The purpose of this work was to investigate whether the phenomenon of metabolic adaptation of HT-29 cells to glucose deprivation and subsequent emergence of differentiated subpopulations (A. Zweibaum et al., J. Cell. Physiol., 122: 21-29, 1985) also applies to anticancer drugs that act at a metabolic level like methotrexate (MTX). Stepwise adaptation of exponentially growing HT-29 cells to increasing concentrations of MTX (10(-7), 10(-6), and 10(-5) mol) results, after a phase of high mortality, in the emergence of subpopulations with stable growth rates and curves close to those of untreated control cells. In contrast to control cells which are heterogenous and contain, after confluency, only a small proportion of differentiated cell types (less than 4%), postconfluent cultures of MTX-adapted cells are totally differentiated. Cells adapted to 10(-7) M MTX form a mixed population of columnar absorptive and mucus cells; at higher concentrations cells are almost exclusively of the mucus-secreting type. All cells, whether mucus-secreting or not, develop an apical brush border which strongly expresses dipeptidylpeptidase IV, carcinoembryonic antigen, and villin. These differentiation features, which resemble those of fetal colon, are associated with decreased rates of glucose consumption and lactic acid production. Both differentiation characteristics and metabolic changes are stably maintained when the cells are subcultured in the absence of the drug. Like the original population, MTX-adapted cells are tumorigenic in nude mice. We propose that cells which are able to differentiate and which are the origin of the small proportion of differentiated cell types found in postconfluent cultures of the original cell line possess an advantage which allows them to be adaptable to "metabolic stress" conditions.

BACKGROUND

The significance of occult hypoperfusion (OH) in the development of respiratory complications (RC), multiple system organ failure (MSOF), and death, and the effect of rapid identification and correction of OH in the severely injured trauma patient was investigated.

METHODS

A pilot retrospective study and the analysis of a prospective protocol to correct OH were performed. Pilot study: all trauma patients admitted to our Level I trauma center between February and December of 1995, who survived greater than 48 hours, had an Injury Severity Score greater than or equal to 20, and intensive care unit stays greater than 48 hours were evaluated. Prospective study: patients admitted between January 1, 1996, and April 30, 1997, who survived greater than 24 hours, with Injury Severity Score greater than or equal to 20, and who were hemodynamically stable (systolic blood pressure greater than 100, pulse rate less than 120, and urine output greater than 1 mL/kg per hour) were included. Serum lactic acid (LA) levels were measured at arrival and at proscribed intervals. In the pilot study, initial LA levels were examined in relation to outcome and complications. In the prospective study, patients with two consecutive LA levels greater than 2.5 mmol/L underwent invasive monitoring and vigorous resuscitation to correct their lactic acidosis.

RESULTS

Among the 31 patients studied in the pilot study, there were 4 deaths, 6 cases of MSOF, and 13 patients with RC. Lactic acidosis and poor cardiac performance, as evidenced by low cardiac index (CI) with normal filling pressures, were seen in all cases of MSOF and RC, as well as in all deaths. From these results, the prospective study was performed. Eighty-five intensive care unit patients met criteria for inclusion in the study. Six additional patients were excluded because of severe, untreatable intracranial hypertension at admission to the intensive care unit. Fifty-eight of these patients had OH in the first 24 hours. Forty-four patients corrected their OH within 24 hours with vigorous resuscitation. There were no deaths, three cases of MSOF, and 10 cases of RC in those patients who corrected OH within 24 hours. Persistent OH (>24 hours) was seen in 14 patients, despite resuscitative efforts, 43% of whom died. MSOF and RC were present in 36% and 50% of cases, respectively (p<0.05).

CONCLUSIONS

Initial lactic acidosis is associated with lower cardiac performance and higher morbidity and mortality. Persistent OH is associated with higher rates of RC, MSOF, and death after severe trauma. Early identification and aggressive resuscitation aimed at correcting continued elevation in serum lactate improves survival and reduces complications in severely injured trauma patients.

Luminescent materials are widely used for imaging and sensing owing to their high sensitivity, rapid response and facile detection by many optical technologies. Typically materials must be chemically tailored to achieve intense, photostable fluorescence, oxygen-sensitive phosphorescence or dual emission for ratiometric sensing, often by blending two dyes in a matrix. Dual-emissive materials combining all of these features in one easily tunable molecular platform are desirable, but when fluorescence and phosphorescence originate from the same dye, it can be challenging to vary relative fluorescence/phosphorescence intensities for practical sensing applications. Heavy-atom substitution alone increases phosphorescence by a given, not variable amount. Here, we report a strategy for modulating fluorescence/phosphorescence for a single-component, dual-emissive, iodide-substituted difluoroboron dibenzoylmethane-poly(lactic acid) (BF(2)dbm(I)PLA) solid-state sensor material. This is accomplished through systematic variation of the PLA chain length in controlled solvent-free lactide polymerization combined with heavy-atom substitution. We demonstrate the versatility of this approach by showing that films made from low-molecular-weight BF(2)dbm(I)PLA with weak fluorescence and strong phosphorescence are promising as 'turn on' sensors for aerodynamics applications, and that nanoparticles fabricated from a higher-molecular-weight polymer with balanced fluorescence and phosphorescence intensities serve as ratiometric tumour hypoxia imaging agents.

During the development of human oral biofilm communities, the spatial arrangement of the bacteria is thought to be driven by metabolic interactions between them. Streptococcus gordonii and Veillonella atypica, two early colonizing members of the dental plaque biofilm, have been postulated to participate in metabolic communication; S. gordonii ferments carbohydrates to form lactic acid, which is a preferred fermentation substrate for V. atypica. We found that, during agar-plate coculture of these organisms, a signaling event occurs that results in increased expression of the S. gordonii alpha-amylase-encoding gene amyB. Confocal scanning laser microscopy of coculture flowcell-grown biofilms using human saliva as the sole nutrient showed that V. atypica caused S. gordonii to increase expression of a PamyB-'gfp transcriptional fusion in a spatially resolved fashion. In this open system, only those streptococci in mixed-species microcolonies containing V. atypica expressed GFP; nearby S. gordonii colonies that lacked V. atypica did not express GFP. In a closed system containing S. gordonii and V. atypica, flow cytometric analysis showed that S. gordonii containing the PamyB-'gfp reporter plasmid exhibited mean fluorescence levels 20-fold higher than did S. gordonii that had not been incubated with V. atypica. Thus, in a closed system where a diffusible signal can accumulate above a required threshold, interspecies signaling mediates a change in gene expression. We provide evidence that, in open systems like those that predominate in natural biofilms, diffusible signals between species are designed to function over short distances, on the order of 1 mum.

Some organic anions are absorbed from the gastrointestinal tract through carrier-mediated transport mechanism(s), which may include proton-coupled transport, anion exchange transport, and others. However, the molecular identity of the organic anion transporters localized at the apical membrane of human intestinal epithelial cells has not been clearly demonstrated. In the present study, we focused on human organic anion transporting polypeptide OATP-B and examined its subcellular localization and functionality in the small intestine. Localization of OATP-B was determined by immunohistochemical analysis. Transport properties of estrone-3-sulfate and the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin by OATP-B-transfected human embryonic kidney 293 cells were measured. OATP-B was immunohistochemically localized at the apical membrane of intestinal epithelial cells in humans. Uptake of [3H]estrone-3-sulfate and [14C]pravastatin by OATP-B at pH 5.5 was higher than that at pH 7.4. [3H]Estrone-3-sulfate transport was decreased by pravastatin, aromatic anion compounds, and the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, but not by small anionic compounds, such as lactic acid and acetic acid. The inhibitory effect of pravastatin on the uptake of [3H]estrone-3-sulfate was concentration-dependent, and the IC50 value was 5.5 mM. The results suggested that OATP-B mediates absorption of anionic compounds and its activity may be optimum at the acidic surface microclimate pH of the small intestine. Accordingly, OATP-B plays a role in the absorption of anionic compounds across the apical membrane of human intestinal epithelial cells, although it cannot be decisively concluded that pH-dependent absorption of pravastatin is determined by OATP-B alone.

The winemaking process includes two main steps: lactic acid bacteria are responsible for the malolactic fermentation which follows the alcoholic fermentation by yeasts. Both types of microorganisms are present on grapes and on cellar equipment. Yeasts are better adapted to growth in grape must than lactic acid bacteria, so the alcoholic fermentation starts quickly. In must, up to ten lactic acid bacteria species can be identified. They belong to the Lactobacillus, Pediococcus, Leuconostoc and Oenococcus genera. Throughout alcoholic fermentation, a natural selection occurs and finally the dominant species is O. oeni, due to interactions between yeasts and bacteria and between bacteria themselves. After bacterial growth, when the population is over 10(6) CFU/ml, malolactic transformation is the obvious change in wine composition. However, many other substrates can be metabolized. Some like remaining sugars and citric acid are always assimilated by lactic acid bacteria, thus providing them with energy and carbon. Other substrates such as some amino acids may be used following pathways restricted to strains carrying the adequate enzymes. Some strains can also produce exopolysaccharides. All these transformations greatly influence the sensory and hygienic quality of wine. Malic acid transformation is encouraged because it induces deacidification. Diacetyl produced from citric acid is also helpful to some extent. Sensory analyses show that many other reactions change the aromas and make malolactic fermentation beneficial, but they are as yet unknown. On the contrary, an excess of acetic acid, the synthesis of glucane, biogenic amines and precursors of ethylcarbamate are undesirable. Fortunately, lactic acid bacteria normally multiply in dry wines; moreover some of these activities are not widespread. Moreover, the most striking trait of wine lactic acid bacteria is their capacity to adapt to a hostile environment. The mechanisms for this are not yet completely elucidated. Molecular biology has provided some explanations for the behaviour and the metabolism of bacteria in wine. New tools are now available to detect the presence of desirable and undesirable strains. Even if much remains unknown, winemakers and oenologists can nowadays better control the process. By acting upon the diverse microflora and grape musts, they are more able to produce healthy and pleasant wines.

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.

This study investigated the in vitro degradation of porous poly(DL-lactic-co-glycolic acid) (PLGA) foams during a 20-week period in pH 7.4 phosphate-buffered saline (PBS) at 37 degrees C and their in vivo degradation following implantation in rat mesentery for up to 8 weeks. Three types of PLGA 85 : 15 and three types of 50 : 50 foams were fabricated using a solvent-casting, particulate-leaching technique. The two types had initial salt weight fraction of 80 and 90%, and a salt particle size of 106-150 microm, while the third type had 90% initial weight fraction of salt in the size range 0-53 microm. The porosities of the resulting foams were 0.82, 0.89, and 0.85 for PLGA 85 : 15, and 0.73, 0.87, and 0.84 for PLGA 50 : 50 foams, respectively. The corresponding median pore diameters were 30, 50, and 17 microm for PLGA 85: 15, and 19, 17, and 17 microm for PLGA 50 : 50. The in vitro and in vivo degradation kinetics of PLGA 85: 15 foams were independent of pore morphology with insignificant variation in foam weight, thickness, pore distribution, compressive creep behavior, and morphology during degradation. The in vitro foam half-lives based on the weight average molecular weight were 11.1 +/- 1.8 (80%, 106-150 microm), 12.0 +/- 2.0 (90%, 106-150 microm), and 11.6 +/- 1.3 (90%, 0-53 microm) weeks, similar to the corresponding values of 9.4 +/- 2.2, 14.3 +/- 1.5, and 13.7 +/- 3.3 weeks for in vivo degradation. In contrast, all PLGA 50 : 50 foams exhibited significant change in foam weight, water absorption, and pore distribution after 6-8 weeks of incubation with PBS. The in vitro foam half-lives were 3.3 +/- 0.3 (80%, 106-150 microm), 3.0 +/- 0.3 (90%, 106-150 microm), and 3.2 +/- 0.1 (90%, 0-53 microm) weeks, and the corresponding in vivo half-lives were 1.9 micro 0.1, 2.2 +/- 0.2, and 2.4 +/- 0.2 weeks. The significantly shorter half-lives of PLGA 50: 50 compared to 85: 15 foams indicated their faster degradation both in vitro and in vivo. In addition, PLGA 50: 50 foams exhibited significantly faster degradation in vivo as compared to in vitro conditions due to an autocatalytic effect of the accumulated acidic degradation products in the medium surrounding the implants. These results suggest that the polymer composition and environmental conditions have significant effects on the degradation rate of porous PLGA foams.

Biodegradable polymeric micelles, self-assembled from a di-block copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG), were prepared to achieve folate receptor targeted delivery of doxorubicin (DOX). In the di-block copolymer structure of PLGA-b-PEG, DOX was chemically conjugated to a terminal end of PLGA to produce DOX-PLGA-mPEG, and folate was separately conjugated to a terminal end of PEG to produce PLGA-PEG-FOL. The two di-block copolymers with different functional moieties at their chains ends were physically mixed with free base DOX in an aqueous solution to form mixed micelles. It was expected that folate moieties were exposed on the micellar surface, while DOX was physically and chemically entrapped in the core of micelles. Flow cytometry and confocal image analysis revealed that folate conjugated mixed micelles exhibited far greater extent of cellular uptake than folate unconjugated micelles against KB cells over-expressing folate receptors on the surface. They also showed higher cytotoxicity than DOX, suggesting that folate receptor medicated endocytosis of the micelles played an important role in transporting an increased amount of DOX within cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that when systemically administered, tumor volume was significantly regressed. Biodistribution studies also indicated that an increased amount of DOX was accumulated in the tumor tissue.

BACKGROUND

Bacterial vaginosis (BV), the most common vaginal condition of reproductive-aged women, is associated with a highly diverse and heterogeneous microbiota. Here we present a proof-of-principle analysis to uncover the function of the microbiota using meta-RNA-seq to uncover genes and pathways that potentially differentiate healthy vaginal microbial communities from those in the dysbiotic state of bacterial vaginosis (BV).

RESULTS

The predominant organism, Lactobacillus iners, was present in both conditions and showed a differing expression profile in BV compared to healthy. Despite its minimal genome, L. iners differentially expressed over 10% of its gene complement. Notably, in a BV environment L. iners increased expression of a cholesterol-dependent cytolysin, and of mucin and glycerol transport and related metabolic enzymes. Genes belonging to a CRISPR system were greatly upregulated suggesting that bacteriophage influence the community. Reflective of L. iners, the bacterial community as a whole demonstrated a preference for glycogen and glycerol as carbon sources under BV conditions. The predicted end-products of metabolism under BV conditions include an abundance of succinate and other short-chain fatty-acids, while healthy conditions are predicted to largely contain lactic acid.

CONCLUSIONS

Our study underscores the importance of understanding the functional activity of the bacterial community in addition to characterizing the population structure when investigating the human microbiome.

Malignant cells characteristically exhibit altered metabolic patterns when compared with normal mammalian cells with increased reliance on anaerobic metabolism of glucose to lactic acid even in the presence of abundant oxygen. The inefficiency of the anaerobic pathway is compensated by increased glucose flux, a phenomenon first noted by Otto Warburg approximately 80 years ago and currently exploited for 2-fluoro-2-deoxy-D-glucose-positron emission tomography imaging in clinical radiology. The latter has demonstrated the glycolytic phenotype is a near-universal phenomenon in human cancers. The potential role of the glycolytic phenotype in facilitating tumor invasion has been investigated through mathematical models of the tumor-host interface. Modified cellular automaton and diffusion reaction models demonstrate protons will diffuse from the tumor into peritumoral normal tissue subjecting nontransformed cells adjacent to the tumor edge to an extracellular pH significantly lower than normal. This leads to normal cell death via p53-dependent apoptosis pathways, as well as degradation of the interstitial matrix, loss of intercellular gap junctions, enhanced angiogenesis, and inhibition of the host immune response to tumor antigens. Transformed cells maintain their proliferative capacity in acidic extracellular pH because of mutations in p53 or some other component in the apoptosis pathways. This allows tumor cells to remain proliferative and migrate into the peritumoral normal tissue producing the invasive phenotype. Mathematical models of invasive cancer based on tumor-induced acidification are consistent with extant data on tumor microenvironment and results from clinical positron emission tomography imaging, including the observed correlation between tumor invasiveness and glucose utilization. Novel treatment approaches focused on perturbation of the tumor microenvironment are predicted from the mathematical models and are supported by recent clinical data demonstrating the benefits of azotemia and metabolic acidosis in survival of patients with metastatic renal cancer. The evolutionary basis for adoption of the glycolytic phenotype during carcinogenesis remains unclear because it appears to confer significant competitive disadvantages on the tumor cells due to of inefficient energy production and expenditure of resources to remove the acid byproducts. We propose that the glycolytic phenotype represents a successful adaptation to environmental selection parameters because it confers the ability to invade. That is, the glycolytic phenotype allows the cell to move from the microenvironment of a premalignant lesion to adjacent normal tissue. There it competes with normal cells that are less fit than the populations within the tumor in a microenvironment of relative substrate abundance. The consequent unrestrained proliferation allows the glycolytic phenotype to emerge simultaneous with the transition from a premalignant lesion to an invasive cancer.

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

Site-specific delivery of angiogenic growth factors from tissue-engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic-co-glycolic acid) microspheres capable of controlling the release of angiogenic factors, such as basic fibroblast growth factor (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac fibroblasts. The controlled delivery of bFGF from the three-dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF-releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 +/- 3/mm(2) and it increased to 70 +/- 7/mm(2) by day 21. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering.

Over the last 50 years, human life expectancy and quality of life have increased dramatically due to improvements in nutrition and the use of antibiotics in the fight against infectious diseases. However, the heyday of antibiotic treatment is on the wane due to the appearance and spread of resistance among harmful microorganisms. At present, there is great concern that commensal bacterial populations from food and the gastrointestinal tract (GIT) of humans and animals, such as lactic acid bacteria (LAB) and bifidobacteria, could act as a reservoir for antibiotic resistance genes. Resistances could ultimately be transferred to human pathogenic and opportunistic bacteria hampering the treatment of infections. LAB species have traditionally been used as starter cultures in the production of fermented feed and foodstuffs. Further, LAB and bifidobacteria are normal inhabitants of the GIT where they are known to exert health-promoting effects, and selected strains are currently been used as probiotics. Antibiotic resistance genes carried by LAB and bifidobacteria can be transferred to human pathogenic bacteria either during food manufacture or during passage through the GIT. The aim of this review is to address well-stated and recent knowledge on antibiotic resistance in typical LAB and bifidobacteria species. Therefore, the commonest antibiotic resistance profiles, the distinction between intrinsic and atypical resistances, and some of the genetic determinants already discovered will all be discussed.

Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.

The survival of four strains of lactic acid bacteria in human gastric juice, in vivo and in vitro, and in buffered saline, pH 1 to 5, has been investigated. The strains studied include two Lactobacillus acidophilus strains, Lactobacillus bulgaricus, and Streptococcus thermophilus. In addition, the adhesion of these strains to freshly collected human and pig small intestinal cells and to pig large intestinal cells has been studied and the effect of milk on both survival and adhesion tested. As a result of these investigations, an in vitro test system for screening potential cultures for use as human dietary adjuncts can be developed. The ability to survive in gastric juice and to adhere varied significantly for the strains tested; L. acidophilus ADH survived and adhered better than the others while S. thermophilus survived and adhered poorly. For all strains, both survival and adhesion was enhanced by milk. As all strains adhered to some extent to both human and pig intestinal cells, the adhesion mechanism is probably a nonspecific attachment as opposed to other reported specific Lactobacillus adhesion to gastric tissue. From the survival and adhesion data it seems feasible to obtain elevated levels of viable Lactobacillus sp. in human intestine by careful selection of the bacterial strains ingested. Furthermore, the in vitro methods used here should be valuable to screen potential strains. The data presented here can then be correlated with human in vivo studies monitoring the beneficial effect of ingestion of these Lactobacillus.

Microbial exopolysaccharides are biothickeners that can be added to a wide variety of food products, where they serve as viscosifying, stabilizing, emulsifying or gelling agents. Numerous exopolysaccharides with different composition, size and structure are synthesized by lactic acid bacteria. The heteropolysaccharides from both mesophilic and thermophilic lactic acid bacteria have received renewed interest recently. Structural analysis combined with rheological studies revealed that there is considerable variation among the different exopolysaccharides; some of them exhibit remarkable thickening and shear-thinning properties and display high intrinsic viscosities. Hence, several slime-producing lactic acid bacterium strains and their biopolymers have interesting functional and technological properties, which may be exploited towards different products, in particular, natural fermented milks. However, information on the biosynthesis, molecular organization and fermentation conditions is rather scarce, and the kinetics of exopolysaccharide formation are poorly described. Moreover, the production of exopolysaccharides is low and often unstable, and their downstream processing is difficult. This review particularly deals with microbiological, biochemical and technological aspects of heteropolysaccharides from, and their production by, lactic acid bacteria. The chemical composition and structure, the biosynthesis, genetics and molecular organization, the nutritional and physiological aspects, the process technology, and both food additive and in situ applications (in particular in yogurt) of heterotype exopolysaccharides from lactic acid bacteria are described. Where appropriate, suggestions are made for strain improvement, enhanced productivities and advanced modification and production processes (involving enzyme and/or fermentation technology) that may contribute to the economic soundness of applications with this promising group of biomolecules.

We compared different lactic acid bacteria for their effect on the immune response to rotavirus in children with acute rotavirus gastroenteritis. After initial oral rehydration, 49 children aged 6 to 35 months with rotavirus gastroenteritis randomly received either Lactobacillus casei subsp. casei strain GG (LGG), L. casei subsp. rhamnosus (Lactophilus), or a combination of Streptococcus thermophilus and L. delbrückii subsp. bulgaricus (Yalacta) twice daily for 5 days. Serum antibodies to rotavirus, total number of immunoglobulin-secreting cells (ISC), and specific antibody-secreting cells (sASC) to rotavirus were measured at the acute stage and at convalescence. The mean (SD) duration of diarrhea was 1.8 (0.8) days in children who received LGG, 2.8 (1.2) days in those receiving Lactophilus, and 2.6 (1.4) days in those receiving Yalacta (F = 3.3, p = 0.04). The ISC response was comparable in the three study groups, but the rotavirus-specific immune responses were different. LGG therapy was associated with an enhancement of IgA sASC to rotavirus and serum IgA antibody level at convalescent stage. We conclude that certain strains of lactic acid bacteria, particularly LGG, promote serum and intestinal immune responses to rotavirus, and thus may be important in establishing immunity against rotavirus reinfections.

We report on ten individuals with a fatal infantile encephalopathy and/or pulmonary hypertension, leading to death before the age of 15 months. Hyperglycinemia and lactic acidosis were common findings. Glycine cleavage system and pyruvate dehydrogenase complex (PDHC) activities were low. Homozygosity mapping revealed a perfectly overlapping homozygous region of 1.24 Mb corresponding to chromosome 2 and led to the identification of a homozygous missense mutation (c.622G>> T) in NFU1, which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. Nine individuals were homozygous for this mutation, whereas one was compound heterozygous for this and a splice-site (c.545 + 5G>> A) mutation. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS). Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases. Upon depletion of NFU1 by RNA interference in human cell culture, LAS and, in turn, PDHC activities were largely diminished. In addition, the amount of succinate dehydrogenase, but no other Fe-S proteins, was decreased. In contrast, depletion of the general Fe-S scaffold protein ISCU severely affected assembly of all tested Fe-S proteins, suggesting that NFU1 performs a specific function in mitochondrial Fe-S cluster maturation. Similar biochemical effects were observed in Saccharomyces cerevisiae upon deletion of NFU1, resulting in lower lipoylation and SDH activity. Importantly, yeast Nfu1 protein carrying the individuals' missense mutation was functionally impaired. We conclude that NFU1 functions as a late-acting maturation factor for a subset of mitochondrial Fe-S proteins.

Fructose, a naturally occurring hexose, is a component of many fruits, vegetables, and sweeteners. Because of the introduction of high fructose corn sweeteners in 1967, the amount of free fructose in the diet of Americans has increased substantially in the last 20 years. Fructose is sweeter, more soluble, and less glucogenic than glucose or sucrose, so it has been recommended as a replacement for these sugars in the diets of diabetic and obese people. Although an acute dose of fructose causes smaller increases in glucose and insulin than a comparable dose of glucose, there are a number of changes after dietary adaptation that may reduce its desirability as a sugar replacement in certain segments of the population. Fructose is absorbed primarily in the jejunum and metabolized in the liver. When consumed in excess of dietary glucose, it may be malabsorbed. Fructose is more lipogenic than glucose or starches, and usually causes greater elevations in triglycerides and sometimes in cholesterol than other carbohydrates. Dietary fructose has resulted in increases in blood pressure, uric acid, and lactic acid. People who are hypertensive, hyperinsulinemic, hypertriglyceridemic, non-insulin-dependent diabetic, or postmenopausal are more susceptible to these adverse effects of dietary fructose than healthy young subjects. Although consumption of fructose as a component of fruits and vegetables is an unavoidable consequence of eating a healthy diet, added fructose seems to provide little advantage over other caloric sweetners and compares unfavorably to complex carbohydrates in susceptible segments of the population.

In yeast, chronological senescence (CS) is defined as loss of viability in stationary culture. Although its relevance to the organismal aging remained unclear, yeast CS was one of the most fruitful models in aging research. Here we described a mammalian replica of yeast CS: loss of viability of overgrown "yellow" cancer cell culture. In a density and time (chronological)-dependent manner, cell culture loses the ability to re-grow in fresh medium. Rapamycin dramatically decelerated CS. Loss of viability was caused by acidification of the medium by lactic acid (lactate). Rapamycin decreased production of lactate, making conditioned medium (CM) less deadly. Both deadly CM and lactate caused loss of viability in low cell density, not preventable by either rapamycin or additional glucose. Also, NAC, LY294002, U0126, GSK733, which all indirectly inhibit mTOR and have been shown to suppress the senescent phenotype in traditional models of mammalian cell senescence, also decreased lactate production and decelerated CS. We discuss that although CS does not mimic organismal aging, the same signal transduction pathways that drive CS also drive aging.

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.

We hypothesize that the efficacy of doxorubicin (DOX) can be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. To test this hypothesis, a unique surface modification technique was used to create PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulating DOX. An avidin-biotin coupling system was used to control poly(ethylene glycol) conjugation to the surface of PLGA nanoparticles, of diameter approximately 130 nm, loaded with DOX to 5% (wt/wt). Encapsulation in nanoparticles did not compromise the efficacy of DOX; drug-loaded nanoparticles were found to be at least as potent as free DOX against A20 murine B-cell lymphoma cells in culture and of comparable efficacy against subcutaneously implanted tumors. Cardiotoxicity in mice as measured by echocardiography, serum creatine phosphokinase (CPK), and histopathology was reduced for DOX-loaded nanoparticles as compared with free DOX. Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening over control animals, whereas nanoparticle-encapsulated DOX produced none of these pathological changes.

UNASSIGNED

The efficacy of doxorubicin (DOX) may be maximized and dose-limiting cardiotoxicity minimized by controlled release from PEGylated nanoparticles. Administration of 18 mg/kg of free DOX induced a sevenfold increase in CPK levels and significant decreases in left ventricular fractional shortening in mice, whereas nanoparticle-encapsulated DOX produced none of these pathological changes.

Understanding the in vivo behavior of nanoparticles is critical for the translation of nanomedicine from laboratory research to clinical trials. In this work, in vivo Forster resonance energy transfer (FRET) imaging was employed to monitor the release of hydrophobic molecules from circulating poly(ethylene glycol)-poly( D, L-lactic acid) (PEG-PDLLA) micelles. A lipophilic FRET pair (DiIC(18) and DiOC(18)) was physically entrapped into micelle cores by mimicking the loading of hydrophobic drugs. The FRET efficiency was found significantly reduced within 15 min after intravenous injection, implying that DiIC(18) and DiOC(18) quickly escaped from the circulating micelles. FRET spectroscopy studies further demonstrated that alpha- and beta-globulins were major factors for the observed fast release, while gamma-globulins, albumin, and red blood cells played minor roles. These results provide useful information for developing blood-stable micelles to deliver hydrophobic drugs to the target site via prolonged circulation and extravasation from the vascular system.