Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system.

The alpha-Gal trisaccharide Gal(alpha)(1-->3)Galbeta(1-->4)GlcNAc 11 was synthesized on a homogeneously soluble polymeric support (polyethylene glycol, PEG) by use of a multi-enzyme system consisting of beta-1,4-galactosyltransferase (EC 2.4.1.38), alpha-1,3-galactosyltransferase (EC 2.4.1.151), sucrose synthase (EC 2.4.1.13) and UDP-glucose-4-epimerase (EC 5.1.3.2). In addition workup was simplified by use of dia-ultrafiltration. Thus the advantages of classic chemistry/enzymology and solid-phase synthesis could be united in one. Subsequent hydrogenolytic cleavage afforded the free alpha-Gal trisaccharide.     

Performance and efficiency of a yeast biofilter for the treatment of a Nigerian fertilizer plant effluent

A biofilter composed of yeasts and cassava peel was used to detoxify fertilizer plant effluent. The biological oxygen demand was reduced on treatment from a range of 1200–1400 mg/l to a range 135–404 mg/l. The ammonia-nitrogen (NH₃–N) and nitrate-nitrogen (NO₃–N) were reduced after treatment from 1000 to 10 mg/l and from 100 to 17.6 mg/l, respectively. The biofilter is simple and easy to handle with high efficiency of 98%.     

Dynamics of concentrations and nutrient fluxes in the xylem of Ricinus communis– diurnal course, impact of nutrient availability and nutrient uptake

Diurnal courses of nutrient transport in the xylem and their response to external availability of nutrients were studied. In soil culture, maximal concentrations in all analysed substances were observed during night‐time. Over experimental periods of up to 20 d, concentrations of some ions increased, most by accumulation in the soil. Stringent nutrient conditions were established in a novel pressure chamber. An aeroponic nutrient delivery system inside allows the sampling of xylem sap from intact plants under full control of the nutrient conditions at the root. Analysis of xylem transport under these highly defined conditions established that (1) diurnal variations in concentrations and fluxes in the xylem are dominated by plant‐internal processes; (2) concentrations of nutrients in the xylem sap are highly but specifically correlated with each other; (3) nitrate uptake and nitrate flux to the shoot are largely uncoupled; and (4) in continuous light, diurnal variations of xylem sap concentrations vanish. Step changes in nitrate concentrations of the nutrient solution established that (5) the concomitant increase in nitrate concentration and flux in the xylem is delayed by 2–3 h and is only transient. Diurnal variations of xylem sap composition and use of the new technique to elucidate xylem‐transport mechanisms are discussed.     

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

The study was performed to compare real-time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR-based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real-time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.     

Accurate method for rapid biomass quantification based on specific absorbance of microalgae species with biofuel importance

The development of microalgae culture technology has been an integral part to produce biomass feedstock to biofuel production. Due to this, numerous attempts have been made to improve some operational parameters of microalgae production. Despite this, specialized research in cell growth monitoring, considered as a fundamental parameter to achieve profitable applications of microalgae for biofuels production, presents some opportunity areas mainly related to the development of specific and accurate methodologies for growth monitoring. In this work, predictive models were developed through statistical tools that correlate a specific micro-algal absorbance with cell density measured by cell count (cells∙per ml), for three species of interest for biofuels production. The results allow the precise prediction of cell density through a logistic model based on spectrophotometry, valid for all the kinetics analysed. The adjusted determination coefficients () for the developed models were 0·993, 0·995 and 0·994 for Dunaliella tertiolecta, Nannochloropsis oculata and Chaetoceros muelleri respectively. The results showed that the equations obtained here can be used with an extremely low error (≤2%) for all the cell growth ranges analysed, with low operational cost and high potential of automation. Finally, a user-friendly software was designed to give practical use to the developed predictive models.     

A Novel Method for Rapid Hybridization of DNA to a Solid Support

Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself.     

Morphological Parameters Associated with Ruptured Posterior Communicating Aneurysms

The rupture risk of unruptured intracranial aneurysms is known to be dependent on the size of the aneurysm. However, the association of morphological characteristics with ruptured aneurysms has not been established in a systematic and location specific manner for the most common aneurysm locations. We evaluated posterior communicating artery (PCoA) aneurysms for morphological parameters associated with aneurysm rupture in that location. CT angiograms were evaluated to generate 3-D models of the aneurysms and surrounding vasculature. Univariate and multivariate analyses were performed to evaluate morphological parameters including aneurysm volume, aspect ratio, size ratio, distance to ICA bifurcation, aneurysm angle, vessel angles, flow angles, and vessel-to-vessel angles. From 2005–2012, 148 PCoA aneurysms were treated in a single institution. Preoperative CTAs from 63 patients (40 ruptured, 23 unruptured) were available and analyzed. Multivariate logistic regression revealed that smaller volume (p = 0.011), larger aneurysm neck diameter (0.048), and shorter ICA bifurcation to aneurysm distance (p = 0.005) were the most strongly associated with aneurysm rupture after adjusting for all other clinical and morphological variables. Multivariate subgroup analysis for patients with visualized PCoA demonstrated that larger neck diameter (p = 0.018) and shorter ICA bifurcation to aneurysm distance (p = 0.011) were significantly associated with rupture. Intracerebral hemorrhage was associated with smaller volume, larger maximum height, and smaller aneurysm angle, in addition to lateral projection, male sex, and lack of hypertension. We found that shorter ICA bifurcation to aneurysm distance is significantly associated with PCoA aneurysm rupture. This is a new physically intuitive parameter that can be measured easily and therefore be readily applied in clinical practice to aid in the evaluation of patients with PCoA aneurysms.     

False negative cytologic diagnosis of breast carcinoma.

OBJECTIVE: To study the reasons for interpretive errors in false negative diagnosis of breast carcinoma on fine needle aspiration cytology material. STUDY DESIGN: We reviewed only those histologically proved malignant cases where the cytologic material was abnormal and to some extent misinterpreted. RESULTS: There were four lobular carcinomas and one each case of in situ, infiltrating duct, medullary and tubular carcinoma. Smears of lobular carcinomas were hypocellular overall, and the cells showed minimal nuclear pleomorphism. In situ, medullary and tubular carcinoma were associated with fibrocystic changes. The presence of bipolar cells and stromal fragments was misleading in cases of infiltrating duct carcinoma. CONCLUSION: The presence of associated fibrocystic disease may be a misleading factor since it may mask a malignancy. Hypocellularity and relatively nuclear monomorphism were the most common reasons for failure to diagnose malignant breast lesions. Careful attention should be paid to extreme nuclear monomorphism and absence of naked bipolar cells. A cytologically atypical or suspicious diagnosis together with radiologic suspicion should suggest a diagnosis of malignancy.     

Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

Type I allergies are immunological disorders that afflict a quarter of the world's population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 microg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.