Oral mesenchymal stem/progenitor cells (MSCs) are renowned in the field of tissue engineering/regeneration for their multilineage differentiation potential and easy acquisition. These cells encompass the periodontal ligament stem/progenitor cells (PDLSCs), the dental pulp stem/progenitor cells (DPSCs), the stem/progenitor cells from human exfoliated deciduous teeth (SHED), the gingival mesenchymal stem/progenitor cells (GMSCs), the stem/progenitor cells from the apical papilla (SCAP), the dental follicle stem/progenitor cells (DFSCs), the bone marrow mesenchymal stem/progenitor cells (BM-MSCs) from the alveolar bone proper, and the human periapical cyst-mesenchymal stem cells (hPCy-MSCs). Apart from their remarkable regenerative potential, oral MSCs possess the capacity to interact with an inflammatory microenvironment. Although inflammation might affect the properties of oral MSCs, they could inversely exert a multitude of immunological actions to the local inflammatory microenvironment. The present review discusses the current understanding about the immunomodulatory role of oral MSCs both in periodontitis and systemic diseases, their “double-edged sword” uniqueness in inflammatory regulation, their affection of the immune system, and the underlying mechanisms, involving oral MSC-derived extracellular vesicles. CD73, as a marker of lymphocyte differentiation, can be coexpressed with CD90 and CD105 in very high concentrations on any potential MSCs. Thus, CD73, CD90, and CD10 are three major makers expressed on the MSCs' surface. CD79a and CD19 are expressed on B cells, and CD34 is a marker of primitive hematopoietic progenitors and endothelial cells. CD14 and CD11b are predominantly expressed on monocytes and macrophages, and CD45 is a marker of pan-leukocyte. HLA-DR molecules are not expressed on MSCs unless stimulated. Accordingly, MSCs lack the expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR antigens [According to the International Society for Cellular Therapy, mesenchymal stem/progenitor cells (MSCs) positively express the surface markers CD73, CD90, and CD105 and negatively express the endothelial as well as the hematopoietic markers CD11b, CD19, CD79MSCs are characterized by their self-renewal and multilineage differentiation capability into osteogenic, adipogenic, chondrogenic, and myogenic- and neurogenic-like lineages. Aside from their remarkable proliferative and multilineage differentiation/regenerative potential , multiplOriginally, MSCs were characterized as fibroblast-like cells isolated from the bone marrow mesenchymal cellular populations . Since t7 BM-MSCs into an injured corneas, surprisingly, less than 10 BM-MSCs appeared in the corneas, with a partial reversal of the deleterious effects of corneal opacity and inflammation [The oral cavity is a distinctive habitat, harbouring an array of microorganism, with more than 700 species , 21. An ammation . This fiammation  to descrammation –30.Escherichia coli lipopolysaccharide (Ec-LPS) inhibited the proliferation of DPSCs significantly at a concentration of 10 μg/ml, while promoted it at 0.1 μg/ml and exerted no influence at the tipping concentration of 1 μg/ml [Ec-LPS enhanced the proliferation rate of BM-MSCs at 1 μg/ml but not at 10 μg/ml [Pg-LPS concentrations, with an increase in proliferation, osteogenic differentiation, and immunomodulatory properties observed at 0.1 μg/ml but a decrease or even apoptosis occurring at 10 μg/ml [Pg-LPS ranging from 1 μg/ml to 50 μg/ml [Multiple investigations exploring the impact of the inflammatory microenvironment on oral MSCs' properties addressed their proliferation potential, migration and homing, multilineage differentiation, and inflammatory cytokines production . Yet, re 1 μg/ml , in a do10 μg/ml . Also, B10 μg/ml . Interes10 μg/ml . Contras50 μg/ml . It appeβ (IL-1β) (5 ng/ml)/tumor necrosis factor- (TNF-) α (10 ng/mL) inflammatory microenvironment upregulated their expression of CXC chemokine receptor 4 (CXCR4), which downregulated the tissue-released chemokine SDF-1/CXCL12 by bonding to it, thereby increasing PDLSCs' homing activity [Cellular proliferation is classically followed by selective migration/homing of MSCs in tissue regenerative/reparative process. This cellular homing and migration ability is principally regulated by local cytokines and chemokines . PDLSCs activity . Moreoveactivity . In concEc-LPS, adipose stem/progenitor cells, DPSCs, and PDLSCs increased their osteogenic mRNA expressions, including alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) [Pg-LPS stimulation [Ec-LPS at 1 μg/ml [β and TNF-α, GMSCs' osteogenic and adipogenic differentiation was suppressed [Interestingly, LPS-challenged oral MSCs altered their regenerative markers' expression profile according to the source of the LPS. After stimulated by in (OCN) , 40, 41.in (OCN) , 42. In mulation  and the  1 μg/ml . With thppressed . Remarkappressed .Ec-LPS stimulation of BM-MSCs, umbilical cord stem/progenitor cells, DFSCs, and DPSCs demonstrated a marked increase in their IL-6 and IL-8, but not in TNF-α secretion [Pg-LPS resulted in a significant increase in cellular IL-1β, IL-6, and IL-8 release [α [Pg-LPS demonstrated no difference in IL-6 expression [On the level of inflammatory cytokine release, a short-termed ecretion –48. A lo release . Comparelease [α . In contpression , 48.Pg-LPS in concentrations ranging from 10 ng/ml to 10 μg/ml upregulated their proliferation while induced minimally their inflammatory response and did not attenuate their regenerative capacity [β, TNF-α, and IFN-γ), the cellular proliferation of GMSCs declined at the initial stage (day 3) but increased significantly at the later stage (day 7), in a time-dependent manner. Their stemness and multilineage differentiation potential kept quite stable under “cocktail” stimulation [Particularly, our group has been addressed in the study of GMSC behavior under inflammatory environment for years and uncovered that they were a unique group of MSCs with the characteristic of inflammatory resistance. Stimulation of GMSCs with capacity . Challenmulation .Toll-like receptors (TLRs) expressed on MSCs were demonstrated to not only affect their migration, proliferation, and differentiation potential but also to play an important role in interaction with inflammatory environment. Therefore, we depicted TLR expression profile of GMSCs, DPSCs, and BM-MSCs under uninflamed –53 and iOxidative stress, defined as the damage caused by an imbalance between oxidants and antioxidants , is two Extracellular matrix (ECM) is also considered a crucial factor to influences oral MSCs by changing its biochemical or physical properties. Compared to collagen type I which is the most commonly purified ECM, decellularized bone extracellular matrix (bECM) hydrogels facilitated a significant upregulation of RUNX-2 and bone sialoprotein (BSP) of DPSCs, indicating their osteogenic differentiation . PhysicaMicro-/nanoparticles have been widely used as bone graft substitute to mimic natural bone tissue. The addition of nanosilicates to poly(glycerol sebacate) could not only modulate the degradation rate and mechanical stiffness of the scaffold but also enhance the adhesion, spreading, proliferation, and osteogenic differentiation potential of preosteoblasts . It was β1, and prostaglandin E2 (PGE2) [Various experimental setups including animal models, cellular coculture system, and conditioned medium application were administrated to explore the impact of MSCs on inflammatory microenvironment, including immune cell infiltration and inflammatory cytokine production. The immunomodulatory effects of oral MSCs are believed to be mediated by direct cell to cell contact as well as through the production of soluble cytokines, including IL-1, IL-6, IL-10, indoleamine 2,3-dioxygenase (IDO), nitric oxide (NO), transforming growth factor (TGF)-2 (PGE2) .γ and TNF-α, while under low concentrations (0.2 ng/ml) of the same cytokines, MSCs promote their cellular proliferation. This “double-edged sword” effect is attributed to the inflammatory-dose-dependent production of NO/IDO by MSCs, which functions as an “on-off” switch, changing MSCs from being immunosuppressive to immune enhancing [γ could boost the antigen-presenting functions of MSCs through upregulating MHC-II and MHC-I, rendering them less susceptible to NK-mediated cell lysis. High doses of IFN-γ, contrastingly suppress the expression of the same MHCs in MSCs [Yet, the immunoregulative capacity of MSCs is largely governed by the surrounding inflammatory intensity . Under lnhancing . Similar in MSCs , 68.Porphyromonas gingivalis total protein extract (Pg-PE), PDLSCs reverse their action, augmenting the ROS production by activating HL-60D [In the periodontium, the inflammatory concentration is directly associated with the severity of periodontitis . PDLSCs g HL-60D –74.−9 M 1α,25(OH)2D3. At higher concentrations of 10−8 M 1α,25(OH)2D3, this effect was not observed [α (5 ng/ml), while inhibited osteoclast formation at high doses of TNF-α (10 ng/ml) [κB ligand (RANKL) is expressed by MSCs during normal physiological remodeling, while under inflammatory conditions the expression of OPG [Similarly, the effects of MSCs on osteoclast also depends on the dose of the surrounding biomolecules as well as the concentration of the inflammatory condition . Osteoclobserved . Similar0 ng/ml) . Recepton of OPG  and IL-1n of OPG  is upregn of OPG  is slighn of OPG .As above, MSCs and the local inflammatory environment could affect each other under the inflammatory stimuli. Although they seem to be two independent biological behavior, underlying correlations exist to maintain tissue equilibrium. The influence of inflammatory environment on MSCs would affect their survival, proliferation, stemness, and differentiation potential, which might impair or boost the process of tissue regeneration/repair. Also, MSCs' actions as immunomodulators could attenuate the inflammatory process.Oral MSCs regulate local inflammatory environment also by interacting with the innate and acquired immune system in a multifaceted way . As primα secretion via an IDO-mediated pathway [Macrophages, which are indispensable components of the innate immune system, contain two major subpopulations, namely proinflammatory M1 phenotype and anti-inflammatory M2 phenotype . DPSCs a pathway . Also, D pathway , 87.α in activated mast cells, which is believed to be partly mediated by TNF-α/PGE2 feedback axis [Mast cells play a significant role in allergy and inflammation. Studies show that GMSCs hinder the production of TNF-ack axis , 86.γ released by activated T cells acts as a regulator in the feedback signaling between T cells and GMSCs [+CD25+FoxP3+ regulatory T cells (Tregs) [β-dependent mechanism [γ and IL-17 by Th1, whereas it enhanced IL-4 secretion by Th2 cells. As to the humoral immunity level, similar to other MSCs, PDLSCs and DPSCs were considered to hinder B cell proliferation, differentiation potential, and antibody production [β1 release [Lymphocytes, consisting of T cells, B cells, and natural killer cells (NK cells), play a vital role in the adaptive immune response. GMSCs were demonstrated to inhibit the proliferation/activation of T cells in vitro through upregulating IL-10 and downregulating tryptophan, via GMSCs' derived indoleamine 2,3-dioxygenase (IDO) , 87. IFNnd GMSCs –91 and p (Tregs) –90, mediechanism , 89. Stuechanism , 93, resoduction , 94 and  release .β, HGF, and IDO [β [Moreover, GMSCs, SCAPs, PDLSCs, DFSCs, and DPSCs are capable of suppressing the proliferation and activation of human peripheral blood mononuclear cells (PBMCs). GMSCs, PDLSCs, and SCAPs were reported to exert an adverse effect on PBMCs , 95, 96,d IDO [β  and TLR4d IDO [β .To date, the intrinsic immunoregulative mechanism of MSCs are not fully elucidated, although paracrine factors have been corroborated to play an important role in this process. The paracrine action may be attributed, at least in part, to EVs released from MSCs in their respective media. EV, known as nanosized membrane structures released by cells in an evolutionally conserved manner, can transfer RNA, microRNA, and even proteins to modulate inflammatory environment –100. EV Pg-LPS, microRNA-155-5p expression in PDLSC-derived exosomes significantly decreased and the exosome was ingested by CD4+ T cells. MicroRNA-155-5p could negatively regulate its downstream target molecule in CD4+ T cells, known as Sirtuin-1. Therefore, the downregulation of microRNA-155-5p in PDLSC-derived exosomes by Pg-LPS rescued the Th17/Treg imbalance via upregulating Sirtuin-1 in CD4+ T cells [Oral MSC-derived exosomes have been reported of therapeutic benefit in a number of inflammatory-related disease, through different molecular mechanisms. BM-MSC-derived exosomes promoted the regeneration/repair of the periodontal ligament and temporomandibular joint, through activation of AKT, ERK, and AMPK signaling pathways that suppress the inflammatory response by chondrocyte and increase the proliferation and migration of periodontal ligament cells , 105. Fo T cells .κB pathways [β-catenin activation [The same exosomes were also demonstrated to suppress the inflammatory cytokines in autoimmune encephalomyelitis, through inactivating NALP3 and NF-pathways , 99. DPSpathways . SHED-depathways . In an apathways . GMSC-depathways . Particutivation .β, IL-6, TNF-α, iNOS, and PTGS2, produced by microglia cells, when cocultured in LPS inflammatory environment. Moreover, the phosphorylation of ERK, JNK, and p38 molecules in microglia cells was also suppressed by BM-MSC-derived microvesicles [α by macrophages via PKR-rich microvesicles [Additionally, BM-MSC-derived microvesicles downregulated the level of IL-1vesicles . In an uvesicles .Ec-LPS-challenged inflammatory environment, monocyte-derived exosomes could promote the osteogenic differentiation of BM-MSCs [Although EVs in the oral MSC-derived conditioned media play a pivotal role in cell-cell communication, the information exchange between oral MSCs and their surrounding inflammatory microenvironment is bidirectional. EVs liberated from oral MSCs could restrain the inflammatory response in immune cells, and conversely immune cells could affect the proliferation, differentiation, and migration potential of MSCs, through their EVs , 114. In BM-MSCs . EVs der BM-MSCs .In this context, EV-based approaches appear to provide a new paradigm for cell-free therapies, overcoming many of the current clinical constrain of cellular transplantation. EV, encapsulating prolific proteins and RNAs, can cross the plasma membrane to deliver their cargo into target cells and are tolerated by the body . MoreoveCompared to other MSCs, oral tissue-derived MSCs are more adaptive to inflammatory environmental challenges, principally because of the bacteria-congregated habitat they usually reside in. The complex subgingival microenvironment is an ideal cradle for bacteria colonization, where it inhabits an array of diverse bacterial species known as subgingival plaque . Still, +  cells and attenuating the level of the proinflammatory cytokines TNF-α and IL-17, as well as increasing the anti-inflammatory cytokine IL-10 and shifting M1 macrophage to a M2 phenotype [α, IL-1α, IL-1β, IL-17, and IFN-γ and an increase in IL-10 [+ (M2) macrophages in periodontal tissues. It further promote the conversion of M1 macrophages which secrete NO, ROS, and TNF-α to CD206+ M2 macrophages which release IL-10, TGF-β, and arginase-1 (Arg1), in a coculture system [α, as well as the IL-6/IL-10 ratio in a rat tooth transplantation model [Under these pathogenic conditions, oral MSCs could exert a variety of anti-inflammatory functions . In inflhenotype , 123. SHin IL-10 , 122–124e system . Similaron model . BM-MSCson model . Additioon model .Thus, through their regenerative and paracrine immunomodulatory actions, oral MSCs augment periodontal tissue regeneration, while ameliorating bone resorption. They decrease inflammatory factor level and RANKL/OPG ratio in periodontal disease by regulating immune cells, resident cells, and osteoclasts, so as to mitigate bone loss. This immunoregulative function and inflammatory resistance in turn maintain their stemness, survival, and multilineage differentiation potential, in order to promote periodontal tissue regeneration/repair.Oral MSCs are applicable not only in oral disease but also in systemic diseases as immunoregulators, mainly through decreasing inflammatory cytokine production, inhibiting bone resorption in a cell-cell contact manner or through a paracrine way  Table 1Table 1.β, IL-6, and TNF-α, as well as upregulating the levels of IL-10 and the macrophage M2 marker Arg-1 in bronchoalveolar lavage fluid [κB activity, and extrainflammatory cytokine release were reversed by BM-MSC administration [Acute lung injury is characterized by an excessive and uncontrolled inflammatory response, which involves the activation of neutrophils and macrophages and the corresponding tissue damage by releasing antimicrobial compounds . Tail inge fluid . Also, tstration .α, COX-2, and iNOS expressions and concomitantly enhancing IL-10 and IL-37 secretion in the experimental autoimmune encephalomyelitis, which is a murine model of multiple sclerosis. Furthermore, PDLSCs were demonstrated to suppress cellular apoptosis by inhibiting the proapoptotic protein caspase-3 and enhancing the antiapoptotic protein Bax and Bcl-2 [Multiple sclerosis is a chronic inflammatory, demyelinating disease of multiple pathogenic factors, which is characterized by myelin degradation and damage of the central nervous system. PDLSCs were corroborated to mitigate the inflammatory response by inhibiting TNF-nd Bcl-2 .Optic neuropathies, the leading cause of irreversible blindness and visual impairment, which result from trauma injury, glaucoma, inflammation, ischemia, or tumor compression, are characterized by the degeneration of retinal ganglion cells (RGCs) and their axons. Intravitreal injection of PDLSCs into the rat vitreous chamber in the optic nerve injury model could promote the survival and neurite regeneration of RGCs by increasing brain-derived neurotrophic factor secretion in a direct cell-cell-contact manner .+ T cells in the pathogenesis of Sjögren's syndrome has been proposed, and multiple studies have demonstrated the role of MSCs in the regulation of T cells. In the murine Sjögren's-like disease model, BM-MSCs restored the salivary flow rate and reduced the lymphocytic infiltration by modulating pro- and anti-inflammatory cytokines INF-γ, TNF-α, IL-10, PGE2, and IL-6 [Sjögren's syndrome is a systemic autoimmune disease that primarily affects the exocrine glands. It is characterized by clinical symptoms including dry eyes and mouth, and histological feature of focal lymphocytic infiltration of the exocrine glands. Currently, the role of CD4and IL-6 .β-cells, involving the upregulation of inflammatory cytokines and the imbalance of Th17 cells and Tregs. GMSCs were demonstrated effective in the treatment for type 1 diabetes, with the better control of blood glucose levels, the delayed diabetes onset, and the ameliorated pathology scores in the pancreas. The mechanism was postulated to be primarily mediated through downregulating the level of IL-17 and IFN-γ expressed by CD4+ and CD8+ T cells and upregulating the number and function of Tregs [Type 1 diabetes is an autoimmune disease caused by the immune-mediated destruction of pancreatic of Tregs . Moreoveof Tregs .α and CII-specific IgG secretion via FasL/Fas pathway [Allergic contact dermatitis, classified as a type IV hypersensitivity reaction, is caused by repeated skin exposure to contact allergens. Contact hypersensitivity (CHS) is a classic murine model of allergic contact dermatitis, in which dermal DCs, allergen-specific T lymphocytes, allergen-specific effector T cells, and mast cells play a significant role. Infusion of GMSCs into the CHS reversed the imbalance of abnormal Th1/Th2 ratio and decreased the infiltration of dendritic cells, mast cells, and Th17. It further increased the number of Tregs in the allergen contact area, with a remission of hypersensitivity. Mechanically, GMSCs inhibited proinflammatory factors and promoted anti-inflammatory factors through COX/PGE2 and PGE2–EP3 signaling pathways , 136. In pathway . Similar pathway .Colitis is an inflammatory-related colonic mucosal injury in the distal small intestine, contributing to the dysfunction of innate and adaptive immunity. GMSCs and DPSCs significantly ameliorated inflammation clinically and histopathologically, by restoring the normal intestinal architecture, reversing weight loss, improving the overall disease score, and suppressing epithelial ulceration. At the cellular and molecular level, GMSCs and DPSCs decreased infiltration of Th1 and Th17 cells, increased Tregs, inhibited IL-6 and IL-17 release, and elevated IL-10 level , 93.Rheumatoid arthritis, a multisystem autoimmune disease characterized by the loss of immunologic self-tolerance and chronic inflammation, could impair the joints of the whole body. GMSCs was reported to mitigate bone and cartilage destruction by remarkably enhanced Tregs number through CD39/CD73 signals in the murine-rheumatoid-arthritis model .α and IL-6 were suppressed while IL-10 was elevated with the administration of GMSCs, with accelerated wound healing, rapid deepithelialization, and increased angiogenesis [Process of cutaneous wound healing can be divided into three stages: inflammation, tissue formation, and remodeling, which involves interactions of resident cells, infiltrating immune cells, and their secretion as well. GMSCs were found to interact with the local inflammatory microenvironment, migrating into the local wound bed and promoting macrophage conversion from M1 to M2 phenotype. Concomitantly, TNF-ogenesis .α level and promoting the differentiation oligodendrocyte progenitor cells. Besides, SHEDs/SHED-conditioned medium were found to promote the fibrotic scar resolution, by suppressing the inflammatory mediators TNF-α, IL-1β, and iNOS and by inducing apoptosis of activated hepatic stellate cells, in the liver fibrosis experimental model [Moreover, spinal cord injury (SCI) normally caused by accident and inflammation is considered a key regulator during the secondary neurodegenerative events . SCAP adal model .As above, oral MSCs contributed to tissue regeneration/repair in a variety of immune/inflammatory disease not only by boosting tissue regeneration but also by ameliorating immune/inflammatory response. However, the majority of studies still remained at the initial stage of observing the variation of inflammatory cytokines and immune cells or detecting the signaling pathway; exact mechanisms are needed to be further elucidated.Due to the immunomodulatory function and low immunogenicity of MSCs, autologous/allogenic MSCs obtain good results in the treatment of human autoimmune and inflammatory diseases. Clinical efficacy and safety have been demonstrated for MSCs applied in patients with systematic lupus erythematosus –145, rheAs for oral MSCs, researchers discovered that GMSCs possess an extraordinary potential in the treatment of autoimmune and inflammatory diseases owing to their high proliferation rate, no tumorigenesis , immunomOral MSCs, with the unique clinical advantages of easy access in large quantities, as well as their remarkable tissue reparative/regenerative potential, have been proposed as ideal candidates for MSC-based tissue regeneration. Moreover, the current knowledge reveals a vivid interaction between the oral MSCs and their inflammatory milieu, both at the cellular and secretomes levels. The interaction largely governs the proliferation potential, migration and homing, multilineage differentiation, and inflammatory response of oral MSCs on the one hand, and on the other hand, regulates the severity of local inflammatory microenvironment. Particularly, the bidirectional regulatory manner of MSCs, including anti-inflammatory and proinflammatory effects, boosts them to become novel natural modulators in maintaining inflammatory balance, not only in periodontitis but also in various systemic diseases. Nevertheless, we still scarcely know the underlying molecular mechanisms, though recently the concept of secretomes-driven cell-free therapeutic potential opens a multitude of very promising perspectives clinically. Thus, a deeper understanding of the underlying mechanisms of oral MSCs' immunomodulatory effects could pave the way to make MSC therapy a clinical reality.


Thapsia L. species that have been used for centuries in folk medicine to treat rheumatic pain, lung diseases, and female infertility. More recently thapsigargin was found to be a potent cytotoxin that induces apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, which is necessary for cellular viability. This biological activity encouraged studies on the use of thapsigargin as a novel antineoplastic agent, which were, however, hampered due to high toxicity of this compound to normal cells. In this review, we summarized the recent knowledge on the biological activity and molecular mechanisms of thapsigargin action and advances in the synthesis of less-toxic thapsigargin derivatives that are being developed as novel anticancer drugs.A sesquiterpene lactone, thapsigargin, is a phytochemical found in the roots and fruits of Mediterranean plants from  Thapsia garganica (Apiaceae), known as “deadly carrot” due to its high toxicity to sheep and cattle. The skin-irritating properties, as well as the medical use of this plant, were known already in ancient times. The resin from the roots and fruits of T. garganica was used for centuries in folk medicine to treat several diseases, such as pulmonary diseases, female infertility, catarrh, fever, and rheumatism [Thapsigargin (Tg), a guaianolide-type sesquiterpene lactone, is abundant in the common Mediterranean weed eumatism ,2.T. garganica, along with other structurally-related guaianolides, by Christensen and co-workers in 1978 [The skin irritating component, named Tg, was isolated from  in 1978 ,4, where in 1978 .S)-carvone, achieved in 42 steps with 0.6% overall yield, was reported by Ley’s group in 2007 [S)-dihydrocarvone Baran and co-workers performed a second, 11-step total synthesis in 2016 [R)-carvone with 5.8% overall yield [In terms of chemical structure, Tg consists of three fused rings, formed by annulation of a cycloheptane, cyclopentene, and a γ-lactone . The str in 2007 . Startin in 2016 . Most rell yield .T. garganica and laborious and expensive chemical synthesis made the availability of Tg very limited. The solution may lie in the development of production platforms for Tg, including agriculture of T. garganica plants and plant tissue cultures producing Tg, which are already in progress by several companies [The interesting biological activities of Tg, especially high cytotoxicity, opened perspectives of its use as an anticancer agent, which caused a growing demand for this compound. The low-yield tedious isolation of Tg from ompanies ,11, as wompanies .Over the past 40 years, several excellent reviews describing Tg and its derivatives at different angles have been published ,12,13,142+-ATPase (SERCA) pump, plays a crucial role in regulation of calcium homeostasis which is pivotal for cell signaling and cell survival [2+ ions from the cytoplasm into the sarcoplasmic reticulum (SR) lumen in skeletal and cardiac muscle cells, and into the endoplasmic reticulum (ER) of non-muscle cells, maintaining low cytosolic Ca2+ concentration [2+ in the cell is responsible for regulation of various cellular functions such as proliferation, contractility, protein folding, gene transcription, and apoptosis [ATP2A1-3 genes [2+ affinity and enzyme kinetics [2+ levels and depletion of Ca2+ in ER stores, triggering ER stress and stress-induced apoptosis [2+ metabolism may cause cardiovascular and muscular diseases, diabetes and also cancer [The sarco/endoplasmic reticulum Casurvival ,16. SERCntration . Such copoptosis ,18,19. S-3 genes . Despitekinetics ,21. Impapoptosis . Dysreguo cancer ,19,20.2+-ATPase or Na+,K+-ATPase [2+ per hydrolyzed ATP molecule via an “alternating-access” mechanism, while the enzyme interconverts between two main conformational states E1 and E2 [2+ ions, in E2 state, two Ca2+ ions are released into the ER lumen. Therefore, E1 and E2 states are characterized by high and low affinity for Ca2+ ions, respectively. Tg interacts stoichiometrically with the SERCA pump in a calcium-free E2 conformational state, forming an irreversible, catalytically inactive ‘dead-end’ inhibitory complex, which prevents Ca2+ binding and ATPase activation [On the other hand, SERCA inhibitors are proposed as novel therapeutic agents in cancer therapy ,16. The +-ATPase . The SER1 and E2 . In E1 stivation ,22,26.2+ storage and dynamics and is a major site for protein synthesis, folding and maturation of eukaryotic cells [The ER plays a key role in Caic cells . Disturbic cells . To alleic cells ,29,30,31Signaling through the UPR increases protein folding, transport and ER-associated protein degradation (ERAD), while inhibiting protein synthesis . The UPRHowever, when ER stress is persistent and unresolved, UPR triggers specific apoptotic pathways to eliminate severely damaged cells ,34,35. EChronic ER stress and defects in UPR signaling may lead to various diseases, including cancer, diabetes, neurodegenerative and autoimmune disorders ,37. UPR Tg is known as an ER stressor that initiates UPR-mediated apoptosis ,39,40 buLinder et al. demonstrated that in human prostate (LNCaP) and colorectal (HCT116) cancer cell lines exposed to Tg, microtubule-associated protein 1A/1B light chain 3B (LC3B), normally associated with autophagy, was required for optimal activation of caspase-8 . The autThe involvement of CHOP in up-regulation of DR5 and promotion of apoptosis in Tg-treated cells was observed also by other authors ,41,43. CIRE1α is yet another UPR sensor, whose role in response to ER-stress is complex and not fully elucidated. It controls both cell survival and the decision to execute apoptosis. Activation of IRE1α leads to production of the alternatively spliced isoform of XBP1 (XBP1s), contributing to cell survival during mild, resolvable ER-stress, whereas activation of c-Jun N-terminal kinase (JNK) late in the ER stress response results in cell death ,48.The involvement of JNK signaling pathway in Tg-mediated ER stress apoptosis was confirmed in several cancer cell lines and in the in vivo models ,44,48. WRecently, Chidawanyika et al.  investig2+ stores and activates UPR, which may eventually result in cell death [2+ stores and their detrimental effects on cell viability. The authors showed that Tg strongly inhibited SERCA1a-mediated Ca2+ transport. They also found that, in prostate  and breast cancer (MCF-7) cells, one-day exposure to Tg caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2–4 days and culminated in cell death. The further studies in PC3 cells focused on extent and duration of ER Ca2+ depletion induced by Tg required for a apoptosis-inducing UPR [2+ levels observed with Tg (1-3 nM) correlated with decreased PC3 cell proliferation in the absence of UPR activation and without succumbing to cell death. The authors showed that a partial depletion of ER Ca2+ is sufficient to reduce cell proliferation. However, cells could tolerate strong and sustained decreases in ER Ca2+ without showing any signs of UPR. At higher cytotoxic concentrations of Tg (> 3 nM), the authors observed a gradual, caspase-dependent increase in cell death, which was likely to be caused by UPR, since an enhanced levels of XBP1s, ATF4, CHOP, BiP, and LC3B were observed. Szalai et al. [2+ depletion, obtained at substantially higher Tg concentrations than those needed to strongly decrease ER Ca2+ levels.As a result of blocking the SERCA pump, Tg depletes ER Call death ,35,39. Hcing UPR . The grai et al.  conclude2+ levels, mediated via store-operated Ca2+ entry (SOCE), were required for ER Ca2+ depletion-induced cell death [2+ levels, as implied previously. The knock-down of two key SOCE components, calcium release-activated calcium channel protein 1 (Orai1) and sensor stromal interaction molecule 1 (STIM1), did not reduce Tg cytotoxicity in PC3, LNCaP or MCF-7 cells, indicating that SOCE-mediated increase in cytosolic Ca2+ was not critical for Tg-induced cell death [2+ levels [Some authors indicated that increases in cytosolic Call death ,50. Howell death . Moreove+ levels ,51.2+ depletion and a sustained UPR activation, whereas high cytostolic Ca2+ concentration and SOCE are not required to start the cell death events [Summing up, it has been shown that Tg-induced apoptosis is initiated by ER Cah events ,46.Detailed structure-activity relationship studies were performed to establish the effect of each functional group of Tg on its activity as a SERCA inhibitor . AccordiThrough the inhibition of SERCA pump, Tg induces cancer cell apoptosis in a proliferation-independent manner ,57. MostProdrugs are inactive compounds that, in the body, are transformed into pharmacologically active therapeutics . The Tg-Prostate-specific antigen (PSA) is a serine protease, produced and secreted only by the prostate luminal epithelial cells in both normal and malignant prostate tissue ,60. ExtrBy coupling Tg derivatives to peptide carriers, which are substrates either for PSA or PSMA, protease-activated prodrugs, selectively affecting prostate cancer cells, were created ,56,57. TG202 was developed as a Tg-based PSMA-activated prodrug that potentially could be used to treat almost all types of solid tumors by targeting PSMA enzymatic activity within tumors and surrounding tumor vasculature . Upon PShttp://clinicaltrials.gov; November 2020).In vivo experiments on a variety of human cancer xenografts in mice reveled that G202 significantly inhibited progression of prostate, breast and bladder cancers, while being minimally toxic for the host animals . After cNOTCH1 gene [Antineoplastic activity of Tg-based prodrugs has been widely described in many types of solid tumors. However, there is almost no data on Tg application for leukemia treatment. Roti et al. demonstrated antileukemic activity of Tg in human T cell acute lymphoblastic leukemia (T-ALL) . This isCH1 gene . NOTCH rCH1 gene . MoreoveCH1 gene . They coCH1 gene . To avoiCH1 gene . For thiCH1 gene , based oCH1 gene . By conjCH1 gene  designedCH1 gene . HoweverNatural products isolated from plants are an important source of chemotherapeutics, in particular, against cancer. However, in general, natural substances cannot be used directly as drugs, either because they have a narrow therapeutic window or because they possess undesired pharmacokinetic properties, such as poor absorption, low solubility, and/or fast metabolism. Therefore, optimization of the properties of natural phytochemicals is required. The solution lies in structural modifications of natural compounds or synthesis of their analogs with improved pharmacological properties.T. garganica over forty years ago. Tg induces apoptosis in a proliferation-independent manner by inhibiting the SERCA pump and emptying the ER Ca2+ stores. A barrier preventing the direct usage of Tg as an anticancer agent is its lack of selectivity, since Tg kills not only cancer but also normal cells. The unique properties of cancer cells were used to develop prodrugs that can transport Tg directly to cancer sites. G115 and G202 are examples of prodrugs obtained by conjugation of Tg to substrates of proteolytic enzymes, that are present only in cancer tissue. JQ-FT is an antileukemic prodrug developed by conjugation of folic acid and Tg derivative. Both described prodrug strategies represent an efficient approach to overcome Tg general cytotoxicity, and are in line with recent directions in targeted cancer therapy.Tg is a potent cytotoxin isolated from


The different senescence-inducing modalities resulted in a lack of the proliferative activity. Nucleomegaly was seen in senescent VSMC as compared to freshly isolated VSMC Phenotypically, senescent VSMC appeared with a significantly larger cell size and polygonal, non-spindle-shaped cell morphology. In line with the supposed switch to a pro-inflammatory phenotype known as the senescence associated secretory phenotype (SASP), we found that both RS and DS upregulated IL-1β and released HMGB-1 from the nucleus, while RS also showed IL-6 upregulation. In regard to cell cycle-regulating molecules, we detected modestly increased p16 levels in both RS and DS, but largely inconsistent p21, p14ARF, and p53 expressions in senescent VSMCs. Since these classical markers of senescence showed insufficient deregulation to warrant senescent VSMC detection, we have conducted a non-biased proteomics and in silico analysis of RS VSMC demonstrating altered RNA biology as the central molecular feature of senescence in this cell type. Therefore, key proteins involved with RNA functionality, HMGB-1 release, LMNB-1 downregulation, in junction with nuclear enlargement, can be used as markers of VSMC senescence, enabling the detection of these pathogenic pro-inflammatory cells in future therapeutic studies in ischemic heart disease and atherosclerosis.The senescence of vascular smooth muscle cells (VSMCs) has been implicated as a causal pro-inflammatory mechanism for cardiovascular disease development and progression of atherosclerosis, the instigator of ischemic heart disease. Contemporary limitations related to studying this cellular population and senescence-related therapeutics are caused by a lack of specific markers enabling their detection. Therefore, we aimed to profile a phenotypical and molecular signature of senescent VSMCs to allow reliable identification. To achieve this goal, we have compared non-senescent and senescent VSMCs from two  Inflammation is a hallmark and potent promoter of cardiovascular disease, in addition to other well-defined risk factors that contribute to the multifactorial processes of disease progression  . ElevateCellular senescence has been proposed the key source of inflammation in atherosclerosis and cardiovascular disease (CVD) . SenesceSenescence of various cell types has been implicated in CVD development . In partTherapeutic options to target these senescent cells have recently emerged . Howeverin vitro, replicative senescence (RS) and DNA-damage-induced senescence (DS). We analyzed classical senescence markers: morphology and structural features of senescent VSMCs cells, cell cycle inhibitor and SASP marker expression, as well as contractile protein expression. To find a more specific signature of VSMC senescence, we conducted an unbiased high-throughput proteomics analysis to reveal novel markers of senescence in VSMCs.To test if the deregulation of previously reported senescence markers applies to senescent human coronary VSMCs, we have performed a detailed characterization of the molecular and phenotypical markers that are common in two separate senescence models Bleomycin sulfate was purchased from Enzo Life Sciences (BML-AP302). The following antibodies were purchased: α-Tubulin , ACTA2 , β-Actin , HMGB1 , MYH11 , Phalloidin-TRITC , p16 , p21 , p53 , smoothelin , TAGLN .3 cells/cm2 until proliferative arrest (passage 6–8). Cumulative population doublings were counted using the formula cPD = X + 3.322∗(log Y−log I), X representing the cumulative population doubling of the subculture used to initiate the culture, Y being the viable cell number on harvest, and I the cell number on inoculation. Low passage VSMCs (passage 2–3) were treated with bleomycin to induce DS, as previously reported  and cultured in growth medium , supplemented with 10% FBS and 1% penicillin-streptomicin (Sigma Aldrich). To achieve RS, cells were serially passaged . Cells wreported .TM cDNA Synthesis Kit  and quantitative expression using the iQTM SYBR® Green Supermix , utilizing target specific human primers: p16INK4a , p14ARF , p21 , IL-1β , IL-6 , TNF-α , LMNB-1 ; TNFRSF10C , GAPDH , FBXO7 .Gene expressions of p16, p14, p21, IL-1β, IL-6, TNF-α, LMNB-1, and TNFRSF10C were measured through RT-qPCR, and normalized to the mean expression of two housekeeping genes, FBXO7 and GAPDH, as previously suggested . Total RTotal protein was obtained from cell pellets, which were previously washed with PBS, via the lysis buffer (Biorad). Samples were loaded on 15% polyacrylamide gels for electrophoresis and transferred to a nitrocellulose membrane (Biorad). After blocking for 1 h in 5% milk-tris buffered saline Tween (TBST), membranes were incubated in 5% milk-TBST solution containing the primary antibody overnight. After washing in TBST, the membranes were incubated with the Secondary IgG-HRP secondary antibody (Santa Cruz) for 2 h, ultimately being rinsed and developed by using the enhanced chemiluminescence (ECL) reagent (Biorad).Cells were fixed using the Cytofix Fixation Buffer  for 15 min, washed with Phosphate Buffered Saline (PBS), incubated with blocking buffer for 30 min . After adding primary antibodies in blocking buffer, cells were incubated overnight in the dark, washed and stained with secondary anti-bodies Alexa Flour 488 and 546 (Thermo Fisher Scientific). Cells were then washed with PBS and stained with DAPI (Thermo Fisher Scientific). Images were acquired using the Nikon Ni-E fluorescence microscope, with four fields of view per condition, containing at least 300 cells. Nuclear or cell size measurements and nuclear morphometric analysis were performed in Image J software (version 1.6), as described previously .2) at 37°C for 16 h. The enzymatic reaction was stopped by washing with cold PBS and cells were counted via bright-field microscopy.The protocol was performed as described by t-test was applied for comparison and visualized via Graph Pad Prism 8. In silico analysis of differentially expressed proteins (mean difference >1/<−1) were analyzed using g:Profiler web tool  by bleomycin, which induces double strand breaks . An analTo find reliable markers of senescence, we have compared NS (1 week of passaging) to RS (7 weeks of passaging) VSMC protein content with unbiased LC-MS technology . The expTo study the functional changes in senescent cells, we have performed a gene enrichment analysis of the top downregulated  and uprein vivo, these studies utilize mice with reporter genes inserted into the CDKN2A gene, which encodes the p16INK4a cell cycle inhibitor  and are thus an interesting anti-inflammatory therapeutic target .in vitro. Whether LMNB1 abundancy is actively regulated  remains unclear.We could show that senescent VSMCs demonstrate nuclear enlargement and cell morphology abnormalities in both RS and DS, a feature of senescence found in various cell types . In linein silico. However, some RS cells remain positive for ACTA2. Furthermore, RS VSMCs retain MYH11 and TAGLN expression, which is also consistent with previous studies for TAGLN  and protein level (LC-MS) shows that senescent VSMCs upregulate several pro-inflammatory factors. IL-1β is overexpressed in both RS and DS, while IL-6 seems to be upregulated in RS and “early-phase” DS. HMGB-1and HMGB-2 are key regulators of the SASP, and show downregulated intracellular levels in senescence . HMGB-1 nt VSMCs . IL-1α cnt VSMCs . HMGB-1 nt VSMCs . Althougnt VSMCs .Based on results above, it appeared that not all classical senescence markers display sufficient consistency and differential regulation to serve as reliable detection tools for senescent VSMCs. To tackle this issue, we have generated a proteomics dataset from RS samples. We could find global RNA metabolism processes highly enriched in our proteomic dataset, with mRNA splicing, transport and catabolism being significantly affected. Indeed, altered RNA splicing has been reported in senescence for specific molecules, such as p53 . This meIn summary, we found that VSMCs display an atypical molecular signature that only partially adheres to the commonly used senescence markers. Altered RNA metabolism is a consistent feature of senescent VSMCs, potentially facilitating their detection in future mechanistic and therapeutic studies.All datasets generated for this study are included in the article/in silico analysis. SS and MF wrote sections of the manuscript. DS and TT equally contributed in the supervision of the process. All authors contributed to manuscript revision, read and approved the submitted version.SS, MF, MK, KX, AJ, AP, JB, JF, DS, and TT contributed to the conception and design of the study. SS performed expression and microscopy experiments and wrote the first draft of the manuscript. AJ and AP performed the mass spectrometry. SS, MF, MK, KX organized the proteomics database. SS, MF, MK, KX, AP, and JF performed the statistical and TT has filed and licensed patents regarding non-coding RNAs in CVD. TT was the founder and shareholder of Cardior Pharmaceuticals GmbH. The remaining 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.


Of the 70,237 drug overdose deaths in the United States in 2017, approximately two thirds  involved an opioid  underlying cause-of-death codes X40–X44 , X60–X64 (suicide), X85 (homicide), or Y10–Y14 (undetermined intent). Among deaths with drug overdose as the underlying cause, the opioid subcategory was determined by the following ICD-10 multiple cause-of-death codes: all opioids Drug overdose deaths were identified in National Vital Statistics System multiple cause-of-death mortality filesChanges from 2017 to 2018 in age-adjusted overdose death ratesDuring 2018, drug overdoses resulted in 67,367 deaths in the United States, a 4.1% decrease from 2017. Among these drug overdose deaths, 46,802 (69.5%) involved an opioid. From 2017 to 2018, opioid-involved death rates decreased 2.0%, from 14.9 per 100,000 population to 14.6 ; decreasPrescription opioid-involved death rates decreased by 13.5% from 2017 to 2018. Rates decreased in males and females, persons aged 15–64 years, non-Hispanic whites, Hispanics, non-Hispanic American Indian/Alaska Natives, and across all urbanization levels. Prescription opioid–involved death rates remained stable in the Northeast and decreased in the Midwest, South, and the West. Seventeen states experienced declines in prescription opioid–involved death rates, with no states experiencing significant increases. The largest relative decrease occurred in Ohio (–40.5%), whereas the largest absolute decrease occurred in West Virginia (–4.1), which also had the highest prescription opioid-involved death rate in 2018 .Heroin-involved death rates decreased 4.1% from 2017 to 2018; reductions occurred among males and females, persons aged 15–34 years, non-Hispanic whites, and in large central metro and large fringe metro areas . Rates dDeath rates involving synthetic opioids increased from 9.0 per 100,000 population in 2017 to 9.9 in 2018 and accounted for 67.0% of opioid-involved deaths in 2018. These rates increased from 2017 to 2018 among males and females, persons aged ≥25 years, non-Hispanic whites, non-Hispanic blacks, Hispanics, non-Hispanic Asian/Pacific Islanders, and in large central metro, large fringe metro, medium metro, and small metro counties. Synthetic opioid–involved death rates increased in the Northeast, South and West and remained stable in the Midwest. Rates increased in 10 states and decreased in two states. The largest relative increase occurred in Arizona (92.5%), and the largest absolute increase occurred in Maryland and Missouri ; the largest relative and absolute decrease was in Ohio . The highest synthetic opioid–involved death rate in 2018 occurred in West Virginia .During 1999–2018, opioids were involved in 446,032 deaths in the United States.The findings in this report are subject to at least five limitations. First, postmortem toxicology testing varies by jurisdiction; improvements in testing might account for some reported increases. Second, the percentage of 2017 and 2018 death certificates with at least one drug specified varied among states and over time, limiting opioid subcategory rate comparisons. Third, because heroin is metabolized to morphine  of drug overdose deaths during 2018, representing decreases from 2017 in overdose death rates involving all opioids (2% decline), prescription opioids (14%), and heroin (4%); rates involving synthetic opioids increased 10%.Surveillance of overdose and polysubstance use trends and the illicit drug supply to track emerging threats, enhancing linkage to treatment, and a multisectoral response are critical to sustaining and accelerating declines in opioid-involved deaths.


Vibrio species of the Harveyi clade are commonly found in free-living and host-associated marine habitats. Here, we report the draft genome sequence for a Harveyi clade bacterium, Vibrio sp. strain LB10LO1, which was isolated from the Atlantic brief squid Lolliguncula brevis. Vibrio species of the Harveyi clade are commonly found in free-living and host-associated marine habitats. Here, we report the draft genome sequence for a Harveyi clade bacterium, Vibrio sp. strain LB10LO1, which was isolated from the Atlantic brief squid Lolliguncula brevis. Vibrio are widely distributed throughout marine systems where they inhabit both nutrient-rich and oligotrophic environments  agar  agar . Cultivaequences , 17, whiA single LB10LO1 colony was picked and streaked onto LBS agar plates and incubated overnight at 24°C. Genomic DNA was extracted from this clonal bacterial growth with a Zymo DNA miniprep kit, and the quantity and quality were determined using an Eppendorf BioSpectrometer. Library preparation was performed using a TruSeq DNA kit , following the manufacturer’s protocol. The library was sequenced using the MiSeq Illumina platform and 300-bp paired-end reads at the University of North Carolina (UNC) High-Throughput Genomic Sequencing Facility, resulting in a total of 1,798,287 paired reads. Raw reads were trimmed using Trimmomatic  using a N50 score of 165,319 bp. A total of 4,995 DNA coding regions were identified, including 4,891 encoding proteins and 104 encoding RNAs. Prokka produced annotated functions for 3,127 of the proteins, while the other 1,764 proteins were assigned as hypothetical. Finally, a MiGA  of >96%.The final draft genome of LB10LO1 is 5,515,790 bp long in 90 contigs , with a G+C content of 45.45%, 97-fold genome coverage, and an , a MiGA  and Geno, a MiGA  analysisPRJNA602499; the Illumina reads are available in the SRA under accession number SRX7614634. Cultures of LB10LO1 are available upon request.This genome sequence is available in GenBank under the BioProject number


Sézary syndrome (SS), an aggressive cutaneous T-cell lymphoma (CTCL) with poor prognosis, is characterized by the clinical hallmarks of circulating malignant T cells, erythroderma and lymphadenopathy. However, highly variable clinical skin manifestations and similarities with benign mimickers can lead to significant diagnostic delay and inappropriate therapy that can lead to disease progression and mortality. SS has been the focus of numerous transcriptomic-profiling studies to identify sensitive and specific diagnostic and prognostic biomarkers. Benign inflammatory disease controls  have served to identify chronic inflammatory phenotypes in gene expression profiles, but provide limited insight into the lymphoproliferative and oncogenic roles of abnormal gene expression in SS. This perspective was recently clarified by a transcriptome meta-analysis comparing SS and lymphocytic-variant hypereosinophilic syndrome, a benign yet often clonal T-cell lymphoproliferation, with clinical features similar to SS. Here we review the rationale for selecting lymphocytic-variant hypereosinophilic syndrome (L-HES) as a disease control for SS, and discuss differentially expressed genes that may distinguish benign from malignant lymphoproliferative phenotypes, including additional context from prior gene expression studies to improve understanding of genes important in SS. Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of skin homing T-cell malignancies, with mycosis fungoides (MF) and its leukemic variant Sézary syndrome (SS) accounting for the majority of cases. These two variants present with highly variable clinical skin inflammation that can be mistaken for benign mimickers, such as psoriasis, atopic dermatitis and other benign inflammatory dermatoses (BID) ,2,3. A cGene expression biomarkers have enormous potential to improve clinical practice for MF/SS, and innovative efforts have been taken to identify sensitive and specific diagnostic and prognostic biomarkers ,10,11,12A remaining challenge to the understanding of SS is the lack of a well-defined, pre/early-neoplastic T-cell population suitable for studies of stage progression, and the evolution of neoplastic phenotypes. However, this role was recently filled by a benign T-cell lymphoproliferative disorder known as lymphocytic-variant hypereosinophilic syndrome (L-HES) , which hIn this review, we will present the clinical and molecular similarities between SS and L-HES, discuss the rationale for using L-HES as a disease control for SS, and review the genes highlighted in a recent meta-analysis of SS and L-HES transcriptomes. We will also incorporate into perspective prior gene expression and functional studies of SS that together may distinguish characteristics of malignant and benign lymphoproliferative phenotypes, and their significance.1, +CD7− cells ≥ 30%, or CD4+CD26− cells ≥ 40% . A . A 33]. −CD4+, with frequent CD7 loss similar to SS  fusion, which is effectively targeted by imatinib therapy [Causes of eosinophilia can be broadly classified as primary/neoplastic or secondary/reactive. Primary eosinophilias are defined by the presence of clonal, neoplastic eosinophils, such as in chronic eosinophilic leukemia or myeloproliferative HES . Neoplas therapy . Seconda therapy . L-HES i therapy . Increas therapy ,48, and  therapy ,50. STAT therapy , and med therapy . The dis therapy . These f therapy . Thus, wSS gene expression profiles have been explored in a number of transcriptomic studies ,74,75,76−CD4+ T cells, with notable changes for growth control genes, including abnormally high expression of IL17RB (IL-25 receptor) and altered expression of transforming growth factor-β superfamily genes. Walker et al. [There are two published transcriptomic studies of atypical T cells from L-HES patients. Ravoet et al.  observedr et al.  describeDNM3, CCR4 and GATA3, which have appeared in diagnostic and prognostic gene-expression panels previously proposed for SS [PLS3, TOX and TWIST1, and reduced expression of STAT4, confirming their association with malignancy. By identifying groups of shared and SS-unique genes associated with benign and malignant phenotypes, respectively, this novel comparison offered a new perspective on abnormal gene expression in SS. The public L-HES data set from Ravoet et al.  was reced for SS ,11,15,16A limitation of the meta-analysis is the small number of cases included for both SS and L-HES. However, the SS and L-HES subjects have well-annotated clinical and immune characteristics that increase confidence in the results. In addition, we conducted a literature review of ten prior SS transcriptome studies  to deterTWIST1, PGN2L1, ANK1, IKZF2, KLHL42, NEDD4L, PLS3, ST8SIA1, TOX, and TPR , which is frequently expressed by cancer cells. The many roles for RANKL-RANK signaling in cancer-niche development, metastatic processes, neoangiogenesis, and immune escape have been reviewed recently [TNFSF11 in SS and L-HES is consistent with the enhanced proliferation of T cells. Increased expression of DNM3 was associated with a better overall survival in a large cohort of SS patients [DNM3 in SS and L-HES is surprising because DNM3 is a component of two multi-gene panels that differentiated SS from MF and BID cases including psoriasis, atopic dermatitis, and benign erythroderma with sensitivity and specificity over 95% [Genes with potential roles in lymphoproliferation were also concordantly regulated in SS and L-HES, including increased expression of ll death , and low in CTCL . TNFSF11recently . The shapatients , which ipatients . The shaover 95% ,16.CDCA7 is a shared gene with a previously unrecognized role in SS, despite its detection in several prior transcriptomic studies of SS [CDCA7 is frequently overexpressed in human cancers [CDCA7 expression may be important to the proliferative nature of SS and L-HES. CDCA7 has weak transforming activity of its own, and is a direct target of C-Myc oncoprotein [CDCA7 impaired lymphoma cell migration and invasion in in vitro and in vivo models [CDCA7 expression was an early event in the evolution of heterogeneous transcriptional states in SS clonal T cells [CDCA7 expression may provide a receptive environment for additional changes. In summary, many gene expression abnormalities shared by SS and L-HES T cells appear to fulfill roles that promote Th2-like and proliferative phenotypes. While SS biomarkers DNM3 and SATB1 may have promise as prognostic biomarkers, they do not reflect processes unique to malignant T cells.es of SS ,72,74. C cancers , includi cancers ,95. Thusoprotein . CDCA7 aoprotein , and silPLS3, TWIST1 and STAT4. In addition, the potential importance of some less well-recognized SS biomarker genes has been elevated by their new SS-unique status. SS-unique genes are associated with several cancer-promoting mechanisms including enhanced survival, oncogenic mi-RNAs, T-cell exhaustion and immunoregulation.The meta-analysis strategy comparing SS and L-HES is supported by the inclusion in the SS-unique category of many genes with rich publication histories in SS, such as PLS3 and TWIST1 are two frequently reported and highly expressed SS-biomarker genes with established roles in the epithelial to mesenchymal transition, and frequent association with disease progression in solid malignancies. PLS3 is one of the earliest SS biomarker genes identified and is consistently upregulated in the majority of SS cases [PLS3 expression is limited to the CD26 negative T-cell population [PLS3 expression correlates with blood disease burden in SS [PLS3 expression is a marker of circulating tumor cells with a metastatic phenotype, and this is associated with a poor prognosis [PLS3 expression has been associated with enhanced migration and apoptosis resistance [PLS3 expression in SS is associated with a better prognosis [PLS3 was expressed in only one of two divergent evolutionary transcriptional states that developed in a malignant T-cell clone [SS cases ,97,98, iSS cases , but is SS cases . Becauseen in SS . In solirognosis ,102. In rognosis . Single ll clone .TWIST1 in SS [Twist1 reduced effector cytokine expression in both Th1- and Th2-polarized cells [TWIST1 overexpression may vary between patients, resulting from promoter hypomethylation [TWIST1 gene [Numerous studies have reported overexpression of T1 in SS ,16,22,72T1 in SS ,104. In T1 in SS . This suT1 in SS . TWIST1 T1 in SS ,108, anded cells . In addihylation , or gainST1 gene .STAT4 expression is one of the most consistent findings in transcriptional studies of SS [STAT4 expression in CTCL lesional skin is associated with progressive disease. However, in a separate study, higher STAT4 expression was associated with HDACi resistance in MF/SS patients [Reduced es of SS . Howeveres of SS  first dees of SS  confirmepatients , suggestSTAT4 mRNA, miR-155 increases mutations by simultaneously interfering with DNA repair and cell cycle checkpoints [STAT4 expression loss in SS appears to be related to STAT5-mediated expression of oncogenic microRNA-155 (miR-155) ,115, as ckpoints , and hasckpoints . Phase 1ckpoints . TWIST1, PLS3 and STAT4 remain some of the most reliable diagnostic biomarkers of SS due to their selective dysregulation in malignant T cells, and absent expression in L-HES. Indeed, the combined altered expression of these three biomarker genes was demonstrated in a multicenter study to distinguish cases of SS from erythrodermic inflammatory dermatoses with 98% sensitivity and 100% specificity [In summary, + malignant T cells have suppressive activity [It has been suggested that the phenotype of SS T cells is plastic and may respond to microenvironmental factors ,120,121.activity , and areactivity .IKZF2, FCRL3, TIGIT and TOX have each been associated with Tregs and/or T-cell exhaustion. FCRL3 is an orphan Fc-like receptor with immunoregulatory properties [+FoxP3+ conventional Tregs [+ T cells in SS [+CD26− T cells expressing FCRL3 [CTCL T cells from lesional skin also exhibit phenotypes of exhausted T cells, potentially due to persistent antigen stimulation in the skin microenvironment. Increased surface expression of compensatory inhibitory receptors has been detected in early stage skin lesions, and a positive correlation was found between increased gene expression for checkpoint receptors and higher disease stage . The SS-operties , and theoperties ,128. Coeal Tregs , and sevls in SS ,122,130.ls in SS ,122, andng FCRL3 , suggestTOX expression correlates with increased disease-specific mortality in SS [TOX expression increases cell cycle regulators and apoptosis in CTCL cell lines, and greatly reduces the growth of CTCL tumor xenografts in mice [TIGIT and TOX was observed during the malignant progression of chronic L-HES to T-cell lymphoma [The transcription factor TOX is required for the thymic development of all CD4 T-cell lineages , while ity in SS , and prety in SS . Suppres in mice . In addilymphoma . These fANK1, GATA6 and HDAC9 have not been well characterized as SS biomarkers, but recent developments provide insight into their potential roles in SS. Overexpression of ANK1 was detected by several gene expression profiling studies [ANK1 harbors MIR486, which is also overexpressed in SS, and is involved in cell survival [ANK1 mRNA and miR-486 is activated in hematopoietic progenitor cells by MYB oncoprotein, in erythroid cells by GATA1 transcription factor, and in muscle cells by myocardin related transcription factor A . In theiewed in , MYB wasrget MAF  was signmotility . In the GATA6 is overexpressed and directly induces expression of CD137L, which promotes proliferation, survival and migration of SS T cells and CTCL cell lines [+, CD137L+ and CD137+ cells have been detected in all stages of MF/SS lesional skin compared to normal skin [GATA6 mRNA in SS T cells and CTCL cell lines is enhanced by DNA hypomethylation and histone acetylation [GATA6 expression is activated by epigenetic dysregulation common to CTCL T cells.The transcription factor GATA6 is widely associated with tumorigenesis , includill lines ,110,140.HDAC9, and the HDAC9 locus is strongly accessible only in leukemic T cells [HDAC9, ANK1, and GATA6 are biomarkers with SS-unique expression discovered by the comparison of SS and L-HES that may illuminate important roles in SS pathogenesis.HDACi therapy differentially modifies the distinct chromatin accessibility signatures observed in leukemic and host T cells from CTCL patients. The only HDAC gene differentially expressed between host and SS cells is  T cells . Several T cells ), but pr T cells , suggest T cells , and HDA T cells ,145. ThuIdentifying highly specific biomarkers for CTCL remains a challenge, but selecting informative controls can improve the outcome. SS and L-HES are skin-tropic diseases that share many clinical and immunological features that are mirrored in their gene expression profiles, which exhibit prominent Th2-like and lymphoproliferative signatures, more so than other inflammatory skin diseases. It is now clear that several previously described SS biomarkers are shared by L-HES, indicating that a reevaluation of their functional significance in oncogenesis unique to SS is needed. The remaining biomarker genes unique to SS are now more focused on malignant gene expression phenotypes, including immunomodulation, EMT, cell survival, and co-expression of oncogenic miRNAs. The refocused SS-unique gene set includes genes that have received little prior attention in SS, presenting an opportunity to gain insight from these less-studied genes  which maIn closing, comparative analysis of SS and L-HES gene expression identified subsets of genes that are unique to each disease, and will serve to improve diagnostic accuracy. A new focus on gene expression associated only with the malignant T-cell phenotype in SS may also illuminate potential therapeutic targets for these T-cell diseases. Understanding the roles of these genes in SS, and the processes by which these genes become dysregulated will yield insight into the mechanisms driving these rare and diagnostically challenging diseases.


SGLT-2 inhibitors demonstrated significantly lower hemoglobin A1c (HbA1c)  and body weight  at end of study compared to baseline level. There were no significant changes in eGFR, serum creatinine, urine protein creatinine ratio, and blood pressure. By subgroup analysis, empagliflozin demonstrated a significant reduction in body mass index (BMI) and body weight. Canagliflozin revealed a significant decrease in HbA1C and systolic blood pressure. In terms of safety profiles, fourteen patients had urinary tract infection. Only one had genital mycosis, one had acute kidney injury, and one had cellulitis. There were no reported cases of euglycemic ketoacidosis or acute rejection during the treatment. Conclusion: Among kidney transplant patients with excellent kidney function, SGLT-2 inhibitors for treatment of DM are effective in lowering HbA1C, reducing body weight, and preserving kidney function without reporting of serious adverse events, including euglycemic ketoacidosis and acute rejection.Background: The objective of this systematic review was to evaluate the efficacy and safety profiles of sodium-glucose co-transporter 2 (SGLT-2) inhibitors for treatment of diabetes mellitus (DM) among kidney transplant patients. Methods: We conducted electronic searches in Medline, Embase, Scopus, and Cochrane databases from inception through April 2020 to identify studies that investigated the efficacy and safety of SGLT-2 inhibitors in kidney transplant patients with DM. Study results were pooled and analyzed utilizing random-effects model. Results: Eight studies with 132 patients (baseline estimated glomerular filtration rate (eGFR) of 64.5 ± 19.9 mL/min/1.73 m Diabetes mellitus (DM) is the leading cause of end-stage kidney disease (ESKD) worldwide . In the Sodium-glucose co-transporter 2 (SGLT-2) inhibitors are novel oral glucose-lowering medications that inhibit glucose reabsorption in the proximal tubule and promote renal excretion of glucose . GlycemiTherefore, we conducted the present systematic review and meta-analysis to assess the efficacy and safety of SGLT-2 inhibitors for treatment of DM among kidney transplant recipients.A comprehensive search of several databases from each database’s inception to 30 April 2020, any language, was conducted. The databases included Ovid MEDLINE(R) and Epub Ahead of Print, In-Process and Other Non-Indexed Citations, Daily, Ovid EMBASE, Ovid Cochrane Central Register of Controlled Trials, Ovid Cochrane Database of Systematic Reviews, and Scopus. The search strategy was designed and conducted by an experienced librarian with input from the study’s principal investigator. Controlled vocabulary supplemented with keywords was used to search for studies of SGLT-2 inhibitors in kidney transplant patients. The actual strategy listing all search terms used is available in the Given limited data, our study included any studies, including case series and randomized controlled trials, examining the efficacy and safety of any SGLT-2 inhibitors among kidney transplant patients. We excluded case reports or animal studies. Case series were defined as a study that included multiple cases receiving SGLT-2 inhibitors without a control group. For cohort studies or randomized controlled trials, we focused only on treatment group with SGLT-2 inhibitors in order to be comparable and consistent with case series. The quality assessment tool for case series studies proposed by the National Heart, Lung, and Blood Institute (NHLBI) was used to rate the quality . StudiesRetrieved articles were individually reviewed for eligibility by two investigators (A.C. and N.P.) utilizing a systematic review management program called Covidence, which has been selected by Cochrane to become a standard production platform for their reviews. Disagreements were addressed and resolved by third investigator (W.C.).The following data were extracted: first author name, year of publication, number of participants in SGLT-2 inhibitors group, duration of follow-up, types of SGLT-2 inhibitors, mean age, sex, and duration of transplantation. Efficacy outcomes included estimated glomerular filtration rate (eGFR), serum creatinine, urine protein-creatinine ratio, HbA1C, systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), and body weight. Safety profile outcomes included incidence of urinary tract infection, genital mycosis, euglycemic ketoacidosis, acute kidney injury, acute rejection, ulcer, and cellulitis.2 statistic and the chi-square test. A value of I2 of 0% to 25% represents insignificant heterogeneity, 26% to 50% low heterogeneity, 51% to 75% moderate heterogeneity, and 76 to 100% high heterogeneity ; p = 0.06, I2 = 0%) (Seven studies with 124 patients treated with SGLT-2 inhibitors did not show a significant decline in eGFR between baseline and the end of the study (WMD = −2.51 mL/min/1.73 mI2 = 0%) .p = 0.21, I2 = 0%). Three studies with 38 participants treated with SGLT-2 inhibitors reported no significant difference in urine protein creatinine ratio between levels at baseline and end of study   .2 ; p = 0.09, I2 = 0%) or between levels at baseline and at 12 months among 4 studies with 68 participants . In terms of urine protein creatinine ratio, there was no significant difference between levels at baseline and at 6 months . For serum creatinine, there was no significant difference between levels at baseline and at 12 months  (There was no significant decline in eGFR comparing between levels at baseline and at either 6 months among 5 studies with 72 participants (WMD = −2.38 mL/min/1.73 mI2 = 0%) .p = 0.006, I2 = 85.2%)  .p = 0.03, I2 = 79.8%). However, comparing HbA1C levels at 6 months and baseline, 5 studies with 72 participants showed no significant difference   .p = 0.18, I2 = 21.3%) and DBP . Eight studies with 132 participants treated with SGLT-2 inhibitors demonstrated a significant decrease in body weight between baseline and the end of study . However, there was no significant change in BMI between at baseline and end of study among 3 studies with 38 participants treated with SGLT-2 inhibitors   .2 ; p = 0.007, I2 = 0%) and body weight  than baseline, while at 12 months, only body weight was significantly lower than baseline  but not BMI   .In total of 8 studies, 14 out of 132 participants had urinary tract infection. One participant out of 72 participants from 5 studies had genital mycosis. No euglycemic ketoacidosis were reported in four studies. Based on 4 studies, 1 out of 28 participants had acute kidney injury. One study reported one small ulcer in lower extremity and another study reported one case of cellulitis. Based on five reported studies, no rejections were found during follow-up period .2 ; p = 0.006, I2 = 0%). Four studies with 48 participants treated with empagliflozin showed a lower body weight at end of study compared to baseline . There were no significant changes in kidney function, HbA1C, SBP, and DBP between baseline and at end of study among participants treated with empagliflozin. On the other hand, 2 studies with 34 participants treated with canagliflozin reported a significant decline in HbA1C  and SBP . There were no significant changes in kidney functions, BMI, and body weight between baseline and at end of study among participants treated with canagliflozin . Moreover, a funnel plot for eGFR of the included studies did not suggest any asymmetry. Therefore, publication bias was less likely to occur in our meta-analysis , small ulcers in lower extremities (10%), cellulitis (10%), AKI (3.6%), and genital fungal infection (1.4%), respectively. No euglycemic ketoacidosis and acute rejection events were reported.SGLT-2 inhibitors inhibit sodium-glucose reabsorption at proximal renal tubules, causing osmotic diuresis and natriuresis . As a reIn terms of blood pressure, theoretically, a decrease in body weight combined with osmotic diuresis and reduction of total body sodium can result in lowering blood pressure around 4–6 mmHg in SBP and 1–2 mmHg in DBP, as shown in non-kidney transplant patients with DM . HoweverIn addition to the effect on diminishing intraglomerular hyperfiltration and hypertension, SGLT-2 inhibitors also demonstrate anti-inflammatory, antifibrotic, and protective effects against deregulation of extracellular matrix evidenced by a reduction in serum levels of TNF receptor 1, IL-6, matrix metalloproteinase 7, and fibronectin 1 . Moreovep = 0.13). This was supported by previous meta-analysis of non-kidney transplant diabetic population, which revealed an increased risk of genital infection but found no significant increased risk of urinary tract infection [2) during the first ~3–6 weeks of treatment SGLT-2 inhibitors has been observed among non-kidney transplant patients, and data has supported this [In light of safety profiles of SGLT-2 inhibitors among kidney transplant recipients with DM, the incidence of urinary tract infection was 43.8% in our study vs. 38.0% among general kidney transplant recipients , which wnfection . In our nfection ,69. Neveted this ,70. Loweted this ,70. Neveted this . While oThere are some limitations in our meta-analysis. Firstly, included studies were mostly case series without a control group, and the sample size was fairly small. Secondly, the median time of follow-up was 12 months. Hence, some complications might not be have been observed during 1–2 years of follow-up. Moreover, we did not have a control group to compare to, so given the design of each study and insufficient data from each study, we could not adjust for potential confounders such as concomitant antidiabetic medications and duration of diabetes. Furthermore, the data on cardiovascular outcomes were lacking. Therefore, large randomized controlled trials with long-term follow-up are required to investigate the effects of SGLT-2 inhibitors on outcomes after kidney transplantation, including cardiovascular events, delayed graft rejection and mortality risk, as well as safety profiles such as hypovolemia . ConversIn conclusion, our study suggested that SGLT-2 inhibitors were effective in lowering HbA1C when treated at least 12 months, reducing body weight when treated at least 6 months, and preserving kidney function among kidney transplant recipients with DM without reported serious adverse events, including euglycemic ketoacidosis and acute rejection. Based on our findings, future randomized controlled trials are required to further investigate the efficacy and safety of SGLT-2 inhibitors, including long-term allograft and patient survival.


Multiple mRNA isoforms are often generated during processing such as alternative splicing of precursor mRNAs (pre-mRNA), resulting in a diversity of generated proteins. Alternative splicing is an essential mechanism for the functional complexity of eukaryotes. Temperature, which is involved in all life activities at various levels, is one of regulatory factors for controlling patterns of alternative splicing. Temperature-dependent alternative splicing is associated with various phenotypes such as flowering and circadian clock in plants and sex determination in poikilothermic animals. In some specific situations, temperature-dependent alternative splicing can be evoked even in homothermal animals. For example, the splicing pattern of mRNA for a cold shock protein, cold-inducible RNA-binding protein (CIRP or CIRBP), is changed in response to a marked drop in body temperature during hibernation of hamsters. In this review, we describe the current knowledge about mechanisms and functions of temperature-dependent alternative splicing in plants and animals. Then we discuss the physiological significance of hypothermia-induced alternative splicing of a cold shock protein gene in hibernating and non-hibernating animals. Alternative splicing of precursor mRNAs (pre-mRNAs) is a process in which exons, parts of exons, and/or parts of introns are combinatorially included into mature RNA ,3,4,5,6.Splicing patterns can be changed in response to environmental factors including temperature. Temperature is a fundamental physical quantity that is involved in all life activities at various levels . In planMultiple mRNA isoforms are often generated by alternative splicing of pre-mRNA, resulting in the diversity of generated proteins ,8,9. VarIn response to changes in ambient temperature, plants show multiple phenotypes such as stem elongation, leaf hyponasty, and flowering ,20,21,22FLM), which is a MADS-domain transcription factor [FLM is subjected to temperature-dependent alternative splicing in Arabidopsis [FLM, FLM-β and FLM-δ, differ in the incorporation of either the second or third cassette exon [FLM-β) and as a promoter (FLM-δ) of flowering [FLM-β to FLM-δ alters in response to ambient temperature [FLM-β is expressed predominantly and FLM-β forms a protein complex with the MADS-domain transcription factor short vegetative phase (SVP), which also functions as a floral repressor [FLM-δ is spliced abundantly and FLM-δ interacts with SVP and works as a dominant-negative isoform of FLM [FLM is a critical molecular action for the FLM-mediated control of flowering in response to thermal change [Flowering time in plants is regulated in response to change in ambient temperature ,26. The n factor ,23,27,28bidopsis ,34,35,36tte exon ,27,37. Tlowering ,23,27,37perature ,29. At aepressor ,23 and cepressor ,27,35. Om of FLM ,27. The m of FLM ,23,27, rl change . MADS affecting flowering 2 (MAF2), which is related to flowering time like FLM in Arabidopsis, also undergoes temperature-dependent alternative splicing to produce two variants, MAF2var1 and MAF2var2 [MAF2var1 is expressed predominantly at a low ambient temperature and its encoded protein forms a repressive complex with SVP to inhibit flowering [MAF2var2 is induced [MAF2var2 cannot interact with SVP, resulting in promotion of flowering [MAF2var2 ,39,40,41lowering ,40,41,42 induced ,40,42. Tlowering .pseudo response regulator (PRR) genes, including PRR7, PRR9 and timing of CAB expression 1  [TOC1 through direct binding to its promoter [The circadian clock produces oscillations of an approximately 24-h period, which allows organisms to synchronize biological phenomena with the day-night cycle ,43. In p1; PRR1) ,44,46,511; PRR1) ,51,52,53promoter ,54,55. CCA1 pre-mRNA produces two splice variants, CCA1α and CCA1β [CCA1β is increased. CCA1β has a dimerization domain but lacks the N-terminal DNA-binding MYB motif [There have been many reports on post-transcriptional regulatory mechanisms of circadian clock genes ,25,56. Cnd CCA1β ,59,60,61YB motif . Thus, CYB motif . In contYB motif ,61,64. CCA1, the circadian clock genes LHY, TOC1, PRR3, PRP5, PRP7, and PRR9 are also subjected to temperature-dependent alternative splicing [LHY, exon 4 skip in PRR7, retention of intron 4 in TOC1, and three and two alternative splicing events in PRR3 and PRR5, respectively [LHY transcript at low temperature [LHY and photoperiod-H1 (PPD-H1) (PRR7 orthologue) in response to a low temperature in barley [PRR7 that retains an intron, whereas PRR9 shows an increase in both full length mRNA and a splice variant with an intron retention after both cold and heat stresses [In addition to splicing ,64. Jameectively . Severalectively . For exaperature . Calixton barley . In addistresses ,64. frequency (FRQ) is subjected to temperature-dependent alternative splicing [FRQ are important for temperature compensation of circadian rhythms. Diernfellner et al. demonstrated that the ratio of l-FRQ versus s-FRQ is regulated by temperature-dependent alternative splicing of intron 6 of FRQ and then expression of l-FRQ increases with elevating temperature, while s-FRQ is expressed at low levels [In addition to investigations of circadian clock genes in plants, it has also been shown that temperature-dependent alternative splicing is essential for the regulation of circadian clock in Neurospora, Drosophila, tilapia and mice ,74,75,76splicing ,68,69. Eperiod (PER) and timeless (TIM) protein and mRNA products undergo daily fluctuations in relation to the circadian clock [PER gene plays a key role in adaptation of a circadian clock to seasonally cold days (low temperatures and short day lengths) in Drosophila [PER mRNA and protein cycles, which contributes to the preferential daytime activity of flies on cold days [TIM in Drosophila [TIM levels are low because of the induction of two cold-specific isoforms, while another isoform is upregulated at 29 °C. The switching of isoform controls the levels and activity of TIM, suggesting that alternative splicing of TIM might act as a thermometer for the circadian clock [PER gene is also affected by cold stress [In Drosophila, an clock ,73,74,77d stress .U2 snRNP auxiliary factor (U2AF) 26 gene is driven by rhythmic body temperature changes, and then skipping of two variable exons (6 and 7) of U2AF26 generates a novel protein variant of U2AF26 that directly controls circadian clock resetting in vivo [Alternative splicing of circadian clock genes has been demonstrated in mice. For example, alternative splicing in  in vivo ,75,76. Taken together, these findings indicate that many clock-related genes are subjected to temperature-dependent alternative splicing. It has been speculated that alternative splicing might contribute to the control of circadian clock functions in a temperature-dependent manner.SRY-box9 (Sox9), Wilms tumor 1(WT1) and doublesex and mab3-related transcription factor 1 (Dmrt1) are involved in TSD [Sox9 in crocodiles, WT1 in turtles, and Dmrt1 in turtles and crocodiles are subjected to alternative splicing and play a role in sex determination [WT1 mRNA, which influences sex determination in the snaping turtle [Sex determination is a pivotal developmental process . There ad in TSD ,84,85,86d in TSD . In accomination ,83,84,86U2AF65A [cyclin-dependent kinase G1 (CDKG1) [SR1 [polypyrimdine tract-binding protein (PTB) 1/2, and suppressor of abi3-5 (SUA) [U2AF26 in mice [serine-arginine-rich (SR) splicing factors in Tilapia [Temperature can modulate the splicing of splicing regulators. For example, U2AF65A , cyclin- (CDKG1) ,88, SR1 G1) [SR1 , polypyr-5 (SUA) , which a in mice ,75,76 an Tilapia  are alsoroot initiation defective1 (RID1) [apetala3 (AP3)-1 [Genes related to plant growth and development are also subjected to temperature-dependent alternative splicing. In Arabidopsis, it has been demonstrated that temperature-dependent alternative splicing of 1 (RID1)  and apet (AP3)-1  affects OsbZIP58 affects grain filling of rice [Bvnpcg2 and Bvnpcg3 are subjected to temperature-dependent alternative splicing, resulting in regulation of the sensitivity to seasonal temperature changes [MtJMJC5, a jumonj C domain-containing demethylase homologue, is involved in the circadian clock [Most of the temperature-dependent alternative splicing studies in plants have focused on Arabidopsis. However, there are several reports about plants other than Arabidopsis. It was shown that temperature-dependent alternative splicing of  of rice . In suga changes . In Median clock . Although mammals have thermosensory molecules such as family of transient receptor potentials (TRPs) ,97,98,99CGE1 as a chloroplast homolog of GrpE in Chlamydomonas and showed that temperature-dependent alternative splicing generates two isoforms of CGE1, CGE1a and CGE1b [CGE1a is expressed predominantly at lower temperatures, but expression of CGE1b increases as well as CGE1a in response to elevated temperatures, which might affect chaperone function [GrpE proteins function as nucleotide exchange factors for DnaK-type Hsp70s, which is a molecular chaperone ,106,107.nd CGE1b . CGE1a iAPE2, which encodes an amino peptidase, is affected by temperature change (heat shock) [In addition to plants and animals, temperature-dependent alternative splicing has been reported in yeast ,110,111.t shock) ,111.Dnml1 and Mff genes [Several studies on temperature-dependent alternative splicing of pre-mRNAs have been performed by using mammalian cultured cells. Gemignani et al. used HeLa and K562 cell lines that stably express thalassemic β-globin genes with mutations inducing aberrant splicing patterns of pre-mRNAs . When thff genes . Although many genes regulated by temperature-dependent alternative splicing have been identified, molecular mechanisms of temperature-dependent alternative splicing are poorly understood. Temperature can influence the speed of RNA polymerase II, which is known to affect splicing ,115. In Temperature-dependent splicing might also be involved in more complex regulatory steps than direct temperature-driven changes in RNA secondary structure . For exaLHY in Arabidopsis. They showed that the splicing and expression of several SFs respond sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of LHY pre-mRNA [It is interesting that temperature can modulate the splicing of splicing regulators such as SR proteins ,118,119.pre-mRNA . Melzer reported that the presence/absence of SFs may contribute to alternative splicing . He alsoTIM in Drosophila, regulating the period of circadian rhythms and circadian behavior phase during temperature cycling [Foley et al. demonstrated that the alternative splicing regulator P-element somatic inhibitor (PSI) regulates the temperature-dependent alternative splicing of  cycling . It has been reported that trimethylated histone H3 at lysine 36 (H3K36me3) is enriched in genes undergoing alternative splicing in mammals ,121,122.Some mammalian species including squirrels and hamsters undergo hibernation ,125. AltOriginally, we were interested in the protective mechanisms operating in hibernating animals. As a molecular candidate involved in the mechanism, we focused on cold shock proteins including as CIRP and RNA-binding motif 3 (RBM3) . This isCIRP mRNA in the heart of a non-hibernating euthermic hamster [CIRP mRNAs were detected, whereas only the shortest variant among them was expressed in hibernating animals (referred to as short-form dominant pattern). The pattern of splicing was commonly observed in various organs [CIRP expression is regulated at the level of pre-mRNA splicing, rather than transcriptional level, when entering torpor. At that stage, we believed that the regulation of alternative splicing is specific for hibernators because it is inconceivable that non-hibernators will be in an extreme hypothermic condition.Owing to the prominent action of cold shock proteins, we considered that these proteins might help to protect organs including the heart against a harmful low temperature during hibernation. At the beginning of our experiments, we simply expected that cold shock proteins are expressed, if any, at very low levels, and that their expression is increased before or when entering a deep hypothermic state (torpor) in hamsters. However, RT-PCR analysis led to two unexpected results. One result was that there was substantial expression of  hamster . The oths organs . These fCIRP during hibernation in hamsters. It is generally considered that the cold resistance property is established during preparatory processes before entering hibernation [CIRP would occur when hamsters were kept in a condition suitable for induction of hibernation. Our experiments, however, showed that the short-form dominant pattern was not induced even after adaptation to a cold environment with short photoperiod [We investigated the factors causing the shift in alternative splicing of ernation . Therefotoperiod , which iWe then tested whether the splicing pattern can be caused in artificial hypothermia. An adenosine A1-receptor agonist was administrated centrally to induce hibernation-like hypothermia ,128. SinCIRP transcripts. If that is the case, the short-form dominant pattern should be reproduced by maintaining hamsters in a state of mild hypothermia for a substantial period of time even in the case of hypothermia induced by anesthetics. Predictably, maintenance of body temperature in a mild hypothermic state (~ 30 °C) for 2 h elicited a shift in the pattern [CIRP is mild hypothermia . Therefore, the control system of CIRP expression at the level of alternative splicing, which was originally found out in hibernating hamsters, may be commonly present in non-hibernators such as mice.At the final stage of a series of experiments, we examined whether the temperature-dependent alternative splicing of hamsters . When mihamsters . In lineroblasts ,138. In CIRP mRNA is expressed in the heart of a non-hibernating euthermic hamster with several different forms probably due to alternative splicing [CIRP transcripts.As mentioned above, splicing ,14. Amonsplicing ,139. Thesplicing . The finCIRP expression at the level of splicing is a fundamental event for survival at a low temperature. In hamsters, the occurrence of cardiac arrhythmias and/or cardiac damage is correlated with the pattern of alternative splicing of CIRP transcripts (CIRP transcripts (We believe that the temperature-dependent switching of the splicing pattern plays a significant role in protection of cells against untoward effects of hypothermia, although no definitive evidence has so far been provided. One of the supportive findings is a simultaneous occurrence of the shift in the splicing pattern in most organs. Taking into consideration the fact that the reaction is commonly observed in mice and rats, it is probable that the regulation of nscripts . Regardlnscripts . It is nTemperature-dependent alternative splicing of pre-mRNAs is widely conserved in eukaryotes including both plants and animals. The post-transcriptional regulatory system affected by the temperature shift plays important roles in many physiological and developmental processes such as flowering, circadian clock, sex determination, thermosensing, stress response and hibernation. Further studies on temperature-dependent alternative splicing may provide a breakthrough in understanding multiple functions that are associated with adaptation to change in ambient or body temperatures.CIRP transcripts is elicited depending on change in body temperature during hibernation [CIRP transcripts is not specific to hibernators but also occurs in non-hibernators. Therefore, the establishment of a method for inducing hibernation-like hypothermia in humans would be feasible.We have demonstrated that a shift in the alternative splicing pattern of ernation ,14. Here