Cytotoxicity Activity of Some meso-Dihydroguaiaretic Acid Derivatives and Mode of Action of the Most Active Compound.

The aim of this study was to screen sixteen meso-1 semi-synthetic derivatives bearing ether, esther, carbamate, phosphate or aminoether functional groups against five cancer cell lines: MCF-7 (breast), A549 (lung), HepG2 (liver), HeLa (cervix), and DU145 (prostate) at 25 µM using the MTT assay. Results from the screening showed that two derivatives had the lowest percentage of cell viability at 25 µM, the aminoether derivative meso-11 and the esther derivative meso-20 against A549 (44.15±0.78 %) and MCF-7 (41.60±0.92 %), respectively. Then, it was determined the IC50 value of each compound against their most sensitive cancer cell line. Results showed that aminoether derivative meso-11 showed potent cytotoxicity against A549 (IC50 =17.11±2.11 µM), whereas it resulted more cytotoxic against the LL-47 lung normal cell line (IC50 =9.49±1.19 µM) having a Selective Index (SI) of 0.55. On the other hand, the esther derivative meso-20 exhibited potent activity against MCF-7 (IC50 =18.20±1.98 µM), whereas it displayed moderate cytotoxicity against the MCF-10 breast normal cell line (IC50 =41.22±2.17 µM) with a SI of 2.2. Finally, studies on the mechanism of action of meso-20 indicated disruption of MCF-7 plasma membrane in vitro and the AMPK activation in silico.

Vaginal Ovule Loaded with Bismuth Lipophilic Nanoparticles and Cetylpyridinium Chloride Inhibits Human Cervical Carcinoma and Candida albicans Growth.

Bismuth lipophilic nanoparticles (BisBAL NPs) and cetylpyridinium chloride (CPC) are antineoplastic and antimicrobial in vitro. As a next pre-clinical step, a clinically viable dosage form for vaginal application was developed. Compendial pharmacopeial tests (mass uniformity, disintegration, and compressive mechanics) and inductively coupled plasma optical emission spectroscopy were conducted on in-house developed glycerinated gelatin (60:15 v/w) vaginal ovules containing BisBAL NP-CPC. The antimycotic activity of BisBAL NP-CPC vaginal ovules was analyzed using disk diffusion and cell viability XTT assays. The antitumor properties of BisBAL NP-CPC vaginal ovules were assessed by cell viability MTT tests. BisBAL NP-CPC and drug-free vaginal ovules deposited into ex vivo porcine vaginas disaggregated without signs of adverse cytotoxicity within the timespan of clinical efficacy. BisBAL NP-CPC vaginal ovules demonstrated antifungal efficacy comparable to miconazole: C. albicans growth inhibition haloes in diffusion tests were 23 ± 0.968 mm (n = 3) for BisBAL NP-CPC and 20.35 ± 0.899 mm (n = 3) for miconazole. Likewise, BisBAL NP-CPC vaginal ovules reduced HeLa cell growth by 81%, outperforming the clinical reference of 500 µM 5-fluouracil, which induced a 70% growth inhibition. BisBAL NP-CPC incorporated into glycerinated gelatin vaginal ovules constitute an innovative drug delivery system for topical antimycotic and anti-cervical carcinoma treatments.

A Polyurethane Electrospun Membrane Loaded with Bismuth Lipophilic Nanoparticles (BisBAL NPs): Proliferation, Bactericidal, and Antitumor Properties, and Effects on MRSA and Human Breast Cancer Cells.

Electrospun membranes (EMs) have a wide range of applications, including use as local delivery systems. In this study, we manufactured a polyurethane Tecoflex™ EM loaded with bismuth-based lipophilic nanoparticles (Tecoflex™ EMs-BisBAL NPs). The physicochemical and mechanical characteristics, along with the antitumor and bactericidal effects, were evaluated using a breast cancer cell line and methicillin-susceptible and resistant Staphylococcus aureus (MRSA). Drug-free Tecoflex™ EMs and Tecoflex™ EMs-BisBAL NPs had similar fiber diameters of 4.65 ± 1.42 µm and 3.95 ± 1.32 µm, respectively. Drug-free Tecoflex™ EMs did not negatively impact a human fibroblast culture, indicating that the vehicle is biocompatible. Tecoflex™ EMs-BisBAL NPs increased 94% more in size than drug-free Tecoflex™ EMs, indicating that the BisBAL NPs enhanced hydration capacity. Tecoflex™ EMs-BisBAL NPs were highly bactericidal against both methicillin-susceptible S. aureus and MRSA clinical isolates, inhibiting their growth by 93.11% and 61.70%, respectively. Additionally, Tecoflex™ EMs-BisBAL NPs decreased the viability of MCF-7 tumor cells by 86% after 24 h exposure and 70.1% within 15 min. Regarding the mechanism of action of Tecoflex™ EMs-BisBAL NPs, it appears to disrupt the tumor cell membrane. In conclusion, Tecoflex™ EMs-BisBAL NPs constitute an innovative low-cost drug delivery system for human breast cancer and postoperative wound infections.

Antimicrobial and antitumor activities of an alginate-based membrane loaded with bismuth nanoparticles and cetylpyridinium chloride.

OBJECTIVE: To evaluate the antitumor and antimicrobial properties of an alginate-based membrane (ABM) loaded with bismuth lipophilic nanoparticles (BisBAL NPs) and cetylpyridinium chloride (CPC) on clinically isolated bacteria and a pancreatic cancer cell line. MATERIAL AND METHODS: The BisBAL NP-CPC ABM was characterized using optical and scanning electron microscopy (SEM). The antimicrobial potential was measured using the disk-diffusion assay, and antibiofilm activity was determined through the live/dead assay and fluorescence microscopy. The antitumor activity was analyzed on the pancreatic cell line (Panc 03.27) using the MTT assay and live/dead assay with fluorescence microscopy. RESULTS: After a 24-h exposure (37°C, aerobic conditions), 5 µM BisBAL NP reduced the growth of K. pneumoniae by 77.9%, while 2.5 µM BisBAL NP inhibited the growth of Salmonella, E. faecalis and E. faecium by 82.9%, 82.6%, and 78%, respectively (p < 0.0001). The BisBAL NPs-CPC ABM (at a ratio of 10:1; 500 and 50 µM, respectively) inhibited the growth of all isolated bacteria, producing inhibition halos of 9.5, 11.2, 7, and 10.3 mm for K. pneumoniae, Salmonella, E. faecalis, and E. faecium, respectively, in contrast to the 6.5, 9.5, 8.5, and 9.8 mm obtained with 100 µM ceftriaxone (p < 0.0001). The BisBAL NPs-CPC ABM also reduced bacterial biofilms, with 81.4%, 74.5%, 97.1%, and 79.5% inhibition for K. pneumoniae, E. faecium, E. faecalis, and Salmonella, respectively. Furthermore, the BisBAL NPs-CPC ABM decreased Panc 03.27 cell growth by 76%, compared to 18% for drug-free ABM. GEM-ABM reduced tumoral growth by 73%. The live/dead assay confirmed that BisBAL NPs-CPC-ABM and GEM-ABM were cytotoxic for the turmoral Panc 03.27 cells. CONCLUSION: An alginate-based membrane loaded with BisBAL NP and CPC exhibits dual antimicrobial and antitumoral efficacy. Therefore, it could be applied in cancer treatment and to diminish the occurrence of surgical site infections.

Geographical approach analysis of the impact of air pollution on newborn intrauterine growth and cord blood DNA damage in Mexico City.

BACKGROUND: Few epidemiologic studies have focused on the specific source of ambient air pollution and adverse health effects in early life. Here, we investigated whether air pollutants from different emission sources were associated with decreased birth anthropometry parameters and increased DNA adduct formation in mother-child pairs residing in the Mexico City Metropolitan Area (MCMA). METHODS: This cross-sectional study included 190 pregnant women recruited during their last trimester of pregnancy from two hospitals at MCMA, and a Modeling Emissions Inventory (MEI) to calculate exposure to ambient air pollutants from different emissions sources (area, point, mobile, and natural) for two geographical buffers 250 and 750 m radii around the participants households. RESULTS: Contaminants were positively correlated with umbilical cord blood (UCB) adducts, but not with maternal blood (MB) adducts. PM10 emissions (area and point sources, overall emissions), PM2.5 (point sources), volatile organic compounds (VOC), total organic compounds (TOC) from point sources were positively correlated with UCB adducts. Air pollutants emitted from natural sources were correlated with a decrease in MB and UCB adducts. PM10 and PM2.5 were correlated (p < 0.05) with a decrease in birth weight (BW), birth length (BL) and gestational age at term (GA). In multivariate analyses adjusted for potential confounders, PM10 was associated with an increase in UCB adducts. PM10 and PM2.5 from overall emissions were associated with a decrease in BW, BL and GA at term. IMPACT: Results suggested higher susceptibility of newborns compared to mothers to damage related to ambient air pollution. PMs are associated with birth anthropometry parameters and DNA damage in adjusted models, highlighting the need for more strict regulation of PM emissions.

Cumulative antitumor effect of bismuth lipophilic nanoparticles and cetylpyridinium chloride in inhibiting the growth of lung cancer.

OBJECTIVE: To determine the combined antitumor effect of bismuth lipophilic nanoparticles (BisBAL NP) and cetylpyridinium chloride (CPC) on human lung tumor cells. MATERIAL AND METHODS: The human lung tumor cells A549 were exposed to 1-100 µM BisBAL NP or CPC, either separately or in a 1:1 combination. Cell viability was measured with the PrestoBlue assay, the LIVE/DEAD assay, and fluorescence microscopy. The integrity and morphology of cellular microtubules were analyzed by immunofluorescence. RESULTS: A 24-h exposure to 1 µM solutions reduced A549 growth with 21.5% for BisBAL NP, 70.5% for CPC, and 92.4% for the combination (p < 0.0001), while a 50 µM BisBAL NP/CPC mixture inhibited cell growth with 99% (p < 0.0001). BisBAL NP-curcumin conjugates were internalized within 30 min of exposure and could be traced within the nucleus of tumor cells within 2 h. BisBAL NP, but not CPC, interfered with microtubule organization, thus interrupting cell replication, similar to the action mechanism of docetaxel. CONCLUSION: The growth inhibition of A549 human tumor cells by BisBAL NP and CPC was cumulative as of 1 µM. The BisBAL NP/CPC combination may constitute an innovative and cost-effective alternative for treating human lung cancer.

Cetylpyridinium chloride inhibits human breast tumor cells growth in a no-selective way.

OBJECTIVE: Analyze the antitumor capacity of cetylpyridinium chloride (CPC) on human breast tumor cells, and the possible action mechanism. MATERIAL AND METHODS: The human breast tumor cells MCF-7 and no-tumor breast cells MCF-10A were exposed to CPC under various condition (concentration and duration). Cell viability was measured with MTT assay, the LIVE/DEAD assay, and fluorescence microscopy. Membrane permeability after CPC exposure was evaluated by Calcein AM assay, mitochondrial morphology with a MitoView staining, and genotoxicity with the comet assay and fluorescence microscopy. RESULTS: CPC was cytotoxic to both MCF-7 and MCF-10A as of a 24-h exposure to 0.1 µM. Cytotoxicity was dose-dependent and reached 91% for MCF-7 and 78% for MCF-10A after a 24-h exposure to 100 µM CPC, which outperformed the positive control doxorubicin in effectiveness and selectivity. The LD50 of CPC on was 6 µM for MCF-7 and 8 µM for MCF-10A, yielding a selectivity index of 1.41. A time response analysis revealed 64% dead cells after only 5 min of exposure to 100 µM CPC. With respect to the action mechanisms, the comet assay did not reveal genome fragmentation. On the other hand, membrane damage was dose-dependent and may also affect mitochondrial morphology. CONCLUSION: Cetylpyridinium chloride inhibits MCF-7 cell growing in a non-selective way as of 5 min of exposure. The action mechanism of CPC on tumor cells involves cell membrane damage without change neither mitochondrial morphology nor genotoxicity.

Bismuth Lipophilic Nanoparticles (BisBAL NP) Inhibit the Growth of Tumor Cells in a Mouse Melanoma Model.

AIM: The objective of this study was to analyze the antitumor effect of BisBAL NP in a mouse melanoma model. MATERIALS AND METHODS: The antitumor activity of BisBAL NP on murine B16-F10 melanoma cells was determined both in vitro (PrestoBlue cell viability assay and Live/Dead fluorescence) and in vivo, in a mouse model, with the following 15-day treatments: BisBAL NP, negative control (PBS), and cell-death control (docetaxel; DTX). Mouse survival and weight, as well as the tumor volume, were recorded daily during the in vivo study. RESULTS: BisBAL NP were homogeneous in size (mean diameter, 14.7 nm) and bismuth content. In vitro, 0.1 mg/mL BisBAL NP inhibited B16-F10 cell growth stronger (88%) than 0.1 mg/mL DTX (82%) (*p<0.0001). In vivo, tumors in mice treated with BisBAL NP (50 mg/kg/day) or DTX (10 mg/kg/day) were 76% and 85% smaller than the tumors of negative control mice (*p<0.0001). The average weight of mice was 18.1 g and no statistically significant difference was detected among groups during the study. Alopecia was only observed in all DTX-treated mice. The survival rate was 100% for the control and BisBAL NP groups, but one DTX- treated mouse died at the end of the treatment period. The histopathological analysis revealed that exposure to BisBAL NP was cytotoxic for tumor tissue only, without affecting the liver or kidney. CONCLUSION: BisBAL NP decreased the tumor growing in a mouse melanoma model without secondary effects, constituting an innovative low-cost alternative to treat melanoma.

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage.

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage.

In vitro evaluation of the antitumor effect of bismuth lipophilic nanoparticles (BisBAL NPs) on breast cancer cells.

AIM: The objective of this study was to evaluate the antitumor activity of lipophilic bismuth nanoparticles (BisBAL NPs) on breast cancer cells. MATERIALS AND METHODS: The effect of varying concentrations of BisBAL NPs was evaluated on human MCF-7 breast cancer cells and on MCF-10A fibrocystic mammary epitheliocytes as noncancer control cells. Cell viability was evaluated with the MTT assay, plasma membrane integrity was analyzed with the calcein AM assay, genotoxicity with the comet assay, and apoptosis with the Annexin V/7-AAD assay. RESULTS: BisBAL NPs were spherical in shape (average diameter, 28 nm) and agglomerated into dense electronic clusters. BisBAL NP induced a dose-dependent growth inhibition. Most importantly, growth inhibition was higher for MCF-7 cells than for MCF-10A cells. At 1 µM BisBAL NP, MCF-7 growth inhibition was 51%, while it was 11% for MCF-10A; at 25 µM BisBAL NP, the growth inhibition was 81% for MCF-7 and 24% for MCF-10A. With respect to mechanisms of action, a 24-hour exposure of 10 and 100 µM BisBAL NP caused loss of cell membrane integrity and fragmentation of tumor cell DNA. BisBAL NPs at 10 µM were genotoxic to and caused apoptosis of breast cancer cells. CONCLUSION: BisBAL NP-induced growth inhibition is dose dependent, and breast cancer cells are more vulnerable than noncancer breast cells. The mechanism of action of BisBAL NPs may include loss of plasma membrane integrity and a genotoxic effect on the genomic DNA of breast cancer cells.

Airborne particulate matter in vitro exposure induces cytoskeleton remodeling through activation of the ROCK-MYPT1-MLC pathway in A549 epithelial lung cells.

Airborne particulate matter with an aerodynamic diameter ≤10µm (PM10) is considered a risk factor for the development of lung cancer. Little is known about the cellular mechanisms by which PM10 is associated with cancer, but there is evidence that its exposure can lead to an acquired invasive phenotype, apoptosis evasion, inflammasome activation, and cytoskeleton remodeling in lung epithelial cells. Cytoskeleton remodeling occurs through actin stress fiber formation, which is partially regulated through ROCK kinase activation, we aimed to investigate if this protein was activated in response to PM10 exposure in A549 lung epithelial cells. Results showed that 10µg/cm2 of PM10 had no influence on cell viability but increased actin stress fibers, cytoplasmic ROCK expression, and phosphorylation of myosin phosphatase-targeting 1 (MYPT1) and myosin light chain (MLC) proteins, which are targeted by ROCK. The inhibition of ROCK prevented actin stress fiber formation and the phosphorylation of MYPT1 and MLC, suggesting that PM10 activated the ROCK-MYPT1-MLC pathway in lung epithelial cells. The activation of ROCK1 has been involved in the acquisition of malignant phenotypes, and its induction by PM10 exposure could contribute to the understanding of PM10 as a risk factor for cancer development through the mechanisms associated with invasive phenotype.

Atmospheric particulate matter (PM10) exposure-induced cell cycle arrest and apoptosis evasion through STAT3 activation via PKCζ and Src kinases in lung cells.

Atmospheric particulate matter with aerodynamic diameter ≤10 µm (PM10) is a risk factor for the development of lung cancer, but cellular pathways are not completely understood. STAT3 is a p21(Waf1/Cip1) transcription factor and is associated with proliferation and cell survival and is upregulated in lung cancer. PM10 exposure induces p21(Waf1/Cip1) expression, which could be related to STAT3 activation. The aims of this work were to investigate whether STAT3 was activated on lung epithelial cells after PM10 exposure and to determine whether or not STAT3 could have an impact on cell cycle distribution and cell survival. Our results showed that PM10 induced STAT3 activation through Src and PKCζ kinases, and it is partially responsible for the p21(Waf1/Cip1) induction that was also observed. Moreover, PM10 induced G1-G0 cell cycle arrest. The inhibition of STAT3 phosphorylation prevented cell cycle arrest and triggered apoptosis. These results suggest that PM10 exposure might activate a survival pathway related to STAT3 activation, similar to what has been described as part of the immune system and apoptosis evasion during tumor promotion and development.

Sampling and composition of airborne particulate matter (PM10) from two locations of Mexico City.

The PM10 airborne particulate matter with an aerodynamic diameter ≤10 µm is considered as a risk factor of various adverse health outcomes, including lung cancer. Here we described the sampling and composition of PM10 collected from an industrial zone (IZ), and a commercial zone (CZ) of Mexico City. The PM10 was collected with a high-volume sampler in the above mentioned locations and both types of PM10 sampled were characterized by the content of polycyclic aromatic hydrocarbons (PAHs), metals, and endotoxin. The endotoxin PM10 content from IZ and CZ displayed 138.4 UE/mg and 170.4 UE/mg of PM10, respectively.

Particulate matter (PM10) induces metalloprotease activity and invasion in airway epithelial cells.

Airborne particulate matter with an aerodynamic diameter ≤ 10 µm (PM10) is a risk factor for the development of lung diseases and cancer. The aim of this work was to identify alterations in airway epithelial (A549) cells induced by PM10 that could explain how subtoxic exposure (10 µg/cm(2)) promotes a more aggressive in vitro phenotype. Our results showed that cells exposed to PM10 from an industrial zone (IZ) and an urban commercial zone (CZ) induced an increase in protease activity and invasiveness; however, the cell mechanism is different, as only PM10 from CZ up-regulated the activity of metalloproteases MMP-2 and MMP-9 and disrupted E-cadherin/ß-catenin expression after 48 h of exposure. These in vitro findings are relevant in terms of the mechanism action of PM10 in lung epithelial cells, which could be helpful in understanding the pathogenesis of some human illness associated with highly polluted cities.

Cytoplasmic p21(CIP1/WAF1), ERK1/2 activation, and cytoskeletal remodeling are associated with the senescence-like phenotype after airborne particulate matter (PM(10)) exposure in lung cells.

The exposure to particulate matter with a mean aerodynamic diameter ≤10 µm (PM10) from urban zones is considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM10 exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remodeling, changes in the subcellular localization of p21(CIP1/WAF1), an increase in ß-galactosidase activity and changes in cell cycle. To test our hypothesis, PM10 from an industrial zone (IZ) and a commercial zone (CZ) were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM10 concentration (10 µg/cm(2)) for 24 h and 48 h. The results showed that PM10 exposure induced an increase in F-actin stress fibers and caused the cytoplasmic stabilization of p21(CIP1/WAF1) via phosphorylation at Thr(145) and Ser(146) and the phosphorylation of ERK1/2 on Thr(202). Changes in the cell cycle or apoptosis were not observed, but an increase in ß-galactosidase activity was detected. The PM10 from CZ caused more dramatic effects in lung cells. We conclude that PM10 exposure induced cytoplasmic p21(CIP1/WAF1) retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotype in lung cells. These alterations could have mechanistic implications regarding the carcinogenic potential of PM10.

Titanium dioxide nanoparticles impair lung mitochondrial function.

Titanium dioxide nanoparticles (TiO(2) NPs) are used in an increasing number of human products such as cosmetics, sunscreen, toothpaste and paints. However, there is clear evidence about effects associated to TiO(2) NPs exposure, which include lung inflammation and tumor formation and these effects are related to reactive oxygen species (ROS) formation. The ROS generation could be attributed to a mitochondrial dysfunction. Even though, it has been shown that TiO(2) NPs exposure can induce some alterations in mitochondria including cytochrome c release to cytosol, change in mitochondrial permeability and decrease of mitochondrial membrane potential (ΔΨ(m)), there is no information about the changes in mitochondrial function induced by TiO(2) NPs. We hypothesized that TiO(2) NPs effects are associated with mitochondrial dysfunction and redox unbalance. To test our hypothesis we isolated mitochondria from lung tissue of rats and exposed them to 10(g TiO(2) NPs (particle size<25nm)/mg protein for 1h. Our results showed that TiO(2) NPs decreases NADH levels and impairs ΔΨ(m) and mitochondrial function accompanied by ROS generation during mitochondrial respiration.

Celecoxib, a cyclooxygenase-2 inhibitor, prevents induction of liver preneoplastic lesions in rats.

BACKGROUND/AIMS: Several studies suggest that cyclooxygenase-2 (COX-2) inhibitors are chemopreventive agents against colon, breast and skin cancer. In this study, we evaluated the chemopreventive effect of celecoxib, a specific COX-2 inhibitor, on the development of liver preneoplastic lesions in rats. METHODS: Male Sprague-Dawley rats were fed during 5 weeks either a control or an experimental diet containing 1500 ppm celecoxib on a medium-term hepatocarcinogenesis protocol. Livers were collected and evaluated by histological and biochemical assays. RESULTS: A reduction by 80 and 90% both in the number and size of altered hepatic foci was observed in the group treated with celecoxib during hepatocarcinogenesis treatment, respectively. No evidence of apoptosis was observed in our present study, however, the expression of the proliferation markers such as PCNA and Ki-67 was drastically reduced. Interestingly, neither COX-2 expression nor prostaglandin-E2 (PGE2) production were altered by the hepatocarcinogenic treatment or celecoxib treatment. Finally, celecoxib inhibited the translocation of Rel A/p65 to the nucleus with significant effect on stability of the repressor IkappaB-alpha. CONCLUSIONS: This is the first demonstration that a specific COX-2 inhibitor, celecoxib, possesses striking chemopreventive activity, inhibiting preneoplastic lesions during hepatocarcinogenesis in vivo, suggesting that celecoxib effects are mediated by PGE2-independent mechanisms.