Problems and solutions of polyethylene glycol co-injection method in multiresidue pesticide analysis by gas chromatography-mass spectrometry: evaluation of instability phenomenon in type II pyrethroids and its suppression by novel analyte protectants.

Polyethylene glycol 300 is commonly used as a base material for "analyte protection" in multiresidue pesticide analysis via gas chromatography-mass spectrometry. However, the disadvantage of the co-injection method using polyethylene glycol 300 is that it causes peak instability in α-cyano pyrethroids (type II pyrethroids) such as fluvalinate. In this study, we confirmed the instability phenomenon in type II pyrethroids and developed novel analyte protectants for acetone/n-hexane mixture solution to suppress the phenomenon. Our findings revealed that among the examined additive compounds, three lipophilic ascorbic acid derivatives, 3-O-ethyl-L-ascorbic acid, 6-O-palmitoyl-L-ascorbic acid, and 6-O-stearoyl-L-ascorbic acid, could effectively stabilize the type II pyrethroids in the presence of polyethylene glycol 300. A mixture of the three ascorbic acid derivatives and polyethylene glycol 300 proved to be an effective analyte protectant for multiresidue pesticide analysis. Further, we designed and evaluated a new combination of analyte protectant compounds without using polyethylene glycol or the troublesome hydrophilic compounds. Consequently, we obtained a set of 10 medium- and long-chain saturated fatty acids as an effective analyte protectant suitable for acetone/n-hexane solution that did not cause peak instability in type II pyrethroids. These analyte protectants will be useful in multiresidue pesticide analysis by gas chromatography-mass spectrometry in terms of ruggedness and reliable quantitativeness. Graphical abstract Comparison of effectiveness of the addition of lipophilic derivatives of ascorbic acid in controlling the instability phenomenon of fluvalinate with polyethylene glycol 300.

[Enhancement of Pesticide Peak Response in GC-MS in the Presence of Multiple Co-Existing Reference Pesticides].

We carried out a collaborative study in six laboratories to confirm the universality of the enhancing effect of co-existing reference pesticides on the GC-MS peak response to a target pesticide (malathion, procymidone, or flucythrinate). First, we confirmed the response enhancement of the target pesticides with increasing numbers of co-existing reference pesticides in solution. Then, using diluted green soybean matrix, we analyzed the target pesticides with two types of matrix-matched calibration, containing the target pesticides or 166 other pesticides. In both cases, the response-enhancing effect of co-existing pesticides was confirmed in all laboratories. The enhancement was reduced by addition of green soybean matrix to the sample and calibration solutions. Our results show that it is necessary to estimate the peak response-enhancing effect of co-existing pesticides in the calibration solution to obtain accurate results with GC-MS determination. The enhancing effect could be reduced by addition of food matrix to the sample and calibration solutions.

Evaluation of the matrix-like effect in multiresidue pesticide analysis by gas chromatography with tandem mass spectrometry.

In multiresidue pesticide analysis using gas chromatography, it has long been recognized that an increase in the number of pesticides present in a standard solution can result in an enhancement of the peak responses of certain pesticides. Despite being widely acknowledged, this phenomenon has been rarely studied and is poorly understood. In this study, the authors have tentatively called this phenomenon the "matrix-like effect" and demonstrated it clearly using gas chromatography with tandem mass spectrometry. Five selected pesticides, namely, omethoate, terbufos, malathion, procymidone, and permethrin, and four internal standard candidates, namely, triphenyl phosphate, naphthalene-d8 , phenanthrene-d10 , and fluoranthene-d10 , were used to evaluate the matrix-like effect following the addition of 58, 108, and 166 other pesticides. With the exception of naphthalene-d8 , the responses of all evaluated pesticides and internal standard candidates were dramatically enhanced by the addition of up to 166 coexisting pesticides. The relative response factors of the five pesticides to each internal standard candidate were not constant under the conditions studied, meaning that these internal standard candidates did not adequately compensate for the matrix-like effect, at least for the five evaluated pesticides. The results revealed that the presence of various mixtures of pesticides in standard solutions might act as an unintentional analyte protectant, that is, some sort of troublesome "quasi-matrix."

Comparison of voice quality after laser cordectomy with that after radiotherapy or chemoradiotherapy for early glottic carcinoma.

PURPOSE OF THE STUDY: The present study aimed to compare the voice quality after each type of cordectomy with that after radiotherapy (RT) or chemoradiotherapy (CRT) for early glottic carcinoma. PROCEDURES: The GRBAS perceptive scale [consisting of 5 domains: grade (G), roughness (R), breathiness (B), asthenia (A), and strain (S)], aerodynamic tests and acoustic analyses, and the Voice Handicap Index questionnaire were evaluated for 58 laser cordectomy cases and 40 RT or CRT cases. Multiple comparison tests were conducted between each type of cordectomy and RT or CRT. RESULTS: No statistical difference was found between type I/II cordectomy and RT for T1 glottic carcinoma (T1RT), whereas T1RT showed a significantly better outcome than type III cordectomy (G score: p = 0.016, maximum phonation time: p < 0.01, mean flow rate: p < 0.01). Type IV cordectomy was equivalent to RT or CRT for T2 glottic carcinoma (T2RT/CRT), while types V and VI showed a worse voice quality than T2RT/CRT (G score: p = 0.038 to type VI, B score: p = 0.025 to type V and p = 0.032 to type VI, A score: p = 0.017 to type VI). CONCLUSIONS: Voice quality after laser cordectomy differs according to the type of cordectomy. Surgeons should inform patients about the expected voice quality after each treatment modality.

The role of sentinel lymph node mapping in colon cancer: detection of micro-metastasis, effect on survival, and driver of a paradigm shift in extent of colon resection.

Unlike in breast cancer and melanoma, sentinel lymph node mapping in colon cancer is primarily used as an aid to the pathologist for accurate nodal staging. The study was undertaken to review the incidence of micro-metastasis and its impact on survival when treated with chemotherapy. The study was also undertaken to see if SLNM could guide limited colon resection in early T stage tumor as a paradigm shift. SLNM was done by subserosal injection of a blue dye. SLNs were ultra-staged by multilevel sectioning and remaining Specimen was then examined by conventional method. For the last 245 patients the specimen was divied ex vivo into two segments as segment A containing the tumor bearing portion of the colon and SLNs with attached mesentery, while segment B include distal part of the colon with attached mesentery. Nodal staging was separately examined. Of the 354 Pts, SLNM was successful in 99.9% of Pts with an average no of SLN/ Pt = 2.8 and total nodes 17.8/pt. Survival was directly related negatively with stage and nodal status. Pts with +ve LN did much better with chemotherapy than without chemotherapy. With 245 Pts, specimen A Vs B, no Pts had +ve node in specimen B with -ve LN in specimen A. SLNM results in more node/Pt, more positive node/Pt ,and more micro-metastasis who when treated with chemotherapy survive longer. Limited segmental resection in early T stage is possible when done with guidance by SLNM without compromising biology.

Clinical features of neuromyelitis optica in a large Japanese cohort: comparison between phenotypes.

OBJECTIVE: To analyse clinicoepidemiological features of neuromyelitis optica in a large cohort and to compare the differences between onset age, gender and clinical phenotypes. METHODS: Antiaquaporin-4 antibody (AQP4-ab) levels were tested in 2366 serum samples of patients diagnosed as having central nervous system inflammatory demyelinating disorders by their referring physicians. AQP4-ab was measured by indirect immunofluorescence staining using human AQP4-transfected HEK 293 cells. A blinded analysis was performed and was combined with clinical information. RESULTS: A total of 583 patients (91.4% women) were AQP4-ab-positive. The average onset age was 42.9±15.9 years. According to MRI studies, spinal-cord lesions were detected in 85.3% of the patients, longitudinally extensive transverse myelitis in 72.7% and cerebral lesions in 51.1%. Unilateral or bilateral blindness was observed in 16.2% of patients, 19.8% were associated with Sjögren syndrome, and 13.6% were associated with thyroid diseases. Myelin basic protein was detected in the cerebrospinal fluid of 57.5% patients. In addition, men presented with an older onset age, a greater number of brainstem MRI lesions and positive myelin basic protein in the cerebrospinal fluid. All child-onset patients (<15 years, n=9) presented with optic neuritis as the first symptom, while older-onset patients presented with myelitis. Twenty patients initially developed limited brain lesions, and seven of these patients did not develop optic or spinal lesions during the 1-5-year follow-up period. CONCLUSIONS: The clinical characteristics of AQP4-ab-positive patients were similar. However, optic neuritis was more common in paediatric patients, while myelitis was more common in older patients. A small number of patients exhibited only cerebral, brainstem, or cerebellar lesions during the initial several years and lower Extended Disability Status Scale scores.

Evaluation of the concordance between GluN1-GluN2 heteromer live-cell-based assay and GluN1 monomer biochip kit assay on anti-NMDAR autoantibody detection.

Anti-N-methyl-d-aspartate receptor (NMDAR) antibodies are most frequently detected in autoantibody-related autoimmune encephalitis. Anti-NMDAR encephalitis mainly affects young women with ovarian teratoma, including acute to subacute onset of psychosis, seizures, consciousness disturbance, dyskinetic involuntary movements, autonomic dysfunction, and others. Diagnosis is based on the detection of anti-NMDAR autoantibodies in cerebrospinal fluid (CSF). The autoantibody recognizes the conformational epitope of the NMDA receptor. NMDA receptors contain hetero-tetramers of GluN1 (NR1) and GluN2/3 (NR2/3), in which GluN1 is essential to form functional receptors on the synaptic membrane in the brain. Thus, the autoantibodies are detected using neurons or culture cells expressing conformational receptors on their cell membrane, the natural form in the brain. The antibodies detected using artificial GluN1 monosubunit expressing cells as the antigens have been widely used for anti-NMDAR-antibody test. In the present study two detection systems were compared, a live-cell-based assay using human embryonic kidney (HEK) 293 cells expressing both of GluN1 and GluN2B, and a commercially available GluN1-monotransfected HEK cell biochip system. As the result, both the methods were equivalent, and the clinical features of both groups were similar, suggesting both tests have equal clinical significance.

Estimation of Irradiation Doses of Raw Beef Liver Samples using 5,6-Dihydrothymidine as an Irradiation Marker.

The quantities of (5S)- and (5R)-5,6-dihydrothymidine (total DHdThd) in frozen beef liver samples were compared between those irradiated by an electron beam (EB) and those subjected to γ-irradiation. DNA extracted from the samples was enzymatically digested to nucleosides and analyzed by liquid chromatography-tandem mass spectrometry for total DHdThd and 2'-deoxythymidine (dThd). Total DHdThd was formed radiospecifically and dose-dependently and the dose-response curves of the ratio of total DHdThd to dThd (total DHdThd/dThd) were similar for both the EB- and γ-irradiated samples. The total DHdThd/dThd was stable after long-term storage (4 months) at -20 °C and the followed heat treatment in a microwave oven. The total DHdThd/dThd could be a robust marker and is equally effective at quantitating both EB- and γ-ray irradiation history. The irradiation doses of raw beef liver samples were estimated using the dose-response curves of the total DHdThd/dThd of other irradiated samples. The ratio of the estimated dose to the actual dose was 0.74-1.30 in the irradiation range of 4.67-7.62 kGy.

Simulated neonatal exposure to DEHP and MEHP from PVC enteral nutrition products.

The leaching of di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) from medical products made of polyvinyl-chloride (PVC) to enteral nutrition (EN) for neonatal patients was determined in a simulated study. The study simulated a typical case of EN administration to a neonatal patient (body weight, 3 kg) in a neonatal care unit (temperature, 25 degrees C); the medical products used were an irrigator and catheter containing DEHP (9.1-31.8%, w/w) as a plasticizer. The worst-case daily exposures of the neonatal patient to DEHP and MEHP by the administration of EN were estimated to be 148 and 3.72 microg/(kg day), respectively, as assessed from the levels of these compounds leaching from the medical products to the EN. The use of DEHP-free medical products reduced the exposure of DEHP and MEHP to the minimum levels contained in the EN at preparation. A transition to DEHP-free medical products for neonatal patients would be effective in reducing the exposure of neonatal patients to DEHP via EN administration.

A rapid and easy multiresidue method for the determination of pesticide residues in vegetables, fruits, and cereals using liquid Chromatography/tandem mass spectrometry.

The applicability of a rapid and easy multiresidue method for determination of pesticide residues in agricultural products by using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was examined. Pesticide residues were extracted with acetonitrile in a disposable tube using a homogenizer, followed by salting out with anhydrous magnesium sulfate and sodium chloride. The extract was purified with a double-layered cartridge column (graphite carbon black/primary-secondary amine silica gel). After removal of the solvent, the extract was resolved in methanol-water and analyzed with LC/MS/MS. Recovery tests of 99 pesticide residues from 7 agricultural products were performed at 20 and 100 ng/g. Throughout all of the agricultural products tested, 47 pesticides exhibited satisfactory recoveries (70-120%) and relative standard deviations (<20%) at both concentrations. The time for processing of 12 samples to test solutions was approximately 2-3 h. This method could be useful for determination of pesticide residues in agricultural products.

The vasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity under both stress and resting conditions.

The neurohypophyseal peptide [Arg(8)]-vasopressin (AVP) exerts major physiological actions through three distinct receptor isoforms designated V1a, V1b, and V2. Among these three subtypes, the vasopressin V1b receptor is specifically expressed in pituitary corticotrophs and mediates the stimulatory effect of vasopressin on ACTH release. To investigate the functional roles of V1b receptor subtypes in vivo, gene targeting was used to create a mouse model lacking the V1b receptor gene (V1bR-/-). Under resting conditions, circulating concentrations of ACTH and corticosterone were lower in V1bR-/- mice compared with WT mice (V1bR+/+). The normal increase in circulating ACTH levels in response to exogenous administration of AVP was impaired in V1bR-/- mice, while corticotropin-releasing hormone-stimulated ACTH release in the V1bR-/- mice was not significantly different from that in the V1bR+/+ mice. AVP-induced ACTH release from primary cultured pituitary cells in V1bR-/- mice was also blunted. Furthermore, the increase in ACTH after a forced swim stress was significantly suppressed in V1bR-/- mice. Our results clearly demonstrate that the V1b receptor plays a crucial role in regulating hypothalamic-pituitary-adrenal axis activity. It does this by maintaining ACTH and corticosterone levels, not only under stress but also under basal conditions.

Simultaneous analysis of 260 pesticide residues in agricultural products by gas chromatography/triple quadrupole mass spectrometry.

A method for simultaneous analysis of about 260 pesticides by gas chromatography coupled to tandem mass spectrometry (GC/MS/MS) with a triple quadrupole analyzer (QqQ) has been studied. The pesticides were extracted with acetonitrile and cleaned up by a bilayer cartridge. A single injection method was developed for the monitoring of all of the targeted pesticides. Two MS/MS transitions were selected for each analyte using the intensity ratio obtained from them as a confirmatory parameter. By using matrix-matched standards, 260 pesticides could be determined in most matrixes with recoveries of 70-120% and a standard deviation of < or = 20 at 2 different fortification levels of 0.02 and 0.1 microg/g. The developed method was applied to the monitoring of 173 agricultural product samples from the local market. The sensitivities of this method were lower than with most of the selective GC detectors, such as flame photometric or single MS. The selectivity of QqQ gives a very clean chromatogram, making compound identification and confirmation easy. The quick and reliable monitoring was achieved by combination with rapid extraction and cleanup.

An Ecological Survey of Mosquitoes and the Distribution of Japanese Encephalitis Virus in Ishikawa Prefecture, Japan, between 2010 and 2014.

Japanese encephalitis virus (JEV) is a flavivirus, responsible for over 30,000 annual cases of encephalitis worldwide, with a mortality rate of approximately 30%. Therefore, it is important to examine the distribution of mosquitos carrying JEV in the fields, even though recently, the number of Japanese encephalitis cases has been approximately 5 per year in Japan. We report the seasonal dynamics of mosquitoes between 2010 and 2014 in Ishikawa Prefecture, Japan. We collected 39,308 female adult mosquitoes, 98.2% of which were classified as Culex tritaeniorhynchus Giles. We identified JEV genomic RNA belonging to genotype 1 from the homogenate of Cx. tritaeniorhynchus, collected during our study using reverse transcription-PCR and nucleotide sequencing techniques. Our results indicate that mosquito vectors for JEV are distributed not only in areas in Ishikawa, but also throughout Japan, and the results suggest that we must be careful regarding JEV outbreaks in Japan in the future.

Rapid and Reliable Method for Determining Irradiation Histories of Ground Beef and Prawns by Measuring 5,6-Dihydrothymidine.

A rapid and reliable method for determining irradiation histories of ground beef and prawns was developed on the basis of a method for determining the irradiation history of beef liver by liquid chromatography-tandem mass spectrometry (LC-MS/MS) of 5,6-dihydrothymidine (DHdThd). Improvements in the method included the following: (1) 50% ethanol precipitation in the DNA extraction step was conducted before the RNase step, (2) snake venom phosphodiesterase I was used for DNA digestion to boost liberation of DHdThd, and (3) a matrix-matched calibration curve was used for determining DHdThd by LC-MS/MS analysis. This method successfully determined irradiation histories of ground beef and prawns. Furthermore, a close correlation between the formation of DHdThd and 2-alkylcyclobutanones, which are an established index of irradiation histories, was observed in ground beef. DHdThd in DNA could be a promising candidate for a new index of irradiation histories of various foods.

Determination of irradiation histories of raw beef livers using liquid chromatography-tandem mass spectrometry of 5,6-dihydrothymidine.

A method for detecting irradiation histories of raw beef livers was developed by measuring 5,6-dihydrothymidine (DHdThd) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Liver DNA was extracted using phenol-chloroform extraction followed by precipitation in 50% ethanol. DNA was then enzymatically digested and nucleosides were purified using an OASIS MCX column. DHdThd and thymidine (dThd) contents of resulting test solutions were analyzed using LC-MS/MS. DHdThd was detected specifically after γ-irradiation. Concentration ratios of DHdThd to dThd in the test solutions increased dose-dependently after irradiation at 1.0-11.3kGy, which included the practical dose for sterilization of 2-7kGy. Dose-response curves from beef livers of individual animals almost overlapped. Thus, this method is a candidate for the detection of irradiation histories of foods from which DNA can be extracted.

Simultaneous analysis of 10 pyrethroid pesticides in natural medicines by GC/MS with negative chemical ionization.

Many methods for the determination of pesticides residues in food have been reported. Although natural medicines should be confirmed as safe as food, only a few methods for the determination of pesticide residues in natural medicines have been reported. In this study, 10 pyrethroid pesticides were detected in natural medicines by GC/MS with negative chemical ionization (NCI). GC/MS with NCI can detect halogenated pesticides selectively, and thus is suitable for the detection of pyrethroid pesticides. This study indicates that GC/MS with NCI is useful for analyzing pyrethroid pesticides in natural medicines.

Vocal fold injury following endotracheal intubation.

Vocal fold scarring results in the formation of fibrous tissue which disturbs the vibratory pattern of the fold during phonation. However, vocal fold scarring in humans is poorly understood because of the lack of clear case reports focusing on voice quality. The authors present a case of vocal fold scarring with changes in voice quality. At the time of injury the pedicle mucosa was cemented with fibrin glue. Phonation was inhibited for two weeks and tranilast (300 mg/day) was given for 3 months. Sixty-nine days later, perceptual evaluation showed a normal result and the phonation time became better, but the mucosal vibration was still lacking. Ninety-seven days later, mucosal vibration was finally restored. We suggest that characterization of vocal fold scarring in humans may be different from that in animals, and recommend that surgical management should be avoided for at least three months after injury.

Binding of the ELAV-like protein in murine autoimmune T-cells to the nonameric AU-rich element in the 3' untranslated region of CD154 mRNA.

The CD154 molecule is important for experimental allergic encephalomyelitis (EAE) which is mediated by autoimmune CD4(+) T-cells. Post-transcriptional instabilization/stabilization of mRNAs, which contain an adenylate uridylate rich element (ARE) in their 3' untranslated region (3'UTR), is regulated in part by binding of ARE-binding proteins to the element. We have investigated the protein which binds to the nonameric ARE in the 3'UTR of CD154 mRNA. A protein which binds to the CD154 ARE was found to exist in a extract prepared from murine autoimmune T-cells activated with myelin basic protein (MBP), and turned out to be mHuR which is a ubiquitous ELAV-like protein. It was found that mHuR was upregulated upon stimulation of the T-cells with a MBP antigen. The CD154 ARE and the ARE in the 3'UTR of tumor necrosis factor-alpha (TNF-alpha) mRNA were competed in binding to mHuR, indicating that both AREs bind to the same site on mHuR. The presence of the CD154 ARE downstream of the luciferase cDNA in a reporter plasmid decreased the translational efficiency, and co-expression of the mHuR slightly increased the translation. These results suggest the possibility that the ELAV-like protein participates in the regulation of the expression of CD154 on the autoimmune T-cells. Modification of the expression of CD154 on autoimmune T-cells by regulating the ELAV-like protein may provide effective therapy for EAE and human multiple sclerosis.

Carvedilol selectively inhibits oscillatory intracellular calcium changes evoked by human alpha1D- and alpha1B-adrenergic receptors.

BACKGROUND: Increasing evidence from clinical trials indicates that carvedilol, an antagonist of alpha1- and beta-adrenergic receptors (ARs), provides an effective treatment for chronic heart failure, whereas nonselective alpha1-AR blockade has an adverse outcome in this disease. It is, however, not clear whether carvedilol exhibits a subtype-dependent impact on three distinct alpha1-adrenergic receptors (alpha1-ARs). METHODS AND RESULTS: We determined binding properties of human ARs for carvedilol using HEK293 human embryonic kidney cells expressing a single AR subtype. Our results showed that the affinities of alpha1D-AR and alpha1B-AR for carvedilol are higher than that of the beta1-AR subtype, a major target in heart failure treatment. The affinity rank order and pKi values of ARs for carvedilol were as follows: alpha1D-AR (8.9)>alpha1B-AR (8.6)>beta1-AR (8.4)>beta2-AR (8.0)>alpha1A-AR (7.9)?alpha2C-AR (5.9)>alpha2B-AR (5.5)>alpha2A-AR (5.3). Furthermore, temporal kinetics of intracellular calcium signaling mediated via alpha1D- and alpha1B-ARs, but not via alpha1A-AR (P<0.01), showed oscillatory patterns with frequencies ranging from 0.3 to 3 per minute in human smooth muscle and HEK293 cells, which were inhibited by the therapeutic concentrations of carvedilol (10 nM) in a subtype-dependent manner. When oscillatory alpha1B-AR and non-oscillatory alpha1A-AR were co-expressed and heteromer receptors were detected with bioluminescence resonance energy transfer and co-immunoprecipitation, carvedilol suppressed only oscillatory component of global cytosolic free calcium change. CONCLUSIONS: These results indicate that in addition to beta-ARs, receptor inhibition by carvedilol is directed to alpha1-ARs, preferably to alpha1D- and alpha1B-AR-mediated signaling events, including intracellular calcium oscillations in vascular smooth muscle.

[Validation Study on a Multi-Residue Method for Determination of Pesticide Residues in Vegetables and Fruits by using General Matrix Standard Solutions].

Quantitative methods using the matrix-matched standard solutions approach are widely used for multi-residue pesticide determination by GC-MS/MS to deal with the issue of matrix effects. However, preparing matrix-matched standard solutions in analyses of many kinds of samples is very time-consuming. In order to solve this problem, a method that employs general matrix standard solutions has been developed using polyethylene glycol (PEG), extract of vegetables-fruit juice (VFJm) and triphenyl phosphate (named the PEG-VFJm method). Here, a validation study for 168 pesticides was performed on three kinds of samples [potato, spinach and apple] at concentrations of 0.010 and 0.050 µg/g. In these three commodities, 144 to 158 pesticides satisfied the required criteria using the matrix-matched method and 129 to 149 pesticides satisfied the same criteria using the PEG-VFJm method. Our results suggest that application of general matrix standard solutions would enable rapid and effective analyses of pesticides.