Gardnerella vaginalis Subgroups Defined by cpn60 Sequencing and Sialidase Activity in Isolates from Canada,Belgium and Kenya

Increased abundance of Gardnerella vaginalis and sialidase activity in vaginal fluid is associated with bacterial vaginosis (BV), a common but poorly understood clinical entity associated with poor reproductive health outcomes. Since most women are colonized with G. vaginalis, its status as a normal member of the vaginal microbiota or pathogen causing BV remains controversial, and numerous classification schemes have been described. Since 2005, sequencing of the chaperonin-60 universal target (cpn60 UT) has distinguished four subgroups in isolate collections, clone libraries and deep sequencing datasets. To clarify potential clinical and diagnostic significance of cpn60 subgroups, we undertook phenotypic and molecular characterization of 112 G. vaginalis isolates from three continents. A total of 36 subgroup A, 33 B, 35 C and 8 D isolates were identified through phylogenetic analysis of cpn60 sequences as corresponding to four “clades” identified in a recently published study, based on sequencing 473 genes across 17 isolates. cpn60 subgroups were compared with other previously described molecular methods for classification of Gardnerella subgroups, including amplified ribosomal DNA restriction analysis (ARDRA) and real-time PCR assays designed to quantify subgroups in vaginal samples. Although two ARDRA patterns were observed in isolates, each was observed in three cpn60 subgroups (A/B/D and B/C/D). Real-time PCR assays corroborated cpn60 subgroups overall, but 13 isolates from subgroups A, B and D were negative in all assays. A putative sialidase gene was detected in all subgroup B, C and D isolates, but only in a single subgroup A isolate. In contrast, sialidase activity was observed in all subgroup B isolates, 3 (9%) subgroup C isolates and no subgroup A or D isolates. These observations suggest distinct roles for G. vaginalis subgroups in BV pathogenesis. We conclude that cpn60 UT sequencing is a robust approach for defining G. vaginalis subgroups within the vaginal microbiome.     

Surface deformation tracking and modelling of soft materials

Biomechanics and Modeling in Mechanobiology - Many computer vision algorithms have been presented to track surface deformations, but few have provided a direct comparison of measurements with other...     

Chemical Characterization of Gallstones: An Approach to Explore the Aetiopathogenesis of Gallstone Disease in Sri Lanka

Introduction

Records on gallstones and associated ailments in Sri Lankan community are scarce, despite frequent detection of gallstone disease. Identification of the chemical composition of gallstones in the local setting is important in defining aetiopathogenic factors which in turn are useful in implementing therapeutic and preventive strategies. This study aimed to describe the chemical composition of gallstones and the socio-demographic factors of a cohort of Sri Lankan patients with gallstone disease.

Materials and Methods

Data on clinical and socio-demographic factors, and gallstones removed at surgery were collected from patients with cholelithiasis admitted to Teaching Hospital, Peradeniya, Sri Lanka from May 2011 to December 2012. External and cross sectional morphological features of gallstones were recorded by naked eye observation. Compositional analysis was carried out by Fourier Transform Infrared Spectroscopy, X - ray Powder Diffraction, and Atomic Absorption Spectrophotometry. Scanning Electron Microscopy was used to identify the microstructure of gallstones.

Results

Data of 102 patients were analyzed. Of them majority (n = 77, 76%) were females with a female: male ratio of 3:1. Mean age of the study group was 46.1±11.6 years. All the patients had primary gallbladder stones. According to the physical and chemical analysis, majority (n = 54, 53%) were pigment gallstones followed by mixed cholesterol gallstones (n = 38, 37%). Only 10 (9%) had pure cholesterol gallstones. Calcium bilirubinate, calcium carbonate and calcium phosphate were the commonest calcium salts identified in pigment gallstones and core of mixed cholesterol gallstones.

Conclusion

Presence of a pigment nidus in gallstones is a common feature in majority of Sri Lankan patients denoting the possible role of elevated unconjugated bilirubin in bile on the pathogenesis of GS. Hence it is imperative to explore this further to understand the aetiopathogenesis of GS among Sri Lankans.     

Separation of taxanes from <Emphasis Type="Italic">Taxus canadensis</Emphasis> using dynamic pressurized liquid extraction

An extraction technique, dynamic pressurized liquid extraction (DPLE), was proposed to extract the taxanes; including 10-DAB III, Baccatin III, 9-DHB III and paclitaxel, from powdered Taxus canadensis needles. A dual-solvent approach was adopted in which the impurities were firstly removed by extraction with hexane, and the taxanes were subsequently extracted with an appropriate solvent. The performance of chloroform, dichloromethane, and mixtures of methanol/dichloromethane was compared for use as the taxane-extracting solvent, and it was found that solvents containing a higher proportion of methanol had higher extraction capabilities. The effect of temperature on DPLE extraction of the taxanes was also studied, and it was found that higher extraction efficiencies could be realized with increasing temperature up to a threshold of 90°C. Based on a progressive conversion model, a kinetic equation for the extraction process was proposed. This model successfully confirmed that smaller needle powder particle sizes would result in higher extraction rates, which is consistent with the data obtained by experimentation.     

Comprehensive molecular phylogeny of the sub-family Dipterocarpoideae (Dipterocarpaceae) based on chloroplast DNA sequences

Dipterocarpoideae, the largest sub-family of well-known plant family Dipterocarpaceae, dominates in South Asian rain forests. Although several previous studies addressed the phylogeny of the Dipterocarpaceae family, relationships among many of its genera from the Dipterocarpoideae sub-family are still not well understood. In particular, little is known about the relationships of the genera Vateriopsis, Stemonoporus, Vateria and inconsistence remains between phylogenetic results and taxonomic classifications of Shorea and Hopea species. We studied molecular phylogeny of the sub-family Dipterocarpoideae using the trnL-trnF spacer, trnL intron and the matK gene sequences of chloroplast DNA (cpDNA). This study is the first comprehensive phylogeny reconstruction for the sub-family Dipterocarpoideae based on cpDNA, as it includes most genera (14) and a large number of species (79) with most species endemic to Sri Lanka, as well as one species from Seychelles and one species from the genus Monotes from Madagascar. Phylogenetic trees were constructed using the Neighbor Joining (NJ) and Maximum Likelihood (ML) methods using combined set of sequences including all three cpDNA regions. The topologies of the NJ and ML trees were to a certain extent, consistent with the current taxonomy of Dipterocarpoideae based on morphology and with previous molecular phylogenies based on cpDNA. Furthermore, our results provided new evidence regarding the relationships of the following genera: Vateriopsis and Stemonoporus and about the validity of the previous morphology based classifications of Shorea species. In addition, the topology of our trees was consistent with the classification of Shorea species proposed by Maury (1978), Maury-Lechon (1979) and Symington (1943). Finally, our results provided evidence for the affinity of the genus Monotes to Asian Dipterocarpoideae rather than to Tiliaceae and indicated that it is a good candidate for outgroup species for future studies of the former sub-family.     

Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.     

Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E. coli

S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s).     

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species'' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.     

Combination of solvent and radiation effects on degradation of aflatoxin B1

Summary Degradation of aflatoxin B₁ in chloroform, ethyl ccetate and coconut oil were examined by exposing the solvents in the form of thin layers to radiation from the sun and from UV and fluorescent tubes. Solar degradation of aflatoxin B₁ occurred in coconut oil leaving no residual aflatoxin B₁ or any new fluorescent derivatives. Other combinations of solvents and radiation produced up to four new fluorescent degradation compounds. Aflatoxin B₁ did not undergo solar degradation in the absence of moisture. Acidity enhanced solar degradation. A fluorescent derivative produced by solar degradation of aflatoxin B₁ in chloroform degraded further on solar irradiation in coconut oil but not in chloroform.

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Resumen Se examinó la degradación de la aflatoxina B1 en cloroformo, acetato de etilo y aceite de coco, exponiendo los disolventes en forma de capa fina a la radiación solar y a radiaciones procedentes de lámparas UV y fluorescentes. La degradación solar de la aflatoxina B1 tuvo lugar en aceite de coco sin dejar residuos de aflatoxina ni de ningún nuevo derivado fluorescente. Otras combinaciones de disolvente y radiaciones produjeron hasta 4 nuevos derivados fluorescentes. La degradación solar de la aflatoxina B1 no se produjo en ausencia de humedad. La acidez potenció la degradación solar. Uno de los derivados fluorescentes obtenidos en la degradación solar de la aflatoxina B1 en cloroformo continuó degradándose bajo la radiación solar en aceite de coco pero no en cloroformo.

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Résumé La dégradation de l'aflatoxine B1 dans le chloroforme, l'acétate d'éthyle et l'huile de noix de coco a été examinée en exposant les solvents sous la forme de films minces à l'irradiation du soleil, des uv et de tubes fluorescents. La dégradation solaire de l'aflatoxine B1 s'est produite dans l'huile de noix de coco en ne laissant ni aflatoxine B1 résiduelle ni nouveaux dérivés fluorescents. D'autres combinaisons de solvents et d'irradiation ont produit jusqu'à 4 nouveaux dérivés fluorescents de dégradation. L'aflatoxine B1 ne subit pas de dégradation solaire en absence d'humidité. L'acidité augmente la dégradation solaire. Un dérivé fluorescent produit par dégradation solaire de l'aflatoxine B1 dans le chloroforme, s'est dégradé davantage sous l'irradiation solaire dans l'huile de noix de coco mais non dans le chloroforme.

     

A pilot plant for detoxification of aflatoxin B1-contaminated coconut oil by solar irradiation

Summary In coconut oil naturally contaminated with aflatoxin B1, more than 85% of the toxin is present in the soluble form, the remainder occuring in the sediment. This aflatoxin is detoxified when the oil, in a static layer less than 15 mm thick is exposed to solar radiation. A pilot plant, designed to take account of the viscosity and flow characteristics of the oil, was constructed for the exposure of thin layers of oil (2 mm or less) flowing under gravity. At aflatoxin concentrations between 166 and 1250 jug/kg, 75% of the toxin was degraded on exposure to solar radiation of 10 cal/cm²; total detoxification was achieved on repeated exposure. The naturally contaminated coconut oil after exposure to solar radiation did not contain any residual aflatoxins or fluorescent compounds which might have been derived from original aflatoxin B1.

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Resumen Planta pilota para el tratramiento de aceite de coco contaminado con aflatoxina B1 mediante radiation solar y centrifugado En aceite de coco contaminado de forma natural con aflatoxina B1, más del 85% de la toxina se encuentra en forma soluble, el resto permaneciendo en el sedimento. Esta aflatoxina pierde sus propiedades tóxicas cuando se expone el aceite en forma de capa estática de 15 mm de grosor a la radiación solar. Se construyó una planta piloto diseñada teniendo en cuenta la viscosidad y las caracteristicas de fluidez del aceite de forma que pudieran exponerse a la radiación solar capas de aceite muy finas (2 mm o menos) fluyendo gracias a la gravedad. Cuando aceite conteniendo aflatoxina en proporciones entre 166 y 1250 /kg se expuso a una radiación solar de 10 cal./cm² la toxina se degradó en un 75%. La detoxificación total se obtuvo mediante repetición del proceso. El aceite de coco contaminado de forma natural, una vez expuesto a la radiación solar no contenía aflatoxinas residuales ni compuestos fluorescentes potencialmente derivados de la aflatoxina B1 contenida originalmente.

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Résumé Usine-pilote pour la détoxification, par irradiation solaire et centrifugation, de l'huile de coprah contaminée par l'aflatoxine B1 Dans l'huile de coprah spontanément contaminée par l'aflatoxine B1, plus de 85% de la toxine est présente sous forme soluble, le reste se trouvant dans le sédiment. Cette aflatoxine est détoxifiée lorsque l'huile est exposée à la radiation solaire en couche statique de moins de 15 mm d'épaisseur. Une usine-pilote, conçue en tenant compte de la viscosité et de l'écoulement de l'huile, a été construite pour irradier une mince couche d'huile (2 mm ou moins) s'écoulant par gravité. Pour des concentrations en aflatoxine allant de 166 à 1250 g/kg, 75% de la toxine est dégradée par exposition à une irradiation solaire de 10 cal./cm² et, en répétant le traitement, on obtient une détoxification complète. Après exposition à la radiation solaire, l'huile de coprah contaminée spontanément ne contient plus d'aflatoxine résiduelle, ni de composés fluorescents dérivés de l'aflatoxine B1 originelle.