Red Blood Cell Membrane-Camouflaged Polydopamine and Bioactive Glass Composite Nanoformulation for Combined Chemo/Chemodynamic/Photothermal Therapy.

Combinations of different therapeutic strategies, including chemotherapy (CT), chemodynamic therapy (CDT), and photothermal therapy (PTT), are needed to effectively address evolving drug resistance and the adverse effects of traditional cancer treatment. Herein, a camouflage composite nanoformulation (TCBG@PR), an antitumor agent (tubercidin, Tub) loaded into Cu-doped bioactive glasses (CBGs) and subsequently camouflaged by polydopamine (PDA), and red blood cell membranes (RBCm), was successfully constructed for targeted and synergetic antitumor therapies by combining CT of Tub, CDT of doped copper ions, and PTT of PDA. In addition, the TCBG@PRs composite nanoformulation was camouflaged with a red blood cell membrane (RBCm) to improve biocompatibility, longer blood retention times, and excellent cellular uptake properties. It integrated with long circulation and multimodal synergistic treatment (CT, CDT, and PTT) with the benefit of RBCms to avoid immune clearance for efficient targeted delivery to tumor locations, producing an "all-in-one" nanoplatform. In vivo results showed that the TCBG@PRs composite nanoformulation prolonged blood circulation and improved tumor accumulation. The combination of CT, CDT, and PTT therapies enhanced the antitumor therapeutic activity, and light-triggered drug release reduced systematic toxicity and increased synergistic antitumor effects.

Microglia Polarization and Antiglioma Effects Fostered by Dual Cell Membrane-Coated Doxorubicin-Loaded Hexagonal Boron Nitride Nanoflakes.

Microglial cells play a critical role in glioblastoma multiforme (GBM) progression, which is considered a highly malignant brain cancer. The activation of microglia can either promote or inhibit GBM growth depending on the stage of the tumor development and on the microenvironment conditions. The current treatments for GBM have limited efficacy; therefore, there is an urgent need to develop novel and efficient strategies for drug delivery and targeting: in this context, a promising strategy consists of using nanoplatforms. This study investigates the microglial response and the therapeutic efficacy of dual-cell membrane-coated and doxorubicin-loaded hexagonal boron nitride nanoflakes tested on human microglia and GBM cells. Obtained results show promising therapeutic effects on glioma cells and an M2 microglia polarization, which refers to a specific phenotype or activation state that is associated with anti-inflammatory and tissue repair functions, highlighted through proteomic analysis.

Cell Membrane Coated pH-Responsive Intelligent Bionic Delivery Nanoplatform for Active Targeting in Photothermal Therapy.

Aim: To produce pH-responsive bionic high photothermal conversion nanoparticles actively targeting tumors for sensitizing photothermal therapy (PTT). Materials and Methods: The bionic nanoparticles (ICG-PEI@HM NPs) were prepared by electrostatic adsorption of indocyanine green (ICG) coupled to polyethyleneimine (PEI) and modified with tumor cell membranes. In vitro and in vivo experiments were conducted to investigate the efficacy of ICG-PEI@HM-mediated PTT. Results: The intelligent responsiveness of ICG-PEI@HM to pH promoted the accumulation of ICG and enhanced the PTT performance of ICG-PEI@HM NPs. Compared with free ICG, NPs exhibited great photothermal stability, cellular uptake, and active tumor targeting for PTT. Conclusion: ICG-PEI@HM NPs can enhance the efficacy of PTT and can be used as a new strategy for the construction of photothermal agents.

Encapsulation of Selenium Nanoparticles and Metformin in Macrophage-Derived Cell Membranes for the Treatment of Spinal Cord Injury.

Spinal cord injury is an impact-induced disabling condition. A series of pathological changes after spinal cord injury (SCI) are usually associated with oxidative stress, inflammation, and apoptosis. These pathological changes eventually lead to paralysis. The short half-life and low bioavailability of many drugs also limit the use of many drugs in SCI. In this study, we designed nanovesicles derived from macrophages encapsulating selenium nanoparticles (SeNPs) and metformin (SeNPs-Met-MVs) to be used in the treatment of SCI. These nanovesicles can cross the blood-spinal cord barrier (BSCB) and deliver SeNPs and Met to the site of injury to exert anti-inflammatory and reactive oxygen species scavenging effects. Transmission electron microscopy (TEM) images showed that the SeNPs-Met-MVs particle size was approximately 125 ± 5 nm. Drug release assays showed that Met exhibited sustained release after encapsulation by the macrophage cell membrane. The cumulative release was approximately 80% over 36 h. In vitro cellular experiments and in vivo animal experiments demonstrated that SeNPs-Met-MVs decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and reduced the expression of inflammatory (TNF-α, IL-1ß, and IL-6) and apoptotic (cleaved caspase-3) cytokines in spinal cord tissue after SCI. In addition, motor function in mice was significantly improved after SeNPs-Met-MVs treatment. Therefore, SeNPs-Met-MVs have a promising future in the treatment of SCI.

Calcium/P53/Ninjurin 1 Signaling Mediates Plasma Membrane Rupture of Acinar Cells in Severe Acute Pancreatitis.

Ninjurin 1 (NINJ1) is a double-transmembrane cell-surface protein that might mediate plasma membrane rupture (PMR) and the diffusion of inflammatory factors. PMR is a characteristic of acinar cell injury in severe acute pancreatitis (SAP). However, the involvement of NINJ1 in mediating the PMR of acinar cells in SAP is currently unclear. Our study has shown that NINJ1 is expressed in acinar cells, and the expression is significantly upregulated in sodium-taurocholate-induced SAP. The knockout of NINJ1 delays PMR in acinar cells and alleviates SAP. Moreover, we observed that NINJ1 expression is mediated by Ca2+ concentration in acinar cells. Importantly, we found that Ca2+ overload drives mitochondrial stress to upregulate the P53/NINJ1 pathway, inducing PMR in acinar cells, and amlodipine, a Ca2+ channel inhibitor, can reduce the occurrence of PMR by decreasing the concentration of Ca2+. Our results demonstrate the mechanism by which NINJ1 induces PMR in SAP acinar cells and provide a potential new target for treatment of SAP.

Can't drop the MIC(A/B): Preventing stress-ligand shedding to enhance pan-cancer targeting.

Chimeric antigen receptor (CAR)-based cellular therapies have achieved remarkable success against hematologic malignancies, but their application against solid tumors remains challenging. In this issue, Goulding et al.1 describe a unique CAR natural killer (NK) cell platform with pan-cancer activity via preservation and recognition of stress ligands on tumor cell membranes.

Application of Cell Membrane-Coated Nanomaterials for Tumor Treatment.

Tumors are a major cause of human mortality worldwide, and the rapid development of nanomaterials (NMs) for tumor therapy and drug delivery has provided new treatment methods. However, NMs' high immunogenicity, short circulation time, and low specificity limit their application in tumor therapy. In recent years, bionanomaterials using cell membranes have emerged to overcome the shortcomings of monomeric NMs. Cell membrane-encapsulated NMs extracted from multiple cells not only retain the physicochemical properties of NMs but also inherit the biological functions of the source cells, aiding in drug delivery. The combination of the cell membrane and drug-loading NMs offers an efficient and targeted drug delivery system tailored to the tumor microenvironment. The research and application of this method have been widely carried out in the academic field of tumor diagnosis and treatment. This review presents the recent research progress of cell membrane-coated NMs as drug carriers in tumor therapy, including cell membrane extraction methods, encapsulation strategies, and the applications of cell membrane-encapsulated NMs in tumor therapy. We believe that biomimetic nanomaterials will be a promising and novel anticancer strategy in the future, and their wide application will certainly bring vitality to the field of tumor diagnosis and treatment. The combination of membrane and drug-loading nanomaterials embodies a highly efficient and target drug delivery system tailored to the tumor microenvironment, which broadens a new path of drug delivery for future cancer treatment. Meanwhile, it is also a perfect combination and application of biomedical nanomaterials, which is of great significance.

Targeting drugs to tumours using cell membrane-coated nanoparticles.

Traditional cancer therapeutics, such as chemotherapies, are often limited by their non-specific nature, causing harm to non-malignant tissues. Over the past several decades, nanomedicine researchers have sought to address this challenge by developing nanoscale platforms capable of more precisely delivering drug payloads. Cell membrane-coated nanoparticles (CNPs) are an emerging class of nanocarriers that have demonstrated considerable promise for biomedical applications. Consisting of a synthetic nanoparticulate core camouflaged by a layer of naturally derived cell membranes, CNPs are adept at operating within complex biological environments; depending on the type of cell membrane utilized, the resulting biomimetic nanoformulation is conferred with several properties typically associated with the source cell, including improved biocompatibility, immune evasion and tumour targeting. In comparison with traditional functionalization approaches, cell membrane coating provides a streamlined method for creating multifunctional and multi-antigenic nanoparticles. In this Review, we discuss the history and development of CNPs as well as how these platforms have been used for cancer therapy. The application of CNPs for drug delivery, phototherapy and immunotherapy will be described in detail. Translational efforts are currently under way and further research to address key areas of need will ultimately be required to facilitate the successful clinical adoption of CNPs.

TCR extracellular domain genetically linked to CD28, 2B4/41BB and DAP10/CD3ζ -engineered NK cells mediates antitumor effects.

NK cells, especially FDA-approved NK-92 cells, could be used for TCR engineering owing to their specialized cytotoxicity against tumors, safety profile and potential use as an off-the-shelf cellular therapy. The TCR complex requires assembly of TCR- α/ ß chains with CD3 molecules (CD3δ, CD3γ, CD3ε, CD3ζ) to be correctly expressed at the cell membrane, and yet NK cells lack expression of these CD3 subunits besides CD3ζ. Since transmembrane regions of TCR α and ß chains are involved in TCR complex assembly, transmembrane regions of TCR replaced by CD28 transmembrane domain could result in the expression of TCR independent of its companion CD3 subunits. However, since the absence of CD3 signaling components can influence the transmission of TCR signals to NK cells, it is necessary to add the signaling molecules of NK cells followed by CD28 transmembrane domain. Both CD3ζ and DAP10 play an important role in the activation and cytotoxicity of NK cells; moreover, 2B4 and 4-1BB are the main costimulatory molecules in NK cells. Therefore, we designed a chimeric TCR that consisted of the extracellular domains of the TCR α and ß chains specific for NYESO-1 fused to the CD28 transmembrane domain followed by the 41BB and CD3ζ signaling domains as well as the 2B4 and DAP10 signaling domain, respectively. The chimeric TCR genetically engineered NK-92 cells exhibit antigen-specific recognition and lysis of tumor cells both in vitro and in vivo. In addition, TCR-28-2B10/BBζ can be feasibly expressed in primary NK cells and exhibit antigen-reactive recognition and effect function. The overall encouraging data highlight the value of NK-92 cells and primary NK cells engineered to express therapeutic chimeric TCR for adoptive immunotherapies.

Negative E-cadherin expression on bone marrow myeloma cell membranes is associated with extramedullary disease.

Background: The loss of E-cadherin expression and the induction of N-cadherin are known as hallmarks of the epithelial-to-mesenchymal transition, an essential initial step in the process of metastasis in solid tumors. Although several studies have reported expressions of these cadherins in patients with multiple myeloma (MM), their clinical significance is unknown as MM cells are non-epithelial. Methods: In this study, we examined the expression of E- and N-cadherins by immunohistochemistry using bone marrow (BM) biopsy specimens from 31 newly diagnosed MM patients and in subsequent biopsy specimens from six of these. Results: Negative E-cadherin expression on BM myeloma cell membranes was significantly associated with the presence of soft-tissue masses arising from bone lesions and breaking through the cortical bone, referred to as extramedullary disease (EMD). Conclusions: Given the aggressive nature of EMD, our study suggests that screening for E-cadherin using BM immunohistochemistry is one measure that could predict the development of EMD in patients with MM.

The ctpF Gene Encoding a Calcium P-Type ATPase of the Plasma Membrane Contributes to Full Virulence of Mycobacterium tuberculosis.

Identification of alternative attenuation targets of Mycobacterium tuberculosis (Mtb) is pivotal for designing new candidates for live attenuated anti-tuberculosis (TB) vaccines. In this context, the CtpF P-type ATPase of Mtb is an interesting target; specifically, this plasma membrane enzyme is involved in calcium transporting and response to oxidative stress. We found that a mutant of MtbH37Rv lacking ctpF expression (MtbΔctpF) displayed impaired proliferation in mouse alveolar macrophages (MH-S) during in vitro infection. Further, the levels of tumor necrosis factor and interferon-gamma in MH-S cells infected with MtbΔctpF were similar to those of cells infected with the parental strain, suggesting preservation of the immunogenic capacity. In addition, BALB/c mice infected with Mtb∆ctpF showed median survival times of 84 days, while mice infected with MtbH37Rv survived 59 days, suggesting reduced virulence of the mutant strain. Interestingly, the expression levels of ctpF in a mouse model of latent TB were significantly higher than in a mouse model of progressive TB, indicating that ctpF is involved in Mtb persistence in the dormancy state. Finally, the possibility of complementary mechanisms that counteract deficiencies in Ca2+ transport mediated by P-type ATPases is suggested. Altogether, our results demonstrate that CtpF could be a potential target for Mtb attenuation.

Plasma membrane lesion type total intestinal eosinophilic enteritis: a case report.

BACKGROUND: Eosinophilic gastroenteritis is a rare gastrointestinal disease that is characterized by diffuse or localized eosinophil infiltration in the gastrointestinal tract, and is accompanied by increased peripheral blood eosinophils. Herein, a case of plasma membrane lesion-type total intestinal eosinophil enteritis is reported. CASE PRESENTATION: We report on a 20-year-old male patient who was admitted to the hospital with "abdominal distension for 15 days". The infiltration of a large number of eosinophils was found by conducting an intestinal biopsy, routine ascites examination, blood routine, smear test, and a bone marrow puncture. A special feature of this patient was that a large number of eosinophils were found in the duodenum, small intestine, and colon. The final diagnosis was plasma membrane lesion type total intestinal eosinophilic enteritis. After four weeks of prednisone treatment, the symptoms disappeared completely and the entire intestinal mucosa was endoscopically observed as smooth. CONCLUSION: Clinical practitioners must pay attention to gastrointestinal endoscopy and biopsy pathology results for patients presenting with abdominal distention and ascites. Combined with an abnormal increase of eosinophils in ascites, bone marrow, and peripheral blood, clinical practitioners must be highly vigilant against plasma membrane lesion type total intestinal eosinophilic enteritis.

Targeting Ion Channels for Cancer Treatment: Current Progress and Future Challenges.

Ion channels are key regulators of cancer cell pathophysiology. They contribute to a variety of processes such as maintenance of cellular osmolarity and membrane potential, motility (via interactions with the cytoskeleton), invasion, signal transduction, transcriptional activity and cell cycle progression, leading to tumour progression and metastasis. Ion channels thus represent promising targets for cancer therapy. Ion channels are attractive targets because many of them are expressed at the plasma membrane and a broad range of existing inhibitors are already in clinical use for other indications. However, many of the ion channels identified in cancer cells are also active in healthy normal cells, so there is a risk that certain blockers may have off-target effects on normal physiological function. This review describes recent research advances into ion channel inhibitors as anticancer therapeutics. A growing body of evidence suggests that a range of existing and novel Na+, K+, Ca2+ and Cl- channel inhibitors may be effective for suppressing cancer cell proliferation, migration and invasion, as well as enhancing apoptosis, leading to suppression of tumour growth and metastasis, either alone or in combination with standard-of-care therapies. The majority of evidence to date is based on preclinical in vitro and in vivo studies, although there are several examples of ion channel-targeting strategies now reaching early phase clinical trials. Given the strong links between ion channel function and regulation of tumour growth, metastasis and chemotherapy resistance, it is likely that further work in this area will facilitate the development of new therapeutic approaches which will reach the clinic in the future.

Neurotherapeutic implications of sense and respond strategies generated by astrocytes and astrocytic tumours to combat pH mechanical stress.

AIMS: Astrocytes adapt to acute acid stress. Intriguingly, cancer cells with astrocytic differentiation thrive even better in an acidic microenvironment. How changes in extracellular pH (pHe) are sensed and measured by the cell surface assemblies that first intercept the acid stress, and how this information is relayed downstream for an appropriate survival response remains largely uncharacterized. METHODS: In vitro cell-based studies were combined with an in vivo animal model to delineate the machinery involved in pH microenvironment sensing and generation of mechanoadaptive responses in normal and neoplastic astrocytes. The data was further validated on patient samples from acidosis driven ischaemia and astrocytic tumour tissues. RESULTS: We demonstrate that low pHe is perceived and interpreted by cells as mechanical stress. GM3 acts as a lipid-based pH sensor, and in low pHe, its highly protonated state generates plasma membrane deformation stress which activates the IRE1-sXBP1-SREBP2-ACSS2 response axis for cholesterol biosynthesis and surface trafficking. Enhanced surface cholesterol provides mechanical tenacity and prevents acid-mediated membrane hydrolysis, which would otherwise result in cell leakage and death. CONCLUSIONS: In summary, activating these lipids or the associated downstream machinery in acidosis-related neurodegeneration may prevent disease progression, while specifically suppressing this key mechanical 'sense-respond' axis should effectively target astrocytic tumour growth.

The polypeptide antibiotic polymyxin B acts as a pro-inflammatory irritant by preferentially targeting macrophages.

Polymyxin B (PMB) is an essential antibiotic active against multidrug-resistant bacteria, such as multidrug-resistant Pseudomonas aeruginosa (MDRP). However, the clinical use of PMB is limited, because PMB causes serious side effects, such as nephrotoxicity and neurotoxicity, probably due to its cytotoxic activity. However, cytotoxic mechanisms of PMB are poorly understood. In this study, we found that macrophages are particularly sensitive to PMB, when compared with other types of cells, including fibroblasts and proximal tubule (PT) cells. Of note, PMB-induced necrosis of macrophages allowed passive release of high mobility group box 1 (HMGB1). Moreover, upon exposure of PMB to macrophages, the innate immune system mediated by the NLR family pyrin domain containing 3 (NLRP3) inflammasome that promotes the release of pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) was stimulated. Interestingly, PMB-induced IL-1ß release occurred in the absence of the pore-forming protein gasdermin D (GSDMD), which supports the idea that PMB causes plasma membrane rupture accompanying necrosis. Emerging evidence has suggested that both HMGB1 and IL-1ß released from macrophages contribute to excessive inflammation that promote pathogenesis of various diseases, including nephrotoxicity and neurotoxicity. Therefore, these biochemical properties of PMB in macrophages may be associated with the induction of the adverse organ toxicity, which provides novel insights into the mechanisms of PMB-related side effects.

Cell membrane breakage and triggering T cell infiltration are involved in human telomerase reverse transcriptase (hTERT) promoter-driven novel peptide KK-64 for liver cancer gene therapy.

Liver cancer is an aggressive malignancy with exhibits both high mortality and morbidity. The current treatment options are associated with several limitations, novel specific anti-cancer drugs are urgently needed to improve liver cancer treatment. In this study, a new peptide KK-64 was designed, and it showed strong cytotoxicity against liver cancer cells. To obtain the tumor targeting property, a plasmid that contains KK-64 DNA fragment and driven by human telomerase reverse transcriptase (hTERT) promoter was constructed. pcTERT-kk-64 plasmid was found to specifically inhibit the viability of liver cancer cells HepG2, induce substantial apoptosis as well as damage to the cell membranes, but had minimal effects toward normal liver HL-7702 cells. Furthermore, pcTERT-kk-64 plasmids was also noted to significantly attenuate migration and invasion of HepG2 cells. The anti-tumor effect of pcTERT-kk-64 plasmid was also observed in H22 cell-bearing mice, and it appeared to cause significant tumor regression, trigger tumor cell apoptosis, and infiltrate cytotoxicity T cells to the tumor tissues after plasmids injection. Thus, pcTERT-kk-64 plasmids showed both strong cytotoxicity and tumor selectivity in vitro and in tumor-bearing mice in liver cancer models.

Current Applications and Discoveries Related to the Membrane Components of Circulating Tumor Cells and Extracellular Vesicles.

In cancer, many analytes can be investigated through liquid biopsy. They play fundamental roles in the biological mechanisms underpinning the metastatic cascade and provide clinical information that can be monitored in real time during the natural course of cancer. Some of these analytes (circulating tumor cells and extracellular vesicles) share a key feature: the presence of a phospholipid membrane that includes proteins, lipids and possibly nucleic acids. Most cell-to-cell and cell-to-matrix interactions are modulated by the cell membrane composition. To understand cancer progression, it is essential to describe how proteins, lipids and nucleic acids in the membrane influence these interactions in cancer cells. Therefore, assessing such interactions and the phospholipid membrane composition in different liquid biopsy analytes might be important for future diagnostic and therapeutic strategies. In this review, we briefly describe some of the most important surface components of circulating tumor cells and extracellular vesicles as well as their interactions, putting an emphasis on how they are involved in the different steps of the metastatic cascade and how they can be exploited by the different liquid biopsy technologies.

Cancer Cell Membrane-Coated Nanosuspensions for Enhanced Chemotherapeutic Treatment of Glioma.

Effective intracerebral delivery is key for glioma treatment. However, the drug delivery system within the brain is largely limited by its own adverse physical and chemical properties, low targeting efficiency, the blood-brain barrier and the blood-brain tumor barrier. Herein, we developed a simple, safe and efficient biomimetic nanosuspension. The C6 cell membrane (CCM) was utilized to camouflaged the 10-hydroxycamptothecin nanosuspension (HCPT-NS) in order to obtain HCPT-NS/CCM. Through the use of immune escape and homotypic binding of the cancer cell membrane, HCPT-NS/CCM was able to penetrate the blood-brain barrier and target tumors. The HCPT-NS is only comprised of drugs, as well as a small amount of stabilizers that are characterized by a simple preparation method and high drug loading. Similarly, the HCPT-NS/CCM is able to achieve targeted treatment of glioma without any ligand modification, which leads it to be stable and efficient. Cellular uptake and in vivo imaging experiments demonstrated that HCPT-NS/CCM is able to effectively cross the blood-brain barrier and was concentrated at the glioma site due to the natural homing pathway. Our results reveal that the glioma cancer cell membrane is able to promote drug transport into the brain and enter the tumor via a homologous targeting mechanism.

A bioelectric model of carcinogenesis, including propagation of cell membrane depolarization and reversal therapies.

As the main theory of carcinogenesis, the Somatic Mutation Theory, increasingly presents difficulties to explain some experimental observations, different theories are being proposed. A major alternative approach is the Tissue Organization Field Theory, which explains cancer origin as a tissue regulation disease instead of having a mainly cellular origin. This work fits in the latter hypothesis, proposing the bioelectric field, in particular the cell membrane polarization state, and ionic exchange through ion channels and gap junctions, as an important mechanism of cell communication and tissue organization and regulation. Taking into account recent experimental results and proposed bioelectric models, a computational model of cancer initiation was developed, including the propagation of a cell depolarization wave in the tissue under consideration. Cell depolarization leads to a change in its state, with the activation and deactivation of several regulation pathways, increasing cell proliferation and motility, changing its epigenetic state to a more stem cell-like behavior without the requirement of genomic mutation. The intercellular communication via gap junctions leads, in certain circumstances, to a bioelectric state propagation to neighbor cells, in a chain-like reaction, till an electric discontinuity is reached. However, this is a reversible process, and it was shown experimentally that, by implementing a therapy targeted on cell ion exchange channels, it is possible to reverse the state and repolarize cells. This mechanism can be an important alternative way in cancer prevention, diagnosis and therapy, and new experiments are proposed to test the presented hypothesis.

Inflammasome-Induced Osmotic Pressure and the Mechanical Mechanisms Underlying Astrocytic Swelling and Membrane Blebbing in Pyroptosis.

Cell swelling and membrane blebbing are characteristic of pyroptosis. In the present study, we explored the role of intracellular tension activity in the deformation of pyroptotic astrocytes. Protein nanoparticle-induced osmotic pressure (PN-OP) was found to be involved in cell swelling and membrane blebbing in pyroptotic astrocytes, and was associated closely with inflammasome production and cytoskeleton depolymerization. However, accumulation of protein nanoparticles seemed not to be absolutely required for pyroptotic permeabilization in response to cytoskeleton depolymerization. Gasdermin D activation was observed to be involved in modification of typical pyroptotic features through inflammasome-induced OP upregulation and calcium increment. Blockage of nonselective ion pores can inhibit permeabilization, but not inflammasome production and ion influx in pyroptotic astrocytes. The results suggested that the inflammasomes, as protein nanoparticles, are involved in PN-OP upregulation and control the typical features of pyroptotic astrocytes.