Engineering Escherichia coli for constitutive production of monophosphoryl lipid A vaccine adjuvant.

During the COVID-19 pandemic, expedient vaccine production has been slowed by the shortage of safe and effective raw materials, such as adjuvants, essential components to enhance the efficacy of vaccines. Monophosphoryl lipid A (MPLA) is a potent and safe adjuvant used in human vaccines, including the Shingles vaccine, Shingrix. 3-O-desacyl-4'-monophosphoryl lipid A (MPL), a representative MPLA adjuvant commercialized by GSK, was prepared via chemical conversion of precursors isolated from Salmonella typhimurium R595. However, the high price of these materials limits their use in premium vaccines. To combat the scarcity and high cost of safe raw materials for vaccines, we need to develop a feasible MPLA production method that is easily scaled up to meet industrial requirements. In this study, we engineered peptidoglycan and outer membrane biosynthetic pathways in Escherichia coli and developed a Escherichia coli strain, KHSC0055, that constitutively produces EcML (E. coli-produced monophosphoryl lipid A) without additives such as antibiotics or overexpression inducers. EcML production was optimized on an industrial scale via high-density fed-batch fermentation, and obtained 2.7 g of EcML (about 135,000 doses of vaccine) from a 30-L-scale fermentation. Using KHSC0055, we simplified the production process and decreased the production costs of MPLA. Then, we applied EcML purified from KHSC0055 as an adjuvant for a COVID-19 vaccine candidate (EuCorVac-19) currently in clinical trial stage III in the Philippines. By probing the efficacy and safety of EcML in humans, we established KHSC0055 as an efficient cell factory for MPLA adjuvant production.

A Fluorescence-Polarization-Based Lipopolysaccharide-Caspase-4 Interaction Assay for the Development of Inhibitors.

Recognition of intracellular lipopolysaccharide (LPS) by Caspase-4 (Casp-4) is critical for host defense against Gram-negative pathogens. LPS binds to the N-terminal caspase activation and recruitment domain (CARD) of procaspase-4, leading to auto-proteolytic activation followed by pro-inflammatory cytokine release and pyroptotic cell death. Aberrant hyper-activation of Casp-4 leads to amplification of the inflammatory response linked to sepsis. While the active site of a caspase has been targeted with peptide inhibitors, inhibition of LPS-Casp-4 interaction is an emerging strategy for the development of selective inhibitors with a new mode of action for treating infectious diseases and sepsis induced by LPS. In this study, a high-throughput screening (HTS) system based on fluorescence polarization (FP) was devised to identify inhibitors of the LPS and Casp-4 interaction. Using HTS and IC50 determination and subsequently showing inhibited Casp-4 activity, we demonstrated that the LPS-Casp-4 interaction is a druggable target for Casp-4 inhibition and possibly a non-canonical inflammatory pathway.

Extracellular pyruvate kinase M2 facilitates cell migration by upregulating claudin-1 expression in colon cancer cells.

Extensive studies have been reported the non-canonical functions of pyruvate kinase M2 (PKM2) as a kinase, transcriptional regulator, and even cell-to-cell communicator, emphasizing its importance in various signaling pathways. However, the role of secreted PKM2 in cancer progression and its signaling pathway is yet to be elucidated. In this study, we found that extracellular PKM2 enhanced the migration of low-metastatic, benign colon cancer cells by upregulating claudin-1 expression and internalizing it to the cytoplasm and nucleus. Knock-down of claudin-1 significantly reduced extracellular PKM2-induced cell migration. Inhibition of either protein kinase C (PKC) or epidermal growth factor receptor (EGFR) resulted in a reduction of extracellular PKM2-mediated claudin-1 expression, suggesting EGFR-PKC-claudin-1 as a signaling pathway in the extracellular PKM2-mediated tumorigenesis of colon cancer cells.

Metabolic engineering of Escherichia coli to produce a monophosphoryl lipid A adjuvant.

Monophosphoryl lipid A (MPLA) species, including MPL (a trade name of GlaxoSmithKline) and GLA (a trade name of Immune Design, a subsidiary of Merck), are widely used as an adjuvant in vaccines, allergy drugs, and immunotherapy to boost the immune response. Even though MPLA is a derivative of lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, bacterial strains producing MPLA have not been found in nature nor engineered. In fact, MPLA generation involves expensive and laborious procedures based on synthetic routes or chemical transformation of precursors isolated from Gram-negative bacteria. Here, we report the engineering of an Escherichia coli strain for in situ production and accumulation of MPLA. Furthermore, we establish a succinct method for purifying MPLA from the engineered E. coli strain. We show that the purified MPLA (named EcML) stimulates the mouse immune system to generate antigen-specific IgG antibodies similarly to commercially available MPLA, but with a dramatically reduced manufacturing time and cost. Our system, employing the first engineered E. coli strain that directly produces the adjuvant EcML, could transform the current standard of industrial MPLA production.

Multi-dimensional histone methylations for coordinated regulation of gene expression under hypoxia.

Hypoxia increases both active and repressive histone methylation levels via decreased activity of histone demethylases. However, how such increases coordinately regulate induction or repression of hypoxia-responsive genes is largely unknown. Here, we profiled active and repressive histone tri-methylations (H3K4me3, H3K9me3, and H3K27me3) and analyzed gene expression profiles in human adipocyte-derived stem cells under hypoxia. We identified differentially expressed genes (DEGs) and differentially methylated genes (DMGs) by hypoxia and clustered the DEGs and DMGs into four major groups. We found that each group of DEGs was predominantly associated with alterations in only one type among the three histone tri-methylations. Moreover, the four groups of DEGs were associated with different TFs and localization patterns of their predominant types of H3K4me3, H3K9me3 and H3K27me3. Our results suggest that the association of altered gene expression with prominent single-type histone tri-methylations characterized by different localization patterns and with different sets of TFs contributes to regulation of particular sets of genes, which can serve as a model for coordinated epigenetic regulation of gene expression under hypoxia.

Sporadic Early-Onset Diffuse Gastric Cancers Have High Frequency of Somatic CDH1 Alterations, but Low Frequency of Somatic RHOA Mutations Compared With Late-Onset Cancers.

BACKGROUND & AIMS: Early-onset gastric cancer, which develops in patients younger than most gastric cancers, is usually detected at advanced stages, has diffuse histologic features, and occurs more frequently in women. We investigated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers (DGCs) from younger patients. METHODS: We conducted whole exome and RNA sequence analyses of 80 resected DGC samples from patients 45 years old or younger in Korea. Patients with pathogenic germline mutations in CDH1, TP53, and ATM were excluded from the onset of this analysis, given our focus on somatic alterations. We used MutSig2CV to evaluate the significance of mutated genes. We recruited 29 additional early-onset Korean DGC samples and performed SNP6.0 array and targeted sequencing analyses of these 109 early-onset DGC samples (54.1% female, median age, 38 years). We compared the SNP6.0 array and targeted sequencing data of the 109 early-onset DGC samples with those from diffuse-type stomach tumor samples collected from 115 patients in Korea who were 46 years or older (late onset) at the time of diagnosis (controls; 29.6% female, median age, 67 years). We compared patient survival times among tumors from different subgroups and with different somatic mutations. We performed gene silencing of RHOA or CDH1 in DGC cells with small interfering RNAs for cell-based assays. RESULTS: We identified somatic mutations in the following genes in a significant number of early-onset DGCs: the cadherin 1 gene (CDH1), TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. None of 109 early-onset DGC cases had pathogenic germline CDH1 mutations. A higher proportion of early-onset DGCs had mutations in CDH1 (42.2%) or TGFBR1 (7.3%) compared with control DGCs (17.4% and 0.9%, respectively) (P < .001 and P = .014 for CDH1 and TGFBR1, respectively). In contrast, a smaller proportion of early-onset DGCs contained mutations in RHOA (9.2%) than control DGCs (19.1%) (P = .033). Late-onset DGCs in The Cancer Genome Atlas also contained less frequent mutations in CDH1 and TGFBR1 and more frequent RHOA mutations, compared with early-onset DGCs. Early-onset DGCs from women contained significantly more mutations in CDH1 or TGFBR1 than early-onset DGCs from men. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times in patients with early-onset DGCs (hazard ratio, 3.4; 95% confidence interval, 1.5-7.7). RHOA activity was reduced by an R5W substitution-the RHOA mutation most frequently detected in early-onset DGCs. Silencing of CDH1, but not RHOA, increased migratory activity of DGC cells. CONCLUSIONS: In an integrative genomic analysis, we found higher proportions of early-onset DGCs to contain somatic mutations in CDH1 or TGFBR1 compared with late-onset DGCs. However, a smaller proportion of early-onset DGCs contained somatic mutations in RHOA than late-onset DGCs. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times of patients, which might account for the aggressive clinical course of early-onset gastric cancer. Female predominance in early-onset gastric cancer may be related to relatively high rates of somatic CDH1 and TGFBR1 mutations in this population.

The ADP-ribose reactive NUDIX hydrolase isoforms can modulate HIF-1α in cancer cells.

The human nucleoside-diphosphate linked moiety-X (NUDIX) hydrolases that utilize ADP-ribose and NADH/NAD+ are overexpressed in cancer cells, but their roles in hypoxia inducible factor-1α (HIF-1α) regulation have not yet been revealed. Here, we showed that these NUDIX hydrolases negatively regulated HIF-1α accumulation by modulating the Ca2+ dependent AMP-activated protein kinase (AMPK) signaling pathway. In specific, knockdown of NUDT9 resulted in accumulation of free ADP-ribose that triggered Ca2+ influx mediated by transient receptor potential cation channel subfamily M member 2 and subsequent activation of Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß). In addition, AMPK activation by CaMKKß was shown to enhance HIF-1α accumulation. Our findings provide insights into the action of NUDIX hydrolases as an additional, discrete modulator of HIF-1α accumulation.

Inflammatory hypoxia induces syndecan-2 expression through IL-1ß-mediated FOXO3a activation in colonic epithelia.

Chronic inflammation is known to be a key causative factor in tumor progression, but we do not yet fully understand the molecular mechanism through which inflammation leads to cancer. Here, we report that the dextran sulfate sodium (DSS)-induced mouse model of chronic colitis is associated with increases in the serum level of IL-1ß and the colonic epithelial expression of the cell-surface heparan sulfate proteoglycan, syndecan-2. We further show that IL-1ß stimulated the transcription of syndecan-2 via NF-κB-dependent FOXO3a activation in CCD841CoN normal colonic epithelial cells and early-stage HT29 colon cancer cells. Inflammatory hypoxia was observed in the colonic epithelia of mice with chronic colitis, suggesting that hypoxic stress is involved in the regulation of syndecan-2 expression. Consistently, experimental inflammatory hypoxia induced hypoxia inducible factor-1α-dependent FOXO3a expression and the p38 MAPK-mediated nuclear localization of FOXO3a. FOXO3a directly mediated syndecan-2 expression in both cell lines and the colonic epithelia of mice with DSS-induced colitis. Moreover, syndecan-2 expression was detected in azoxymethane/DSS-induced colon tumors. Together, these data demonstrate that inflammatory hypoxia up-regulates syndecan-2 via the IL-1ß-NF-κB-FOXO3a pathway. These findings provide new mechanistic insights into inflammatory hypoxia-mediated syndecan-2 expression to connect chronic inflammation and the development of colon cancer.-Choi, S., Chung, H., Hong, H., Kim, S. Y., Kim, S.-E., Seoh, J.-Y., Moon, C. M., Yang, E. G., Oh, E.-S. Inflammatory hypoxia induces syndecan-2 expression through IL-1ß-mediated FOXO3a activation in colonic epithelia.

p21-activated kinase 4 regulates HIF-1α translation in cancer cells.

The p21-activated kinases (Paks) interact with Rac/Cdc42 GTPases to regulate the actin cytoskeleton as well as various signaling pathways. Although activation of Paks in many human cancers is known to mediate cancer progression, the role of Pak proteins in hypoxia is poorly understood. In this study, we found that both Pak1 and Pak4 are highly expressed in HeLa cervical cancer cells, but only Pak4 knockdown attenuates expression of hypoxia-inducible factor-1α (HIF-1α) in hypoxia. We further discovered that Pak4 regulates HIF-1α translation via the Akt-mTOR-4E-BP1 pathway under hypoxic conditions. These results support a novel connection between HIF-1α and Pak4 in hypoxic cancer cells, and provide insights into mechanisms whereby tumors respond to and thrive under oxygen-deficient conditions.

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer-related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R(2) > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.

Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry.

While human plasma serves as a great source for disease diagnosis, low-molecular-weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we employ top-down mass spectrometry to analyze the LMW proteoforms present in four types of human plasma samples pooled from three healthy controls (HCs) without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. The LMW proteoforms were first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC 3.0. As a result, a total of 442 LMW proteins and cleaved products, including those with post-translational modifications and single amino acid variations, were identified. From additional comparative analysis of plasma samples without immunoaffinity depletion between HCs and colorectal cancer (CRC) patients via top-down approach, tens of LMW proteoforms, including platelet factor 4, were found to show >1.5-fold changes between the plasma samples of HCs and CRC patients, and six of the LMW proteins were verified by Western blot analysis.

Protein Kinase C Isoforms Differentially Regulate Hypoxia-Inducible Factor-1α Accumulation in Cancer Cells.

Hypoxia-inducible factor-1α (HIF-1α) is one of the key transcription factors that mediate adaptation to hypoxia. Despite increasing evidence implicating the PKC family as potential modulators of HIF-1α, the molecular mechanisms of PKC isoform-dependent HIF-1α activity under hypoxic conditions have not been systematically elucidated in cancer cell lines. Here, we collectively investigated how each isoform of the PKC family contributes to HIF-1α accumulation in the human cervical cancer cell line HeLa. Among the abundant PKC isoforms, blockade of either PKCα or PKCδ was found to substantially reduce HIF-1α accumulation and transcriptional activity in hypoxic cells. Knockdown of PKCδ resulted in a reduction of HIF-1α mRNA levels, whereas the HIF-1α mRNA level was unchanged regardless of PKCα knockdown. Upon searching for the downstream effectors of these kinases, we found that PKCα controls HIF-1α translation via AKT-mTOR under hypoxic conditions. On the other hand, one of the well-known transcriptional regulation pathways of HIF-1α, nuclear factor-κB (NF-κB) is identified as a downstream effector of PKCδ. Taken together, our findings provide insights into the roles of PKC isoforms as additional, discrete modulators of hypoxia-stimulated HIF-1α accumulation through different signaling pathways.

Pyrithione Zn selectively inhibits hypoxia-inducible factor prolyl hydroxylase PHD3.

Increasing evidence emphasizes the role of the hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) isoforms in regulating non-HIF substrates, but isoform selective PHD inhibitors under physiological conditions have not yet been reported. Here we have identified pyrithione Zn (PZ) as a potent, isoform-selective PHD3 inhibitor. The IC50 value of PZ was determined as 0.98 µM for PHD3, while it did not show any inhibitory activity toward full length and truncated PHD2 up to 1 mM. The selective efficacy of PZ was further demonstrated at the cellular level by observing inhibition of the PHD3-dependent DNA damage response pathway without stabilization of HIF-1α.

A facile method to prepare large quantities of active caspase-3 overexpressed by auto-induction in the C41(DE3) strain.

Since human Caspase-3, a member of the cysteine protease family, plays important roles not only in the apoptosis pathway as an executioner protein, but also in neurological disorders as a critical factor, biomedical researchers have been interested in the development of modulators of caspase-3 activity. Such studies require large quantities of purified active caspase-3. So far, purification of soluble caspase-3 from full-length human caspase-3 in Escherichia coli (E. coli) yields only several mg from a liter of culture media. Therefore, a number of alternative strategies to purify active caspase-3 have been described in the literature, including refolding and protein engineering. In this study, we systematically study the effects of host E. coli strains and growth conditions on purifications of active caspase-3 from full-length human caspase-3. Using a combination of conditions that include use of the C41(DE3) strain, low-temperature expression, and auto-induction that induces caspase-3 expression depending on metabolic state of the individual host cell, we are able to obtain 14-17 mg caspase-3 per liter of culture, an amount that is about 7 times larger than published results. This optimized expression and purification method for caspase-3 can be easily scaled up to facilitate the demand for active enzyme.

The action of HIF-3α variants on HIF-2α-HIF-1ß heterodimer formation is directly probed in live cells.

Hypoxia-inducible factors (HIFs), consisting of α and ß subunits, activate various genes to adapt to low oxygen environments through their heterodimeric complex formation in the nucleus. While most of the studies have been extensively focused on the HIF-1α isoform, the effect of HIF-α isoforms on the complex formation between HIF-2α and HIF-1ß in live cells has not been reported in detail. To probe these interactions in a physiological condition, we established a fluorescence resonance energy transfer (FRET) assay by introducing fluorescent reporter proteins onto the N-termini of HIF-2α and HIF-1ß in live PC3 cells. After thorough validations of our FRET assay system, we showed that both HIF-1α and HIF-3α variants likely function as negative regulators on the heterodimer formation of HIF-2α with HIF-1ß in cells. We also characterized the localization and stabilization of HIF-3α variants and measured the interaction between HIF-3α variants and other HIF isoforms in live cells. In contrast to the previous results showing HIF-3α-mediated blockage of HIF-1α translocation, the presence of HIF-3α did not affect the localization of HIF-2α, suggesting distinct roles of HIF-3α in regulation of two HIF-α isoforms.

Both targeted mass spectrometry and flow sorting analysis methods detected the decreased serum apolipoprotein E level in Alzheimer's disease patients.

Apolipoprotein E (ApoE) polymorphism has been appreciated as a valuable predictor of Alzheimer disease (AD), and the associated ε4 allele has been recognized as an indicator of susceptibility to this disease. However, serum ApoE levels have been a controversial issue in AD, due to the great variability regarding the different target detection methods, ethnicity, and the geographic variations of cohorts. The aim of this study was to validate serum ApoE levels in relation to AD, particularly using two distinct detection methods, liquid chromatography-selected reaction monitoring (SRM) mass spectrometry and microsphere-based fluorescence-activated cell sorting (FACS) analysis, to overcome experimental variations. Also, comparison of serum ApoE levels was performed between the level of protein detection by FACS and peptide level by SRM in both control and AD patients. Results from the two detection methods were cross-confirmed and validated. Both methods produced fairly consistent results, showing a significant decrease of serum ApoE levels in AD patients relative to those of a control cohort (43 control versus 45 AD, p < 0.0001). Significant correlation has been revealed between results from FACS and SRM (p < 0.0001) even though lower serum ApoE concentration values were measured in protein by FACS analysis than in peptide-level detections by SRM. Correlation study suggested that a decrease of the serum ApoE level in AD is related to the mini-mental state exam score in both results from different experimental methods, but it failed to show consistent correlation with age, gender, or clinical dementia rating.

Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications.

Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD) and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

Visualization of hypoxia-inducible factor 1α-p300 interactions in live cells by fluorescence resonance energy transfer.

Hypoxia-inducible factor (HIF)-1α mediates the hypoxia response signaling pathway essential for maintaining cellular homeostasis in low oxygen environments through its complex formation with CBP/p300 in the nucleus. Employing fluorescence resonance energy transfer (FRET), we devised a live-cell interaction assay based on reporter proteins by tagging fluorescent proteins onto the carboxy termini of HIF-1α and p300. The nature of the constructed reporter protein was verified by observing localized distribution, degradation, and stabilization kinetics in cells transfected with the HIF-1α containing plasmid. A mutant HIF-1α incapable of binding to p300 was then utilized to demonstrate insignificant FRET efficiency, thereby confirming that our constructs could effectively probe the direct interaction between HIF-1α and p300. We further examined the effects of small molecules known to modulate the HIF-1α-p300 interaction and transcriptional activity on FRET. Finally, by inhibiting activities of two HIF-specific hydroxylases, HIF-specific prolyl hydroxylase (PHD) 2 and factor inhibiting HIF-1 (FIH-1) with their specific siRNAs, we explored how these HIF-specific hydroxylases contribute to the HIF-1α-p300 interaction by FRET measurements along with HIF-1 mediated transcriptional activation. Therefore, this technique would provide a way to study selective inhibition of either PHD2 or FIH-1 within living cells, and to screen specific inhibitors of HIF-mediated transcription activity for therapeutic applications.

Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis.

The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe(2+)/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.

Probing enzymatic activity inside living cells using a nanowire-cell "sandwich" assay.

Developing a detailed understanding of enzyme function in the context of an intracellular signal transduction pathway requires minimally invasive methods for probing enzyme activity in situ. Here, we describe a new method for monitoring enzyme activity in living cells by sandwiching live cells between two vertical silicon nanowire (NW) arrays. Specifically, we use the first NW array to immobilize the cells and then present enzymatic substrates intracellularly via the second NW array by utilizing the NWs' ability to penetrate cellular membranes without affecting cells' viability or function. This strategy, when coupled with fluorescence microscopy and mass spectrometry, enables intracellular examination of protease, phosphatase, and protein kinase activities, demonstrating the assay's potential in uncovering the physiological roles of various enzymes.