Characterization of an ssDNA ligase and its application in aptamer circularization.

Aptamers are widely used in various biomedical areas as novel molecular recognition elements, however, short single-stranded DNA (ssDNA) or RNA oligonucleotides are easily degraded by nucleases in biological fluids. This problem can be solved by circularizing aptamers with circular ligases. Herein, a moderately thermostable ssDNA ligase was expressed and purified. The purified ligase showed good circularization activity for different length substrates and much higher circularization efficiency than T4 RNA ligase 1. Biochemical characterization revealed that the enzyme showed optimal circularization activity at pH 7.5 and 50 ᵒC. Mn2+ and Mg2+ increased enzyme circularization activity, with Mn2+ having higher activity than Mg2+. The optimal concentrations of Mn2+ and ligase were 1.25-2.5 mM and 0.02 nM, respectively. The kinetic parameters Km, Vmax and Kcat of ssDNA ligase were 1.16 µM, 10.71 µM/min, and 10.7 min-1, respectively. The ssDNA ligase efficiency was nucleotide-dependent, and 5'-G and 3'-T were the most ligase-favored terminal nucleotides. In addition, the affinity and stability of the circular aptamer were determined. The affinity constant (KD) was 4.9 µM, and the stability increased compared to its linear form. Molecular docking results showed that the circular aptamer bound to the target via two hydrogen bonds. This study provides a simple and efficient aptamer circularization modification method for improving aptamer stability and expanding its applications.

Current Research Status of Azaspiracids.

The presence and impact of toxins have been detected in various regions worldwide ever since the discovery of azaspiracids (AZAs) in 1995. These toxins have had detrimental effects on marine resource utilization, marine environmental protection, and fishery production. Over the course of more than two decades of research and development, scientists from all over the world have conducted comprehensive studies on the in vivo metabolism, in vitro synthesis methods, pathogenic mechanisms, and toxicology of these toxins. This paper aims to provide a systematic introduction to the discovery, distribution, pathogenic mechanism, in vivo biosynthesis, and in vitro artificial synthesis of AZA toxins. Additionally, it will summarize various detection methods employed over the past 20 years, along with their advantages and disadvantages. This effort will contribute to the future development of rapid detection technologies and the invention of detection devices for AZAs in marine environmental samples.

Enantioselective Copper-Catalyzed Sequential Hydrosilylation of Arylmethylenecyclopropanes.

Despite impressive advances in the construction of enantioenriched silacarbocycles featuring silicon-stereogenic centers via a selection of well-defined sila-synthons, the development of a more convenient and economic method with readily available starting materials is significantly less explored and remains a considerable challenge. Herein, we report the first example of copper-catalyzed sequential hydrosilylation of readily accessible methylenecyclopropanes (MCPs) and primary silanes, affording an efficient and convenient route to a wide range of chiral silacyclopentanes bearing consecutive silicon- and carbon-stereogenic centers with excellent enantio- and diastereoselectivities (generally ≥98% ee, > 25:1 dr). Mechanistic studies reveal that these reactions combine copper-catalyzed intermolecular ring-opening hydrosilylation of aryl MCPs and intramolecular asymmetric hydrosilylation of the resultant Z/E mixture of homoallylic silanes.

Interaction between Heavy Water and Single-Strand DNA: A SERS Study.

The structure and function of biological macromolecules change due to intermolecular deuterium bond formation or deuterium substitution with environmental D2O. In this study, surface-enhanced Raman spectroscopy (SERS) was used to detect interaction sites between D2O and ssDNA and their action mechanisms. SERS peaks of ssDNA changed with increasing D2O proportions, and the site of action mainly involved A and G bases, whose number strengthened the interaction between sequences and D2O and hence the SERS peak intensities. Fixing the number of A and G bases prevented changes in their positions from significantly altering the map. We also identified the interaction between ssDNA sequences that easily formed a G-quadruplex structure and D2O. The amplitude of the SERS peak intensity change reflected the ssDNA structural stability and number of active sites. These findings are highly significant for exploring genetic exchanges and mutations and could be used to determine the stability and structural changes of biological macromolecules.

Rapid and sensitive detection of nodularin-R in water by a label-free BLI aptasensor.

Contamination of freshwater with nodularin-R (NOD-R) represents a significant global environmental and public health concern. However, ethical problems and technical difficulties surrounding the current detection methods for NOD-R necessitate further studies to devise appropriate alternatives within a regulatory monitoring regime. In this work, we employed an aptamer as a specific recognition element and developed a biolayer interferometry (BLI) biosensor platform for NOD-R detection. The aptasensor we propose displayed a broad detection range from 40 to 600 nM NOD-R (and a linear response range from 40 to 200 nM), and achieved a detection limit as low as 167 pM. In addition, the aptamer-based biosensor was shown to possess high selectivity, as well as good reproducibility and stability. We believe that this novel aptamer-based biosensor provides a potential alternative for the sensitive and rapid detection of NOD-R.

S5a binds to death receptor-6 to induce THP-1 monocytes to differentiate through the activation of the NF-κB pathway.

Analyses of supernatants from apoptotic cells have helped in the identification of many signals that modulate the states of cell activation and differentiation. However, the current knowledge about the soluble factors that are released during apoptosis is rather limited. Previous studies have shown that S5a and angiocidin (both encoded by PSMD4) induce human acute monocytic leukemia cells (THP-1 cells) to differentiate into macrophages, but the cell-surface receptor of S5a has not been identified. In this study, we show that apoptotic THP-1 cells release endogenous S5a that binds to death receptor-6 (DR6, also known as TNFRSF1), which was identified as an orphan receptor, to induce THP-1 cells to differentiate. Furthermore, we found that the NF-κB pathway is activated, and that the transcription factors WT1 (Wilms' tumor 1) and c-myb mediate S5a-induced THP-1 differentiation. We also show that differentiation is blocked by anti-DR6 antibody, DR6 siRNA, DR6-Fc, NF-κB inhibitor or WT1 siRNA treatment. Our findings indicate that the interaction between cells can determine their differentiation, and we provide evidence for a functional interaction between S5a and DR6, which provides a novel potential mechanism to induce the differentiation of cancer cells, especially during biotherapy for leukemia.

Post-SELEX optimization of aptamers.

Aptamers are functional single-stranded DNA or RNA oligonucleotides, selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can fold into stable unique three-dimensional structures that bind their target ligands with high affinity and specificity. Although aptamers show a number of favorable advantages such as better stability and easier modification when compared with the properties of antibodies, only a handful of aptamers have entered clinical trials and only one, pegaptanib, has received US Food and Drug Administration approval for clinical use. The main reasons that limit the practical application of aptamers are insufficient nuclease stability, bioavailability, thermal stability, or even affinity. Some aptamers obtained from modified libraries show better properties; however, polymerase amplification of nucleic acids containing non-natural bases is currently a primary drawback of the SELEX process. This review focuses on several post-SELEX optimization strategies of aptamers identified in recent years. We describe four common methods in detail: truncation, chemical modification, bivalent or multivalent aptamer construction, and mutagenesis. We believe that these optimization strategies should improve one or more specific properties of aptamers, and the type of feature(s) selected for improvement will be dependent on the application purpose.

Study of the binding mechanism between aptamer GO18-T-d and gonyautoxin 1/4 by molecular simulation.

GTX1/4 can induce the formation of an antiparallel G-quadruplex structure in aptamer GO18-T-d and combine steadily in the groove at the top of the G-quadruplex structure. The complex structures and special induced fit mechanism between aptamer and small molecules provide a reference for aptamer development in molecular diagnostics and therapeutic application.

Escin Increases the Survival Rate of LPS-Induced Septic Mice Through Inhibition of HMGB1 Release from Macrophages.

BACKGROUND: Previous studies have described the effects of Escin on improving the survival rate of endotoxemic animals. The purpose of this study was to explore the molecular mechanisms of this potentially beneficial treatment. METHODS: First, the survival rate of endotoxemic mice was monitored for up to 2 weeks after Escin pretreatment, Escin post-treatment, or Escin post-treatment + rHMGB1. The effects of Escin on the release of pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6 and HMGB1 in the serum of endotoxemic mice and LPS-induced macrophages were evaluated by ELISA. Furthermore, the mRNA and protein levels of HMGB1 in LPS-induced macrophages were measured by qRT-PCR and Western blot, respectively. Additionally, the release of pro-inflammatory cytokines such as TNF-α, IL-1ß, IL-6 was evaluated by ELISA in rHMGB1-induced macrophages. Finally, the protein levels and the activity of NF-κB in macrophages were checked by Western blot and ELISA, respectively. RESULTS: Both pretreatment and post-treatment with Escin could improve the survival rate of endotoxemic mice, while exogenous rHMGB1 reversed this effect. In addition, Escin decreased the level of the pro-inflammatory cytokinesTNF-α,IL-1ß, IL-6 and HMGB1 in endotoxemic mice and in LPS-induced macrophages. Escin could also inhibit the mRNA levels and activity of HMGB1. The release of the pro-inflammatory cytokinesTNF-α,IL-1ß, IL-6 could be suppressed in rHMGB1-induced macrophages by Escin. Finally, Escin could suppress the activation of NF- κB in LPS-induced macrophages. CONCLUSION: Escin could improve the survival of mice with LPS-induced endotoxemia. This effect maybe meditated by reducing the release of HMGB1, resulting in the suppression of the release of pro-inflammatory cytokines.

Targeted induction of apoptosis in glioblastoma multiforme cells by an MRP3-specific TRAIL fusion protein in vitro.

Single-chain Fv fragments (scFvs) consist of the variable heavy-chain (VH) and variable light-chain (VL) domains, which are the smallest immunoglobulin fragments containing the whole antigen-binding site. Human soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) proves to acquire a potent pro-apoptotic activity only after selective binding to a predefined tumor cell surface antigen and has no off-target effects towards normal cells. Glioblastoma multiforme (GBM) is the most frequent and aggressive type of brain tumor and overexpresses human multidrug resistance protein 3 (MRP3). In this study, we designed a novel fusion protein, termed scFvM58-sTRAIL, in which the MRP3-specific scFv antibody M58 was genetically fused to the N-terminus of human soluble TRAIL (sTRAIL). The recombinant scFvM58-sTRAIL fusion protein, expressed in Escherichia coli, was purified by chromatography and tested for cytotoxicity. scFvM58-sTRAIL showed a significant apoptosis-inducing activity towards MRP3-positive GBM cells in vitro. The pro-apoptotic activity of scFvM58-sTRAIL towards GBM cells was strongly inhibited in the presence of the parental scFvM58 antibody, suggesting that cytotoxic activity is MRP3-restricted. In a control experiment with MRP3-negative Jurkat cells, scFvM58-sTRAIL did not induce apparent apoptosis. In addition, through target antigen-restricted binding, scFvM58-sTRAIL was capable of activating not only TRAIL-R1 but also TRAIL-R2. In conclusion, our results suggest that fusion protein scFvM58-sTRAIL with specificity for MRP3 is a highly selective therapeutic agent and may provide an alternative therapy for human GBM.

HIFs, angiogenesis, and cancer.

Tumor hypoxia was first described in the 1950s by radiation oncologists as a frequent cause of failure to radiotherapy in solid tumors. Today, it is evident that tumor hypoxia is a common feature of many cancers and the master regulator of hypoxia, hypoxia-inducible factor-1 (HIF-1), regulates multiple aspects of tumorigenesis, including angiogenesis, proliferation, metabolism, metastasis, differentiation, and response to radiation therapy. Although the tumor hypoxia response mechanism leads to a multitude of downstream effects, it is angiogenesis that is most crucial and also most susceptible to molecular manipulation. The delineation of molecular mechanisms of angiogenesis has revealed a critical role for HIF-1 in the regulation of angiogenic growth factors. In this article, we review what has been described about HIF-1: its structure, its regulation, and its implication for cancer therapy and we focus on its role in angiogenesis and cancer.

Disorders in angiogenesis and redox pathways are main factors contributing to the progression of rheumatoid arthritis: a comparative proteomics study.

OBJECTIVE: To clarify the pathogenesis of rheumatoid arthritis (RA) by comparing protein expression in fibroblast-like synoviocytes (FLS) from patients with RA with that in FLS from normal subjects, using proteomics analysis. METHODS: Proteins extracted from primary cultures of FLS obtained from 50 patients with RA and 10 normal subjects were analyzed by automated 2-dimensional nano-electrospray ionization liquid chromatography tandem mass spectometry. Differentially expressed proteins were screened by 2-sample t-test (P < 0.05) and fold change (>1.5), based on the bioinformatics analysis. The expression of vasculature development-related proteins (Thy-1, connective tissue growth factor [CTGF], and thrombospondin 1 [TSP-1]) and redox-related proteins (superoxide dismutase 2 [SOD2]) in synovial tissue was confirmed by real-time polymerase chain reaction and Western blotting. The effect of Thy-1 and CTGF knockdown on Thy-1, CTGF, TSP-1, and vascular endothelial growth factor (VEGF) was analyzed by RNA interference experiments. RESULTS: According to the criteria of having >1 unique peptide per protein present and a false discovery rate of ≤5%, 1,060 proteins were identified from patients with RA, and 1,292 proteins were identified from normal subjects, from which 100 differentially expressed proteins were screened out from the RA proteins. Of these, 46 proteins were up-regulated, and the remaining 54 proteins were down-regulated. Gene ontology and pathway analyses showed that 6 vasculature development-related proteins were up-regulated, including Thy-1, CTGF, and TSP-1, while 11 redox-related proteins were down-regulated, including SOD2. The results were consistent with those obtained using mass spectrometry. Thy-1, VEGF, CTGF, and TSP-1 were down-regulated after Thy-1 knockdown, and VEGF and CTGF were down-regulated after CTGF knockdown. Recombinant human CTGF could enhance proliferation and Transwell migration of human umbilical vein endothelial cells. CONCLUSION: Up-regulation of vasculature development-related proteins and down-regulation of redox-related proteins in FLS are predominant factors that may contribute to the pathogenesis of RA.

Expression and purification of human ARP1 protein and rapid preparation of polyclonal antibody.

Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Scraping Therapy Improved Muscle Regeneration through Regulating GLUT4/Glycolytic and AMPK/mTOR/4EBP1 Pathways in Rats with Lumbar Multifidus Injury.

Background: High morbidity of nonspecific low back pain (NLBP) and large consumption of medical resources caused by it have become a heavy social burden. There are many factors inducing NLBP, among which the damage and atrophy of multifidus (MF) are most closely related to NLBP. Scraping therapy can have significant treatment effects on NLBP with fewer adverse reactions and less medical fund input than other modalities or medications. However, the mechanism of scraping therapy treating NLBP remains unclarified. Here, we wanted to investigate the effects of scraping therapy on promoting MF regeneration and the underlying mechanisms. Methods: A total of 54 male rats (SD, 6-7 weeks old) were randomly divided into nine groups, namely, K, M6h, M1d, M2d, M3d, G6h, G1d, G2d, and G3d, with six rats in each group. They were injected with bupivacaine (BPVC) to intentionally induce MF injury. We then performed scraping therapy on the rats that had been randomly chosen and compared treatment effects at different time points. In vitro data including skin temperature and tactile allodynia threshold were collected and histological sections were analyzed. mRNA sequencing was applied to distinguish the genes or signaling pathways that had been altered due to scraping therapy, and the results were further verified through reverse transcription polymerase chain reaction and Western blot analysis. Results: Transitory petechiae and ecchymosis both on and beneath the rats' skin raised by scraping therapy gradually faded in about 3 d. Cross-sectional area (CSA) of MF was significantly smaller 30 h, 2 d, and 4 d after modeling (P=0.007, P=0.001, and P=0.015, respectively, vs. the blank group) and was significantly larger in the scraping group 1 d after treatment (P=0.002 vs. the model 1d group). Skin temperature significantly increased immediately after scraping (P < 0.001) and hindlimb pain threshold increased on the 2nd day after scraping (P=0.046 and P=0.028, respectively). 391 differentially expressed genes and 8 signaling pathways were characterized 6 h after scraping; only 3 differentially expressed genes and 3 signaling pathways were screened out 2 d after treatment. The amounts of mRNAs or proteins for GLUT4, HK2, PFKM, PKM, LDHA (which belong to the GLUT4/glycolytic pathway), p-mTOR, p-4EBP1 (which belong to the AMPK/mTOR/4EBP1 pathway), and BDH1 were enhanced, and p-AMPKα was decreased after scraping therapy. Conclusions: Scraping therapy has therapeutic effects on rats with multifidus injury by promoting muscle regeneration via regulating GLUT4/glycolytic and AMPK/mTOR/4EBP1 signaling pathways.

Prognostic and immunological significance of an M1 macrophage-related gene signature in osteosarcoma.

As the most abundant infiltrating immune cells in the tumor microenvironment (TME), tumor-associated macrophages (TAMs) are pivotal in tumor development and treatment. The present investigation endeavors to explore the potential of M1 macrophage-related genes (MRGs) as biomarkers for assessing risk in individuals with osteosarcoma. RNA-sequence data and clinical data were derived from TCGA and GEO databases. The CIBERSORT method was utilized to discern subtypes of tumor-infiltrating immune cells. Identification of MRGs was achieved through Pearson correlation analysis. A prognostic risk model for MRGs was developed using Cox and LASSO regression analyses. A tripartite gene signature comprising CD37, GABRD, and ARHGAP25 was an independent prognostic indicator and was employed to develop a risk score model. The internal and external validation cohort confirmed the results. The area under the ROC curve (AUC) was determined for survival periods of 1 year, three years, and five years, yielding values of 0.746, 0.839, and 0.850, respectively. The C-index of the risk score was found to be superior to clinicopathological factors. GO/KEGG enrichment showed that the differences between high- and low-risk groups were predominantly associated with immune response pathways. Immune-related analysis related to proportions of immune cells, immune function, and expression levels of immune checkpoint genes all showed differences between the high- and low-risk groups. The qRT-PCR and Western blotting results indicate that CD37 expression was markedly higher in MG63 and U2OS cell lines when compared to normal osteoblast hFOB1.19. In U2OS cell line, GABRD expression levels were significantly upregulated. ARHGAP25 expression levels were elevated in both 143B and U2OS cell lines. In summary, utilizing a macrophage genes signature demonstrates efficacy in predicting both the prognosis and therapy response of OS. Additionally, immune analysis confirms a correlation between the risk score and the tumor microenvironment. Our findings, therefore, provide a cogent account for the disparate prognoses observed among patients and furnish a justification for further inquiry into biomarkers and anti-tumor treatment strategies.

Inflammation amplification by Versican: the first mediator.

The effects of inflammation may not always benefit the individual. Its amplifying nature represents a highly regulated biological program, and the inflammatory microenvironment is its essential component. Growing evidence suggests that the ECM (extracellular matrix) is important for the early steps of inflammation. Versican, a ubiquitous component of the ECM, contributes to the formation of the inflammatory response and is highly regulated by cytokines. Certain cytokines exert their initial effects on versican to alter the homeostasis of the inflammatory milieu, and inappropriate production of versican may promote the next inflammatory response. Therefore, versican could be the first step in the amplification of the inflammatory response, and ongoing research of this molecule may help to explain the pathogenesis of inflammation.

[Expression of leptin and p-mTOR and their clinicopathological significance in human colon carcinoma].

OBJECTIVE: To investigate the relationship between the expression of leptin, p-mTOR protein and the pathogenesis, development and clinicopathological features in colon carcinoma. METHODS: The expression of leptin and p-mTOR protein was evaluated by immunohistochemical methods in 40 normal colon mucosas, 40 colon adenomatous polyps and 108 cases of colon carcinomas. The relationship between the staining pattern and clinicopathogical features was examined. RESULTS: The positive rates of detection of leptin in normal colon mucosa, adenomatous polyps and colon carcinomas were 10% (4/40), 27.5% (11/40), and 71.3% (77/108), respectively; with significant differences among the three groups (P<0.05). The positive rates of p-mTOR protein in the normal colon mucosa, the adenomatous polyps, and the colon carcinomas were 2.5% (1/40), 20% (8/40), and 61.1% (66/108), respectively; with significant differences among the three groups (P<0.05). The expression of leptin and p-mTOR proteins were related to invasive depth, TNM stages, lymph node metastasis, distant metastasis and tumor differentiation (P<0.05), but not to age, sex, or site (P>0.05). In colon carcinoma tissues, leptin expression was positively correlated with p-mTOR expression (P<0.01). CONCLUSION: Leptin and p-mTOR proteins may play important roles in the occurrence and development of colon carcinoma. The detection of leptin and p-mTOR may be helpful for evaluation of the prognosis of the patient with colon carcinoma.

[Computer-aided aptamers screening technologies: a review].

The computer information technology that has penetrated into every aspect of our lives, can not only assist the screening of drugs, but also simulate the effect of drugs. At present, computer-aided technologies have been used to screen aptamers, which play an important role in improving the screening efficiency and screening high affinity binding aptamers. This review summarized the screening methods of aptamers through computer-aided sequence evaluation, structural analysis and molecular docking.

Proteomic Studies of the Mechanism of Cytotoxicity, Induced by Palytoxin on HaCaT Cells.

Palytoxin (PLTX) is a polyether marine toxin isolated from sea anemones. It is one of the most toxic nonprotein substances, causing many people to be poisoned every year and to die in severe cases. Despite its known impact on Na+,K+-ATPase, much still remains unclear about PLTX's mechanism of action. Here, we tested different concentrations of PLTX on HaCaT cells and studied its distributions in cells, its impact on gene expression, and the associated pathways via proteomics combined with bioinformatics tools. We found that PLTX could cause ferroptosis in HaCaT cells, a new type of programmed cell death, by up-regulating the expression of VDAC3, ACSL4 and NCOA4, which lead to the occurrence of ferroptosis. PLTX also acts on the MAPK pathway, which is related to cell apoptosis, proliferation, division and differentiation. Different from its effect on ferroptosis, PLTX down-regulates the expression of ERK, and, as a result, the expressions of MAPK1, MAP2K1 and MAP2K2 are also lower, affecting cell proliferation. The genes from these two mechanisms showed interactions, but we did not find overlap genes between the two. Both ferroptosis and MAPK pathways can be used as anticancer targets, so PLTX may become an anticancer drug with appropriate modification.

Selection, Characterization, and Optimization of DNA Aptamers against Challenging Marine Biotoxin Gymnodimine-A for Biosensing Application.

Gymnodimines (GYMs), belonging to cyclic imines (CIs), are characterized as fast-acting toxins, and may pose potential risks to human health and the aquaculture industry through the contamination of sea food. The existing detection methods of GYMs have certain defects in practice, such as ethical problems or the requirement of complicated equipment. As novel molecular recognition elements, aptamers have been applied in many areas, including the detection of marine biotoxins. However, GYMs are liposoluble molecules with low molecular weight and limited numbers of chemical groups, which are considered as "challenging" targets for aptamers selection. In this study, Capture-SELEX was used as the main strategy in screening aptamers targeting gymnodimine-A (GYM-A), and an aptamer named G48nop, with the highest KD value of 95.30 nM, was successfully obtained by screening and optimization. G48nop showed high specificity towards GYM-A. Based on this, a novel aptasensor based on biolayer interferometry (BLI) technology was established in detecting GYM-A. This aptasensor showed a detection range from 55 to 1400 nM (linear range from 55 to 875 nM) and a limit of detection (LOD) of 6.21 nM. Spiking experiments in real samples indicated the recovery rate of this aptasensor, ranging from 96.65% to 109.67%. This is the first study to report an aptamer with high affinity and specificity for the challenging marine biotoxin GYM-A, and the new established aptasensor may be used as a reliable and efficient tool for the detection and monitoring of GYMs in the future.