Even though degradation products of biodegradable polymers are known to be largely non-cytotoxic, little detailed information is available regarding the degradation rate-dependent acidic byproduct effect of the scaffold. In vitro and in vivo scaffold degradation rate could be differentiated using a fast degrading polymer (e.g., poly D, L-lactic-glycolic acid co-polymer, PLGA, 50:50) and a slow degrading polymer (e.g., poly epsilon-caprolactone, PCL). We applied a new method to develop uniform 10 microm thickness of high porous scaffolds using a computer-controlled knife coater with a motion stage and exploiting phase transition properties of a combination of salts and water in salt-leaching method. We then verified in vitro the effect of fast degradation by assessing the viability of primary mouse aortic smooth muscle cell cultured in the three-dimensional scaffolds. We found that cell viability was inversely related to degradation rate and was dependent on the depth from the seeding (upper) surface toward the lower surface. The pH measurement of culture medium using fluorescence probes showed time-dependent decrease in pH in the PLGA scaffolds, corresponding to PLGA degradation, and closely related to cell viability. In vivo analysis of scaffolds implanted subcutaneously into the back of mice, showed significant differences in inflammation and cell invasion into PLGA vs. PCL. Importantly, these were correlated with the degree of the functional angiogenesis within the scaffolds. Again, PLGA scaffolds demonstrated less cell mobilization and less angiogenesis, further supporting the negative effect of the acidic environment created by the degradation of biocompatible polymers.

Tissue engineering aims at resolving problems such as donor shortage and immune rejection faced by transplantation. Scaffolds (artificial extracellular matrices) have critical roles in tissue engineering. Recently, we developed nano-fibrous poly(L-lactic acid) scaffolds under the hypothesis that synthetic nano-fibrous scaffolding, mimicking the structure of natural collagen fibers, could create a more favorable microenvironment for cells. This is the first report that the nano-fibrous architecture built in three-dimensional scaffolds improved the features of protein adsorption, which mediates cell interactions with scaffolds. Scaffolds with nano-fibrous pore walls adsorbed four times more serum proteins than scaffolds with solid pore walls. More interestingly, the nano-fibrous architecture selectively enhanced protein adsorption including fibronectin and vitronectin, even though both scaffolds were made from the same poly(L-lactic acid) material. Furthermore, nano-fibrous scaffolds also allowed >1.7 times of osteoblastic cell attachment than scaffolds with solid pore walls. These results demonstrate that the biomimetic nano-fibrous architecture serves as superior scaffolding for tissue engineering.

Wine is the product of complex interactions between fungi, yeasts and bacteria that commence in the vineyard and continue throughout the fermentation process until packaging. Although grape cultivar and cultivation provide the foundations of wine flavour, microorganisms, especially yeasts, impact on the subtlety and individuality of the flavour response. Consequently, it is important to identify and understand the ecological interactions that occur between the different microbial groups, species and strains. These interactions encompass yeast-yeast, yeast-filamentous fungi and yeast-bacteria responses. The surface of healthy grapes has a predominance of Aureobasidium pullulans, Metschnikowia, Hanseniaspora (Kloeckera), Cryptococcus and Rhodotorula species depending on stage of maturity. This microflora moderates the growth of spoilage and mycotoxigenic fungi on grapes, the species and strains of yeasts that contribute to alcoholic fermentation, and the bacteria that contribute to malolactic fermentation. Damaged grapes have increased populations of lactic and acetic acid bacteria that impact on yeasts during alcoholic fermentation. Alcoholic fermentation is characterised by the successional growth of various yeast species and strains, where yeast-yeast interactions determine the ecology. Through yeast-bacterial interactions, this ecology can determine progression of the malolactic fermentation, and potential growth of spoilage bacteria in the final product. The mechanisms by which one species/strain impacts on another in grape-wine ecosystems include: production of lytic enzymes, ethanol, sulphur dioxide and killer toxin/bacteriocin like peptides; nutrient depletion including removal of oxygen, and production of carbon dioxide; and release of cell autolytic components. Cell-cell communication through quorum sensing molecules needs investigation.

The genus Enterococcus is the most controversial group of lactic acid bacteria. Studies on the microbiota of many traditional cheeses in the Mediterranean countries have indicated that enterococci play an important role in the ripening of these cheeses, probably through proteolysis, lipolysis, and citrate breakdown, hence contributing to their typical taste and flavour. Enterococci are also present in other fermented foods, such as sausages and olives. However, their role in these products has not been fully elucidated. Furthermore, the production of bacteriocins by enterococci is well documented. Moreover, enterococci are nowadays used as probiotics. At the same time, however, enterococci have been associated with a number of human infections. Several virulence factors have been described and the number of vancomycin-resistant enterococci is increasing. The controversial nature of enterococci has prompted an enormous increase in scientific papers and reviews in recent years, where researchers have been divided into two groups, namely pro and contra enterococci. To the authors' impression, the negative traits have been focused on very extensively. The aim of the present review is to give a balanced overview of both beneficial and virulence features of this divisive group of microorganisms, because it is only acquaintance with both sides that may allow their safe exploitation as starter cultures or co-cultures.

Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

Biodegradable polymer/bioceramic composite scaffolds can overcome the limitations of conventional ceramic bone substitutes such as brittleness and difficulty in shaping. However, conventional methods for fabricating polymer/bioceramic composite scaffolds often use organic solvents (e.g., the solvent casting and particulate leaching (SC/PL) method), which might be harmful to cells or tissues. Furthermore, the polymer solutions may coat the ceramics and hinder their exposure to the scaffold surface, which may decrease the likelihood that the seeded osteogenic cells will make contact with the bioactive ceramics. In this study, a novel method for fabricating a polymer/nano-bioceramic composite scaffold with high exposure of the bioceramics to the scaffold surface was developed for efficient bone tissue engineering. Poly(D,L-lactic-co-glycolic acid)/nano-hydroxyapatite (PLGA/HA) composite scaffolds were fabricated by the gas forming and particulate leaching (GF/PL) method without the use of organic solvents. The GF/PL method exposed HA nanoparticles at the scaffold surface significantly more than the conventional SC/PL method does. The GF/PL scaffolds showed interconnected porous structures without a skin layer and exhibited superior enhanced mechanical properties to those of scaffolds fabricated by the SC/PL method. Both types of scaffolds were seeded with rat calvarial osteoblasts and cultured in vitro or were subcutaneously implanted into athymic mice for eight weeks. The GF/PL scaffolds exhibited significantly higher cell growth, alkaline phosphatase activity, and mineralization compared to the SC/PL scaffolds in vitro. Histological analyses and calcium content quantification of the regenerated tissues five and eight weeks after implantation showed that bone formation was more extensive on the GF/PL scaffolds than on the SC/PL scaffolds. Compared to the SC/PL scaffolds, the enhanced bone formation on the GF/PL scaffolds may have resulted from the higher exposure of HA nanoparticles at the scaffold surface, which allowed for direct contact with the transplanted cells and stimulated the cell proliferation and osteogenic differentiation. These results show that the biodegradable polymer/bioceramic composite scaffolds fabricated by the novel GF/PL method enhance bone regeneration compared with those fabricated by the conventional SC/PL method.

Tissue engineering has shown great promise for creating biological alternatives for implants. In this approach, scaffolding plays a pivotal role. Hydroxyapatite mimics the natural bone mineral and has shown good bone-bonding properties. This paper describes the preparation and morphologies of three-dimensional porous composites from poly(L-lactic acid) (PLLA) or poly(D,L-lactic acid-co-glycolic acid) (PLGA) solution and hydroxyapatite (HAP). A thermally induced phase separation technique was used to create the highly porous composite scaffolds for bone-tissue engineering. Freeze drying of the phase-separated polymer/HAP/solvent mixtures produced hard and tough foams with a co-continuous structure of interconnected pores and a polymer/HAP composite skeleton. The microstructure of the pores and the walls was controlled by varying the polymer concentration, HAP content, quenching temperature, polymer, and solvent utilized. The porosity increased with decreasing polymer concentration and HAP content. Foams with porosity as high as 95% were achieved. Pore sizes ranging from several microns to a few hundred microns were obtained. The composite foams showed a significant improvement in mechanical properties over pure polymer foams. They are promising scaffolds for bone-tissue engineering.

The switch of cellular metabolism from mitochondrial respiration to glycolysis is the hallmark of cancer cells and associated with tumor malignancy. However, the mechanism of this metabolic switch remains largely unknown. Herein, we reported that hypoxia-inducible factor-1 (HIF-1) induced pyruvate dehydrogenase kinase-3 (PDK3) expression leading to inhibition of mitochondrial respiration. Promoter activity assay, small interference RNA knockdown assay, and chromatin immunoprecipitation assay demonstrated that hypoxia-induced PDK3 gene activity was regulated by HIF-1 at the transcriptional level. Forced expression of PDK3 in cancer cells resulted in increased lactic acid accumulation and drugs resistance, whereas knocking down PDK3 inhibited hypoxia-induced cytoplasmic glycolysis and cell survival. These data demonstrated that increased PDK3 expression due to elevated HIF-1alpha in cancer cells may play critical roles in metabolic switch during cancer progression and chemoresistance in cancer therapy.

A Mn(II)-dependent peroxidase found in the extracellular medium of ligninolytic cultures of the white rot fungus, Phanerochaete chrysosporium, was purified by DEAE-Sepharose ion-exchange chromatography, Blue Agarose chromatography, and gel filtration on Sephadex G-100. Sodium dodecyl sulfate-gel electrophoresis indicated that the homogeneous protein has an Mr of 46,000. The absorption spectrum of the enzyme indicates the presence of a heme prosthetic group. The pyridine hemochrome absorption spectrum indicates that the enzyme contained one molecule of heme as iron protoporphyrin IX. The absorption maximum of the native enzyme (406 nm) shifted to 433 nm in the reduced enzyme and to 423 nm in the reduced-CO complex. Both CN- and N-3 readily bind to the native enzyme, indicating an available coordination site and that the heme iron is high spin. The absorption spectrum of the H2O2 enzyme complex, maximum at 420 nm, is similar to that of horseradish peroxidase compound II. P. chrysosporium peroxidase activity is dependent on Mn(II), with maximal activity attained above 100 microM. The enzyme is also stimulated to varying degrees by alpha-hydroxy acids (e.g., malic, lactic) and protein (e.g., gelatin, albumin). The peroxidase is capable of oxidizing NADH and a wide variety of dyes, including Poly B-411 and Poly R-481. Several of the substrates (indigo trisulfonate, NADH, Poly B-411, variamine blue RT salt, and Poly R-481) are oxidized by this Mn(II)-dependent peroxidase at considerably faster rates than those catalyzed by horseradish peroxidase. The enzyme rapidly oxidizes Mn(II) to Mn(III); the latter was detected by the characteristic absorption spectrum of its pyrophosphate complex. Inhibition of the oxidation of the substrate diammonium 2,2-azino-bis(3-ethyl-6-benzothiazolinesulfonate) (ABTS) by Na-pyrophosphate suggests that Mn(III) plays a role in the enzyme mechanism.

The most common synthetic biodegradable polymers being investigated for tissue engineering applications are FDA approved, clinically used poly(alpha-hydroxy esters). To better assess the applicability of the electrospinning technology for scaffold fabrication, six commonly used poly(alpha-hydroxy esters) were used to prepare electrospun fibrous scaffolds, and their physical and biological properties were also characterized. Our results suggest that specific, optimized fabrication parameters are required for each polymer to produce scaffolds that consist of uniform structures morphologically similar to native extracellular matrix. Scanning electron microscopy (SEM) revealed a highly porous, three-dimensional structure for all scaffolds, with average fiber diameter ranging from 300nm to 1.5microm, depending on the polymer type used. The poly(glycolic acid) (PGA) and poly(d,l-lactic-co-glycolic acid 50:50) (PLGA5050) fibrous structures were mechanically stiffest, whereas the poly(l-lactic acid) (PLLA) and poly(epsilon-caprolactone) (PCL) scaffolds were most compliant. Upon incubation in physiological solution, severe structural destruction due to polymer degradation was found in the PGA, poly(d,l-lactic acid) (PDLLA), PLGA5050, and poly(d,l-lactic-co-glycolic acid 85:15) (PLGA8515) fibrous scaffolds, whereas PLLA and PCL fibrous scaffolds maintained a robust scaffold structure during the same time period, based on macroscopic and SEM observations. In addition, PLLA scaffolds supported the highest rate of proliferation of seeded cells (chondrocytes and mesenchymal stem cells) than other polymeric scaffolds. Our findings showed that PLLA and PCL based fibrous scaffolds exhibited the most optimal structural integrity and supported desirable cellular response in culture, suggesting that such scaffolds may be promising candidate biomaterials for tissue engineering applications.

OBJECTIVE

Low extracellular pH is a hallmark of solid tumors. It has long been thought that this acidity is mainly attributable to the production of lactic acid. In this study, we tested the hypothesis that lactate is not the only source of acidification in solid tumors and explored the potential mechanisms underlying these often-observed high rates of acid production.

METHODS

We compared the metabolic profiles of glycolysis-impaired (phosphoglucose isomerase-deficient) and parental cells in both in vitro and two in vivo models (dorsal skinfold chamber and Gullino chamber).

RESULTS

We demonstrated that CO(2), in addition to lactic acid, was a significant source of acidity in tumors. We also found evidence supporting the hypothesis that tumor cells rely on glutaminolysis for energy production and that the pentose phosphate pathway is highly active within tumor cells. Our results also suggest that the tricarboxylic acid cycle is saturable and that different metabolic pathways are activated to provide for energy production and biosynthesis.

CONCLUSIONS

These results are consistent with the paradigm that tumor metabolism is determined mainly by substrate availability and not by the metabolic demand of tumor cells per se. In particular, it appears that the local glucose and oxygen availabilities each independently affect tumor acidity. These findings have significant implications for cancer treatment.

A novel method was developed to produce highly porous sponges for potential use in tissue engineering, without the use of organic solvents. Highly porous sponges of biodegradable polymers are frequently utilized in tissue engineering both to transplant cells or growth factors, and to serve as a template for tissue regeneration. The processes utilized to fabricate sponges typically use organic solvents, but organic residues remaining in the sponges may be harmful to adherent cells, protein growth factors or nearby tissues. This report describes a technique to fabricate macroporous sponges from synthetic biodegradable polymers using high pressure carbon dioxide processing at room temperature. Solid discs of poly (D,L-lactic-co-glycolic acid) were saturated with CO2 by exposure to high pressure CO2 gas (5.5 MPa) for 72 h at room temperature. The solubility of the gas in the polymer was then rapidly decreased by reducing the CO2 gas pressure to atmospheric levels. This created a thermodynamic instability for the CO2 dissolved in the polymer discs, and resulted in the nucleation and growth of gas cells within the polymer matrix. Polymer sponges with large pores (approximately 100 microns) and porosities of up to 93% could be fabricated with this technique. The porosity of the sponges could be controlled by the perform production technique, and mixing crystalline and amorphous polymers. Fibre-reinforced foams could also be produced by placing polymer fibres within the polymer matrix before CO2 gas processing.

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

One of the most common signatures of highly malignant tumors is their capacity to metabolize more glucose to lactic acid than their tissues of origin. Hepatomas exhibiting this phenotype are dependent on the high expression of type II hexokinase, which supplies such tumors with abundant amounts of glucose 6-phosphate, a significant carbon and energy source especially under hypoxic conditions. Here we report that the distal region of the hepatoma type II hexokinase promoter displays consensus motifs for hypoxia-inducible factor (HIF-1) that overlap E-box sequences known to be related in other gene promoters to glucose response. Moreover, we show that subjecting transfected hepatoma cells to hypoxic conditions activates the type II hexokinase promoter almost 3-fold, a value that approaches 7-fold in the presence of glucose. Consistent with these findings is the induction under hypoxic conditions of the HIF-1 protein. Reporter gene analyses with a series of nested deletion mutants of the hepatoma type II hexokinase promoter show that a significant fraction of the total activation observed under hypoxic conditions localizes to the distal region where the overlapping HIF-1/E-box sequences are located. Finally, DNase I footprint analysis with a segment of the promoter containing these elements reveals the binding of several nuclear proteins. In summary, these novel studies identify and characterize a marked glucose-modulated activation response of the type II hexokinase gene to hypoxic conditions within highly glycolytic hepatoma cells, a property that may help assure that such cells exhibit a growth and survival advantage over their parental cells of origin.

The genomic revolution has identified therapeutic targets for a plethora of diseases, creating a need to develop robust technologies for combination drug therapy. In the present work, we describe a self-assembled polymeric nanoparticle (NP) platform to target and control precisely the codelivery of drugs with varying physicochemical properties to cancer cells. As proof of concept, we codelivered cisplatin and docetaxel (Dtxl) to prostate cancer cells with synergistic cytotoxicity. A polylactide (PLA) derivative with pendant hydroxyl groups was prepared and conjugated to a platinum(IV) [Pt(IV)] prodrug, c,t,c-[Pt(NH(3))(2)(O(2)CCH(2)CH(2)COOH)(OH)Cl(2)] [PLA-Pt(IV)]. A blend of PLA-Pt(IV) functionalized polymer and carboxyl-terminated poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer in the presence or absence of Dtxl, was converted, in microfluidic channels, to NPs with a diameter of ∼100 nm. This process resulted in excellent encapsulation efficiency (EE) and high loading of both hydrophilic platinum prodrug and hydrophobic Dtxl with reproducible EEs and loadings. The surface of the NPs was derivatized with the A10 aptamer, which binds to the prostate-specific membrane antigen (PSMA) on prostate cancer cells. These NPs undergo controlled release of both drugs over a period of 48-72 h. Targeted NPs were internalized by the PSMA-expressing LNCaP cells via endocytosis, and formation of cisplatin 1,2-d(GpG) intrastrand cross-links on nuclear DNA was verified. In vitro toxicities demonstrated superiority of the targeted dual-drug combination NPs over NPs with single drug or nontargeted NPs. This work reveals the potential of a single, programmable nanoparticle to blend and deliver a combination of drugs for cancer treatment.

Gut microbiota shows host-specific diversity and temporal stability and significantly contributes to maintenance of a healthy gut. However, in inflammatory bowel disease, this microbiota has been implicated as a contributory factor to the illness. This study compared bacterial dynamics in Crohn's disease patients to those in a control group using a culture-independent method to assess the temporal stability, relative diversity, and similarity of the dominant fecal microbiota, Clostridium spp., Bacteroides spp., Bifidobacterium spp., and lactic acid bacteria spp. (LAB) for all individuals. Fecal samples were collected over several time points from individuals with Crohn's disease who were in remission (n = 11), from Crohn's disease patients who relapsed into an active Crohn's disease state (n = 5), and from a control group (n = 18). Denaturing gradient gel electrophoresis profiles were generated for the different microbial groups by specifically targeting different regions of the 16S rRNA gene and were compared on the basis of similarity and diversity. The temporal stability of dominant species for all Crohn's disease patients was significantly lower (P < 0.005) than that for the control group. Analysis of group-specific profiles for Bifidobacterium spp. found that they were similar in all samples, while the diversity of the LAB varied significantly between the groups, but temporal stability was not significantly altered. We observed significant changes in two functionally important mutualistic groups of bacteria, viz., Clostridium and Bacteroides spp., which may have implications for the host's gut health, since some genera are involved in production of short-chain fatty acid, e.g., butyrate.

Injectable biodegradable and biocompatible copolymers of lactic and glycolic acid (PLGA) are an important advanced delivery system for week-to-month controlled release of hydrophobic drugs (e.g., from biopharmaceutical classification system class IV), which often display poor oral bioavailability. The basic principles and considerations to develop such microparticle formulations is reviewed here based on a comprehensive study of papers and patents from the beginnings of hydrophobic drug encapsulation in polylactic acid and PLGA up through the very recent literature. Challenges with the diversity of drug properties, microencapsulation methods, and organic solvents are evaluated in light of the precedence of commercialized formulations and with a focus on decreasing the time to lab-scale encapsulation of water-insoluble drug candidates in the early stage of drug development. The influence of key formulation variables on final microparticle characteristics, and how best to avoid undesired microparticle properties, is analyzed mechanistically. Finally, concepts are developed to manage the common issues of maintaining sink conditions for in vitro drug release assays of hydrophobic compounds. Overall, against the backdrop of an increasing number of new, poorly orally available drug entities entering development, microparticle delivery systems may be a viable strategy to rescue an otherwise undeliverable substance.

Bacteriocins are ribosomally-synthesized peptides or proteins with antimicrobial activity, produced by different groups of bacteria. Many lactic acid bacteria (LAB) produce bacteriocins with rather broad spectra of inhibition. Several LAB bacteriocins offer potential applications in food preservation, and the use of bacteriocins in the food industry can help to reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods which are more naturally preserved and richer in organoleptic and nutritional properties. This can be an alternative to satisfy the increasing consumers demands for safe, fresh-tasting, ready-to-eat, minimally-processed foods and also to develop "novel" food products (e.g. less acidic, or with a lower salt content). In addition to the available commercial preparations of nisin and pediocin PA-1/AcH, other bacteriocins (like for example lacticin 3147, enterocin AS-48 or variacin) also offer promising perspectives. Broad-spectrum bacteriocins present potential wider uses, while narrow-spectrum bacteriocins can be used more specifically to selectively inhibit certain high-risk bacteria in foods like Listeria monocytogenes without affecting harmless microbiota. Bacteriocins can be added to foods in the form of concentrated preparations as food preservatives, shelf-life extenders, additives or ingredients, or they can be produced in situ by bacteriocinogenic starters, adjunct or protective cultures. Immobilized bacteriocins can also find application for development of bioactive food packaging. In recent years, application of bacteriocins as part of hurdle technology has gained great attention. Several bacteriocins show additive or synergistic effects when used in combination with other antimicrobial agents, including chemical preservatives, natural phenolic compounds, as well as other antimicrobial proteins. This, as well as the combined use of different bacteriocins may also be an attractive approach to avoid development of resistant strains. The combination of bacteriocins and physical treatments like high pressure processing or pulsed electric fields also offer good opportunities for more effective preservation of foods, providing an additional barrier to more refractile forms like bacterial endospores as well. The effectiveness of bacteriocins is often dictated by environmental factors like pH, temperature, food composition and structure, as well as the food microbiota. Foods must be considered as complex ecosystems in which microbial interactions may have a great influence on the microbial balance and proliferation of beneficial or harmful bacteria. Recent developments in molecular microbial ecology can help to better understand the global effects of bacteriocins in food ecosystems, and the study of bacterial genomes may reveal new sources of bacteriocins.

In the past few years, an increasing number of in situ-forming systems have been reported in the literature for various biomedical applications, including drug delivery, cell encapsulation, and tissue repair. There are several possible mechanisms that lead to in situ gel formation: solvent exchange, UV-irradiation, ionic cross-linkage, pH change, and temperature modulation. The thermosensitive approach can be advantageous for particular applications as it does not require organic solvents, co-polymerization agents, or an externally applied trigger for gelation. In the last 2 decades, several thermosensitive formulations have been proposed. This manuscript focuses on aqueous polymeric solutions that form implants in situ in response to temperature change, generally from ambient to body temperature. It mainly reviews the characterization and use of polysaccharides, N-isopropylacrylamide copolymers, poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) (poloxamer) and its copolymers, poly(ethylene oxide)/(D,L-lactic acid-co-glycolic acid) copolymers, and thermosensitive liposome-based systems.

The term "lactic acid bacteria" is discussed. An overview of the following topics is given: main pathways of homo- and heterofermentation of hexoses, i.e. glycolysis, bifidus pathway, 6-phosphogluconate pathway; uptake and dissimilation of lactose (tagatose pathway); fermentation of pentoses and pentitols; alternative fates of pyruvate, i.e. splitting to formate and acetate, CO2 and acetate or formation of acetoin and diacetyl; lactate oxidation; biochemical basis for the formation of different stereoisomers of lactate.

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibres also use lactic acid as a respiratory fuel. The stereoselective transport of L-lactic acid across the plasma membrane of muscle fibres has been shown to involve a proton-linked monocarboxylate transporter (MCT) similar to that described in erythrocytes and other cells. This transporter plays an important role in the pH regulation of skeletal muscle. A family of eight MCTs has now been cloned and sequenced, and the tissue distribution of each isoform varies. Skeletal muscle contains both MCT1 (the only isoform found in erythrocytes but also present in most other cells) and MCT4. The latter is found in all fibre types, although least in more oxidative red muscles such as soleus, whereas expression of MCT1 is highest in the more oxidative muscles and very low in white muscles that are almost entirely glycolytic. The properties of MCT1 and MCT2 have been described in some detail and the latter shown to have a higher affinity for substrates. MCT4 has been less well characterized but has a lower affinity for L-lactate (i.e. a higher Km of 20 mM) than does MCT1 (Km of 5 mM). MCT1 expression is increased in response to chronic stimulation and either endurance or explosive exercise training in rats and humans, whereas denervation decreases expression of both MCT1 and MCT4. The mechanism of regulation is not established, but does not appear to be accompanied by changes in mRNA concentrations. However, in other cells MCT1 and MCT4 are intimately associated with an ancillary protein OX-47 (also known as CD147). This protein is a member of the immunoglobulin superfamily with a single transmembrane helix, whose expression is known to be increased in a range of cells when their metabolic activity is increased.

A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours.

Subacute ruminal acidosis (SARA) is a metabolic disease in dairy cattle that occurs during early and mid-lactation and has traditionally been characterized by low rumen pH, but lactic acid does not accumulate as in acute lactic acid acidosis. It is hypothesized that factors such as increased gut permeability, bacterial lipopolysaccharides, and inflammatory responses may have a role in the etiology of SARA. However, little is known about the nature of the rumen microbiome during SARA. In this study, we analyzed the microbiome of 64 rumen samples taken from eight lactating Holstein dairy cattle using terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA genes and real-time PCR. We used rumen samples from two published experiments in which SARA had been induced with either grain or alfalfa pellets. The results of TRFLP analysis indicated that the most predominant shift during SARA was a decline in gram-negative Bacteroidetes organisms. However, the proportion of Bacteroidetes organisms was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA (35.4% versus 26.0% and 16.6%, respectively). This shift was also evident from the real-time PCR data for Prevotella albensis, Prevotella brevis, and Prevotella ruminicola, which are members of the Bacteroidetes. The real-time PCR data also indicated that severe grain-induced SARA was dominated by Streptococcus bovis and Escherichia coli, whereas mild grain-induced SARA was dominated by Megasphaera elsdenii and alfalfa pellet-induced SARA was dominated by P. albensis. Using discriminant analysis, the severity of SARA and degree of inflammation were highly correlated with the abundance of E. coli and not with lipopolysaccharide in the rumen. We thus suspect that E. coli may be a contributing factor in disease onset.