Cyclization Of Squalene Research Articles

Impact of steroid biosynthesis on the aerobic adaptation ofeukaryotes.

Steroids are indispensable components of the eukaryotic cellular membrane and the acquisition of steroid biosynthesis was a key factor that enabled the evolution of eukaryotes. The polycyclic carbon structures of steroids can be preserved in sedimentary rocks as chemical fossils for billions of years and thus provide invaluable clues to trace eukaryotic evolution from the distant past. Steroid biosynthesis consists of (1) the production of protosteroids and (2) the subsequent modifications toward "modern-type" steroids such as cholesterol and stigmasterol. While protosteroid biosynthesis requires only two genes for the cyclization of squalene, complete modification of protosteroids involves ~10 additional genes. Eukaryotes universally possess at least some of those additional genes and thus produce modern-type steroids as major final products. The geological biomarker records suggest a prolonged period of solely protosteroid production in the mid-Proterozoic before the advent of modern-type steroids in the Neoproterozoic. It has been proposed that mid-Proterozoic protosteroids were produced by hypothetical stem-group eukaryotes that presumably possessed genes only for protosteroid production, even though in modern environments protosteroid production as a final product is found exclusively in bacteria. The host identity of mid-Proterozoic steroid producers is crucial for understanding the early evolution of eukaryotes. In this perspective, we discuss how geological biomarker data and genetic data complement each other and potentially provide a more coherent scenario for the evolution of steroids and associated early eukaryotes. We further discuss the potential impacts that steroids had on the evolution of aerobic metabolism in eukaryotes, which may have been an important factor for the eventual ecological dominance of eukaryotes in many modern environments.

Biosynthetic pathways of triterpenoids and strategies to improve their Biosynthetic Efficiency.

“Triterpenoids” can be considered natural products derived from the cyclization of squalene, yielding 3-deoxytriterpenes (hydrocarbons) or 3-hydroxytriterpenes. Triterpenoids are metabolites of these two classes of triterpenes, produced by the functionalization of their carbon skeleton. They can be categorized into different groups based on their structural formula/design. Triterpenoids are an important group of compounds that are widely used in the fields of pharmacology, food, and industrial biotechnology. However, inadequate synthetic methods and insufficient knowledge of the biosynthesis of triterpenoids, such as their structure, enzymatic activity, and the methods used to produce pure and active triterpenoids, are key problems that limit the production of these active metabolites. Here, we summarize the derivatives, pharmaceutical properties, and biosynthetic pathways of triterpenoids and review the enzymes involved in their biosynthetic pathway. Furthermore, we concluded the screening methods, identified the genes involved in the pathways, and highlighted the appropriate strategies used to enhance their biosynthetic production to facilitate the commercial process of triterpenoids through the synthetic biology method.

A squalene–hopene cyclase in Schizosaccharomyces japonicus represents a eukaryotic adaptation to sterol-limited anaerobic environments

Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop-17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes.

Natural Compounds in Sex Hormone-Dependent Cancers: The Role of Triterpenes as Therapeutic Agents.

Sex hormone-dependent cancers currently contribute to the high number of cancer-related deaths worldwide. The study and elucidation of the molecular mechanisms underlying the progression of these tumors was a double-edged sword, leading to the expansion and development of new treatment options, with the cost of triggering more aggressive, therapy resistant relapses. The interaction of androgen, estrogen and progesterone hormones with specific receptors (AR, ER, PR) has emerged as a key player in the development and progression of breast, ovarian, prostate and endometrium cancers. Sex hormone-dependent cancers share a common and rather unique carcinogenesis mechanism involving the active role of endogenous and exogenous sex hormones to maintain high mitotic rates and increased cell proliferation thus increasing the probability of aberrant gene occurrence and accumulation highly correlated with abnormal cell division and the occurrence of malignant phenotypes. Cancer related hormone therapy has evolved, currently being associated with the blockade of other signaling pathways often associated with carcinogenesis and tumor progression in cancers, with promising results. However, despite the established developments, there are still several shortcomings to be addressed. Triterpenes are natural occurring secondary metabolites biosynthesized by various pathways starting from squalene cyclization. Due to their versatile therapeutic potential, including the extensively researched antiproliferative effect, these compounds are most definitely a cornerstone in the research and development of new natural/semisynthetic anticancer therapies. The present work thoroughly describes the ongoing research related to the antitumor activity of triterpenes in sex hormone-dependent cancers. Also, the current review highlights both the biological activity of various triterpenoid compounds and their featured mechanisms of action correlated with important chemical structural features.

Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae.

Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast Saccharomyces cerevisiae therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid "sterol surrogate" tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene TtTHC1 from the ciliate Tetrahymena thermophila enables synthesis of tetrahymanol by S. cerevisiae Moreover, expression of TtTHC1 enabled exponential growth of anaerobic S. cerevisiae cultures in sterol-free synthetic media. After deletion of the ERG1 gene from a TtTHC1-expressing S. cerevisiae strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of TtTHC1-expressing S. cerevisiae on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to S. cerevisiae under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts.IMPORTANCE The laboratory experiments described in this report simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote Saccharomyces cerevisiae therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This report provides a proof of principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale anaerobic industrial processes. The sterol-independent yeast strains described in this report provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.

Diatoms synthesize sterols by inclusion of animal and fungal genes in the plant pathway

Diatoms are ubiquitous microalgae that have developed remarkable metabolic plasticity and gene diversification. Here we report the first elucidation of the complete biosynthesis of sterols in the lineage. The study has been carried out on the bloom-forming species Skeletonema marinoi and Cyclotella cryptica that synthesise an ensemble of sterols with chemotypes of animals (cholesterol and desmosterol), plants (dihydrobrassicasterol and 24-methylene cholesterol), algae (fucosterol) and marine invertebrates (clionasterol). In both species, sterols derive from mevalonate through cyclization of squalene to cycloartenol by cycloartenol synthase. The pathway anticipates synthesis of cholesterol by enzymes of the phytosterol route in plants, as recently reported in Solanaceae. Major divergences stem from reduction of Δ24(28) and Δ24(25) double bonds which, in diatoms, are apparently dependent on sterol reductases of fungi, algae and animals. Phylogenetic comparison revealed a good level of similarity between the sterol biosynthetic genes of S. marinoi and C. cryptica with those in the genomes of the other diatoms sequenced so far.

Origin and Evolution of Polycyclic Triterpene Synthesis.

Polycyclic triterpenes are members of the terpene family produced by the cyclization of squalene. The most representative polycyclic triterpenes are hopanoids and sterols, the former are mostly found in bacteria, whereas the latter are largely limited to eukaryotes, albeit with a growing number of bacterial exceptions. Given their important role and omnipresence in most eukaryotes, contrasting with their scant representation in bacteria, sterol biosynthesis was long thought to be a eukaryotic innovation. Thus, their presence in some bacteria was deemed to be the result of lateral gene transfer from eukaryotes. Elucidating the origin and evolution of the polycyclic triterpene synthetic pathways is important to understand the role of these compounds in eukaryogenesis and their geobiological value as biomarkers in fossil records. Here, we have revisited the phylogenies of the main enzymes involved in triterpene synthesis, performing gene neighborhood analysis and phylogenetic profiling. Squalene can be biosynthesized by two different pathways containing the HpnCDE or Sqs proteins. Our results suggest that the HpnCDE enzymes are derived from carotenoid biosynthesis ones and that they assembled in an ancestral squalene pathway in bacteria, while remaining metabolically versatile. Conversely, the Sqs enzyme is prone to be involved in lateral gene transfer, and its emergence is possibly related to the specialization of squalene biosynthesis. The biosynthesis of hopanoids seems to be ancestral in the Bacteria domain. Moreover, no triterpene cyclases are found in Archaea, invoking a potential scenario in which eukaryotic genes for sterol biosynthesis assembled from ancestral bacterial contributions in early eukaryotic lineages.

Synthesis and Anticancer Activity of CDDO and CDDO-Me, Two Derivatives of Natural Triterpenoids.

Triterpenoids are natural compounds synthesized by plants through cyclization of squalene, known for their weak anti-inflammatory activity. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO), and its C28 modified derivative, methyl-ester (CDDO-Me, also known as bardoxolone methyl), are two synthetic derivatives of oleanolic acid, synthesized more than 20 years ago, in an attempt to enhance the anti-inflammatory behavior of the natural compound. These molecules have been extensively investigated for their strong ability to exert antiproliferative, antiangiogenic, and antimetastatic activities, and to induce apoptosis and differentiation in cancer cells. Here, we discuss the chemical properties of natural triterpenoids, the pathways of synthesis and the biological effects of CDDO and its derivative CDDO-Me. At nanomolar doses, CDDO and CDDO-Me have been shown to protect cells and tissues from oxidative stress by increasing the transcriptional activity of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2). At doses higher than 100 nM, CDDO and CDDO-Me are able to modulate the differentiation of a variety of cell types, both tumor cell lines or primary culture cell, while at micromolar doses these compounds exert an anticancer effect in multiple manners; by inducing extrinsic or intrinsic apoptotic pathways, or autophagic cell death, by inhibiting telomerase activity, by disrupting mitochondrial functions through Lon protease inhibition, and by blocking the deubiquitylating enzyme USP7. CDDO-Me demonstrated its efficacy as anticancer drugs in different mouse models, and versus several types of cancer. Several clinical trials have been started in humans for evaluating CDDO-Me efficacy as anticancer and anti-inflammatory drug; despite promising results, significant increase in heart failure events represented an obstacle for the clinical use of CDDO-Me.

Biosynthesis of ambrein in ambergris: evidence from isotopic data and identification of possible intermediates

Ambrein is found in ambergris, a coprolith occurring in the rectum of the sperm whale. In vitro, ambrein is produced by enzymatic cyclisation of squalene, via a monocyclic intermediate. However, little is known of the in vivo process. In order to find evidence for the reaction in vivo, a comparison was made of the δ13C relative isotopic ratios of ambrein in ambergris with those of co-occurring sterols. A statistically significant difference was noted. This suggests that ambrein originates via a different biosynthetic mechanism from that of the sterols. Examination of the minor constituents of a hydrogenolysed extract of ambergris revealed compounds with a bicyclic polypodane nucleus, rather than those with monocyclic structures. It is hypothesised that in vivo biosynthesis of ambrein proceeds, at least in some cases, via bacterial production of bicyclic polypodenols. The latter are known products of non-concerted squalene (or squalene oxide) cyclisations in other organisms.

Squalene Cyclases and Cycloartenol Synthases from Polystichum polyblepharum and Six Allied Ferns.

Ferns are the most primitive of all vascular plants. One of the characteristics distinguishing them from flowering plants is its triterpene metabolism. Most cyclic triterpenes in ferns are hydrocarbons derived from the direct cyclization of squalene by squalene cyclases (SCs). Both ferns and more complex plants share sterols and biosynthetic enzymes, such as cycloartenol synthases (CASs). Polystichum belongs to Dryopteridaceae, and is one of the most species-rich of all fern genera. Several Polystichum ferns in Japan are classified as one of three possible chemotypes, based on their triterpene profiles. In this study, we describe the molecular cloning and functional characterization of cDNAs encoding a SC (PPH) and a CAS (PPX) from the type species Polystichum polyblepharum. Heterologous expression in Pichia pastoris revealed that PPH and PPX are hydroxyhopane synthase and CAS, respectively. By using the PPH and PPX sequences, we successfully isolated SC- and CAS-encoding cDNAs from six Polystichum ferns. Phylogenetic analysis, based on SCs and oxidosqualene cyclase sequences, suggested that the Polystichum subclade in the fern SC and CAS clades reflects the chemotype—but not the molecular phylogeny constructed using plastid molecular markers. These results show a possible relation between triterpenes and their biosynthetic enzymes in Polystichum.

MD Simulations Reveal Complex Water Paths in Squalene-Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site.

Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.

Preservation of hopenol-B in Eocene dammar resins: Insights into the evolution of biochemical synthesis of angiosperm metabolites

Plants synthesize various types of triterpenoids as secondary metabolites to achieve greater fitness with respect to their surrounding environment. In last few decades, interest in biochemical synthesis and regulation of plant triterpenoids has been increased due to their scientific and economic significance. However, until now evolution of such triterpenoid synthesis over geological time is not clearly understood. In order to better understand the biosynthesis of triterpenoid in deep time, the present study reports hopenol-B (hop-22[29]-en-3β-ol), a plant secondary metabolite from fossil dammar resin of Eocene time, derived from Dipterocarpaceae family. The studied fossil resins were obtained from two lignite-bearing sequences of early and middle Eocene age from the Cambay and Kutch Basin, western India, respectively. This bioactive metabolite is recorded from several modern angiosperm trees. Hopenol-B is synthesized by the enzyme (oxidosqalene cyclase containing hopanoid skeleton)-mediated cyclization of 2,3-oxidosqualene by which other angiosperm-derived triterpenoids are also formed. In contrast, hopanoid class triterpenoids of bacterial and non-vascular plants origin are formed by the cyclization of squalene, catalyzed by squalene-hopene cyclases (SHCs). The present study provides the earliest signature of hopenol-B found so far and its occurrence in the analyzed samples suggests that the biosynthetic pathway of this compound in angiosperms had already been evolved during the early Eocene.

Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step.

Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl-β-curcumene (C35 ) but a bicyclic scaffold from squalene (C30 ) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TCY167A produced an unnatural tricyclic triterpenol, but TCY167L , TCY167F , and TCY167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large-scale production of an onoceroid for which natural sources are limited.

Computational studies on the cyclization of squalene to the steroids and hopenes

A review of computational studies of the related biosyntheses of steroids and hopenes reported during the last two decades is presented. Computations in the gas phase and those that include the enzyme are covered. Based on the numerical results conclusions have been drawn about the biosynthetic mechansims of these all important biological reactions.

A distinct pathway for tetrahymanol synthesis in bacteria

Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.

Antimicrobial activity of oleanolic and ursolic acids: an update.

Triterpenoids are the most representative group of phytochemicals, as they comprise more than 20,000 recognized molecules. These compounds are biosynthesized in plants via squalene cyclization, a C30 hydrocarbon that is considered to be the precursor of all steroids. Due to their low hydrophilicity, triterpenes were considered to be inactive for a long period of time; however, evidence regarding their wide range of pharmacological activities is emerging, and elegant studies have highlighted these activities. Several triterpenic skeletons have been described, including some that have presented with pentacyclic features, such as oleanolic and ursolic acids. These compounds have displayed incontestable biological activity, such as antibacterial, antiviral, and antiprotozoal effects, which were not included in a single review until now. Thus, the present review investigates the potential use of these triterpenes against human pathogens, including their mechanisms of action, via in vivo studies, and the future perspectives about the use of compounds for human or even animal health are also discussed.

ChemInform Abstract: Cyclization of Squalene from Both Termini: Identification of an Onoceroid Synthase and Enzymatic Synthesis of Ambrein (I).

ChemInform Abstract: Cyclization of Squalene from Both Termini: Identification of an Onoceroid Synthase and Enzymatic Synthesis of Ambrein (I).

Cyclization of Squalene from Both Termini: Identification of an Onoceroid Synthase and Enzymatic Synthesis of Ambrein

The onoceroids are triterpenoids biosynthesized from squalene or (3S)-2,3-oxidosqualene by cyclization from both termini. We recently revealed that tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-β-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. Here, we reveal that BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is the first onoceroid synthase. We also report the first onoceroids from bacterial origin. Our discoveries suggest that symmetric and asymmetric onoceroids could be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene. Furthermore, the new function of BmeTC enabled the synthesis of (+)-ambrein, a major constituent of ambergris that is difficult to obtain naturally, via a mutated squalene-hopene cyclase-catalyzed reaction from easily available squalene.

Activation-Independent Cyclization of Monoterpenoids

The biosynthesis of cyclic monoterpenes (C(10)) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C(30)), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase from Zymomonas mobilis (ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.

Medicago truncatula CYP716A12 Is a Multifunctional Oxidase Involved in the Biosynthesis of Hemolytic Saponins

Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis.