Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.

Current strategies to limit macrophage adhesion, fusion and fibrous capsule formation in the foreign body response have focused on modulating material surface properties. We hypothesize that topography close to biological scale, in the micron and nanometric range, provides a passive approach without bioactive agents to modulate macrophage behavior. In our study, topography-induced changes in macrophage behavior was examined using parallel gratings (250 nm-2 mum line width) imprinted on poly(epsilon-caprolactone) (PCL), poly(lactic acid) (PLA) and poly(dimethyl siloxane) (PDMS). RAW 264.7 cell adhesion and elongation occurred maximally on 500 nm gratings compared to planar controls over 48 h. TNF-alpha and VEGF secretion levels by RAW 264.7 cells showed greatest sensitivity to topographical effects, with reduced levels observed on larger grating sizes at 48 h. In vivo studies at 21 days showed reduced macrophage adhesion density and degree of high cell fusion on 2 mum gratings compared to planar controls. It was concluded that topography affects macrophage behavior in the foreign body response on all polymer surfaces examined. Topography-induced changes, independent of surface chemistry, did not reveal distinctive patterns but do affect cell morphology and cytokine secretion in vitro, and cell adhesion in vivo particularly on larger size topography compared to planar controls.

Phenolic compounds are important constituents of food products of plant origin. These compounds are directly related to sensory characteristics of foods such as flavour, astringency, and colour. In addition, the presence of phenolic compounds on the diet is beneficial to health due to their chemopreventive activities against carcinogenesis and mutagenesis, mainly due to their antioxidant activities. Lactic acid bacteria (LAB) are autochthonous microbiota of raw vegetables. To get desirable properties on fermented plant-derived food products, LAB has to be adapted to the characteristics of the plant raw materials where phenolic compounds are abundant. Lactobacillus plantarum is the commercial starter most frequently used in the fermentation of food products of plant origin. However, scarce information is still available on the influence of phenolic compounds on the growth and viability of L. plantarum and other LAB species. Moreover, metabolic pathways of biosynthesis or degradation of phenolic compounds in LAB have not been completely described. Results obtained in L. plantarum showed that L. plantarum was able to degrade some food phenolic compounds giving compounds influencing food aroma as well as compounds presenting increased antioxidant activity. Recently, several L. plantarum proteins involved in the metabolism of phenolic compounds have been genetically and biochemically characterized. The aim of this review is to give a complete and updated overview of the current knowledge among LAB and food phenolics interaction, which could facilitate the possible application of selected bacteria or their enzymes in the elaboration of food products with improved characteristics.

Brock, Thomas D. (Indiana University, Bloomington), Barbara Peacher, and Deborah Pierson. A survey of the bacteriocines of enterococci. J. Bacteriol. 86:702-707. 1963.-A survey has been made of bacteriocine production by a wide variety of well-characterized strains of group D streptococci. On the basis of spectrums and sensitivity to chloroform, heat, and proteolytic enzymes, five distinct bacteriocines can be defined. Type 1 is produced by all Streptococcus zymogenes (S. faecalis var. zymogenes) strains, is active against a wide variety of gram-positive bacteria, and is also a hemolysin. Type 2 is produced by some S. liquefaciens (S. faecalis var. liquefaciens) strains, and acts on many enterococci as well as on certain other lactic acid bacteria. Type 3 is produced by certain strains of both S. faecalis and S. faecium, and inhibits a wide variety of group D streptococci, but is inactive against all other lactic acid bacteria tested except Leuconostoc citrovorum. Type 4 is produced by certain S. faecium strains and resembles in certain ways the type 3 activity, but differs from it in other ways. Type 5 has been found to be produced by only one proteolytic strain of S. zymogenes, and this bacteriocine has a very narrow spectrum. The strain that produces this bacteriocine also produces type 1 activity. No strain is sensitive to a bacteriocine of the type it produces.

While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.

Acquired antibiotic resistance, i.e. resistance genes located on conjugative or mobilizable plasmids and transposons can be found in species living in habitats (e.g. human and animal intestines) which are regularly challenged with antibiotics. Most data are available for enterococci and enteric lactobacilli. Raw material from animals (milk and meat) which are inadvertantly contaminated with fecal matters during production will carry antibiotic resistant lactic acid bacteria into the final fermented products such as raw milk cheeses and raw sausages. The discovered conjugative genetic elements of LAB isolated from animals and food are very similar to elements studied previously in pathogenic streptococci and enterococci, e.g. theta-type replicating plasmids of the pAMbeta1, pIP501-family, and transposons of the Tn916-type. Observed resistance genes include known genes like tetM, ermAM, cat, sat and vanA. A composite 29,871 bp resistance plasmid detected in Lactococcus lactis subsp. lactis isolated from a raw milk soft cheese contains tetS previously described in Listeria monocytogenes, cat and str from Staphylococcus aureus. Three out of five IS elements on the plasmid are almost or completely identical to IS1216 present in the vanA resistance transposon Tn1546. These data support the view that in antibiotic challenged habitats lactic acid bacteria like other bacteria participate in the communication systems which transfer resistance traits over species and genus borders. The prevalence of such bacteria with acquired resistances like enterococci is high in animals (and humans) which are regularly treated with antibiotics. The transfer of antibiotic resistant bacteria from animals into fermented and other food can be avoided if the raw substrate milk or meat is pasteurized or heat treated. Antibiotic resistance traits as selectable markers in genetic modification of lactic acid bacteria for different purposes are presently being replaced, e.g. by metabolic traits to generate food-grade vectors.

Neonatal rat calvarial osteoblasts were cultured in 90% porous, 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) foam scaffolds for up to 56 days to examine the effects of the cell seeding density, scaffold pore size, and foam thickness on the proliferation and function of the cells in this three-dimensional environment. Osteoblasts were seeded at either 11.1 x 10(5) or 22.1 x 10(5) cells per cm2 onto PLGA scaffolds having pore sizes in the range of 150-300 or 500-710 microm with a thickness of either 1.9 or 3.2 mm. After 1 day in culture, 75.6 and 68.6% of the seeded cells attached and proliferated on the 1.9 mm thick scaffolds of 150-300 microm pore size for the low and high seeding densities, respectively. The number of osteoblasts continued to increase throughout the study and eventually leveled off near 56 days, as indicated by a quantitative DNA assay. Osteoblast/foam constructs with a low cell seeding density achieved comparable DNA content and alkaline phosphatase (ALPase) activity after 14 days, and mineralization results after 56 days to those with a high cell seeding density. A maximum penetration depth of osseous tissue of 220+/-40 microm was reached after 56 days in the osteoblast/foam constructs of 150-300 microm pore size initially seeded with a high cell density. For constructs of 500-710 microm pore size, the penetration depth was 190+/-40 microm under the same conditions. Scaffold pore size and thickness did not significantly affect the proliferation or function of osteoblasts as demonstrated by DNA content, ALPase activity, and mineralized tissue formation. These data show that comparable bone-like tissues can be engineered in vitro over a 56 day period using different rat calvarial osteoblast seeding densities onto biodegradable polymer scaffolds with pore sizes in the range of 150-710 microm. When compared with the results of a previous study where similar polymer scaffolds were seeded and cultured with marrow stromal cells, this study demonstrates that PLGA foams are suitable substrates for osteoblast growth and differentiated function independent of cell source.

The end products of the metabolism of phenylalanine, tyrosine and tryptophan by growing cultures of clostridia have been identified. The species used were Clostridium aminovalericum; C. bifermentans; C. botulinum proteolytic type A; C. botulinum proteolytic type B; C. cochlearium; C. difficile; C. ghoni; C. histolyticum; C. lentoputrescens; C. limosum; C. lituseburense; C. malenomenatum; C. mangenoti; C. propionicum; C. putrefaciens; C. sordellii; C. sporogenes; C. sporosphaeroides; C. sticklandii; C. subterminale; C. tetani; C. tetanomorphum. The mixture of aromatic compounds formed, which depended upon the species, included phenyl acetic acid, phenyl propionic acid, phenyl lactic acid, phenol, p-cresol, p-hydroxy phenyl acetic acid, p-hydroxy phenyl propionic acid, indole, indole acetic acid and indole propionic acid.

The aim of this study was to determine if probiotics reduce epithelial injury following exposure to Escherichia coli O157:H7 and E. coli O127:H6. The pretreatment of intestinal (T84) cells with lactic acid-producing bacteria reduced the pathogen-induced drop in transepithelial electrical resistance. These findings demonstrate that probiotics prevent epithelial injury induced by attaching-effacing bacteria.

Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and alpha-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with alpha-amylase enzymes (family GH13), with a predicted permuted (beta/alpha)(8) barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of alpha-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize beta-fructan polymers with either beta-(2-->6) (inulin) or beta-(2-->1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed beta-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either beta-(2-->6) or beta-(2-->1) linkages, degree and type of branching, and fructan molecular mass remain to be identified.

Antibacterial bonding agents could combat recurrent caries at the tooth-composite margins. The objectives of this study were to develop novel antibacterial dentin primers containing quaternary ammonium dimethacrylate (QADM) and nanoparticles of silver (NAg), and to investigate the effects on dentin bond strength and dental plaque microcosm biofilms for the first time. Scotchbond Multi-Purpose ("SBMP") bonding agent was used. QADM and NAg were incorporated into SBMP primer, yielding 4 primers: SBMP primer (control), control + 10% QADM (mass), control + 0.05% NAg, and control + 10% QADM + 0.05% NAg. Human saliva was collected to grow microcosm biofilms. The NAg particle size (mean ± SD; n = 100) was 2.7 ± 0.6 nm. Dentin shear bond strengths (n = 10) with human third molars were approximately 30 MPa for all groups (p>> 0.1). QADM-NAg-containing primer increased the bacteria inhibition zone by 9-fold, compared with control primer (p < 0.05). QADM-NAg-containing primer reduced lactic acid production and colony-forming units of total micro-organisms, total streptococci, and mutans streptococci by an order of magnitude. In conclusion, novel QADM-NAg-containing primers were strongly antibacterial without compromising dentin bond strength, and hence are promising to inhibit biofilms and secondary caries. The processing method of incorporating QADM and NAg together into the same primer produced the strongest antibacterial effect, which could have a wide applicability to other bonding systems.

The aim of this study is to investigate the effect of the cell culture conditions of three-dimensional polymer scaffolds seeded with rat marrow stromal cells (MSCs) cultured in different bioreactors concerning the ability of these cells to proliferate, differentiate towards the osteoblastic lineage, and generate mineralized extracellular matrix. MSCs harvested from male Sprague-Dawley rats were culture expanded, seeded on three-dimensional porous 75:25 poly(D,L-lactic-co-glycolic acid) biodegradable scaffolds, and cultured for 21 days under static conditions or in two model bioreactors (a spinner flask and a rotating wall vessel) that enhance mixing of the media and provide better nutrient transport to the seeded cells. The spinner flask culture demonstrated a 60% enhanced proliferation at the end of the first week when compared to static culture. On day 14, all cell/polymer constructs exhibited their maximum alkaline phosphatase activity (AP). Cell/polymer constructs cultured in the spinner flask had 2.4 times higher AP activity than constructs cultured under static conditions on day 14. The total osteocalcin (OC) secretion in the spinner flask culture was 3.5 times higher than the static culture, with a peak OC secretion occurring on day 18. No considerable AP activity and OC secretion were detected in the rotating wall vessel culture throughout the 21-day culture period. The spinner flask culture had the highest calcium content at day 14. On day 21, the calcium deposition in the spinner flask culture was 6.6 times higher than the static cultured constructs and over 30 times higher than the rotating wall vessel culture. Histological sections showed concentration of cells and mineralization at the exterior of the foams at day 21. This phenomenon may arise from the potential existence of nutrient concentration gradients at the interior of the scaffolds. The better mixing provided in the spinner flask, external to the outer surface of the scaffolds, may explain the accelerated proliferation and differentiation of marrow stromal osteoblasts, and the localization of the enhanced mineralization on the external surface of the scaffolds.

Vaginal bacterial communities are thought to help prevent sexually transmitted infections. Bacterial vaginosis (BV) is a common clinical syndrome in which the protective lactic acid-producing bacteria (mainly species of the Lactobacillus genus) are supplanted by a diverse array of anaerobic bacteria. Epidemiologically, BV has been shown to be an independent risk factor for adverse outcomes including preterm birth, development of pelvic inflammatory disease, and acquisition of sexually transmitted infections. Longitudinal studies of the vaginal microbiome using molecular techniques such as 16S ribosomal DNA analysis may lead to interventions that shift the vaginal microbiota toward more protective states.

The use of non-culture gene amplification techniques has improved our understanding of the composition of the vaginal bacterial ecosystem. In most healthy women in the reproductive period the predominant vaginal bacteria are one or more of the following species of Lactobacillus: L. crispatus, L. iners and L. gasseri. However, in other apparently healthy women lactobacilli may be deficient or absent, being replaced by other lactic-acid-producing bacteria: Atopobium, Megasphaera and/or Leptotrichia species. Infection and/or proliferation of pathogenic bacteria in the vagina is suppressed by lactic acid production, bacteria-generated antimicrobial products, and the local activities of the innate and cell-mediated immune systems. Vaginal epithelial cells produce a range of compounds with antimicrobial activities. These cells also possess membrane-bound Toll-like receptors that recognize pathogen-associated molecular patterns. Recognition leads to pro-inflammatory cytokine production and antigen-specific immunity. Local production of IgG and IgA antibodies can also be initiated in the endocervix and vagina in response to infection.

The use of plants is as old as the mankind. Natural products are cheap and claimed to be safe. They are also suitable raw material for production of new synthetic agents. Rosemary (Rosmarinus officinalis Linn.) is a common household plant grown in many parts of the world. It is used for flavouring food, a beverage drink, as well as in cosmetics; in folk.medicine it is used as an antispasmodic in renal colic and dysmenorrhoea, in relieving respiratory disorders and to stimulate growth of hair. Extract of rosemary relaxes smooth muscles of trachea and intestine, and has choleretic, hepatoprotective and antitumerogenic activity. The most important constituents of rosemary are caffeic acid and its derivatives such as rosmarinic acid. These compounds have antioxidant effect. The phenolic compound, rosmarinic acid, obtains one of its phenolic rings from phenylalanine via caffeic acid and the other from tyrosine via dihydroxyphenyl-lactic acid. Relatively large-scale production of rosmarinic acid can be obtained from the cell culture of Coleus blumei Benth when supplied exogenously with phenylalanine and tyrosine. Rosmarinic acid is well absorbed from gastrointestinal tract and from the skin. It increases the production of prostaglandin E2 and reduces the production of leukotriene B4 in human polymorphonuclear leucocytes, and inhibits the complement system. It is concluded that rosemary and its constituents especially caffeic acid derivatives such as rosmarinic acid have a therapeutic potential in treatment or prevention of bronchial asthma, spasmogenic disorders, peptic ulcer, inflammatory diseases, hepatotoxicity, atherosclerosis, ischaemic heart disease, cataract, cancer and poor sperm motility.

Therapeutic nanoparticles (TNPs) have shown heterogeneous responses in human clinical trials, raising questions of whether imaging should be used to identify patients with a higher likelihood of NP accumulation and thus therapeutic response. Despite extensive debate about the enhanced permeability and retention (EPR) effect in tumors, it is increasingly clear that EPR is extremely variable; yet, little experimental data exist to predict the clinical utility of EPR and its influence on TNP efficacy. We hypothesized that a 30-nm magnetic NP (MNP) in clinical use could predict colocalization of TNPs by magnetic resonance imaging (MRI). To this end, we performed single-cell resolution imaging of fluorescently labeled MNPs and TNPs and studied their intratumoral distribution in mice. MNPs circulated in the tumor microvasculature and demonstrated sustained uptake into cells of the tumor microenvironment within minutes. MNPs could predictably demonstrate areas of colocalization for a model TNP, poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG), within the tumor microenvironment with >85% accuracy and circulating within the microvasculature with >95% accuracy, despite their markedly different sizes and compositions. Computational analysis of NP transport enabled predictive modeling of TNP distribution based on imaging data and identified key parameters governing intratumoral NP accumulation and macrophage uptake. Finally, MRI accurately predicted initial treatment response and drug accumulation in a preclinical efficacy study using a paclitaxel-encapsulated NP in tumor-bearing mice. These approaches yield valuable insight into the in vivo kinetics of NP distribution and suggest that clinically relevant imaging modalities and agents can be used to select patients with high EPR for treatment with TNPs.

The microencapsulation process in which the removal of the hydrophobic polymer solvent is achieved by evaporation has been widely reported in recent years for the preparation of microspheres and microcapsules based on biodegradable polymers and copolymers of hydroxy acids. The properties of biodegradable microspheres of poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) have been extensively investigated. The encapsulation of highly water soluble compounds including proteins and peptides presents formidable challenges to the researcher. The successful encapsulation of such entities requires high drug loading in the microspheres, prevention of protein degradation by the encapsulation method, and predictable release of the drug compound from the microspheres. To achieve these goals, multiple emulsion techniques and other innovative modifications have been made to the conventional solvent evaporation process.

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of beta-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known beta-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The beta-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.

BACKGROUND

Certain lactic acid bacteria may accelerate recovery from acute diarrhea. Lactobacillus reuteri is a commonly occurring Lactobacillus species with therapeutic potential in diarrhea.

METHODS

Prospective, randomized, placebo-controlled trial in two hospitals.

METHODS

Children between 6 and 36 months of age admitted for rotavirus-associated diarrhea were randomized into three groups to receive either 10(10) or 10(7) colony-forming units (cfu) of L. reuteri or a matching placebo once a day for up to 5 days.

RESULTS

The main effect of L. reuteri was on the duration of watery diarrhea. The mean (+/-SD) duration of watery diarrhea after initiation of treatment was 2.5 (1.5) days in the placebo group (n = 25) vs. 1.9 (0.9) days in the small dosage (n = 20) and 1.5 (1.1) days in the large dosage (n = 21) L. reuteri recipients (P = 0.01). By the second day of treatment watery diarrhea persisted in 80% of the placebo, 70% of the small dosage and 48% of the large dosage L. reuteri recipients (P = 0.04, large dosage vs. placebo). Stool cultures for lactobacilli confirmed that administration of L. reuteri resulted in good colonization of the GI tract. The mean (+/-SD) of total Lactobacillus count 2 days after treatment initiation was 2.8 (1.6) log 10 cfu/g in the placebo group, 4.5 (2.0) log 10 cfu/g in the small dosage L. reuteri group and 6.1 (1.2) log 10 cfu/g in the large dosage L. reuteri group (P = 0.0004).

CONCLUSIONS

L. reuteri effectively colonized the gastrointestinal tract after administration and significantly shortened the duration of watery diarrhea associated with rotavirus. There was a correlation between the dosage of L. reuteri and the clinical effect.

Excessive inflammation and failed resolution of the inflammatory response are underlying components of numerous conditions such as arthritis, cardiovascular disease, and cancer. Hence, therapeutics that dampen inflammation and enhance resolution are of considerable interest. In this study, we demonstrate the proresolving activity of sub-100-nm nanoparticles (NPs) containing the anti-inflammatory peptide Ac2-26, an annexin A1/lipocortin 1-mimetic peptide. These NPs were engineered using biodegradable diblock poly(lactic-co-glycolic acid)-b-polyethyleneglycol and poly(lactic-co-glycolic acid)-b-polyethyleneglycol collagen IV-targeted polymers. Using a self-limited zymosan-induced peritonitis model, we show that the Ac2-26 NPs (100 ng per mouse) were significantly more potent than Ac2-26 native peptide at limiting recruitment of polymononuclear neutrophils (56% vs. 30%) and at decreasing the resolution interval up to 4 h. Moreover, systemic administration of collagen IV targeted Ac2-26 NPs (in as low as 1 µg peptide per mouse) was shown to significantly block tissue damage in hind-limb ischemia-reperfusion injury by up to 30% in comparison with controls. Together, these findings demonstrate that Ac2-26 NPs are proresolving in vivo and raise the prospect of their use in chronic inflammatory diseases such as atherosclerosis.

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.

Exenatide once-weekly (EQW [2 mg s.c.]) is under development as monotherapy as an adjunct to diet and exercise or as a combination therapy with an oral antidiabetes drug(s) in adults with type 2 diabetes. This long-acting formulation contains the active ingredient of the original exenatide twice-daily (EBID) formulation encapsulated in 0.06-mm-diameter microspheres of medical-grade poly-(D,L-lactide-co-glycolide) (PLG). After mechanical suspension and subcutaneous injection by the patient, EQW microspheres hydrate in situ and adhere to one another to form an amalgam. A small amount of loosely bound surface exenatide, typically less than 1%, releases in the first few hours, whereas drug located in deeper interstices diffuses out more slowly (time to maximum, ~2 weeks). Fully encapsulated exenatide (i.e., drug initially inaccessible to diffusion) releases over a still longer period (time to maximum, ~7 weeks) as the PLG matrix hydrolyzes into lactic acid and glycolic acid, which are subsequently eliminated as carbon dioxide and water. For EQW, plasma exenatide concentrations reach the therapeutic range by 2 weeks and steady state by 6-7 weeks. This gradual approach to steady state seems to improve tolerability, as nausea is less frequent with EQW than EBID. EQW administrations may be associated with palpable skin nodules that generally resolve without further medical intervention. In comparative trials, EQW improved hemoglobin A1c more than EBID, sitagliptin, pioglitazone, or insulin glargine and reduced fasting plasma glucose more than EBID. Weight loss due to EQW or EBID was similar. EQW is the first glucose-lowering agent that is administered once weekly.

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.

BACKGROUND

Chemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies. Curcumin is a naturally occurring compound with anti-cancer activity in multiple cancers; however, its chemo/radio-sensitizing potential is not well studied in ovarian cancer. Herein, we demonstrate the effectiveness of a curcumin pre-treatment strategy for chemo/radio-sensitizing cisplatin resistant ovarian cancer cells. To improve the efficacy and specificity of curcumin induced chemo/radio sensitization, we developed a curcumin nanoparticle formulation conjugated with a monoclonal antibody specific for cancer cells.

METHODS

Cisplatin resistant A2780CP ovarian cancer cells were pre-treated with curcumin followed by exposure to cisplatin or radiation and the effect on cell growth was determined by MTS and colony formation assays. The effect of curcumin pre-treatment on the expression of apoptosis related proteins and beta-catenin was determined by Western blotting or Flow Cytometry. A luciferase reporter assay was used to determine the effect of curcumin on beta-catenin transcription activity. The poly(lactic acid-co-glycolic acid) (PLGA) nanoparticle formulation of curcumin (Nano-CUR) was developed by a modified nano-precipitation method and physico-chemical characterization was performed by transmission electron microscopy and dynamic light scattering methods.

RESULTS

Curcumin pre-treatment considerably reduced the dose of cisplatin and radiation required to inhibit the growth of cisplatin resistant ovarian cancer cells. During the 6 hr pre-treatment, curcumin down regulated the expression of Bcl-XL and Mcl-1 pro-survival proteins. Curcumin pre-treatment followed by exposure to low doses of cisplatin increased apoptosis as indicated by annexin V staining and cleavage of caspase 9 and PARP. Additionally, curcumin pre-treatment lowered beta-catenin expression and transcriptional activity. Nano-CUR was successfully generated and physico-chemical characterization of Nano-CUR indicated an average particle size of ~70 nm, steady and prolonged release of curcumin, antibody conjugation capability and effective inhibition of ovarian cancer cell growth.

CONCLUSIONS

Curcumin pre-treatment enhances chemo/radio-sensitization in A2780CP ovarian cancer cells through multiple molecular mechanisms. Therefore, curcumin pre-treatment may effectively improve ovarian cancer therapeutics. A targeted PLGA nanoparticle formulation of curcumin is feasible and may improve the in vivo therapeutic efficacy of curcumin.