Tumor Susceptibility Gene 101 (TSG101) is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer. The multidomain protein encoded by the  Tumor Susceptibility Gene 101 (Tsg101). Designating this gene as a TSG seemed appropriate at the time since the exogenous expression of the Tsg101 cDNA in both antisense and sense orientation resulted in a transformed phenotype. In 1997, TSG101 had become more widely known as a tumor suppressor when it was reported that this gene is mutated, or its expression is lost in a significant subset of sporadic breast cancers [In the mid-1990s, the laboratory of Stanley Cohen at Stanford University conducted an insertional mutagenesis screen on immortalized mouse embryonic fibroblasts NIH3T3 cells) to identify candidate tumor suppressor genes [ cancers . These fT3 cells  cancers ,5,6, andTSG101 mRNAs are 86% identical on the nucleotide level [Tsg101 locus is highly conserved between mouse and human [TSG101 implied that the majority of truncated transcripts that were observed in cancer and nonmalignant tissues are true alternative splice products that originate from exon skipping, rather than aberrant transcripts from cryptic splice sites as proposed earlier [TSG101 is expressed in all tissues and cell types [Tsg101 mRNA is already present in 1-cell and 2-cell stage mouse embryos. The expression of Tsg101 in germ and stem cells may also explain the origin of a processed Tsg101 pseudogene in the mouse genome, which made it challenging to identify the actual Tsg101 locus and isolate genomic DNA sequences for the construction of gene targeting vectors to generate knockout mice [Tsg101 mRNA levels were observed in the brain and the lactating mammary gland [The coding sequences of the mouse and human de level . Sanger  earlier ,9,10,11. earlier . The 5′ ll types ,8, and tout mice ,14,15. DTsg101 as a housekeeping gene had several implications. First, a complete knockout of Tsg101 might cause early embryonic lethality [mRNA expression levels from microarray and RNA-sequencing data are frequently being used to judge the importance of genes in cancer development and patient survival. As discussed later in this review, a tight post-translational control of the TSG101 protein level that balances variations in mRNA expression also imposes challenges for the generation of genetically engineered models to assess the effects of TSG101 gain- or loss-of-function in normal organogenesis and cancer development.The designation of ethality . More imTSG101 transcripts encode proteins of approximately 50 kDa in size with more than 94% similarity. The TSG101 protein contains several conserved domains  in Saccharomyces cerevisiae, which is also known as STP22. Subsequent studies revealed that TSG101/VPS23 is a central member of a subset of VPS class E proteins  that form the Endosomal Sorting Complex Required for Transport I (ESCRT-I). As part of this complex, TSG101 is suggested to selectively bind ubiquitinated cargo proteins and direct their sorting into multivesicular endosomes [Two reports from the laboratories of Duane Jenness and Scott Emr ,34 were ndosomes . In linendosomes ,35,36,37ndosomes , and, asndosomes ,15,39.Insight into the multifaceted functions of TSG101 as a member of ESCRT-I was gained from a series of studies on the association of TSG101 with retroviral proteins and their intracellular movement . TSG101 The specific functions of TSG101 in retroviral particle assembly and budding might be exemplary for a broader role in the biosynthesis and release of exosomes or, as now generally defined, secreted extracellular vesicles (EVs). Using a proteomic approach, Thery et al.  were firTSG101 locus have not been widely employed. As reviewed in the next section, the phenotypes of defined TSG101 genomic knockout models deviate from observations in cell lines where TSG101 is only knocked down with siRNAs. It is unclear to date which of the known molecular mechanisms, if any, are responsible for the indispensable functions of TSG101 for the proliferation and survival of mammalian cells.In summary, the TSG101 protein is suggested to mediate a variety of intracellular processes, but the majority of published articles only highlight its proposed roles in endosomal trafficking. Notably, many of the reports that solely focus on the molecular, biochemical, and intracellular functions of TSG101 emphasize the importance of their specific findings for diseases like cancer and neurodegeneration, but the proposed mechanisms are rarely validated in genetically defined in vivo disease models or in primary human tissue samples. Consequently, the biological relevance of these multifaceted functions of TSG101 for normal development, differentiation, and tissue homeostasis, as well as potential roles of TSG101 in cancer are poorly defined. On the experimental level, most published studies that examined the intracellular roles of TSG101 relied exclusively on RNA interference, overexpression of TSG101 mutants, or the use of chemical compounds that disrupt entire cellular processes. CRISPR/Cas-based targeted knockouts or other gene editing methods to introduce mutations into the endogenous Tsg101 gene promoter sequence and expression profiling, we predicted that a conventional knockout of this gene causes embryonic lethality [Tsg101 in germ cells and differentiated tissues during embryonic and postnatal development as well as in cultured cells that are primary, immortalized, or tumorigenic [p53 did not extend the survival of TSG101-deficient cells [Tsg101. More interestingly, we noted that individual rescue clones expressed exogenous TSG101 at the same levels as the endogenous protein in parental controls [TSG101 cDNA can fully restore normal growth of mouse cells that lack endogenous TSG101, suggesting that both mammalian proteins function in an identical manner. In contrast to the full-length cDNA, expression of TSG101 mRNA splice variants or deletion mutants lacking individual domains of TSG101 did not rescue the deleterious knockout phenotype  [From the analysis of the ethality . Indeed,ethality  were firorigenic ,15,39. Torigenic . As contnt cells ,39. Re-econtrols , supportcontrols . We haveblished) .Tsg101 in the germline resulted in an earlier embryonic lethality prior to or during implantation [Whey Acidic Protein gene promoter (WAP- Cre) demonstrated that TSG101 is equally required for cell survival and tissue homeostasis in adult animals. Secretory epithelial cells that have completed their functional differentiation show a significantly reduced proliferation rate and it is therefore evident that crucial functions of TSG101 for cell survival are independent of its suggested roles in cell division. Re-expression of exogenous TSG101 in the lactating mammary gland restores normal mammary gland development and lactation [In contrast to mutants that lack exons 8 and 9, the Cre-mediated excision of the promoter and exon 1 of antation . A tissuactation . Similaractation .TSG101 conditional knockout mice were also utilized to examine biologically relevant functions of TSG101 for normal tissue homeostasis in organ systems other than the mammary gland. Using a cardiac-specific and tamoxifen-inducible knockout of TSG101, Essandoh et al.  recentlyLike the mammary epithelium and cardiomyocytes, the deletion of TSG101 in oligodendroglia was reported to cause apoptosis, demyelination, vacuolation, and severe spongiform encephalopathy. Mice lacking TSG101 in parts of the central nervous system developed a tremor and had significantly reduced body weight by 8 weeks of age . In a paCip1 is a downstream mediator of the p53-dependent cell cycle arrest in response to the deletion of Tsg101 [Cdkn2a (p16Ink4a/p19Arf knockout) did not alter the ability of MDM2 to sequester p53 and restore the p21-mediated G1 arrest. The collective results obtained from the analysis of Tsg101/p53, Tsg101/p21, and Tsg101/Cdkn2a conditional double-knockout cell lines that were generated in our team suggested that a biologically significant interaction of TSG101 with the MDM2/p53 circuit seems unlikely. The p53/p21-controlled G1 arrest might, therefore, be an indirect consequence of an activation of stress response pathways that are being triggered by the knockout of TSG101 [In summary, the complete loss of TSG101 triggers an accumulation of p53 and a cell cycle arrest at the G1 phase followed by cell death, which is independent of p53 function and proliferation. This finding is not unique to mammals and can be extended to other vertebrate species. A knockdown of TSG101 with antisense morpholinos causes a growth delay and lethality of zebrafish embryos [f Tsg101 ,53. Althf Tsg101 , we demof TSG101 .Fibroblasts with a conditional knockout of TSG101 exhibit several characteristics of stressed cells: a) phosphorylation of mitogen-activated protein (MAP) kinases independent of growth factor stimulation, b) widespread redistribution of actin filaments, and c) induction of autophagy prior to cell death . TSG101 Tsg101 gene causes a substantial decline in the steady-state levels of the EGFR and ERBB2 [Although the precise cellular process that causes TSG101 knockout cells to die is still unknown, one important consequence that may trigger an intense stress-response is growth factor deprivation. Unlike previous observations that a siRNA-mediated knockdown of TSG101 resulted in enhanced recycling or retention of the EGFR within early endosomes , the defnd ERBB2 . This mand ERBB2 , and thind ERBB2 . It remaTsg101 alleles in surviving knockout rescue clones was confirmed by Southern blot (fl/flTsg101) as well as knockout rescue clones expressing HA-tagged exogenous, wildtype (wt) TSG101 AP motif-containing proteins at different sites. Pornillos and coworkers  demonstr) TSG101 B. Given Tsg101 gene causes cell cycle arrest and cell death instead of accelerated growth and neoplastic transformation, which would support its proposed function as a tumor suppressor. This is also the case for other vertebrate animal models such as zebrafish [p53 and Cdkn2a. TSG101 is also essential for the growth of neoplastic cells that are capable of forming tumors in mice [Tsg101 alleles in somatic tissues of genetically engineered mice, such as the mammary epithelium, led to the development of benign neoplasms or cancer [Tsg101 was deleted in the mammary epithelium that lacked both copies of the tumor suppressor p53 [Tsg101 in established, ERBB2-transformed mammary cancer cells causes cell death  [Despite a wealth of information about multifaceted intracellular functions of TSG101, it is still a conundrum why a knockout of the mammalian ebrafish . The ess in mice ,15,39. Nr cancer ,57. Moressor p53 . This wablished) . These fblished) ,61,62. Iblished) . Silenciblished) , which, blished) . On the blished) .TSG101 ortholog Erupted in Drosophila causes excessive cell growth, albeit in a non- cell- autonomous manner through the expression of the Notch target gene Unpaired, which is the secreted ligand that stimulates JAK/STAT signaling in the fly. It is obvious that a mechanism of a non-cell-autonomous role of TSG101 in growth suppression in the fly does not correspond to the phenotypes of a knockdown of TSG101 in normal and neoplastic cells of human and mouse origin. Although the proposed function of Erupted as a paracrine growth suppressor seems interesting, it needs to be explained why the phenotype of the Erupted-deficient fly is very different from a knockout of dVps28, which results in early embryonic lethality [Erupted and dVps28 in mutant flies might suggest that the mechanism that controls the post- translational cross-regulation of the levels of TSG101 and VPS28 in mammalian cells may not exist in Drosophila. Regardless of the nature of these discrepancies, we and others have never observed any palpable tumors in TSG101 conditional knockout mice where one or both copies of Tsg101 were deleted in a mosaic fashion in selected cell types with Cre recombinase. The same was true for a morpholino-mediated knockdown of TSG101 in zebrafish [In support of a tumor-suppressive role of TSG101, Moberg and coworkers  reportedethality . TSG101 ethality , and morethality ,69. The ebrafish . Hence, TSG101 gene in human cancers could not be confirmed. Moreover, alternative splice variants of TSG101, which have been subject to intense investigations in the late 1990s, can only be detected by nested RT-PCR, and they are also present in normal tissues [In conclusion, the various genetically engineered animal models, as well as observations from human cancer cell lines where TSG101 was knocked down with siRNAs, do not support a tumor- suppressive role of its encoded protein, either in a cell-autonomous manner or by means of juxtracrine or paracrine signaling. As stated in the opening paragraph of this review, initial reports about a loss of the  tissues ,6,8. As www.proteinatlas.org), most human cancers exhibit a moderate cytoplasmic and membranous immunoreactivity against TSG101, and the intensity of the staining might serve as a prognostic marker for selected cancer types such as hepatocellular and renal tumors. Using immunohistochemistry staining on a limited set of primary human breast cancers and normal breast tissues, our team observed that TSG101 is elevated in 50% of invasive breast cancers [TSG101 across all cases may not serve as a prognostic marker . In contrast, higher levels of TSG101 transcripts correlate significantly with reduced survival in luminal type A  and in luminal type B  breast cancers . This implies that any studies that are designed to examine the biological significance of TSG101 and associated molecular mechanisms for a given tumor type  should be conducted on cell lines and animal models that resemble the main characteristics of a particular cancer-intrinsic subtype .Investigations from various research teams including our own have shown that TSG101 is overexpressed rather than lost in a significant subset of malignancies, including breast, lung, thyroid, ovarian, and colon cancers ,71,72,73 cancers . HoweverSimilar to breast cancer, it has been reported that TSG101 is upregulated in human ovarian epithelial cells that express oncogenic HRAS or KRAS as well as in 70% of human ovarian carcinomas analyzed on tissue arrays ,72,74. WTSG101 as a transforming oncogene when an expression library of a highly metastatic lung adenocarcinoma cell line was introduced into mouse NIH3T3 cells. To our knowledge, the transforming ability of TSG101 overexpression has not been assessed in any other cell lines, in particular, those of human origin. As mentioned earlier, TSG101 protein levels are being maintained within a relatively narrow range of a given cell type, which may complicate the generation of suitable overexpression models. A minor or moderate expression of exogenous TSG101 results in a compensatory downregulation of the endogenous protein on the post-translational level [Whey Acidic Protein (Wap) promoter can be achieved in functionally differentiated epithelial cells of the lactating mammary gland [The elevated expression of TSG101 in cancer compared to normal tissues may support the notion that TSG101 plays a role at particular stages of cancer initiation or progression. Experimental evidence that TSG101 overexpression might contribute to neoplastic transformation was provided in the inaugural paper by Li and Cohen , who isoal level . Possiblal level . It is ary gland . TSG101 ry gland . In anotry gland  achievedWap gene promoter developed mammary tumors after a relatively long latency [Wap gene promotor is suitable to drive a tissue-specific expression of genes almost exclusively to the mammary epithelium, a high activation occurs predominantly in functionally differentiated alveolar cells during pregnancy and lactation, and the majority of these cells die during post-lactational mammary gland remodeling following the weaning of the offspring. As such, exogenous TSG101 is not persistently overexpressed in most epithelial cells throughout the lifetime of a female mouse. It is therefore not known whether a persistently high expression of TSG101 in diverse epithelial subtypes of the mammary gland may cause an earlier onset of mammary cancer. To our knowledge, the WAP-TSG101 transgenic mouse strain is currently the only published in vivo model that demonstrated that TSG101 can function as a transforming oncogene that causes a sporadic occurrence of preneoplastic lesions and mammary cancer. Given the long latencies of tumor formation, it is evident that secondary genetic or epigenetic changes must occur that drive the progression of bona fide cancers in this model.In contrast to the heart-specific overexpression model, a subset of transgenic females (approximately 20%) that express exogenous TSG101 under control of the  latency . At the TSG101 possesses characteristics of a housekeeping gene, and significant variations in the expression of its encoded protein between diverse cell types are, in part, a consequence of post-translational mechanisms. Given the stringent control of the TSG101 protein levels in a particular cell type, it is likely that the deregulated expression of TSG101 before or during neoplastic progression is primarily caused by an interference with its post-translational control mechanisms. The precise molecular processes that regulate the amount of TSG101 within cells are less well defined and have not been associated with any biologically relevant functions of TSG101 as an oncoprotein.As reviewed earlier, Arf. Evidently, TSG101 may not function as an oncoprotein under these physiological conditions. In contrast, TSG101 may only exert oncogenic activities in the MDM2/p53 circuit when it is upregulated and able to stabilize MDM2 without being ubiquitinated and degraded by this ubiquitin ligase.MDM2, LRSAM1/TAL, and MGRN1 are three E3 ubiquitin ligases that have been reported to control TSG101 levels in mammalian cells. Li and coworkers  have shoUsing a yeast two-hybrid system, Amit et al.  identifiMGRN1 locus has been reported to be amplified in osteosarcomas [Like the PTAP–PSAP double motif in TAL, the Mahogunin Ring Finger-1 (MGRN1) E3 ubiquitin ligase possesses a single, evolutionarily conserved PSAP amino acid sequence that can bind to the UEV domain of TSG101 . MGRN1 msarcomas  and, accAlthough TSG101 is suggested to function in a variety of cellular processes, the vast majority of published reports over the past 20 years have highlighted specific molecular mechanisms by which TSG101 may control the intracellular trafficking of cargo proteins such as RTKs and viral particles. Key observations from these studies are often highlighted as proof of the molecular underpinnings by which TSG101 may function as a growth suppressor. Then again, the validity of the proposed mechanisms is rarely, if at all, tested in genetically defined disease models or in primary human cancer tissues. At present, there is no conclusive evidence from experimental models or human tissues in support of the notion that deficiency in TSG101 or a functional loss of the protein could contribute to the genesis and progression of cancer. It has been established that TSG101 may function as a protooncogene in certain cell types. In the context of a whole organism, the biological significance of the proposed multifaceted roles of TSG101 in developmental processes and normal tissue homeostasis is still unclear. None of the proposed molecular roles sufficiently explains why cells that completely lack TSG101 enter a cell cycle arrest and die. Many of the critical functions in the intracellular trafficking of cargos as well as the binding of factors that control the post-translational modification and functionality of the TSG101 protein have been attributed to two structural features, i.e., PTAP amino acid motif-binding and ubiquitin-binding domains. Future studies using mouse models with CRISPR/Cas9-mediated targeted knock-in mutations might show whether these two domains or additional amino acid residues of TSG101 have a biologically significant role in normal development and disease.


The nucleus of a differentiated cell can be reprogrammed to a totipotent state by exposure to the cytoplasm of an enucleated oocyte, and the reconstructed nuclear transfer embryo can give rise to an entire organism. Somatic cell nuclear transfer (SCNT) has important implications in animal biotechnology and provides a unique model for studying epigenetic barriers to successful nuclear reprogramming and for testing novel concepts to overcome them. While initial strategies aimed at modulating the global DNA methylation level and states of various histone protein modifications, recent studies use evidence-based approaches to influence specific epigenetic mechanisms in a targeted manner. In this review, we describe—based on the growing number of reports published during recent decades—in detail where, when, and how manipulations of the epigenome of donor cells and reconstructed SCNT embryos can be performed to optimize the process of molecular reprogramming and the outcome of nuclear transfer cloning. The genome sequence includes the principal instructions to build, develop, and maintain an organism, but epigenetic mechanisms determine how this information is used in specific cell types during development and differentiation as well as in physiological and pathological processes. Whereas the DNA sequence is almost identical in all cells of an organism, epigenetic determinants—summarized as the “epigenome”—vary widely across different cell types and developmental stages and thereby modulate the cellular gene activity profile. Epigenetic mechanisms, i.e., DNA methylation, histone protein modifications, and effects associated with non-coding RNAs, are influenced by endogenous physiological and pathological stimuli, but also by exogenous environmental effects. Experimental studies in model organisms, but also epidemiological studies in humans indicate that epigenetic mechanism are—at least in part—heritable (reviewed in ).Nuclear transfer (NT) experiments were originally designed to answer one of the most intriguing questions of developmental biology: is embryonic development and differentiation and subsequent fetal growth associated with irreversible modifications in the resulting somatic cells? NT experiments were first performed in amphibians, where transfer of a differentiated cell into an enucleated oocyte resulted in the development of an adult animal, demonstrating that totipotency of somatic cells can be restored . In mammCloning offers great perspectives for agricultural and biomedical applications as well as for basic research. In agriculture, cloning provides great possibilities to rescue endangered species, to protect the genetic resources of commercially important species, and to accelerate the propagation of breeding livestock . In cHowever, practical application of SCNT technology is hindered by its low efficiency. Losses occur throughout preimplantation, postimplantation, and perinatal development. Surviving animals often exhibit various abnormalities, such as large offspring syndrome (LOS), enlarged placentas and organ defects , or obesity in mouse and abnormal teat numbers and cleft lips in pigs ,21,22,23Epigenetics refers to the modulation of gene expression through the physical and biochemical properties of chromatin without changing the DNA sequence. Epigenetic mechanisms include DNA methylation, post-translational modification of DNA-binding proteins and the integration of chromatin-binding proteins to maintain chromatin in either an active or repressed configuration . During Many attempts have been made to improve the development of SCNT embryos by manipulating their epigenome. In this review, we describe and discuss in detail where, when and how these manipulations may occur and show examples of global and specific manipulations of the epigenome of nuclear donor cells or SCNT embryos from the growing number of reports published during the last decades. Finally, we discuss the future perspectives of manipulating the epigenome for improving cloning efficiency.DNA methylation within gene promoter regions is associated with a repressive transcriptional state and plays a pivotal role in mammalian development. The most prominent methylation site is the 5th carbon of cytosine (5mC). DNA methylation is established and maintained by DNA methyltransferases (DNMTs). Demethylation occurs by ten-eleven translocation (TET) protein-mediated oxidation .Histone proteins are key players in nucleosome formation and DNA packaging. They are subject to many post-translational modifications affecting mainly the N-terminal tails of core histones, including acetylation (lysine), methylation (lysine and arginine), phosphorylation (serine and threonine), ADP ribosylation, sumoylation (lysine), ubiquitylation (lysine), butyrylation, citrullination, crotonylation, formylation, proline isomerization, and propionylation .Acetylation of histone tails loosens the histone-DNA interactions and enables gene expression, whereas deacetylation of histone tails strengthens the interactions and is generally associated with gene repression. Histone acetylation is regulated by the balanced action of histone acetyltransferases (HATs) and histone deacetylases (HDACs) .The effect of histone methylation depends on both the modified residue and the extent of methylation. Histone lysine (K) methylation can exist in one of three states: mono-, di- or tri-methylation. Di- and tri-methylations at H3K4, H3K36, and H3K79 are typically gene-activating, with H3K4 tri-methylation (H3K4me3) marking promoters, and H3K36 and H3K79 methylations occurring primarily over gene bodies. Mono-methylation of H3K4 is an activating mark unique to enhancers. H3K9 and H3K27 methylations are generally gene-repressive, but serve unique functions. H3K27me3 is considered easily reversible and marks dynamically regulated genes, rendering it especially important in development, when genes need to be switched on and off in a highly dynamic fashion depending on developmental signals. H3K9me3 is characteristic of heterochromatin, whereas H3K9me2 is found more commonly at silent or lowly expressed genes in euchromatin. Histone K methylation is regulated by histone methyltransferases and histone demethylases (reviewed in ).Embryos, fetuses and offspring generated by SCNT may suffer from a variety of epigenetic abnormalities, which are attributed to insufficient or aberrant epigenetic reprogramming of the somatic donor nucleus. These epigenetic barriers to SCNT cloning are summarized in Oct4  an anXist lIt is reasonable to speculate that simultaneous manipulation of several key players in the process of nuclear remodeling and reprogramming using a combination of approaches might provide a better option for removing multiple epigenetic barriers and for improving the developmental competence of cloned embryos. The combined manipulation or treatment can be performed by various means, including treatment of donor cells and/or cloned embryos with different compounds affecting more than one epigenetic modification simultaneously to obtain a synergistic effect on the development of cloned embryos .In rabbits, treatment of cloned embryos with the combination of two HDACis, TSA and Scriptaid, was more beneficial than the use of a single HDACi for improving cloning efficiency .Treatment of donor cells with 5-aza-dC and TSA improved cloning efficiency in pigs , but notOCT4, SOX2, H19, IGF2, and DNMT1 genes in treated cloned embryos were more similar to IVF embryos than without treatment.Combination of DNMTi and HDACi, RG108 and Scriptaid , ZebularXist KO donor cells coupled with Kdm4d mRNA injection resulted in very high efficiencies (up to more than 20%) of mouse cloning with different types of donor cells. However, many of the cloned embryos still suffered from postimplantation developmental arrest and surviving embryos had an abnormally large placenta [placenta .A study of Mizutani, et al.  presentsOne of the most efficient mouse cloning protocols used the combination of TSA and antioxidant vitamin C in culture medium with deionized bovine serum albumin. This resulted in activation of reprogramming-resistant genes, demethylation of H3K9me3/2/1 and in 15% of cloned embryos developing to term, indicating that this treatment can overcome major epigenetic barriers to successful nuclear reprogramming .Dnmt3a and Dnmt3b with overexpression of histone demethylases by injecting enucleated oocytes with Kdm4b and Kdm5b mRNAs led to stronger reductions in inappropriate DNA methylation and synergistic enhancement of full-term development of cloned embryos [Aberrant DNA methylation as a critical epigenetic barrier to mouse cloned embryo development can be rescued by inactivation of DNMTs, resulting in improved cloning efficiency, which is further enhanced by simultaneous removal of additional epigenetic barriers. Combining inhibition of DNMTs by siRNAs for  embryos .Macaca fascicularis) have been successfully cloned by NT using fetal fibroblasts and injection of H3K9me3 demethylase and KDM4D mRNA together with TSA treatment at the one-cell stage [Cynomolgus monkeys  may silence as many genes as they activate, likely due to direct and indirect effects on other transcriptional regulators and cell signaling pathways ,153.A second group of tools involves the use of traditional genetic knockout/knock-in, transgenic, viral, and/or RNA interference (RNAi) technologies to manipulate specific epigenetic enzymes. While these approaches typically allow improved substrate specificity, including isoforms or subclasses and even limited cellular specificity, they still operate universally within a given cell and therefore lack the ability to modulate the epigenome in specific ways . Thus, tNew tools and strategies to promote gene-specific epigenetic modifications, referred to as epigenome editing, are now available and open the perspective of precisely targeting epigenetic deregulations. This allows the development of new hypotheses regarding epigenetic function and drastically reduces undesired side effects. Epigenome editing would perfectly complement recent developments in genomic, proteomic, and metabolomic profiling of embryos to link their viability and reproductive potential to specific signatures. Application of spectroscopy and bioinformatics for noninvasive metabolomic profiling of embryo culture media revealed a unique footprint for embryos with high reproductive potential compared to those failing to implant. Future efforts should focus on associating specific metabolomic or proteomic signatures with normal patterns of epigenetic marks in in vitro culture models, which will greatly impact our ability to identify and generate embryos with high reproductive potential (reviewed in ).As a concluding remark, NT cloning will have enormous benefits for basic research, preservation, and multiplication of desired genotypes, and for the generation of tailored animal models for human diseases.


Unconjugated monoclonal antibodies (mAb) have revolutionized the treatment of B-cell malignancies. These targeted drugs can activate innate immune cytotoxicity for therapeutic benefit. mAb activation of the complement cascade results in complement-dependent cytotoxicity (CDC) and complement receptor-mediated antibody-dependent cellular phagocytosis (cADCP). Clinical and laboratory studies have showed that CDC is therapeutically important. In contrast, the biological role and clinical effects of cADCP are less well understood. This review summarizes the available data on the role of complement activation in the treatment of mature B-cell malignancies and proposes future research directions that could be useful in optimizing the efficacy of this important class of drugs. Unconjugated monoclonal antibodies (mAb) have an important clinical role in the management of mature B-cell malignancies ,2,3,4,5.We will review the clinical data on the role of complement activation by mAb in the treatment of mature B-cell lymphoid malignancies and our current understanding of the role of activation of complement in killing malignant B lymphocytes.2/human IgG1 Fc) anti-CD20 mAb, was the culmination of a multi-decade effort to utilize mAbs to treat malignancies of the immune system and autoimmune disease [The development of rituximab, the prototype unconjugated chimeric  was selected for improved CD20 binding properties (decreased off rate) and proximity of binding to the cell membrane, both of which increased complement activation ,29. In cAlemtuzumab , a humanized rat anti-CD52 mAb utilizing wild-type human IgG1 Fc , is highDaratumomab  is a fully human IgG1 mAb targeting CD38 that could have a role in treatment of other CD38-positive B-cell malignancies . In contmAbs are now an integral component of targeted therapy of B-cell malignancies based largely on very promising data from clinical trials. However, a better understanding of the mechanisms of action of these valuable drugs and the B-cell characteristics that determine sensitivity and resistance to mAb-mediated cytotoxicity is needed to continue to improve their efficacy. This knowledge should then allow for rational development of combination therapy with other drugs. These data will be critical for designing and testing regimens that can exploit orthogonal cytotoxic mechanisms so as to eliminate resistant subclones of malignant B cells and improve patient outcomes.Although mAb-containing treatment regimens are now standard of care for many diseases, their mechanisms of action and how pathological B cells evade their cytotoxic effects are not well defined. The mAb described above have limited or no direct cytotoxic effects and their therapeutic effects result primarily from activation of innate immune cytotoxicity ,44,45,46Complement activation results in generation of MAC that can cause target cell lysis or necrosis. This has been demonstrated in vitro for mAb with wild-type IgG1 constant regions including rituximab, ofatumumab, alemtuzumab and daratumumab ,28,39,45Initial in vitro studies of rituximab-mediated cellular cytotoxicity demonstrated circulating human mononuclear cell-mediated ADCC of rituximab-opsonized B cells . SubsequThe recognition that the major cytotoxic mechanism of anti-CD20 mAb is the innate immune system has profound consequences. Foremost among these is the likelihood that the finite cytotoxic capacity of the innate immune system limits the therapeutic effect of each dose of mAb and that subsequent “exhaustion” could have deleterious effects on therapy. We recently reported that hypophagia, the autoregulatory macrophage feedback mechanism that limits phagocytic capacity , could iThe therapeutic effect of mAb activation of complement is determined by mAb pharmacokinetics and pharmacodynamics, availability of complement, target cell antigen expression and intrinsic target lymphocyte susceptibility to complement-mediated cytotoxicity.Human Fc wild-type IgG mAbs are cleared from the circulation by the liver and spleen in a manner similar to the mechanism of clearance of endogenous IgG and in addition by ligation to target cells . In patimAb currently used to treat B-cell malignancies vary widely in their ability to activate complement. mAb that bind ligand epitopes closer to the cell membrane  are more likely to mediate membrane capture of activated complement components. Although ofatumumab has similar binding affinity for CD20 compared to rituximab, it has a slower off rate, resulting in more durable binding and binding stoichiometry that causes closer association of multiple mAb Fc regions which further increases its ability to activate complement ,55,56,57mAb ability to activate complement is determined by target cell membrane ligand density and the ability to generate the hexameric Fc configuration that efficiently activates C1q ,58. For In summary, characteristics of mAb that contribute to efficient activation of complement are high antigen affinity and low off rate, binding to antigen epitopes close to the cell membrane, antigen binding stoichiometry that facilitates Fc association, and lipid rafts.Patients with some B-cell malignancies  can have defective complement function which could decrease mAb-induced cytotoxicity . Serum cTherapy with fresh frozen plasma (FFP) has been reported to increase activity of rituximab in CLL patients but this was not formally demonstrated to be mediated by increased complement availability . FFP conCLL cells have low expression of the CRP CD46 and high expression of CD55 and CD59 . InhibitCDC requires higher levels of mAb ligation of cell membrane antigens than ADCP ,50,64. CThe terminal event of activation of the classical complement pathway by mAb is generation of MACs that insert in the cell membrane and cause cell necrosis or lysis. In vitro mAbs that are highly efficient at activating complement such as alemtuzumab are cytotoxic to over 90% of CLL cells . CytotoxAs already noted, mAb ligation on the target cell surface can also lead to deposition of complement fragments that trigger ADCP upon binding to CRs on phagocytes. While cADCP can mediate rapid clearance of mAb-ligated tumor cells in vitro, the relative contribution of cADCP to tumor cell clearance in vivo is likely a very important but still poorly understood killing mechanism of mAbs.Mβ2 or CD11b, is the best-studied cADCP receptor. The signaling events that trigger phagocytosis downstream of CR3 are distinct from those of FcγR signaling. Activation of Rho family small GTPases  is critical for driving the cytoskeletal reorganization that leads to target cell internalization [Activation of complement by mAb on target cells leads to the covalent binding of C3 activation fragments  that effectively opsonize the target cell surface. These opsonins can be recognized by CR1, CR3, and CR4 on phagocytes to trigger a form of engulfment that is biochemically distinct from FcγR-mediated ADCP . CR3 andlization . Unlike lization . Active lization ,77. Withlization . To datePart of understanding the relative contributions of both FcγR- and CR-activated ADCP pathways in therapeutic mAb-mediated cell clearance involves understanding the extent of crosstalk between these two pathways . Early iWhile in vitro studies have been useful in establishing the ability of different mAbs to activate complement and trigger cADCP by macrophages, our understanding of the role of cADCP in vivo is much less developed. Using C1q-deficient mice, Di Gaetano et al. showed that activation of complement was essential for anti-CD20 mAbs to control tumor burden in a lymphoma model . IntriguThe development of mAbs targeting B lymphocytes has significantly improved therapy for patients with B-cell malignancies. Despite the impressive empiric clinical data supporting the use of these mAbs both as monotherapy and in combination regimens, there is limited data about how mAbs cause malignant lymphocyte cytotoxicity and how a subpopulation of these cells resist this cytotoxicity. Available data support complement activation as an important innate immune cytotoxic mechanism for rituximab, ofatumumab, daratumumab, and alemtuzumab. We propose that additional studies of the roles of CDC and cADCP in the clinical efficacy of these mAbs are required to provide the data needed to optimize the use of these drugs; these studies should allow for the design of new drugs to enhance activity and overcome resistance. These investigations could focus on defining the cytotoxic capacity of the innate immune system in order to develop clinical regimens that prevent immune “exhaustion”. It will be important to examine how nucleated cells can survive MAC-mediated calcium fluxes, and to determine the role of cADCP which has only limited overlap (and thus cross resistance) with CDC and ADCP. Current mAb-based therapeutic developments include engineered Fc with enhanced ability to form hexameric complement-activating structures , and bis


Morus has long been appreciated as an antiphlogistic, diuretic and expectorant drug in Chinese herbal medicine, albeit with barely known targets and mechanisms of action. In the 1970s, the development of analytic chemistry allowed for the discovery of morusin as one of 7 different isoprene flavonoid derivatives in the root bark of Morus. However, the remarkable antioxidant capacity of morusin with the unexpected potential for health benefits over the other flavonoid derivatives has recently sparked scientific interest in the biochemical identification of target proteins and signaling pathways and further clinical relevance. In this review, we discuss recent advances in the understanding of the functional roles of morusin in multiple biological processes such as inflammation, apoptosis, metabolism and autophagy. We also highlight recent in vivo and in vitro evidence on the clinical potential of morusin treatment for multiple human pathologies including inflammatory diseases, neurological disorders, diabetes, cancer and the underlying mechanisms.The root bark of  Morus) as an antiphlogistic, diuretic and expectorant drug has long been acknowledged in Chinese herbal medicine [Morus including Morus alba L. and Morus nigra L. Such discoveries, taken together with the anti-inflammatory properties of flavonoid species, significantly provoked scientific attention to the potential application of Morus-derived flavonoids to a myriad of human pathologies beyond its known benefits, leading to subsequent biochemical studies from multiple research groups in which the chemical and bioactive properties of the phytochemicals were tested.The therapeutic relevance of the root bark of the mulberry tree  a prenyl unit at position 3, (2) hydroxy groups at 5, 2′′ and 4′ and (3) a 2,2-dimethyl pyran group across positions 7 and 8 , matrix metalloproteinases (MMP-9 and MMP-2), cytochrome P450 and HIV reverse transcriptase (further discussed in later sections) ,15,16,17We briefly mentioned the chemical properties of morusin here, since this review mainly focuses on the biological aspects of morusin treatment with various clinical relevance. More detailed physiochemical properties of flavonoids including morusin and their purification processes are well described elsewhere ,6,7.Multiple physiological processes have been proposed as effectors of morusin treatment in various biochemical studies . In this4 in A23187-stimulated MC/9 mast cells [mycoplasma pneumonia [Many studies have confirmed the anti-inflammatory capacity of morusin in different in vivo and in vitro contexts—(1) Morusin treatment inhibits secretion of cytokines such as CCL5 and CCL17 in TNFα- and IFN-γ-stimulated keratinocytes and also inhibits the release of histamine and LTCst cells . (2) Morst cells . (3) Morst cells . (4) Morst cells . (5) Morst cells . (6) Morneumonia .This explosion of observations on the anti-inflammatory effects of morusin have raised questions of the molecular and biochemical targets of morusin and the underlying mechanisms. In this context, the antioxidant capacity of morusin has been linked to the suppression of iNOS induction, thereby reducing nitric oxide formation ,30,31. TAlthough cytotoxicity upon morusin treatment has been observed across multiple cancer cell lines, biochemical characterization of the underlying mechanisms and clinical potential in vivo are still active areas of research. In particular, multiple recent studies have performed a thorough assessment of the biochemical targets of morusin, allowing for the identification of apoptosis as a critical tumor-killing effect of morusin.Apoptosis is a type of tightly regulated programmed cell death, characterized by cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation . ApoptosIn addition, several studies have suggested the possibility of STAT3 as a component of the mechanisms underlying the pro-apoptotic effects of morusin ,38,47,48Turning off signaling pathways implicated in cell survival and proliferation has been functionally associated with the activation of apoptosis . In thisFinally, some evidence has suggested that morusin treatment also results in the activation of extrinsic apoptotic pathways, in which morusin strongly increased expression of the death receptor DR5 at the transcriptional level and conferred sensitization of glioblastoma to TRAIL signaling ,38. HoweMetabolism plays key roles in energy homeostasis and signal transduction, which are fundamental aspects of organisms. One of the unique aspects of metabolism is the remarkable metabolic flexibility in response to various stimuli ; metabolMorus alba root bark extract including morusin reduces lipid peroxidation, thereby leading to hypoglycemic effects in vivo in a streptozotocin-induced mouse model of type I diabetes. Such beneficial effects may be associated with decreasing oxidative stress and preservation of pancreatic β-cell integrity by reducing cell death [Morusin treatment was shown to exert beneficial effects on systemic and cellular metabolism. Those effects have also been mostly associated with the antioxidant capacity of morusin. Multiple studies have shown that morusin treatment reduces reactive oxygen species in metabolically relevant organs, in which redox homeostasis is tightly associated with general metabolic fitness including increased mitochondrial performance and the amelioration of dysregulated fuel metabolism . For exall death ,60.per se [It has recently been shown that morusin also serves as an allosteric inhibitor of metabolic enzymes including pancreatic lipase, UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) ,61. Suchper se ,20, althde novo lipogenesis inhibitor as well as a lipolysis stimulator [Morusin has also been highlighted as a potent imulator . The stuimulator . A similimulator . The detAutophagy is a physiological process through which unnecessary and dysfunctional cellular components are removed or recycled . VariousCells have evolved multiple physiological strategies to cope with diverse environmental and intracellular stresses. Among these is stress granule (SG) formation, during which macromolecular aggregates of mRNA and proteins are assembled, leading to the attenuation of translation and activation of various physiological pathways for DNA damage repair and cell survival . Since sAlthough our study only highlighted the potential connection between SG formation and the intrinsic apoptotic pathway, it may also be of interest to study the biochemical mechanism(s) and the pathological relevance of interplay with other pathways including the extrinsic apoptotic pathways also activated by morusin, as well as inflammatory signaling, given the involvement of PKR in this mechanism.Exploding number of causality studies linking specific cellular processes to human pathologies, together with morusin implication in the processes, warranted assessment of the clinical potential of morusin in various disease settings in vivo and in vitro. Here, we discuss recent advances in the understanding of the potential application of morusin in treating common human diseases.The anti-inflammatory capacity of morusin has been tested in various models of inflammatory diseases, which was initiated with several in vitro studies on morusin-mediated inhibition of iNOS, cyclooxygenases and lipoxygenases ,41—(1) Lper se; Pang et al. and We et al., demonstrated that morusin has the remarkable capacity to target Staphylococcus aureus and clinical methicillin-resistant Staphylococcus aureus (MRSA) in vitro and in vivo [Numerous microbes and viruses take advantage of host cellular processes and machineries to maximize their infection capacities. One such strategy is to provoke cellular and systemic inflammatory responses. Morusin has been administered to a few models of infectious diseases, such as an in vivo mouse model of mycoplasma pneumonia infection, in which morusin treatment efficiently suppressed mycoplasma pneumonia via the inhibition of Wnt/β-catenin and NF-κB signaling . Morusin in vivo ,76. The  in vivo . Mechani in vivo  and SARS in vivo .Morus alba has been used as a neuroprotective herb. The neuroprotective functions of morusin and the implications for disease have recently been appreciated at the cellular and molecular levels. For example, morusin treatment protects neuroblastoma SH-SY5Y cells from nitric oxide-induced cell death [3), showing that morusin ameliorates the impaired memory and learning capacity by decreasing the AlCl3-induced rise in brain acetylcholinesterase (AChE) activity and brain oxidative stress levels. Although in vivo studies of the effects of morusin on neurological disorders are very limited, morusin may exert beneficial functions against neurodegenerative diseases including Alzheimer’s disease, given that (1) morusin shows inhibitory activity against AChE, butyrylcholinesterase (BChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), which play important roles in the prevention and treatment of Alzheimer’s disease [In Chinese medicine, ll death . This inll death . In the  disease ,81 and  pancreatic β-cell dysfunction followed by reduced serum levels of insulin (Type I diabetes), and/or (2) reduced insulin sensitivity of systemic cells and relatively reduced insulin secretion from pancreatic β-cells (Type II diabetes) . Althougob/ob, db/db mouse or HFD-fed mice), morusin may also exert anti-diabetes effects in this context, considering that morusin potentially regulates systemic ROS levels, lipogenesis, lipolysis, C/EBPβ and PPARγ signaling, arachidonic acid metabolism and ER-resident proteins implicated in ER stress [Although no studies have yet tested the effects of morusin on the pathogenesis of type II diabetes using relevant in vivo models  cancer stem cells (GSCs) were targeted by morusin treatment in vivo and in vitro . This obMorusin may also employ other various cellular processes independent of NF-κB for targeting cancers—(1) An in vitro study of gastric cancer showed that morusin governs the expression of c-Myc and multiple downstream genes including CDKs and cyclins implicated in cell proliferation, thereby exerting antitumor capacity . (2) TheA cancer cell continuously undergoes dynamic remodeling of numerous cellular processes, which confer various benefits such as drug resistance. In this context, the two recent studies from our lab have highlighted the biochemical mechanisms by which cancer cells gain resistance against the cytotoxicity of morusin ,74. As dMorus in Chinese herbal medicine have been expanded to the potential application of individual phytochemicals extracted from such arcane plants to treat multiple chronic diseases common in modern society. Morusin has been appreciated as a phytochemical with the unique physiochemical properties of isoprene flavonoids, allowing for versatile salutary effects including antioxidant and tumor killing activities. Morusin reduces ROS formation and inflammation at non-cytotoxic concentrations (lower than 10 μM), while it induces ROS formation and cytotoxicity against cancer cells at high concentrations (higher than 20 μM). A growing body of evidence, described here, highlighted the biological properties of morusin in multiple in vitro and in vivo pathophysiological settings and proposed clinical potential. The as yet unraveled clinical potential of morusin is waiting for thorough scientific assessment of the effects of morusin on human pathology with an accompanying understanding of the underlying biochemical mechanisms and effective platforms, which may allow for bona fide application of morusin in patients with various pathologies in the near future.The well-acknowledged functions of the root bark of


Foot-and-mouth disease (FMD) is a highly infectious viral disease, recognised to affect animals in the order Artiodactyla. The disease is rarely fatal in adult animals, however high mortality is associated with neonatal and juvenile infection.Five puppies died after being fed lamb carcases, the lambs having died during an outbreak of FMD in Iran. Following a post-mortem examination, cardiac tissue from one of the dead puppies was subjected to virus isolation, antigen ELISA, real-time RT-PCR, sequencing and confocal microscopy to assess the presence and characteristics of any FMD virus. The virological and microscopic examination of the cardiac tissue provided evidence of FMD virus replication in the canine heart.The data generated in this study demonstrate for the first time that FMD virus can internalise and replicate in dogs and may represent an epidemiologically significant event in FMD transmission, highlighting the dangers of feeding diseased animal carcases to other species. The reporting of this finding may also focus attention on similar disease presentations in dogs in FMD endemic countries allowing a better understanding of the prevalence of such events. Syncerus caffer caffer). Infection of non-artiodactyla species has been reported in natural and/or experimental settings such as in hedgehogs, rats, cats, kangaroos and dogs [Foot-and-mouth disease (FMD) is an important transboundary animal disease, the aetiological agent of which is a picornavirus FMD virus (FMDV). The most commonly recognised signs of infection are vesicles on the feet and in the mouth, often associated with pyrexia. Acute myocarditis and death may also occur during infection of young susceptible animals. FMD directly impacts the livelihoods of farming communities in sub-Saharan Africa and large parts of Asia via reduced productivity and loss of draft-power, as well as imposing barriers on access to lucrative export markets for animals and animal products . In FMD-and dogs , 4, althand dogs , 6, howeand dogs , Germanyand dogs  demonstrand dogs . Due to and dogs .Canis lupus familiaris isolate CAN001). RNA was extracted from the submitted tissues, and FMDV RNA was detected by real-time RT-PCR targeting the 5′ untranslated and 3D polymerase regions [In February-March 2016, cases of FMD due to serotype O were reported in Iranian provinces to the south and east of Tehran resulting in widespread disease in livestock, including reports of FMDV-associated mortality among young animals. In February, the Iran Veterinary Organisation (IVO) were informed that 2 puppies in Esfahan Province, and 3 in Kurdistan Province had become lethargic and weak 2 days after being fed carcases of lambs which themselves had died during an outbreak of FMD. During the following 1–2 days all 5 puppies died. Subsequent post mortem examination of these puppies by the IVO revealed lesions in heart tissue suggestive of FMD infection, and cardiac tissue from one case was collected and found to be positive for FMDV RNA via RT-PCR. This tissue was included in a batch of clinical samples collected from the region and submitted to the FAO World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD), UK, for FMDV confirmation and characterisation. The canine origin of this sample was confirmed by using PCR amplification and sequencing of a fragment of the cytochrome c oxidase subunit 1 (COX1) gene  which de regions . The Ct  regions  and the QOM − 15 lineage closely related to FMDVs circulating concurrently in the region . The 3A region of the FMDV genome has previously been associated with host specificity, with deletions present in the 3A region of isolates of swine origin found to be attenuated in cattle. Interestingly, a reverse mutation in the same position (I248T) has previously been observed in an FMDV isolate experimentally adapted to the guinea pig which may indicate importance in this site for species specific tropism [Complete FMDV genomes (8189 nucleotides) obtained from both the canine heart sample and isolate IRN/9/2016 (GenBank accession numbers MT944980 and MT944981 respectively) were determined using next-generation sequencing . PhylogeThe presence of FMDV proteins and microscopic cardiac damage was imaged using confocal microscopic examination. The cardiac tissue, which had been stored in a 50:50 v/v PBS:glycerol solution at -20 °C, was placed in PBS at 4 °C overnight to remove the glycerol and the following day was frozen in a cryomould containing optimal cutting temperature (OCT) compound. Tissue sections were immunolabelled using a mouse IgG2a monoclonal antibody (mAb), 2C2, targeting the FMDV non-structural protein 3A and a mouse IgG1 mAb, IB11, previously described as targeting the FMDV capsid . PrimaryIn summary, the data presented here support the conclusion that canine fatalities occurred as a result of FMDV infection in puppies which had fed upon infected carcasses in an outbreak of serotype O FMDV in Iran. This represents the first description of natural infection of dogs by FMDV. Specifically, evidence is provided to show that FMDV can infect and damage the cardiac myocytes and was the cause of death in at least one of these animals. The wider epidemiological importance of this finding is unknown. However, wider awareness of this case report should ensure that a provenance of FMDV infection is given greater consideration in scenarios entailing canine deaths associated with FMD outbreaks in endemic countries. Importantly there is a clear risk of feeding young dogs the carcases of animals which have been infected with FMDV, and therefore this practice should be avoided.


A misdirected or imbalanced local immune composition is often one of the reasons for unsuccessful regeneration resulting in scarring or fibrosis. Successful healing requires a balanced initiation and a timely down-regulation of the inflammation for the re-establishment of a biologically and mechanically homeostasis. While biomaterial-based approaches to control local immune responses are emerging as potential new treatment options, the extent to which biophysical material properties themselves play a role in modulating a local immune niche response has so far been considered only occasionally. The communication loop between extracellular matrix, non-hematopoietic cells, and immune cells seems to be specifically sensitive to mechanical cues and appears to play a role in the initiation and promotion of a local inflammatory setting. In this review, we focus on the crosstalk between ECM and its mechanical triggers and how they impact immune cells and non-hematopoietic cells and their crosstalk during tissue regeneration. We realized that especially mechanosensitive receptors such as TRPV4 and PIEZO1 and the mechanosensitive transcription factor YAP/TAZ are essential to regeneration in various organ settings. This indicates novel opportunities for therapeutic approaches to improve tissue regeneration, based on the immune-mechanical principles found in bone but also lung, heart, and skin. The immune response is essential for successful healing after an injury. Even under sterile conditions, the immune system reacts to injury with a pro-inflammatory reaction as a first step elasticity, pore size, patterns, shape, and geometrical organization. Interestingly, matching for example the stiffness of the biomaterial to the surrounding tissue could already minimize the foreign body response  niche, which gives rise to the myeloid and lymphoid compartment of the immune system  abundant reticular (CAR) cells, were shown to contribute to the HSC niche by expressing CXCL12, a chemokine with important functions in the homing and retention of hematopoietic stem/progenitor cells in the bone marrow   and cathepsin K, both playing a role in the degradation and inactivation of CXCL12  cells in the bone marrow have been found to be associated with immune system pathologies.Conversely, both lymphoid and myeloid immune cells influence the behavior of bone cells in many bone pathologies by altering the balance of bone formation and resorption  analog has been shown to improve fracture healing by downregulating pro-inflammatory cytokine release from CD8+ and CD4+ T cells while promoting the anti-inflammatory response via an increased numbers of M2 macrophages in the fracture gap  cell/Treg ratio  most likely leads to direct bone formation, a moderately fixed fracture may heal by endochondral ossification and an unstable fixed fracture gap almost suppresses the bone healing cascade , or its more severe form, acute respiratory distress syndrome (ARDS) ; the enzyme that produces LMW-HA . HA exists as a more immunological inert high molecular weight form (HMW-LA), and as pro-inflammatory low molecular weight hyaluronic acid (LMW-HA) form. Mechanical ventilation of rat lungs with high tidal volumes but not low tidal volumes led to an accumulation of LMW-HA in lungs / WW domain-containing transcription regulator 1  pathway integrates mechanical stimuli, soluble signals, and metabolic information to regulate cell proliferation, cell plasticity and stemness . When activated, TRPV4 causes a Ca2+ ion channel involved in mechanically triggered inflammation. PIEZO1 is highly expressed on macrophages and other immune cells enabling them to sense omnidirectional pressure  -1 and MCP-3, and triggers typical inflammatory pathways  the concentrations of TNF-α and IL-1β were significantly elevated after 3 days and increased levels of the chemokines MCP-1, MIP-2, and TGF-β were found after 7 days  connects mechanical forces with skin fibrosis via inflammatory signals. In fibroblasts, FAK became activated after a skin injury and its activity can be potentiated by mechanical stretching  on the cell surface reduced fibronectin mediated signaling in osteoblast and might explain the loss of bone despite higher fibronectin concentrations (Yang et al., Forces acting on the fracture hematoma/callus  likely iAnother ECM component that is found in bone and is involved in the regulation of the immune system is agrin (Bezakova and Ruegg, Moreover, the mechanosensitive ion channels PIEZO1 and TRPV4 might also play a role in bone healing. Similar to the findings in lung tissue, IL-8 also attracts PMNs in bone tissue, which have been shown to be a critical player in bone fracture healing (Kovtun et al., in vivo (Geißler et al., In addition, signaling via TRPV4 in lung macrophages has been found to increase ROS levels (Hamanaka et al., During bone healing, the local mechanical niche changes a lot. The fracture hematoma is soft and relatively dense. While in the subsequent healing phases the stiffness increases steadily. This might have different implications on the cellular behavior of non-hematopoietic and immune cells. On soft and confined substrates, cells tend to show spherical morphology and cytoplasmic localization of YAP/TAZ, while more open and stiff substrates allows for cytoskeletal re-arrangements, cell spreading and nuclear localization of YAP/TAZ (Panciera et al., These examples encourage that also during bone regeneration the immune response is modulated by the ECM composition and the mechanical niche. In particular, the ECM components fibronectin and agrin could also have immunomodulatory functions in bone, and TRPV4 and PIEZO1 on macrophages could be important mechano-transducers and thereby influence the outcome of bone healing.The understanding of regeneration has come a long way. Today, both the adaptive and the innate immune system are now considered to play a central role in healing and regeneration (Medzhitov, In most situations, unphysiological mechanical stimuli or mechanical overstimulation resulted in the release of common pro-inflammatory mediators. Specific ECM components are actively involved in immune regulation, either by acting as DAMPs [LMW-HA (Jiang et al., In all the tissues described, non-hematopoietic cells often respond to abnormal mechanical stimuli by secreting chemoattractants and cytokines that lure immune cells out from the vasculature and in most cases the secreted cytokines create a pro-inflammatory niche. Within the cell, calcium mediated signaling pathways, FAK and YAP/TAZ are often involved in the translation of the perceived mechanical environment into a cellular response. It is already known that YAP/TAZ plays an important role in the regulation of cell proliferation, and a targeted manipulation of this signaling pathway could help to increase the number of cells after injury (Moya and Halder, Immune cells most often perceived the mechanical environment via mechanosensitive ion channels such as PIEZO1 (Solis et al., in vivo micro-niche also depends on the mechanical environment. In vivo, the biomaterial is not mechanically isolated, but connected to the mechanical environment. It is conceivable that when cells are encapsulated in the biomaterial, not only the intrinsic mechanical properties of the biomaterial determine the cell behavior, but also how much of the external mechanical stimuli are transmitted through the biomaterial. Biomaterials may not only reduce the diffusion of unwanted DAMPs or pro-inflammatory mediators but may also protect cells from pathological mechanical over-stimulation. These protected cells could then help to downregulate a too pronounced pro-inflammatory reaction.The above findings suggest that the success of using biomaterials for cell delivery or as a cell modulating The examples described here, are possibly just the tip of the iceberg. It seems likely that the described concepts are not only found in the lung, heart, and skin, but also in various tissues throughout the body. Since immune cells are the first cells that interact with a biomaterial and often determine the amount of a foreign body response (Anderson et al., RK compiled the literature, prepared, and wrote the manuscript. CB, SV, CT, KS-B, and GD outlined the manuscript and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Notably, vessel enlargement can occur as fast as in few hours in response to changes in flow and pressure. The high plasticity and responsiveness of blood vessels rely on endothelial cells. Changes within the bloodstream, such as increasing shear stress in a narrowing vessel or lowering blood flow in redundant vessels, are sensed by endothelial cells and activate downstream signaling cascades, promoting behavioral changes in the involved cells. This way, endothelial cells can reorganize themselves to restore normal circulation levels within the vessel. However, the dysregulation of such processes can entail severe pathological circumstances with disturbances affecting diverse organs, such as human hereditary telangiectasias. There are different pathways through which endothelial cells react to promote vessel enlargement and mechanisms may differ depending on whether remodeling occurs in the adult or in developmental models. Understanding the molecular mechanisms involved in the fast-adapting processes governing vessel enlargement can open the door to a new set of therapeutical approaches to be applied in occlusive vascular diseases. Therefore, we have outlined here the latest advances in the study of vessel enlargement in physiology and pathology, with a special insight in the pathways involved in its regulation.From developmental stages until adulthood, the circulatory system remodels in response to changes in blood flow in order to maintain vascular homeostasis. Remodeling processes can be driven by  During development, vascular beds often form as honeycombed shaped capillary plexus that then become perfused and remodel to form a hierarchical vessel structure. One of the most visible changes that occur in this process is the enlargement of both arteries and veins. Vessel enlargement can occur remarkably quickly. In the embryo, this occurs within a single day after the onset of blood flow. As such, vessel enlargement has therapeutic potential in occlusive vascular diseases that slower processes, such as angiogenesis, lack. However, so far, most research on therapeutic strategies has been focused on angiogenesis and significantly less advancement has been made to exploit vessel enlargement as a potential therapy in ischemic diseases. In strokes and heart attacks, for example, local collateral vessels immediately dilate to restore blood flow, but angiogenesis is a latter process in the body’s attempt to restore proper perfusion . Vessel Though occlusive vascular diseases are important pathologies where inducing vessel enlargement could provide therapeutic benefits, when vessels enlarge ectopically, there can be devastating negative consequences. Arterial-venous malformations (AVMs) are a form of anomalous vessel enlargement where enlarged shunts bypass the capillary bed and directly connect arteries and veins. Because the venous network is then exposed to arterial blood pressure, AVMs are prone to hemorrhage, which can be fatal depending on the organ where they occur. For instance, cerebral AVMs account for 50% of childhood strokes . Though Despite the importance of vessel enlargement, we are just beginning to understand how diameter changes occur. It was initially assumed that vessels enlarged by proliferation and while this may be true in some vascular beds, proliferation is a slow process and therefore could not restore blood flow on the timescales needed after stroke or heart attack. Vessel dilation, followed by outward remodeling can, at least partially, mitigate the slow process of cell proliferation. More recently, however, migration and capillary fusion have been proposed as methods by which a vascular network can increase the diameter of vessels. It is important to note, however, that it is unlikely that any of these processes happen in isolation. As such, vascular networks likely use a combination of means to increase vessel diameters. We therefore review here the processes and pathways by which vessels enlarge, and highlight differences and similarities between vascular development, vasculopathy and enlargement in adult vascular beds.Four mechanisms of vessel enlargement have been identified, though the prevalence or relative importance of each of these mechanisms is still not clear. First, endothelial cell migration can lead to the accumulation of cells in one region, leading to regional enlargement of a vessel. Second, smaller vessels can fuse with each other, thereby rapidly increasing the diameter. Vessels can also increase in diameter because of either local proliferation of endothelial cells (third mechanism), or by local hypertrophy of endothelial cells (fourth). Vessel enlargement occurring in developmental vascular networks refers to the enlargement of capillaries to form arterioles/venules. In the adult, vessel enlargement has mostly only been studied with respect to enlargement of larger vessels (arterioles and venules) and of collateral vessels. Differences in the type of vessel which is enlarging can also account for differences in mechanism highlighted below.2  or in dyne/cm2. Shear stress can be thought of as friction against the endothelium. It relates both to the speed of the blood flow and vessel geometry. In the migration paradigm of vessel enlargement, endothelial cells migrate against flow when shear stress levels are decreased compared to physiological normal levels (which are 15 dyn/cm2 in humans or 30 dyn/cm2 in mouse) and stop migrating in the presence of physiological levels −/− embryos, which have severely reduced flow and do not undergo vessel enlargement, have the same levels of endothelial cell proliferation and apoptosis as control wild-type embryos  cause Hereditary Hemorrhagic Telangiectasia (HHT), which is a genetic form of AVM development  control the expression of the gene (GJA4) for the gap junction protein Cx37  pathway is also required for polarized migration in response to flow . Mutatioelopment . Loss ofelopment  and ALK1elopment  in mouseFLT1; also known as VEGFR1, Vascular Endothelial Growth Factor Receptor 1) and Notch, thereby promoting endothelial cell proliferation −/− embryo, involved in repulsive endothelial cell guidance, shows a transient phenotype whereby a plexus is present that eventually coalesces to paired dorsal aortae. This suggests that vessel coalescence may be retarded in these mutants and, therefore, observable  and Mitogen-activated protein kinase (MAPK) pathways promoting proliferation . VEGF exlaterals . Thus, alaterals , the rol2  that activates the downstream PI3K pathway in a ligand-independent manner . This le2 , however2 . Oscilla2 . Further2 .S-nitrosylation. NO leads to S-nitrosylation of MAPK phosphatase 7 (MKP7), rendering it inactive which then prevents the inactivation of c-Jun N-terminal Kinase 3  and its increased activity induced by shear stress was one of the first studied effects of mechanotransduction . Nitric  3 JNK3; . The con 3 JNK3; . At conc 3 JNK3; .SMAD4 and in the Growth differentiation factor 2 (GDF2) genes (encoding for SMAD4 and BMP9) had been also reported later on to cause variants of HHT. Both the endothelial-specific ablation of SMAD4 (a transcription factor in the ALK1/ENG pathway) and of ENG show increased proliferation of endothelial cells within the developing shunt  is involved in the maintenance of the endothelium in a nonactivated state  and prot2) activated the phosphorylation of p53 protein through the JNK pathway. The increased levels of p53 in turn activated Growth Arrest and DNA Damage 45 (GADD45) and p21 proteins, inhibiting the Cyclin-Dependent Kinase (CDK) and, thus, arresting endothelial cell proliferation  expression and the subsequent shear stress-induced arrest of the cell cycle  under control of a somite muscle-specific promoter (PLGFmusc), cell size was found to contribute to vessel enlargement but, importantly, alone could not account for the diameter increase  impairs migration, and enlarged veins and capillaries form without arterial enlargement  receptor . Constitat E10.5 . If TIE2s do not . This isin vitro . HoweverENG knockout model, imaging of developing AVMs showed that these initiated in arterioles and grew toward the venous vasculature , Hairy/enhancer-of-split related with YRPW motif protein 2 (HEY2), and EPHB2 , the response is outward remodeling (2) for vessel enlargement during development, but an acute non-physiological level of shear stress (above 30 dyn/cm2) in models of vessel enlargement in the adult.In femoral artery ligation, arteriogenesis occurs far away from where ischemia is occurring . As suchmodeling . Thus, dThe activated endothelium produces NO that is essential for collateral growth. Both eNOS and iNOS  are upregulated in remodeling collateral vessel . When NOAnother difference between adult and developmental vessel enlargement is the involvement of immune cells. Immune cells, especially monocytes and macrophages, are essential for adult collateral growth and vessel enlargement in general . Inhibitpu.1 morpholino inhibits collateral growth in the gridlock mutant embryo  or nanoparticles within 2–4 h of the occlusion . DegradaThe presence of this extensive basement membrane is one of the main reasons that post-natal vessel enlargement is unlike to occur by vascular fusion. The presence of extensive matrix and mural cells from the arterioles onward would be a physical barrier for fusion. Hence, vascular fusion could only occur on the capillary level allowing arterioles to grow, which would then have to further increase in diameter by combination with another mechanism.Vessel enlargement plays a critical role both during development as well as in the adult vasculature, with a high capacity for adapting to flow changes. Through an extremely fast responsiveness and interconnected processes such as fusion, endothelial cell migration and proliferation, vessels reshape in order to accommodate changes in flow rates such as to restore physiological levels. Because these processes are so much present along all the vasculature lifetime, dysregulations may entail critical pathologies. However, more and more pathways and molecular interconnections are being uncovered, shedding light to a better understanding and control over these pathologies.LG-R and EAVJ contributed to writing, editing, and making figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, cannabis has been gaining increasing interest in both the medical research and clinical fields, with regard to its therapeutic effects in various disorders. One of the major fields of interest is its role as an anticonvulsant for refractory epilepsy, especially in the pediatric population. This paper presents and discusses the current accumulated knowledge regarding artisanal cannabis and Epidiolex®, a United States Food and Drug Administration (FDA)-approved pure cannabidiol (CBD), in epilepsy management in pediatrics, by reviewing the literature and raising debate regarding further research directions. Childhood epilepsy may be coarsely divided into (1) a large group (70%) of benign epileptic syndromes and easily controlled symptomatic epilepsies, and (2) a smaller but significant group of drug-resistant epilepsies which includes idiopathic and genetic epileptic encephalopathies and various symptomatic acquired epilepsies. The burden of intractable epilepsy on infants and children and their families is enormous, and in addition to the risks carried by the actual seizures it significantly affects the children’s development and quality of life. Most of these children are on polytherapy, which has its own consequences. This devastating situation has led to a quest for additional solutions. This quest, with regard to the use of cannabis, has been led by parents and caretakers in parallel to the medical authorities.Various ancient cultures have mentioned cannabis as a useful tool to treat epileptic convulsions. There are historical records from ancient China dating back to 2700 BC4Despite the Marijuana Tax Act of 1937, which led to the removal of cannabis from the US pharmacopeia in 1941, and its classification as a schedule 1 substance, several researchers and physicians renewed investigation on the biological effects and medicinal use of its various components in the 1970s. Several animal studies and small-scale clinical trials examined its use. Studies focusing mainly on purified cannabidiol (CBD) in epilepsy management in drug-resistant patients were published.11In the last decade social media, patient and family advocacy groups, and Internet activity have led to significant public interest in cannabis as an alternative treatment for those living with epilepsy. This public demand revived both basic and clinical research into cannabis and CBD use for epilepsy treatment particularly in the pediatric population. This review provides an overview and discussion of the current data relating to artisanal cannabis and pure CBD use in epilepsy.Cannabis sativa, and Cannabis indica contain up to 500 chemical species, with more than 100 different phytocannabinoid compounds.14The marijuana plant, Biologically, THC’s mechanism of action is primarily related to its effect on endogenous cannabinoid receptors in the brain, mainly cannabinoid receptor type 1 (CB1), and to the extra central nervous system (CNS) receptor, CB2.17The CBD anticonvulsant activity is most probably multifactorial and relates to: gamma-aminobutyric acid (GABA)-mediated inhibition of glutaminergic forebrain neurons21Cannabidiol has been tested in several animal epileptic models, including maximal electroshock, pentylenetetrazol, pilocarpine, penicillin, audiogenic seizures, 6-Hz, subcutaneous metrazol threshold test, and cobalt implantation,27Because of the specific interest given to the positive effect of CBD in Dravet syndrome patients, this compound was studied in an SCN1A knockout mouse model showing decreased spontaneous seizure frequency and duration, as well as decreased severity of heat-induced seizures. Autistic-like social interaction deficits improved with low-dose CBD but failed to improve with the higher dosages required for seizure control.28Studying the effect of THC on seizures in various animal models showed conflicting results—including anticonvulsant, no effect, and proconvulsant responses—making it less attractive for clinical epilepsy treatment.29Cannabidiol has poor oral bioavailability of around 6%, which is related to its lipophilic structure, variable absorption rate, and extensive hepatic first-pass metabolism by isozymes CYP2C19 and CYP3A4. Its bioavailability can be increased or decreased by exposure to a strong enzyme inhibitor or inducer, respectively.32Over the last six years, medical publications regarding epilepsy treatment with cannabis oil extracts and pure CBD can be divided into several groups: retrospective surveys and chart reviews of patients independently treated with artisanal cannabis by their caretakers which was reported to physicians; retrospective chart reviews of CBD-enriched cannabis oil use, as directed by physicians; and open-label followed by placebo-controlled prospective studies of US Food and Drug Administration (FDA)-approved pure CBD oil (Epidiolex®); in addition to anecdotal reports of other “pure CBD” compounds used.33Porter et al. surveyed parents who belong to a Facebook group that used CBD extracts to treat their children’s seizures. Nineteen out of 150 participants in the group responded: 84% reported a reduction in seizure frequency; of these, 11% (2/16) experienced a positive effect and became seizure-free.A retrospective medical chart review was performed on 75 children and adolescents who received various types and dosages of oral cannabis extracts to treat their epilepsy. Based on parental reports, within the subgroup of responders (33%), more than a 50% reduction of seizures was noted in children with Lennox–Gastaut syndrome (LGS) and Dravet syndrome. Interestingly there was a significantly higher response rate from families who had moved to Colorado in order access artisanal cannabis treatment for their child as compared to the families who were already Colorado residents (47% versus 22%), suggesting a higher placebo effect in those with higher treatment expectations.37A retrospective multicenter study was performed with data from three epilepsy clinics in Israel treating 74 children for intractable epilepsy with one of two well-controlled cannabis oil extracts . The authors found that 52% of patients experienced more than 50% reduction in seizure frequency; only 7% of patients experienced seizure aggravation.39While the use of artisanal cannabis preparations can be criticized as being inaccurate and not as precise as expected for other drug treatments in the medical community, a more well-controlled process leading to FDA and European Medicines Agency (EMA) approval was performed for a pure CBD extract produced by GW Pharma called “Epidiolex.”The first published study using Epidiolex as an add-on treatment for children with intractable epilepsy was by Devinsky et al.The following study41Two multicenter double-blind placebo-controlled trials investigated the short-term effect of CBD in LGS patients: the first one looked at the efficacy of CBD (20 mg/kg/day) as an add-on therapy for drop seizures in 171 patients (2–55 years) with treatment-resistant LGS and found a statistically significant reduction in drop seizure number in the CBD compared to the placebo group.43Equally important to double-blind placebo-controlled studies, which are usually short term, is investigation into a drug’s long-term effect on epilepsy. This collected data was lately reported by Szaflarski et al.Based on these positive trials the FDA approved pharmaceutical-grade CBD Epidiolex as an oral solution (100 mg CBD/mL) for treating seizures in LGS and Dravet syndrome patients over the age of two years. However, other CBD products remain as schedule I substances under the Controlled Substances Act. The above-mentioned approval was followed by EMA approval of Epidiolex for the same indications and age group, but only when added to clobazam, based on the assumption that because CBD increases blood levels of clobazam active metabolite, its antiepileptic effect in many patients in the randomized studies that led to the FDA approval could be attributed to this effect only.Smaller-scale studies looked at the effect of Epidiolex in other rare epileptic syndromes and disorders with severe epilepsy, including its positive effect on seizures in tuberous sclerosis patients47Cannabidiol is a potent inhibitor of CYP2C19, CYP2D6, and CYP2C9, which leads to an increase in the level of several antiepileptic drugs, with the most significant effect being on clobazam and its metabolite N-desmethylclobazam, and a less prominent effect on topiramate, eslicarbazepine, zonisamide, rufinamide, and brivaracetam.50Abnormal elevation in liver enzymes (transaminases) can occur with concomitant use of valproate and CBD without significant changes in the valproate levels, suggesting a pharmacodynamic rather than a pharmacokinetic interaction.32The adverse events reported in the well-controlled trials of both artisanal cannabis use and Epidiolex were qualitatively similar and included fatigue, decreased appetite, somnolence, vomiting, diarrhea, and seizures. Somnolence was more frequently reported in patients treated with Epidiolex in addition to clobazam.43Elevations of liver transaminases were reported only in the Epidiolex studies, mainly during the first two months after treatment initiation, and were primarily dose-related; delayed transaminase elevations have also been noted, particularly with concomitant valproate use, and less frequently with concomitant clobazam use. The transaminase elevation was reversible with discontinuation or reduction of CBD oil and/or concomitant valproate.32The significantly higher rate of adverse events reported in the Epidiolex well-controlled trials compared to the open-label artisanal cannabis reports may be partially related to bias effect of the artisanal retrospective reports; the high rate of adverse events reported in the placebo group of the Epidiolex studies supports this assumption. But it could also be related to the much higher dose and rapid titration rate of CBD used in the Epidiolex studies as compared to artisanal cannabis use. The possibility that differing amounts of other constituents in artisanal cannabis oil, such as THC, other cannabinoids, and terpenes, have a protective effect merits further investigation.It is interesting to note that the artisanal cannabis reports and the short- and long-term Epidiolex studies reported other positive side effects not directly related to seizure control. The beneficial effects of CBD-enriched cannabis, other than reduced seizures, were reported in all studies related to artisanal cannabis use and included: increased alertness (>50%); improved sleep (25%–68%), behavior (33%), language (10%), and motor skills (10%–20%); and decreased self-stimulation (32%).51Pure CBD (i.e. Epidiolex) and CBD-enriched cannabis oil extracts were found to be effective for epileptic seizure control in pediatric patients and young adults, particularly in the specific epileptic syndromes, Dravet syndrome and LGS, as was the case in larger more varied groups of patients with intractable epilepsy. Based on the current data, it is essential that drug formulations contain as low a THC content as possible, since the anti-seizure activity of THC is equivocal and it can potentially aggravate seizures; moreover, it can be associated with additional short- and long-term side effects, especially related to memory as well as other aspects of cognition and behavior.52We should be aware that artisanal and other commercially available products are currently not well controlled. This has been proven through in-depth chemical profiling of cannabinoids, terpenes, and oxidation products of commercially available CBD oils used for treating epilepsy in the United States, which found that 9/14 of the samples studied (64%) had concentrations that differed from the declared amount, with only five maintaining optimal concentrations.On the other hand, future research on the role of cannabis in epilepsy should keep in mind that the controlled, randomized trials have revealed that the actual reduction in seizure frequency in response to CBD is comparable to that achieved in response to other antiepileptic drugs, and have failed to meet the 60%–85% responder rates in unblended web-based surveys and chart reviews based on parental reports. In addition the rate of side effects was higher in the well-controlled Epidiolex studies compared to artisanal cannabis use. Although these differences may primarily be related to the positive bias of the open-label retrospective studies and higher CBD dosages used in the controlled pure CBD trials, there is still room for debate regarding the role of other phytocannabinoids present in artisanal cannabis extracts, which may have an “entourage effect” on both the anticonvulsant potency and their protective role, which points to the need for further research in this direction.54