A growing number of studies have suggested that microRNAs exert an essential role in the development and occurrence of multiple tumours and act as crucial regulators in various biological processes. However, the expression and function of miRNA-140 in hepatocellular carcinoma (HCC) cells are not yet adequately identified and manifested.  The expression of miRNA-140 was determined in HCC tissues and adjacent nontumour tissues by quantitative real-time polymerase chain reaction (qRT-PCR). Kaplan–Meier survival analysis and Cox regression analysis were performed to explore the correlation between miRNA-140 expression level and the survival rate of patients with HCC. Additionally, overexpression experiments were conducted to investigate the biological role of miRNA-140 in HCC cells. Bioinformatics was used to predict the related target genes and pathways of miRNA-140. P < 0.0001). Compared with the control group, the SMMC-7721 HCC cells in the miRNA-140 mimic group had a decrease in proliferation, migration, and invasion (P < 0.05), whereas those in the miRNA-140 inhibitor group had an increase in proliferation, migration, and invasion (P < 0.05). Cell cycle arrest occurred in the G0/1 phase. Prognosis analysis showed that the expression level of miRNA-140 was not related to the prognosis of HCC. Furthermore, the Kaplan–Meier test revealed that patients with lower miRNA-140 expression levels in liver cancer tissue had significantly shorter disease-free survival  and overall survival (OS) times (P = 0.010) after hepatectomy. Cox regression analysis further indicated that miRNA-140 was an independent risk factor that may affect the DFS (P = 0.004) and OS times (P = 0.014) of patients after hepatectomy. Our results suggested that miRNA-140 might be a crucial regulator involved in the HCC progression and is thus considered a potential prognostic biomarker and therapeutic target for HCC. QRT-PCR results signified that the expression level of miRNA-140 in HCC was lower than that of adjacent normal tissues ( Hepatocellular carcinoma (HCC) is one of the most common malignant tumours. HCC ranks second amongst the causes of death in patients with cancer because of its high morbidity, high mortality, and poor prognosis , 2. HCC β receptor 1 in HCC [miRNA is an endogenous, evolutionarily conserved, single-stranded, and noncoding RNA . Its mai1 in HCC .miRNAs are abnormally expressed in HCC and participate in the growth, development, and metastasis of HCC by acting as oncogenes or tumour suppressors . In the Eighty specimens of cancer and adjacent normal tissues were collected from the Department of Hepatobiliary Surgery of the Affiliated Tumour Hospital of Guangxi Medical University. The 80 patients met the following conditions: underwent radical hepatectomy, had pathologically diagnosed HCC, had no other treatments before surgery, had no other tumours, and had complete clinical and follow-up data. The corresponding adjacent normal liver tissues were collected more than 2 cm away from the tumour boundary, and normality was confirmed by pathological examination. The tissue samples were surgically removed from the patients with HCC, quickly placed into cryopreservation tubes with RNA protection solution, labelled, placed into a liquid nitrogen tank, and later transferred to a −80°C laboratory freezer for further preservation.The HCC tissues and corresponding adjacent normal liver tissues of 198 patients with HCC were collected during hepatectomy from January 2014 to December 2016 in the Hepatobiliary Surgery Department of Guangxi Medical University Affiliated Tumour Hospital. The patients were followed up every 3 months within 1 year by outpatient reexamination and in the long-term by telephone. The follow-up deadline was September 2019. The study protocol was approved by the Hospital Ethics Committee. All patients signed the relevant informed consent form. The corresponding adjacent normal liver tissues were collected more than 2 cm away from the tumour boundary, and normality was confirmed by pathological examination. The tissues were stored at −80°C.The clinical data of the patients with HCC, including data on their biological sex, age, smoking and drinking history, family history, body mass index (BMI), alpha-fetoprotein (AFP) value, liver cirrhosis, number of tumours, metastasis, Barcelona clinic liver cancer (BCLC) stage, and portal vein tumour thrombus (PVTT) were collected during hepatectomy from January 2014 to December 2016 in the Hepatobiliary Surgery Department of Guangxi Medical University Affiliated Tumour Hospital. The study was approved by the ethics committee of the Affiliated Tumour Hospital of Guangxi Medical University, and all patients signed the relevant informed consent forms.The reagents include RNA protection solution ; TRIzol total RNA extraction reagent ; isopropanol, ethanol, and chloroform ; miRNA cDNA kit and miRNA PCR kit (Beijing Tiangen Co.); phosphate-buffered saline (PBS) powder, penicillin double-antibody, EDTA R-trypsin digestive fluid and EDTA-free protease digestive fluid (Solabo Company of China); Dulbecco's modified Eagle's media (DMEM), high-glucose medium, foetal bovine serum ; Opti-MEM serum-free medium (Semefei Company); Hsa-miRNA-140 mimic, mimic negative control (NC), Hsa-miRNA-140 inhibitor, and inhibitor NC ; Lipo6000 transfection reagent ; cell cycle detection kit and annexin V–fluorescein isothiocyanate (FITC) cell apoptosis detection kit ; transwell chamber ; and Matrigel-coated membrane .ΔΔCt− method using U6 as the internal reference.The specific method for total RNA extraction is as follows. SMMC-7721 cells were extracted based on the instructions of the Tiangen total RNA extraction kit. The concentration and purity of the extracted RNA were detected using a UV spectrophotometer. The RNA was reverse transcribed into cDNA using Takara's reverse transcription kit, and the qRT-PCR results were later analysed by the 2μg/mL streptomycin and cultured in a humidified incubator with 5% CO2 at 37°C. The cells were transfected in a 6-well plate. According to the relevant reagent specifications, miRNA-140 mimic, mimic NC, miRNA-140 inhibitor, and inhibitor NC, as well as Lipo6000, were separately added into the pretransfection pores of a 6-pore plate. The expression of miRNA-140 in SMMC-7721 cells was subsequently detected by qRT-PCR after 48 h of transfection.Human hepatoma cell line SMMC-7721 was purchased from the cell bank of Shanghai Life Science Research Institute. The cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 4 per 100 μL/well) after 48 h of transfection and then at 0, 24, 48, 72, and 96 h after inoculation. The original medium was discarded, and 120 μL of medium containing 3--2, 5-diphenyltetrazolium bromide  was added into each well. The culture process was subsequently terminated after 4 h at 37°C in 5% CO2, and the in-hole medium was extracted with careful deliberation. In addition, 150 μL of dimethyl sulfoxide was later added into each well. The viability of HCC cells was measured by measuring the optical density of the cells at 490 nm wavelength.SMMC-7721 cells were inoculated into a 96-well plate  were inoculated into each pore of the 12-pore plate and then transfected with miRNA-140 mimic, mimic NC, miRNA-140 inhibitor, and inhibitor NC. A set of 200-mL pipette tips, perpendicular to each hole above the surface of HCC cells, were labelled 48 h later. The floating cells were further rinsed with PBS and replaced with DMEM. Photos were captured at 0 and 24 h after scratches.Logarithmic cells  was added to the lower chamber. The chamber was removed after 18–24 h. The cells that did not penetrate the membrane inside the chamber were wiped out. The remaining cells were fixed with methanol and stained with 0.1% crystal violet solution for 30 min. The number of transmembrane cells in five fields was counted under a microscope.According to the experimental protocol, the Transwell cell was used to measure the migration or invasion ability of HCC cells. The polycarbonate film of the Transwell cell was precoated with or without Matrigel glue, placed in a 24-well culture plate, and inoculated with 5 × 105 cells were collected and centrifuged at 1,200 rpm for 5 min prior to the removal of the supernatant. Next, 1 mL of precooled 75% ethanol was placed into each tube. All the collected cells were again suspended, fixed for more than 2.5 h at 4°C without light, and centrifuged for 5 min at 1,200 rpm. The relevant supernatant was discarded. The cells were cleansed with 1 mL of PBS and centrifuged. The supernatant was discarded. Finally, each sample was stained with 500 μL of propidium iodide (PI)/RNase dye and incubated for 15 min. The whole incubation process was carried out at room temperature and protected from light. The entire process was completed within 0.5 h.A total of 5 × 105) were centrifuged at 1,200 rpm for 5 minutes, and the supernatant was removed. Each tube was added with 50 μL 1× binding buffer. The cells were resuspended and added with dye according to the following groups: the negative tube was not added with dye, and the sample tube was added with 5 μL of annexin V–FITC and 10 μL of PI. The solutions were shaken gently, incubated at room temperature (25°C) in the dark for 15 min, and added with 200 μL of 1× binding buffer. Cell apoptosis was determined using a computer software within 1 h.Cell apoptosis was detected by Annexin V–FITC/PI double staining. Cells (10http://www.mirdb.org/index.html), miRTarBase (http://mirtarbase.cuhk.edu.cn/php/index.php), miRWalk , and TargetScan (http://www.targetscan.org/vert_72/). Gene Ontology  functional annotation and Kyoto Encyclopaedia of Genes and Genomes  pathway enrichment analysis were performed on miRNA-140 target genes.The target genes of miRNA140 were predicted by miRDB  rates. Two-way P < 0.05 indicates a statistical difference.The SPSS 22.0 software  was used for statistical analysis, and GraphPad Prism 8 Project software  was used for drawing. The measurement data in the experiment are expressed as mean ± standard deviation. Student's P < 0.0001; P < 0.0001; QRT-PCR was used to determine the expression of miRNA-140 in 80 HCC tissues and adjacent normal tissues. The subsequent results signified that the expression level of miRNA-140 in adjacent normal tissues was significantly higher than that in the corresponding HCC tissues  but not with other factors, such as biological sex, age, drinking history, family history, BMI, AFP, liver cirrhosis, number of tumours, and PVTT  or miRNA-140 inhibitor than those transfected with inhibitor NC  and that the miRNA-140 inhibitor markedly promoted the proliferation of SMMC-7721 cells compared with the inhibitor NC and BL , fewer cells in the G2/M phase , and fewer cells in the S phase  compared with the control group. The miRNA-140 inhibitor and inhibitor NC groups had fewer cells in the G0/G1 phase , more cells in the G2/M phase , and more cells in the S phase  than the control group. The results indicated that miRNA-140 overexpression blocked the G1 phase of the HCC cell cycle and inhibited the HCC cell cycle from the G0/G1 phase to the S phase. Therefore, the overexpression of miRNA-140 inhibits the proliferation of HCC cells . Although the inhibitor group and inhibitor NC group had a significant difference (P = 0.0405), this result was not enough to show that miRNA-140 has an effect on apoptosis. Therefore, we speculate that the miRNA-140 gene may not play an important role in the apoptosis of HCC cells (We detected the effect of the upregulation and downregulation of miRNA-140 on SMMC-7721 cell apoptosis by flow cytometry to detect whether miRNA-140 affects apoptosis. The results showed no significant difference between the miRNA-140 mimic group and the mimic NC group (CC cells .β signalling pathway, MAPK signalling pathway, relaxin signalling pathway, miRNAs in cancer, and apelin signalling pathway (We predicted 751, 322, 14,701, and 1,386 miRNA-140 target genes through miRDB, miRTarbase, miRWalk, and TargetScan. Thirty-six genes were found in the intersection of the four databases . A miRNA pathway .As one of the common malignant tumours, HCC can participate in the abnormal activation of signal pathways involved in gene mutations, chromosomal aberrations, abnormal secretion of growth factors, and epigenetic changes –23. Howeα signalling pathway can downregulate the expression of miRNA-140, and the target of miRNA-140 to regulate the initiation of breast cancer cells is the transcription factor Sox2 [The anticancer effect of miRNA-140 is reflected in a variety of tumour cells and tissues and affects the occurrence and development of tumours. Iorio et al. discovered that the expression of miRNA-140 decreases in ovarian cancer and its related cell lines by prediction analysis of microarrays . miRNA-1tor Sox2 . Furthertor Sox2 . miRNA-1tor Sox2 . Our expβ and MAPK by regulating the downstream target gene TGF-β receptor 1 and FGF9 and thus affects the transformation and proliferation of hepatoma cells. Yang et al. [ACER2 and ACVR2B, by using the intersection method of the miRDB, miRTarBase, miRWalk, and TargetScan databases. In KEGG enrichment analysis, these 36 genes were remarkably enriched in cellular senescence, PI3K Akt signalling pathway, TGF-β signalling pathway, and MAPK signalling pathway. These results suggested the critical role played by miRNA-140 in HCC.A variety of existing studies have confirmed the correlation between miRNA-140-5p and the metastases of ovarian cancer , colorecg et al.  discoverg et al.  demonstrg et al.  uncovereOur results suggested that miRNA-140 was significantly downregulated in HCC tissues and that a high level of miRNA-140 expression was critical to achieve the long-term survival of patients with HCC. miRNA-140 might inhibit HCC cell proliferation, migration, and invasion. We predicted the target genes and pathways of miRNA-140 through bioinformatics, but further research and studies are needed to better understand the specific types of targeted regulatory genes and their molecular mechanisms. miRNA-140 could be a potential prognostic biomarker and therapeutic target for HCC.


Hematopoietic stem cells (HSCs) develop at several anatomical locations and are thought to undergo different niche regulatory cues originating from highly conserved cell signaling pathways, such as Wnt, Notch, TGF-β family, and Hedgehog signaling. Most insight into these pathways has been obtained by reporter models and loss- or gain of function experiments, yet results differ in many cases according to the approach. In this review, we discuss existing murine reporter models regarding these pathways, considering the genetic constructs and reporter proteins in the context of HSC studies; yet these models are relevant for all other stem cell systems. Lastly, we describe a multi-reporter model to properly study and understand the cross-pathway interaction and how reporter models are highly valuable tools to understand complex signaling dynamics in stem cells. It is well-established that a small number of signaling pathways control the development of all cell types in the mammalian body . The dosPositional structure is particularly important for HSC development, heretofore evident at the exclusive residence of the first definitive HSCs during embryogenesis in the ventral area of the aorta . FurtherAlthough most in vitro and in vivo gain-of-function studies validate a role of conserved signaling pathways in HSC regulation, many in vivo loss-of-function models of these signaling pathways suggest that none of these pathways are indispensable for adult HSC function. Nonetheless, the complexity of niche interactions with HSCs and the complexity of intracellular signaling pathway cross-talk make the interpretation and combination of these results difficult and prejudiced. Rather, a combination between suppression and activation of these pathways will ultimately solve the balance of quiescence and proliferation in HSCs. Careful experiments will be needed to elucidate such effects; reporter systems for these pathways serve as invaluable tools in such analyses.Reporter systems are useful models to visualize the genetic regulation of a cellular response. Most signaling pathways initiate a cascade by several intracellular signaling molecules that end up placing transcriptional activator or repressor proteins to bind at a specific DNA sequence. This binding enhances the rate of transcriptional initiation in the promoter region of selective target genes, which in many cases is a complex convergence of multiple signals on the same promoter. The discovery of transcriptional activators and target genes have offered researchers means to genetically build-in reporter molecules to measure signaling pathway activity. Various reporter assays ranging from stable cell lines, transduction vectors, target gene-knock-ins, or multimerized transcription factor binding sites have been employed, but can give different outcomes when studying the same pathway.luc and lux gene, respectively) and jellyfish Green Fluorescent Protein (GFP). Nowadays, more advanced fluorescent proteins offer easier detection methods, expanding the possibilities of understanding quantitative and qualitative cell signaling. Nevertheless, caution should be taken with interpretation of reporter models mostly due to their genetic context. Barolo, commented on several important factors to consider when interpreting the data of Wnt/β-catenin/TCF reporters, which are in fact applicable to every reporter model [The most widely used detection systems in living organisms are bioluminescent proteins genetically engineered into reporter systems. The most commonly used reporter proteins in signaling pathway assays are firefly and bacterial luciferase , among others. Axin2 is well known for also being a negative regulator of the Wnt signaling pathway and plays an important role in signaling regulation [Secreted Wnt proteins activate the signaling cascade by binding to their corresponding Frizzled receptors and LRP co-receptors, leading to the nuclear translocation of cytoplasmic β-catenin . Withoutgulation .LacZ for a GFP fluorescent protein in the already existing TCF/Lef-LacZ and BAT-gal reporters, but was only successful in the TCF/Lef-LacZ derived variant. They steadily proposed that their new TCF/Lef:H2B-GFP reporter expression is independent of the DNA integration site, even though no confirmative data contributed this claim [Axin2 promoter, exon 1, and intron 1, reported by a reduced stable enhanced GFP [+/lacZ reporter, to study colorectal and liver tumors [Axin2 heterozygote, but no negative effects were reported. The Conductin+/lacZ reporter is considered to be the most reliable Wnt reporter to date [Axin2 targeting construct but with an mTurquoise2 fluorescent marker: Axin2-mTurquoise2 [TCF/Lef:H2B-GFP reporter and respects the endogenous genetic context of the Axin2 Wnt target gene. Lastly, the group of Roel Nusse, created Wnt reporters with cell lineage tracing capacity by adopting an Axin2 responsive element with a Cre-recombining reporter cassette in the Rosa26 locus [P2A-rtTA3-T2A-3xNLS-SGFP2 murine model, makes use of a knock-in multi-cystronic cassette upstream of the Axin2 stop codon containing an rtTA3 doxycycline-inducible driver, a triple nuclear localization signal GFP fusion protein and self-cleaving 2A peptides, which preserves both functional alleles of the Axin2 gene [Axin2 remains to be the best Wnt reporting gene, motivating for the generation of smarter transgenic models to overcome the low-level and low-amplitude oscillations of Axin2 expression [Several canonical Wnt signaling reporters have intended to make Wnt signaling visible and understandable . Most ofis claim . Nonetheis claim ,14 are tis claim . These mnced GFP . Althougr tumors . As a re to date , howeverrquoise2 . This fl26 locus . The mosin2 gene . Notwithpression .Hes and Hey. Nonetheless, Notch activity is highly context-dependent due to modulation through accessory proteins on the extracellular and intracellular side, resulting in elaborate receptor complexes. Various murine reporters have been published attempting to faithfully visualize Notch signaling  . The NCIignaling . The besll state . By combll state ,41. Addiand Dtx1 .Hes1-EmGFPSAT mice, which is a knock-in of the Hes1 region, showed real-time Notch activity in the intestine through administration of a pharmacological Notch inhibitor dibenzazepine (DBZ) to show GFP abolishment in intestinal crypts, but residual signaling in the villi. Oh et al., used the same Hes1-EmGFPSAT model together with tamoxifen-inducible CreER knock-in mice for individual Notch receptors to study Notch signaling in hematopoiesis [Hes1 expression in long term HSCs. Additionally, they reported molecular priming towards the erythrocytic lineage in Hes1 expressing Linneg/cKit+/Sca1+ cells, whereas Notch negative cells tend to differentiate towards megakaryocytic and granulocyte-monocytic subsets. Alternatively, the Hes1-GFP reporter [Less synthetic Notch endogenous murine reporters are available, where either Notch related promoters or directed knock-in strategies have been used respectively ,28. The opoiesis . They shreporter  was usedreporter .The most recent Notch reporter is a derivative from the TNR reporter with a human histone H2B–Venus (YFP) fusion fluorescent protein; CBF:H2B-Venus . This moThe transforming growth factor-β family include two distinct signaling pathways known as the TGF-β and BMP (Bone Morphogenetic Proteins) signaling pathways. Either pathway makes use of different SMAD proteins, but have SMAD4 in common to exert transcription of target genes. A third pathway, Activin, shares most of the intracellular regulatory and transcriptional proteins of TGF-β signaling but will not be further discussed in this review. TGF-β family signaling is initiated by the binding of either TGF-β, Activin or BMP proteins to their corresponding receptor . To dateCAGA sequence elements first detected in the human PAI-1 promoter [CAGA boxes, bind with the TGF-β /activin pathway-restricted SMAD3/SMAD4 complex but not with the SMAD2/SMAD4 complex [CAGA box reporters for the study of HSC regulation, due to their differing genetic background or lack of reporter activity verification in non-hematopoietic organs. The CAGA12-eGFP reporter, for example, is made on a fibrillin-1-deficient BL6/SJL background [12-eGFP reporter to be unfit for HSC studies. On the other hand, the SBE-lucRT mice are composed out of a trifusion detection protein containing Luciferase, red fluorescent protein (RFP), and thymidine kinase for noninvasive in vivo imaging [c-2J genetic background, which could be a point to consider before using this reporter. The role of TGF-β signaling in HSC biology is highly debated due to inconsistencies between studies . To datepromoter  , suggesting an association with TGF-β signaling. Activin signaling may additionally mask exclusive TGF-β signaling and other activated downstream Smad-independent pathways are feasible options upon receptor binding as well ,55. A coThe hedgehog (Hh) signaling pathway is well known for its function in organ patterning during embryogenesis by controlling cell proliferation, differentiation and maturation. Despite it being largely inactive in adult tissues, it has been implicated in several types of cancer. Its potential role in HSC maintenance has been studied to a lesser extent and usually hints towards a synergistic action with other conserved signaling pathways mentioned previously in this review. Gli1 and other target gene expression. Hh signaling is believed to induce different gene sets depending on the ligand activation gradient and tissue [Gli1 to be a reliable Hh transcriptional target gene, although Gli1 is not required for proper Hh signaling [Hh cell signaling is regulated by the GLI-Kruppel proteins (Gli1-3) which act as transcriptional repressors or activators. These zinc-finger containing proteins are highly conserved, but differ at the N-terminal and C-terminal domains which determines their functional role. Generally, Gli1 is known to be a constitutive transcriptional activator, Gli2 can be either a transcriptional activator or repressor, and Gli3 is a transcriptional repressor. The transformation of Gli proteins to their A-Gli (activator) of R-Gli (repressor) form is directed by the protein Suppressor of fused (Sufu) which directs proteasomal cleavage activity of the Gli-protein terminal ends. Gli1 on the other hand, is transcribed depending on the balance between A-Gli2 and R-Gli3. The Hh receptor, Patched (Ptch), responds to three secreted ligands: Sonic, Desert, and Indian . When thd tissue . Bai et ignaling . Neverthignaling , suggestlz reporter [Gli1 locus with a lacZ reporter protein, resulting in a dysfunctional Gli1. The Gli1 knock-out is the only non-lethal mutant of the Gli family members. This same reporter was used as a Gli1null (homozygote) model to study the role of Gli1 in adult HSC maintenance [CreERT x Rosa26-EGFP [lz reporter [Ptch is considered to be a negative regulator and a possible gene target of the Hh pathway, no murine reporter models are known to date using Ptch. Given the possible interference of other pathways on the downstream effector Gli1 [Ptch expression [Ptch is a less ideal gene for a Hh reporter.Most studies on Hh signaling have been performed in Drosophila and Zebrafish models in embryonic development. The first known Hh murine reporter was the Gli1reporter  , Hes1/5 , BRE, and CAGA endogenous promoters  and Gli . Secondly, the chosen fluorescent protein should match the contextual pathway dynamics while considering the maturation and degradation time (how quickly is it turned on and off). Cai, for instance studied a set of 15 XFPs on in vivo aggregate formation, photostability, and paraformaldehyde fixation stability before opting for a new Brainbow multi-fluorescent toolbox design [Before developing suitable murine reporter models, a knowledgeable reporter gene and fluorescent protein strategy must be defined. Caution should be taken when merely interpreting gene expression profiles for the selection of pathway-specific genes; hence they do not reflect actual protein effector function, especially when analyzing a complete signaling pathway. For instance, Gupta showed that ignaling . Therefox design . Thirdlyx design , since tx design ,72. In gx design .Id1-mKOκ and PAI-1-mScarlet-I transgenic approach would have attractive properties. Considering that BMP signaling is highly present in bone tissue and close to matrix components, a far-red XFP could diminish the existence of autofluorescence for easier investigation of HSCs. Both the orange-protein mKOκ [Gli1-mNeonGreen variant. mNeonGreen [lz reporter. Even insight into the cross-pathway interaction of Wnt and Hg signaling can be studied with the proposed FRET pairs mTurquoise2 and mNeonGreen [We envision a fluorescent reporter protein strategy adequate for flow cytometry technology based on the contextual setting of the pathway, brightness and maturation of the XFP  and FRETein mKOκ  and far-ein mKOκ  are exciein mKOκ ,81,82. LeonGreen  is a brieonGreen . The proposed set of reporter mice do not only have individual practicality, but furthermore have been envisaged for the combination of a multi-XFP mouse combining all five pathway reporters. In view of excitation and emission spectra, an accomplishable combination is feasible without unsolvable discriminative measurement of the XFP expression. By careful election of longpass and bandpass filters, all five XFP expression profiles can be evaluated while still being able to use phenotypic makers to determine specific hematopoietic cell subsets. A standard flow cytometer has an ultra-violet (405 or 407 nm), blue (488 nm), and yellow-green (561 nm) laser and the possibility to change filter sets according to the XFP combination. For proper XFP reporter distinction, mTurquoise2 is best excited by 405 or 407 nm, mNeonGreen and mVenus by 488 nm, and mKOκ and mScarlet-I by 561 nm . PossiblThe regulation of HSC homeostasis or even stress hematopoiesis makes the interpretation of the entirety of involved signaling pathways complicated. For therapeutic purposes, an adequate understanding on how to conduct clinically prepared HSCs to the desired state will only be feasible with more coherent and interconnective understanding on the role of signaling pathways. Merely stimulating one or two signaling routes, as often is done in in vitro HSC priming prior to transplantation, will not solve the equation of the intracellular pathway crosstalk which is strictly regulated in the physiological bone marrow niche. Although several attempts haven been completed to mimic the HSC niche in for example (3D) scaffolds or in bioreactors, researchers have not reached the point of re-enacting the cues required in HSC regulation. Before coming to that accomplishment, more knowledge should be gained on how HSCs respond to HSC niche ligands under physiological and transplantation settings in vivo . In part


Hyperuricemia is an important risk factor that contributes to atrium injury and AF, but the underlying molecular mechanism remains to be elucidated. In this review, we discussed the scientific evidence for clarifying the role of hyperuricemia in the pathogenesis of AF. Experimental and Clinical evidence endorse hyperuricemia as an independent risk factor for the incidence of AF. Various  Atrial fibrillation (AF) is considered to be the most frequent cardiac arrhythmia and its prevalence is increasing substantially. In 2016, 46.3 million individuals had prevalent AF/atrial flutter globally, and the prevalence of AF has been estimated between 2 and 4% in adults .Chronic diseases such as rheumatic heart disease, hypertension, hyperthyroidism, chronic kidney disease, and diabetes mellitus have all been regarded as risk factors for AF , 7. AlthA recent review on the role of UA in cardiovascular diseases (CVD) has focused on summarizing the association between uric acid and various cardiovascular diseases, mainly from experimental evidence . Also, tin vitro and in vivo studies regarding UA acting on several cellular signaling pathways in Bearing in mind that the extensive overlap exists between comorbidities and risk factors of hyperuricemia and arrhythmias, multivariable analyses of epidemiologically collected data cannot substitute proof generated from basic and clinical studies. As such, there is a need for further basic research to establish a causal relationship between UA and AF and to identify the mechanism by which UA is involved in AF pathology. Elevated UA can cause arrhythmia either directly or indirectly, depending on the availability of other risk factors. In this review, we have summarized Clinically, hypertension is an independent risk factor for AF. In the past, it was well-established that UA and nitrate concentrations were positively associated with elevated blood pressure . Some rein vitro has been found to involve Ikbα phosphorylation via NF-kB activated inflammatory signaling pathway. This leads to the up-regulation of inducible NO synthase (iNOS) expression and excessive NO production, which subsequently contribute to the injury of cells  to create a mild hyper-UA model ficantly . What's ficantly . Studiesficantly , 30, whiper se is a cytoplasmic enzyme consisting of at least one catalytic transmembrane-spanning NOX subunit, which produces ROS by transferring electrons from NADPH to molecular oxygen. In addition, the reduction of bioavailability can result in the down-regulation of NO and an increase in protein nitrosylation and lipid oxidation  and obesity , 31. Earxidation , 19. Conxidation .A substantial body of evidence suggests that oxidative stress plays a key role in the pathophysiology of AF. However, the molecular pathways of this pathologic process are complex. Therefore, oxidative stress and its modulation in AF require the development of strategies that target specific sources of ROS implicated in atrial remodeling . XO is dCell experiments have been conducted to reveal the effect of UA on cardiac remolding by stimulating the vascular Renin-Angiotensin System (RAS) . The stuNote, elevated UA levels cannot only increase the risk of myocardial oxidative damage through activating RAS but also lead to cellular damage by activating other pathways. For instance, a shred of evidence has shown that high UA can promote the up-regulation of NOX4 expression in renal proximal tubule cells and result in an increase in ROS production via activating P38 and ERK1/2 phosphorylation. Such ROS production through activation of P38 and ERK1/2 phosphorylation can further inhibit PI3K and Akt activation, and unbalance Bax/Bcl-2 equilibrium, which could eventually contribute to increased apoptosis and decreased cell activity .in vivo, consistent with those obtained in vitro. The phosphorylation of ERK and P38 was up-regulated in mice with acute hyperuricemia model  in humans . Recentl+ and UA can trigger an overproduction of TNF, IL-1, IL-6, and KC  and human vascular smooth muscle cells have been reported to produce a certain degree of vascular inflammation and vascular remodeling, mainly in the up-regulation of the expression of C-reactive protein, one of the independent risk factors for cardiovascular diseases and an important marker of inflammation . Anotherper se promotes its cellular release and acts as an autocrine and paracrine to activate both proinflammatory and pro-reparative mediators  through calcium mobilization and MERK/ERK pathway. A study that involved treatment of HUVEC with UA resulted in increased HMGB1 mRNA expression and acetylation of nuclear HMGB1 . UA afteediators . Besidesediators .in vitro and AF induced by hyperuricemic rats occurred primarily via induction of atrial remodeling , caspase-3, α-smooth muscle actin, and TGF-β1. Also, the study reported that uric acid significantly induced primary rat cardiomyocyte apoptosis and fibrosis modeling . Also, amodeling . Thus, Upressure . Likewispressure . Importapressure . These rA significant association has been reported between UA and cell apoptosis. Of note, Bcl2 family proteins are key regulators of apoptosis cell death. An experimental study suggested a high concentration of UA downregulates B-cell lymphoma 2 (Bcl-2) expression in pancreatic β-cells . This coImmune cells may play a vital role in the immunological pathogenesis of AF , and UA P < 0.001) (p < 0.001] in patients with type 2 diabetes after adjusted for multiple confounding factors (P < 0.001), even after adjustment for age and gender  had a higher risk of AF  . A singl factors . Also, a factors . Over a d gender . Anotherd gender . This evDifferent treatment modalities have shown different results. A cohort study tested the use of allopurinol and the risk of atrial fibrillation in the elderly in 8,569 beneficiaries using Medicare data. After adjustment for age, sex, race, Charlson–Romano comorbidity index, and use of statins, diuretics, ACE inhibitors, and β-blockers, the use of allopurinol for 6 months or more was associated with a reduced risk of incident AF in the elderly  . On the P = 0.001)  . However= 0.001) . FurtherP < 0.05) after adjusting for traditional risk factors such as hypertension, diabetes, nephropathy, and lipid metabolism disorders  level of serum UA were associated with increased risks of AF in comparison to patients with the lowest level of serum UA  but not in men   . Similar > 0.05) . Similar > 0.05) , althoug > 0.05) . Recentl > 0.05) .P < 0.001) and weighted genetic risk score (wGRS)  were significantly associated with increased UA levels. The mendelian randomized analysis was causally associated with rs1165196 , but not with wGRS  , IL-6, and TNF-α and IL-1β, especially in women . This ev= 0.832) , confirmExisting studies strongly suggest that hyperuricemia is independently associated with the increasing incidence of AF. Epidemiological and clinical studies highlight a close association between various conditions including hypertension, metabolic syndrome, DM, and other CVD comorbidities and increased risk of AF. Therefore, the mechanistic links between UA and AF are complex with several underlying diseases and conditions. Experimental and clinical data indicate that UA is implicated in the pathophysiology of AF via activation of inflammation, oxidative stress, and fibrosis induced atrial remodeling. Briefly, atrial remodeling involves electrophysiological and structural abnormalities that promote the development of UA induced AF. Also, UA induced AF activates apoptosis and immune system. There is still a need for further investigation to obtain a more comprehensive understanding of the role of UA in the pathophysiology of AF.YD and FL were contributed to literature researches, data collection, and were involved in the draft of the manuscript. XY and YX were contributed to the coordination and designing of the review and writing of the final draft of the manuscript. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RHOB – and identified possible signaling pathway crosstalk underlying cranial neural crest migration. The results reveal novel putative targets of canonical Wnt signaling during cranial neural crest EMT and highlight important intersections across signaling pathways involved in craniofacial development.Canonical Wnt signaling plays an essential role in proper craniofacial morphogenesis, at least partially due to regulation of various aspects of cranial neural crest development. In an effort to gain insight into the etiology of craniofacial abnormalities resulting from Wnt signaling and/or cranial neural crest dysfunction, we sought to identify Wnt-responsive targets during chick cranial neural crest development. To this end, we leveraged overexpression of a canonical Wnt antagonist, Draxin, in conjunction with RNA-sequencing of cranial neural crest cells that have just activated their epithelial–mesenchymal transition (EMT) program. Through differential expression analysis, gene list functional annotation, hybridization chain reaction (HCR), and quantitative reverse transcription polymerase chain reaction (RT-qPCR), we validated a novel downstream target of canonical Wnt signaling in cranial neural crest –  The migratory pathways and derivatives formed by the neural crest are regionalized according to their axial level of origin, such that cells from a given axial level give rise to a characteristic array of progeny and follow distinct pathways from those arising at other axial levels . The mosthe neck . Importathe neck .Many facets of cranial neural crest development are regulated by Wnt signaling . FurtherHere, we took advantage of a canonical Wnt signaling inhibitor, Draxin, to identify Wnt-responsive targets during chick cranial neural crest development. As Draxin overexpression impedes cranial neural crest EMT in a β-catenin-dependent mechanism , 2019, hGallus gallus) eggs , as previously described , then incubated tissue in Accumax  at 37°C for 15 min, with trituration every 5 min. Following dissociation, cells were washed with Hanks’ Balanced Salt Solution (Thermo Fisher Scientific #88284), filtered through a 20 μM nylon net mesh filter (Millipore Product #NY2004700), and resuspended in Hanks’ supplemented with 0.25% bovine serum albumin and 5% RQ1 DNase (Promega #M6101). GFP + cells were then collected using fluorescence activated cell sorting (FACS) at the Caltech Flow Cytometry Cell Sorting Facility.G. gallus genome  with Bowtie2  were fixed in 4% paraformaldehyde 1 h at room temperature, then dehydrated in graded methanol washes and stored at least one overnight at −20°C. HCR was performed as previously described , with cuTFAP2β. Integrated density measurements were quantified for ROIs on the control electroporated (left) and experimental electroporated (right) sides from the same embryo. Relative fluorescence intensity was then calculated by dividing the integrated density measurements for the experimental versus the control side of the same embryo. Statistical analyses were performed using Prism . P values are defined in the text, and significance was established with P < 0.05. P values were calculated for embryos using one-tailed paired t-tests with integrated density measurements for control versus experimental sides, and for qRT-PCR using two-tailed one sample t-tests for ΔΔCT values.Confocal images were acquired with an upright Zeiss LSM 880 at the Caltech Biological Imaging Facility. Images were minimally processed for brightness/contrast and pseudocolored using Fiji  and AdobT with normalization against 18S ribosomal RNA (ΔCT = Target CT − 18S CT) for Draxin, SNAI2, and RHOB for samples, then calculated ΔΔCT values (ΔΔCT = Average Control ΔCT − Perturbation ΔCT) for each target and replicate. The gene-specific primers used for qPCR were: Draxin-F 5′-CTACGCTGTTATGCCAAATTCC; Draxin-R 5′-GAATGATCCCTGCTCTCCATT; SNAI2-F 5′-GCA ACAAGACCTATTCCACTTTC; SNAI2-R 5′-GTACTTG CAGCTGAACGATTTC; RHOB-F 5′-CGTGATCCTCATGT GCTTCT; RHOB-R 5′-TGCGCAGGTCTTTCTTGT; 18S-F 5′-CCATGATTAAGAGGGACGGC; 18S-R 5′-TGGCAAA TGCTTTCGCTTT.RNA was extracted from sorted cells (Draxin overexpression) and dissected HH9+ embryonic half heads  using the RNAqueous-Micro Total RNA Isolation Kit (Invitrogen), according to manufacturer instructions. Following RNA isolation in elution buffer, cDNA was reverse transcribed using the SuperScriptIII First-Strand Synthesis System (Invitrogen) with oligo-dT priming. Quantitative PCR (qPCR) was performed using gene-specific primers with FastStart Universal SYBR Green Master Mix with Rox (Roche) and cDNA (diluted 1:10) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems) in triplicate. We determined ΔCWe have previously shown that the secreted protein Draxin functions as a potent inhibitor of cranial neural crest cell migration during EMT , 2019. IDraxin, as expected due to its experimental overexpression. Interestingly, we also detected significant downregulation of the Notch pathway effector HES5 (and related genes), and BMP4 (as well as its downstream target MSX1) , resulting in a filtered list of 134 differentially expressed genes  . Using Pet MSX1) , suggestet MSX1) , furtherCYTL1, ILK, NOV), a critical function of cranial neural crest, and genes involved in ribosome biogenesis , which has implications for craniofacial development  ,b. Consielopment  , consistent with predicted trends based on our transcriptomic analyses. This is consistent with our previously published work indicating that Draxin acts upstream of Snail2 protein expression (SNAI2 and RHOB using quantitative reverse transcription PCR (RT-qPCR) with sorted cells collected alongside sequenced cells from SNAI2 and RHOB with Draxin overexpression . Importantly, we observed upregulation of SNAI2, an established direct target of canonical Wnt signaling , and suggest that Draxin downregulation, and subsequent activation of Wnt signaling, is essential for crosstalk and feedback of signaling pathways that alter cranial neural crest transcriptional activation, and ultimately EMT.Taken together, our data identify a novel target of Draxin and canonical Wnt signaling during cranial neural crest EMT  or based on signaling pathway crosstalk  remains to be explored.Using transcriptome profiling of Draxin-responsive targets, we identified likely gene targets of canonical Wnt signaling during cranial neural crest EMT. Consistent with our previously published work examining protein expression , we verimination ; its mismination . Interesmination . Further1 ligand . This is1 ligand . Interes1 ligand . Thus, wSOX10 and ETS1, genes associated with neural crest EMT  can be found below: Ethical review and approval was not required for the animal study because our study uses chicken embryos at E1-2. These are not considered vertebrate embryos until E10, and thus we do not require ethics committee approvals or protocols, as they are not considered vertebrates at the stages we work.EH, MP, and MB conceived the project and conducted the experimental design and data interpretation. EH and MP performed the cell dissociations, library preparations, and RNA-seq analyses. EH performed the functional annotation, hybridization chain reaction experiments, imaging, quantitation, and statistical analyses. EH and MB wrote the manuscript with editing by MP. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read‐out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter‐copper electron transfer catalyzed by CuNiRs. Cryoreduction‐EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox‐center containing proteins . Metalloenzyme catalysis: Through the use of cryoreduction and annealing, a method of studying electron transfer in redox active metalloenzymes is developed. Combined with electron paramagnetic resonance spectroscopy, active states in the catalytic cycle of the copper containing nitrite reductases are probed. Transition metals  are ubiquitous in biology and play key roles in redox enzymes that are vital for life and the next generation of biofuels.Cryoreduction‐EPR involves the use of ionizing radiation to reduce frozen solutions of protein e.g. 77 K). Then, by annealing the frozen protein solution to higher temperatures, reaction intermediates are formed and trapped, enabling the capture of transient enzyme species, which if paramagnetic, can be investigated using EPR spectroscopy. Active catalytic intermediates in a number of enzymes containing heme catalytic centers7 K. ThenCuNiRs catalyze the reduction of soluble nitrite to produce gaseous nitric oxide, a key step in denitrification and the global nitrogen cycle:c or azurin partner proteins fused at their C‐ or N‐termini.Two‐domain CuNiRs were first identified over 40 years ago, and have been the subject of extensive biochemical and biophysical studies.Alcaligenes xylosoxidans CuNiR; AxNiR) and three‐domain cytochrome c‐fused CuNiRs  are trimeric enzymes.2O), nitrite (NO2−) or nitric oxide (NO) molecule coordinate the T2Cu .Two‐domain CuNiRs  collected on the RpNiR core protein, it is observed that the EPR signals at the perpendicular orientation is split into four hyperfine lines, a feature that is due to the strong hyperfine coupling of the 63, 65Cu nuclei with the electron spin of T1Cu center. This splitting is absent in the “nitrite free” AxNiR sample, suggesting subtle differences in the electronic structures of the T1Cu centers present in both of these CuNiR proteins .AxNiR and RpNiR proteins at 77 K with ϒ‐rays from a 60Co source, a reduction in the T1Cu EPR signal is observed , it has recently been reported that it is unable to transfer electrons between the copper centers in the absence of bound nitrite.RpNiR core protein should in theory remain oxidized during the annealing process.Solvated electrons generated from the ϒ‐radiation in principle could directly reduce the T1 and the T2Cu centers of RpNiR core samples are shown in Figure AxNiR protein, there are few or no alterations in the intensity of the T2Cu signal of RpNiR core during temperature ramping , indicating that solvated electrons produced from ϒ‐radiation do not directly reduce the T2Cu of the CuNiR proteins during the annealing. We do however observe a change in the electronic properties of the T2Cu center during annealing, with a second paramagnetic T2Cu species (T2Cu[2]) forming over the experimental temperature range . Between 170–250 K, the signal attributed to the T1Cu continuously decreases until it plateaus at a minimum. Unexpectedly, between 250 and 270 K, we observe what we hypothesize to be electron transfer from the fully reduced T1Cu to the T2Cu[2] species, causing a shift in the T2Cu equilibrium position towards the “resting” T2Cu species (T2Cu[1]) accompanied by a decrease in the T2Cu hyperfine intensity .Temperature‐dependent changes in the T1 and T2Cu signals of the cryolytically reduced RpNiR core protein in absence of nitrite.RpNiR core protein, which is formed upon reduction of the T1Cu and can facilitate inter‐copper electron transfer. We must note that in our cryoreduction‐EPR experiments performed on the RpNiR core protein, the percentage signal change attributed to the reduction of the T1Cu site during annealing is far lower than that seen in the AxNiR sample (approx. 15 % in RpNiR core and 40 % in the AxNiR sample). We attribute this to a larger percentage of T1Cu reduced in the RpNiR core protein during the initial cryolytic reduction process , and also plausibly, a result of redox potential differences for the T1Cu centers in the different constructs ; ≈370 MHz with g=2.290; AxNiR). This is indicative of the nitrite being bound to the catalytic T2Cu site. For the “nitrite‐bound” RpNiR core protein, there are subtle changes in the T2Cu hyperfine features. Based on our new EPR spectral simulations , approx. 20 % of the T2Cu hyperfine features have shifted from a “nitrite‐free” to a “nitrite‐bound” state when the oxidized RpNiR core was incubated with 5 mm nitrite. No additional changes in the hyperfine features of the T2Cu center were observed upon the addition of supplementary nitrite, suggesting that in an oxidized state only a fraction of the RpNiR core population can accept nitrite. Previous studies have shown that a conserved tyrosine residue, present on a cyt c linking region, blocks nitrite from binding to the T2Cu site of the oxidized full‐length RpNiR protein.RpNiR core protein, but occupies an alternative state in the X‐ray structure, allowing nitrite to bind.We also performed cryolytic reduction‐EPR measurements on “nitrite‐bound” forms of II) of “nitrite‐bound”, cryolytically reduced AxNiR are presented in Figure I−NO+] or [CuII−NO] redox state at the T2Cu, neither of which are EPR active. The observation of a decrease in intensity in T2Cu EPR signal with A∥; ≈370 MHz with g=2.290 also rules out the formation of [CuI−NO] redox state, which would be observed g≤2.00 region, as in the case of Rhodobacter sphaeroides NiR (RsNiR),I−NO inorganic model complex,14N nucleus of paramagnetic NO center; . Alternatively, as hypothesized previously,II−OH] redox state at the T2Cu center. Between 218 and 238 K, the signal associated with oxidized nitrite‐bound T2Cu “grows in” once again  T1 and T2Cu signals of the “nitrite‐bound”, cryolytically reduced RpNiR core protein are presented in Figure RpNiR core protein occurs in the absence of protein motions. Above 180 K, there is an inverse trend in the signals attributed to the oxidized T1 and T2Cu centers . From 180–210 K, the intensity of the oxidized T1Cu decreases, while the T2CuII signal increases. However, between 210–270 K, a decrease in the oxidized T2Cu signal and an increase in the population of oxidized T1Cu are observed. Based on these data, we propose the following mechanism for the RpNiR core protein. In the oxidized form of the enzyme, 20 % of the T2Cu centers are occupied with nitrite. The reducing conditions available for both T1Cu and T2Cu centers lead to the depopulation of the resting state EPR signal of the T2Cu center. This reduction of the T1Cu site appears to support binding of the nitrite substrate at the T2Cu site . Following this, electrons transfer from the T1Cu to the T2Cu site, which is now fully occupied with nitrite.Oxidized m)In recent work, we proposed that electron delivery to the tethered In summary, the cryoreduction‐EPR method can be used to track inter‐copper electron transfer in the copper nitrite reductase family of enzymes. Our approach highlights the importance of protein dynamics in inter‐copper electron transfer catalyzed by two‐domain copper nitrite reductases. To the best of our knowledge, this is the first spectroscopic study that has enabled direct monitoring of electron delivery, nitrite‐binding and nitric oxide production at the T2Cu in this family of enzymes. Our work has also enabled simultaneous observation of electron transfer between the T1 and T2 coppers in CuNiRs. The approach we have developed is general and could be used to further understand intra‐protein electron transfer in other multi‐center copper‐containing enzymes that have a minimal UV/Vis optical signal associated with the T2Cu sites such as laccases,The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The Wnt signaling pathway plays important roles in embryonic development, homeostatic processes, cell differentiation, cell polarity, cell proliferation, and cell migration via the β-catenin binding of Wnt target genes. Dysregulation of Wnt signaling is associated with various diseases such as cancer, aging, Alzheimer’s disease, metabolic disease, and pigmentation disorders. Numerous studies entailing the Wnt signaling pathway have been conducted for various cancers. Diverse signaling factors mediate the up- or down-regulation of Wnt signaling through post-translational modifications (PTMs), and aberrant regulation is associated with several different malignancies in humans. Of the numerous PTMs involved, most Wnt signaling factors are regulated by ubiquitination and deubiquitination. Ubiquitination by E3 ligase attaches ubiquitins to target proteins and usually induces proteasomal degradation of Wnt signaling factors such as β-catenin, Axin, GSK3, and Dvl. Conversely, deubiquitination induced by the deubiquitinating enzymes (DUBs) detaches the ubiquitins and modulates the stability of signaling factors. In this review, we discuss the effects of ubiquitination and deubiquitination on the Wnt signaling pathway, and the inhibitors of DUBs that can be applied for cancer therapeutic strategies. MITF gene, which plays an important role in melanogenesis. Also, it is known that MITF is phosphorylated by ERK, and ubiquitinated and regulated by hUBC9 . Th. Th46]. y cancer . In humal cancer . APC is  gastric , liver, Axin1 by X-ray irradiation inhibits lung cancer cell proliferation and invasion . C. C197]. Inhibitors for USP7 have been well studied among the DUB families, since USP7 regulates cancer related proteins such as MDM2, p53, PTEN, and FOXO . USP7 isFurthermore, the USP14 inhibitor b-AP15 is known to induce ER stress and represses liver cancer cell growth through regulation of the Wnt signaling pathway . EffectsIn addition to USP7 and USP14, a number of inhibitors for DUB involved in the deubiquitination of Wnt signaling components have been identified and developed . HoweverOver the past few decades, numerous researches have focused on the association of Wnt signaling pathway and human cancers. It has been found that the modification of stability or activity of each component in the Wnt signaling pathway through gene mutation or PTM causes and inhibits diverse human cancers. Thus, a mechanism controlling Wnt signaling has an important implication for targeting cancers by dysregulation of Wnt signaling factors. Ubiquitination and deubiquitination are usually known to regulate protein levels by ubiquitin mediated proteasomal degradation. Diverse E3 ligases and DUBs are associated with Wnt signaling factors, resulting in upregulation or downregulation of Wnt target genes such as c-Myc and Cyclin D1, which are related to tumor growth. Many researches have determined the E3 ligase and DUB target Wnt signaling factors, as well as sites of target proteins which undergo ubiquitination and deubiquitination. However, many aspects remain unclear in the ubiquitination and deubiquitination mechanism for Wnt signaling factors, and future studies require a full understanding of the adjustment of Wnt signaling through ubiquitination and deubiquitination. The balance between these mechanisms will be significant for modulating the Wnt signaling pathway and deriving potent therapeutic strategies in different contexts.


Achieving rapid genetic progress while maintaining adequate genetic diversity is one of the main challenges facing the dairy industry. The increase in inbreeding can be used to monitor the loss of genetic diversity. Inbreeding tends to increase the proportion of homozygous loci, some of which cause homozygosity of recessive alleles that results in reduced performance. This phenomenon is known as inbreeding depression and tends to be most prominent on fitness-related traits, such as male fertility. Traditionally, inbreeding has been monitored using pedigree information, or more recently, genomic data. Alternatively, it can be quantified using runs of homozygosity (ROH), i.e., contiguous lengths of homozygous genotypes observed in an individual’s chromosome.The objective of this study was to evaluate the association between ROH and sire conception rate. ROH were evaluated using 268 k genetic markers in 11,790 US Holstein bulls. Interestingly, either the sum, mean, or maximum length of ROH were negatively associated with bull fertility. The association analysis between ROH and sire fertility was performed comparing 300 high-fertility vs. 300 low-fertility bulls. Both the average and sum of ROH length were higher in the low-fertility group. The enrichment of ROH regions in bulls with low fertility was assessed using a Fisher’s exact test. Nine regions were significantly enriched in low-fertility compared to high-fertility bulls. Notably, these regions harbor genes that are closely related to sperm biology and male fertility, including genes exclusively or highly expressed in testis.The results of this study can help not only to manage inbreeding in genomic selection programs by designing custom mating schemes, but also to better understand the mechanisms underlying male fertility in dairy cattle. Inbreeding is the mating of individuals that share at least one common ancestor, and therefore, making the number of direct ancestors in the pedigree to be smaller than expected. Inbreeding cannot be avoided in populations of limited size. In addition, in livestock species, only a limited number of parents are used to produce the offspring for the next generation. In dairy cattle, the extensive use of semen from elite bulls and common breeding practices such as mating half-brother and half-sister or first or second cousins, has led to a decrease in effective population size along with a steady increase in inbreeding coefficients for the last decades .Increased levels of inbreeding leads to inbreeding depression, which is the reduction of the mean phenotypic value for any trait under selection . InbreedInbreeding depression can arise from three basic mechanisms: (i) the partial dominance hypothesis refers to the expression of deleterious recessive alleles in homozygous individuals and therefore expressing the deleterious genotype; (ii) the over-dominance hypothesis where the heterozygous alleles expressing the superior genotype are underrepresented in a population with increased inbreeding; and (iii) the epistasis hypothesis where a combination of heterozygous alleles expressing a superior genotype is less frequent as inbreeding arises . In geneAs high-throughput genotyping became available, different methods using genome-wide molecular information were developed to estimate the realized proportion of the genome shared by two individuals. This information can be used to calculate inbreeding levels even for animals with no pedigree information , 14. It High density single nucleotide polymorphism (SNP) arrays allow detecting long IBS segments within an individual’s chromosome. These continuous homozygous segments assumed to be inherited from a recent common ancestor are called runs of homozygosity (ROH) and provide a potential solution for many of the problems mentioned with the pedigree and genomic methods. Indeed, ROH has been proven to be the most powerful method to calculate inbreeding coefficients . The usen = 11,790). The mean of ROH length was 5187 kilobases, equivalent to 543 consecutive homozygous SNP. The maximum length of ROH was 140,731 kilobases, equivalent to 13,417 SNP. The number of ROH segments per bull ranged from 13 to 104, with an average of 58.7. The percentage of homozygous regions for each chromosome is shown in Fig. A total of 692,131 runs of homozygosity (ROH) were found in the entire US Holstein bull population . The Pearson’s correlation coefficient between SCR and sum of ROH was equal to −0.09, while the correlation between ROH and pedigree inbreeding was equal to 0.74. The relationship between SCR and ROH was also evaluated for each individual autosome, and the same trend was observed for each chromosome (data not shown).Association between homozygosity and male fertility was assessed by plotting the total homozygous regions for each animal (sum of ROH) versus sire conception rate records Fig. b. The reP-value ≤ 0.01) between fertility groups. Mean values of ROH were 4950 kb and 5360 kb for high-fertility and low-fertility bulls, respectively  were found between groups. Indeed, the mean of total ROH length was 323 Mb for low-fertility bulls and 280 Mb for high-fertility bulls  and the t-values  can be found in Table The significant association between these nine ROH segments and dairy bull fertility was validated using the entire US Holstein bull population. From the original 9 overlapping ROH regions, eight of them were significantly associated with sire conception rate records in the whole population , with the exception of the region located in chromosome 2 that showed a Inbreeding is a growing concern for the dairy cattle industry. Traditionally, inbreeding has been assessed using either pedigree or genomic relationships, but these methods are not perfect, mainly because their inability to explore specific regions of high homozygosity in the genome. The impact of inbreeding and increased homozygosity on fitness traits, such as longevity and fertility, has been studied in dairy cows, but the impact on dairy bull fertility has received less attention, despite the fact that one sire is generally used to inseminate hundreds or even thousands of cows. To the best of our knowledge, this is the first study investigating how local inbreeding affects dairy bull fertility. Our goal was to investigate how specific homozygous regions of the genome impact male fertility in dairy cattle and reveal the putative candidate genes located in these regions. First, we evaluated the association between runs of homozygosity (ROH) and sire conception rate (SCR) records using 11,790 Holstein bulls with 268 k SNP markers. Second, we separated the entire population into 300 high-fertility and 300 low-fertility bulls in order to investigate the potential enrichment of ROH segments in subfertile sires, i.e., bulls with very low SCR values. Finally, we mapped the significant ROH regions to the bovine genome in order to identify candidate genes that may affect male fertility in cattle.n ≈ 59, was higher than the average reported by Kim and collaborators [n ≈ 40), probably because we used a 100 SNP window threshold rather than the 50 SNP window, although very similar results were found for total autozygosity (~ 10%). The distribution of total homozygosity among chromosomes was relatively even, but higher in chromosomes 2, 7, 10 and 20. The distribution of ROH is correlated with genomic features such as GC content, but also selection and recombination rate, with longer ROH occurring more often in low recombination areas of the genome [Runs of homozygosity were detected in all the bulls evaluated in this study. The average length of ROH was longer than 5 Mb, and hence, it can be defined as a long ROH. Long ROH are likely to arise from a recent common ancestor, less than 10 generations ago . The aveborators  (n ≈ 40)e genome , 22. Sime genome  found thThe impact of homozygosity on service sire fertility was assessed by calculating the relationship between total ROH and SCR values. The correlation was consistently negative for all autosomes. These results are in agreement with the premise that bull fertility is a complex phenotype which is influenced by several features in the genome with relatively small effects. Runs of homozygosity has been previously used to study the negative impact of local homozygosity in dairy cow fertility, including increased intervals from first to last insemination  and embrThe 300 top and 300 bottom Holstein bulls in the SCR distribution clearly represent highly fertile and subfertile sires, respectively. Notably, there was a clear difference in the levels of homozygosity between these two fertility groups, in terms of average length of ROH and also in terms of total homozygosity, represented as the sum of ROH. Moreover, although none of the identified ROH regions were exclusive to low-fertility bulls, nine ROH segments were found to be significantly enriched in the subfertile bulls. Importantly, eight out of the nine ROH regions were statistically validated in the entire population, and therefore, these results are not false positives due to population structure. It is also worth noting that some of the identified regions are located near previously reported regions associated with dairy bull fertility, either regions with additive effects  or non-aTAF12, PAFR, and RF00026. Gene TAF12 encodes the TATA-box binding protein associated factor 12, which regulates a testis-specific gene expression program in primary spermatocytes required for terminal differentiation of male germ cells [RF00026 encodes the U6 spliceosomal RNA and homologs of this gene has been associated with testicular hypoplasia in Nellore cattle [The region in BTA2 harbors three strong candidate genes directly implicated in male fertility, namely rm cells . Gene RFe cattle  and alsoDDX46, CATSPER3, TGFB1, SMAD, and PKD2L2. Gene DDX46 encodes a member of the DEAD box protein family and is implicated in a number of cellular processes involving alteration of RNA secondary structure. Interestingly, DDX46 is highly expressed in testis where it plays a critical role in the final step of the first meiotic prophase in male germ cells [CATSPER3 is exclusively expressed in the testis and encodes a sperm-specific ion channel. This gene has been associated with sperm function and male fertility in several species including cattle [TGFB1 and SMAD5 are clustered together in BTA7 and encode ligands that play important roles in the regulation of cell development and growth [PKD2L2 is also located in this significant region of BTA7, and although its role in sperm biology and male fertility is not well understood, it is highly expressed in mammalian testis [The region in BTA7 contains several candidate genes, including rm cells . Gene CAg cattle . Abnormag cattle . Both ged growth . Interesd growth  and abnod growth . Finallyn testis .PIP5K1B, FXN, and TJP2. Gene PIP5K1B along with PIP5K1A play an important role in spermatogenesis in mice, since knockout males lacking both proteins exhibit complete infertility due to decreased sperm number and a 25% reduction in testes weight compared to wild-type males [FXN encodes a mitochondrial protein that regulates mitochondrial iron transport and respiration. Interestingly, a knockout mutation in FXN produces embryonic lethality in mice [TJP2 encodes a tight junction protein that is critical for the blood-testis barrier, and its deficiency has been associated with reduced fertility and pathological changes in the testis [The significant ROH segment in BTA8 harbors at least 3 putative genes related to male fertility, namely pe males . Gene FX in mice . Gene TJe testis .ARID4A, TOMM20L, and DAAM1. Gene ARID4A plays an important role in regulating Sertoli cells in spermatogenesis [TOMM20L is mostly expressed in testis and regulates different sperm functions, such as motility and viability [DAAM1 has a fundamental role in cytoskeletal organization in testis and sperm production in rats [The region in BTA10 contains three strong candidate genes, ogenesis . Of specogenesis . Gene TOiability . Finally in rats .MYO3. The protein encoded by MYO3 belongs to the myosin superfamily and its expressed only in testis. During spermiogenesis, myosins participate in acrosomal formation, nuclear morphogenesis, mitochondrial translocation and spermatid individualization [TBX3 and TBX5. These T-box genes encode transcription factors that are directly involved in the regulation of different developmental processes. These two transcript factors are highly expressed in testis and prostate, and play key roles in the development of the mammalian reproductive system [The region in BTA13 harbors gene lization . Finallye system .This study evaluated the association between runs of homozygosity and male fertility in dairy cattle. Notably, runs of homozygosity were more prevalent in low-fertility compared to high-fertility Holstein bulls, suggesting that inbreeding and increased homozygosity have a negative impact on dairy bull fertility. Genome-wide mapping of ROH can help to find putative genes affecting bull fertility, and hence, provide a better understanding of the molecular mechanisms underlying male fertility. Here, most of the ROH segments enriched in low-fertility bulls harbor genes directly implicated in testis development, spermatogenesis, and sperm biology. It should be noted that our bull fertility dataset, based on cow field records, is subjected to pre-selection given that only bulls with decent sperm quantity and quality parameters are marketed, and hence, our results should be considered in the context of subfertile bulls in field conditions. Overall, the findings of this study can contribute to the design of mating programs that avoid the production of homozygous offspring which may carry deleterious alleles for male fertility.Sire conception rate (SCR) was used as a measured of bull fertility. It represents the US national phenotypic evaluation of service sire fertility, and it is based on confirmed pregnancy records. The statistical model for evaluating dairy bull fertility considers both variables related to the sire (including age of the bull and AI organization), and also variables associated with the cow that receives the unit of semen, including herd-year-season, cow age, parity and milk yield , 46.https://www.uscdcb.com/). Since there are Holstein bulls in the dataset with more than one male fertility evaluation, the most reliable value, i.e. the SCR record with most breedings was used for the analyses [The SCR records from 11,790 US Holstein bulls were used in this study. The SCR values ranged from − 18.4 to + 11.4%, and the number of breedings per bull ranged from 300 to 136,001. These SCR records were released from August 2008 to April 2018 and are freely available in the Council on Dairy Cattle Breeding (CDCB) website  for the high-fertility and low-fertility groups were + 3.77% (0.73) and − 6.98% (2.28), respectively. The average and the sum of ROH lengths were compared between fertility groups.https://www.ensembl.org/) was used to retrieve the list of genes within each genomic region of interest.The next step was to identify genomic regions where ROH overlapped across individuals. We defined consensus ROH as segments of overlapping ROH that had a minimum of five SNP. The goal was to determine if the proportion of individuals with overlapping ROH was different in low-fertility vs high-fertility group. For each ROH of interest, a Fisher’s exact test using a 2 × 2 table was performed to determine if there was a statistical difference, i.e., if there was a significant enrichment of this ROH region in the low-fertility group. All significant ROH regions were mapped to the latest bovine refence genome assembly (ARS-UCD1.2) in order to identify candidate genes affecting male fertility. The Ensembl BioMart MartView ; b is the vector of fixed effects including each of the significant ROH regions as binary trait (presence/absence); X and Z are the design matrices relating SCR records to fixed and random effects, respectively; u is the vector of animal genetic effects  and e is the vector of random residuals. The random effects u and e were distributed as G is the additive genomic relationship matrix and R is an identity matrix. The association of each ROH region with SCR was evaluated using t-test and those ROH with |t-value| ≥ 2 were declared as significantly associated with male fertility, and therefore were considered as validated. This analysis was performed using the BLUPF90 family programs from Ignacy Misztal and collaborators, University of Georgia.The set of significant ROH regions identified in the previous step was validated in the entire US Holstein bull population  using the following linear mixed model:Additional file 1. List of overlapping ROH detected across the entire genome.Additional file 2. List of ROH segments enriched in low-fertility bulls.


In the absence of clinical data on Human T leukaemia Type 1 and COVID-19 infection, we are providing guidance to clinicians who look after people living with HTLV-1. Human T Leukaemia Type -1 (HTLV-1) is one of the most oncogenic human viruses . Most peAs clinicians, what advice should we provide to our patients who are either asymptomatic HTLV-1 carriers or those who suffer from chronic inflammatory conditions such as HAM/TSP and/or ATL?To our knowledge, there are no clinical data available on people who live with HTLV-1 and have suffered from mild or severe COVID-19 infection, but there have been several statements released on how to provide care for people who live with HIV during the COVID-19 pandemic. Reassuringly, the morbidity and mortality of people who live with HIV suffering from COVID-19 infection do not differ from people without HIV infection, if they have been sufficiently immune reconstituted while on HIV antiviral medication .However, for several reasons, it will be challenging to extrapolate guidance provided for people living with HIV infection for people living with HTLV-1. There is no effective HTLV-1 antiviral therapy which can allow immune reconstitution as well as reduce tissue inflammation. People suffering from HAM/TSP often receive life-long immune-suppressive drugs such as corticosteroids, methotrexate, cyclosporine and azathioprine. People affected by ATL are already severely immunosuppressed and receive further immunosuppressive therapy such as corticosteroids, interferon, chemotherapy or myeloablation as preparation for stem-cell transplantation, and are then on life-long immunosuppressive therapy.To date, there is no peer-reviewed publication on HTLV-1 and COVID-19 coinfection to rely on. Therefore, we make the following recommendations based on our clinical experience, scientific knowledge, recent data on risk factors for developing severe COVID-19 infection, and common sense—having worked as HTLV-1 clinical experts for an accumulated 45 years.A person living with HTLV-1 suffers from an HTLV-1 disease, for example ATL, HAM/TSP, broncho-alveolitis or uveitis.A person living with HTLV-1 suffers from other co-morbidities such as diabetes, chronic cardiac, lung, liver or kidney disease.We recommend shielding if:Reducing clinician appointments to a safe minimum and ideally held through telehealth.Arrange for regular food and medication delivery, to avoid having to leave their home.Using hand sanitation, wearing a mask and maintain a distance of 1.5–2 m from people.Continue to exercise or engage in physical therapy at home on their own or supervised by a physical therapist via telehealth.In a shared household, where other co-habitants access the outside world, the person living with HTLV-1 should stay in their own room as much as possible. They should use their personal towels, bathroom, and toilet if available, and arrange to use shared spaces, such as the kitchen, only when on their own.Shielding is when a person tries to minimize any contact with the outside world for as long as COVID-19 is endemic where they live. This means:We recommend limiting the number of persons a patient comes in contact with, implementing sensible personal distancing and wearing masks if a person living with HTLV-1 has no other co-morbidities but isMaleOver 60 years of ageHave a Body Mass Index of 29.9For all people living with HTLV-1, it is crucial to stop smoking and to keep alcohol consumption to a healthy minimum to support the immune system and to limit lung injury. It is advisable to offer eligible persons influenza and/or pneumococcal vaccination.It is vital to rapidly seek health care advice if a person living with HTLV-1 develops symptoms associated with COVID-19, and to disclose information about their HTLV-1 infection and medication to their health care team. Ideally the patient’s HTLV physician will immediately be contacted for additional clinical input.These recommendations differ from our routine recommendations to patients suffering from HAM/TSP, where daily exercise and stretching in- and out-doors are paramount to keep the musculoskeletal and nervous system simulated. However, we believe that sufficient stimulation can be achieved through small-dose daily exercise at home.Furthermore, many HAM/TSP and ATL patients are wheelchair- or bed-bound, and, traditionally, some people living with HTLV-1 may sleep on the floor and not on beds, which makes mobilization on their own difficult. It may be difficult for these patients to self-isolate, since they have to rely on family, friends and carers for their daily needs. Whenever possible, these patients should be cared for by a limited number of people at home, ensuring that they are regularly mobilized to avoid pressure ulcers, infections and sepsis.People in isolation need support with their physical needs and mental health. It can be daunting for people living with HTLV-1 to limit their routine exercise and human–human interactions. Establishing a healthy daily routine to support the mental health of a person living in isolation can be as important as regular healthy meals. Regular remote socialization via phone and video calls with friends and family supports well-being and daily meditation may strengthen the immune-system and reduce stress ,15.Finally, trying to expose the skin to mid-day sunshine by sitting by an open window, on the balcony, or in private garden can help maintain good bone health and the immune system through regular Vitamin D production .We are aware that these recommendations are far from being evidence-based the way we would like to see. These limitations are because there are no data available that we are aware of on HTLV-1 and COVID-19 infection. Besides, unlike for HIV, there is no HTLV Health Topic provided by the World Health Organisation so that updates could be offered regularly . FurtherNevertheless, we hope this information can be of use and provide clinicians with proactive actions they can take to make sure people living with HTLV-1 are safe from catching COVID-19, while maintaining good physical and mental health.


Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in the innate immune response. These receptors are expressed on a wide variety of immune and non-immune cells, and they help shape the immune response to infection and injury through the recognition of pathogen-associated molecular patterns (PAMPs) as well as endogenous damage-associated molecular patterns (DAMPs). Accumulating evidence suggests that, in addition to regulating mature effector immune cells, TLRs can influence the immune response from the level of the hematopoietic stem cell (HSC). HSCs express TLRs, and exposure to TLR ligands influences the cycling, differentiation, and function of HSCs, with chronic TLR stimulation leading to impairment of normal HSC repopulating activity. Moreover, enhanced TLR expression and signaling is associated with myelodysplastic syndromes (MDS), a heterogenous group of HSC disorders characterized by ineffective hematopoiesis and a high risk of transformation to acute leukemias. In this review, we will discuss the role of TLR signaling in the pathogenesis of MDS, focusing on the known direct and indirect effects of this type of signaling on HSCs, the mechanisms of TLR signaling upregulation in MDS, the changes in TLR expression with disease progression, and the therapeutic implications for modulating TLR signaling in the treatment of MDS. The TLRs are a family of pattern recognition receptors that recognize foreign pathogen-associated molecular patterns (PAMPs) as well as endogenous byproducts of cellular damage  , 2, and The TLR family is comprised of 10 members in humans (TLR1-TLR10) , 12–15, Tlr2−/−, Tlr4−/−, Tlr9−/−, and MyD88−/− mice) display enhanced repopulating activity in competitive transplantation experiments  and indirect (cell non-autonomous) mechanisms. Supporting a direct effect of TLR signaling on HSCs, for example, TLR stimulation via LPS or PAM3CSK4 leads to proliferation and myeloid differentiation of sorted HSPCs (Lineage- c-Kit+ Sca-1+ cells) in culture  recipients (thus minimizing indirect effects of TLR stimulation) differentiate into macrophages upon treatment with TLR ligands (Tlr4−/− and WT cells transplanted into Tlr4−/− recipients) to show that LPS directly stimulates HSC proliferation and impairs HSC self-renewal. Further, they found that the LPS-induced loss of HSC self-renewal is TRIF-, and not MyD88-, dependent, and is mediated by the production of reactive oxygen species (ROS) and activation of p38 MAPK  , 35, 36,eriments , 40. Coneriments . Similar culture , and pur ligands  used chip38 MAPK . Zhang ep38 MAPK  similarlp38 MAPK . Our ownThe myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by abnormal hematopoiesis and a high risk of transformation to acute myeloid leukemia (AML) , 44. TheThe pathogenesis of MDS is thought to involve a combination of genetic mutations  –55, whicRegarding the contribution of hyper-activated innate immune signaling, numerous studies have shown that TLR signaling is enhanced in the CD34+ HSPCs of patients with MDS, with multiple components of the pathway (TLRs themselves or downstream effectors) either overexpressed or aberrantly activated compared to healthy controls –70. Wei Not only are the TLRs overexpressed in MDS, but multiple genetic abnormalities and epigenetic events in MDS have been found to contribute to enhanced sensitivity to TLR ligands. In patients with deletions of 5q (del(5q) MDS), for example, loss of a copy of the micro-RNAs mir-145 and miR-146a result in activated TLR signaling. These micro-RNAs normally inhibit the TLR signaling intermediates TIRAP (mir-145) and TRAF6 (mir-146a), and knockdown of these miRs or enforced expression of TRAF6 in mouse hematopoietic stem and progenitor cells recapitulates some of the features of human del(5q) MDS including elevated serum IL-6 levels, megakaryocytic dysplasia, and progression to marrow failure or acute myeloid leukemia . EnhanceAdditionally, spliceosome mutations , which are common in MDS , have bein vitro, as well as engraftment of the MDS-L cell line into NSG mice. Further, inhibition of HMGB1 resulted in enhanced cell death, with increased expression of p53-upregulated modulator of apoptosis (PUMA) and activation of caspase-3. Interestingly, this inhibition also reduced the expression of TLRs themselves in MDS CD34+ cells, suggesting that TLR stimulation may amplify its own expression in MDS.In addition to increased expression of TLRs and components of the TLR signaling pathways, as well as enhanced sensitivity to TLR ligands in the HSPCs of patients with MDS, some of the known TLR ligands are more abundant in the bone marrow and/or serum of individuals with MDS compared to healthy controls. High mobility group box 1 (HMGB1), for example, a nuclear DAMP released upon cell death that stimulates TLR2 and TLR4, is increased in the serum of patients with MDS . Kam et in vitro , which contribute to inflammasome assembly and inflammatory cytokine production . In a reNUP98-HOXD13 (NHD13) mouse model of MDS led to a reduction in premalignant cell death and more rapid leukemic transformation (NHD13 mice (expressing the NUP98-HOXD13 fusion from the hematopoietic Vav-1 promoter) display cytopenias, bone marrow dysplasia, and increased HSPC cell death, and the mice die of acute leukemia (predominately myeloid) or bone marrow failure by about a year of life  on leukemic blasts, suggesting that TLR2 is downregulated at some point during the transformation process. This finding is consistent with the human studies showing higher expression of TLR2 in low-risk patients compared to high-risk  has now been trialed as second-line therapy for patients with lower-risk MDS (NCT02363491) . In addi3CSK4 induces p38-dependent apoptosis and NF-κB-dependent differentiation of both murine and primary human AML cells (Finally, while inhibition of TLR signaling has been the general strategy for MDS, multiple studies suggest that stimulation of this signaling may have therapeutic efficacy in AML, with TLR agonists promoting the death and differentiation of myeloid blasts –126. ForML cells . SimilarML cells  found thML cells .In addition to directly promoting AML cell death and differentiation, TLR agonists have also been considered for use as anti-tumor agents for their indirect effects on leukemia cells via promotion of the natural immune response to these cells –130. ForEnhanced innate immune signaling, and in particular TLR signaling, is a common feature of MDS. The etiology of this TLR signaling upregulation is multifactorial, involving increased expression of TLRs and downstream signaling components, enhanced sensitivity to TLR stimulation, and increased availability of TLR ligands in the bone marrow and blood of patients with MDS. In many cases, the mechanism of TLR upregulation is not clear. Furthermore, TLR expression differs between low- and high-risk patients, suggesting that the role of TLR signaling may change as MDS disease progresses . FurtherLP and LS wrote this review manuscript and generated the figure together. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.