Cunninghamella echinulata DSM1905 biofilm-based L-asparaginase production in pneumatically-driven bioreactors.

We evaluated by comparing the performance of three pneumatically-driven bioreactors in the production of L-asparaginase (L-ASNase), an enzyme used to treat leukaemia and lymphoma. A two-step screening process was conducted to detect Cunninghamella spp. strains producing L-ASNase. Cunninghamella echinulata DSM1905 produced the highest levels of L-ASNase during screening assays. Subsequently, fermentations were performed in bubble column (BCR), airlift (ALR), and hybrid fixed-bed airlift (FB-ALR) bioreactors to determine the best upstream bioprocess. Mycelial biomass production was higher in BCR than in ALR and FB-ALR (p ≤ 0.0322). The activity of L-ASNase produced in FB-ALR, in which the fungus grew as a consistent biofilm, was significantly higher (p ≤ 0.022) than that from ALR, which was higher than that of BCR (p = 0.036). The specific activity of ALR and FB-ALR presented no differences (p = 0.073), but it was higher than that of BCR (p ≤ 0.032). In conclusion, C. echinulata DSM1905, grown under the biofilm phenotype, produced the highest levels of L-ASNase, and FB-ALR was the best upstream system for enzyme production.

Score of fear of COVID-19 and physical activity level are related to the habitual consumption of dietary supplements.

The measures implemented to contain the COVID-19 pandemic resulted in both behavioral and lifestyle changes. The "Changes in Lifestyle-Related Behavior" instrument was developed to assess lifestyle-related behaviors in the Indian population. However, considering current knowledge, this instrument was not adapted for the Brazilian population. In addition, the relationship between fear of COVID-19 and consumption of dietary supplements has not yet been evaluated. Thus, we aimed to investigate the relationship between the use of dietary supplements with lifestyle behavior and the fear of COVID-19, as well as assess the psychometric properties of the Brazilian version of the "Changes in Lifestyle-Related Behavior" instrument. An online questionnaire assessed sociodemographic, occupational, anthropometric, physical activity (International Physical Activity Questionnaire-short form), fear of COVID-19, and lifestyle behavior data from 416 Brazilian adults (237 females; 18-60y). Mann-Whitney, Chi-square test, exploratory, and confirmatory analyses were applied. Exploratory and confirmatory analyses showed a satisfactory adequacy level of the questionnaire (CMIN/DF = 2.689; Cronbach's α = 0.60) with 5 domains ('Bad eating behavior'; 'Healthy eating'; 'Sleep quality'; 'Interest in cooking'; 'Number of portions and meals'). Lower fear of COVID-19 scores and higher levels of physical activity were found in participants who reported previous dietary supplement intake during the pandemic; in addition, the group that did not ingest dietary supplements reported greater changes in stress and anxiety levels during the pandemic (p<0.05). The intake of dietary supplements before the pandemic was associated with greater energy expenditure and better coping with the fear of COVID-19 during the pandemic. Additionally, the Changes in Lifestyle-Related Behavior tool can be used to assess lifestyle-related variables during the pandemic.

Monoterpene antifungal activities: evaluating geraniol, citronellal, and linalool on Candida biofilm, host inflammatory responses, and structure-activity relationships.

Introduction: Despite the rising concern with fungal resistance, a myriad of molecules has yet to be explored. Geraniol, linalool, and citronellal are monoterpenes with the same molecular formula (C10H18O), however, neither the effect of these compounds on inflammatory axis induced by Candida spp. nor the antibiofilm Structure-Activity Relationship (SAR) have been well-investigated. Herein we analyzed geraniol, linalool and citronellal antifungal activity, cytotoxicity, and distinctive antibiofilm SAR, also the influence of geraniol on Candida spp induced dysregulated inflammatory axis, and in vivo toxicity. Methods: Minimal inhibitory (MIC) and fungicidal (MFC) concentrations against Candida spp were defined, followed by antibiofilm activity (CFU-colony forming unit/mL/g of dry weight). Cytotoxic activity was assessed using human monocytes (THP-1) and oral squamous cell (TR146). Geraniol was selected for further analysis based on antifungal, antibiofilm and cytotoxic results. Geraniol was tested using a dual-chamber co-culture model with TR146 cells infected with C. albicans, and THP-1 cells, used to mimic oral epithelium upon fungal infection. Expression of Candida enzymes (phospholipase-PLB and aspartyl proteases-SAP) and host inflammatory cytokines (interleukins: IL-1ß, IL-6, IL-17, IL-18, IL-10, and Tumor necrosis factor-TNF) were analyzed. Lastly, geraniol in vivo toxicity was assessed using Galleria mellonella. Results: MIC values obtained were 1.25-5 mM/mL for geraniol, 25-100 mM/mL for linalool, and 100-200 mM/mL for citronellal. Geraniol 5 and 50 mM/mL reduced yeast viability during biofilm analysis, only 500 mM/mL of linalool was effective against a 72 h biofilm and no biofilm activity was seen for citronellal. LD50 for TR146 and THP-1 were, respectively: geraniol 5.883 and 8.027 mM/mL; linalool 1.432 and 1.709 mM/mL; and citronellal 0.3006 and 0.1825 mM/mL. Geraniol was able to downregulate expression of fungal enzymes and host pro-inflammatory cytokines IL-1ß, IL-6, and IL-18. Finally, safety in vivo parameters were observed up to 20 mM/Kg. Discussion: Despite chemical similarities, geraniol presented better antifungal, antibiofilm activity, and lower cytotoxicity when compared to the other monoterpenes. It also showed low in vivo toxicity and capacity to downregulate the expression of fungal enzymes and host pro-inflammatory cytokines. Thus, it can be highlighted as a viable option for oral candidiasis treatment.

Anadenanthera colubrina regulated LPS-induced inflammation by suppressing NF-κB and p38-MAPK signaling pathways.

We aimed to determine the chemical profile and unveil Anadenanthera colubrina (Vell.) Brenan standardized extract effects on inflammatory cytokines expression and key proteins from immunoregulating signaling pathways on LPS-induced THP-1 monocyte. Using the RT-PCR and Luminex Assays, we planned to show the gene expression and the levels of IL-8, IL-1ß, and IL-10 inflammatory cytokines. Key proteins of NF-κB and MAPK transduction signaling pathways (NF-κB, p-38, p-NF-κB, and p-p38) were detected by Simple Western. Using HPLC-ESI-MSn (High-Performance Liquid-Chromatography) and HPLC-HRESIMS, we showed the profile of the extract that includes an opus of flavonoids, including the catechins, quercetin, kaempferol, and the proanthocyanidins. Cell viability was unaffected up to 250 µg/mL of the extract (LD50 = 978.7 µg/mL). Thereafter, the extract's impact on the cytokine became clear. Upon LPS stimuli, in the presence of the extract, gene expression of IL-1ß and IL-10 were downregulated and the cytokines expression of IL-1ß and IL-10 were down an upregulated respectively. The extract is involved in TLR-4-related NF-κB/MAPK pathways; it ignited phosphorylation of p38 and NF-κB, orchestrating a reduced signal intensity. Therefore, Anadenanthera colubrina's showed low cytotoxicity and profound influence as a protector against the inflammation, modulating IL-1ß and IL-10 inflammatory cytokines gene expression and secretion by regulating intracellular NF-κB and p38-MAPK signaling pathways.

Syzygium aromaticum essential oil and its major constituents: Assessment of activity against Candida spp. and toxicity.

Syzigium aromaticum essential oil (EO), eugenol, and ß-caryophyllene were evaluated regarding antifungal, antibiofilm, and in vitro toxicity. Additionally, in vivo toxicity of EO was observed. Anti-Candida activity was assessed through broth microdilution assay for all compounds. Time-kill assay (0, 1, 10, 30 min, 1, 2, and 4 h) was used to determine the influence of EO and eugenol on Candida Growth kinetics. Thereafter, both compounds were evaluated regarding their capacity to act on a biofilm formation and on mature biofilm, based on CFU/ml/g of dry weight. Cell Titer Blue Viability Assay was used for in vitro cytotoxicity, using oral epithelial cells (TR146) and human monocytes (THP-1). Lastly, Galleria mellonella model defined the EO in vivo acute toxicity. All compounds, except ß-cariofilene (MIC > 8000 µg/ml), presented antifungal activity against Candida strains (MIC 500-1000 µg/ml). The growth kinetics of Candida was affected by the EO (5xMIC 30 min onward; 10xMIC 10 min onward) and eugenol (5xMIC 10 min onward; 10xMIC 1 min onward). Fungal viability was also affected by 5xMIC and 10xMIC of both compounds during biofilm formation and upon mature biofilms. LD50 was defined for TR146 and THP1 cells at, respectively, 59.37 and 79.54 µg/ml for the EO and 55.35 and 84.16 µg/ml for eugenol. No sign of toxicity was seen in vivo up to 10mg/ml (20 x MIC) for the EO. S. aromaticum and eugenol presented antifungal and antibiofilm activity, with action on cell growth kinetics. In vivo acute toxicity showed a safe parameter for the EO up to 10 mg/ml.

Identification of the Neoaspergillic Acid Biosynthesis Gene Cluster by Establishing an In Vitro CRISPR-Ribonucleoprotein Genetic System in Aspergillus melleus.

Filamentous fungi are an essential source of bioactive mycotoxins. Recent efforts have focused on developing antifungal agents that are effective against invasive yeasts, such as Candida spp. By screening fungal strains isolated from regions surrounding the Chernobyl nuclear power plant disaster for antifungal activity against Candida albicans, we found that Aspergillus melleus IMV 01140 produced compounds that inhibited the growth of the yeast. The active compound produced by A. melleus was isolated and found to be neoaspergillic acid, a compound that is closely related to aspergillic acid. While aspergillic acid and its derivatives have been characterized and were found to have antibacterial and antifungal properties, neoaspergillic acid has been much less studied. Even though neoaspergillic acid and related compounds were found to have antibacterial and antitumoral effects, further investigation into this group of compounds is limited by challenges associated with large-scale production, isolation, and purification. The production of neoaspergillic acid has been shown to require co-cultivation methods or special growth conditions. In this work, neoaspergillic acid and related compounds were found to be produced by A. melleus under laboratory growth conditions. The biosynthetic gene cluster of neoaspergillic acid was predicted using the aspergillic acid gene cluster as a model. The biosynthetic pathway for neoaspergillic acid was then confirmed by establishing an in vitro CRISPR-ribonucleoprotein system to individually delete genes within the cluster. A negative transcriptional factor, mcrA, was also eliminated to further improve the production of neoaspergillic acid and the related compounds for future studies.

Racial and ethnic disparities in periodontal health among adults seeking dental care in rural North Carolina communities: A retrospective study.

BACKGROUND: Population studies consistently demonstrate a greater prevalence of chronic diseases, including oral diseases, among underrepresented minorities. This retrospective study aimed to measure and describe the prevalence and extent of periodontitis among adults seeking dental care within an academic practice-based network in rural North Carolina. METHODS: This study used de-identified electronic health record (EHR) data from 2011 to 2017 of adult dentate patients (aged ≥30 years) seeking dental care who received a comprehensive periodontal examination at one of nine networked clinical centers. Periodontitis prevalence was calculated using CDC/AAP case definitions, along with extent (%) scores for periodontal parameters. Comparisons focused on age, sex, race, ethnicity, tobacco use, diabetes status, payer or insurance status, plaque scores, and the number of teeth. RESULTS: EHR data for 10,544 adult patients (60.5% female) indicated 79.8% had some form of periodontitis. This patient population was diverse: 22.6% Black, 4.4% American Indian, and 53.8% White, with 4.8% self-identified as Hispanic. Patients 50 years and older showed greater mean extent scores for clinical attachment levels relative to patients 30 to 49 years. Males exhibited greater periodontitis than females (p = 0.001). Blacks showed significantly (p < 0.001) greater periodontitis prevalence relative to Whites. Hispanics also showed a greater prevalence of periodontitis (p < 0.001) relative to non-Hispanics. Significantly greater periodontitis was also noted for tobacco users (p < 0.001) but not for diabetes or payer status. A multiple logistic regression analysis of periodontitis prevalence confirmed significant associations for periodontitis for age, sex, race, ethnicity, tobacco use, high plaque scores, and the number of teeth (p < 0.001), but not diabetes or payer status. CONCLUSIONS: The data document that racial and ethnic inequalities in periodontal health occur within the population of adults residing in rural communities in North Carolina and seeking dental care.

Biological and social aspects of Coronavirus Disease 2019 (COVID-19) related to oral health

Abstract The expansion of coronavirus disease 2019 (COVID-19) throughout the world has alarmed all health professionals. Especially in dentistry, there is a growing concern due to it's high virulence and routes of transmission through saliva aerosols. The virus keeps viable on air for at least 3 hours and on plastic and stainless-steel surfaces up to 72 hours. In this sense, dental offices, both in the public and private sectors, are high-risk settings of cross infection among patients, dentists and health professionals in the clinical environment (including hospital's intensive dental care facilities). This manuscript aims to compile current available evidence on prevention strategies for dental professionals. Besides, we briefly describe promising treatment strategies recognized until this moment. The purpose is to clarify dental practitioners about the virus history and microbiology, besides guiding on how to proceed during emergency consultations based on international documents. Dentists should consider that a substantial number of individuals (including children) who do not show any signs and symptoms of COVID-19 may be infected and can disseminate the virus. Currently, there is no effective treatment and fast diagnosis is still a challenge. All elective dental treatments and non-essential procedures should be postponed, keeping only urgent and emergency visits to the dental office. The use of teledentistry (phone calls, text messages) is a very promising tool to keep contact with the patient without being at risk of infection.

Isoflavonoids from Brazilian red propolis down-regulate the expression of cancer-related target proteins: A pharmacogenomic analysis.

Vestitol and neovestitol are bioactive isoflavonoids isolated from Brazilian red propolis, a unique Apis melifera type of propolis botanically originated from Dalbergia ecastophyllum. Although these molecules have relevant biological effects, including anticancer and immunomodulatory activities, their mechanism(s) of action and the affected pathways remain largely unknown. Here, we carried out a pharmacogenomic analysis to investigate the effects of vestitol and neovestitol on the whole-genome expression in human tumor cells, particularly cancer-related target proteins. HeLa cells were exposed to the compounds at IC20 and genomic information of treated cells was analyzed using the Illumina transcriptome system and GeneGo MetaCore software. Our results showed that vestitol (IC20  = 214.7 µM) reduced the expression of genes enrolled with the alpha tubulin (fold -3.7), tubulin in microtubules (fold -3.7), and histone h3 (fold = -3.03), and that treatment with neovestitol (IC20  = 102.91 µM) downregulated prostaglandin E synthase gene (fold = -3.12), which are considered ideal targets for anticancer therapy. These data open avenues for the study of vestitol and neovestitol as potential promising candidates for anticancer therapy. Toxicological, non-clinical, and clinical validation of the findings presented herein is needed.

Oral microbe-host interactions: influence of ß-glucans on gene expression of inflammatory cytokines and metabolome profile.

BACKGROUND: The aim of this study was to evaluate the effects of ß-glucan on the expression of inflammatory mediators and metabolomic profile of oral cells [keratinocytes (OBA-9) and fibroblasts (HGF-1) in a dual-chamber model] infected by Aggregatibacter actinomycetemcomitans. The periodontopathogen was applied and allowed to cross the top layer of cells (OBA-9) to reach the bottom layer of cells (HGF-1) and induce the synthesis of immune factors and cytokines in the host cells. ß-glucan (10 µg/mL or 20 µg/mL) were added, and the transcriptional factors and metabolites produced were quantified in the remaining cell layers and supernatant. RESULTS: The relative expression of interleukin (IL)-1-α and IL-18 genes in HGF-1 decreased with 10 µg/mL or 20 µg/mL of ß-glucan, where as the expression of PTGS-2 decreased only with 10 µg/mL. The expression of IL-1-α increased with 20 µg/mL and that of IL-18 increased with 10 µg/mL in OBA-9; the expression of BCL 2, EP 300, and PTGS-2 decreased with the higher dose of ß-glucan. The production of the metabolite 4-aminobutyric acid presented lower concentrations under 20 µg/mL, whereas the concentrations of 2-deoxytetronic acid NIST and oxalic acid decreased at both concentrations used. Acetophenone, benzoic acid, and pinitol presented reduced concentrations only when treated with 10 µg/mL of ß-glucan. CONCLUSIONS: Treatment with ß-glucans positively modulated the immune response and production of metabolites.

Review of flavonoids: A diverse group of natural compounds with anti-Candida albicans activity in vitro.

Flavonoids are a subdivision of polyphenols, a versatile class of natural compounds that represent secondary metabolites from higher plants and are abundant in human diet. Various protective effects of flavonoids have been reported, including antimicrobial and antifungal activities. Due to the nature of oral candidiasis and the increased use of antifungal agents, several drug-resistant strains have emerged making it impractical to rely on one standard therapeutic regime. The aim of this review is to summarize the antifungal activity of some examples of the major subclasses of flavonoids in pure extract forms against C. albicans in vitro, as reported in literature over the past 10 years (2004-2015). In addition, this review outlines the potential mechanism of actions of flavonoids studied in vitro, which may contribute to a better understanding of flavonoids as multi-targets agents in the treatment and/or prevention of oral candidiasis in clinical settings.

In Vitro and In Vivo Antifungal Activity of Lichochalcone-A against Candida albicans Biofilms.

Oral candidiasis (OC) is an opportunistic fungal infection with high prevalence among immunocompromised patients. Candida albicans is the most common fungal pathogen responsible for OC, often manifested in denture stomatitis and oral thrush. Virulence factors, such as biofilms formation and secretion of proteolytic enzymes, are key components in the pathogenicity of C. albicans. Given the limited number of available antifungal therapies and the increase in antifungal resistance, demand the search for new safe and effective antifungal treatments. Lichochalcone-A is a polyphenol natural compound, known for its broad protective activities, as an antimicrobial agent. In this study, we investigated the antifungal activity of lichochalcone-A against C. albicans biofilms both in vitro and in vivo. Lichochalcone-A (625 µM; equivalent to 10x MIC) significantly reduced C. albicans (MYA 2876) biofilm growth compared to the vehicle control group (1% ethanol), as indicated by the reduction in the colony formation unit (CFU)/ml/g of biofilm dry weight. Furthermore, proteolytic enzymatic activities of proteinases and phospholipases, secreted by C. albicans were significantly decreased in the lichochalcone-A treated biofilms. In vivo model utilized longitudinal imaging of OC fungal load using a bioluminescent-engineered C. albicans (SKCa23-ActgLUC) and coelenterazine substrate. Mice treated with lichochalcone-A topical treatments exhibited a significant reduction in total photon flux over 4 and 5 days post-infection. Similarly, ex vivo analysis of tongue samples, showed a significant decrease in CFU/ml/mg in tongue tissue sample of lichochalcone-A treated group, which suggest the potential of lichochalcone-A as a novel antifungal agent for future clinical use.

Malva sylvestris Inhibits Inflammatory Response in Oral Human Cells. An In Vitro Infection Model.

The aim of this study was to investigate the in vitro anti-inflammatory activity of Malva sylvestris extract (MSE) and fractions in a co-culture model of cells infected by Aggregatibacter actinomycetemcomitans. In addition, we evaluated the phytochemical content in the extract and fractions of M. sylvestris and demonstrated that polyphenols were the most frequent group in all samples studied. An in vitro dual-chamber model to mimic the periodontal structure was developed using a monolayer of epithelial keratinocytes (OBA-9) and a subepithelial layer of fibroblasts (HGF-1). The invasive periodontopathogen A. actinomycetemcomitans (D7S-1) was applied to migrate through the cell layers and induce the synthesis of immune factors and cytokines in the host cells. In an attempt to analyze the antimicrobial properties of MSE and fractions, a susceptibility test was carried out. The extract (MIC 175 µg/mL, MBC 500µg/mL) and chloroform fraction (MIC 150 µg/mL, MBC 250 µg/mL) were found to have inhibitory activity. The extract and all fractions were assessed using a cytotoxicity test and results showed that concentrations under 100 µg/mL did not significantly reduce cell viability compared to the control group (p > 0.05, viability > 90%). In order to analyze the inflammatory response, transcriptional factors and cytokines were quantified in the supernatant released from the cells. The chloroform fraction was the most effective in reducing the bacterial colonization (p< 0.05) and controlling inflammatory mediators, and promoted the down-regulation of genes including IL-1beta, IL-6, IL-10, CD14, PTGS, MMP-1 and FOS as well as the reduction of the IL-1beta, IL-6, IL-8 and GM-CSF protein levels (p< 0.05). Malva sylvestris and its chloroform fraction minimized the A. actinomycetemcomitans infection and inflammation processes in oral human cells by a putative pathway that involves important cytokines and receptors. Therefore, this natural product may be considered as a successful dual anti-inflammatory-antimicrobial candidate.

ß-Glucans (Saccharomyces cereviseae) Reduce Glucose Levels and Attenuate Alveolar Bone Loss in Diabetic Rats with Periodontal Disease.

The objective of this study was to assess the effects of oral ingestion of ß-glucans isolated from Saccharomyces cereviseae on the metabolic profile, expression of gingival inflammatory markers and amount of alveolar bone loss in diabetic rats with periodontal disease. Diabetes mellitus was induced in 48 Wistar rats by intraperitoneal injection of streptozotocin (80 mg/kg). After confirming the diabetes diagnosis, the animals were treated with ß-glucans (by gavage) for 28 days. On the 14th day of this period, periodontal disease was induced using a ligature protocol. ß-glucans reduced the amount of alveolar bone loss in animals with periodontal disease in both the diabetic and non-diabetic groups (p < 0.05). ß-glucans reduced blood glucose, cholesterol and triacylglycerol levels in diabetic animals, both with and without periodontal disease (p < 0.05). Furthermore, treatment with ß-glucans reduced the expression of cyclooxygenase-2 and receptor activator of nuclear factor kappa-B ligand and increased osteoprotegerin expression in animals with diabetes and periodontal disease (p < 0.05). It was concluded that treatment with ß-glucans has beneficial metabolic and periodontal effects in diabetic rats with periodontal disease.

Coriandrum sativum L. (Coriander) essential oil: antifungal activity and mode of action on Candida spp., and molecular targets affected in human whole-genome expression.

Oral candidiasis is an opportunistic fungal infection of the oral cavity with increasingly worldwide prevalence and incidence rates. Novel specifically-targeted strategies to manage this ailment have been proposed using essential oils (EO) known to have antifungal properties. In this study, we aim to investigate the antifungal activity and mode of action of the EO from Coriandrum sativum L. (coriander) leaves on Candida spp. In addition, we detected the molecular targets affected in whole-genome expression in human cells. The EO phytochemical profile indicates monoterpenes and sesquiterpenes as major components, which are likely to negatively impact the viability of yeast cells. There seems to be a synergistic activity of the EO chemical compounds as their isolation into fractions led to a decreased antimicrobial effect. C. sativum EO may bind to membrane ergosterol, increasing ionic permeability and causing membrane damage leading to cell death, but it does not act on cell wall biosynthesis-related pathways. This mode of action is illustrated by photomicrographs showing disruption in biofilm integrity caused by the EO at varied concentrations. The EO also inhibited Candida biofilm adherence to a polystyrene substrate at low concentrations, and decreased the proteolytic activity of Candida albicans at minimum inhibitory concentration. Finally, the EO and its selected active fraction had low cytotoxicity on human cells, with putative mechanisms affecting gene expression in pathways involving chemokines and MAP-kinase (proliferation/apoptosis), as well as adhesion proteins. These findings highlight the potential antifungal activity of the EO from C. sativum leaves and suggest avenues for future translational toxicological research.

Antiproliferative effect of benzophenones and their influence on cathepsin activity.

The antiproliferative activity of two prenylated benzophenones isolated from Rheedia brasiliensis, the triprenylated garciniaphenone and the tetraprenylated benzophenone 7-epiclusianone, was investigated against human cancer cell lines. The antiproliferative activity on melanoma (UACC-62), breast (MCF-7), drug-resistant breast (NCI-ADR), lung/non-small cells (NCI460), ovarian (OVCAR 03), prostate (PC03), kidney (786-0), lung (NCI-460) and tongue (CRL-1624 and CRL-1623) cancer cells was determined using spectrophotometric quantification of the cellular protein content. The effect of these benzophenones on the activity of cathepsins B and G was also investigated. Garciniaphenone displayed cytostatic activity in all cell lines, whereas 7-epiclusianone showed a dose-dependent cytotoxic effect. The IC(50) values for cell proliferation revealed that 7-epiclusianone is more active than garciniaphenone against most of the cell lines. Furthermore, the antiproliferative effects demonstrated by garciniaphenone and 7-epiclusianone were related to their cathepsin inhibiting properties. In conclusion, 7-epiclusianone is a promising naturally occurring agent which displays multiple inhibitory effects which may be working in concert to inhibit cancer cell proliferation in vitro. The putative pathway by which 7-epiclusianone affects cancer cell development may involve cathepsin inhibition.