The innovative development of azapeptide analogues of growth hormone releasing peptide-6 (GHRP-6) has produced selective modulators of the cluster of differentiation 36 receptor (CD36). The azapeptide CD36 modulators curb macrophage-driven inflammation and mitigate atherosclerotic and angiogenic pathology. In macrophages activated with Toll-like receptor-2 heterodimer agonist, they reduced nitric oxide production and proinflammatory cytokine release. In a mouse choroidal explant microvascular sprouting model, they inhibited neovascularization. In murine models of cardiovascular injury, CD36-selective azapeptide modulators exhibited cardioprotective and anti-atherosclerotic effects. In subretinal inflammation models, they altered activated mononuclear phagocyte metabolism and decreased immune responses to alleviate subsequent inflammation-dependent neuronal injury associated with retinitis pigmentosa, diabetic retinopathy and age-related macular degeneration. The translation of GHRP-6 to potent and selective linear and cyclic azapeptide modulators of CD36 is outlined in this review which highlights the relevance of turn geometry for activity and the biomedical potential of prototypes for the beneficial treatment of a wide range of cardiovascular, metabolic and immunological disorders. In modern biomedical science, the transformation of biologically active peptides into potent and selective therapeutic prototypes is a challenge requiring an interplay of chemical synthesis and biological analysis. Over more than a decade of such collaborative research, unselective peptide hits exhibiting binding affinity for the cluster of differentiation 36 receptor (CD36) were transformed into potent and selective azapeptide leads with promising medicinal potential. In the absence of structural data for CD36 alone nor in complex with small molecules, lead optimization relied on structure–activity relationship data obtained from different assays designed to assess the pleiotropic effects of the receptor. During this research novel methods were conceived for the synthesis of linear and cyclic azapeptides. Certain azapeptides were shown to reduce inflammation by a mechanism featuring binding of CD36 and disruption of its interaction with the Toll-like receptor (TLR)-2/6 heterodimer to subsequently perturb downstream signaling and attenuate the pro-inflammatory cascade. In choroidal explant models, some azapeptides reduced neovascularization mediated by the TLR-2 heterodimer. Other CD36-selective azapeptides exhibited cardioprotective effects associated with an increase in circulating adiponectin levels. This review focuses on the chemistry leading to a better understanding of the topological requirements for binding and regulating CD36 activity towards the discovery of novel prototypes for treating cardiovascular, metabolic and immunological conditions. N- and C-termini with short cytosolic domains that are anchored into the membrane by four palmitoylation sites. Functioning as a class B scavenger receptor, CD36 is involved in the regulation of inflammatory processes, inhibition of microvascular angiogenesis, transport of oxidized lipoproteins and phospholipids, phagocytosis of microorganisms and cells, and autophagy indol-4-one-2-carboxylic acid) exhibited cardioprotective activity, reduced myocardial fatty acid uptake and prevented non-esterified fatty acid mobilization from adipose tissue  revealed that the lysine-rich Asn132-Gln177 extracellular domain which interacted with GHRP-6 ligands overlapped with the oxLDL binding site [164 and Lys166 have been found to directly contribute to oxLDL ligand binding to CD36 [Topographical mapping of the binding hotspot domains of CD36 with a benzophenone alaninyl (Bpa) hexarelin derivative [Tyr-Bpa-Ala-His-D-(2-Me)Trp-Ala-Trp-D-Phe-Lys-NHing site . Within  to CD36 . Conside to CD36 .50 values) of peptide ligands for CD36 and the GHS-R1a receptor (GHS-R1a) were respectively determined by competitive binding assays using [125I]-radiolabeled photoactivatable Bpa-GHRP-6 derivative 3 as tracer and by a ghrelin binding assay with [125I]-ghrelin as radiotracer GHRP-6 (6) provided analogues which maintained CD36 binding affinity with improved selectivity . Although micromolar affinity to both the CD36 and GHSR-1a receptors was conserved upon deletion of Lys6 from [azaLeu3]GHRP-6 (6), further truncation was unfruitful, and replacement of His1 for proline, propionic acid, glycine and D-alanine, all reduced binding affinity by ~103 for the GHSR-1a receptor, but did not improve CD36 binding affinity (data not shown) GHRP-6 derivatives were synthesized using a variety of substituted benzyl halides in the semicarbazone alkylation step and their structure–activity relationships (SAR) were examined in vitro. Moreover, the submonomer approach gave access to a broader diversity of azapeptides by arylation, Michael addition and conjugate addition-elimination chemistries (vide infra) [In an example of the “submonomer” method  27], be, be27], e infra) .ex vivo microvascular sprouting assay using mouse choroid explants. For example, although [azaTyr4]GHRP-6 (9) and GHRP-6 (15) displayed similar receptor binding affinities and CD spectra with curve shapes indicative of β-turn conformations [9 exhibited anti-angiogenic activity and aza-phenylalanine analogue 15 had limited if not a slightly pro-angiogenic effect on the choroidal explants. Quantification of microvascular sprouting after 4-day treatment with [azaTyr4]GHRP-6 (9) revealed 17% choroid sprouting, validating its antiangiogenic properties compared to the control, vehicle condition which produced 53% neovascularization. On the other hand, -GHRP-6 (15), which exhibited high binding affinity and CD36 selectivity, increased slightly neovascularization compared to the control, likely due to the activation of Src kinase recruitment and a vascular endothelial growth factor (VEGF)-driven protein kinase B (Akt) phosphorylation pathway [4]-GHRP-6 analogues possessing the parent His1 residue, an Ala1 substitution, and diverse side-chain modifications at the azaPhe4 residue GHRP-6 (15) series 28} in lieu of histidine {e.g., [azaPhe4]GHRP-6 (8) series 29} to examine the influence of the N-terminal residue. Intrigued by the similar β-turn CD spectra and divergent effects on angiogenesis of lead azaPhe-residue . AromatiR)-fibroblast-stimulating lipopeptide (R-FSL-1). The physiological mediator NO is a marker of inflammation that can be readily trapped to provide a measurable fluorescent adduct [4]-GHRP-6 (8) and -GHRP-6 (15) showed respectively no effect and the most significant ability to decrease NO production, but neither influenced vascular growth in the angiogenesis assay -GHRP-6 analogues exhibited divergent effects on choroidal neovascularization. Nitric oxide (NO) production by immune cells such as monocytes and macrophages has been considered an index of the inflammatory process in response to stimuli such as TLR agonists, proinflammatory cytokines and interferon γ. Azapeptide analogues were examined for their ability to reduce NO production in macrophages activated with the TLR-2 agonist (t adduct . The redis assay . The CD34]- and [aza(4-F)Phe4]-GHRP-6 (9 and 29b). Moreover, anti-angiogenic trends were exhibited by analogues with other 4-position electronegative groups [29d (4-Br) and 29f (4-MeO)]. In contrast, [aza(4-n-PrO)Phe4]-GHRP-6 (29h) increased neovascularization. The subtle effects of the N-terminal residue were indicated further by the anti- and pro-angiogenic trends exhibited respectively by -GHRP-6 (28c) and [aza(4-Cl)Phe4]-GHRP-6 (29c). In contrast to the relatively high binding affinity and anti-angiogenic effects of [azaTyr4]GHRP-6 (9), [aza(3-HO)Phe4]GHRP-6 (29n) exhibited about 15-fold lower CD36 binding affinity and no effect on microvascular growth. Anti-angiogenic activity was demonstrated by [azaTyr4]GHRP-6 analogues by synthetic approaches that delivered respectively aza-arylglycine and aza-1-aryl-2,3-triazole-3-alanine residues . This chemistry featured respectively Cu-catalyzed N-arylation of azaGly semicarbazones GHRP-6 analogues 33 and 34 exhibited Kd values around 2-10-fold lower than GHRP-6 (1) in the SPR binding assay (unpublished results). Side chain diversity was expanded beyond the benzyl substituents in the GHRP-6 (35b) and [aza-(ethyl diethylphosphoryl)Gly3]GHRP-6 (35e). In contrast, [azaGlu3]GHRP-6 (35a) and [aza-(ethyl methylphosphoryl)Gly3]GHRP-6 (35c) bound with ~10-fold lower affinity, and [aza-(ethyl dimethylphosphoryl)Gly3]GHRP-6 (35d) exhibited > 500-lower CD36 binding affinity. At the Trp4 position, compared to hexarelin (38), less significant decreases in CD36 binding affinity were exhibited by azaGlu analogues {e.g., [azaGlu4]GHRP-6  and [aza-(E/Z)-(2-carboxy-p-nitrocinnamyl)Gly4]GHRP-6 } compared to non-ionic aliphatic aza-residues: [aza-(ethyl methylphosphoryl)Gly4]- (36b), [aza-(ethyl diethylphosphoryl)Gly4]- (36d) and [aza-(E/Z)-(2-cyano-p-methoxycinnamyl)Gly4]GHRP-6 (37b) all showing >103-fold drops in binding affinity. The relatively better binding affinity of the azaGlu4 analogues may be due to potential to engage in salt bridges with CD36 GHRP-6 (47a) was found to be inactive and used subsequently as a negative control in studies of azapeptide CD36 modulator activity -GHRP-6 (49f) exhibited therapeutic potential in a model of atherosclerosis (vide infra). In addition, intramolecular variations of the A3-coupling reaction were developed to access cyclic azapeptide derivatives of GHRP-6 with promising results GHRP-6 analogues with various substituents on the amino group, different degrees of side chain saturation and chain length, as well as azaLys residues placed respectively at the Alaositions . Analysiactivity . Moreove results ,38,39.4]GHRP-6 (9) -GHRP-6 (9) -GHRP-6, which exhibited respectively similar and no influence at 10–6 M and 10–7 M, cyclic azapeptides 57b–d caused significant inhibition of R-FSL-1-induced NO production at 10–7 M [59 and 60 reduced NO overproduction with less than half the activity as azapeptide counterparts 56 and 57c with the (S)-isomers exhibiting a stronger influence than the (R)-counterparts [Biological screening demonstrated that azacyclopeptide GHRP-6 analogues RP-6 (9)  and [N-At 10–7 M . Cyclic terparts .57c exhibited unprecedented CD36 binding affinity (IC50 0.08 μM) in the competitive binding assay against photoactivatable [125I]-Tyr-Bpa-Ala-hexarelin (3) as radiotracer . Pr. Pr65 wa4]-GHRP-6 {[(4-F)AsF4]-GHRP-6, 65c} exhibited significant ability to reduce NO overproduction [125I]-Tyr-Bpa-Ala-hexarelin (3), [(4-F)AsF4]-GHRP-6  had respectively 1.3-fold lower and 2.4-fold higher CD36 binding affinities relative to the semicarbazide counterparts, [azaTyr4]- and [aza(4-F)Phe4]-GHRP-6 (9 and 29b). The subtle switch from a carbonyl group to a sulfuryl moiety caused relatively little change on ability to mediate a TLR-2-triggered inflammatory response and on binding affinity; however, as seen earlier with [azaPhe4]-GHRP-6 analogues possessing different aromatic substituents [4]- and [aza(4-F)Phe4]-GHRP-6 (9 and 29b) had exhibited significant anti-angiogenic activity, azasulfuryl-GHRP-6 analogue 65c featured no anti-angiogenic activity azasulfurylphenylalaninyloduction . Moreove-6 9 and b. The suactivity . 42]. . 4]-GHRPN-aminosulfamide residue indicated that turn geometry may influence angiogenic activity . . N-termid-Trp2-Ala3-Trp4-d-Phe5 region of GHRP-6 by aza-proline (azaPro scan) afforded azapeptides 70–73. Among the four, only replacement of the Ala residue with azaPro gave an analogue that retained micromolar CD36 receptor binding affinity. In the competition assay against [125I]-Tyr-Bpa-Ala-hexarelin (3), [azaPro3]-GHRP-6   cycloaddition reactions with butadiene and 2,3-dimethylbutadiene - and [azaPip4]GHRP-6 derivatives (78 and 80–82) decreased NO production in macrophages with activities similar to that of [azaTyr4]GHRP-6 (9). On the other hand, [azaPip3]GHRP-6 (79) was inactive -GHRP-6 [(S)- 87b] which retained CD36 selectivity (13.4 μM), replacements of the D- and L-Trp residues with lactams diminished significantly binding affinity especially for the ghrelin receptor, likely due to the loss of the aromatic side chain. Among the selective CD36 ligands, the notable binding affinity of [(S)-Agl3]-GHRP-6  and [(R)-Bgl3]-GHRP-6  indicated that a turn conformer about the Ala3 residue may favor receptor engagement, because complementary bent structures may be achieved by Agl and Bgl residues of opposite stereochemistry. Finally, replacement of D-Phe5 with (S)-Agl and (R)- and (S)-Bgl provided GHRP-6 analogues which bound selectively CD36 (21.4 μM–94.0 μM) -GHRP-6 (9) and -GHRP-6 (15). Azapeptides 9 and 15 have been respectively examined in models of ischemia-reperfusion injury and subretinal inflammation induced by exposure to blue-light.The biomedical potential of specific azapeptide analogues has been further examined in mouse models featuring both myocardial and subretinal inflammation. Examples of these ongoing studies are presented using -GHRP-6 (15) was found to exert cardioprotective effects against myocardial damage and dysfunction following transient left coronary artery ligation in mice -GHRP-6 (15) enhanced the cardioprotective effect of adiponectin in a CD36-dependent manner to prevent injury in induced murine models -GHRP-6 (49f) was shown to diminish aortic lesion progression and reduce lesions in the aortic sinus of atherosclerotic mice below pre-existing levels GHRP-6 (9) exhibited cytoprotective effects and preserved retinal function under photooxidative stress conditions simulating chronic inflammation [9 reduced proinflammatory cytokine levels and mononuclear phagocyte recruitment in wild-type  mice exposed to blue LED-light for five days at an illuminance of 6000 lux (9 in WT mice [The CD36-selective modulator [azaTyrammation . Azapept6000 lux . A mecha WT mice . 9 mitigated the activation of the CD36-TLR-2/6 complex after induction by R-FSL as diacyl peptide TLR-2-agonist. The influence of [azaTyr4]GHRP-6 (9) on the CD36-TLR-2 complex was ascertained by measuring Förster resonance energy transfer (FRET) efficiency using antibodies for the respective proteins coupled to energy donor and acceptor Cy3 and Cy5 dyes. The TLR-2 agonist R-FSL1 induced an increase in FRET indicating a rapid association between CD36 and TLR-2 on the membranes of cultured mononuclear phagocytes. Aza-tyrosine analogue 9 attenuated the R-FSL1-induced energy transfer efficiency between Cy3 and Cy5 indicating the dissociation of the CD36-TLR-2/6 heterodimer complex at the mononuclear phagocyte cell membrane. Disruption of the heterodimer complex by azapeptide 9 resulted in alteration of the downstream signaling of the activated TLR-2 heterodimer as observed in the photo-oxidative stress-triggered pro-inflammatory cascade GHRP-6 (9) modulated the metabolic homeostasis of the macrophages by activating peroxisome proliferator-activated receptor-γ (PPAR-γ). Selective allosteric modulation of the interaction between CD36 and TLR-2 by azapeptide 9 decreased the immune response and reduced inflammation. Within the retina, the decreased inflammatory response caused by azapeptide 9 correlated with the preservation of neuronal cells GHRP-6 analogues , exhibit typically curve shapes indicative of β-turn conformations with negative maxima centered at around 190 and 230 nm and a positive maximum band at 215 nm [4]GHRP-6 derivatives for CD36 binding . Th. Th3-macCD36 is involved in a wide range of biological processes including the regulation of inflammation, microvascular angiogenesis, and transport of oxidized lipoproteins and phospholipids. Moreover, CD36 is implicated in microorganism and cell phagocytosis, and autophagic cell cycle events. As a clinically relevant biological marker, CD36 is associated with cardiovascular biology , cardiac metabolism in the uptake of free fatty acids, and inflammatory related pathologies. Regulatory ligands of CD36 offer promise for various biomedical applications. Although unselective, peptide ligands related to the synthetic growth hormone releasing peptide-6 (GHRP-6) demonstrated interesting CD36 modulatory activity. Azapeptide GHRP-6 analogues bind selectively CD36 presumably due to the stabilization of β-turn conformers. The structure–activity relationship studies of CD36-selective azapeptides provided ligands exhibiting divergent effects on angiogenesis. Moreover, azapeptides can curb neovascularization, reduce nitric oxide production, and decrease immune responses mediated by mononuclear phagocytes, therefore mitigating potential neuronal injury associated with outer- and sub-retinal disorders such as retinitis pigmentosa, diabetic retinopathy and AMD. Azapeptides also exhibited cardioprotective effects and anti-atherosclerotic activity in a CD36-dependent manner. With proven beneficial activity in models of a wide range of cardiovascular, metabolic and immunological disorders, CD36-selective azapeptides are valuable for the development of peptide-based drugs. The translation of CD36 ligands into therapeutic agents is an on-going focus of our research program which is also investigating pharmacokinetic properties towards the design of optimal prototypes. Considering the challenges of conceiving drug-like ligands of receptors for which structural characterization is lacking, the synthetic methods described in this review offer pathways for the development of various peptidomimetics with selective properties for different specific indications.


Human hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region during Carnegie stages (CS) 14–17. Although we previously reported that these HSCs can generate no less than 300 daughter HSCs, their actual number has never been established. Here, we show that a single human AGM region HSC can generate 600–1,600 functional daughter HSCs. The presence of HSCs in the CS 17 liver in one case gave us a unique opportunity to describe a reduction of HSC self-renewal potential after liver colonization. From a clinical perspective, the efficacy of long-term hematopoietic regeneration depends on HSC self-renewal capacity. We quantitatively show that this capacity dramatically declines in the umbilical cord blood compared with HSCs in the AGM region. A full appreciation of the vast regenerative potential of the first human embryo-derived HSCs sets a new bar for generation of clinically useful HSCs from pluripotent stem cells. •A single human AGM region HSC can generate 600–1,600 fully functional daughter HSCs•HSC self-renewal potential may decline by the time of liver colonization•Transplanted umbilical cord blood HSCs self-renew without significant amplification•This study sets a new bar for the self-renewal potential of human HSCs In this article, A. Medvinsky and colleagues show that the first human hematopoietic stem cells (HSCs) emerging in the embryonic aorta-gonad-mesonephros region can generate up to 1,600 functional daughter HSCs upon transplantation into immunocompromized NSG mice. The vast self-renewal/regeneration potential of the first HSCs dramatically drops by the end of fetal development in umbilical cord blood HSCs. PrkdcscidIl2rgtm1Wjl/Sz (NSG) mice showed that the first human hematopoietic stem cells (HSCs) emerge in the aorta-gonad-mesonephros (AGM) region during Carnegie stages (CS) 14–17  and possess a substantial self-renewal potential  was transplanted. The engrafted primary recipient showed human multilineage hematopoietic chimerism  . Five moIn experiment 2, the AGM region  was transplanted. The engrafted recipient showed high-level human multilineage chimerism  . Six monIn experiment 3, the AGM region  transplantation resulted in high-level human multilineage hematopoietic engraftment of one recipient  . Nine moUnexpectedly, the transplantation of CS 17 embryonic liver cells (from the embryo used in experiment 2) into two NSG recipients gave long-term multilineage hematopoietic repopulation in both mice. Previously, we observed a complete lack of HSCs in the liver up until at least CS 17 in 26 human embryos analyzed . In this+ cells isolated from fresh UCB using anti-human CD34 magnetic beads. Each primary recipient received 2,500 CD34+ cells, a dose containing only one or two HSCs . In each experiment, 50,000 UCB CD34+ cells were transplanted per recipient, which is equivalent to 10–20 HSCs per mouse . Previously, except for one case of multilineage long-term engraftment by cultured CS 17 liver , liver cin vitro . Animals were transplanted with cells intravenously via the tail vein.NSG mice were bred within the University of Edinburgh  according to the provisions of the Animal (Scientifi Procedures) Act 1986 under the project licence granted by the Home Office (UK). Mice were kept under specific-pathogen-free conditions in individually ventilated cages. Up to 6 h before transplantation with human cells, 6–8 week old female NSG mice received a sublethal total body irradiation dose of 3.5 Gy at a rate of 0.75 Gy/min from a The BM was flushed out from the bones and a single-cell suspension was prepared by gentle pipetting. A cell fraction associated with endosteum was obtained from bones after treatment with 1 mg/mL collagenase/dispase and 0.12 mg/mL DNase I at 37°C for 40 min with gentle shaking and added to the flushed-out BM fraction. To calculate the proportion of total BM transplanted into secondary recipients, we used previously reported data on the distribution of BM cells in different mouse bones .The following mouse anti-human monoclonal antibodies  were used for flow-cytometry analysis: CD3-APC, PE and PerCP (clones SK7 and SP34-2), CD4-APC and APC-Cy7 (clone RPA-T4), CD8-PE and PE-Cy7 (clone RPA-T8), CD11b-PE-Cy7 (clone ICRF44), CD13-APC (clone WM15), CD14-APC and APC-Cy7 (clones M5E2 and MϕP9), CD19-PE (clone HIB19), CD33-PE (clone WM53), CD34-APC (clone 8G12), CD38-FITC and PE (clone HIT2), CD41a-FITC (clone HIP8), CD45-Biotin, FITC, and V450 (clone HI30), CD66b-FITC (clone G10F5), CD94-APC (clone HP-3D9), CD235a-APC (clone GA-R2), IgM-Biotin (clone G20-127), αβ TCR-FITC (clone WT31), and γδ TCR-PE (clone 11F2). The Human FcR Blocking Reagent (Miltenyi Biotec) and anti-mouse CD16/32 purified monoclonal antibody (clone 93) (eBioscience) were used to prevent unwanted binding of antibodies to Fc receptors. All antibodies and reagents listed above were used at final concentrations either recommended by the manufacturer or determined by titration in-house. 7-Amino-actinomycin (eBioscience) was used for dead cell exclusion. A FACSCalibur, LSRFortessa (both from BD), or CyAn ADP (Dako) instrument was used for flow-cytometry analysis. Flow-cytometry data were analyzed with FlowJo v.7.6.1 software (Tree Star).http://bioinf.wehi.edu.au/software/elda/ . A.I. and S.R. performed the experiments, interpreted experimental data, and wrote the manuscript. R.A.A. provided human embryonic material and feedback on data analysis. A.M. directed the study, interpreted experimental data, and wrote the manuscript.


Dexamethasone (DEX) can exert a cytotoxic effect on cultured osteoblasts. The current study explored the potential osteoblast cytoprotective effect of fibroblast growth factor 23 (FGF23). In OB-6 human osteoblastic cells and primary murine osteoblasts, FGF23 induced phosphorylation of the receptor FGFR1 and activated the downstream Akt-S6K1 signaling. FGF23-induced FGFR1-Akt-S6K phosphorylation was largely inhibited by FGFR1 shRNA, but augmented with ectopic FGFR1 expression in OB-6 cells. FGF23 attenuated DEX-induced death and apoptosis in OB-6 cells and murine osteoblasts. Its cytoprotective effects were abolished by FGFR1 shRNA, Akt inhibition or Akt1 knockout. Conversely, forced activation of Akt inhibited DEX-induced cytotoxicity in OB-6 cells. Furthermore, FGF23 activated Akt downstream nuclear-factor-E2-related factor 2 (Nrf2) signaling to alleviate DEX-induced oxidative injury. On the contrary, Nrf2 shRNA or knockout almost reversed FGF23-induced osteoblast cytoprotection against DEX. Collectively, FGF23 activates FGFR1-Akt and Nrf2 signaling cascades to protect osteoblasts from DEX-induced oxidative injury and cell death. Dexamethasone (DEX) is a medication routinely prescribed to patients with chronic inflammatory and auto-immune diseases . In UnitFibroblast growth factor 23 (FGF23), a phosphaturic factor, is a member of the endocrine FGF19 family. It is produced by osteoblasts in the bone –18. Its heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), γ-glutamyl cysteine ligase catalytic subunit (GCLC), and the modifier subunit (GCLM) [The nuclear-factor-E2-related factor 2 (Nrf2) cascade is recognized as one of the most important endogenous antioxidant mechanism –27. Witht (GCLM) , will prt (GCLM) , 30. We In the differentiated OB-6 human osteoblastic cells, FGFR1, the main functional receptor of FGF23 , is exprTo silence FGFR1, two lentiviral FGFR1 shRNAs, against non-overlapping sequences of FGFR1 (“shFGFR1-Seq1/Seq2”), were individually transduced to OB-6 cells. Via selection by puromycin-containing medium, the stable cells were established. Testing FGFR1 expression, we show that the applied FGFR1 shRNAs induced over 90% knockdown of FGFR1 in stable cells . ImportaIn the primary murine osteoblasts, FGFR1 expression was also detected . FurtherNext, experiments were performed to test the potential effect of FGF23 in DEX-treated osteoblasts. OB-6 cells were treated with DEX. In line with the previous findings , 31–33, In the primary murine osteoblasts DEX treatment similarly induced viability reduction , cell deAkt is a key pro-survival factor, protecting cells against various stress stimulations . To blocNext a constitutively-active Akt1  was traStudies have shown that DEX induces reactive oxygen species (ROS) production and oxidative injury in osteoblasts, contributing to subsequent cell death and apoptosis , 37–39. In the primary murine osteoblasts, FGF23 exerted a similar antioxidant activity, inhibiting DEX-induced superoxide accumulation , lipid pHO1, NQO1 and GCLC, was significantly increased following FGF23 treatment in OB-6 cells, with Nrf2 mRNA levels unchanged    and 5H, nd GCLC) . Taken tHO1, NQO1 and GCLC, was blocked by Nrf2 shRNA or KO  were utNA or KO . SignifiNA or KO  and cellNA or KO . TherefoKeap1 depletion should induce Nrf2 cascade activation and mimic FGF23-induced osteoblastic cytoprotection. Therefore, a lentiviral CRISPR/Cas9-Keap1-KO construct was transduced to OB-6 cells, and stable cells established with puromycin selection (“ko-Keap1” cells). CRISPR/Cas9-induced Keap1 depletion  resultedThese studies further support that Nrf2 cascade activation is required for FGF23-inducd osteoblast cytoprotection.in vitro induced phosphorylation of FGFR1 and its downstream Akt-S6K1 in OB-6 cells and murine osteoblasts. FGFR1 silencing in OB-6 cells, by targeted shRNAs, largely inhibited FGF23-induced Akt-S6K1 phosphorylation, but augmented with ectopic overexpression of FGFR1. Importantly, FGF23-induced osteoblast cytoprotection against DEX is mediated by FGFR1-Akt signaling. With FGFR1 silencing (by shRNA), Akt inhibition (LY294002) or Akt1 KO (by CRISPR/Cas9d) FGF23 was ineffective against DEX-induced cell death and apoptosis. Conversely, forced Akt activation, by caAkt1, mimicked FGF23’s action and inhibited DEX-induced cytotoxicity in osteoblasts. These results suggest that FGF23 activated FGFR1-Akt signaling to protect osteoblasts from DEX-induced cell death and apoptosis.Xiao et al., have shown that FGF23 could activate FGFR1-Akt signaling without full length α-Klotho . The resDEX treatment in cultured osteoblastic cells/osteoblasts shall provoke significant ROS production and oxidative injury, which is responsible for subsequent cell death and apoptosis. Contrarily, ROS scavengers or oxidative stress inhibition could protect osteoblastic cells/osteoblasts from DEX-induced cell death , 42, 43.However the uses of traditional Nrf2 activators are limited due to their high-concentrations and possible off-target toxicities. In the present study we showed that FGF23, at only ng/mL concentrations, induced significant Nrf2 cascade activation, causing Nrf2 protein stabilization, expression of Nrf2-pathway genes and an increase of NQO1 activity in OB-6 cells and primary murine osteoblasts. Functional studies showed that FGF23 efficiently attenuated DEX-induced oxidative injury in osteoblasts, suppressing superoxide accumulation, lipid peroxidation and mitochondrial depolarization. Therefore, Nrf2 signaling activation by FGF23 exerted anti-DEX osteoblast cytoprotection in osteoblasts.HO1 and NQO1 in OB-6 cells. Contrarily, HO1 and NQO1 expression was significantly increased in OB-6 cells with caAkt1. These results indicated that FGF23-induced Akt activation severed as the upstream signaling for Nrf2 cascade activation in OB-6 cells.Studies have implied that Akt (and its downstream mTOR) could be an important upstream molecule of Nrf2 signaling cascade. Lee et al., found that Nrf2 activation by sulforaphane was dependent on activation of the upstream PI3K-Akt . Xu et aCollectively, these results suggest that FGF23 activates FGFR1-Akt signaling cascade to protect osteoblasts from DEX-induced oxidative injury and cell death.FGF23, DEX, LY294002, Trizol reagents, polybrene and puromycin were obtained from Sigma-Aldrich Chemicals Co. . The cell culture reagents were provided by Gibco Co. . FGFR1 antibody (#3472) and all other antibodies were purchased from Cell Signaling Technology .OB-6 human osteoblastic cells  and primAfter treatment of cells, total cellular lysates (30-40 μg per lane) were separated by 10% SDS-PAGE gels, thereafter transferred onto polyvinylidene difluoride (PVDF) blots. Afterwards, the blots were blocked and subsequently incubated with the applied primary and secondary antibodies. Enhanced chemiluminescence (ECL) reagents  were utilized to visualize the targeted bands (based on the molecular weights) using x-ray films –15. An IFrom Shanghai Genechem Co.  a set of non-overlapping lentiviral shRNAs (“Seq1/Seq2”) against human FGFR1  were designed and synthesized. The shRNA lentivirus was added to cultured OB-6 cells for 48h. Puromycin  was then added to select stable cells for another 6 days. FGFR1 silencing was confirmed by Western blotting. Control OB-6 cells were infected with lentiviral scramble control shRNA .The full-length FGFR1 cDNA was synthesized by Shanghai Genechem Co. It was inserted into a GV369 construct . The construct and the lentivirus-packing plasmids  were co-transfected to HEK-293T cells, establishing FGFR1-expressing lentivirus (“lv-FGFR1”). The viruses were enriched, filtered, and added to cultured OB-6 cells (cultured in complete medium with polybrene) for 24h. Puromycin was added in the complete medium for 6 days to select stable cells. FGFR1 over-expression in the resulting stable cells was verified by Western blotting.Cells were initially seeded into the 96-well plates at 4, 000 cells per well. After the applied DEX treatment, a Cell Counting Kit-8  assay kit  was utilAs described previously –15, foll4 cells per well). Following the applied DEX treatment, a TUNEL In Situ Cell Death Detection Kit  was applied to quantitatively test cell apoptosis [vs. DAPI).OB-6 cells or the primary murine osteoblasts were initially seeded into the 24-well tissue-culture plates  were reverse-transcribed (RT). qPCR was performed by the SYBR green kit under the ABI-7600 fast-PCR system (Applied Biosystems). The 2iversity –54.3 cells per well). Following the applied DEX stimulation, a superoxide colorimetric assay kit  was utilized to examine the cellular superoxide contents using the attached protocols, with the superoxide’s absorbance tested at the 450 nm [OB-6 cells or the primary murine osteoblasts were initially seeded into the 96-well tissue-culturing plates (4 × 10e 450 nm .Lipid peroxidation5 cells per well). Following the applied DEX stimulation, the lipid peroxidation assay kit  was utilized to examine and quantify cellular lipid peroxidation levels, tested by the thiobarbituric acid reactive (TBAR) concentration using the described protocols [As reported previously  OB-6 celrotocols , 55.Testing the relative NQO1 activity in osteoblastic cells or murine osteoblasts with or without DEX treatment was described previously, using menadione as the substrate . NQO1 acThe recombinant adenovirus constitutively-active Akt1  construct was provided by Dr. Zhang . Ad-caAkvia GFP FACS sorting. Akt1 knockout (KO) in the single stable cells was verified by Western blotting.The lenti-CRISPR-GFP Akt1-KO construct was provided by Dr. Zhang at Soochow University . OB-6 ceThe Nrf2 shRNA lentiviral particles  were adThe lenti-CRISPR-GFP-Nrf2 knockout (KO) construct (from Dr. Xu ) was traThe lentivirus with Keap1 CRISPR/Cas9 KO plasmid was provided by Dr. Liu at Jiangsu University , added tExperiments were repeated at least three times throughout the study. Quantitative data were expressed as mean ± standard deviation (SD). Statistics were analyzed by two-way ANOVA using a Scheffe’s f-test (SPSS 21.0). To test significance between two treatment groups, a two-tailed unpaired T test (Excel 2007) was utilized. p values < 0.05 were considered statistically significant.Supplementary Figure 1


Those lesions are not affected by PSMA-RLT and a change in therapy management is needed. To enable early mismatch detection, possible blood parameters as indicators for the occurrence of [18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings on PET/CT were evaluated.PSMA-targeted radioligand therapy (PSMA-RLT) yielded impressive results in the metastasized castration-resistant prostate carcinoma (mCRPC) setting. High expression of PSMA is essential for successful PSMA-RLT. However, some patients develop [N = 66 advanced mCRPC patients with dual [68Ga]Ga-PSMA-11 and [18F]FDG PET/CT imaging within 4 weeks, who were referred for or received [177Lu]Lu-PSMA-617 radioligand therapy. Prostate-specific antigen (PSA), neuron-specific enolase (NSE), gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP) were tested as indicators for the occurrence of [18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings. Additional to absolute values, relative changes  over a period of 4 ± 1 weeks prior to [18F]FDG PET/CT were analyzed.Retrospective study of 18F]FDG/[68Ga]Ga-PSMA-11 mismatch finding on PET/CT. These mismatch findings were detected in 13/41 (32%) patients by screening for and in 28/41 (68%) patients during PSMA-RLT. NSE serum level  and ΔNSE  were significantly higher in the mismatch group than in the non-mismatch group. No significant differences were found for serum PSA (p = 0.424), ΔPSA (p = 0.417), serum ALP (p = 0.937), ΔALP (p = 0.611), serum GGT (p = 0.773), and ΔGGT (p = 0.971). For NSE and ΔNSE, the maximum value of the Youden index in ROC analysis was at a cut-off level of 26.8 μg/l  and at + 13.9% , respectively. An introduced scoring system of both parameters achieved a sensitivity of 90% and a specificity of 88% for the occurrence of [18F]FDG/[68Ga]Ga-PSMA-11 mismatch.In total, 41/66 (62%) patients revealed at least one [18F]FDG-avid and insufficient PSMA expressing metastases ([18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings on PET/CT) in our cohort. NSE might be used as a potential laboratory indicator for [18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings, if this observation is confirmed in future, ideally prospective, studies in larger patient cohorts.We observed a significantly higher absolute serum concentration and a higher relative increase of NSE in advanced mCRPC patients with [ Mostly, prostate carcinoma cells have a low glucose metabolism due to energy gain by lipids and other energetic molecules but in advanced late-stage disease the glucose metabolism is highly increased by the Warburg effect and shifting to aerobic glycolysis after numerous mutation events [18F]FDG and PSMA-targeted PET/CT may be able to detect clinically relevant glucose metabolic viable tumor lesions with low or no PSMA expression (mismatch lesions) in the advanced mCRPC setting. A recently prospective phase-II trial of [177Lu]Lu-PSMA-617 RLT excluded 16% of screened patients due to missing or low PSMA expression in [18F]FDG-avid metastases [18F]FDG PET/CT in addition to PSMA-targeted PET/CT is very cost intensive and associated with additional radiation exposure for the patient, which should be avoided even in a palliative setting.With over 1,000,000 new cases and approximately 300,000 deaths worldwide in 2012, prostate carcinoma is one of the most frequent malignant diseases in men . In addi effects , 12. PSM routine . PSMA-ta routine , 15 and  routine . High ex routine , 18. [18n events . Therefo18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings on PET/CT in this study, including the prostate-specific antigen (PSA) as the routine control and response parameter Ga-PSMA-11 and [18F]FDG PET/CT imaging within 4 weeks, who were referred for or received [177Lu]Lu-PSMA-617 radioligand therapy at the clinic of nuclear medicine at Saarland University Medical Center from October 2015 till August 2019. Patients with secondary malignancies were excluded to avoid potential interference of image interpretation.Retrospective monocenter study of mCRPC patients with dual FDG PET/CT. The patients received a [68Ga]Ga-PSMA-11 PET/CT and [18F]FDG PET/CT within a short time period prior to intended commencement of PSMA-RLT (n = 14/66) or in the course of PSMA-RLT (n = 52/66). The mean time between both PET/CT scans was 7.3 ± 10.7 days  ). The mean age of the patients was 69 years [range 45–89 years]. All patients received several pretreatments. Detailed information about the pretreatments and the patient characteristics is presented in Table 68Ga]Ga-PSMA-11 and [18F]FDG PET/CT were performed on a compassionate use basis under the German Pharmaceutical Act §13 (2b). Patients gave written consent after being thoroughly informed about the risks and potential side effects of this intervention. Additionally, patients consented to publication of any resulting data in accordance with the Declaration of Helsinki. Retrospective analysis approval was waived by the local institutional review board.68Ga]Ga-PSMA-11  and 268.6 ± 28.7 MBq [18F]FDG  was administered, followed by a 500-ml infusion of NaCl 0.9%. Fasting mean blood glucose value was 98.1 ± 17.3 mg/dl  before administration of [18F]FDG. The mean uptake time was approximately 60 min  for [68Ga]Ga-PSMA-11 according to standard procedures for prostate cancer imaging [18F]FDG, according to the our standard procedure and German guideline for tumor imaging [18F]FDG and [68Ga]Ga-PSMA-11 PET/CT were read simultaneously by three experienced physicians (at least 5 years of experience in PET reading) searching for mismatch findings. A mismatch finding was defined as metastasis with remarkable [18F]FDG uptake and no or considerably less concordant [68Ga]Ga-PSMA-11 uptake based on visual analysis. The decision of a mismatch finding was taken in consensus of all PET readers.For PET imaging, a mean activity of 124.1 ± 14.4 MBq [ imaging  and 90 m18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings in PET/CT: prostate-specific antigen (PSA), neuron-specific enolase (NSE), gamma-glutamyltransferase (GGT), and alkaline phosphatase (ALP). Absolute values were tested in all 66 patients. Additionally, relative changes  over a period of 4 ± 1 weeks prior to [18F]FDG PET/CT were analyzed in 55/66 patients (83.3%). In 11/66 cases (16.7%) no laboratory data from previous blood samples were available.Selected serum parameters used in our clinical practice were tested as indicators for the occurrence of FDG/[68Ga]Ga-PSMA-11 mismatch finding on PET/CT, and 25/66 (38%) had no mismatch findings. Overall, 390 mismatch lesions were detected: 211 in bone (in 24/41 patients), 62 in lymph nodes (in 21/41 patients), 100 in the liver (in 20/41 patients), 4 in the lung (in 1/41 patients), and 13 in other locations (in 9/41 patients). Figure 68Ga]Ga-PSMA-11 PET/CT before starting PSMA-RLT, 29/269 (11%) were initially intensely PSMA-positive, 52/269 (19%) were moderately PSMA-positive, and the majority, 188/269 (70%), were not identifiable on initial [68Ga]Ga-PSMA-11 PET/CT. Figure 68Ga]Ga-PSMA-11 PET/CT.In total, 41/66 62%) patients included in this analysis revealed at least one FDG/[68Ga]Ga-PSMA-11 mismatch lesions, whereas the maximum value of the Youden Index for ΔNSE (J = 0.59) was at + 13.9% increase with a sensitivity of 84% and a specificity of 75%. Only one patient had mismatch findings with serum NSE < 15 μg/l. On the other hand, we observed several mismatch (13/41) and non-mismatch (16/25) patients with NSE in the range between 15 and 30 μg/l  in our cohort.We observed a significantly higher absolute serum concentration and a higher relative increase of NSE in advanced mCRPC patients with FDG PET/CT might be an important imaging tool to examine neuroendocrine prostate carcinoma [18F]FDG PET/CT in neuroendocrine prostate carcinoma [18F]FDG PET/CT has clinical benefit and reported a high detection rate of metastatic disease, especially of lymph node and visceral metastases FDG-avid lesions with low or no PSMA expression, have a poor prognosis with very short survival time FDG/[68Ga]Ga-PSMA-11 mismatch. Statistical analysis of our data proposed an NSE cut-off value for a mismatch occurrence of 26.8 μg/l  and of + 13.9 % increase for ΔNSE . A combination of both parameters may improve the predictive power. We achieved a sensitivity and specificity of almost 90% with our introduced Combined NSE Score . Consequently, more mismatch patients (62% in our cohort) than non-mismatch patients were included in our study, resulting in a preselected cohort of mCRPC patients not representing the normal incidence of mismatch in candidates referred for PSMA-RLT. In a prospective phase-II trial for PSMA-RLT, Hofman et al. (2019) reported an incidence of 16.3% (n = 7/43) for [18F]FDG/[68Ga]Ga-PSMA-11 mismatch at time of screening for PSMA-RLT [68Ga]Ga-PSMA-11 PET/CT was set, which is also a global problem in literature suffering from defined cut-offs for adequate PSMA expression. Furthermore, additional serum parameters, which were recently reported as prognostic markers in the mCRPC setting for PSMA-RLT, such as the neuroendocrine marker chromogranin A or the lactate dehydrogenase (LDH) FDG-avid and insufficient PSMA expressing metastases ([18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings on PET/CT) in our cohort. NSE might be used as a potential laboratory indicator for [18F]FDG/[68Ga]Ga-PSMA-11 mismatch findings, if this observation is confirmed in future, ideally prospective, studies in larger patient cohorts.We observed a significantly higher absolute serum concentration and a higher relative increase of NSE in advanced mCRPC patients with [


The Diabetes Prevention Program (DPP) lifestyle intervention demonstrated that meeting the weight loss (WL) and activity goals prevents/delays type 2 diabetes. Older DPP participants, 60-85 years, reduced the risk of developing diabetes by 71% versus 58% in those <60 years. Currently, community translated DPP-based lifestyle interventions including Group Lifestyle Balance (DPP-GLB), are reimbursed by Medicare for overweight/obese older adults with prediabetes. This effort examined the impact of age group  on both DPP-GLB maintenance session attendance (months 7-12) and achieving the 5% WL goals at 6- and 12-months. Data were combined from two identical 12-month DPP-GLB intervention trials involving overweight/obese adults with prediabetes and/or metabolic syndrome. Participants ≥60 years attending ≥4 sessions (months 0-6), with complete data on session attendance and WL were included . Participants aged 66-70 years (N=46) were more likely to meet the 6-month 5% WL goal (67.4%) vs. 60-65 years . Participants aged 66-70 (69.6%) and ≥71 years  were more likely to meet the 12-month WL goal vs. 60-65 years . Maintenance attendance did not vary by age group with approximately 30% of each group attending ≥4 of 6 maintenance sessions (p=0.55). In conclusion, adults 66+ vs. 60-65 years more successfully met the clinically meaningful 5% WL goals at 6 and 12 months. With nationwide implementation of community-based “real-world” DPP-GLB lifestyle interventions, better understanding of program success across older adult age groups will enhance program reach and effectiveness.


H19 and its derived miRNA miR-675-5p were enriched in EVT cells. Although miR-675-5p acts as a placental/trophoblast growth suppressor, there is little information on the involvement of miR-675-5p in trophoblast cell invasion. Next, we evaluated a possible role of miR-675-5p in EVT cell invasion using the EVT cell lines HTR-8/SVneo and HChEpC1b; overexpression of miR-675-5p significantly promoted the invasion of both EVT cell lines. The transcription factor gene GATA2 was shown to be a target of miR-675-5p; moreover, small interfering RNA-mediated GATA2 knockdown significantly promoted cell invasion. Furthermore, we identified MMP13 and MMP14 as downstream effectors of miR-675-5p/GATA2-dependent EVT cell invasion. These findings suggest that miR-675-5p-mediated GATA2 inhibition accelerates EVT cell invasion by upregulating matrix metalloproteinases.The invasion of extravillous trophoblast (EVT) cells into the maternal decidua, which plays a crucial role in the establishment of a successful pregnancy, is highly orchestrated by a complex array of regulatory mechanisms. Non-coding RNAs (ncRNAs) that fine-tune gene expression at epigenetic, transcriptional, and post-transcriptional levels are involved in the regulatory mechanisms of EVT cell invasion. However, little is known about the characteristic features of EVT-associated ncRNAs. To elucidate the gene expression profiles of both coding and non-coding transcripts , and microRNAs (miRNAs)) expressed in EVT cells, we performed RNA sequencing analysis of EVT cells isolated from first-trimester placentae. RNA sequencing analysis demonstrated that the lncRNA  During early placentation, the invasion of extravillous trophoblast (EVT) cells into the maternal decidua and subsequent remodeling of the spiral arteries play crucial roles in the establishment of a successful pregnancy . InsuffiH19 and its derived miRNA miR-675-5p were enriched in EVT cells. The sncRNA miR-675-5p is generated from the lncRNA H19, which serves as a primary miRNA precursor of MIR675 [miR-675-5p acts as a placental growth suppressor [miR-675-5p inhibits trophoblast cell proliferation [miR-675-5p in the regulatory mechanisms of trophoblast cell invasion. This miRNA may also play a role in cancer cell invasion and metastasis [miR-675-5p plays a role in EVT cell invasion in the human placenta during early pregnancy. To test our hypothesis, we evaluated the role of miR-675-5p in EVT cell invasion using EVT cell lines. We found that miR-675-5p accelerated EVT cell invasion by direct suppressing the transcription factor gene GATA2. Furthermore, we revealed that matrix metalloproteinases  were downstream effectors of miR-675-5p/GATA2-dependent EVT cell invasion.Non-coding RNAs (ncRNAs) that are not translated into protein have recently attracted much attention for their ability to fine-tune gene expression at epigenetic, transcriptional, and post-transcriptional levels . These nf MIR675 . Previouppressor ,15. Addiferation ,17. Howetastasis ,19. HencEGFR, TEAD4, and TP63; EVT cells have lost these markers and instead express HLA-G, MCAM, MYC, and CDH5 [ITGA6 and ITGB4), and the gain of ITGA1, ITGA5, and ITGB1 [CDH5, were significantly upregulated in the isolated cells compared with CVT cells; five cytotrophoblast cell marker genes were significantly downregulated in the isolated cells  cells, 17,194 protein-coding genes (mRNAs), 4370 lncRNAs, and 1560 mature miRNAs were defined as expressed. A statistical summary of clean reads from RNA sequencing of sample libraries is provided in and CDH5 ,21,22. End ITGB1 ,24,25. Sed cells . Our RNAq < 0.05 and an absolute log2 fold change (log FC) ≥ 1. Of 23,124 genes, 5092 mRNAs, 422 lncRNAs, and 401 mature miRNAs were identified as differentially expressed. Details of the DEGs expressed in EVT cells can be found in DLK1-DIO3 region [http://www.geneimprint.com/site/genes-by-species), 68 genes were found to be DEGs; the percentages of the maternally and paternally imprinted genes that were significantly downregulated in EVT cells were 63%  and 89% , respectively  were identified based on a 3 region , were siectively .Using Ingenuity Pathway Analysis (IPA), we performed Core Analysis to interpret our dataset of DEGs filtered by the IPA microRNA Target Filter tool . The top canonical pathways, as shown in H19 was encoded by a DEG  and the most highly expressed lncRNA (70.47%) in EVT cells  mimic into the EVT cell lines, miR-675-5p significantly promoted the invasion of EVT cells compared with the control  mimic-transfected cells , CDKN2A, CHMP1A, DDB2, DRD2, EMILIN1, GATA2, ING5, LCN2, NOG, RUNX1, and SMAD5) [miR-675-5p in HTR-8/SVneo cells. Among the 12 genes, BHLHE41, CHMP1A, GATA2, and NOG were significantly downregulated in miR-675-5p-overexpressing cells compared with control cells  was mutated  ,37,38,39ol cells . FurtherATA2/mut C, a repo mutated C. LucifeR-675-5p . We alsomiR-675-5p-mediated GATA2 inhibition accelerates EVT cell invasion, we analyzed invasion by cells in which GATA2 was downregulated via small interfering RNA (siRNA)-mediated knockdown (designated as siGATA2). First, we evaluated the efficiency of siGATA2 using qPCR and Western blotting. In EVT cell lines, siGATA2 inhibited the expression of GATA2 mRNA and protein  previously reported to be associated with cell invasion [miR-675-5p-overexpressing and siGATA2-transfected EVT (HTR-8/SVneo and HChEpC1b) cells compared with control cells, we investigated their expression levels using qPCR . First, we assessed the efficiency of siMMP13 and siMMP14 using qPCR and Western blotting in EVT cells and found that siMMP13 and siMMP14 significantly inhibited both the mRNA and protein levels of MMP13 and MMP14, respectively . We identified approximately 6000 DEGs between EVT and CVT cells. Bioinformatics analysis revealed that DEG-containing signaling pathways/gene sets are associated with the migratory/invasive phenotype of trophoblast cells. This EVT phenotype revealed by RNA-sequencing is consistent with previous findings from microarray experiments ,43. SingmiR-675-5p may play a role in EVT cell invasion during early pregnancy. In the present study, we showed that miR-675-5p, an EVT-associated miRNA, was involved in the regulation of EVT cell invasion. Our data further revealed that miR-675-5p accelerated EVT cell invasion by direct suppressing GATA2 and subsequently activating MMP13 and MMP14.We originally hypothesized that H19-derived miR-675-5p in trophoblast cells, although some researchers have reported on its role in cell proliferation, we could not find a significant association between miR-675-5p and the proliferation of EVT cell lines in this study  involved in a variety of physiological and pathological processes [Gata2/Gata3 conditional gene deletion study revealed the importance of functional redundancy of Gata2 and Gata3 in mouse trophoblast development [miR-675-5p/GATA2 axis in EVT cell invasion, the possible involvement of miR-675-5p/GATA2 axis in the GATA2/GATA3-dependent transcriptional mechanisms in EVT cells requires further investigation. Although various studies have investigated the role of miR-675-5p in cancer cell invasion [miR-675-5p remain controversial. Zheng et al. reported that miR-675-5p promoted glioma cell invasion, migration, and proliferation via negative regulation of the tumor suppressor gene RB1 [miR-675-5p accelerated cell invasion, migration, and proliferation of esophageal squamous cell carcinoma cells by inhibiting REPS2, which is involved in growth factor signaling [RB1 and REPS2 may be potential miR-675-5p target genes in EVT cell invasion. In contrast, He et al. reported that miR-675-5p suppressed cell invasion, migration, and proliferation of non-small cell lung cancer cells by targeting the proto-oncogene gene GPR55 [miR-675-5p has been shown to inhibit cell invasion, migration, and the proliferation of papillary thyroid cancer cells by suppressing MAPK1 [miR-675-5p in cancer cell invasion depend on the type of cancer, as target mRNA and protein levels are differentially modulated.Within the context of trophoblast cell invasion, many studies have reported that various miRNAs and their target genes are associated with trophoblast cell invasion ,46,47. Irocesses . GATA2 arocesses . Both GAelopment . Since tinvasion ,51,52,53ignaling . In thatMMP2 expression was not regulated by miR-675-5p in EVT cell lines  via MMP2 [Trophoblast cell invasion is tightly controlled by triggering or inhibiting various signaling pathways, including MAPK, PI3K/AKT, JAK-STAT, Wnt, FAK and Rho/ROCK, and TGFB superfamily signaling ,55. Twenll lines A. Althounockdown A,B. Thusvia MMP2 , there avia MMP2 ,63. Activia MMP2  and degrvia MMP2 ,65. Althvia MMP2 ,67,68, tvia MMP2 ,69,70, avia MMP2 ,72,73. Mvia MMP2 ,75,76. Wvia MMP2 . Majali-via MMP2 . Our finvia MMP2 , EVT celDLK1-DIO3 region). The DLK1-DIO3 cluster contains three paternally expressed protein coding genes  and multiple maternally expressed ncRNAs ) [Dlk1-Dio3 region is correlated with pluripotency levels of mouse stem cells [DLK1-DIO3 region in EVT cells suggests the loss of stemness of CVT cells and subsequent differentiation into EVT cells. Greife et al. reported epigenetic silencing across the DLK1-DIO3 imprinted gene cluster due to aberrant epigenetic regulation , which resulted in the simultaneous downregulation of oppositely expressed and imprinted genes of the region in urothelial carcinoma [DLK1-DIO3 region in EVT cells remain to be elucidated. Although C14MC miRNAs are expressed predominantly in human placental trophoblast cells , there are two other placenta-associated miRNA clusters in chromosome 19: the chromosome-19 miRNA cluster  and the miR-371-3 cluster (three miRNA precursors in the vicinity of C19MC) [In terms of RNA sequencing, we found significant downregulation of DEGs in the chromosome region 14q32.2 ) . These imiRNAs)) ,80,81. Lem cells . Severalarcinoma . The epif C19MC) ,85. The f C19MC) . As descH19, except for its role as a primary miRNA of MIR675, in EVT cells are still unknown. LncRNA H19 is located in an imprinted region of human chromosome 11, along with the IGF2 gene [H19 and IGF2 are reciprocally imprinted  and regulated by the H19 differentially methylated region (H19-DMR). H19 and IGF2 are important for proper placental development function [H19 promoting EVT cell invasion by acting as an endogenous miRNA sponge [H19 and miR-675-5p in EVT cells during first-trimester pregnancy may cause inadequate EVT cell differentiation, invasion, and subsequent placenta dysfunction [This study has some limitations. We employed two EVT cell lines (HTR8/SVneo and HChEpC1b) for in vitro functional analysis since the numbers of obtainable first-trimester placentas after legal abortions were being limited in our institutes. Therefore, future work should perform using primary first-trimester EVT cells. In addition, the specific roles of lncRNA GF2 gene . H19 andfunction ,88. TherA sponge ,90. The function ,92. Placfunction . BesidesDLK1-DIO3 region). RNA-sequencing analysis also revealed that the lncRNA H19 and its derived miRNA miR-675-5p were abundantly expressed in EVT cells. We evaluated the possible role of miR-675-5p in EVT cell invasion using EVT cell lines. In vitro analysis demonstrated that the lncRNA H19-derived miR-675-5p accelerated EVT cell invasion by direct suppressing GATA2. Moreover, MMP13 and MMP14 were shown to be downstream effectors of miR-675-5p/GATA2. Thus, our study suggests that the miR-675-5p-GATA2-MMP14/MMP13 axis may play an important role in EVT cell invasion . The immortalized human lymphocyte cell line Jurkat was employed for the construction of luciferase reporter vectors; Jurkat cells were cultured in RPMI1640 medium supplemented with 10% FBS  [HTR-8/SVneo and HChEpC1b cells were used as human EVT model cell lines ,95. The  5% CO2) .n = 3) were obtained after legal abortions. The gestational age was determined by the last menstrual period and confirmed by ultrasound measurement in the first-trimester of pregnancy.Human placentae from pregnant women who provided informed consent were obtained using protocols approved by the Jichi Medical University Ethics Committee and the Nippon Medical School Ethics Committee. First-trimester placental tissues . For small RNA sequencing, the TruSeq Small RNA Sample Prep Kit-Set A  was used to prepare small RNA-sequencing libraries, according to the manufacturer’s instructions. Briefly, 1 µg of total RNA was processed for adaptor ligation and subsequent cDNA amplification. A 145–160-bp size selection was then performed on a 6% polyacrylamide gel. For mRNA/lncRNA sequencing, libraries were initially prepared with 1 μg of total RNA. Following the removal of ribosomal RNA, the remaining RNA was fragmented and used for cDNA library construction  with the TruSeq Stranded Total RNA Library Prep Kit  according to the manufacturer’s instructions. Indexed cDNA libraries were pooled at an equimolar ratio and used at a final concentration of 10–16 pM with 1% PhiX Control. The miRNA and mRNA/lncRNA sequencing analyses were carried out on an Illumina MiSeq system using MiSeq v2 Reagent Kit 50cycle  and MiSeq v2 Reagent Kit 300cycle , respectively.The 26 bp single-end reads for miRNA sequencing and the 151 bp paired-end reads for mRNA/lncRNA sequencing were obtained as fastq files and imported into CLC Genomics Workbench  for the analysis of raw data, including trimming, filtering, and the cleaning up of contaminated reads based on default parameters ). Clean 26 bp and 151 bp reads were aligned to the human genome sequence retrieved from the Ensembl database  and the microRNA database miRbase (release 21), respectively, using the CLC Genomics Workbench “Map Reads to Reference” tool (length fraction = 0.95 and similarity fraction = 0.9).https://www.bioconductor.org/). Statistically significant differentially expressed genes were identified using edgeR [p-value  of < 0.05 and an absolute log2 fold change (log FC) ≥ 1 were considered significantly differentially expressed  [https://www.ncbi.nlm.nih.gov/geo/).Normalization of gene expression data was performed according to the trimmed mean of M-values (TMM) method with the edgeR package  ,100. Thehsa-miR-675-5p (designated as 675-5p mimic) and cel-miR-239b-5p as a negative control  were purchased from Ajinomoto Bio-Pharma . For mRNA-knockdown analysis, the following siRNA oligos were employed: siRNA for GATA2 (siGATA2), sense: 5′-UUCUUGGACUUGUUGGACAUCUUCC-3′ and antisense: 5′-GGAAGAUGUCCAACAAGUCCAAGAA-3′; siRNA for MMP13 (siMMP13), sense: 5′-GGAGAUAUGAUGAUACUAATT-3′ and antisense: 5′-UUAGUAUCAUCAUAUCUCCTT-3′; siRNA for MMP14 (siMMP14), sense: 5′- CCAGAAGCUGAAGGUAGAATT-3′ and antisense: 5′- UUCUACCUUCAGCUUCUGGTT-3′; and nonspecific control siRNA (siNC), sense: 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense: 5′-ACGUGACACGUUCGGAGAATT-3′ (Ajinomoto Bio-Pharma). Each miRNA and siRNA oligo  was transfected into cells using Lipofectamine 2000  at 37 °C for 4 h.For miRNA transfection analysis, miRNA mimic oligos for H19 and mRNAs are shown in Total RNA was isolated from the samples using RNAiso Plus. Then, qPCR was performed with the Applied Biosystems 7300 Real-Time PCR System  according to the manufacturer’s instructions. TB Green Premix Ex Taq  was employed for the quantitative analysis of mRNA level. To normalize the expression levels of mRNAs, 18S ribosomal RNA (designated as 18S) was used. Three independent analyses of the expression levels of each mRNA and miRNA were performed. Primers for the lncRNA Cell lysates were obtained using mammalian protein extraction reagent  supplemented with 1% Halt protease inhibitor cocktail . Twenty micrograms of cell lysate protein were separated using Mini-PROTEAN TGX gels  with a PowerPac Basic power supply  . The proteins were then transferred onto polyvinylidene fluoride membranes using Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Packs  with the Trans-Blot Turbo Transfer System . Blotted proteins were incubated with a primary antibody , rabbit anti-MMP13 , rabbit anti-MMP14 , or mouse anti-GAPDH ) at room temperature for 2 h, followed by incubation with horseradish peroxidase-conjugated anti-mouse  or anti-rabbit  secondary antibody at room temperature for 1 h. Signals were detected with Clarity Western ECL Substrate  and visualized using an Amersham Imager 680 .4 HTR-8/SVneo or 7.0 × 104 HChEpC1b) in 250 μL of each culture medium without FBS were placed in the upper chamber. Then, 800 µl of each culture medium with FBS  was added to the lower chamber. After incubation for 24 h (HTR-8/SVneo) or 48 h (HChEpC1b), the non-invading cells at the top of each Transwell chamber were scraped off using cotton swabs. The invading cells at the bottom of the membrane were fixed with 2.5% glutaraldehyde -PBS, stained with 0.5% crystal violet , and counted manually using a light microscope  under 200× (HTR-8/SVneo) or 100× (HChEpC1b) magnification. The average numbers of invading cells in five areas were counted to reflect invasive ability. Three invasion assays were conducted under each experimental condition.The cell invasion assay was performed as described previously . BrieflyEvaluation of cell proliferation ability was performed using a CellTiter-Glo Luminescent Cell Viability Assay kit . Briefly, 7500 HTR-8/SVneo or 15,000 HChEpC1b cells were placed in 96-well plates for 24 h and transfected with 675-5p or NC mimic (50 nM). After transfection for 24, 48, and 72 h, the cells were lysed using a reagent from the CellTiter-Glo Luminescent Cell Viability Assay kit, and the ATP activities of the cell lysates were measured using the Glomax-Multi Detection System . ATP activity was measured five times for each experimental condition.http://www.targetscan.org/vert_72/; accessed on 7 May 2018) was employed to predict miRNA target genes.The TargetScanHuman database . Total RNA isolated from Jurkat cells was reverse-transcribed to cDNA using PrimeScript reverse transcriptase . The portion of the GATA2 3′-UTR (327 bp), including the target candidate sequence of miR-675-5p was then amplified from the cDNA with the following primers: GAGGGAGCTCACCCTTAGCAGCCCAGCAT (SacI site underlined) and ACGCACGCGTCACCAAGTCTCCAAGTCCTTGTT (MluI site underlined). After sequence verification, the GATA2 3′-UTR (327 bp) was cloned into pMIR-REPORT via the SacI and MluI restriction sites. This final construct was designated as pMIR-GATA2/wild. To construct a reporter plasmid with a mutated miR-675-5p recognition site for the GATA2 3′-UTR . PCR amplification was carried out using the previously cloned vector ZERO Blunt TOPO containing the GATA2 3′-UTR. Plasmid DNA was digested by DpnI. Amplified DNA was transformed into Escherichia coli . After sequence verification, the mutated 3′-UTR sequence was cloned into pMIR-REPORT via the SacI and MluI restriction sites. The final construct was designated as pMIR-GATA2/mut.The luciferase reporter assay for 2 3′-UTR C, an invRenilla luciferase expression plasmid), together with 20 nM 675-5p or NC mimic using Lipofectamine 2000 in 24-well plates. At 24 h after transfection, luciferase assays were performed using the Dual Luciferase Reporter Assay System . Firefly luciferase (from the pMIR-REPORT vector) and Renilla luciferase (from the phRL-TK vector) activities in the cell lysates were measured using the Glomax-Multi Detection System . Firefly luciferase activity was normalized to Renilla luciferase activity. Luciferase activity was measured three times for every experimental condition.For the reporter assay, HTR-8/SVneo cells were transfected with pMIR-GATA2/wild or pMIR-GATA2/mut and the control vector pRL-TK . The significance of between-group differences was assessed using a Student’s


Collectively, our data suggest that the TAC5 series of compounds are potential candidates for treating autoimmune diseases such as psoriasis or SLE.Toll-like receptors (TLRs) play a fundamental role in the inflammatory response against invading pathogens. However, the dysregulation of TLR-signaling pathways is implicated in several autoimmune/inflammatory diseases. Here, we show that a novel small molecule TLR-inhibitor (TAC5) and its derivatives TAC5-a, TAC5-c, TAC5-d, and TAC5-e predominantly antagonized poly(I:C) (TLR3)-, imiquimod (TLR7)-, TL8-506 (TLR8)-, and CpG-oligodeoxynucleotide (TLR9)-induced signaling pathways. TAC5 and TAC5-a significantly hindered the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), reduced the phosphorylation of mitogen-activated protein kinases, and inhibited the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6. Besides, TAC5-a prevented the progression of psoriasis and systemic lupus erythematosus (SLE) in mice. Interestingly, TAC5 and TAC5-a did not affect Pam Drosophila Toll protein, which plays an important role during dorso-ventral partitioning of the embryo and confers immune resistance in the fly -mediated TNF-α secretion  on shaved back skin for six consecutive days A. TAC5-aFurthermore, the immunohistochemical analysis indicated a greater reduction of macrophage (CD68) and TH17 (CD4 and IL-17) cells infiltration in the dermis . This suThe therapeutic potential of TAC5-a against systemic autoimmunity was further studied using MRL/lpr mice by intraperitoneally injecting daily doses of 10 nmol/g of TAC5-a (dissolved in 1% dimethyl sulfoxide [DMSO]) for 11 days. The inhibitor affected mice lymphoproliferation; lymphoid expansion was significantly prevented in the TAC5-a-treated mice as compared to that in vehicle-treated mice A. The prAfter confirming the in vitro and in vivo efficacy of TAC5-a, we sought to predict its binding mode and interaction pattern with TLR7 and TLR8. As the agonist/antagonist binding locations on these receptors have been well defined by X-ray crystallography studies ,39, we cTLRs located on the endosomal membrane  are crucial for protecting the host against various viral and bacterial infections. Notably, TLRs 7, 8, and 9 co-expression are essential for a sustained defense against pathogenic components or self-antigens released from damaged or stressed tissues/cells . MalfuncHere, we discovered a pyridine-containing chemical compound (TAC5) by utilizing QSAR models and obtained its equipotent 3-amino derivative (TAC5-a) via a synthetic route. Both compounds inhibited the secretion of proinflammatory cytokines triggered by TLR3/7/8/9 without affecting the cell surface TLRs . Based on sequence homology, TLRs 7/8/9 are grouped into one subfamily ,51; thus−/− deficient mice were hypersensitive to TLR7/8 agonist treatment (R848), showing several indicators of inflammation, such as splenomegaly, increased serum levels of autoantibodies against dsDNA or ribonucleoprotein, and GN [Recent medicinal chemistry efforts have resulted in the discovery of several antagonists ,23,25,54, and GN . Further, and GN . Researc, and GN ,67. There are numerous cases where TLR7/8, and occasionally TLR7/8/9, can bind to a common agonist or antagonist. For instance, Dynavax Technologies, Inc. designed oligonucleotide antagonists for TLR7/8/9 that are foreseen as treatments for autoimmune and inflammatory diseases . In a no


Keeping a balance between DNA methylation and demethylation balance is central for mammalian development and cell function, particularly in the hematopoietic system. In various mammalian cells, Tet methylcytosine dioxygenase 2 (Tet2) catalyzes oxygen transfer to a methyl group of 5-methylcytosine (5mC), yielding 5-hydroxymethylcytocine (5hmC). Tet2 mutations drive tumorigenesis in several blood cancers as well as in solid cancers. Here I discuss recent studies that elucidate mechanisms and biological consequences of Tet2 dysregulation in blood cancers. I focus on recent findings concerning Tet2 involvement in lymphoid and myeloid cell development and its functional roles, which may be associated with tumorigenesis. I also discuss how Tet2 activities are modulated by microRNAs, metabolites, and other interactors, including vitamin C and 2-hydroxyglutarate (2-HG), and review the clinical relevance and potential therapeutic applications of Tet2 targeting. Finally, I propose key unanswered hypotheses regarding Tet2 in the cancer-immunity cycle. Shuai Jiang discusses the regulation and functional role of the Tet2 enzyme, a regulator of lymphoid and myeloid cell development frequently mutated in haematopoietic cancers. He highlights how Tet2 activity may be modulated to influence cancer immune responses. The first identified gene of the Tet family, Tet1, acts as a fusion regulator in cases of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL)2. Two other Tet genes, Tet2 and Tet3, were later identified based on sequence homology with Tet13. In mammalian cells, all Tet family members, Tet1, Tet2, and Tet3, catalyze the successive oxidation of 5mC, yielding 5hmC, 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC)4.DNA methylation/demethylation is dynamically coordinated throughout hematopoietic differentiation, and Tet proteins play crucial roles in adjusting gene expression levels through balancing DNA methylation in hematopoiesis and during immune-cell activation and expansion6. DNA modification by Tet2 is fundamental for gene control in both cancer cells and immune-cell subtypes8. Tet2 protein and its downstream effectors-5hmC/5mC-mediated DNA modification are constitutional in immune cells, including T cells, B cells, and macrophages in both physiological and pathological conditions9 , or double KO (DKO) of Tet2 and Tet3, causes myeloid or lymphoid cell spread and the progression of fully infiltrating aggressive tumors13.The Tet2 gene is subjected to frequent somatic mutations in an extensive range of hematopoietic cancers, including myeloid and lymphoid cancer, and several solid cancersns9 Fig. . In blooThis review discusses Tet2-related profiles in hematopoietic cancers, different subtypes of adaptive and innate immune cells, and Tet2 regulation by various interactors. I particularly emphasize the potential applications of targeting Tet2 to influence normal and malignant cancer-immunity crosstalk.14. Tet2 mRNA is expressed in hematopoietic cell subpopulations, including progenitors and mature immune cells, displaying detectable levels of 5hmC and 5mC15. In mice, Tet2 deletion enhances the Lin−Sca-1+c-Kit+ (LSK) cell subpopulation, which exhibits boosted hematopoietic repopulation competence and biased cell differentiation toward monocyte/macrophage lineages, followed by myeloid cancer progression16. In vitro tissue culture work supports that RNA interference (RNAi)-mediated Tet2 silencing in murine hematopoietic precursors can alter their differentiation toward monocyte/macrophage lineages17. In mice, Tet2 is pivotal for modulating normal hematopoiesis, and negatively influences hematopoietic stem cell (HSC) homeostasis and differentiation. Tet2 loss reshapes the HSC niche and HSC proliferation/differentiation potential in vitro14, whereas Tet2-KO HSCs show elevated ability to reconstitute hematopoiesis in vivo17. Tet2 deletion results in reduced genomic 5hmC levels and expands the hematopoietic progenitor cell population in a cell-intrinsic manner16. Tet2-KO HSCs are capable of multilineage reconstitution and show a competitive advantage over wild-type HSCs, promoting increased hematopoiesis into myeloid and lymphoid lineages15. Tet2 disorder also results in DNA hypermethylation of enhancers in granulocyte–monocyte progenitor (GMP) and embryonic stem (ES) cells18. Overall, Tet2 plays vital roles in modulating HSC expansion and function, presumably by controlling 5hmC levels in genes crucial for HSC self-renewal, proliferation, and differentiation, which warrants future examination of the detailed mechanisms.Tet2 epigenetically controls gene expression by modulating methylation-driven gene silencing and is expressed in diverse populations of mouse and human hematopoietic cells19. Tet2 mutations may be caused by splice site mutations, out-of-frame insertions or deletions and base substitutions. Tet2-KO mice recapitulate characteristics of patients with myeloid cancer, indicating that Tet2 plays a functional role as a tumor suppressor to sustain hematopoietic cell homeostasis. Tet2-KO mice show a phenotype that is similar to CMML, including splenomegaly and enhanced white blood cell (WBC) counts with unequal numbers of monocytes14. Moreover, Tet2-KO mice develop an extensive spectrum of myeloid cancers, which occur within the first year of life in ~30% of these animals17. Mechanistically, Tet2 blocks leukemic transformation by keeping enhancers free of aberrant DNA methylation that would lead to enhanced stem cell expansion and leukemogenesis20. RNAi-mediated Tet2 silencing reduces 5hmC levels in cord blood CD34+ cells and promotes progenitor differentiation toward the granulomonocytic lineage18. CD34+ stem cells from MPN patients with Tet2 mutations reconstitute hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice and display skewed differentiation toward myeloid lineages15.In humans, Tet2 mutations occur in ~10–30% of myeloid cancers, including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), and myeloproliferative neoplasm (MPN)21. Adult T-cell leukemia/lymphoma (ATLL) progression accompanies genomic 5hmC loss, suggesting that Tet2 downregulation is the vital mechanism underlying 5hmC regulation during ATLL progression22. Tet2-KO mice develop B-cell tumors, and B-cell conditional Tet2 knockout (cKO) mice show B1-cell subset abnormalities and B-cell tumor development after long latency23. Tet2 deletion may prompt alterations of peripheral B-cell subsets23. Certain Tet2-KO cancers have AID-mediated somatic mutations23. Future work is required to investigate whether and how dysregulation of Tet2 impacts on B-cell activation upon various stimuli. Young Tet2-KO mice exhibit a moderately enhanced B1a subset population in the spleen23. On the other hand, aging Tet2-KO mice expand clonal CD19+B220low immunoglobulin (Ig)M+ B-cell subpopulations, which are transplantable and display similarities to chronic lymphocytic leukemia (CLL), including sensitivity to ibrutinib-induced B-cell receptor signaling blockage24. Further investigations are needed to examine the molecular signaling pathways controlling B1a-cell development and proliferation in young adult Tet2-KO mice.Tet2 mutations are also common in both T-cell and B-cell lymphomas, with a 40–70% incidence observed among patients with peripheral T-cell lymphoma (PTCL), particularly in subsets of angioimmunoblastic T-cell lymphoma (AITL)25. Compared with control mice, middle-aged Tet2 gene trap mice (Tet2gt/gt) show Tfh-like cell over-expansion in the spleen25. Aging Tet2gt/gt mice eventually generate T-cell lymphoma with Tfh-like features25. In future research, it will be intriguing to examine how Tet2 works in Tfh cells. A mouse model with conditional Tet2-KO (cKO) in Tfh cells would be valuable for in vivo and in vitro analyses.In mice, attenuated Tet2 function results in T-cell lymphoma with follicular helper T-cell-like (Tfh) featuresBoth Tet1 and Tet3 are expressed in hematopoietic cells, without alterations in Tet2-KO cells, demonstrating that Tet1/Tet3 is inadequate to compensate for Tet2 deletion. It will be interesting to dissect their physiological and pathological roles in various immune-cell subsets. Tet2 and Tet3 may cooperate to inhibit abnormal hematopoiesis, including hematopoietic transformation. Further investigations are necessary to test whether and how Tet2 works together with other Tet proteins in distinct blood cancer cells. Deletion of Tet2 in hematopoietic cells or various immune-cell subsets might prompt a specific and progressive enhancer hypermethylation feature linked to altered gene expression, enhanced cell accumulation rate, and leukemia progression, which also warrants future investigation. A better understanding of Tet proteins’ physiological and pathological functional roles may highlight potential novel avenues to develop epigenetic therapies to treat hematological cancers.26. Tet1 and Tet2 are simultaneously downregulated in B-cell acute lymphoblastic leukemia26. Although double (DcKO) or triple (TcKO) conditional KO mice would be valuable, it might be challenging to conditionally KO all three Tet proteins in certain immune-cell sub-settings. Identifying Tet2-targeted genes/molecules in myelopoiesis and lymphomagenesis will be crucial for future studies aimed to uncover the molecular mechanisms through which Tet2 modulates hematopoiesis and serves as a tumor-suppressor gene in myelopoiesis and T/B-cell lymphomas. It is intriguing to hypothesize that inactivating somatic Tet2 mutations might latently remain in hematopoietic cells for a long period, possibly even years. Specifically targeting Tet2 or its downstream molecular signaling pathways might offer novel therapeutic strategies for blood cancer prevention.Intriguingly, in mice, the combined deletion of Tet1 and Tet2 apparently boosts B-cell cancer, without influencing myeloid cancers27. In mice, Tet2 and Tet3 KO at an early B-cell stage halts the transition of pro-B cells to pre-B cells in the bone marrow (BM), and diminishes the rearrangement of Igk locus28. Tet2/Tet3 DKO pro-B cells exhibit enhanced CpG methylation levels at the Ig kappa 3ʹ and distal enhancers29. Tet2/Tet3 modulates the kappa-chain expression level, and the DNA modification status of the Ig kappa locus, both in vivo and in vitro29. In mice, Tet2 deletion causes myeloid leukemia after a long duration. Compared to Tet2 deficiency, Tet1/Tet2 deletion results in B-cell lymphoma with delayed disease progression26. The Tet2/Tet3 deletion in developing B cells leads to B-cell lymphoma development. These mice caused the pathological disorders within six months of age, which is earlier than in Tet1/Tet2-KO mice (~20 months)7. Moreover, regulatory B cells (Bregs) play central roles in boosting inflammation and carcinogenesis30, but the evidence is lacking regarding whether and how Tet2 protein functions in Bregs both in vitro and in vivo. Continued efforts to investigate the probable anticancer impact of Tet2 in Bregs will guide novel Tet2-based immune therapy. Additionally, in future studies, it would be useful to use B-cell-specific conditional (such as CD19-cKO) Tet2 cKO mice to examine whether and how Tet2 epigenetically affects antibody production in vivo.Tet2 is also essential for B-cell development and function+ T-cell memory differentiation. Deletion of Tet2 advocates early gain of a memory CD8+ T-cell fate, in a cell-intrinsic manner, without affecting antigen-driven cell proliferation or effector behavour31. After secondary stimulation, Tet2-KO memory CD8+ T cells possess remarkable pathogen control capacity, both before and after re-challenge. CD8+ T cells with Tet2 conditional KO exhibit full effector functional role upon acute viral infection, and Tet2 deletion enhances memory CD8+ T-cell formation31. CD8+ cytotoxic T lymphocytes (CTLs) are favored immune cells for treating cancer progression32. Programmed death‐1 receptor (PD‐1) ligand (PD‐L1) and CTL‐associated antigen 4 (CTLA‐4) are checkpoint receptors that can be targeted to re-activate anticancer CD8+ T-cell responses in cancer-immune therapy33. It would be interesting to study whether Tet2 might be involved in CTLA-4 or PD-1 pathway-mediated immune checkpoint inhibition for cancer treatment. Further studies are required to explore whether and how Tet2 acts in CD8+ CTLs, and how it cooperates with PD-L1/CTLA-4 to modulate anticancer CD8+ T-cell responses.Tet2 deletion augments CD8flox/floxCD4Cre+ mice have intact thymic and peripheral T-cell subpopulations31. Tet2 enhances DNA demethylation and cytokine gene activation, for instance, IL17a, in CD4+ T cells. In autoimmune diseases, Tet2 modulates T-cell cytokine production in vivo, and specifically influences IL-10, IFN-g, and IL-17 expression levels34. Tet2 depletion in CD4+ T cells is linked to reduced cytokine expression, and diminished p300 recruitment34. Moreover, Tet2/Tet3 DcKO CD4+ T cells induce pathological diseases in healthy mouse35. Tet2 enhances typical cytokine gene expression level in Th1 and Th17 cells in vitro, and modulates Th1 and Th17 cell differentiation34.Tet2flox/flox/Tet3flox/floxFoxp3Cre+ DcKO mice) show severe inflammation, resulting in inflammatory diseases, and Tregs from these DcKO mice exhibit adjusted expression levels of Treg-typical cytokine genes and dysregulation of genes involved in cancer progression35. Overall, these studies reveal that both Tet2 and Tet3 maintain Treg stability and homeostasis. Tet2/Tet3 DcKO in Tregs enhances T-cell activation. Moreover, Tet2/Tet3 DcKO Tregs show an altered cell surface feature and fair hypermethylation of conserved noncoding sequence 2 (CNS2)35. Wild-type (WT) Tregs are not able to regulate autoimmunity progression in Tet2-deficient mice35. Double Tet1/Tet2 deletion also causes Treg inactivation, differentiation, and even autoimmune diseases36. Notably, Tregs hamper anticancer immune surveillance in a healthy person, and prevent effective antitumor immunity in cancer patients, boosting tumor progression37. Thus, methods for specific targeting Tet2/Tet3 in Treg cells might be valuable for modulating anticancer immune responses.Tet2/Tet3 loss in regulatory T cells (Tregs) contributes to effector behave phenotypes. Treg-cell-specific Tet2/Tet3 double conditional KO mice  proliferation and cell-lineage differentiation9. Compared to other Tets, Tet2 is the most profoundly expressed in murine macrophage differentiation. Tet2 is essential for controlling inflammation through recruiting Hdac2, inhibiting IL-6 levels40. Tet2-KO macrophages and dendritic cells (DCs) produce more IL-6 in response to bacterial activation in vitro and in vivo. Thus, compared to wild-type (WT) mice, Tet2-KO mice show increased susceptibility to endotoxin-induced shock, DSS-induced colitis40 and Salmonella infections41. Tet2 transcription is induced by LPS activation, likely in a nuclear factor κβ (NF-κβ)-dependent way42. In vitro, Tet2 loss in BMDMs does not influence early LPS-induced gene activities but enhances arginase 1 (Arg1) mRNA level during later activation stages. Tet2 deletion does not dramatically alter alternative macrophage (M2) gene expression levels in response to IL-4 stimuli39. Tet2 expression is sustained in differentiated murine macrophages, and Tet2 deletion does not negatively alter markers of macrophage differentiation42. Resting Tet2-KO peritoneal macrophages reveal atypical induction of LPS-associated genes, and the Tet2-deficiency peritoneal macrophage features are linked to inflammation across the whole body40.Tet2 is further essential for innate immune-cell functional roles43. Thus, modulating Tet2 expression levels in M1 macrophages might contribute to cancer-immune therapy , Tet2 expression is activated along the IL-1R/MyD88 axis39. Myeloid-specific Tet2 loss causes higher numbers of tumor-infiltrating T cells39. During melanoma growth, Tet2 expression is enhanced in myeloid-derived suppressor cells (MDSCs) and TAMs39. Further, Tet2 preserves immunosuppressive gene expression levels in TAMs. Mice with myeloid cell-specific Tet2 deletion exhibit diminished tumor growth, and enhanced tumor-infiltrating T cells; moreover, T-cell depletion suppresses the attenuated tumor progression39. Whether Tet2 plays a similar or different role in myeloid cell-mediated tumor growth in cancers other than melanoma awaits further investigation.M1 macrophages are essential for tumor cell eradicationHere, I have summarized our current understanding of the functional roles of Tet proteins in B cells, various types of T-cell subsets, and M1/M2 macrophages in both physiological and pathological conditions. Further study is essential to investigate how all Tet proteins and 5hmC/5mC modulate DNA modification, gene expression, and transcriptional networks in various types of innate and adaptive immune cells, and to dissect the underlying molecular mechanisms and potential for cancer-immune therapy in this field.44. Vitamin C interacts with the core catalytic domain of Tet2, directly boosting its catalytic activities4. Additionally, Tet2 expression can be directly altered at the post-transcriptional level45. Numerous microRNAs (miRs) post-transcriptionally inhibit Tet expression and certain miRs work together to control the expression of Tet genes. Tet2 genes contain conserved elements in the three prime untranslated regions (3′-UTR) predicted to be directly regulated by miR-2246. High-throughput screening has validated that the Tet2 expression level in hematopoietic cells is regulated by multiple miRs, including miR-7, miR-125b, miR-29b/c, miR-26, and miR-10147. Moreover, Tet2 can be directly and indirectly modulated by the miR let-7adf in macrophages41. O-GlcNAc transferase (OGT) transfers a single O linked-GlcNAc moiety to serine/threonine residues in multiple proteins48. OGT co-localizes with Tet2 in the genome, mainly at GC-rich promoters and transcriptional start sites (TSS) containing CpG islands, and reduces Tet2’s enzymatic activities by enhancing its nuclear export49. In the context of metabolic pressure, Tet2 can also be modulated by alpha-ketoglutaric acid (α-KG)50. Tet2 deletion through CRISPR-CAS9 causes globally enhanced DNA methylation at enhancers, thus blocking the estrogen responses51, indicating that Tet2 acts as a transcriptional coactivator in the estrogen responses. Fe2+ and HIF-1a also support to maintain Tet2 activities52. Thus, various cofactors  and noncoding RNAs (such as miRs) can affect Tet2 functional roles, constituting an additional level of regulation. Moreover, the epigenetic regulator CXXC5 and Tet2 are essential in the fight against viral infections. CXXC5 enrolls Tet2 to modulate TLR7/9-elicited IFN responses, and to influence DNA methylation, in human plasmacytoid DCs53. RUNX1 alters DNA demethylation by recruiting Tet2 and other enzymes with demethylation functional roles to its attaching sites in various hematopoietic cells54. All of these interactors, and other small-molecule activators of Tet2, would be helpful methods for stabilizing in vitro-generated immune cells or modified tumor cells for potential clinical utilization.Numerous regulatory mechanisms have either directly or indirectly been demonstrated to regulate Tet2 activities or expression levels in immune-cell subsets and cancer cells , generated by IDH mutation in immune cells, could also halt Tet2 activities in immune cells, , which would trigger less-positive antitumor immune responses. Thus, it might also be valuable for future preclinical studies to investigate the restoration of Tet2 in these immune cells, for instance, by directly adding vitamin C or a-KG. Future work will focus on the accurate targeting of Tet2 in the context of different specific cell types, among both cancer and immune cells.There may be several potential ways of specifically targeting Tet2 activity in immune-cell subpopulations and cancer cells (Fig. Investigations in both mouse models and human clinical samples have demonstrated that Tet2 loss is not essential to cause cancer, but rather influences cancer progression. This may be by reason for the behavior of Tet2 in various types of immune-cell subsets, and it might explain why some patients with Tet2 mutations stay healthy, whereas others develop either myeloid or lymphoid cancer. Future studies are needed to examine therapies targeting cytosine modifications, DNA methyltransferase inhibitors, and IDH inhibitors, which might be valuable for future cancer-immune therapy.Collectively, Tet2 is critical to the cancer-immunity crosstalk, which is essential for both cancer development and anticancer immune responses Fig. . It appe