Mosquito Identification From Bulk Samples Using DNA Metabarcoding: a Protocol to Support Mosquito-Borne Disease Surveillance in Canada.

Approximately 80 species of mosquitoes (Diptera: Culicidae) have been documented in Canada. Exotic species such as Aedes albopictus (Skuse) (Diptera: Culicidae) are becoming established. Recently occurring endemic mosquito-borne diseases (MBD) in Canada including West-Nile virus (WNV) and Eastern Equine Encephalitis (EEE) are having significant public health impacts. Here we explore the use of DNA metabarcoding to identify mosquitoes from CDC light-trap collections from two locations in eastern Canada. Two primer pairs (BF2-BR2 and F230) were used to amplify regions of the cytochrome c oxidase subunit I (CO1) gene. High throughput sequencing was conducted using an Illumina MiSeq platform and GenBank-based species identification was applied using a QIIME 1.9 bioinformatics pipeline. From a site in southeastern Ontario, Canada, 26 CDC light trap collections of 72 to >300 individual mosquitoes were used to explore the capacity of DNA metabarcoding to identify and quantify captured mosquitoes. The DNA metabarcoding method identified 33 species overall while 24 species were identified by key. Using replicates from each trap, the dried biomass needed to identify the majority of species was determined to be 76 mg (equivalent to approximately 72 mosquitoes), and at least two replicates from the dried biomass would be needed to reliably detect the majority of species in collections of 144-215 mosquitoes and three replicates would be advised for collections with >215 mosquitoes. This study supports the use of DNA metabarcoding as a mosquito surveillance tool in Canada which can help identify the emergence of new mosquito-borne disease potential threats.

A Wearable Wireless Armband Sensor for High-Density Surface Electromyography Recording.

This paper presents a portable and modular wireless multichannel sensor system for high-density surface electromyography (HD-sEMG) signals acquisition. Featuring low-power and high-quality off-the-shelf components such as the Intan Technologies RHD2132 digital electrophysiology interface chip, the current iteration of the proposed sensor system allows the recording of 32 surface electromyography (sEMG) channels, each at a sampling rate of 1 kHz, and a sample resolution of 16 bits. It features the RHD2132's typical input-referred noise of 2.4 µVrms, with <; 15% variation with amplifier bandwidth as specified by the manufacturer, and a total power consumption of 49.5 mW. Data is sent in real-time to a base station using a 2.4-GHz industrial, scientific and medical (ISM) wireless link. Along with the recording platform, the integrated sensor system includes a dry surface electrodes array prototype directly built on a printed circuit board. Intended for complex muscles activity patterns detection on the forearm, the flexible 32 surface electrodes array is designed to be placed flat or to fit a curved area like the forearm in a hand gestures recognition prosthetic system. In such applications, this device will offer improved prosthesis control scheme intuitiveness and ease-of-use. Among other core features of the system are its compact, light-weight and easy to install physical design. The complete system fits on a 2 by 6.5 cm2 printed circuit board mounted on a 7.6 by 11.8 cm2 electrodes array. HD-sEMG user forearm output data collected with the system is presented with a proposed frequency-time-space cross-domain preprocessing method for visualization of HD-EMG data and building training datasets.

Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates.

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as beta-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons.

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.

Absence of deep-water formation in the Labrador Sea during the last interglacial period.

The two main constituent water masses of the deep North Atlantic Ocean-North Atlantic Deep Water at the bottom and Labrador Sea Water at an intermediate level-are currently formed in the Nordic seas and the Labrador Sea, respectively. The rate of formation of these two water masses tightly governs the strength of the global ocean circulation and the associated heat transport across the North Atlantic Ocean. Numerical simulations have suggested a possible shut-down of Labrador Sea Water formation as a consequence of global warming. Here we use micropalaeontological data and stable isotope measurements in both planktonic and benthic foraminifera from deep Labrador Sea cores to investigate the density structure of the water column during the last interglacial period, which was thought to be about 2 degrees C warmer than present. Our results indicate that today's stratification between Labrador Sea Water and North Atlantic Deep Water never developed during the last interglacial period. Instead, a buoyant surface layer was present above a single water mass originating from the Nordic seas. Thus the present situation, with an active site of intermediate-water formation in the Labrador Sea, which settled some 7,000 years ago, has no analogue throughout the last climate cycle.

Quartz exposure, retention, and early silicosis in sheep.

The purposes of this study were (1) to investigate the chronology of events in cellular and biochemical changes thought to be important in the development of silicosis, (2) to relate these to changes in lung function and radiograph, and (3) to evaluate the relation of quartz exposure and retention to individual response leading to early silicosis. Thirty-six sheep were exposed by repeated intratracheal infusion at 10-day intervals to 100 mg Minusil-5 in 100 ml saline (Si group), and 10 sheep were exposed at the same intervals to 100 ml saline (control). All sheep were investigated at 3-month intervals by chest radiograph, lung function, and lung lavage. At month 9, chest radiograph score of parenchymal opacities was significantly increased at 2.8 +/- 0.6 versus 0.4 +/- 0.4 in the Si group (p less than .05), establishing early radiologic silicosis. Lung function was significantly altered with reduction in lung compliance, vital capacity, and diffusion capacity (p less than .05). Lung lavage cellularity revealed significant increase in total cells (X 2.5), macrophages (X3), and neutrophils (X3). Albumin in BAL remained at the control level. Fibronectin production was significantly increased, as was the fibroblast growth activity, without significant change in procollagen 3 at this early stage of disease. Total phospholipids were significantly elevated in the Si-exposed sheep, and the profile demonstrated an increase in all the phospholipid components. Spontaneous release of hydrogen peroxide by alveolar cells was not increased, but in the presence of phorbol myristate acetate (PMA) higher levels of peroxide were found in the quartz-exposed sheep (p less than .05). The cellular and biochemical alterations of lung lavage preceded other changes. At month 12, there were good correlations (r greater than .49, p less than .001) between parameters evaluating related phenomena but poor correlations between measurements evaluating different aspects of the disorder. To investigate the heterogeneity in the individual response of sheep to the same exposure (susceptibility), individual quartz retention levels at month 12 were measured and found to correlate well with individual parameters of disease activity. We concluded that in early silicosis of sheep, cellular and biochemical changes in lung lavage preceded derangements of pulmonary function and radiographic abnormalities. Thereafter, parameters of lung lavage, lung function, and radiograph were significantly interrelated, but for a given exposure the degree of quartz retention appeared to determine the intensity of the silicotic process.

Granulocyte elastase-mediated proteolysis of alpha 1-antitrypsin in cystic fibrosis bronchopulmonary secretions.

Airway secretions of patients with cystic fibrosis (CF) contain large amounts of alpha 1-antitrypsin (alpha 1-AT), yet elastase activity is also often detectable, suggesting that airway alpha 1-AT may not be functional in some CF patients. It is unknown whether in CF sputum alpha 1-AT is inactivated by oxidants, neutrophil metalloproteinases, bacterial elastase, or neutrophil elastase. To investigate the mechanism(s) by which alpha 1-AT may be inactivated in CF airway secretions, sputum samples were obtained from nine patients during respiratory physiotherapy. alpha 1-AT was measured by radial immunodiffusion. Sputum-alpha 1-AT was purified by antibody affinity chromatography. Electrophoresis of alpha 1-AT from seven patients with acute infectious exacerbations revealed two distinct components: a minor band corresponding to an elastase/alpha 1-AT complex and a major band typical of proteolysed alpha 1-AT (Mr = 48 kD). Each patient had large amounts of sputum elastase activity. In contrast, two patients without free sputum elastase activity had intact sputum alpha 1-AT; however, alpha 1-AT was partially truncated by porcine pancreatic elastase suggesting that the alpha 1-AT may have been partially oxidized. Adding alpha 1-AT purified from normal serum to alpha 1-AT-depleted sputum containing elastase activity resulted in a small alpha 1-AT/elastase complex with most alpha 1-AT being truncated. The serine proteinase inhibitor phenylmethylsulfonyl fluoride but not the metalloproteinase inhibitor EDTA prevented alpha 1-AT proteolysis, thus granulocyte elastase can mediate alpha 1-AT degradation in CF. Apparently, the large granulocyte elastase burden in some acutely ill patients with cystic fibrosis can proteolytically inactivate alpha 1-AT.

Aluminum inhalation reduces silicosis in a sheep model.

In recent studies, we have documented that the biologic activity of quartz can be substantially reduced by surface chemistry modification with aluminum lactate treatment of the particles. In the present study, we evaluated the efficacy of aluminum lactate inhalation to reduce the biologic activity of experimental silicosis in the sheep tracheal lobe model. Four groups of 10 sheep were exposed once to either 100 ml phosphate-buffered saline (PBS) followed by aerosol inhalation of 10 ml PBS at monthly intervals (PBS-PBS group), to 100 ml PBS followed by inhalation of 100 mg aluminum lactate in 10 ml PBS (PBS-Al group), to 100 mg of quartz in 100 ml PBS followed by inhalation of 10 ml PBS (Si-PBS group), or to 100 mg of quartz in 100 ml PBS followed by inhalation of 100 mg aluminum lactate in 10 ml PBS (Si-Al group). Bronchoalveolar lavage (BAL) was repeated at monthly intervals for 6 months from before exposure (Month 0), and all sheep were autopsied at Month 6. All aerosol inhalations were carried out 24 h after BAL starting at Month 1 and monthly thereafter. In the PBS-PBS group and PBS-Al group, all BAL analyses remained at control levels and lung histology remained normal. In the Si-PBS group, BAL analyses documented significant sustained 3- to 10-fold increases in macrophages, lymphocytes, neutrophils, immunoglobulins, lactate dehydrogenase, glycosaminoglycans, lecithin, and phosphatidylglycerol, with histopathologic changes of nodular silicosis (pathologic score, 2.9 +/- 0.9) and mean retention of quartz at 2.83 +/- 0.98 micrograms/mg lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-reactive monoclonal antibodies to alcohol dehydrogenases.

Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.

Monoclonal antibodies against metallothioneins and metalloproteins.

Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

Post-myringotomy care: a prospective study.

Children with tympanostomy tubes have always been considered somewhat handicapped in regard to swimming and bathing. Their parents had to maintain constant surveillance to prevent then from getting water in their ears. A prospective study involving more than 1,000 children was conducted between June 1981 and August 1982 on two groups of randomly selected patients to determine the prevalence of suppurative otitis media and its relationship to bathing and swimming. One group had to follow strict rules to prevent water entering the ear (bathing caps, earplugs) whereas the other group was allowed to bathe and swim without any precaution upon the condition of using a polymyxin B/gramicidin ear drop combination at bedtime on the day they swam. The study shows no increase in prevalence of suppurative otitis media in the "open canal" group as compared to the "closed canal" group. Furthermore, the monthly distribution of infections shows a relatively evan distribution throughout the year. This study implies that swimming and bathing are safe for the vast majority of children with tympanostomy tubes and thus simplifies enormously the post-myringotomy care for the child, the parents, and the physician.

Asbestos-related fibrin formation in human plasma.

This study was designed to test the hypothesis that asbestos is responsible for the activation of clotting, for the reduction in activity of clotting factors and for the formation of fibrin when human plasma is exposed to asbestos chrysotile fibers in vitro. The recalcification time was accelerated in the early phase and was found to be greatly prolonged at 24 hours. The activated partial thromboplastin time showed marked changes at 24 hours only. One group of clotting factors, consisting of factors V, IX and X, showed the greatest decrease in their plasmatic activity. The factors least changed were factors XII and VIII; the other clotting factors were found in between these groups. Histologic examination demonstrated fibrin fibers in close proximity to the asbestos. Thus, chrysotile asbestos fibers activate clotting with the subsequent decrease in activity of some coagulation factors resulting in the formation of fibrin.

Gel chromatography of heparin.

It is assumed that heparin is a heterogeneous substance. In order to further investigate the purification of heparin, a column chromatographic technique for the fractionation of heparin is described using various diameters of bead form cross-linked dextran gels and an automated apparatus. It was observed that Sephadex G-50 resulted in the separation of three well formed peaks and provided superior resolution compared to all other gels. One of the peaks, representing 51% of the original material, possessed strong anticoagulant activity as measured by the recalcification time, partial thromboplastin time, thrombin time and the anti-Xa test. This peak also possessed strong metachromasia after electrophoresis as well as having a very potent anticoagulant effect in vivo. This technique may have a significant role in the purification of this agent from tissue sources.

Heparin excretion in intact and hepatectomized rats.

Metabolism and kinetics of 3H-heparin were compared in intact and hepatectomized rats. Rats were divided into three groups: 1) intact rats with biliary fistulas and cystostomies 2) intact rats with only cystostomies and 3) hepatectomized rats with cystostomies. Radioactivity in blood, bile and urine besides anticoagulant activity in blood and urine were examined. In addition, column chromatography of urine was used to isolate possible metabolites. Seventy percent and 80% of the radioactive dose was found in the urine of intact rats at 24 hr and 48 hr. Close to 5% of the radioactivity was found in bile or rats with a biliary fistula after 48 hr. The APTT declined to near normal values at 1 hr whether rats had a biliary fistula or not. In contrast, only 25% of the radioactivity could be exerted into the urine of hepatectomized rats in 24 hr; the APTT did not decline as fast and at 5 hr, it was still 100 seconds. Only one radioactive component could be isolated on chromatography from all urines of these animals and appears to be similar to the original heparin. Thus, the liver has no important role to play in regulating the anticoagulant effects and excretion of heparin.

Levels of antithrombin III, alpha 2-macroglobulin, and alpha 1-antitrypsin in acute ischemic heart disease.

Since measurements of AT III and other possible coagulation inhibitors might provide an index of hypercoagulability, the goal was to measure over a 3-month period individual changes in blood levels of AT III, alpha 2-microglobulin, and alpha 1-antitrypsin in 51 patients with acute ischemic heart disease who were admitted to a Coronary Care Unit with the following diagnosis: unstable angina (26 patients), acute transmural myocardial infarction (20 patients), or subendocardial myocardial infarction (5 patients). Some patients received prophylactic antithrombotic therapy. AT III was measured by the von Kaulla, Owen, Thrombo-Screen, chromogenic, immunodiffusion, and immunoelectrophoretic methods. Alpha 2-macroglobulin and alpha 1-antitrypsin were measured by immunodiffusion. All inhibitors were measured on three different occasions: (1) on admission to hospital, (1) day of departure from hospital, and (3) at 3 months after hospitalization. Alpha 1-antitrypsin showed a significant increase compared to the control and remained elevated during the 3-month interval. Compared to normal control values, AT III was found to be significantly diminished when measured by one functional method (von Kaulla) in all three blood samples from patients with unstable angina and transmural myocardial infarction and by one immunological method (immunodiffusion) in patients with unstable angina, transmural myocardial infarction, and subendocardial myocardial infarction. Most methods determining functional AT III detected a significant increase 3 months after the acute ischemic episode; this rise was observed more often in patients with myocardial infarction than in those with unstable angina. Stepwise discriminant analysis separated the patients of the three groups. Subcutaneous heparin has no significant effect on AT III levels. Unexplained discrepancies still exist between the results obtained by various functional and immunological methods for determining AT III. It appears, however, that methods measuring functional AT III seem to be more suited than immunological methods to detect changes in AT III levels that might occur during and after an acute episode of ischemic heart disease.