Compound-Specific Isotope Analysis Provides Direct Evidence for Identifying the Source of Residual Pesticides Diazinon and Procymidone in the Soil-Plant System.

Compound-specific isotope analysis stands as a promising tool for unveiling the behavior of pesticides in agricultural environments. Using the commercial formulations of persistent fungicide procymidone (PRO) and less persistent insecticide diazinon (DIA), respectively, we analyzed the concentration and carbon isotope composition (δ13C) of the residual pesticides through soil incubation experiments in a greenhouse (for 150 days) and lab conditions (for 50-70 days). Our results showed that the magnitude of δ13C variation depends on pesticide specificity, in which PRO in the soil exhibited little variation in δ13C values over the entire incubation times, while DIA demonstrated an increased δ13C value, with the extent of δ13C variability affected by different spiking concentrations, plant presence, and light conditions. Moreover, the pesticides extracted from soils were isotopically overlapped with those from crop lettuce. Ultimately, the isotope composition of pesticides could infer the degradation and translocation processes and might contribute to identifying the source(s) of pesticide formulation in agricultural fields.

3-Methylcatechol mediates anti-fecundity effect by inhibiting estrogen-related receptor-induced glycolytic gene expression in Myzus persicae.

Aphids are a major problem in agriculture, horticulture, and forestry by feeding on leaves and stems, causing discoloration, leaf curling, yellowing, and stunted growth. Although urushiol, a phenolic compound containing a catechol structure, is known for its antioxidant and anticancer properties, using small molecules to control aphids via catechol-mediated mechanisms is poorly understood. In this study, we investigated the effects of 3-methylcatechol (3-MC) on Myzus persicae fecundity. Our results showed that treatment with 3-MC significantly reduced the intrinsic transcriptional activity of the aphid estrogen-related receptor (MpERR), which regulates the expression of glycolytic genes. Additionally, 3-MC treatment suppressed the promoter activity of MpERR-induced rate-limiting enzymes in glycolysis, such as phosphofructokinase and pyruvate kinase, by inhibiting MpERR binding. Finally, 3-MC also suppressed MpERR-induced glycolytic gene expression and reduced the number of offspring produced by viviparous female aphids. Overall, our findings suggest that 3-MC has the potential to be used as a new strategy for managing aphid populations by controlling their offspring production.

Stable Isotope Analysis of Residual Pesticides via High Performance Liquid Chromatography and Elemental Analyzer-Isotope Ratio Mass Spectrometry.

To broaden the range of measurable pesticides for stable isotope analysis (SIA), we tested whether SIA of the anthranilic diamides cyantraniliprole (CYN) and chlorantraniliprole (CHL) can be achieved under elemental analyzer/isotope ratio mass spectrometry with compound purification in high-performance liquid chromatography (HPLC). Using this method, carbon isotope compositions were measured in pesticide residues extracted from plants (lettuce) grown indoors in potting soil that were treated with 500 mg/kg CHL and 250 mg/kg CYN and were followed up for 45 days. Our results show that the CYN and CHL standard materials did not have significant isotope differences before and after clean-up processing in HPLC. Further, when applied to the CYN product and CHL product in soil, stable isotope differences between the soil and plant were observed at <1.0‱ throughout the incubation period. There was a slight increase in the variability of pesticide isotope ratio detected with longer-term incubation (CHL, on average 1.5‱). Overall, we measured the carbon isotope ratio of target pesticides from HPLC fraction as the purification and pre-concentration step for environmental and biological samples. Such negligible isotopic differences in pesticide residues in soils and plants 45 days after application confirmed the potential of CSIA to quantify pesticide behavior in environments.

Development and validation of a gas chromatography method for the determination of ß-caryophyllene in clove extract and its application.

The purpose of this study is to check the effectiveness of the analysis method that separates and quantifies ß-caryophyllene among clove extracts and validate according to current ICH guidelines. The ß-caryophyllene was active constituent of clove buds. The developed method gave a good detection response. In the specificity test, the standard solution was detected at about 17.32 min, and the test solution was detected at 17.32 min. The linearity of ß-caryophyllen was confirmed, and at this time, the correlation coefficient (R2) of the calibration curve showed a high linearity of 0.999 or more in the concentration range. The levels of LOD and LOQ were 1.28 ug/mL and 3.89 ug/mL, respectively. The accuracy was confirmed to be 101.6-102.2% and RSD 0.95 ~ 1.31%. As a result of checking the repeatability and inter-tester reproducibility to confirm the precision, the RSD was found to be 1.34 ~ 2.69%. This validated GC method was successfully applied to a soft capsule containing clove extract and other materials for clinical trials. Therefore, this method can be used as an analytical tool for quality control of various samples, including clove extracts and their products of food and pharmaceutical uses.

Aphid estrogen-related receptor controls glycolytic gene expression and fecundity.

Aphids, the major insect pests of agricultural crops, reproduce sexually and asexually depending upon environmental factors such as the photoperiod and temperature. Nuclear receptors, a unique family of ligand-dependent transcription factors, control insect development and growth including morphogenesis, molting, and metamorphosis. However, the structural features and biological functions of the aphid estrogen-related receptor (ERR) are largely unknown. Here, we cloned full-length cDNA encoding the ERR in the green peach aphid, Myzus persicae, (Sulzer) (Hemiptera: Aphididae) (MpERR) and demonstrated that the MpERR modulated glycolytic gene expression and aphid fecundity. The phylogenetic analysis revealed that the MpERR originated in a unique evolutionary lineage distinct from those of hemipteran insects. Moreover, the AF-2 domain of the MpERR conferred nuclear localization and transcriptional activity. The overexpression of the MpERR significantly upregulated the gene expression of rate-limiting enzymes involved in glycolysis such as phosphofructokinase and pyruvate kinase by directly binding to ERR-response elements in their promoters. Moreover, ERR-deficient viviparous female aphids showed decreased glycolytic gene expression and produced fewer offspring. These results suggest that the aphid ERR plays a pivotal role in glycolytic transcriptional control and fecundity.

Biosurfactants Induce Antimicrobial Peptide Production through the Activation of TmSpatzles in Tenebrio molitor.

Biosurfactant immunomodulatory activities in mammals, nematodes, and plants have been investigated. However, the immune activation property of biosurfactants in insects has not been reported. Therefore, here, we studied the defense response triggered by lipopeptides (fengycin and iturin A), glycolipids (rhamnolipid), and cyclic polypeptides (bacitracin) in the coleopteran insect, mealworm Tenebrio molitor. The in vitro antimicrobial activities against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans) were assessed by mixing these pathogens with the hemolymph of biosurfactant-immune-activated larvae. E. coli growth was remarkably inhibited by this hemolymph. The antimicrobial peptide (AMP) induction results also revealed that all biosurfactants tested induced several AMPs, exclusively in hemocytes. The survivability analysis of T. molitor larvae challenged by E. coli (106 CFU/µL) at 24 h post biosurfactant-immune activation showed that fengycin, iturin A, and rhamnopid significantly increased survivability against E. coli. Biosurfactant-induced TmSpatzles activation was also monitored, and the results showed that TmSpz3 and TmSpz-like were upregulated in the hemocytes of iturin A-injected larvae, while TmSpz4 and TmSpz6 were upregulated in the fat bodies of the fengycin-, iturin A-, and rhamnolipid-injected larvae. Overall, these results suggest that lipopeptide and glycolipid biosurfactants induce the expression of AMPs in T. molitor via the activation of spätzle genes, thereby increasing the survivability of T. molitor against E. coli.

Soluble RANKL expression in Lactococcus lactis and investigation of its potential as an oral vaccine adjuvant.

BACKGROUND: To initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer's patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant. RESULTS: For efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB. CONCLUSIONS: Our results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.

PRMT1 and PRMT4 Regulate Oxidative Stress-Induced Retinal Pigment Epithelial Cell Damage in SIRT1-Dependent and SIRT1-Independent Manners.

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is involved in the progression of diabetic retinopathy. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has emerged as an important histone modification involved in diverse diseases. Sirtuin (SIRT1) is a protein deacetylase implicated in the onset of metabolic diseases. Therefore, we examined the roles of type I PRMTs and their relationship with SIRT1 in human RPE cells under H2O2-induced oxidative stress. H2O2 treatment increased PRMT1 and PRMT4 expression but decreased SIRT1 expression. Similar to H2O2 treatment, PRMT1 or PRMT4 overexpression increased RPE cell damage. Moreover, the H2O2-induced RPE cell damage was attenuated by PRMT1 or PRMT4 knockdown and SIRT1 overexpression. In this study, we revealed that SIRT1 expression was regulated by PRMT1 but not by PRMT4. Finally, we found that PRMT1 and PRMT4 expression is increased in the RPE layer of streptozotocin-treated rats. Taken together, we demonstrated that oxidative stress induces apoptosis both via PRMT1 in a SIRT1-dependent manner and via PRMT4 in a SIRT1-independent manner. The inhibition of the expression of type I PRMTs, especially PRMT1 and PRMT4, and increased SIRT1 could be therapeutic approaches for diabetic retinopathy.

Epidemiologic differences of four major respiratory viruses between children, adolescents, and adults in Korea.

The purpose of this study was to investigate the epidemiology of four major respiratory viruses among the Korean population. This retrospective study was conducted over four years, from January 2005 to December 2008. Among a total of 23,806 specimens, 5512 virus isolates underwent culture for influenza A and B viruses (IFA/B), parainfluenza virus (PIV), respiratory syncytial virus (RSV), and adenovirus (ADV). Patients were divided into two groups: children/adolescents and adults. The viruses detected in specimens from children/adolescents included PIV (7.8%), RSV (7.3%), ADV (4.0%), IFA (2.9%), and IFB (2.2%). In adults, IFB (5.6%), IFA (4.4%), RSV (1.1%), PIV (0.5%), and ADV (0.2%) were detected, thus demonstrating two distinct patterns of virus infection. Influenza viruses had similar seasonal patterns and periods of infection among children/adolescents and adults; however, the isolation rate in adults was slightly higher than that in children and adolescents. Correlation coefficient analysis based on weekly seasonal patterns indicated that influenza viruses were detected a week earlier in children than in adults. RSV, PIV, and ADV did not show similar trends between the two age groups due to low detection rates and sporadic isolations among adult patients. Of note, different respiratory viruses should be considered depending on patient age when a clinical respiratory viral infection is suspected. Furthermore, in the case of influenza, a preceding epidemic among a pediatric population could be useful to predict a subsequent epidemic among adults.

Etiology and clinical outcomes of acute respiratory virus infection in hospitalized adults.

BACKGROUND: Etiologies and clinical profiles of acute respiratory viral infections need to be clarified to improve preventive and therapeutic strategies. MATERIALS AND METHODS: A retrospective observational study at a single, university-affiliated center was performed to evaluate the respiratory viral infection etiologies in children compared to that in adults and to document the clinical features of common viral infections for adults from July 2009 to April 2012. RESULTS: The common viruses detected from children (2,800 total patients) were human rhinovirus (hRV) (31.8%), adenovirus (AdV) (19.2%), respiratory syncytial virus (RSV) A (17.4%), RSV B (11.7%), and human metapneumovirus (hMPV) (9.8%). In comparison, influenza virus A (IFA) had the highest isolation rate (28.5%), followed by hRV (15.5%), influenza virus B (IFB) (15.0%), and hMPV (14.0%), in adults (763 total patients). Multiple viruses were detected in single specimens from 22.4% of children and 2.0% of adults. IFA/IFB, RSV A/B, and hMPV exhibited strong seasonal detection and similar circulating patterns in children and adults. Adult patients showed different clinical manifestations according to causative viruses; nasal congestion and rhinorrhea were more common in hRV and human coronavirus (hCoV) infection. Patients with RSV B, hRV, or AdV tended to be younger, and those infected with RSV A and hMPV were likely to be older. Those with RSV A infection tended to stay longer in hospital, enter the intensive care unit more frequently, and have a fatal outcome more often. The bacterial co-detection rate was 26.5%, and those cases were more likely to have lower respiratory tract involvement (P = 0.001), longer hospital stay (P = 0.001), and higher mortality (P = 0.001). CONCLUSIONS: The etiologic virus of an acute respiratory infection can be cautiously inferred based on a patient's age and clinical features and concurrent epidemic data. Large-scale prospective surveillance studies are required to provide more accurate information about respiratory viral infection etiology, which could favorably influence clinical outcomes.

Cancer of unknown primary finally revealed to be a metastatic prostate cancer: a case report.

The vast majority of patients with metastatic prostate cancer present with bone metastases and high prostate specific antigen (PSA) level. Rarely, prostate cancer can develop in patients with normal PSA level. Here, we report a patient who presented with a periureteral tumor of unknown primary site that was confirmed as prostate adenocarcinoma after three years with using specific immunohistochemical examination. A 64-year old man was admitted to our hospital with left flank pain associated with masses on the left pelvic cavity with left hydronephrosis. All tumor markers including CEA, CA19-9, and PSA were within the normal range. After an exploratory mass excision and left nephrectomy, the pelvic mass was diagnosed as poorly differentiated carcinoma without specific positive immunohistochemical markers. At that time, we treated him as having a cancer of unknown primary site. After approximately three years later, he revisited the hospital with a complaint of right shoulder pain. A right scapular mass was newly detected with a high serum PSA level (101.7 ng/ml). Tissues from the scapular mass and prostate revealed prostate cancer with positive immunoreactivity for P504S, a new prostate cancer-specific gene. The histological findings were the same as the previous pelvic mass; however, positive staining for PSA was observed only in the prostate mass. This case demonstrates a patient with prostate cancer and negative serological test and tissue staining that turned out to be positive during progression. We suggest the usefulness of newly developed immunohistochemical markers such as P504S to determine the specific primary site of metastatic poorly differentiated adenocarcinoma in men.

Identification of an ISR-related metabolite produced by rhizobacterium Klebsiella oxytoca C1036 active against soft-rot disease pathogen in tobacco.

BACKGROUND: Klebsiella oxytoca C1036 (C1036) causes induced systemic resistance (ISR) activity against the soft-rot pathogen Pectobacterium carotovorum subsp. carotovorum SCC1 (SCC1). However, microbial metabolites from C1036 involved in ISR activity remain unknown. The present study was performed to identify an ISR-related metabolite produced by C1036. RESULTS: The supernatants of C1036 cultures grown on Luria-Bertani medium were subjected to solvent extraction, repeated column chromatography and preparative liquid chromatography for isolation of an ISR-related metabolite. High-resolution mass spectrometer analysis of the isolated metabolite indicated a C9H15O3N compound with a mass of 185.11. Low-resolution mass spectrometer analysis of the metabolite showed a molecular ion peak at 185 and its fragment ions at 84 and 56. Nuclear magnetic resonance spectrometer analyses characterised all protons and carbons of the isolated metabolite. Based on the data, the isolated metabolite was determined to be butyl 2-pyrrolidone-5-carboxylate (BPC). BPC at 12 mM significantly suppressed the disease symptoms in ISR bioassays against SCC1. CONCLUSION: This is the first report identifying BPC as an ISR-related metabolite produced by C1036. C1036 may play a role in promoting plant growth because it produces ISR-related metabolites against the plant pathogen SCC1.

Lupane glycosides from the leaves of Acanthopanax koreanum.

Three new lupane-type saponins, acankoreosides F--H (1--3) were isolated from the methanol extract of the leaves of Acanthopanax koreanum NAKAI. The structures of these three saponins were established by chemical and spectroscopic analysis as 3alpha,30-dihydroxylup-20(29)-en-23,28-dioic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester (1), 3alpha,30-dihydroxylup-23-al-20(29)-en-28-oic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester (2), and (20S) 3alpha-hydroxylup-23-al-28,29-dioic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester (3), respectively. The effects of the isolates (1-3) on the lipopolysaccharide-induced production of nitric oxide and prostaglandin E2 were evaluated in RAW 264.7 macrophages.

Identification of an ISR-related metabolite produced by Pseudomonas chlororaphis O6 against the wildfire pathogen pseudomonas syringae pv.tabaci in tobacco.

Pseudomonas chlororaphis O6 exhibits induced systemic resistance (ISR) against P. syringae pv. tabaci in tobacco. To identify one of the ISR metabolites, O6 cultures were extracted with organic solvents, and the organic extracts were subjected to column chromatography followed by spectroscopy analyses. The ISR bioassay-guided fractionation was carried out for isolation of the metabolite. Highresolution mass spectrometric analysis of the metabolite found C(9)H(9)O(3)N with an exact mass of 179.0582. LC/MS analysis in positive mode showed an (M+H)(+) peak at m/zeta 180. Nuclear magnetic resonance ((1)H, (13)C) analyses identified all protons and carbons of the metabolite. Based on the spectroscopy data, the metabolite was identified 4-(aminocarbonyl) phenylacetate (4-ACPA). 4-ACPA applied at 68.0 mM exhibited ISR activity at a level similar 1.0 mM salicylic acid. This is the first report to identify an ISR metabolite produced by P. chlororaphis O6 against the wildfire pathogen P. syringae pv. tabaci in tobacco.

Soybean oil-degrading bacterial cultures as a potential for control of green peach aphids (Myzus persicae).

Microorganisms capable of degrading crude oil were isolated and grown in soybean oil as a sole carbon source. The microbial cultures were used to control green peach aphids in vitro. Approximately 60% mortality of aphids was observed when the cultures were applied alone onto aphids. To examine the cultures as a pesticide formulation mixture, the cultures were combined with a low dose of the insecticide imidacloprid (one-fourth dose of recommended field-application rate) and applied onto aphids. The cultures enhanced significantly the insecticidal effectiveness of imidacloprid, which was higher than imidacloprid alone applied at the low dose. The isolated microorganisms exhibited high emulsifying index values and decreased surface tension values after being grown in soybean oil media. GC/MS analyses showed that microorganisms degraded soybean oil to fatty acids. The cultures were suggested to play the roles of wetting, spreading, and sticking agents to improve the effectiveness of imidacloprid. This is the first report on the control of aphids by using oil-degrading microbial cultures.

Analysis of volatile compounds in fresh healthy and diseased peppers (Capsicum annuum L.) using solvent free solid injection coupled with gas chromatography-flame ionization detector and confirmation with mass spectrometry.

The characteristic volatile flavor compounds in healthy peppers (Capsicum annuum L.) were evaluated using a solvent-free solid injector coupled with a-gas chromatography-flame ionization detector (SFSI-GC-FID) and the results of evaluation were confirmed using GC-mass spectrometry (GC-MS). These compounds were compared with those obtained from peppers that were naturally infected or artificially inoculated with Colletotrichum spp. Parameters influencing the vaporization efficiency, including the injector temperature, pre-heating time and holding time, were optimized to improve the analytical efficiency. A total of 96 compounds (excluding eight capillary compounds), 17 of which were identified in healthy peppers, 49 of which were found in naturally infected peppers, and 61 of which were identified in artificially inoculated peppers, were separated and identified under the optimal conditions of an injector temperature of 250 degrees C and 7-min preheating and holding times. Acetic acid and 2-furanmethanol were the major compounds detected in the volatiles of the healthy and diseased peppers. The major compound detected in both the healthy and naturally infected peppers was 3-hydroxypyridine, while hexadecanoic acid was the primary compound identified in the artificially inoculated peppers. Indole derivatives (1H-indole, 4-methylindole and 1-ethylindole) were suggested to be the key factors contributing to the pepper infection caused by Colletotrichum spp. We conclude that SFSI in combination with GC is a suitable approach for distinguishing between healthy and diseased peppers by the investigation of their volatile compounds. It does not require the use of solvents and complicated equipment.

Production of indole-3-acetic acid in the plant-beneficial strain Pseudomonas chlororaphis O6 is negatively regulated by the global sensor kinase GacS.

Certain plant growth-promoting bacteria, such as Pseudomonas fluorescens 89B61 and Bacillus pumilus SE34, secreted high levels of indole-3-acetic acid (IAA) in tryptophan-amended medium in stationary phase as determined by chromogenic analysis and high-performance liquid chromatography. Two other growth-promoting strains, P. chlororaphis O6 and Serratia marcescens 90-166, did not produce these high levels of IAA. However, when the gacS mutant of P. chlororaphis O6 was grown in tryptophan-supplemented medium, IAA was detected in culture filtrates. IAA production by the gacS mutant in P. chlororaphis O6 was repressed in the tryptophan medium by complementation with the wild-type gacS gene. Thus, the global regulatory Gac system in P. chlororaphis O6 acts as a negative regulator of IAA production from trypophan.