Development and Validation of Micro-Azocasein Assay for Quantifying Bromelain.

The proteolytic activity of enzymes may be evaluated by a colorimetric method with azocasein. Hence, we developed a micro-assay to quantify bromelain using azocasein. A total of 250 µL of 1.0% azocasein in dH2O was added to 250 µL of test solution, vortexed and incubated at ambient room temperature/30 min. The reaction was terminated with 1500 µL of 5% trichloroacetic acid, vortexed and centrifuged. A total of 150 µL of 0.5M NaOH was added to 150 µL of supernatant in triplicates, and absorbance was recorded at 410 nm. The linearity of the calibration curve was tested with 200-800 µg/mL serial dilutions. The detection limit, precision, accuracy, and robustness were tested along with the substrate enzyme reaction time and solvent matrix effect. Good linearity was seen with serially diluted 200 µg/mL bromelain. The limit of quantification and limit of detection were 5.412 and 16.4 µg/mL, respectively. Intra-day and inter-day analyses showed a relative standard deviation below 2.0%. The assay was robust when tested over 400-450 nm wavelengths. The assays performed using dH2O or PBS diluents indicated a higher sensitivity in dH2O. The proteolytic activity of bromelain was enhanced with L-cysteine or N-acetylcysteine. Hence, this micro-azocasein assay is reliable for quantifying bromelain.

Intraperitoneal BromAc® Does Not Interfere with the Healing of Colon Anastomosis.

A combination of bromelain and acetylcysteine, BromAc®, is an efficient intraperitoneal mucolytic for thick mucus secreted in pseudomyxoma peritonei (PMP). Patients with PMP quite often undergo colon anastomosis. Hence, we investigated the effect of the intraperitoneal delivery of BromAc® on colon-anastomosis healing in a rat model. Sixteen Wistar rats were divided into two groups (N = 8). The controls received intraperitoneal saline after anastomosis, whilst the other group received BromAc®. They were monitored for body-weight and general health parameters. Half the rats in each group (N = 4) were culled at 4 or 13 days post-surgery for assessment. The healing process of the tissues was assessed by burst pressure and collagen density with histology to assess the integrity of the internal organs. The results indicated that there was a similar pattern of weight fluctuation during the experiment, although the rats treated with the BromAc® showed slightly greater weight loss during the first 4 days. Although the burst pressure was similar in both groups, the BromAc® group at day 13 showed a slightly higher burst pressure, which was complemented by a higher collagen density (albeit not statistically significant). The histology of the internal organs was comparable to those of the controls. This study indicates that the intraperitoneal delivery of BromAc® in a rat model does not interfere with the healing process of colonic anastomosis.

Effect of Nebulized BromAc on Rheology of Artificial Sputum: Relevance to Muco-Obstructive Respiratory Diseases.

Respiratory diseases such as cystic fibrosis, COPD, and COVID-19 are difficult to treat owing to viscous secretions in the airways that evade mucocilliary clearance. Earlier studies have shown success with BromAc as a mucolytic agent. Hence, we tested the formulation on two gelatinous airway representative sputa models, to determine whether similar efficacy exist. Sputum lodged in an endotracheal tube was treated to aerosol N-acetylcysteine, bromelain, or their combination (BromAc). After measuring the particle size of aerosolized BromAc, the apparent viscosity was measured using a capillary tube method, whilst the sputum flow was assessed using a 0.5 mL pipette. Further, the concentration of the agents in the sputa after treatment were quantified using chromogenic assays. The interaction index of the different formulations was also determined. Results indicated that the mean particle size of BromAc was suitable for aerosol delivery. Bromelain and N-acetylcysteine affected both the viscosities and pipette flow in the two sputa models. BromAc showed a greater rheological effect on both the sputa models compared to individual agents. Further, a correlation was found between the rheological effects and the concentration of agents in the sputa. The combination index using viscosity measurements showed synergy only with 250 µg/mL bromelain + 20 mg/mL NAC whilst flow speed showed synergy for both combinations of bromelain (125 and 250 µg/mL) with 20 mg/mL NAC. Hence, this study indicates that BromAc may be used as a successful mucolytic for clearing airway congestion caused by thick mucinous immobile secretions.

Physical and chemical factors affecting the loading and release of bromelain from DC beads.

Doxorubicin loaded DC beads (microspheres) has been used for treating un-resectable tumours by transarterial chemoembolization (TACE). We have shown that bromelain, an enzyme from the pineapple plant, enhances the cytotoxic effect of a number of chemotherapeutic drugs and in an earlier study we have demonstrated that it can be loaded into DC beads. Therefore, in the current study we have investigated how certain physical and chemical parameters affect its loading and release for future development of DC beads in cancer therapy. Aliquots of 40-60 µL of DC beads (100-300 µm) were treated to bromelain in distilled water and various parameters such as pH of solution, bromelain concentration, temperature, loading period, presence/absence of agitation and the cytotoxic effect of bromelain loaded beads were investigated. Further release kinetics was also studied with additional investigation of pH effect on the proteolytic activity of bromelain. Results indicate that higher loading of bromelin was achieved in the beads at lower pH, higher concentration of bromelain, with agitation, 24 hours loading and ambient room temperature. Proteolytic activity of bromelain was maximal at pH 4.5 whilst cytotoxicity was at par if not better in the bromelain loaded DC beads. Release kinetics indicated that bromelain can be delivered over several hours. Hence, we conclude that bromelain can be loaded more efficiently with manipulation of certain parameters with noticeable cytotoxicity in tumour cells.

A novel method for potentiation of chemotherapy in soft tissue sarcomas with BromAc.

Single-agent doxorubicin currently forms part of standard care for patients with sarcomas. However, efficacy is limited by the presence of dose-dependent cardiotoxicity and toxicity to renal, hepatic, and neurological systems. Therefore, there is a pressing need for novel drug regimens which can provide increased efficacy and safety. BromAc is a novel drug combination developed as a mucolytic agent which has demonstrated anticancer activity both in vitro and in vivo in several cancers. Here, we investigated the efficacy of BromAc in combination with doxorubicin for four subtypes of sarcoma. Cell proliferation, alongside western blot for a variety of cell cycle, apoptosis, and autophagy biomarkers assays was performed following treatment of cell lines in vitro at various concentrations of BromAc and doxorubicin. The impact of drug treatment on MUC1 and MUC4 levels was assessed through immune-cytological methods. Drug agent synergy was assessed through the Chou-Talalay framework. BromAc treatment in combination with doxorubicin was more efficacious than single-agent doxorubicin, with synergistic effects observed. The immuno-cytological analysis demonstrated significant mucin depletion following treatment with BromAc and doxorubicin used in combination, providing a potential mechanistic underpinning for the observed anticancer effects.

Role of human nucleoside transporters in pancreatic cancer and chemoresistance.

The prognosis of pancreatic cancer is poor with the overall 5-year survival rate of less than 5% changing minimally over the past decades and future projections predicting it developing into the second leading cause of cancer related mortality within the next decade. Investigations into the mechanisms of pancreatic cancer development, progression and acquired chemoresistance have been constant for the past few decades, thus resulting in the identification of human nucleoside transporters and factors affecting cytotoxic uptake via said transporters. This review summaries the aberrant expression and role of human nucleoside transports in pancreatic cancer, more specifically human equilibrative nucleoside transporter 1/2 (hENT1, hENT2), and human concentrative nucleoside transporter 1/3 (hCNT1, hCNT3), while briefly discussing the connection and importance between these nucleoside transporters and mucins that have also been identified as being aberrantly expressed in pancreatic cancer. The review also discusses the incidence, current diagnostic techniques as well as the current therapeutic treatments for pancreatic cancer. Furthermore, we address the importance of chemoresistance in nucleoside analogue drugs, in particular, gemcitabine and we discuss prospective therapeutic treatments and strategies for overcoming acquired chemoresistance in pancreatic cancer by the enhancement of human nucleoside transporters as well as the potential targeting of mucins using a combination of mucolytic compounds with cytotoxic agents.

Monepantel antitumor activity is mediated through inhibition of major cell cycle and tumor growth signaling pathways.

In women, epithelial ovarian cancer is the leading cause of gynaecological malignancy-related deaths. Development of resistance to standard platinum and taxane based chemotherapy and recurrence of the disease necessitate development of novel drugs to halt disease progression. An established concept is to target molecular and signaling pathways that substantially contribute to development of drug resistance and disease progression. We have previously shown that, monepantel (MPL) a novel small molecule acetonitrile derivative is highly effective in suppressing growth, proliferation and colony formation of ovarian cancer cells. These effects are achieved through inhibition of the mTOR/p70S6K pathway in cancer cells. The present study was conducted to find in vivo corroboration and explore the effect of MPL om other growth stimulating putative signaling pathways. Here, female nude mice with subcutaneous OVCAR-3 xenografts were treated with 25 and 50 mg/kg doses of MPL administered (IP) three times weekly for 2 weeks. At the doses employed, MPL was modestly effective at suppressing tumor growth, but highly effective in inhibiting, mTOR, P70S6K and 4EBP1. There were also modest reductions in tumor cyclin D1 and retinoblastoma protein expression. Furthermore, it was found that MPL treatment causes down-regulation of IGF-1R, and c-MYC thus unveiling new dimensions to the growing antitumor actions of this potential anticancer drug. MPL treatment led to reduced tumor volume and weights without causing any detectable side effects. Coupled with the recent human safety data published on this molecule, expanded future trials are highly anticipated.

Comparison of proteolytic, cytotoxic and anticoagulant properties of chromatographically fractionated bromelain to un-fractionated bromelain.

Bromelain consisting of a number of proteolytic enzymes possess anticancer and thrombotic properties. Hence, four chromatically separated fractions were examined for their proteolytic, anticancer and antithrombotic activity. Bromelain fractions were separated using ion-exchange column chromatography. Proteolytic properties were assessed using standard azocasein assay. Anticancer properties were first assessed using four different cell lines PANC-1, HEP 2B, HEP 3G and OVCAR-3 on cells grown in 96 well plates. Subsequently, fraction 2 and fraction 3 combined with gemcitabine were tested in ASPC-1 cells. Then cytotoxicity of fraction 3 was compared to bromelain in combination with doxorubicin and N-acetylcysteine on HEP G2 and HEP 3B cells. Finally, the anticoagulation effect of fraction 3 or bromelain combined with N-acetylcysteine was evaluated using human blood. Fraction 3 showed the highest proteolytic activity (5% greater than standard bromelain) whilst others were less active. Cytotoxicity as assessed by IC50 indicated fraction 3 to be the most potent whilst the others did not follow their proteolytic potency order. OVCAR-3 was the most sensitive amongst the cell lines. Fraction 3 showed higher potency in combination with gemcitabine in ASPC-1 cells compared to fraction 2. Similarly, fraction 3 in combination with doxorubicin showed higher toxicity when compared to bromelain. Fraction 3 or bromelain only showed thrombolytic activity in combination with N-acetylcysteine. Fraction 3 may be developed for clinical use since it showed better cytotoxicity compared to bromelain.

Addition of bromelain and acetylcysteine to gemcitabine potentiates tumor inhibition in vivo in human colon cancer cell line LS174T.

The combinations of Bromelain and Acetylcysteine (BromAc®) with cytotoxics such as Gemcitabine, 5-Fluorouracil or Oxaliplatin have shown a dramatic reduction in IC50 values in a variety of cancers, including colon cancer, suggesting the possibility of effective treatment without undesired side effects. In the current study, we investigated whether a similar effect is present in vivo using the colorectal cell line LS174T. Animals after acclimatization were randomized and allocated equally in the groups for the different studies (safety, dose-escalation, and efficacy). Drugs were delivered by the intraperitoneal route and animals were monitored for wellbeing. Separately, an efficacy study was conducted with intraperitoneal drug delivery after intraperitoneal tumor induction. At the termination of the experiment, tumors and other tissues were collected for evaluation. BromAc® was safe when delivered intraperitoneally in a rat model at the concentrations used. Subsequent investigations of these adjuvants in combination with Gemcitabine, Oxaliplatin, and 5-Fluorouracil in mice were also proven to be safe. Preliminary efficacy studies with Oxaliplatin and 5-Fluorouracil on tumor growth (LS174T) were negative. Gemcitabine was assessed with BromAc® showing an almost 71% tumor inhibition compared to controls. This in vivo study indicates that Gemcitabine at 2 mg/kg in combination with BromAc® 3 mg/300 mg/Kg was effective and safe, supporting its potential for future clinical application.

The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

The effect of intraperitoneal administration of BromAc on blood parameters: phase 1 study.

Intraperitoneal administration of BromAc (bromelain + acetylcysteine) is currently undergoing a phase 1 clinical trial for pseudomyxoma peritonei at our institution. This study reports on analysis of routine blood parameters before and after treatment for a series of 25 patients in this trial. Blood parameters assessed included full blood count, electrolytes, urea, and creatinine, liver function tests, coagulation studies, as well as inflammatory markers (CRP). Certain parameters such as CRP, and white cell count, were significantly elevated after treatment whilst serum albumin level was reduced indicating an inflammatory reaction. However, liver enzymes, coagulation studies, and other parameters were not affected. Therefore, there are no additional safety signals evident upon analysis of routine blood parameter testing.

Enhancing the potency of chemotherapeutic agents by combination with bromelain and N-acetylcysteine - an in vitro study with pancreatic and hepatic cancer cells.

Current systemic dosages of chemotherapeutic drugs such as gemcitabine, 5-FU, cisplatin, doxorubicin are administered every 7 days over 4 cycles due to systemic toxicity. An increase in potency of the drugs will result in dosage reduction with more frequent administration and efficacy increase. Hence, we investigated how the drugs potency can be increased by combining with bromelain and N-acetylcysteine. Tumour cells (5,000/well) were seeded into a 96 well plate and treated 24 hrs later with either single agents or in combinations at various concentrations. Cell survival was assessed by the sulforhodamine B assay after 72 hours of exposure. LD 50 was determined for each treatment and the Combination Index (CI) was assessed to determine synergy using Tallarida's method. CI indicated that synergy was dependent on the concentration of the agents used and was cell line specific. For bromelain and N-acetylcysteine, certain ratio of the two agents gave very good synergy that was prevalent in almost all cell lines. Gemcitabine and 5-FU and doxorubicin reacted favourably with most concentrations of bromelain and NAC investigated. Cisplatin and oxaliplatin were not very compatible with NAC. A value of CI <0.5 indicated that the current clinical chemotherapeutic dosage can be dramatically reduced. Bromelain with NAC showed synergy in all tumour cell lines and acting synergistically with chemotherapeutic drugs. Synergistic combinations resulting in considerable dosage reduction of chemotherapeutic agents may enable more frequent treatment with higher efficacy.

Targeting CD300f to enhance hematopoietic stem cell transplantation in acute myeloid leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) significantly reduces the rate of relapse in acute myeloid leukemia (AML) but comes at the cost of significant treatment-related mortality. Despite the reduction in relapse overall, it remains common, especially in high-risk groups. The outcomes for patients who relapse after transplant remains very poor. A large proportion of the morbidity that prevents most patients from accessing allo-HSCT is due to toxic nonspecific conditioning agents that are required to remove recipient hematopoietic stem and progenitor cells (HSPCs), allowing for successful donor engraftment. CD300f is expressed evenly across HSPC subtypes. CD300f has transcription and protein expression equivalent to CD33 on AML. We have developed an anti-CD300f antibody that efficiently internalizes into target cells. We have generated a highly potent anti-CD300f antibody-drug conjugate (ADC) with a pyrrolobenzodiazepine warhead that selectively depletes AML cell lines and colony forming units in vitro. The ADC synergizes with fludarabine, making it a natural combination to use in a minimal toxicity conditioning regimen. Our ADC prolongs the survival of mice engrafted with human cell lines and depletes primary human AML engrafted with a single injection. In a humanized mouse model, a single injection of the ADC depletes CD34+ HSPCs and CD34+CD38-CD90+ hematopoietic stem cells. This work establishes an anti-CD300f ADC as an attractive potential therapeutic that, if validated in transplant models using a larger cohort of primary AML samples, will reduce relapse rate and toxicity for patients with AML undergoing allo-HSCT.

Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway.

We have recently shown that the novel anthelmintic drug monepantel (MPL) inhibits growth, proliferation and colony formation, arrests the cell cycle and induces cleavage of PARP-1 in ovarian cancer cell lines. Here we report on the mechanism behind the anticancer properties of MPL. The cytotoxic effect of MPL on ovarian cancer cells (OVCAR-3 and A2780) was investigated employing a panel of tests used for the detection of apoptosis and autophagy. Apoptosis and autophagy were defined by caspase activity, DNA-laddering, Annexin-V and acridine orange (AO) staining. Autophagy markers such as LC3B, SQSTM1/p62 and mammalian target of rapamycin (mTOR) pathway related proteins were assessed by western blotting and ELISA techniques. MPL did not activate caspases 3 or 8, nor did it alter the percentage of Annexin V positive stained cells. Failure to cause DNA laddering and the inability of z-VAD-fmk to block the MPL antiproliferative effects led to the ruling out of apoptosis as the mechanism behind MPL-induced cell death. On the other hand, accumulation of acidic vacuoles with distinct chromatin morphology and an increase in punctuate localization of green fluorescent protein-LC3B, and MPL-induced changes in the expression of SQSTM1/p62 were all indicative of MPL-induced autophagy. Consistent with this, we found inhibition of mTOR phosphorylation leading to suppression of the mTOR/p70S6K signalling pathway. Our findings provide the first evidence to show that MPL triggers autophagy through the deactivation of mTOR/p70S6K signalling pathway.

Human Sprouty1 suppresses growth, migration, and invasion in human breast cancer cells.

Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Expression of human Sprouty1 (hSpry1) gene is downregulated in most breast cancer patients, implicating it as an important tumor suppressor gene. So, we hypothesized that overexpression of hSpry1 gene may suppress breast cancer cell growth, migration, and invasion. Here, we demonstrate that in breast cancer cell lines, MDA-MB-231 and T47D, transfection-induced overexpression of hSpry1 reduced cell population, proliferation, and colony formation in vitro without affecting cell apoptosis. Adhesion molecules act as both positive and negative modulators of cellular migration and invasion. Here, we found that overexpression of hSpry1 enhances the initial establishment events in breast cancer cell adhesion to type IV collagen and vitronectin. Moreover, the overexpression of hSpry1 in the highly invasive MDA-MB-231 breast cancer cells causes a significant reduction in cellular migration and invasion through Matrigel membranes. In addition, we showed that hSpry1 overexpression prevents VEGF secretion. VEGF is essential for primary tumor growth, migration, and invasion. Thus, our study provides a novel mechanism of tumor suppression activity of hSpry1.

Involvement of urokinase-type plasminogen activator system in cancer: an overview.

Currently, there are several studies supporting the role of urokinase-type plasminogen activator (uPA) system in cancer. The association of uPA to its receptor triggers the conversion of plasminogen into plasmin. This process is regulated by the uPA inhibitors (PAI-1 and PAI-2). Plasmin promotes degradation of basement membrane and extracellular matrix (ECM) components as well as activation of ECM latent matrix metalloproteases. Degradation and remodeling of the surrounding tissues is crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, release of tumor growth factors, activation of cytokines as well as induction of tumor cell proliferation, migration, and invasion. Hence, many tumors showed a correlation between uPA system component levels and tumor aggressiveness and survival. Therefore, this review summarizes the structure of the uPA system, its contribution to cancer progression, and the clinical relevance of uPA family members in cancer diagnosis. In addition, the review evaluates the significance of uPA system in the development of cancer-targeted therapies.

Human sprouty1 suppresses urokinase receptor-stimulated cell migration and invasion.

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in several processes in tumor progression including cell migration and invasion in addition to initiation of signal transduction. Since uPAR lacks a transmembrane domain, it uses the interaction with other proteins to modulate intracellular signal transduction. We have previously identified hSpry1 as a partner protein of uPAR, suggesting a physiological role for hSpry1 in the regulation of uPAR signal transduction. In this study, hSpry1 was found to colocalize with uPAR upon stimulation with epidermal growth factor (EGF), urokinase (uPA), or its amino terminal fragment (uPA-ATF), implicating a physiological role of hSpry1 in regulation of uPAR signalling pathway. Moreover, hSpry1 was able to inhibit uPAR-stimulated cell migration in HEK293/uPAR, breast carcinoma, and colorectal carcinoma cells. In addition, hSpry1 was found to inhibit uPAR-stimulated cell invasion in breast carcinoma and osteosarcoma cell lines. Increasing our understanding of how hSpry1 negatively regulates uPAR-stimulated cellular functions may determine a distinctive role for hSpry1 in tumour suppression.

HAX1 Augments Cell Proliferation, Migration, Adhesion, and Invasion Induced by Urokinase-Type Plasminogen Activator Receptor.

The urokinase-type plasminogen activator receptor (uPAR) is a cell surface receptor which has a multifunctional task in the process of tumorigenesis including cell proliferation, adhesion, migration, and invasion. Many of the biological functions of uPAR necessitate interactions with other proteins. We have shown previously that uPAR interacts with HAX1 protein (HS-1-associated protein X-1). In the current study, to gain insight into the possible role of HAX1 overexpression in regulation of uPAR signal transduction pathway, several function assays were used. We found that, upon stimulation of uPAR, HAX1 colocalizes with uPAR suggesting a physiological role for HAX1 in the regulation of uPAR signal transduction. HAX1 overexpression augments cell proliferation and migration in uPAR-stimulated cells. Moreover, HAX1 over-expression augmented uPAR-induced cell adhesion to vitronectin as well as cellular invasion. Our results suggest that HAX1 over-expression may underlay a novel mechanism to regulate uPAR-induced functions in cancer cells.

Minocycline inhibits growth of epithelial ovarian cancer.

OBJECTIVE: These studies were designed to determine whether minocycline inhibits ovarian cancer growth in vitro and in vivo and the molecular mechanisms involved. MATERIALS AND METHODS: The effect of minocycline on ovarian cancer cell proliferation, cell cycle progression and apoptosis was assessed using human ovarian cancer cell lines OVCAR-3, SKOV-3 and A2780. Then, the capacity of minocycline to inhibit growth of OVCAR-3 xenografts in female nude mice was examined. RESULTS: Minocycline inhibited cell proliferation and colony formation, down-regulated cyclins A, B and E leading to arrest of cells in the G(0) phase of the cycle and suppression of DNA synthesis. Furthermore, exposure of these cells to minocycline led to DNA laddering, activation of caspase-3 and cleavage of PARP-1. In nude mice bearing sub-cutaneous tumors, minocycline suppressed tumor proliferation index, angiogenesis and tumor growth. CONCLUSION: These findings provide the initial basis for further evaluation of minocycline in the treatment of ovarian cancer.

Novel protein interactors of urokinase-type plasminogen activator receptor.

The urokinase-type plasminogen activator receptor (uPAR) has been implicated in tumor growth and metastasis. The crystal structure of uPAR revealed that the external surface is largely free to interact with a number of proteins. Additionally, due to absence of an intracellular cytoplasmic protein domain, many of the biological functions of uPAR necessitate interactions with other proteins. Here, we used yeast two-hybrid screening of breast cancer cDNA library to identify hSpry1 and HAX1 proteins as putative candidate proteins that interact with uPAR bait constructs. Interaction between these two candidates and uPAR was confirmed by GST-pull down, co-immunoprecipitation assays and confocal microscopy. These novel interactions that have been identified may also provide further evidence that uPAR can interact with a number of other proteins which may influence a range of biological functions.