from the bone marrow (BM) into the peripheral blood (PB) in rats. METHODS: GM-CSF was administered subcutaneously to rats at 50 µg/kg body weight for 5 consecutive days. The BM and PB of rats were collected at 1, 3, and 5 days during the administration for analysis. RESULTS: Upon GM-CSF administration, the number of mononuclear cells increased rapidly at day 1 both in the BM and PB. This number decreased gradually over time in the BM to below the initial amount by day 5, but was maintained at a high level in the PB until day 5. The colony-forming unit-fibroblasts were increased in the PB by 10.3-fold at day 5 of GM-CSF administration, but decreased in the BM. Compared to GM-CSF, granulocyte-colony stimulating factor (G-CSF) stimulated lower levels of MSC mobilization from the BM to the PB. Immunohistochemical analysis revealed that GM-CSF induced a hypoxic and proteolytic microenvironment and increased C-X-C chemokine receptor type 4 (CXCR4) expression in the BM. GM-CSF added to BM MSCs in vitro dose-dependently increased CXCR4 expression and cell migration. G-CSF and stromal cell derived factor-1 (SDF-1) showed similar results in these in vitro assays. Know-down of CXCR4 expression with siRNA significantly abolished GM-CSF- and G-CSF-induced MSC migration in vitro, indicating the involvement of the SDF-1-CXCR4 interaction in the mechanism. CONCLUSION: These results suggest that GM-CSF is a useful tool for mobilizing BM MSCs into the PB.

Overexpression of the laeA gene leads to increased production of cyclopiazonic acid in Aspergillus fumisynnematus.

To explore novel bioactive compounds produced via activation of secondary metabolite (SM) gene clusters, we overexpressed an ortholog of laeA, a gene that encodes a global positive regulator of secondary metabolism in Aspergillus fumisynnematus F746. Overexpression of the laeA gene under the alcA promoter resulted in the production of less pigment, shorter conidial head chains, and fewer conidia. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis revealed that SM production in OE::laeA was significantly increased, and included new metabolites that were not detected in the wild type. Among them, a compound named F1 was selected on the basis of its high production levels and antibacterial effects. F1 was purified by column chromatography and preparative TLC and identified as cyclopiazonic acid (CPA) by LC/MS, which had been previously known as mycotoxin. As A. fumisynnematus was not known to produce CPA, these results suggest that overexpression of the laeA gene can be used to explore the synthesis of useful bioactive compounds, even in a fungus for which the genome sequence is unavailable.

Effects of low-intensity ultrasound on oxidative damage in retinal pigment epithelial cells in vitro.

Oxidative stress in retinal pigment epithelium (RPE) is one of the key causative factors of RPE injury in age-related macular degeneration (AMD). Low-intensity ultrasound (LIUS) less than 1 W/cm(2) in intensity has been found to have cytoprotective and anti-inflammatory effects in many cell types and diseases. In this study, we investigated for the first time the feasibility of using LIUS to protect RPE cells from oxidative damage. ARPE-19 cells were treated with H2O2 (an exogenous source of reactive oxygen species) or L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthase inhibitor, and exposed immediately to LIUS at intensities of 50, 100 and 200 mW/cm(2) and a frequency of 1 MHz for 20 min. Both H2O2 and BSO increased the percentage of cells positive for mitochondrial reactive oxygen species at 1 h, but not at 24 h. Co-treatment with LIUS clearly repressed these cells similarly at all intensities by approximately 34%-43% for H2O2 and 24%-25% for BSO (p < 0.05). The percentage of cells with mitochondrial membrane depolarization also increased with H2O2 and BSO treatment, particularly at 1 h, and decreased by approximately 60% with LIUS at 100 mW/cm(2) (p < 0.05). The amount of intracellular calcium ion ([Ca(2+)]i) was elevated only by BSO at 24 h and was also significantly diminished, by approximately 45%, by LIUS at 100 mW/cm(2) (p < 0.05). Both H2O2 and BSO significantly hampered cell viability at 24 h, but LIUS at 100 mW/cm(2) restored only BSO-induced cell viability by approximately 2.7-fold (p < 0.05). This study illustrated that LIUS has a protective effect on RPE cells against oxidative damage caused by BSO, an endogenous mitochondrial reactive oxygen species generator. We speculate that LIUS has the potential to treat oxidative damage and related pathologic changes in RPE.

Survey of Yogurt Powder Storage in Ambient Export Countries A Safety Evaluation Standard Compliance and Comparative Analysis.

Yogurt powder is fermented milk processed in the form of dry yogurt, and has advantages such as stability, storability, convenience, and portability. China and Vietnam are important export target countries because of the increased demand for dairy products. Therefore, we surveyed dairy product standardization in order to establish an export strategy. Lactic acid bacteria counts are unregulated in Korea and Vietnam. In China, lactic acid bacteria counts are regulated at 1×10(6) colony-forming units (CFU)/mL and detected at 6.24±0.33 Log CFU/mL. All three countries have regulated standards for total bacterial counts. In China, total bacterial counts of milk powder are regulated to n=5, c=2, m=50,000, M=200,000 and detected at 6.02±0.12 Log CFU/mL, exceeding the acceptable level. Lactic acid bacterial counts appeared to exceed total bacterial counts. Coliform group counts, Staphylococcus aureus, Listeria monocytogenes, and Salmonella species were not detected. Acidity is not regulated in Korea and Vietnam. In China, acidity was regulated to over 70°T and detected 352.38±10.24°T. pH is unregulated in all three countries. pH was compared to that of general fermented milk, which is 4.2, and that of the sample was 4.28±0.01. Aflatoxin levels are not regulated in Korea and China. In Vietnam, aflatoxin level is regulated at 0.05 ppb. Therefore, all ingredients of the yogurt powder met the safety standards. This data obtained in this study can be used as the basic data in assessing the export quality of yogurt powder.

Determination of Shelf Life for Butter and Cheese Products in Actual and Accelerated Conditions.

The aim of this study was to estimate the shelf life of butter and cheese products, with shelf life being a guide used to determine the storage period of food before deterioration. Butter and cheese samples stored at 10℃ and 15℃ had a shelf life of 221 d, while those stored at 25℃ and 35℃ had a shelf life of 109 d. Quality changes, including total cell count, coliform counts, Listeria monocytogenes counts, acid value, moisture content, pH, acidity and overall sensory evaluation, were monitored. In order to pass the overall sensory evaluation, a quality score of 5 points on a 9-point scale was required. For other quality criteria, legal quality limits were established based on the "Process Criteria and Ingredient Standard of Livestock Products" by the Animal, Plant and Fisheries Quarantine and Inspection Agency (Republic of Korea). The nonlegal quality limit was estimated by regression analysis between non-quality criteria (y) and overall sensory evaluation (x). The shelf life was estimated based on the number of days that the product passed the quality limit of the quality criteria. The shelf life of samples stored at 10℃, 15℃, 25℃ and 35℃ was 21.94, 17.18, 6.10 and 0.58 mon, respectively, for butter and 10.81, 9.47, 4.64 and 0.20 mon, respectively, for cheese.

Determination of the Authenticity of Dairy Products on the Basis of Fatty Acids and Triacylglycerols Content using GC Analysis.

Milk fat is an important food component, and plays a significant role in the economics, functional nutrition, and chemical properties of dairy products. Dairy products also contain nutritional resources and essential fatty acids (FAs). Because of the increasing demand for dairy products, milk fat is a common target in economic fraud. Specifically, milk fat is often replaced with cheaper or readily available vegetable oils or animal fats. In this study, a method for the discrimination of milk fat was developed, using FAs profiles, and triacylglycerols (TGs) profiles. A total of 11 samples were evaluated: four milk fats (MK), four vegetable oils (VG), two pork lards (PL), and one beef tallow (BT). Gas chromathgraphy analysis were performed, to monitor the FAs content and TGs composition in MK, VG, PL, and BT. The result showed that qualitative determination of the MK of samples adulterated with different vegetable oils and animal fats was possible by a visual comparision of FAs, using C14:0, C16:0, C18:1n9c, C18:0, and C18:2n6c, and of TGs, using C36, C38, C40, C50, C52, and C54 profiles. Overall, the objective of this study was to evaluate the potential of the use of FAs and TGs in the detection of adulterated milk fat, and accordingly characterize the samples by the adulterant oil source, and level of adulteration. Also, based on this preliminary investigation, the usefulness of this approach could be tested for other oils in the future.

Reduction in programmed cell death and improvement in functional outcome of transient focal cerebral ischemia after administration of granulocyte-macrophage colony-stimulating factor in rats. Laboratory investigation.

OBJECT: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent hematopoietic growth factor that both enhances the survival and drives the differentiation and proliferation of myeloid lineage cells. Recent studies have suggested that GM-CSF has a neuroprotective effect against CNS injury. In this paper, the authors investigated the neuroprotective effect of GM-CSF on neuron survival and locomotor behavior in a rat model of focal cerebral ischemic injury. MATERIALS: To understand its neuroprotective effect in vitro, GM-CSF was administered to a glutamate-induced excitotoxicity neuronal injury cell culture model that mimics the pathophysiology of focal hypoxic cerebral injury. In the animal study, the authors prepared a rat focal cerebral ischemia model by occluding the unilateral middle cerebral artery. They then examined the effects of GM-CSF administration on changes in infarct volume, apoptosis-related gene expression, and improvement in locomotor behavior. RESULTS: Treatment with GM-CSF significantly increased cell viability in a cell culture model of glutamate-induced neuronal injury. Furthermore, in vivo administration of GM-CSF at 60 microg/kg body weight daily for 5 consecutive days beginning immediately after injury decreased infarction volume, altered the expression of several apoptosis-related genes (Bcl-2, Bax, caspase 3, and p53), and improved locomotor behavior in the focal cerebral ischemia model. CONCLUSIONS: The GM-CSF had neuroprotective effects in in vitro and in vivo experiments and resulted in decreased infarction volume and improved locomotor behavior. Although the specific mechanism involved in stroke recovery was not fully elucidated as it was not the primary focus of this study, administration of GM-CSF appeared to decrease the extent of neuronal apoptosis by modulating the expression of several apoptosis-related genes such as Bcl-2, Bax, caspase 3, and p53. Further investigations are necessary to better understand the role of GM-CSF on neural regeneration during the recovery phase of a stroke, as well as the intracellular signal transduction pathways that mediate neuroprotection.