Research on the mechanism of Guanyu Zhixie Granule in intervening gastric ulcers in rats based on network pharmacology and multi-omics.

Objective: Guanyu Zhixie Granule (GYZXG) is a traditional Chinese medicine compound with definite efficacy in intervening in gastric ulcers (GUs). However, the effect mechanisms on GU are still unclear. This study aimed to explore its mechanism against GU based on amalgamated strategies. Methods: The comprehensive chemical characterization of the active compounds of GYZXG was conducted using UHPLC-Q/TOF-MS. Based on these results, key targets and action mechanisms were predicted through network pharmacology. GU was then induced in rats using anhydrous ethanol (1 mL/200 g). The intervention effects of GYZXG on GU were evaluated by measuring the inhibition rate of GU, conducting HE staining, and assessing the levels of IL-6, TNF-α, IL-10, IL-4, Pepsin (PP), and epidermal growth factor (EGF). Real-time quantitative PCR (RT-qPCR) was used to verify the mRNA levels of key targets and pathways. Metabolomics, combined with 16S rRNA sequencing, was used to investigate and confirm the action mechanism of GYZXG on GU. The correlation analysis between differential gut microbiota and differential metabolites was conducted using the spearman method. Results: For the first time, the results showed that nine active ingredients and sixteen targets were confirmed to intervene in GU when using GYZXG. Compared with the model group, GYZXG was found to increase the ulcer inhibition rate in the GYZXG-M group (p < 0.05), reduce the levels of IL-6, TNF-α, PP in gastric tissue, and increase the levels of IL-10, IL-4, and EGF. GYZXG could intervene in GU by regulating serum metabolites such as Glycocholic acid, Epinephrine, Ascorbic acid, and Linoleic acid, and by influencing bile secretion, the HIF-1 signaling pathway, and adipocyte catabolism. Additionally, GYZXG could intervene in GU by altering the gut microbiota diversity and modulating the relative abundance of Bacteroidetes, Bacteroides, Verrucomicrobia, Akkermansia, and Ruminococcus. The differential gut microbiota was strongly associated with serum differential metabolites. KEGG enrichment analysis indicated a significant role of the HIF-1 signaling pathway in GYZXG's intervention on GU. The changes in metabolites within metabolic pathways and the alterations in RELA, HIF1A, and EGF mRNA levels in RT-qPCR experiments provide further confirmation of this result. Conclusion: GYZXG can intervene in GU induced by anhydrous ethanol in rats by regulating gut microbiota and metabolic disorders, providing a theoretical basis for its use in GU intervention.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

Mechanisms predictive of Tibetan Medicine Sophora moorcroftiana alkaloids for treatment of lung cancer based on the network pharmacology and molecular docking.

BACKGROUND: Leguminous Sophora moorcroftiana (SM) is a genuine medicinal material in Tibet. Many research results have reveal the Sophora moorcroftiana alkaloids (SMA), as the main active substance, have a wide range of effects, such as antibacterial, antitumor and antiparasitic effects. However, there are few reports on the inhibition of lung cancer (LC) and its inhibitory mechanism, and the pharmacological mechanism of SMA is still unclear, Therefore, exploring its mechanism of action is of great significance. METHODS: The SMA active components were obtained from the literature database. Whereas the corresponding targets were screened from the PubChem and PharmMapper database, UniProt database were conducted the correction and transformation of UniProt ID on the obtained targets. The GeneCards and OMIM databases identified targets associated with LC. Venny tools obtained the intersection targets of SMA and LC. R language and Cytoscape software constructed the visual of SMA - intersection targets - LC disease network. The intersection targets protein-protein interaction (PPI) network were built by the STRING database. The functions and pathways of the common targets of SMA and LC were enriched by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Subsequently, molecular docking And A549 cells vitro experiment were performed to further validate our finding. RESULTS: We obtained six kinds of alkaloids in SM, 635 potential targets for these compounds, and 1,303 genes related to LC. SMA and LC intersection targets was 33, including ALB, CCND1, ESR1, NOTCH1 and AR. GO enrichment indicated that biological process of SMA was mainly involved in the positive regulation of transcription and nitric oxide biosynthetic process, and DNA-templated, etc. Biological functions were mainly involved in transcription factor binding and enzyme binding, etc. Cell components were mainly involved in protein complexes, extracellular exosome, cytoplasm and nuclear chromatin, etc., Which may be associated with its anti-LC effects. KEGG enrichment analysis showed that main pathways involved in the anti-LC effects of SMA, including pathway in cancer, non small-cell lung cancer, p53, PI3K-Akt and FOXO signaling pathways. Molecular docking analyses revealed that the six active compounds had a good binding activity with the main therapeutic targets 2W96, 2CCH and 1O96. Experiments in vitro proved that SMA inhibited the proliferation of LC A549 cells. CONCLUSIONS: Results of the present study, we have successfully revealed the SMA compounds had a multi-target and multi-channel regulatory mechanism in treatment LC, These findings provided a solid theoretical reference of SMA in the clinical treatment of LC.

Lonicerae Japonicae Caulis: a review of its research progress of active metabolites and pharmacological effects.

Lonicerae Japonicae Caulis is the aboveground stem part of the Lonicera Japonica Thunb, which belongs to the medicine food homology species in China. It has the effects of clearing away heat, toxic material, dredging wind and unblocking collaterals. Modern research shows that it contains various active metabolites and a wide range of pharmacological effects, which is of great research and clinical application value. It mainly contains organic acids, volatile oils, flavonoids, triterpenes, triterpene saponins and other active metabolites. Its pharmacological effects mainly include anti-inflammatory, antibacterial, antitumor, antioxidant, and repairing bone and soft tissue. Based on the literature reports in recent years, the active metabolites, pharmacological effects and mechanisms of Lonicerae Japonicae Caulis were sorted out and summarized. It lays a foundation for explaining the efficacy material basis and application value of Lonicerae Japonicae Caulis. It aims to provide a reference for the in-depth research, development and utilization of Lonicerae Japonicae Caulis.

Metabonomics study on the hepatoprotective effect mechanism of polysaccharides from different processed products of Angelica sinensis on layer chickens based on UPLC-Q/TOF-MS/MS, multivariate statistical analysis and conjoint analysis.

Chicken colibacillosis is one of the most severe diseases in the poultry industry. Ceftiofur sodium (CS) is often used to treat it in clinical practice and lipopolysaccharide (LPS) accumulates in the chicken's body. Previous experimental studies found that CS combined with LPS could induce liver injury in layer chickens, and polysaccharides from charred Angelica sinensis(CASP) had a better hepatoprotective effect than polysaccharides from unprocessed Angelica sinensis(UASP). However, the intervention mechanism was unclear. Thus, UPLC-Q/TOF-MS/MS-based metabonomics and transcriptomics were used in this study to clarify the hepatoprotective effect mechanism of CASP and UASP in layer chickens. Transcriptomics and enzyme-linked immunosorbent assay were used for biological verification of some critical mutual metabolic pathways screened with metabonomics. The comprehensive analysis results showed that in a layer chicken liver injury model built with LPS and CS, 12 critical metabolic pathways were disturbed, involving 10 important differential metabolites. The hepatoprotective effect mechanism of CASP is related to the arachidonic acid metabolism and mTOR signaling pathways, involving nine important differential metabolites. In contrast, the hepatoprotective effect mechanism of UASP is related to the arachidonic acid metabolism pathway, involving six important differential metabolites.

A new method providing complementary explanation of the blood-enriching function and mechanism of unprocessed Angelica sinensis and its four kinds of processed products based on tissue-integrated metabolomics and confirmatory analysis.

Angelica sinensis (AS) is a common Traditional Chinese Medicine used for tonifying blood in China. Unprocessed AS and its four kinds of processed products (ASs) are used to treat blood deficiency syndrome in the country. The different blood-tonifying mechanisms of ASs remain unclear. In this work, a novel method integrating metabolomics and hematological and biochemical parameters was established to provide a complementary explanation of blood supplementation mechanism of ASs. Our results revealed that different ASs exhibited various blood supplementation effect, and that AS parched with alcohol demonstrated the best blood supplementation effect. Eight metabolites from liver tissue and 12 metabolites from spleen tissue were considered to be potential biomarkers. These biomarkers were involved in four metabolic pathways. Correlation analysis results showed that l-aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) exhibited a high positive or negative correlation with the aforesaid biochemical indicators. The blood-supplementation effect mechanism of ASs were related to four metabolic pathways. l-Aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) were the four key metabolites associated with the blood supplementation effect of ASs. This study gives a complementary explanation of the blood supplementation effect and mechanism of action of ASs.

Mycobacterium tuberculosis Thymidylyltransferase RmlA Is Negatively Regulated by Ser/Thr Protein Kinase PknB.

Ser/Thr phosphorylation by serine/threonine protein kinases (STPKs) plays significant roles in molecular regulation, which allows Mycobacteria to adapt their cell wall structure in response to the environment changes. Identifying direct targets of STPKs and determining their activities are therefore critical to revealing their function in Mycobacteria, for example, in cell wall formation and virulence. Herein, we reported that RmlA, a crucial L-rhamnose biosynthesis enzyme, is a substrate of STPK PknB in Mycobacterium tuberculosis (M. tuberculosis). Mass spectrometry analysis revealed that RmlA is phosphorylated at Thr-12, Thr-54, Thr-197, and Thr-12 is located close to the catalytic triad of RmlA. Biochemical and phenotypic analysis of two RmlA mutants, T12A/T12D, showed that their activities were reduced, and cell wall formation was negatively affected. Moreover, virulence of RmlA T12D mutant was attenuated in a macrophage model. Overall, these results provide the first evidence for the role of PknB-dependent RmlA phosphorylation in regulating cell wall formation in Mycobacteria, with significant implications for pathogenicity.

Antibody Binding Epitope Mapping (AbMap) of Hundred Antibodies in a Single Run.

Antibodies play essential roles in both diagnostics and therapeutics. Epitope mapping is essential to understand how an antibody works and to protect intellectual property. Given the millions of antibodies for which epitope information is lacking, there is a need for high-throughput epitope mapping. To address this, we developed a strategy, Antibody binding epitope Mapping (AbMap), by combining a phage displayed peptide library with next-generation sequencing. Using AbMap, profiles of the peptides bound by 202 antibodies were determined in a single test, and linear epitopes were identified for >50% of the antibodies. Using spike protein (S1 and S2)-enriched antibodies from the convalescent serum of one COVID-19 patient as the input, both linear and potentially conformational epitopes of spike protein specific antibodies were identified. We defined peptide-binding profile of an antibody as the binding capacity (BiC). Conceptually, the BiC could serve as a systematic and functional descriptor of any antibody. Requiring at least one order of magnitude less time and money to map linear epitopes than traditional technologies, AbMap allows for high-throughput epitope mapping and creates many possibilities.

A novel dark septate fungal endophyte positively affected blueberry growth and changed the expression of plant genes involved in phytohormone and flavonoid biosynthesis.

Dark septate endophytes (DSEs) are one of the most studied groups of root fungal endophytes in recent years. However, the effects of DSE on host plant are still under debate, and the molecular mechanisms are poorly understood. In this study, we identified a DSE fungus of the genus Anteaglonium, named T010, from the wild blueberry. When inoculated into Vaccinium corymbosum L. plants, T010 could enhance root growth and promote shoot branching, leading to increased plant growth. By comparative transcriptome analysis, we obtained 1948 regulated differentially expressed genes (DEGs) from the V. corymbosum plants treated by T010. Further functional enrichment analysis identified a series of DEGs enriched in transcriptional regulation, material transport, phytohormone biosynthesis and flavonoid biosynthesis. Moreover, the comparative analysis of liquid chromatography-mass spectrometry verified that T010 treatment induced the changes in the contents of various phytohormones and flavonoids. This is the first report on the isolation of DSE fungi of the genus Anteaglonium from blueberry roots. Moreover, our results suggested that T010 colonization could result in a series of changes in cell metabolism, biosynthesis and signal pathways, thereby promoting plant growth. Particularly, the changes of phytohormone and flavonoid metabolism induced by T010 colonization might contribute to the promotion of blueberry growth. Our results will provide new insights into understanding of the interaction of DSE fungi and host plants, as well as the development and utilization of DSE preparations.

Global discovery the PstP interactions using Mtb proteome microarray and revealing novel connections with EthR.

Mycobacterium tuberculosis (Mtb) serine/threonine protein phosphatase PstP plays an important role in regulating Mtb cell division and growth by reversible phosphorylation signaling. However, the substrates of Mtb with which the PstP interacts, and the underlying molecular mechanisms are still largely unknown. In this study, we performed an Mtb proteome microarray to globally identify the PstP bindings. In this way, we discovered 78 interactors between PstP and Mtb proteins, and found a novel connections with EthR. The interaction between PstP and EthR has been validated by Bio-Layer interferometry and Yeast-two-hybrid. And functional studies showed that PstP significantly enhances the binding between EthR and related DNA domain through its interaction with EthR. Phenotypically, overexpression of PstP promoted the resistance of Mycobacterium smegmatis with the antibiotic of ethionamide. Overall, we hopefully wish that the PstP interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PstP plays in the regulation of Mtb dephosphorylation. SIGNIFICANCE: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which is responsible of ~1.5 million death per year. Understanding the knowledge about the basic biological regulation pathways in Mtb is an effective approach to discover the novel drug targets for cure TB. PstP is a serine/threonine protein phosphatase in Mtb, and plays important roles in regulating Mtb cell division and growth by reversible phosphorylation signaling. In this study, we identified 78 PstP interacting Mtb proteins using Mtb proteome microarray, which could preliminarily explain the roles of PstP played in Mtb. Moreover, functional analysis showed that a novel transcription factor EthR had been found regulated by PstP through binding, which could enhance the resistance to the antibiotic ETH. Overall, this network constructed with PstP-Mtb proteins could serve as a valuable resource for studying Mtb growth.

Identification of Serum Biomarkers for Systemic Lupus Erythematosus Using a Library of Phage Displayed Random Peptides and Deep Sequencing.

Systemic lupus erythematosus (SLE) is one of the most serious autoimmune diseases, characterized by highly diverse clinical manifestations. A biomarker is still needed for accurate diagnostics. SLE serum autoantibodies were discovered and validated using serum samples from independent sample cohorts encompassing 306 participants divided into three groups, i.e. healthy, SLE patients, and other autoimmune-related diseases. To discover biomarkers for SLE, a phage displayed random peptide library (Ph.D. 12) and deep sequencing were applied to screen specific autoantibodies in a total of 100 serum samples from 50 SLE patients and 50 healthy controls. A statistical analysis protocol was set up for the identification of peptides as potential biomarkers. For validation, 10 peptides were analyzed using enzyme-linked immunosorbent assays (ELISA). As a result, four peptides (SLE2018Val001, SLE2018Val002, SLE2018Val006, and SLE2018Val008) were discovered with high diagnostic power to differentiate SLE patients from healthy controls. Among them, two peptides, i.e. SLE2018Val001 and SLE2018Val002, were confirmed between SLE with other autoimmune patients. The procedure we established could be easily adopted for the identification of autoantibodies as biomarkers for many other diseases.

Global Profiling of PknG Interactions Using a Human Proteome Microarray Reveals Novel Connections with CypA.

Mycobacterium tuberculosis (Mtb) serine/threonine kinase PknG plays an important role in the Mtb-host interaction by facilitating the survival of Mtb in macrophages. However, the human proteins with which the PknG interacts, and the underlying molecular mechanisms are still largely unknown. In this study, a HuProt array is been applied to globally identify the host proteins to which PknG binds. In this way, 125 interactors are discovered, including a cyclophilin protein, CypA. This interaction between PknG and CypA is validated both in vitro and in vivo, and functional studies show that PknG significantly reduces the protein levels of CypA through phosphorylation, which consequently inhibit the inflammatory response through downregulation of NF-κB and ERK1/2 pathways. Phenotypically, overexpression of PknG reduces cytokine levels and promotes the survival of Mycobacterium smegmatis (Msm) in macrophages. Overall, it is expected that the PknG interactors identified in this study will serve as a useful resource for further systematic studies of the roles that PknG plays in the Mtb-host interactions.

Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis.

Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA.

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity.

Mycobacterium tuberculosis (Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, Mycobacterium smegmatis, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis.

Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide.

Tuberculosis is still on the top of infectious diseases list on both mobility and mortality, especially due to drug-resistance of Mycobacterium tuberculosis (M.tb). Ethionamide (ETH) is one of effective second line anti-TB drugs, a synthetic compound similar to isoniazid (INH) structurally, with existing severe problem of ETH resistance. ETH is a prodrug, which is activated by Etha inside M.tb, and etha is transcriptionally repressed by Ethr. We found that c-di-GMP could bind Ethr, enhanced the binding of Ethr to the promoter of etha, and then repressed the transcription of etha, thus caused resistance of M.tb to ETH. Through docking analysis and in vitro validation, we identified that c-di-GMP binds 3 amino acids of Ethr, i.e., Q125, R181 and E190, while the first 2 were the major binding sites. Homology analysis showed that Ethr was highly conservative among mycobacteria. Further docking analysis showed that c-di-GMP preferentially bound proteins of TetR family at the junction hole of symmetric dimer or tetramer proteins. Our results suggest a possible drug-resistance mechanism of ETH through the regulation of Ethr by c-di-GMP.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb.

Mycobacterium tuberculosis proteome microarray for global studies of protein function and immunogenicity.

Poor understanding of the basic biology of Mycobacterium tuberculosis (MTB), the etiological agent of tuberculosis, hampers development of much-needed drugs, vaccines, and diagnostic tests. Better experimental tools are needed to expedite investigations of this pathogen at the systems level. Here, we present a functional MTB proteome microarray covering most of the proteome and an ORFome library. We demonstrate the broad applicability of the microarray by investigating global protein-protein interactions, small-molecule-protein binding, and serum biomarker discovery, identifying 59 PknG-interacting proteins, 30 bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding proteins, and 14 MTB proteins that together differentiate between tuberculosis (TB) patients with active disease and recovered individuals. Results suggest that the MTB rhamnose pathway is likely regulated by both the serine/threonine kinase PknG and c-di-GMP. This resource has the potential to generate a greater understanding of key biological processes in the pathogenesis of tuberculosis, possibly leading to more effective therapies for the treatment of this ancient disease.

Global identification of CobB interactors by an Escherichia coli proteome microarray.

Protein acetylation is one of the most abundant post-translational modifications and plays critical roles in many important biological processes. Based on the recent advances in mass spectrometry technology, in bacteria, such as Escherichia coli, tremendous acetylated proteins and acetylation sites have been identified. However, only one protein deacetylase, i.e. CobB, has been identified in E. coli so far. How CobB is regulated is still elusive. One right strategy to study the regulation of CobB is to globally identify its interacting proteins. In this study, we used a proteome microarray containing ∼4000 affinity-purified E. coli proteins to globally identify CobB interactors, and finally identified 183 binding proteins of high stringency. Bioinformatics analysis showed that these interacting proteins play a variety of roles in a wide range of cellular functions and are highly enriched in carboxylic acid metabolic process and hexose catabolic process, and also enriched in transferase and hydrolase. We further used bio-layer interferometry to analyze the interaction and quantify the kinetic parameters of putative CobB interactors, and clearly showed that CobB could strongly interact with TopA and AccC. The novel CobB interactors that we identified could serve as a start point for further functional analysis.