Effects of the Temperature-Time Regime of Curing of Composite Patch on Repair Process Efficiency.

Repair procedures with the use of composite patches are considered to be the most effective among the current technologies of repair of the structures of various applications. In the process of moulding-on of a patch made of polymeric composite material by means of curing, technological stresses arise in the patch. Determination of residual technological stresses is a priority task for the modelling of the repair process. Reduction of residual stresses can be achieved by optimization of the mode of repair patch curing. For meeting this objective, the method for determination of technological stresses, which arise in the structure under repair in the process of curing of a composite patch, has been developed. The method takes into account the shrinkage, change in physico-mechanical characteristics, rheological processes occurring in the binder during moulding process, and determination of stresses in the structure under repair at any time. Therefore, premature failure of the repair joint at the stage of repair can be avoided. It is shown that the method adequately describes the level of deformations and stresses in the structure being repaired at the stage of heating and holding of the composite patch. Increase in the moulding temperature leads to a reduction in residual stresses in the structure under repair. However, current stresses at the stages of heating and temperature holding are increased significantly. Reliability of assumptions and developed method is confirmed by the comparison with the experimental data. The obtained experimental graph of total deformation of the composite patch allowed us to clearly determine the moment of residual stress occurrence in the structure under repair. This moment matches quite exactly (with the discrepancy not exceeding 5 min) the gel point determined analytically based on dependence of the degree of curing on the moulding mode. Consequently, the research together with the results previously obtained allows making an integrated choice of geometric parameters of the repair composite patch and temperature-time regime of its curing in order to ensure the specified level of strength and stiffness of the structure under repair.

Towards the First Principles in Biology and Cancer: New Vistas in Computational Systems Biology of Cancer.

These days many leading scientists argue for a new paradigm for cancer research and propose a complex systems-view of cancer supported by empirical evidence. As an example, Thea Newman (2021) has applied "the lessons learned from physical systems to a critique of reductionism in medical research, with an emphasis on cancer". It is the understanding of this author that the mesoscale constructs that combine the bottom-up as well as top-down approaches, are very close to the concept of emergence. The mesoscale constructs can be said to be those effective components through which the system allows itself to be understood. A short list of basic concepts related to life/biology fundamentals are first introduced to demonstrate a lack of emphasis on these matters in literature. It is imperative that physical and chemical approaches are introduced and incorporated in biology to make it more conceptually sound, quantitative, and based on the first principles. Non-equilibrium thermodynamics is the only tool currently available for making progress in this direction. A brief outline of systems biology, the discovery of emergent properties, and metabolic modeling are introduced in the second part. Then, different cancer initiation concepts are reviewed, followed by application of non-equilibrium thermodynamics in the metabolic and genomic analysis of initiation and development of cancer, stressing the endogenous network hypothesis (ENH). Finally, extension of the ENH is suggested to include a cancer niche (exogenous network hypothesis). It is expected that this will lead to a unifying systems-biology approach for a future combination of the analytical and synthetic arms of two major hypotheses of cancer models (SMT and TOFT).

From biotechnology principles to functional and low-cost metallic bionanocatalysts.

Chemical, physical and mechanical methods of nanomaterial preparation are still regarded as mainstream methods, and the scientific community continues to search for new ways of nanomaterial preparation. The major objective of this review is to highlight the advantages of using green chemistry and bionanotechnology in the preparation of functional low-cost catalysts. Bionanotechnology employs biological principles and processes connected with bio-phase participation in both design and development of nano-structures and nano-materials, and the biosynthesis of metallic nanoparticles is becoming even more popular due to; (i) economic and ecologic effectiveness, (ii) simple one-step nanoparticle formation, stabilisation and biomass support and (iii) the possibility of bio-waste valorisation. Although it is quite difficult to determine the precise mechanisms in particular biosynthesis and research is performed with some risk in all trial and error experiments, there is also the incentive of understanding the exact mechanisms involved. This enables further optimisation of bionanoparticle preparation and increases their application potential. Moreover, it is very important in bionanotechnological procedures to ensure repeatability of the methods related to the recognised reaction mechanisms. This review, therefore, summarises the current state of nanoparticle biosynthesis. It then demonstrates the application of biosynthesised metallic nanoparticles in heterogeneous catalysis by identifying the many examples where bionanocatalysts have been successfully applied in model reactions. These describe the degradation of organic dyes, the reduction of aromatic nitro compounds, dehalogenation of chlorinated aromatic compounds, reduction of Cr(VI) and the synthesis of important commercial chemicals. To ensure sustainability, it is important to focus on nanomaterials that are capable of maintaining the important green chemistry principles directly from design inception to ultimate application.

The impact of Ivan Málek's continuous culture concept on bioprocessing.

This paper summarizes research results and their industrial applications obtained by continuous culture in the former Czechoslovakia. Past achievements as well as recent trends and developments worldwide are presented. The term "Prague School of continuous culture" is put forward and its international activity is outlined. The impact of this school was pervasive across the entire field of applied microbiology and biotechnology in Czechoslovakia and, perhaps, even beyond the country's borders. Continuous culture is a very mature field, and since its establishment it has become a powerful research tool. The present activity in this field amounts to a renaissance of continuous culture, emphasizing new dimensions in bioinformatics and systems biology.

Nanovehicular intracellular delivery systems.

This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood-brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list "elementary" phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach.

Multifunctional nanoparticulate polyelectrolyte complexes.

Water-soluble, biodegradable, polymeric, polyelectrolyte complex dispersions (PECs) have evolved because of the limitations, in terms of toxicity, of the currently available systems. These aqueous nanoparticulate architectures offer a significant advantage for products that may be used as drug delivery systems in humans. PECs are created by mixing oppositely charged polyions. Their hydrodynamic diameter, surface charge, and polydispersity are highly dependent on concentration, ionic strength, pH, and molecular parameters of the polymers that are used. In particular, the complexation between polyelectrolytes with significantly different molecular weights leads to the formation of water-insoluble aggregates. Several PEC characteristics are favorable for cellular uptake and colloidal stability, including hydrodynamic diameter less than 200 nm, surface charge of >30 mV or <-30 mV, spherical morphology, and polydispersity index (PDI) indicative of a homogeneous distribution. Maintenance of these properties is critical for a successful delivery vehicle. This review focuses on the development and potential applications of PECs as multi-functional, site-specific nanoparticulate drug/gene delivery and imaging devices.

Kinetic analysis of nanoparticulate polyelectrolyte complex interactions with endothelial cells.

A non-toxic, nanoparticulate polyelectrolyte complex (PEC) drug delivery system was formulated to maintain suitable physicochemical properties at physiological pH. Toxicity, binding, and internalization were evaluated in relevant microvascular endothelial cells. PEC were non-toxic, as indicated by cell proliferation studies and propidium iodide staining. Inhibitor studies revealed that PEC were bound, in part, via heparan sulfate proteoglycans and internalized through macropinocytosis. A novel, flow cytometric, Scatchard protocol was established and showed that PEC, in the absence of surface modification, bind cells non-specifically with positive cooperativity, as seen by graphical transformations.

Development of improved nanoparticulate polyelectrolyte complex physicochemistry by nonstoichiometric mixing of polyions with similar molecular weights.

Water-based, biodegradable polyelectrolyte complex dispersions (PECs) prepared by mixing oppositely charged polyions are advantageous drug delivery systems due to constituent biocompatibility and nanoparticulate architectures. Reaction phase environmental parameters dictate PEC physicochemical properties, and specifically, complexation between polyelectrolytes having significantly different molecular weights leads to formation of water-insoluble aggregates. Starting with this fact, four-component similar and dissimilar molecular weight PEC chemistries were applied and compared with and without frequency-induced dispergation. The goal was to define nanoparticulate PEC systems with desirable characteristics for use in biological systems. Results show PEC formulations from precursors with similar low molecular weights yielded dispersions with suitable physicochemical characteristics, as verified by photon correlation spectroscopy and TEM, presumably due to efficient ion pairing. Similar low molecular weight PECs fabricated with dispergation exhibited pH-independent stability, as validated by charge and size measurements. These physicochemical advantages lead to an ideal delivery platform.

A proposed mechanism for detergent-assisted foam fractionation of lysozyme and cellulase restored with beta-cyclodextrin.

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as beta-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to beta-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.

Effect of beta-cyclodextrin in artificial chaperones assisted foam fractionation of cellulase.

Foam fractionation has the potential to be a low-cost protein separation process; however, it may cause protein denaturation during the foaming process. In previous work with cellulase, artificial chaperones were integrated into the foam fractionation process in order to reduce the loss of enzymatic activity. In this study, other factors were introduced to further reduce the loss of cellulase activity: type of cyclodextrin, cyclodextrin concentration, dilution ratio cyclodextrin to the foamate and holding time. alpha-Cyclodextrin was almost as effective as beta-cyclodextrin in refolding the foamed cellulase-Cetyltrimethylammonium bromide mixture. beta-Cyclodextrin (6.5 mM) was almost as effective as 13 mM beta-cyclodextrin in refolding. The dilution ratio, seven parts foamate and three parts beta-cyclodextrin solution, was found to be most effective among the three ratios tested (7:3, 1:1, and 3:7). The activity after refolding at this dilution ratio is around 0.14 unit/mL. The refolding time study showed that the refolding process was found to be most effective for the short refolding times (within 1 h).

Enhancing cellulase foam fractionation with addition of surfactant.

Foam fractionation cannot be used to recover cellulase from an aerated water solution effectively because cellulase by itself can produce only a small amount of foam. The addition of a surfactant can, however, increase the foamate volume and enhance the concentration of cellulase. We studied three detergents individually added to a 200 mg/L cellulase solution to promote foaming. These detergents were anionic, cationic, and nonionic surfactants, respectively. Although contributing to foam production, it was observed that nonionic surfactant (Pluronic F-68) barely concentrated cellulase, leaving the enrichment ratio unchanged, near 1. With anionic surfactant, sodium dedecyl sulfate, and cationic surfactant, cetyltrimethylammonium bromide (CTAB), the enrichment ratio became much larger, but cellulase denaturation occurred, reducing the activity of the enzyme. When CTAB was used to help foam cellulase, beta-cyclodextrin was subsequently added to the foamate to help restore the enzyme activity.

Nanoparticulate system for efficient gene transfer into refractory cell targets.

A biocompatible, nanoparticulate formulation has been designed to retain, protect, and deliver adenoviral gene constructs over an extended time course. Such devices can be administered locally or systemically with low toxicity. A multipolymeric nanoparticulate system, featuring very high stability in physiologic media, was designed to allow efficient in vitro gene transfer. The efficacy of nanoparticulate delivery is effective in cell systems that are normally refractory to gene transfer, such as pancreatic islets and antigen-presenting cells. The findings suggest a nonspecific uptake system that permits adenoviral particle release within the transfected cells. A comparison with literature data revealed that our system is efficient at much lower levels (at least three orders of magnitude) of infectious viral particles.

NanoLiterBioReactor: long-term mammalian cell culture at nanofabricated scale.

There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. We fabricated NBR prototypes, each of which incorporates a culture chamber, inlet and outlet ports, and connecting microfluidic conduits. The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via plasma activation against a glass slide, which served as the primary culture substrate in the NBR. The input and outlet ports were punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10-100 nL volume), as well as cell seeding. Because of the intrinsically high oxygen permeability of the PDMS material, no additional CO(2)/air supply was necessary. The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay. We have arrived at several intermediate designs. One is termed "circular NBR with a central post (CP-NBR)," another, "perfusion (grid) NBR (PG-NBR)," and a third version, "multitrap (cage) NBR (MT-NBR)," the last two providing total cell retention. Three cells lines were tested in detail: a fibroblast cell line, CHO cells, and hepatocytes. Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed in the NBR design. To delineate the effect of cell seeding density on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes. In both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 1-5 days following the seeding. Low seeding densities resulted in the absence of cell proliferation for some cell lines because of the deficiency of cell-cell and extracellular matrix (ECM)-cell contacts. High viabilities were achieved in all designs. We conclude that an instrumented microfluidics-based NanoBioReactor (NBR) will represent a dramatic departure from the standard culture environment. The employment of NBRs for mammalian cell culture opens a new paradigm of cell biology, so far largely neglected in the literature.

Degeneration of beta-glucosidase activity in a foam fractionation process.

Foam fractionation is a promising technique for concentrating proteins because of its simplicity and low operating cost. One such protein that can be foamed is the enzyme cellulase. The use of inexpensively purified cellulase may be a key step in the economical production of ethanol from biomass. We conducted foam fractionation experiments at total reflux using the cellulase component beta-glucosidase to study how continuous shear affects beta-glucosidase in a foam such as a fermentation or foam fractionation process. The experiments were conducted at pH 2.4, 5.4, and 11.6 and airflow rates of 3, 6, 15, 20, and 32 cc/min to determine how beta-glucosidase activity changes in time at these different conditions. This is apparently a novel and simple way of testing for changes in enzyme activity within a protein foam. The activity did not degenerate during 5 min of reflux at pH 5.4 at an airflow rate of 10 cc/min. It was established that at 10 min of refluxing, the beta-glucosidase denatured more as the flow rate increased. At pH 2.4 and a flow rate of 10 cc/min, the activity remained constant for at least 15 min.

A microphysiometer for simultaneous measurement of changes in extracellular glucose, lactate, oxygen, and acidification rate.

A microphysiometer capable of measuring changes in extracellular glucose, lactate, oxygen, and acidification rate has been developed by incorporating modified electrodes into a standard Cytosensor Microphysiometer plunger. Glucose and lactate are measured indirectly at platinum electrodes by amperometric oxidation of hydrogen peroxide, which is produced from catalysis of glucose and lactate at films containing their respective entrapped oxidase. Oxygen is measured amperometrically at a platinum electrode coated with a Nafion film, while the acidification rate is measured potentiometrically by a Cytosensor Microphysiometer. Analytical information is obtained during the Cytosensor stop-flow cycles, where the electrodes measure changes in the extracellular medium corresponding to the consumption or production of the analyte by the cells. Modification of the Cytosensor plunger for multianalyte determination is described, and the operation of the technique is illustrated by the simultaneous measurement of all four analytes during the addition of fluoride and DNP to Chinese hamster ovary cells and fluoride and antimycin A to mouse fibroblast cells. Cell metabolic recovery and dynamics after exposure to agents can also be observed in specific cases.

Variation of bubble size distribution in a protein foam fractionation column measured using a capillary probe with photoelectric sensors.

Bubble size is used to characterize not only bubble-specific interfacial area but also bubble coalescence in a foam column. The bubble size distributions were obtained in a continuous foam fractionation process for concentrating ovalbumin using a developed photoelectric probe. When the continuous process reached steady state, the bubble size distribution pattern remained stable. Bubble size distribution data above (+1 cm) or below (-1 cm) the bulk liquid-foam interface showed symmetry along the diameter of the column (14 cm ID). The bubble size distribution was affected by the column wall. The nearly constant protein concentration distribution across the column cross-section indicated that the bubble flow distribution approached a flat profile across the column. A log-normal bubble distribution pattern best fit the weighted range of bubbles in the column at column lengths above and below the liquid-foam interface. These observations may prove to be useful in understanding the mechanisms underlying the foam fractionation of proteins.

The effect of pH on the foam fractionation of beta-glucosidase and cellulase.

The surface tension-pH profile of beta-glucosidase was established to determine its relationship to the corresponding profile of cellulase and to the foam fractionation of that cellulase. The goal of this work was to determine the optimal foaming points for both cellulase and cellobiase. This data may prove useful in the separation of certain components of cellulase, since the non-foaming hydrophilic beta-glucosidase does not foam as well as the hydrophobic components of cellulase at low concentrations. A key finding from these experiments was that there are two local minima in the surface tension-pH trajectory for Trichoderma reesei cellulase, as contrasted to the usual single minimum. The lower of these minimum points corresponds to the cellulase isoelectric point. The double minimum surface tension-pH profile was also observed for cellobiase alone. The optimal foaming pH for cellobiase alone was determined to be around 10.5, while for cellulase it was between 6 and 9.

Effect of bubble size on foam fractionation of ovalbumin.

The bubble size distribution and void fraction (epsilong) (at two bulk liquid pool positions below the bulk liquid-foam interface and one lower foam phase position) in a continuous foam fractionation column containing ovalbumin were obtained using a photoelectric capillary probe. The bubble size and epsilong data were gathered for different operating conditions (including the changes in the superficial gas velocity and feed flow rate) at a feed solution of pH 6.5 and used to calculate the specific area, a, of the bubbles. Thus, local enrichment (ERl), values of ovalbumin could be estimated and compared with directly obtained experimental results. The ERl results were also correlated with the bubble size and epsilong to understand better the concentration mechanisms of foam fractionation. The high ERl in the lower foam phase was largely attributable to the abrupt increase in epsilong (from 0.25 to 0.75), or the a (from about 12 to 25 cm2/cm3) from the bulk liquid to the foam phase. These changes correspond with enhanced gravity drainage. With an increase in the superficial gas velocity, the bubble size increased and the a decreased in both the bulk liquid and lower foam phases, resulting in a decrease in the local experimentally determined enrichments at high superficial gas velocities. At intermediate feed flow rates, the bubble size reached the maximum. The epsilong and a, on the other hand, were the largest for the largest feed flow rate. The ERl in the lower foam phase was maximized at the lowest feed flow rate. It follows, therefore, that a alone is not sufficient to determine the magnitude of the ERl in the foam phase.

Foam fractionation of a dilute solution of bovine lactoferrin.

Lactoferrin (Lf), a protein found in human and bovine milk, tears, blood, and other secretory fluids, has been used to prevent infection from potential microbial pathogens by its ability to bind with iron (Fe3+). Currently, bovine lactoferrin can be purified from milk using ion exchange resin, which is a costly procedure making lactoferrin expensive. The purpose of this work was to investigate a low-cost foam fractionation process as the first step in separating lactoferrin from milk.