An investigation of alkaline phosphatase enzymatic activity after electrospinning and electrospraying

The high target specificity and multifunctionality of proteins has led to great interest in their clinical use. To this end, the development of delivery systems capable of preserving their bioactivity and improving bioavailability is pivotal to achieve high effectiveness and satisfactory therapeutic outcomes. Electrohydrodynamic (EHD) techniques, namely electrospinning and electrospraying, have been widely explored for protein encapsulation and delivery. In this work, monoaxial and coaxial electrospinning and electrospraying were used to encapsulate alkaline phosphatase (ALP) into poly(ethylene oxide) fibres and particles, respectively, and the effects of the processing techniques on the integrity and bioactivity of the enzyme were assessed. A full morphological and physicochemical characterisation of the blend and core-shell products was performed. ALP was successfully encapsulated within monolithic and core-shell electrospun fibres and electrosprayed particles, with drug loadings and encapsulation efficiencies of up to 21% and 99%, respectively. Monoaxial and coaxial electrospinning were equally effective in preserving ALP function, leading to no activity loss compared to fresh aqueous solutions of the enzyme. While the same result was observed for monoaxial electrospraying, coaxial electrospraying of ALP caused a 40% reduction in its bioactivity, which was attributed to the high voltage (22.5 kV) used during processing. This demonstrates that choosing between blend and coaxial EHD processing for protein encapsulation is not always straightforward, being highly dependent on the chosen therapeutic agent and the effects of the processing conditions on bioactivity

Effect of surface conditioning with cellular extracts on Escherichia coli adhesion and initial biofilm formation

Bacterial adhesion and subsequent biofilm formation start with surface conditioning by molecules originating from the surrounding medium and from cell lysis. Different cell extracts e.g. total cell extract (TCE), cytoplasm with cellular debris (CCDE) and periplasmic extract (PE) were tested in agitated 96-well microtiter plates and in a flow cell. Crystal violet assay demonstrated that a polystyrene substratum conditioned with TCE or CCDE decreased initial biofilm formation, however cell adhesion generally increased when PE was used. These results were dependent on conditioning film concentration. Using a parallel plate flow chamber, the use of optimal conditioning film concentrations resulted in all the different cellular extracts reducing biofilm formation. Multifractal analysis was used to generate quantitative data on the number of cell clusters. Surface conditioning with cellular components affected the amount and clustering of bacteria on polystyrene surfaces and their propensity to induce biofilm formation. To the best of our knowledge, this is the first study addressing the effect of cellular surface conditioning of cellular compartments on E. coli adhesion and initial biofilm formation. This work leads to a greater understanding of the factors that influence biofilm formation under flow conditions which are prevalent in food industry

Factors Influencing Performance of Cholangioscopy-Guided Lithotripsy Including Available Different Technologies: A Prospective Multicenter Study with 94 Patients

Background: Peroral cholangioscopy (POC)-guided lithotripsy is an effective treatment for difficult biliary stones. A clear definition of factors associated with the efficacy of POC-guided lithotripsy in one session and the performance of electrohydraulic lithotripsy (EHL) and laser lithotripsy (LL) have not clearly emerged. Methods: This was a non-randomized prospective multicenter study of all consecutive patients who underwent POC lithotripsy (using EHL and/or LL) for difficult biliary stones. The primary endpoint of the study was the number of sessions needed to achieve complete ductal clearance and the factors associated with this outcome. Secondary endpoints included the evaluated efficacies of LL and EHL. Results: Ninety-four patients underwent 113 procedures of EHL or LL. Complete ductal clearance was obtained in 93/94 patients (98.94%). In total, 80/94 patients (85.11%) achieved stone clearance in a single session. In the multivariate analysis, stone size was independently associated with the need for multiple sessions to achieve complete ductal clearance (odds ratio = 1.146, 95% confidence interval: 1.055-1.244; p = 0.001). Using ROC curves and the Youden index, 22 mm was found to be the optimal cutoff for stone size (95% confidence interval: 15.71-28.28; p < 0.001). The majority of the patients (62.8%) underwent LL in the first session. Six patients failed the first session with EHL after using two probes and therefore were crossed over to LL, obtaining ductal clearance in a single additional session with a single LL fiber. EHL was significantly associated with a larger number of probes (2.0 vs. 1.02) to achieve ductal clearance (p < 0.01). The mean procedural time was significantly longer for EHL than for LL [72.1 (SD 16.3 min) versus 51.1 (SD 10.5 min)] (p < 0.01). Conclusions: POC is highly effective for difficult biliary stones. Most patients achieved complete ductal clearance in one session, which was significantly more likely for stones < 22 mm. EHL was significantly associated with the need for more probes and a longer procedural time to achieve ductal clearance.info:eu-repo/semantics/publishedVersio

Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system

We present a setup of optical tweezers combined with linear and nonlinear microspectroscopies that enhances the capabilities of capture and analysis of both techniques. We can use either a continuous-wave (cw) Ti:sapphire laser for Raman measurements or a pulsed femtosecond Ti:sapphire laser that permitted the observation of nonlinear results such as hyper-Raman, hyper-Rayleigh, and two-photon luminescence. Only the high peak intensity of the femtosecond laser allows the observation of all these nonlinear spectroscopies. The sensitivity of our system also permitted the observation of morphology-dependent resonance (MDR) modes of a single stained trapped microsphere of 6 mu m. The possibility of performing spectroscopy in a living microorganism optically trapped in any desired neighborhood would mean that one can dynamically observe the chemical reactions and/or mechanical properties changing in real time.721

Diversity of lactic acid bacteria of the bioethanol process

<p>Abstract</p> <p>Background</p> <p>Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil.</p> <p>Results</p> <p>A total of 489 LAB isolates were obtained from four distilleries in 2007 and 2008. The abundance of LAB in the fermentation tanks varied between 6.0 × 10⁵and 8.9 × 10⁸CFUs/mL. Crude sugar cane juice contained 7.4 × 10⁷to 6.0 × 10⁸LAB CFUs. Most of the LAB isolates belonged to the genus <it>Lactobacillus </it>according to rRNA operon enzyme restriction profiles. A variety of <it>Lactobacillus </it>species occurred throughout the bioethanol process, but the most frequently found species towards the end of the harvest season were <it>L. fermentum </it>and <it>L. vini</it>. The different rep-PCR patterns indicate the co-occurrence of distinct populations of the species <it>L. fermentum </it>and <it>L. vini</it>, suggesting a great intraspecific diversity. Representative isolates of both species had the ability to grow in medium containing up to 10% ethanol, suggesting selection of ethanol tolerant bacteria throughout the process.</p> <p>Conclusions</p> <p>This study served as a first survey of the LAB diversity in the bioethanol process in Brazil. The abundance and diversity of LAB suggest that they have a significant impact in the bioethanol process.</p

Phenoloxidase activity acts as a mosquito innate immune response against infection with semliki forest virus

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses