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PubMed2024

Adeno-Associated Virus Engineering and Load Strategy for Tropism Modification, Immune Evasion and Enhanced Transgene Expression.

Gene therapy aims to add, replace or turn off genes to help treat disease. To date, the US Food and Drug Administration (FDA) has approved 14 gene therapy products. With the increasing interest in gene therapy, feasible gene delivery vectors are necessary for inserting new genes into cells. There are different kinds of gene delivery vectors including viral vectors like lentivirus, adenovirus, retrovirus, adeno-associated virus et al, and non-viral vectors like naked DNA, lipid vectors, polymer nanoparticles, exosomes et al, with viruses being the most commonly used. Among them, the most concerned vector is adeno-associated virus (AAV) because of its safety, natural ability to efficiently deliver gene into cells and sustained transgene expression in multiple tissues. In addition, the AAV genome can be engineered to generate recombinant AAV (rAAV) containing transgene sequences of interest and has been proven to be a safe gene vector. Recently, rAAV vectors have been approved for the treatment of various rare diseases. Despite these approvals, some major limitations of rAAV remain, namely nonspecific tissue targeting and host immune response. Additional problems include neutralizing antibodies that block transgene delivery, a finite transgene packaging capacity, high viral titer used for per dose and high cost. To deal with these challenges, several techniques have been developed. Based on differences in engineering methods, this review proposes three strategies: gene engineering-based capsid modification (capsid modification), capsid surface tethering through chemical conjugation (surface tethering), and other formulations loaded with AAV (virus load). In addition, the major advantages and limitations encountered in rAAV engineering strategies are summarized.

Dependovirus↗
PubMed1989

Completing the transition from clinical engineering to technology management.

Comprehensive technology management has a bright, important future in the nation's hospitals as they move into the 1990s in an environment that emphasizes high-quality, cost-effective health care. Going from equipment control to technology management will not be easy for some hospitals and some clinical engineers, but it is important to take whatever steps are necessary to accomplish the transition. The speed with which hospitals complete the transition to technology management will vary. A hospital may wish to broaden its clinical engineering department's role immediately but be realistically limited by the number of qualified personnel or the funds available. If it is not possible to accomplish them in a short time, technology management functions can be phased in. But they must not be forgotten. In many hospitals, clinical engineering has made substantial progress toward the ideal Level 5 department described in the previous article. Embracing the concept of comprehensive technology management and performing its functions will enable clinical engineering to achieve a place in the hospital that is more important and valuable than simply equipment support, and will help to prepare the hospital for the 1990s.

Biomedical Engineering↗
PubMed2006

Cartilage and bone tissue engineering using hydrogels.

Tissue engineering is an emerging field of regenerative medicine which holds promise for the restoration of tissues and organs affected by chronic diseases, age-linked degeneration, congenital deformity and trauma. During the past decade, tissue engineering has evolved from the use of naked biomaterials, which may just replace small area of damaged tissue, to the use of controlled three-dimensional scaffolds in which cells can be seeded before implantation. These cellularized constructs aims at being functionally equal to the unaffected tissue and could make possible the regeneration of large tissue defects. Among the recently developed scaffolds for tissue engineering, polymeric hydrogels have proven satisfactory in cartilage and bone repair. Major technological progress and advances in basic knowledge (physiology and developmental biology) are today necessary to bring this proof of concept to clinical reality. The present review focuses on the recent advances in hydrogel-based tissue engineered constructs potentially utilizable in bone and cartilage regenerative medicine.

Animals↗
PubMed2006

[Cell culture, tissue engineering and regenerative medicine. Part I].

Tissue engineering is an interdisciplinary field that applies the principles and methods of engineering and the cell culture toward the development of biomaterials that restore, maintain or improve tissue function. The amalgamation of engineering and medicine has interested many scientists for at last two hundred years. What was the goal of cell culture? First, for progress in life sciences achievement and subsequent for virology and toxicology development. In vitro studies are done because of many problems with carrying out animal experiments. In this work the authors present the attempts of physicians, anatomopathologists, embryologists and biologists which contributed to fast development of new area in medicine--tissue engineering.

Animals↗
PubMed1991

An engineered liver glycogen phosphorylase with AMP allosteric activation.

Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.

Adenosine Monophosphate↗
PubMedDate not supplied

Clinical engineering education in the high technology hospital.

The application of high technology in the critical care field has created two potential approaches: (1) development of critical care technicians to coordinate clinical engineering educational support; and (2) modification of the hospital organization to streamline communications between the clinical engineers and those they support. At one university hospital studied from 1978 to 1982, both the use of high technology equipment and the number of critical care technicians approximately doubled. With emphasis on technician coordination of engineering support, the in-service education lectures increased 134%, while the engineering staff and the corrective maintenance workload remained unchanged.

Biomedical Engineering↗
PubMedDate not supplied

Muscle tissue engineering for partial glossectomy defects.

BACKGROUND: Tongue reconstruction represents a difficult reconstructive problem, based on the tongue's complex multimodality function. Existing methods of tongue reconstruction often result in significant deficits in speech and deglutition. A functional neotongue requires adequate soft tissue bulk and restoration of coordinated muscle function. Tissue engineering, a scientific approach that allows introduction of isolated cell populations of interest within 3-dimensional polymer scaffolds to create new tissue, may allow the generation of more highly functional tissue in tongue reconstruction. OBJECTIVES: To apply muscle-tissue engineering techniques in the reconstruction of partial glossectomy defects in rats, and to compare the gross and histological nature of tissue found after reconstruction of the hemiglossectomy defect with acellular vs tissue-engineered composite material. MATERIALS AND METHODS: Thirty mature Lewis rats underwent a left-sided mucosa-sparing partial glossectomy. The defects were then filled with 1 of the following 3 substances: isotonic sodium chloride solution, a collagen-rich hydrogel, or hydrogel containing a suspension of neonatal myoblasts from syngeneic rats. The animals were killed after 6 weeks and the tongues were harvested. The control and operated-on tongue halves were evaluated for weight differences and histological features. RESULTS: The group receiving the hydrogel-myoblast composite injections demonstrated a statistically significant increase in tongue weight of the operated-on side compared with the control side. In contrast, the isotonic sodium chloride solution and hydrogel groups demonstrated loss of tongue weight. These findings correlated with the results of the histological evaluation. Hemitongues from the composite group demonstrated formation of new tissue with areas of musclelike tissue extending from islands of residual hydrogel, and we found evidence of neovascularization and possible neurotization. In contrast, the isotonic sodium chloride solution group exhibited dense fibrous scar with loss of muscle architecture and dramatic loss of tongue volume. The hydrogel group demonstrated preservation of tongue volume with persistent islands of gel, but no clear evidence of new tissue formation. CONCLUSIONS: The introduction of a hydrogel into the rat hemiglossectomy pocket appears to promote volume preservation and/or muscle regeneration. The addition of myoblasts suspended in collagen gel supports the development of new tissue that preserves weight and volume after hemiglossectomy and may possess muscle properties similar to the tissue desired. This tissue-engineering approach represents a promising new strategy in tongue reconstruction and merits further investigation into the possible functional advantages it offers compared with current techniques.

Animals↗
PubMedDate not supplied

Quantitative study of tissue-engineered cartilage with human bone marrow mesenchymal stem cells.

OBJECTIVES: To assess the possibility of cartilage tissue engineering using human mesenchymal stem cells (hMSCs) and to investigate the quantitative relationship between hMSCs and engineered cartilage. DESIGN: Human mesenchymal stem cells were cultured, cryopreserved, and expanded in vitro. Surface antigens were detected by flow cytometry. In vitro chondrogenesis of hMSCs and cryopreserved hMSCs was performed. The chondrogenesis-induced hMSCs were seeded onto polyglycolic acid scaffolds, cultured in vitro for 3 weeks in chondrogenic medium, and then implanted into nude mice. The implants were harvested after 10 weeks and examined with histologic and immunochemical staining. RESULTS: The construction of cartilages was identified grossly and histologically: 1.9 to 2.5 x 10(7) nucleated cells were obtained from 1 mL of bone marrow, and about 1 to 2 x 10(6) hMSCs were obtained from the primary culture. The number of hMSCs tripled at every passage and reached 1.4 to 2.8 x 10(12) at passage 15. The purity of hMSCs was 95% and 98% at the primary and the fourth passages, respectively. Twenty-one days was the optimal (induction rate, 95%) induction time, with no apparent differences in induction rates among different passages. Based on our findings, hMSCs from 0.07 to 0.14 mL of bone marrow, expanded during 4 passages and induced for 21 days, would be sufficient to engineer 1 cm(2) of cartilage, 3-mm thick. CONCLUSION: Quantitative standards of hMSCs as seed cells for cartilage tissue engineering were established and may have value for later clinical work.

Animals↗
PubMed2001

Advances in biomedical engineering.

The most visible contributions of biomedical engineering to clinical practice involve instrumentation for diagnosis, therapy, and rehabilitation. Cell and tissue engineering also have emerged as clinical realities. In the next 25 years, advances in electronics, optics, materials, and miniaturization will accelerate development of more sophisticated devices for diagnosis and therapy, such as imaging and virtual surgery. The emerging new field of bioengineering-engineering based in the science of molecular cell biology-will greatly expand the scope of biomedical engineering to tackle challenges in molecular and genomic medicine.

Biocompatible Materials↗
PubMed1998

Collagen in tissue-engineered cartilage: types, structure, and crosslinks.

The function of articular cartilage as a weight-bearing tissue depends on the specific arrangement of collagen types II and IX into a three-dimensional organized collagen network that can balance the swelling pressure of the proteoglycan/water gel. To determine whether cartilage engineered in vitro contains a functional collagen network, chondrocyte-polymer constructs were cultured for up to 6 weeks and analyzed with respect to the composition and ultrastructure of collagen by using biochemical and immunochemical methods and scanning electron microscopy. Total collagen content and the concentration of pyridinium crosslinks were significantly (57% and 70%, respectively) lower in tissue-engineered cartilage that in bovine calf articular cartilage. However, the fractions of collagen types II, IX, and X and the collagen network organization, density, and fibril diameter in engineered cartilage were not significantly different from those in natural articular cartilage. The implications of these findings for the field of tissue engineering are that differentiated chondrocytes are capable of forming a complex structure of collagen matrix in vitro, producing a tissue similar to natural articular cartilage on an ultrastructural scale.

Amino Acids↗
PubMed1998

Genetic engineering for high methionine grain legumes.

Methionine (Met) is the primary limiting essential amino acid in grain legumes. The imbalance in amino acid composition restricts their biological value (BV) to 55 to 75% of that of animal protein. So far improvement of the BV could not be achieved by conventional breeding. Therefore, genetic engineering was employed by several laboratories to resolve the problem. Three strategies have been followed. A) Engineering for increased free Met levels; B) engineering of endogenous storage proteins with increased numbers of Met residues; C) transfer of foreign genes encoding Met-rich proteins, e.g. the Brazil nut 2S albumin (BNA) and its homologue from sunflower, into grain legumes. The latter strategy turned out to be most promising. In all cases the gene was put under the control of a developmentally regulated seed specific promoter and transferred into grain legumes using the bacterial Agrobacterium tumefaciens-system. Integration into and copy numbers in the plant genome as well as Mendelian inheritance and gene dosage effects were verified. After correct precursor processing the mature 2S albumin was intracellularly deposited in protein bodies which are part of the vacuolar compartment. The foreign protein amounted to 5 to 10% of the total seed protein in the best transgenic lines of narbon bean (Vicia narbonensis L., used in the authors' laboratories), lupins (Lupinus angustifolius L., used in CSIRO, Australia), and soybean (Glycine max (L.) Merr., used by Pioneer Hi-Bred, Inc., USA). In the narbon bean the increase of Met was directly related to the amount of 2S albumin in the transgenic seeds, but in soybean it remained below the theoretically expected value. Nevertheless, trangenic soybean reached 100%, whereas narbon bean and lupins reached approximately 80% of the FAO-standard for nutritionally balanced food proteins. These results document that the Met problem of grain legumes can be resolved by genetic engineering.

Amino Acids, Essential↗
PubMed2000

Tissue engineering: challenges and opportunities.

This article reviews the key developments in the tissue engineering field over the past several years. The issues related to the development of the components of tissue-engineered products including cells, biomaterials, and biomolecules, and their integration into safe and effective products are presented. Moreover, the article outlines the challenges to the commercialization of tissue-engineered products, and highlights the ongoing efforts by the American Society for Testing and Materials (ASTM) in developing standards for tissue-engineered medical products. Furthermore, funding opportunities at the Advanced Technology Program at NIST are presented. Published 2000 John Wiley & Sons, Inc.

Animals↗
PubMed2002

Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction.

OBJECTIVE: An important role in joint and cartilage homeostasis in adults has been demonstrated recently for morphogenetic factors of the transforming growth factor beta family. Therefore, this study was undertaken to investigate the potential of bone morphogenetic proteins (BMPs) in chondrocyte differentiation using current technologies of tissue engineering. METHODS: Complementary DNAs of recombinant human BMPs 2, 4, 5, 6, and 7 were transfected into primary bovine articular chondrocytes. Transgenic chondrocytes were assembled 3-dimensionally in alginate or in bioresorbable co-polymer fleeces of vicryl and polydioxanon embedded in low-melting-point agarose. Redifferentiation and formation of cartilage tissue in vitro or after subcutaneous transplantation into nude mice were assayed by semiquantitative reverse transcriptase-polymerase chain reaction, histology, and in situ hybridization, and findings were compared with those in unmodified or control-transfected primary chondrocytes. RESULTS: Compared with other BMPs and control vector, BMP-7 induced a decrease in type I collagen expression in artificial cartilage, while transcription of the cartilage-specific type II collagen remained stable. In transplantation experiments, BMP-7 transgenic cartilage revealed the greatest amount of matrix synthesis, and BMP-7 was the only morphogen to suppress the infiltrative response of mouse fibroblastic cells into engineered cartilage, thereby preventing transplant destruction. CONCLUSION: Cartilage differentiation and matrix maturation are promoted by BMPs in cartilage engineering. The inhibitory effect of BMP-7 on a nonspecific infiltrative response in immunocompromised nude mice further suggests that individual morphogens not only may contribute to cartilage maturation, but also may protect it from nonspecific inflammation and invasive destruction. These properties advance BMPs as promising tools for engineering of cartilaginous joint bioprostheses and as candidate biologic agents or genes for cartilage stabilization in arthritis.

Animals↗
PubMed2001

Engineering of vascular ingrowth matrices: are protein domains an alternative to peptides?

Anastomotic intimal hyperplasia and surface thrombogenicity are the main reasons for the high failure rate of prosthetic small-diameter vascular grafts. While anastomotic intimal hyperplasia is a multifactorial event, ongoing surface thrombogenicity is primarily caused by the lack of an endothelium, even after years of clinical implantation. After decades of poorly performing synthetic artery-grafts, tissue engineering has emerged as a promising approach to generate biologically functional bio-synthetic hybrid grafts mimicking native arteries regarding the presence of an endothelial lining on the blood surface. "In vitro endothelialization" represented the first generation of such tissue-engineered vascular grafts, utilising cell culture techniques for the creation of a confluent autologous endothelium on ePTFE grafts. The clinical long-term results with this method in almost 200 patients are highly encouraging, showing patencies equal to vein grafts. Since "in vitro endothelialization" requires cell culture facilities, it will always be confined to large centres. Therefore, research of the 1990s turned to the development of spontaneously endothelializing implants, to make tissue-engineered grafts amenable to the entire vascular-surgical community. Apart from scaffold designs allowing transmural ingrowth, biological signalling through a facilitating ingrowth matrix holds a key to spontaneous endothelialization. In biological signalling, the increasingly deeper understanding of bio-active molecules and the discovery of domains and peptide sequences during the 1980s created the expectation in the 1990s that peptide signalling may be all that is needed. This present review highlights the possible problems associated with such a reductionist approach. Using the fibronectin molecule, we demonstrated that domains may be more suitable modules in tissue engineering than peptide sequences.

Animals↗
PubMed2003

Inhibition of cartilage degradation: a combined tissue engineering and gene therapy approach.

OBJECTIVE: To determine if tissue-engineered cartilage can be protected from cytokine-induced degradation using a gene therapy approach. METHODS: Chemical and pantropic retroviral gene transfer methodologies were compared for their ability to introduce a luciferase reporter gene into adult bovine cartilage chondrocytes grown in monolayer. Pantropic retrovirus was then used to transduce these cells with human tissue inhibitor of metalloproteinases 1 (TIMP-1), and the stability of expression in monolayer or pellet culture was monitored for 6 weeks. Untransduced and TIMP-1-transduced cells were also used to tissue engineer 3-dimensional cartilage constructs that were then challenged with interleukin-1 (IL-1) for 4 weeks. Conditioned media and residual cartilage were collected for analysis of matrix components, including type II collagen and proteoglycans, and for TIMP-1 production and matrix metalloproteinase (MMP) activity. RESULTS: Chemical transfection of adult bovine chondrocytes gave rise to short-lived reporter expression that was virtually undetectable after 4 weeks of culture. In contrast, pantropic retroviral transduction gave rise to stable expression that persisted at a high level for at least 6 weeks. Pantropic transduction of the cells with TIMP-1 gave rise to similar long-term expression, both in monolayer and pellet cultures. TIMP-1-transduced tissue-engineered cartilage also retained TIMP-1 expression for an additional 4 weeks of culture in the presence of IL-1. Compared with control samples, TIMP-1-transgenic cartilage resisted the catabolic effects of IL-1, with MMP activity reduced to basal levels and a decreased loss of type II collagen. CONCLUSION: Pantropic retroviral transduction permits long-term expression of potentially therapeutic transgenes in adult tissue-engineered cartilage. While TIMP-1 transduction could be used to prevent collagen breakdown, alternative transgenes may be necessary to protect cartilage proteoglycans.

Animals↗
PubMed2005

Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants.

Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.

Biological Assay↗
PubMed2005

Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature?

In this review we consider how cell specific recombinant monoclonal antibody (Mab) production by engineered mammalian cells can be improved. Whilst it is generally recognized that Mab production is limited post-transcriptionally at folding and assembly reactions, genetic engineering strategies based on overexpression of individual chaperones or foldases in mammalian cells have not reliably increased cell specific Mab production. Given that recent studies have established that chaperones and foldases themselves exist in a large multiprotein complex, which may coordinate the sequential processing of Mabs, we propose that global expansion of all components of the secretory pathway will likely be necessary to generically improve recombinant Mab production by mammalian cells. In this context, what can be learnt from nature? Important recent studies have delineated some of the main cellular pathways involved in the differentiation of B-cells into nature's own high level Mab producers, plasma cells. This is achieved by a dramatic re-programming of cellular function where the coordinated expansion of metabolic and secretory machinery precedes Ig production, then is maintained by induction of a key intracellular signaling pathway, the unfolded protein response (UPR). Here we review genetic engineering strategies to increase cell specific production rate and discuss whether manipulation of intracellular signaling systems such as the UPR will provide a novel means to engineer mammalian cells for high level recombinant Mab production.

Animals↗
PubMed2005

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development.

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.

Adipocytes↗