Three-Dimensional Cell Culture Micro-CT Visualization within Collagen Scaffolds in an Aqueous Environment.

Among all of the materials used in tissue engineering in order to develop bioequivalents, collagen shows to be the most promising due to its superb biocompatibility and biodegradability, thus becoming one of the most widely used materials for scaffold production. However, current imaging techniques of the cells within collagen scaffolds have several limitations, which lead to an urgent need for novel methods of visualization. In this work, we have obtained groups of collagen scaffolds and selected the contrasting agents in order to study pores and patterns of cell growth in a non-disruptive manner via X-ray computed microtomography (micro-CT). After the comparison of multiple contrast agents, a 3% aqueous phosphotungstic acid solution in distilled water was identified as the most effective amongst the media, requiring 24 h of incubation. The differences in intensity values between collagen fibers, pores, and masses of cells allow for the accurate segmentation needed for further analysis. Moreover, the presented protocol allows visualization of porous collagen scaffolds under aqueous conditions, which is crucial for the multimodal study of the native structure of samples.

Direct and cell-mediated EV-ECM interplay.

Extracellular vesicles (EV) are a heterogeneous group of lipid particles excreted by cells. They play an important role in regeneration, development, inflammation, and cancer progression, together with the extracellular matrix (ECM), which they constantly interact with. In this review, we discuss direct and indirect interactions of EVs and the ECM and their impact on different physiological processes. The ECM affects the secretion of EVs, and the properties of the ECM and EVs modulate EVs' diffusion and adhesion. On the other hand, EVs can affect the ECM both directly through enzymes and indirectly through the modulation of the ECM synthesis and remodeling by cells. This review emphasizes recently discovered types of EVs bound to the ECM and isolated by enzymatic digestion, including matrix-bound nanovesicles (MBV) and tissue-derived EV (TiEV). In addition to the experimental studies, computer models of the EV-ECM-cell interactions, from all-atom models to quantitative pharmacology models aiming to improve our understanding of the interaction mechanisms, are also considered. STATEMENT OF SIGNIFICANCE: Application of extracellular vesicles in tissue engineering is an actively developing area. Vesicles not only affect cells themselves but also interact with the matrix and change it. The matrix also influences both cells and vesicles. In this review, different possible types of interactions between vesicles, matrix, and cells are discussed. Furthermore, the united EV-ECM system and its regulation through the cellular activity are presented.

Interaction of Serum and Plasma Proteins with Polyelectrolyte Microparticles with Core/Shell and Shell-Only Structures.

Polyelectrolyte microparticles (MPs) synthesized on calcium carbonate cores are considered a promising basis for new drug delivery systems. It is known that microparticles entering a physiological environment absorb proteins on their surface, which can change the properties of the microparticles and alter their functional activity. This study aimed to compare the compositions of the adsorbed protein layer formed on microparticles with the core/shell and shell structures obtained by layer-by-layer deposition. The difference in the microparticle structure was associated with changes in their surface topography and ζ-potential. These microparticles were incubated with human serum or plasma at 37°C for 24 h. The adsorbed proteins were eluted and analyzed by means of SDS-PAGE. The protein composition of the eluates was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS); a total of 357 proteins were identified, and 183 of them were detected in all samples. Our results demonstrate that the relative abundance of proteins of different functional groups (immunoglobulins, complement proteins, and apolipoproteins) varied depending on the structure and surface characteristics of the polyelectrolyte microparticles and the incubation medium. Our findings expand the understanding of the influence of the physicochemical properties of the microparticles on their interaction with proteins, which can help to improve the design of microparticles for drug delivery.

Modulation of the skin and gut microbiome by psoriasis treatment: a comprehensive systematic review.

The microbiome is intricately linked to the development of psoriasis, serving as both a potential cause and consequence of the psoriatic process. In recent years, there has been growing interest among psoriasis researchers in exploring how psoriasis treatments affect the skin and gut microbiome. However, a comprehensive evaluation of the impact of modern treatment approaches on the microbiome has yet to be conducted. In this systematic review, we analyze studies investigating alterations in the skin and gut microbiome resulting from psoriasis treatment, aiming to understand how current therapies influence the role of the microbiome in psoriasis development. The systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. PubMed and Scopus databases were searched for eligible studies from the inception dates until July 5, 2023. Study selection, data extraction, and risk of bias assessment were carried out by three overlapping pairs of reviewers, resolving any disagreements through consensus. Our analysis of various treatments, including biologics, conventional medications, phototherapy, and probiotics, reveals significant shifts in microbial diversity and abundance. Importantly, favorable treatment outcomes are associated with microbiota alterations that approach those observed in healthy individuals. While the studies reviewed exhibit varying degrees of bias, underscoring the need for further research, this review supports the potential of microbiome modulation as both a preventive and therapeutic strategy for psoriasis patients. The findings underscore the importance of personalized therapeutic approaches, recognizing the profound impact of treatment on the microbiome. They also highlight the promise of probiotics, prebiotics, and dietary interventions in psoriasis management.

Prg4-Expressing Chondroprogenitor Cells in the Superficial Zone of Articular Cartilage.

Joint-resident chondrogenic precursor cells have become a significant therapeutic option due to the lack of regenerative capacity in articular cartilage. Progenitor cells are located in the superficial zone of the articular cartilage, producing lubricin/Prg4 to decrease friction of cartilage surfaces during joint movement. Prg4-positive progenitors are crucial in maintaining the joint's structure and functionality. The disappearance of progenitor cells leads to changes in articular hyaline cartilage over time, subchondral bone abnormalities, and the formation of ectopic ossification. Genetic labeling cell technology has been the main tool used to characterize Prg4-expressing progenitor cells of articular cartilage in vivo through drug injection at different time points. This technology allows for the determination of the origin of progenitor cells and the tracking of their progeny during joint development and cartilage damage. We endeavored to highlight the currently known information about the Prg4-producing cell population in the joint to underline the significance of the role of these cells in the development of articular cartilage and its homeostasis. This review focuses on superficial progenitors in the joint, how they contribute to postnatal articular cartilage formation, their capacity for regeneration, and the consequences of Prg4 deficiency in these cells. We have accumulated information about the Prg4+ cell population of articular cartilage obtained through various elegantly designed experiments using transgenic technologies to identify potential opportunities for further research.

Mechanical characterization of soft biomaterials: which time and spatial scale to choose?

The mechanical properties of soft gels hold significant relevance in biomedicine and biomaterial design, including the development of tissue engineering constructs and bioequivalents. It is important to adequately characterize the gel's mechanical properties since they play a role both in the overall structural properties of the construct and the physiological responses of cells. The question remains which approach for the mechanical characterization is most suitable for specific biomaterials. Our investigation is centered on the comparison of three types of gels and four distinct mechanical testing techniques: shear rheology, compression, microindentation, and nanoindentation by atomic force microscopy. While analyzing an elastic homogeneous synthetic hydrogel (a polyacrylamide gel), we observed close mechanical results across the different testing techniques. However, our findings revealed more distinct outcomes when assessing a highly viscoelastic gel (Ecoflex) and a heterogeneous biopolymer hydrogel (enzymatically crosslinked gelatin). To ensure precise data interpretation, we introduced correction factors to account for the boundary conditions inherent in many of the testing methods. The results of this study underscore the critical significance of considering both the temporal and spatial scales in mechanical measurements of biomaterials. Furthermore, they encourage the employment of a combination of diverse testing techniques, particularly in the characterization of heterogeneous viscoelastic materials such as biological samples. The obtained results will contribute to the refinement of mechanical testing protocols and advance the development of soft gels for tissue engineering.

Bovine Serum Albumin Effect on Collapsing PNIPAM Chains in Aqueous Solutions: Spin Label and Spin Probe Study.

The influence of bovine serum albumin (BSA) on collapsing poly(N-isopropylacrylamide) (PNIPAM) chains was studied with turbidimetry and spin probe and spin label electron paramagnetic resonance spectroscopy. An increased ratio of collapsed chains in aqueous solutions in the narrow temperature region near the LCST appeared in the presence of 2.5-10 wt% BSA. The spin probe EPR data indicate that the inner cavities of the BSA dimers are probably responsive to the capture of small hydrophobic or amphiphilic molecules, such as TEMPO nitroxyl radical. The observed features of the structure and dynamics of inhomogeneities of aqueous PNIPAM-BSA solutions, including their mutual influence on the behavior of the polymer and protein below the LCST, should be considered when developing and investigating PNIPAM-based drug delivery systems.

Translational Approach Using Advanced Therapy Medicinal Products for Huntington's Disease.

Current therapeutic approaches for Huntington's disease (HD) focus on symptomatic treatment. Therefore, the unavailability of efficient disease-modifying medicines is a significant challenge. Regarding the molecular etiology, targeting the mutant gene or advanced translational steps could be considered promising strategies. The evidence in gene therapy suggests various molecular techniques, including knocking down mHTT expression using antisense oligonucleotides and small interfering RNAs and gene editing with zinc finger proteins and CRISPR-Cas9-based techniques. Several post-transcriptional and post-translational modifications have also been proposed. However, the efficacy and long-term side effects of these modalities have yet to be verified. Currently, cell therapy can be employed in combination with conventional treatment and could be used for HD in which the structural and functional restoration of degenerated neurons can occur. Several animal models have been established recently to develop cell-based therapies using renewable cell sources such as embryonic stem cells, induced pluripotent stem cells, mesenchymal stromal cells, and neural stem cells. These models face numerous challenges in translation into clinics. Nevertheless, investigations in Advanced Therapy Medicinal Products (ATMPs) open a promising window for HD research and their clinical application. In this study, the ATMPs entry pathway in HD management was highlighted, and their advantages and disadvantages were discussed.

Alteration in DNA methylation patterns: Epigenetic signatures in gastrointestinal cancers.

Abnormalities in epigenetic modifications can cause malignant transformations in cells, leading to cancers of the gastrointestinal (GI) tract, which accounts for 20% of all cancers worldwide. Among the epigenetic alterations, DNA hypomethylation is associated with genomic instability. In addition, CpG methylation and promoter hypermethylation have been recognized as biomarkers for different malignancies. In GI cancers, epigenetic alterations affect genes responsible for cell cycle control, DNA repair, apoptosis, and tumorigenic-specific signaling pathways. Understanding the pattern of alterations in DNA methylation in GI cancers could help scientists discover new molecular-based pharmaceutical treatments. This study highlights alterations in DNA methylation in GI cancers. Understanding epigenetic differences among GI cancers may improve targeted therapies and lead to the discovery of new diagnostic biomarkers.

GBM Immunotherapy: Macrophage Impacts.

BACKGROUND: Glioblastoma (GBM) is an extremely aggressive form of brain tumor with low survival rates. Current treatments such as chemotherapy, radiation, and surgery are problematic due to tumor growth, invasion, and tumor microenvironment. GBM cells are resistant to these standard treatments, and the heterogeneity of the tumor makes it difficult to find a universal approach. Progression of GBM and acquisition of resistance to therapy are due to the complex interplay between tumor cells and the TME. A significant portion of the TME consists of an inflammatory infiltrate, with microglia and macrophages being the predominant cells. METHODS: Analysis of the literature data over a course of 5 years suggest that the tumor-associated macrophages (TAMs) are capable of releasing cytokines and growth factors that promote tumor proliferation, survival, and metastasis while inhibiting immune cell function at the same time. RESULTS: Thus, immunosuppressive state, provided with this intensively studied kind of TME cells, is supposed to promote GBM development through TAMs modulation of tumor treatment-resistance and aggressiveness. Therefore, TAMs are an attractive therapeutic target in the treatment of glioblastoma. CONCLUSION: This review provides a comprehensive overview of the latest research on the nature of TAMs and the development of therapeutic strategies targeting TAMs, focusing on the variety of macrophage properties, being modulated, as well as molecular targets.

Review on Kidney-Liver Crosstalk: Pathophysiology of Their Disorders.

Kidney-liver crosstalk plays a crucial role in normal and certain pathological conditions. In pathologic states, both renal-induced liver damage and liver-induced kidney diseases may happen through these kidney-liver interactions. This bidirectional crosstalk takes place through the systemic conditions that mutually influence both the liver and kidneys. Ischemia and reperfusion, cytokine release and pro-inflammatory signaling pathways, metabolic acidosis, oxidative stress, and altered enzyme activity and metabolic pathways establish the base of this interaction between the kidneys and liver. In these concomitant kidney-liver diseases, the survival rates strongly correlate with early intervention and treatment of organ dysfunction. Proper care of a nephrologist and hepatologist and the identification of pathological conditions using biomarkers at early stages are necessary to prevent the complications induced by this complex and potentially vicious cycle. Therefore, understanding the characteristics of this crosstalk is essential for better management. In this review, we discussed the available literature concerning the detrimental effects of kidney failure on liver functions and liver-induced kidney diseases.

Radical-Generating Activity, Phagocytosis, and Mechanical Properties of Four Phenotypes of Human Macrophages.

Macrophages are the major players and orchestrators of inflammatory response. Expressed proteins and secreted cytokines have been well studied for two polar macrophage phenotypes-pro-inflammatory M1 and anti-inflammatory regenerative M2, but little is known about how the polarization modulates macrophage functions. In this study, we used biochemical and biophysical methods to compare the functional activity and mechanical properties of activated human macrophages differentiated from monocyte with GM-CSF (M0_GM) and M-CSF (M0_M) and polarized into M1 and M2 phenotypes, respectively. Unlike GM-CSF, which generates dormant cells with low activity, M-CSF confers functional activity on macrophages. M0_M and M2 macrophages had very similar functional characteristics-high reactive oxygen species (ROS) production level, and higher phagocytosis and survival compared to M1, while M1 macrophages showed the highest radical-generating activity but the lowest phagocytosis and survival among all phenotypes. All phenotypes decreased their height upon activation, but only M1 and M2 cells increased in stiffness, which can indicate a decrease in the migration ability of these cells and changes in their interactions with other cells. Our results demonstrated that while mechanical properties differ between M0 and polarized cells, all four phenotypes of monocyte-derived macrophages differ in their functional activities, namely in cytokine secretion, ROS production, and phagocytosis. Within the broad continuum of human macrophages obtained in experimental models and existing in vivo, there is a diversity of phenotypes with varying combinations of both markers and functional activities.

Advanced Therapeutic Medicinal Products in Bone and Cartilage Defects.

The number of patients with functional loss of bone and cartilage tissue has shown an increasing trend. Insufficient or inappropriate conventional treatments applied for trauma, orthopedic diseases, or other bone and cartilage-related disorders can lead to bone and cartilage damage. This represents a worldwide public health issue and a significant economic burden. Advanced therapeutic medicinal products (ATMPs) proposed promising alternative therapeutic modalities by application of cell-based and tissue engineering approaches. Recently, several ATMPs have been developed to promote bone and cartilage tissue regeneration. Fifteen ATMPs, two related to bone and 13 related to cartilage, have received regulatory approval and marketing authorization. However, four ATMPs were withdrawn from the market for various reasons. However, ATMPs that are still on the market have demonstrated positive results, their broad application faced limitations. The development and standardization of methodologies will be a major challenge in the coming decades. Currently, the number of ATMPs in clinical trials using mesenchymal stromal cells or chondrocytes indicates a growing recognition that current ATMPs can be improved. Research on bone and cartilage tissue regeneration continues to expand. Cell-based therapies are likely to be clinically supported by the new ATMPs, innovative fabrication processes, and enhanced surgical approaches. In this study, we highlighted the available ATMPs that have been used in bone and cartilage defects and discussed their advantages and disadvantages in clinical applications.

Collagen Matrix to Restore the Tympanic Membrane: Developing a Novel Platform to Treat Perforations.

Modern otology faces challenges in treating tympanic membrane (TM) perforations. Instead of surgical intervention, alternative treatments using biomaterials are emerging. Recently, we developed a robust collagen membrane using semipermeable barrier-assisted electrophoretic deposition (SBA-EPD). In this study, a collagen graft shaped like a sponge through SBA-EPD was used to treat acute and chronic TM perforations in a chinchilla model. A total of 24 ears from 12 adult male chinchillas were used in the study. They were organized into four groups. The first two groups had acute TM perforations and the last two had chronic TM perforations. We used the first and third groups as controls, meaning they did not receive the implant treatment. The second and fourth groups, however, were treated with the collagen graft implant. Otoscopic assessments were conducted on days 14 and 35, with histological evaluations and TM vibrational studies performed on day 35. The groups treated with the collagen graft showed fewer inflammatory changes, improved structural recovery, and nearly normal TM vibrational properties compared to the controls. The porous collagen scaffold successfully enhanced TM regeneration, showing high biocompatibility and biodegradation potential. These findings could pave the way for clinical trials and present a new approach for treating TM perforations.

Four sides to the story: A proteomic comparison of liquid-phase and matrix-bound extracellular vesicles in 2D and 3D cell cultures.

Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.

Electrophoretically Co-Deposited Collagen-Lactoferrin Membranes with Enhanced Pro-Regenerative Properties for Oral Soft Tissue Regeneration.

The quality of soft tissue defect regeneration after dental surgeries largely determines their final success. Collagen membranes have been proposed for the healing of such defects, but in some cases, they do not guarantee a sufficient volume of the regenerated tissue and vascularization. For this purpose, lactoferrin, a protein with natural pro-regenerative, anti-inflammatory, and pro-angiogenic activity, can be added to collagen. In this article, we used a semipermeable barrier-assisted electrophoretic deposition (SBA-EPD) method for the production of collagen-lactoferrin membranes. The membrane structure was studied by SEM, and its mechanical properties were shown. The lactoferrin release kinetics were shown by ELISA within 75 h. When tested in vitro, we demonstrated that the collagen-lactoferrin membranes significantly increased the proliferation of keratinocytes (HaCaT) and fibroblasts (977hTERT) compared to blank collagen membranes. In vivo, on the vestibuloplasty and free gingival graft harvesting models, we showed that collagen-lactoferrin membranes decreased the wound inflammation and increased the healing rates and regeneration quality. In some parameters, collagen-lactoferrin membranes outperformed not only blank collagen membranes, but also the commercial membrane Mucograft®. Thus, we proved that collagen-lactoferrin membranes produced by the SBA-EPD method may be a valuable alternative to commercially used membranes for soft tissue regeneration in the oral cavity.

Oral Galvanism Side Effects: Comparing Alloy Ions and Galvanic Current Effects on the Mucosa-like Model.

The interaction of different dental alloys with the oral environment may cause severe side effects (e.g., burning sensation, inflammatory reactions, carcinogenesis) as a result of oral galvanism. However, the pathogenesis of side effects associated with oral galvanism is still unclear, and the effects of direct current and alloy corrosion ions are considered potentially contributing factors. Therefore, the aim of this study was to systemically compare the damaging effects of (1) galvanism as a synergistic process (direct current + corrosion ions), (2) direct current separately, and (3) corrosion ions separately on an in vitro mucosa-like model based on a cell line of immortalized human keratinocytes (HaCaTs) to reveal the factors playing a pivotal role in dental alloys side effects. For this, we chose and compared the dental alloys with the highest risk of oral galvanism: Ti64-AgPd and NiCr-AgPd. We showed that galvanic current may be the leading damaging factor in the cytotoxic processes associated with galvanic coupling of metallic intraoral appliances in the oral cavity, especially in the short-term period (28 days). However, the contribution of corrosion ions (Ni2+) to the synergistic toxicity was also shown, and quite possibly, in the long term, it could be no less dangerous.

Resveratrol's Impact on the Chondrogenic Reagents' Effects in Cell Sheet Cultures of Wharton's Jelly-Derived MSCs.

Human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest in tissue engineering. We obtained hWJ-MSCs from four patients, and then we stimulated their chondrogenic phenotype formation in vitro by adding resveratrol (during cell expansion) and a canonical Wnt pathway activator, LiCl, as well as a Rho-associated protein kinase inhibitor, Y27632 (during differentiation). The effects of the added reagents on the formation of hWJ-MSC sheets destined to repair osteochondral injuries were investigated. Three-dimensional hWJ-MSC sheets grown on P(NIPAM-co-NtBA)-based matrices were characterized in vitro and in vivo. The combination of resveratrol and LiCl showed effects on hWJ-MSC sheets similar to those of the basal chondrogenic medium. Adding Y27632 decreased both the proportion of hypertrophied cells and the expression of the hyaline cartilage markers. In vitro, DMSO was observed to impede the effects of the chondrogenic factors. The mouse knee defect model experiment revealed that hWJ-MSC sheets grown with the addition of resveratrol and Y27632 were well integrated with the surrounding tissues; however, after 3 months, the restored tissue was identical to that of the naturally healed cartilage injury. Thus, the combination of chondrogenic supplements may not always have additive effects on the progress of cell culture and could be neutralized by the microenvironment after transplantation.

Empowering Renal Cancer Management with AI and Digital Pathology: Pathology, Diagnostics and Prognosis.

Renal cell carcinoma is a significant health burden worldwide, necessitating accurate and efficient diagnostic methods to guide treatment decisions. Traditional pathology practices have limitations, including interobserver variability and time-consuming evaluations. In recent years, digital pathology tools emerged as a promising solution to enhance the diagnosis and management of renal cancer. This review aims to provide a comprehensive overview of the current state and potential of digital pathology in the context of renal cell carcinoma. Through advanced image analysis algorithms, artificial intelligence (AI) technologies facilitate quantification of cellular and molecular markers, leading to improved accuracy and reproducibility in renal cancer diagnosis. Digital pathology platforms empower remote collaboration between pathologists and help with the creation of comprehensive databases for further research and machine learning applications. The integration of digital pathology tools with other diagnostic modalities, such as radiology and genomics, enables a novel multimodal characterization of different types of renal cell carcinoma. With continuous advancements and refinement, AI technologies are expected to play an integral role in diagnostics and clinical decision-making, improving patient outcomes. In this article, we explored the digital pathology instruments available for clear cell, papillary and chromophobe renal cancers from pathologist and data analyst perspectives.

Carbonic Anhydrase Inhibitors Induce Ferroptosis through Inhibition of AKT/FTH1 Signaling in Ewing Sarcoma Tumor Cells.

Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.