Differential effects of commensal bacteria on progenitor cell adhesion, division symmetry and tumorigenesis in the Drosophila intestine.

Microbial factors influence homeostatic and oncogenic growth in the intestinal epithelium. However, we know little about immediate effects of commensal bacteria on stem cell division programs. In this study, we examined the effects of commensal Lactobacillus species on homeostatic and tumorigenic stem cell proliferation in the female Drosophila intestine. We identified Lactobacillus brevis as a potent stimulator of stem cell divisions. In a wild-type midgut, L.brevis activates growth regulatory pathways that drive stem cell divisions. In a Notch-deficient background, L.brevis-mediated proliferation causes rapid expansion of mutant progenitors, leading to accumulation of large, multi-layered tumors throughout the midgut. Mechanistically, we showed that L.brevis disrupts expression and subcellular distribution of progenitor cell integrins, supporting symmetric divisions that expand intestinal stem cell populations. Collectively, our data emphasize the impact of commensal microbes on division and maintenance of the intestinal progenitor compartment.

Enzymatically derived sunflower protein hydrolysate and peptides inhibit NFκB and promote monocyte differentiation to a dendritic cell phenotype.

Enzymatic hydrolysis of proteins produces bioactive peptides that have the potential to provide health benefits. This study examined the inflammatory- and immune-modulating properties of a flavourzyme-derived sunflower protein hydrolysate (SPH) and peptides. The SPH was fractionated into <1, 1-3, 3-5, and >5 kDa peptides by membrane ultrafiltration. The SPH blunted IL-1ß stimulated NFκB activation and boosted IL-4/GM-CSF induced expression of surface markers CD14 and CD86, indicating maturation into a dendritic cell (DC) phenotype. Testing of SPH membrane ultrafiltration and HPLC fractions indicated that smaller and non-polar peptides were the most potent, respectively. Four novel peptides (YFVP, SGRDP, MVWGP and TGSYTEGWS) were identified and all of them blunted IL-1ß stimulated NFκB activation. The peptides also boosted IL-4/GM-CSF induction of CD14, while only MVWGP and TGSYTEGWS boosted the expression of CD86. MVWGP was the most potent immune-modulatory peptide across all cellular assays, which was attributed to the presence of a methionine residue.

Genomic and phenotypic effects of inbreeding across two different hatchery management regimes in Chinook salmon.

Genomic approaches permit direct estimation of inbreeding and its effect on fitness. We used genomic-based estimates of inbreeding to investigate their relationship with eight adult traits in a captive-reared Pacific salmonid that is released into the wild. Estimates were also used to determine whether alternative broodstock management approaches reduced risks of inbreeding. Specifically, 1,100 unlinked restriction-site associated (RAD) loci were used to compare pairwise relatedness, derived from a relationship matrix, and individual inbreeding, estimated by comparing observed and expected homozygosity, across four generations in two hatchery lines of Chinook salmon that were derived from the same source. The lines are managed as "integrated" with the founding wild stock, with ongoing gene flow, and as "segregated" with no gene flow. While relatedness and inbreeding increased in the first generation of both lines, possibly due to population subdivision caused by hatchery initiation, the integrated line had significantly lower levels in some subsequent generations (relatedness: F2 -F4 ; inbreeding F2 ). Generally, inbreeding was similar between the lines despite large differences in effective numbers of breeders. Inbreeding did not affect fecundity, reproductive effort, return timing, fork length, weight, condition factor, and daily growth coefficient. However, it delayed spawn timing by 1.75 days per one standard deviation increase in F (~0.16). The results indicate that integrated management may reduce inbreeding but also suggest that it is relatively low in a small, segregated hatchery population that maximized number of breeders. Our findings demonstrate the utility of genomics to monitor inbreeding under alternative management strategies in captive breeding programs.

Vibrio cholerae-Symbiont Interactions Inhibit Intestinal Repair in Drosophila.

Pathogen-mediated damage to the intestinal epithelium activates compensatory growth and differentiation repair programs in progenitor cells. Accelerated progenitor growth replenishes damaged tissue and maintains barrier integrity. Despite the importance of epithelial renewal to intestinal homeostasis, we know little about the effects of pathogen-commensal interactions on progenitor growth. We find that the enteric pathogen Vibrio cholerae blocks critical growth and differentiation pathways in Drosophila progenitors, despite extensive damage to epithelial tissue. We show that the inhibition of epithelial repair requires interactions between the Vibrio cholerae type six secretion system and a community of common symbiotic bacteria, as elimination of the gut microbiome is sufficient to restore homeostatic growth in infected intestines. This work highlights the importance of pathogen-symbiont interactions for intestinal immune responses and outlines the impact of the type six secretion system on pathogenesis.

Maintaining a wild phenotype in a conservation hatchery program for Chinook salmon: The effect of managed breeding on early male maturation.

In many salmonid species, age and size at maturation is plastic and influenced by the interaction between genetic and environmental factors. Hatchery reared salmon often mature at an earlier age and smaller size than wild fish. Modern salmon conservation efforts have focused on managing the level of gene flow between hatchery and natural origin fish to minimize potential genotypic and phenotypic change. In salmonids, maturation probability is dependent on exceeding a genetically set threshold in growth rate and energetic status (and by association, body size) referred to as the probabalisitic maturation reaction norm (PMRN). Over fourteen years, we monitored the frequency of age-2 precocious male maturation (common term: age-2 minijack rate) and the PMRN of natural founder (FNDR), integrated natural-hatchery (INT), and segregated hatchery (SEG) broodlines of spring Chinook salmon, Oncorhynchus tshawytscha. The average age-2 minijack rate (± SEM) of the FNDR, INT and SEG broodlines was 48.2 ± 5.2%, 41.9 ± 3.6% and 30.9 ± 4.7%, respectively. Additionally, the PMRN WP50 (predicted weight at 50% maturation) of the SEG broodline was significantly greater (20.5 g) than that of the FNDR/INT broodlines (18.2 g). We also conducted a common garden experiment exploring the effects of less than one [INT (0-1)], one [SEG (1)] or two [SEG (2)] generations of hatchery culture on the age-2 minijack rate and PMRN WP50. Growth was not significantly different among broodlines, but age-2 minijack rates were significantly lower following two consecutive generations of hatchery culture: [INT (0-1): 68.3 ± 1.7%], [SEG (1): 70.3 ± 1.8%] and [SEG (2): 58.6 ± 0.4%] and the PMRN WP50 was significantly higher by 6.1 g after two generations of SEG culture. These results indicate that managed gene flow reduces phenotypic divergence, but may serve to maintain potentially undesirably high age-2 minijack rates in salmon conservation hatchery programs.

Monoassociation with Lactobacillus plantarum Disrupts Intestinal Homeostasis in Adult Drosophila melanogaster.

Adult Drosophila melanogaster raised in the absence of symbiotic bacteria have fewer intestinal stem cell divisions and a longer life span than their conventionally reared counterparts. However, we do not know if increased stem cell divisions are essential for symbiont-dependent regulation of longevity. To determine if individual symbionts cause aging-dependent death in Drosophila, we examined the impacts of common symbionts on host longevity. We found that monoassociation of adult Drosophila with Lactobacillus plantarum, a widely reported fly symbiont and member of the probiotic Lactobacillus genus, curtails adult longevity relative to germfree counterparts. The effects of Lactobacillus plantarum on life span were independent of intestinal aging. Instead, we found that association with Lactobacillus plantarum causes an extensive intestinal pathology within the host, characterized by loss of stem cells, impaired epithelial renewal, and a gradual erosion of epithelial ultrastructure. Our study uncovers an unknown aspect of Lactobacillus plantarum-Drosophila interactions and establishes a simple model to characterize symbiont-dependent disruption of intestinal homeostasis.IMPORTANCE Under homeostatic conditions, gut bacteria provide molecular signals that support the organization and function of the host intestine. Sudden shifts in the composition or distribution of gut bacterial communities impact host receipt of bacterial cues and disrupt tightly regulated homeostatic networks. We used the Drosophila melanogaster model to determine the effects of prominent fly symbionts on host longevity and intestinal homeostasis. We found that monoassociation with Lactobacillus plantarum leads to a loss of intestinal progenitor cells, impaired epithelial renewal, and disruption of gut architecture as flies age. These observations uncover a novel phenotype caused by monoassociation of a germfree host with a common symbiont and establish a simple model to characterize symbiont-dependent loss of intestinal homeostasis.

Genomewide association analyses of fitness traits in captive-reared Chinook salmon: Applications in evaluating conservation strategies.

A novel application of genomewide association analyses is to use trait-associated loci to monitor the effects of conservation strategies on potentially adaptive genetic variation. Comparisons of fitness between captive- and wild-origin individuals, for example, do not reveal how captive rearing affects genetic variation underlying fitness traits or which traits are most susceptible to domestication selection. Here, we used data collected across four generations to identify loci associated with six traits in adult Chinook salmon (Oncorhynchus tshawytscha) and then determined how two alternative management approaches for captive rearing affected variation at these loci. Loci associated with date of return to freshwater spawning grounds (return timing), length and weight at return, age at maturity, spawn timing, and daily growth coefficient were identified using 9108 restriction site-associated markers and random forest, an approach suitable for polygenic traits. Mapping of trait-associated loci, gene annotations, and integration of results across multiple studies revealed candidate regions involved in several fitness-related traits. Genotypes at trait-associated loci were then compared between two hatchery populations that were derived from the same source but are now managed as separate lines, one integrated with and one segregated from the wild population. While no broad-scale change was detected across four generations, there were numerous regions where trait-associated loci overlapped with signatures of adaptive divergence previously identified in the two lines. Many regions, primarily with loci linked to return and spawn timing, were either unique to or more divergent in the segregated line, suggesting that these traits may be responding to domestication selection. This study is one of the first to utilize genomic approaches to demonstrate the effectiveness of a conservation strategy, managed gene flow, on trait-associated-and potentially adaptive-loci. The results will promote the development of trait-specific tools to better monitor genetic change in captive and wild populations.

Commensal pathogen competition impacts host viability.

While the structure and regulatory networks that govern type-six secretion system (T6SS) activity of Vibrio cholerae are becoming increasingly clear, we know less about the role of T6SS in disease. Under laboratory conditions, V. cholerae uses T6SS to outcompete many Gram-negative species, including other V. cholerae strains and human commensal bacteria. However, the role of these interactions has not been resolved in an in vivo setting. We used the Drosophila melanogaster model of cholera to define the contribution of T6SS to V. cholerae pathogenesis. Here, we demonstrate that interactions between T6SS and host commensals impact pathogenesis. Inactivation of T6SS, or removal of commensal bacteria, attenuates disease severity. Reintroduction of the commensal, Acetobacter pasteurianus, into a germ-free host is sufficient to restore T6SS-dependent pathogenesis in which T6SS and host immune responses regulate viability. Together, our data demonstrate that T6SS acts on commensal bacteria to promote the pathogenesis of V. cholerae.

Gromwell (Lithospermum erythrorhizon) root extract protects against glycation and related inflammatory and oxidative stress while offering UV absorption capability.

Glycation and advanced glycation end products (AGE) damage skin which is compounded by AGE-induced oxidative stress and inflammation. Lip and facial skin could be susceptible to glycation damage as they are chronically stressed. As Gromwell (Lithospermum erythrorhizon) root (GR) has an extensive traditional medicine history that includes providing multiple skin benefits, our objective was to determine whether GR extract and its base naphthoquinone, shikonin, might protect skin by inhibiting glycation, increasing oxidative defenses, suppressing inflammatory responses and offering ultraviolet (UV) absorptive potential in lip and facial cosmetic matrices. We show GR extract and shikonin dose-dependently inhibited glycation and enhanced oxidative defenses through nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element activation. Inflammatory targets, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and tumor necrosis factor alpha, were suppressed by GR extract and shikonin. Glyoxalase 1 (GLO1) and glutathione synthesis genes were significantly upregulated by GR extract and shikonin. GR extract boosted higher wavelength UV absorption in select cosmetic matrices. Rationale for the use of GR extract and shikonin are supported by our research. By inhibiting glycation, modulating oxidative stress, suppressing inflammation and UV-absorptive properties, GR extract and shikonin potentially offer multiple skin benefits.

A versatile method for the determination of photochemical quantum yields via online UV-Vis spectroscopy.

We have developed a simple method for determining the quantum yields of photo-induced reactions. Our setup features a fibre coupled UV-Vis spectrometer, LED irradiation sources, and a calibrated spectrophotometer for precise measurements of the LED photon flux. The initial slope in time-resolved absorbance profiles provides the quantum yield. We show the feasibility of our methodology for the kinetic analysis of photochemical reactions and quantum yield determination. The typical chemical actinometers, ferrioxalate and ortho-nitrobenzaldehyde, as well as riboflavin, a spiro-compound, phosphorus- and germanium-based photoinitiators for radical polymerizations and the frequently utilized photo-switch azobenzene serve as paradigms. The excellent agreement of our results with published data demonstrates the high potential of the proposed method as a convenient alternative to the time-consuming chemical actinometry.

Star-shaped Polymers through Simple Wavelength-Selective Free-Radical Photopolymerization.

Star-shaped polymers represent highly desired materials in nanotechnology and life sciences, including biomedical applications (e.g., diagnostic imaging, tissue engineering, and targeted drug delivery). Herein, we report a straightforward synthesis of wavelength-selective multifunctional photoinitiators (PIs) that contain a bisacylphosphane oxide (BAPO) group and an α-hydroxy ketone moiety within one molecule. By using three different wavelengths, these photoactive groups can be selectively addressed and activated, thereby allowing the synthesis of ABC-type miktoarm star polymers through a simple, highly selective, and robust free-radical polymerization method. The photochemistry of these new initiators and the feasibility of this concept were investigated in unprecedented detail by using various spectroscopic techniques.

Wavelength-Dependent Photochemical Stability of Photoinitiator-Derived Macromolecular Chain Termini.

Herein, we report the unique-and first time-wavelength-dependent investigation with strictly monochromatic light of 305-405 nm wavelength into the stability of photoinitiator-derived chain termini of poly(methyl methacrylate) using a tunable laser system fused with pulsed-laser irradiation and size exclusion chromatography hyphenated to high-resolution electrospray mass spectrometry (PLI-SEC-ESI-MS). We assess several substitution patterns of methyl groups on the common benzoyl-type radical fragment. Critically, methyl substitution in the 2- and 6-positions of the benzoyl moiety, i.e., in both ortho-positions, resulted in stable chain ends up to approximately 350 nm. The stability can be attributed to a blue-shift of the n-π* transitions (relevant for the end group reactivity) as predicted by earlier density functional theory (DFT) calculations on model species. In sharp contrast, our experiments show a far reduced stability of the end groups commencing from 400 nm onwards, when the dual ortho-methyl substitution in the benzoyl fragment is missing. Thus, we demonstrate that the substitution pattern on the phenyl ring of the benzoyl group dictates the chain end stability as a function of wavelength in excellent agreement with the quantum chemical predictions. Our study thus provides critical insights into selecting suitable photoinitiation systems for specific wavelength regimes.

Comparative evaluation of the genomes of three common Drosophila-associated bacteria.

Drosophila melanogaster is an excellent model to explore the molecular exchanges that occur between an animal intestine and associated microbes. Previous studies in Drosophila uncovered a sophisticated web of host responses to intestinal bacteria. The outcomes of these responses define critical events in the host, such as the establishment of immune responses, access to nutrients, and the rate of larval development. Despite our steady march towards illuminating the host machinery that responds to bacterial presence in the gut, there are significant gaps in our understanding of the microbial products that influence bacterial association with a fly host. We sequenced and characterized the genomes of three common Drosophila-associated microbes: Lactobacillus plantarum, Lactobacillus brevis and Acetobacter pasteurianus For each species, we compared the genomes of Drosophila-associated strains to the genomes of strains isolated from alternative sources. We found that environmental Lactobacillus strains readily associated with adult Drosophila and were similar to fly isolates in terms of genome organization. In contrast, we identified a strain of A. pasteurianus that apparently fails to associate with adult Drosophila due to an inability to grow on fly nutrient food. Comparisons between association competent and incompetent A. pasteurianus strains identified a short list of candidate genes that may contribute to survival on fly medium. Many of the gene products unique to fly-associated strains have established roles in the stabilization of host-microbe interactions. These data add to a growing body of literature that examines the microbial perspective of host-microbe relationships.

Bis(mesitoyl)phosphinic acid: photo-triggered release of metaphosphorous acid in solution.

Bis(mesitoyl)phosphinic acid and its sodium salt display a unique photo-induced reactivity: both derivatives stepwise release two mesitoyl radicals and, remarkably, metaphosphorous acid (previously postulated as transient species in the gas phase), providing a new phosphorus-based reagent.

Bone health nutraceuticals alter microarray mRNA gene expression: A randomized, parallel, open-label clinical study.

BACKGROUND AND OBJECTIVE: Dietary intake of fruits and vegetables has been suggested to have a role in promoting bone health. More specifically, the polyphenols they contain have been linked to physiological effects related to bone mineral density and bone metabolism. In this research, we use standard microarray analyses of peripheral whole blood from post-menopausal women treated with two fixed combinations of plant extracts standardized to polyphenol content to identify differentially expressed genes relevant to bone health. METHODS: In this 28-day open-label study, healthy post-menopausal women were randomized into three groups, each receiving one of three investigational fixed combinations of plant extracts: an anti-resorptive (AR) combination of pomegranate fruit (Punica granatum L.) and grape seed (Vitis vinifera L.) extracts; a bone formation (BF) combination of quercetin (Dimorphandra mollis Benth) and licorice (Glycyrrhiza glabra L.) extracts; and a fixed combination of all four plant extracts (AR plus BF). Standard microarray analysis was performed on peripheral whole blood samples taken before and after each treatment. Annotated genes were analyzed for their association to bone health by comparison to a gene library. RESULTS: The AR combination down-regulated a number of genes involved in reduction of bone resorption including cathepsin G (CTSG) and tachykinin receptor 1 (TACR1). The AR combination also up-regulated genes associated with formation of extracellular matrix including heparan sulfate proteoglycan 2 (HSPG2) and hyaluronoglucosaminidase 1 (HYAL1). In contrast, treatment with the BF combination resulted in up-regulation of bone morphogenetic protein 2 (BMP-2) and COL1A1 (collagen type I α1) genes which are linked to bone and collagen formation while down-regulating genes linked to osteoclastogenesis. Treatment with a combination of all four plant extracts had a distinctly different effect on gene expression than the results of the AR and BF combinations individually. These results could be due to multiple feedback systems balancing activities of osteoblasts and osteoclasts. CONCLUSION: In summary, this ex-vivo microarray study indicated that the pomegranate, grape seed, quercetin and licorice combinations of plant extracts modulated gene expression for both osteoclastic and osteogenic processes.

Effectiveness of managed gene flow in reducing genetic divergence associated with captive breeding.

Captive breeding has the potential to rebuild depressed populations. However, associated genetic changes may decrease restoration success and negatively affect the adaptive potential of the entire population. Thus, approaches that minimize genetic risks should be tested in a comparative framework over multiple generations. Genetic diversity in two captive-reared lines of a species of conservation interest, Chinook salmon (Oncorhynchus tshawytscha), was surveyed across three generations using genome-wide approaches. Genetic divergence from the source population was minimal in an integrated line, which implemented managed gene flow by using only naturally-born adults as captive broodstock, but significant in a segregated line, which bred only captive-origin individuals. Estimates of effective number of breeders revealed that the rapid divergence observed in the latter was largely attributable to genetic drift. Three independent tests for signatures of adaptive divergence also identified temporal change within the segregated line, possibly indicating domestication selection. The results empirically demonstrate that using managed gene flow for propagating a captive-reared population reduces genetic divergence over the short term compared to one that relies solely on captive-origin parents. These findings complement existing studies of captive breeding, which typically focus on a single management strategy and examine the fitness of one or two generations.

Echinacea purpurea root extract inhibits TNF release in response to Pam3Csk4 in a phosphatidylinositol-3-kinase dependent manner.

Polysaccharides derived from Echinacea have historically been shown to be immunostimulatory. We describe in this work however the anti-inflammatory effect of a water extract of Echinacea purpurea roots (EPRW) that inhibited Pam3Csk4 stimulated production of TNFα by human monocytic THP-1 cells. The polyphenols and alkylamides typically found in Echinacea extracts were absent in EPRW suggesting that the anti-inflammatory component(s) was a polysaccharide. This anti-inflammatory activity was shown to be mediated by the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway as chemical inhibition of PI3K abolished the EPRW anti-inflammatory effect. Demonstration of phosphorylation of Akt and ribosomal S6 proteins, downstream targets of PI3K confirmed EPRW-mediated activation of this pathway. In conclusion, this observation suggests that non-alkylamide/non-polyphenolic phytochemicals from Echinacea may contribute in part to some of the anti-inflammatory therapeutic effects such as reduced severity of symptoms that have been observed in vivo in the treatment of upper respiratory tract infections with Echinacea.

Oncolytic activity of reovirus in HPV positive and negative head and neck squamous cell carcinoma.

BACKGROUND: The management of patients with advanced stages of head and neck cancer requires a multidisciplinary and multimodality treatment approach which includes a combination of surgery, radiation, and chemotherapy. These toxic treatment protocols have significantly improved survival outcomes in a distinct population of human papillomavirus (HPV) associated oropharyngeal cancer. HPV negative head and neck squamous cell carcinoma (HNSCC) remains a challenge to treat because there is only a modest improvement in survival with the present treatment regimens, requiring innovative and new treatment approaches. Oncolytic viruses used as low toxicity adjunct cancer therapies are novel, potentially effective treatments for HNSCC. One such oncolytic virus is Respiratory Orphan Enteric virus or reovirus. Susceptibility of HNSCC cells towards reovirus infection and reovirus-induced cell death has been previously demonstrated but has not been compared in HPV positive and negative HNSCC cell lines. OBJECTIVES: To compare the infectivity and oncolytic activity of reovirus in HPV positive and negative HNSCC cell lines. METHODS: Seven HNSCC cell lines were infected with serial dilutions of reovirus. Two cell lines (UM-SCC-47 and UM-SCC-104) were positive for type 16 HPV. Infectivity was measured using a cell-based ELISA assay 18 h after infection. Oncolytic activity was determined using an alamar blue viability assay 96 h after infection. Non-linear regression models were used to calculate the amounts of virus required to infect and to cause cell death in 50% of a given cell line (EC50). EC50 values were compared. RESULTS: HPV negative cells were more susceptible to viral infection and oncolysis compared to HPV positive cell lines. EC50 for infectivity at 18 h ranged from multiplicity of infection (MOI) values (PFU/cell) of 18.6 (SCC-9) to 3133 (UM-SCC 104). EC50 for cell death at 96 h ranged from a MOI (PFU/cell) of 1.02×10(2) (UM-SCC-14A) to 3.19×10(8) (UM-SCC-47). There was a 3×10(6) fold difference between the least susceptible cell line (UM-SCC-47) and the most susceptible line (UM-SCC 14A) EC50 for cell death at 96 h. CONCLUSIONS: HPV negative HNSCC cell lines appear to demonstrate greater reovirus infectivity and virus-mediated oncolysis compared to HPV positive HNSCC. Reovirus shows promise as a novel therapy in HNSCC, and may be of particular benefit in HPV negative patients.

Immunomodulatory effects of Lippia sidoides extract: induction of IL-10 through cAMP and p38 MAPK-dependent mechanisms.

Lippia sidoides is an aromatic shrub that grows wild in the northeastern region of Brazil. In local traditional medicine, the aerial portions of this species are used as anti-infectives, antiseptics, spasmolytics, sedatives, hypotensives, and anti-inflammatory agents. In this research, we evaluate the potential immunological properties of Lippia extract through in vitro analysis of its ability to modulate intracellular cyclic adenosine monophosphate (cAMP) levels and interleukin-10 (IL-10) production. These results show that Lippia extract increases intracellular cAMP through the inhibition of phosphodiesterase activity. They also demonstrate that Lippia extract increases IL-10 production in THP-1 monocytes through both an increase in intracellular cAMP and the activation of p38 MAPK. These results suggest that the Lippia-mediated inhibition of phosphodiesterase activity and the subsequent increase in intracellular cAMP may explain some of the biological activities associated with L. sidoides. In addition, the anti-inflammatory activity of L. sidoides may also be due, in part, to its ability to induce IL-10 production through the inhibition of cyclic nucleotide-dependent phosphodiesterase activity and by its activation of the p38 MAPK pathway.

A targeted approach for evaluating preclinical activity of botanical extracts for support of bone health.

Using a sequential in vitro/in vivo approach, we tested the ability of botanical extracts to influence biomarkers associated with bone resorption and bone formation. Pomegranate fruit and grape seed extracts were found to exhibit anti-resorptive activity by inhibiting receptor activator of nuclear factor-κB ligand (RANKL) expression in MG-63 cells and to reduce IL-1ß-stimulated calvarial (45)Ca loss. A combination of pomegranate fruit and grape seed extracts were shown to be effective at inhibiting bone loss in ovariectomised rats as demonstrated by standard histomorphometry, biomechanical and bone mineral density measurements. Quercetin and licorice extract exhibited bone formation activity as measured by bone morphogenetic protein-2 (BMP-2) promoter activation, increased expression of BMP-2 mRNA and protein levels, and promotion of bone growth in cultured mouse calvariae. A combination of quercetin and licorice extract demonstrated a potential for increasing bone mineral density in an intact female rat model as compared with controls. The results from this sequential in vitro/in vivo research model yielded botanical extract formulas that demonstrate significant potential benefits for bone health.