In vitro fertilisation with frozen-thawed bovine sperm sexed by flow cytometry and validated for accuracy by real-time PCR

The methodologies used for cytometric sorting of fresh spermatozoa never allowed a clear resolution of sexual chromosomes of frozen-thawed semen. To devise a novel method for the production of bovine predefined sexed embryos using frozen-thawed semen, sorting efficiency of different protocols was studied using a new quantitative real-time PCR method to verify the purity of sexed semen. To this aim, after Percoll separation, frozen-thawed samples were stained at different temperatures and concentrations of Hoechst 33342 using a short-incubation time. The concentration of Hoechst 33342 of 500 mug/ml at a temperature of 37 degrees C provided good and stable fluorescence signals. Preventing the sperm clustering by adding 0.6% BSA in the 90% Percoll fraction led to X-bearing sperms purity of 91+/-2%. Thereafter, sorted sperms were used for in vitro fertilisation (IVF). Despite the lower cleavage rates reported in the sorted groups when compared with the control groups (40 vs 48%, P<0.01), blastocyst formation in the sorted and control groups was not different (27 vs 24% of the cleaved respectively). The PCR analysis of 30 blastocysts confirmed 26 embryos to be correctly sexed (87%). Transfer of two embryos per recipient into six synchronised heifers resulted in four pregnancies. Two abortions occurred at day 60, while two pregnancies went to term delivering two female calves. In conclusion, high purity and repeatability of sorting was obtained with frozen-thawed bull semen that was subsequently used for IVF giving rise to viable embryos and offspring. In addition, real-time PCR revealed to be an optimal support for these studies, providing a rapid and reliable estimation of flow cytometric efficiency.     

Combination of X-ray and digital image correlation for the analysis of moisture-induced strain in wood: opportunities and challenges

In this present study, the moisture-induced deformation behaviour of a spruce sample was analysed one- and two-dimensionally with high resolution on the radial-tangential surface. For this purpose, an artificial speckle pattern was applied to the surface which was then recorded by a CCD camera during the deformation. The generated TIF images were analysed with a strain mapping software (VIC 2D) that computed the two-dimensional strain field from the surface deformation. Selected options to evaluate two-dimensional data generated with X-ray imaging and digital image correlation are presented. Combining and correlating these techniques enables detailed analysis of structure-function relationships during swelling (and shrinkage) processes in wood. However, several issues still have to be solved to enhance effectiveness and user-friendliness of such investigations, as elucidated in detail in this paper.     

Larks: Dynamic Matchmaking Among Heterogeneous Software Agents in Cyberspace

Service matchmaking among heterogeneous software agents in the Internet is usually done dynamically and must be efficient. There is an obvious trade-off between the quality and efficiency of matchmaking on the Internet. We define a language called Larks for agent advertisements and requests, and present a flexible and efficient matchmaking process that uses Larks. The Larks matchmaking process performs both syntactic and semantic matching, and in addition allows the specification of concepts (local ontologies) via ITL, a concept language. The matching process uses five different filters: context matching, profile comparison, similarity matching, signature matching and constraint matching. Different degrees of partial matching can result from utilizing different combinations of these filters. We briefly report on our implementation of Larks and the matchmaking process in Java. Fielded applications of matchmaking using Larks in several application domains for systems of information agents are ongoing efforts.     

A Markov chain model to describe fluidization of particles with time-varying properties

A one-dimensional mathematical model of particulate solids thermal treatment in a fluidized bed based on the theory of Markov chains is proposed. A feature of the model takes into account the variation of particle properties that takes place during the fluidization. This variation occurs due to thermo-physical and chemical processes that take place during particulate solids treatment in the fluidized bed. These variations affect the particle settling velocity which is the main parameter that forms the bed expansion and local velocity of flow around the particles that strongly influences the thermo-physical and chemical processes in the bed. A generalized correlation for the drag force coefficient is proposed and tested for various materials. It was necessary to have more precise calculation of the particle settling velocity in the model. The model was developed to describe the drying process in the bed. A good correlation between computed and experimental data was achieved.     

Adding security and trust to multiagent systems

Multiagent systems (MASs) are societies whose individuals are software delegatees (agents) acting on behalf of their owners or delegators (people or organizations). When deployed in an open network such as the Internet, MASs face some trust and security issues. Agents comeand go, and interact with strangers. Assumptions about security and general trustworthiness of agents and their deployers are inadequate in this context. In this paper, the design of a security infrastructure is presented applicable to MASs in general. This design addresses both security threats and trust issues. In this design, there are mechanisms for ensuring secure communication among agents and secure naming and resource location services. And two types of trusts are addressed: trust that agents will not misbehave and trust that agents are really delegatees of whom they claim to be. To establish the first type of trust, deployers of agents are made liable for the actions of their agents; to establish the second type of trust, it is proposed that agents prove that they know secrets that only their delegators know.     

Optimization of a Fabric Softener Formulation with an Electrochemical Sensor and Streaming Potential Measurements

An electrochemical, cationic, surfactant-selective sensor based on multiwalled carbon nanotubes (MWCNT) functionalized with a sulfate group and the cetylpyridinium ion (MWCNT–OSO₃⁻CP⁺) as a sensing material was used for optimization of the formulation of a fabric softener. Potentiometric titrations were performed and response measurements were obtained using four cationic surfactants (CS) of technical grade and four CS of analytical grade. The slope closest to Nernstian was obtained for di-(tallow carboxyethyl) hydroxyethyl methylammonium methosulfate (MAS) (59.5 ± 1.1 mV/decade of activity in water and 57.5 ± 1.3 mV/decade of activity in CaCl₂). When using CS as analytes in potentiometric titrations, the best accuracy (99.8%) was obtained when using MAS; therefore, it was chosen as the CS for the fabric softener formulation. Due to the better properties of fabric softeners with silicone in their formulations, four silicones at several concentration levels were used as potential additives. Based on the stability and viscosity of the system, the diquaternary polydimethylsiloxane (DPS) (w = 0.19%) was chosen for the fabric softener formulation. The pH did not significantly influence the potential when in the range of 3–8 or the recovery of potentiometric titrations when in the range of 3–7. The application profile of the CS was assessed through streaming potential measurements of reference fabrics in an electrokinetic analyzer. The obtained electrokinetic parameters indicated on lag in adsorption of model fabric softener (MFS) based on MAS (w = 9%), with the addition of silicone DPS (MFS 3), on cotton and polyester fabrics, but advantage in stability when compared with other MFS investigated.     

Effect of the mold temperature on the electrical properties of carbon-black-loaded polystyrene/SB block copolymer blends

The electrical resistivities of a carbon-black-filled styrene–butadiene block copolymer (SB) and their blends with polystyrene were measured as a function of carbon content for specimens compression-molded at 200 and 250°C. The insulator–conductor point transition was greatly influenced by the mold temperature. This behavior associated to the scanning electronic microscopy investigations suggests the presence of some amount of the filler at the interface. A strong interaction between the filler and the polymer was also observed. The formation of bound rubber and a coherent rubber–filler gel depend on the molding conditions and the carbon black content in the composites. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 69: 825–833, 1998     

Poly(vinyl alcohol)‐Based Film Potentially Suitable for Antimicrobial Packaging Applications

This work aimed at developing a thin and water‐resistant food‐grade poly(vinyl alcohol) (PVOH)‐based matrix able to swell when in contact with high moisture content food products without rupturing to release antimicrobial agents onto the food surface. This film was prepared by blending PVOH and 7.20% (wt/wt of PVOH) of poly(ethylene glycol) (PEG) with citric acid as crosslinking agent. The film‐forming solution was then casted onto a flat surface and the obtained film was 60 μm in thickness and showed a good transparency (close to T = 100%) in the visible region (400 to 700 nm). After immersion in water for 72 h at room temperature, the crosslinked matrix loses only 19.2% of its original weight (the percentage includes the amount of unreacted crosslinking agent, antimicrobial in itself). Water content, degree of swelling, and crosslinking density of the film prove that the presence of PEG diminishes the hydrophilic behavior of the material. Also the mechanical properties of the wet and dry film were assessed. Alongside this, 2.5% (wt/wt of dry film) of grapefruit seed extract (GSE), an antimicrobial agent, was added to the film‐forming solution just before casting and the ability of the plastic matrix to release the additive was then evaluated in vitro against 2 GSE‐susceptible microorganisms, Salmonella enteritidis and Listeria innocua. The results indicate that the developed matrix may be a promising food‐grade material for the incorporation of active substances.     

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions Preserves the Chemical Characteristics and Biological Activity of Lyo-Secretome Isolated by Ultrafiltration

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.