The angiotensin-converting enzyme 2 (ACE2) is a type I integral membrane that exists in two forms: the first is a transmembrane protein; the second is a soluble catalytic ectodomain of ACE2. The catalytic ectodomain of ACE2 undergoes shedding by a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), in which calmodulin mediates the calcium signaling pathway that is involved in ACE2 release, resulting in a soluble catalytic ectodomain of ACE2 that can be measured as soluble ACE2 plasma activity. The shedding of the ACE2 catalytic ectodomain plays a role in cardiac remodeling and endothelial dysfunction and is a predictor of all-cause mortality, including cardiovascular mortality. Moreover, considerable evidence supports that the ACE2 catalytic ectodomain is an essential entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Additionally, endotoxins and the pro-inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-alpha (TNFα) all enhanced soluble catalytic ectodomain ACE2 shedding from the airway epithelia, suggesting that the shedding of ACE2 may represent a mechanism by which viral entry and infection may be controlled such as some types of betacoronavirus. In this regard, ACE2 plays an important role in inflammation and thrombotic response, and its down-regulation may aggravate COVID-19 via the renin-angiotensin system, including by promoting pathological changes in lung injury. Soluble forms of ACE2 have recently been shown to inhibit SARS-CoV-2 infection. Furthermore, given that vitamin D enhanced the shedding of ACE2, some studies reported that vitamin D treatment is associated with prognosis improvement in COVID-19. This is an updated review on the evidence, clinical, and therapeutic applications of ACE2 for COVID-19. On December 2019, at Wuhan, China had reported the first cases of pneumonia due to a novel coronavirus that was identified as a betacoronavirus in the same subgenus as the severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs) attachment . The WorACE2 binding triggers a spike protein-mediated fusion of the viral envelope with the cell plasma membrane or intracellular endosomal membranes. The spike protein is synthesized as a single polypeptide that is cleaved by the cellular protease furin into S1 and S2 subunits in the endoplasmic reticulum and then further processed by transmembrane serine protease 2 (TMPRSS2) on target cells ,9. The SThe first known human homolog of the angiotensin-converting enzyme (ACE) was discovered in 2000 by two independent groups and was designated as ACE2 ,15. The The catalytic active ectodomain of ACE2 is located at the extracellular region and undergoes shedding by a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), also known as a tumor necrosis factor alpha-converting enzyme, which was first discovered as the sheddase responsible for the cleavage of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α) . ThereafACE2 activity has a strong pH dependence under acidic conditions, such that the enzyme is almost inactive at pH 5.0 and has optimal activity at pH 6.5 . HoweverProspective cohorts have demonstrated that a high equilibrium and circulating levels of angiotensin 1–7 (AT1-7)/AT2 ratios were associated with improved survival, lower hospitalization duration, and better New York Heart Association (NYHA) class irrespective of left ventricular ejection fraction (LVEF) ,25. ThisThe chymase is a chymotrypsin-like serine protease that is synthesized and stored in the cardiac mast cells and is an alternative source of AT2 that is increased in failing human hearts . A studyn = 10,743), showed that being male, having a higher body mass index, DM, older age, higher low-density lipoprotein (LDL) cholesterol, smoking, and higher blood pressure were all associated with increased levels of circulating plasma ACE2. Additionally, a higher plasma ACE2 concentrations were associated with a greater risk of incident HF, MI, stroke, and DM, as well with increased risk of total deaths with similar increases in cardiovascular and non-cardiovascular deaths [In the last years, studies showed that increasing soluble ACE2 plasma activity is associated with clinical HF in patients with ischemic and without ischemic cardiomyopathies, and is correlated with increasing B-type natriuretic peptide levels and a worsening NYHA functional class ,30. Morer deaths . Althougr deaths ,34,35,36n = 8271) revealed that the overall deep vein thrombosis (DVT) rate was 20%, in the intensive care unit (ICU), it was 28%, and postmortem, it was 35%. The overall pulmonary embolism (PE) rate was 13%, in the ICU, it was 19%, and postmortem, it was 22%. The overall arterial thromboembolism rate was 2%, and in the ICU, it was 5%. Moreover, the pooled odds of mortality were 74% higher among patients who developed thromboembolism compared to those who did not, with an odds ratio (OR) of 1.74; 95% confidence interval (CI) 1.01–2.98 (p = 0.04) [n = 74,814) showed that PE in COVID-19 patients at emergency department (ED) presentation was about 0.5%. The standardized incidence of PE in the COVID-19 population resulted in 310 per 100,000 person-years vs. 35 per 100,000 person-years in non-COVID-19 population . Thus, even though the PE in COVID-19 patients at ED presentation was low, the incidence is approximately ninefold higher than in the general (non-COVID-19) population. Furthermore, several characteristics in COVID-19 patients were independently associated with PE; the strongest direct associations were D-dimer levels >1000 ng/mL and chest pain, and chronic HF had an inverse associatio [p =  0.910). This notwithstanding, when patients had D-dimer levels greater than 3.0 μg/mL, those receiving unfractionated heparin (UFH) had lower mortality rates than those not receiving unfractionated heparin , suggesting that only patients with markedly elevated D-dimer may benefit from anticoagulant treatment [n = 4389) demonstrated that therapeutic AC was associated with a 47% reduction in the hazard ratio (HR) of in-hospital mortality 0.53  compared with no AC. Similarly, prophylactic AC was associated with lower mortality compared with no AC. Moreover, therapeutic AC was associated with a 31% reduction in the hazard of intubation , while prophylactic AC was also associated with a reduced incidence of intubation compared with no AC. Additionally, the administration of AC within 48 hours of admission showed that therapeutic AC was associated with a 14% reduction in hazard of mortality compared with prophylactic AC, though this did not reach statistical significance (p = 0.08). This notwithstanding, the proportion of patients with bleeding events after the initiation of AC treatment was highest in patients on therapeutic AC (3.0%) compared with patients on prophylactic AC (1.7%) and no AC (1.9%). Bleeding rates were higher in those on low molecular weight heparin (LMWH) compared with direct oral anticoagulants (DOACs) (2.6% vs. 1.3%). Bleeding rates were higher in those on UFH compared with LMWH (1.7% vs. 0.7%). Therefore, the study confirmed that AC lowered mortality among hospitalized COVID-19 patients [It is well known by many cohort studies that the incidence of venous thromboembolism (VTE) is higher among patients with severe COVID-19, relative to its presence among patients with respiratory failure related to other causes ,38. A me = 0.04) . A Spanisociatio . In contsociatio . The coareatment . Furtherpatients . In contn = 1245) reported that in patients with severe or critical COVID-19, the pooled mean serum IL-6 concentration was 36.7 pg/mL. Mean IL-6 concentrations were nearly 100 times higher in patients with cytokine release syndrome (3110.5 pg/mL), 27 times higher in patients with sepsis (983.6 pg/mL), and 12 times higher in patients with ARDS unrelated to COVID-19 (460 pg/mL) [Pre-clinical studies showed that the lower activity of ACE2, AT1-7, and ACE2/ACE ratio were associated with higher thrombus formation in vessels ,45. More0 pg/mL) . This suThe calcium signaling pathway is involved in the catalytic ectodomain of the ACE2 shedding process regulated by calmodulin (CaM) ,20. CaM A diverse array of target proteins has been identified, such as CaM kinases and ion channels, which are known to interact with CaM with or without calcium . CaM alsp = 0.013) [p = 0.007). Variables that were independently associated with higher odds of having a headache in COVID-19 patients were anosmia, myalgia, and female sex. Conversely, variables that were associated with lower odds of having headache were increased CRP, D-dimer, and lymphopenia on admission [In epidemiological registries, reported smell or taste dysfunction in 56.9% of COVID-19 cases , and in = 0.013) . In addi= 0.013) . Other sdmission . Contrasdmission , and whedmission . This wadmission . Hence, dmission ,84. NRP1dmission . Additiodmission . NRP1 isdmission . NRP-1 idmission . NRP1 exdmission . A Chinedmission . A repordmission . Moreovedmission . Additiodmission . Similardmission . This sudmission . Furtherdmission ,95,96. Idmission . The lowdmission , but witdmission , suggestdmission . Moreovedmission  supportsdmission . Many prdmission . The readmission . In contdmission , this sudmission ,100. Sigdmission , and he dmission ,99,100.Gdmission , hypertedmission , and DM dmission , and thedmission , the higdmission . NeverthThe ACE2 is a type I integral membrane that exits in two forms. The first is as a transmembrane domain that can be measured as ACE2 mRNA expression. The second is a soluble form of ACE2. The catalytic ectodomain of ACE2 undergoes shedding because of ADAM17, in which calmodulin mediates the calcium signaling pathway that is involved. CaM is a ubiquitous calcium-binding protein that is known to bind other transmembrane proteins and regulate their cell surface expression. The ACE2 release results in a soluble catalytic ectodomain of ACE that can be measured as soluble ACE2 plasma activity. The shedding of the ACE2 catalytic ectodomain plays a role in cardiac remodeling and endothelial dysfunction. It is unclear whether circulating soluble ACE2 in plasma indicates the cause or effect of an adaptive or maladaptive physiological process operating in HF and cardiovascular diseases. Notwithstanding, at the present time, it is proven by many studies that high soluble ACE2 plasma activity is a predictor of MACE, cardiovascular mortality, and all-cause mortality. Moreover, many studies have demonstrated that the catalytic ectodomain of ACE2 is an entry receptor for SARS-CoV-2 infection. In addition, endotoxins and the pro-inflammatory cytokines, IL-1β and TNFα, all enhanced soluble catalytic ectodomain ACE2 shedding from the airway epithelia, suggesting that the shedding of ACE2 may represent a mechanism by which viral entry and infection may be controlled, such as with SARS-COV-1 and SARS-COV-2. ACE2 plays an important role in inflammation and the thrombotic response, and its down-regulation may aggravate COVID-19 via the renin-angiotensin system, including by promoting pathological changes in lung injury. This ACE2 down-regulation induced by the cell entry of the virus may be particularly detrimental in patients with preexisting ACE2 deficiency due, for example, to older age, DM, and hypertension. Furthermore, some studies suggest that vitamin D plays an essential role in CaM function and causes a slight increase in ACE2 shedding. Hence, studies demonstrated that either enhancing the shedding of the soluble catalytic ectodomain of ACE2 or the administration of transgenic soluble catalytic ectodomain of ACE2 forms inhibit entry and the replication of SARS-COV-2, which confirm the important role of the soluble catalytic ectodomain of ACE2 against SARS-CoV-2 infection. Observational studies showed that deficient vitamin D status was associated with increased COVID-19 risk. Similarly, those studies revealed that low vitamin levels are associated with poor prognosis in COVID-19. In addition, some clinical trials reported that the administration of vitamin D treatment was associated with prognosis improvement in COVID-19. Therefore, the high ACE2 cell expression reserve and the capacity to produce an appropriate ACE2 shedding process could be implicated in the COVID-19 prognosis. Nevertheless, more studies are required to confirm the therapeutic role of the soluble catalytic ectodomain of ACE2 in COVID-19, as well the ACE2 expression, and ACE2 plasma activity levels, and their prognostic significance needs, to be clarified with further studies.


Cardiovascular diseases (CVDs) have emerged as a major threat to global health resulting in a decrease in life expectancy with respect to humans. Thrombosis is one of the foremost causes of CVDs, and it is characterized by the unwanted formation of fibrin clots. Recently, microbial fibrinolytic enzymes due to their specific features have gained much more attention than conventional thrombolytic agents for the treatment of thrombosis. Marine microorganisms including bacteria and microalgae have the significant ability to produce fibrinolytic enzymes with improved pharmacological properties and lesser side effects and, hence, are considered as prospective candidates for large scale production of these enzymes. There are no studies that have evaluated the fibrinolytic potential of marine fungal-derived enzymes. The current review presents an outline regarding isolation sources, production, features, and thrombolytic potential of fibrinolytic biocatalysts from marine microorganisms identified so far. Thrombosis is a major cause of cardiovascular diseases (CVDs) including acute myocardial infarction, ischemic heart disease, valvular heart disease, peripheral vascular disease, arrhythmias, high blood pressure and stroke, and it is a leading cause of death worldwide . With poFibrinolytic enzymes are deemed as the most promising medication for the clinical treatment of thrombosis . They caPlasminogen activators hydrolyze fibrin by the production of plasmin, and the latter can directly break down fibrin clots . To dateIn the last decades, many fibrinolytic enzymes from natural resources, such as snakes , earthwoThe current review presents an overview regarding the resources, production, properties and thrombolytic activity of fibrinolytic enzymes from marine microbes identified so far.Streptomyces lusitanus [Streptomyces radiopugnans VITSD8 [Streptomyces violaceus VITYGM [Pseudomonasaeruginosa KU1 [Alteromonas piscicida [Pseudoalteromonas sp. IND11 [Marinobacter aquaeolei MS2-1 [Bacillus flexus [Bacillus subtilis [Bacillus subtilis HQS-3 [Bacillus vallismortis [Bacillus subtilis D21-8 [Bacillus pumilus BS15 [Bacillus subtilis WR350 [Bacillus subtilis JS2 [Bacillus velezensis BS2 [Bacillus subtilis ICTF-1 [Shewanella sp. IND20 [Serratia rubidaea KUAS001 [Serratia marcescens subsp. sakuensis [Arthrospira platensis [Chlorella vulgaris [Dunaliella tertiolecta [Tetraselmis subcordiformis [Bacillus are considered as the most valuable resources for the production of fibrinolytic enzymes, while there are no studies that have evaluated the fibrinolytic potential of marine fungal-derived enzymes. It should be noted that the catalytic activity of fibrinolytic enzymes can be improved by chemical modifications and mutant selection [Marine microorganisms are important resources of fibrinolytic enzymes. These enzymes possess potential efficacy for health augmentation and nutraceutical use, and their application could prevent cardiovascular diseases effectively . Marine usitanus , Streptos VITSD8 , Streptos VITYGM , Pseudomnosa KU1 ,47, Alteiscicida , Pseudoap. IND11 , bacterip. IND11 , Marinobei MS2-1 , Bacillus flexus , Bacillusubtilis , Bacilluis HQS-3 , Bacilluismortis , Bacilluis D21-8 , Bacillulus BS15 , Bacilluis WR350 , Bacilluilis JS2 , Bacillunsis BS2 , Bacillus ICTF-1 , Shewanep. IND20 , Serrati KUAS001  and Serrakuensis ,63,64, Alatensis ) and micvulgaris ,67, Dunatiolecta  and Tetrdiformis ), are suelection ,71.The main purpose of purifying enzymes is to remove other contaminating proteins and other interfering biomolecules. Furthermore, enzyme purification allows the acquisition of insights about structural and functional features of the purified enzyme, as well as foretells its applications . The reqCurrently, several approaches have been used for separating and purifying fibrinolytic enzymes from marine microorganisms . These aArthrospira platensis. The eluted enzyme showed a specific activity of 7,988 U/mg with 32.42-fold purification [Bacillus subtilis ICTF-1 was purified by a three-step procedure. As a first step, ammonium sulfate precipitation was adopted for providing suitable protein concentration, followed by UnoQ Sepharose Strong Anion Exchanger and Butyl Sepharose FF chromatography. The enzyme had a molecular mass of 72 kDa [Several purification strategies are listed in fication . A fibrif 72 kDa .Streptomyces lusitanus) [Arthrospira platensis) [Streptomyces radiopugnans VITSD8) [Bacillus flexus) [sitanus)  to as hiatensis) . Most maatensis)  to 7 [43atensis) ,62 and fatensis) ,58,59,60atensis) ,75. The  VITSD8)  to 60 °C flexus) .Bacillus spp. belongs to serine proteases, and their activity is inhibited by PMSF (Phenyl Methyl Sulphonyl Fluoride). Proteolytic enzymes possessing an active group (OH) from serine amino acid in the catalytic site are recognized as serine proteases. During inhibition of catalytic activity, sulfonyl group of PMSF binds irreversibly to the serine OH group in the active site [Pseudomonas aeruginosa KU1 is repressed by some metal ions, e.g., Mn2+, Fe2+ and Zn2+ [2+, Mg2+ and Zn2+, for enzymes from Serratia marcescens subsp. sakuensi [2+ for enzymes from Arthrospira platensis [Chlorella vulgaris [N,N,N′,N′-tetraacetic acid). Moreover, some studies have focused on the effect of chemical reagents and metal ions to delineate and characterize catalysis by these novel fibrinolytic enzymes. ive site . In addiand Zn2+ . Similarsakuensi , Fe2+ folatensis  and Chlovulgaris ; thus, tBacillus enzymes are generally directly acting on fibrin forming or fibrin degradation products. AprEBS2 isolated from Bacillus velezensis BS2 revealed high Aα fibrinolytic activity, followed by moderate Bβ and mild γ chains fibrinolysis [Bacillus pumilus BS15 [Bacillus subtilis JS2 [Bacillus licheniformis KJ-31 was one of the microorganisms that only produced fibrinolytic enzymes with high Aα fibrinogen lytic activity [The efficacy of fibrinolytic enzymes is determined by two different mechanisms: indirectly activating the plasminogen and directly acting on fibrins . In Tablinolysis . Neverthlus BS15  and Baciilis JS2  displayeactivity .max) and the turnover number (kcat) help understand the specificity and affinity of an enzyme for a particular substrate [Microbial fibrinolytic enzymes display amidolytic (or pro-coagulant) activity, which is assessed using different synthetic chromogenic substrates . Most ofubstrate . Table 6Bacillus pumilus BS15 [Bacillus subtilis [Gene cloning, mutagenesis and recombinant DNA technology have also been employed for the overexpression of fibrinolytic enzymes in bacterial hosts and to engineer their catalytic properties. For example, Yao and colleagues (2018) achieved significantly higher fibrinolytic activity of the recombinant fibrinolytic enzyme from lus BS15 . In anotsubtilis . Hence, Serratia rubidaea KUAS001 obtained from marine milieus [Bacillus subtilis D21-8 [5) [18-orthogonal array method [Bacillus flexus using a solid state fermentation process. They demonstrated an enhanced production of fibrinolytic enzymes up to 3.5-fold [The production cost of an enzyme is one of the challenging factors for the industrial sector. The commercial obtainability of microbial fibrinolytic enzymes needs high yield at the lowest possible costs . Hence,  milieus . In addiis D21-8 . Moreoveis D21-8 , two-lev21-8 [5) ,52,60, r21-8 [5) ,81, Plac21-8 [5) ,81, one-21-8 [5) , L18-orty method  and centy method ,67, are Serratia marcescens resulted in 38% clot lysis, which was significantly higher than that reported by streptokinase and heparin [Bacillus subtilis suggest its potential use for the treatment and/or prevention of thrombosis [Pseudomonas aeruginosa KU1, has exceptional potential for being developed as a therapeutic agent to treat CVDs [The effective treatment of CVDs relies on thrombolysis agents, such as microbial fibrinolytic enzymes ,83. Micr heparin . In addi heparin . The charombosis . Moreoveeat CVDs .The scientific community already effectively utilizes all available information on fibrinolytic enzymes. As a future prospective, the community should focus on the exploration of novel sources of fibrinolytic enzymes, especially from the marine environment. Marine microbial fibrinolytic enzymes have immense therapeutic potential as target drugs to prevent or cure CVDs. Extensive studies on these enzymes promises to develop cost effective, safe and preventive solutions for the management of cardiac diseases. The new trend for developing and improving thrombolytic agents is to enhance its fibrin specificity and binding efficacy. Further optimization of production parameters is also required to design economical, effective and safe drugs. Thus, the use of marine microbial fibrinolytic enzymes as thrombolytic agents might be auspicious and a safe option in future.


Pin1 knockout (KO) mice exhibited significant attenuation of DSS-induced colitis compared to wild-type (WT) mice, based on various parameters, including body weight, colon length, microscopic observation of the intestinal mucosa, inflammatory cytokine expression, and cleaved caspase-3. In addition, a role of Pin1 in inflammation was suggested because the percentage of M1-type macrophages in the colon was decreased in the Pin1 KO mice while that of M2-type macrophages was increased. Moreover, Pin1 KO mice showed downregulation of both Il17 and Il23a expression in the colon, both of which have been implicated in the development of colitis. Finally, oral administration of Pin1 inhibitor partially but significantly prevented DSS-induced colitis in mice, raising the possibility of Pin1 inhibitors serving as therapeutic agents for IBD.Inflammatory bowel diseases (IBDs) are serious disorders of which the etiologies are not, as yet, fully understood. In this study, Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) protein was shown to be dramatically upregulated in the colons of dextran sodium sulfate (DSS)-induced ulcerative colitis model mice. Interestingly,  Inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), are serious disorders. The pathophysiology underlying these diseases has yet to be fully clarified. IBDs are chronic relapsing disorders characterized pathologically by inflammation and epithelial injury involving the gastrointestinal tract. Symptoms include abdominal pain, diarrhea, rectal bleeding, weight loss and fatigue in both Crohn’s disease and ulcerative colitis ,2.Recent studies have identified a major role of both genetic and environmental factors in the pathogenesis of IBD. Genetic and environmental risk factors trigger epithelial barrier function deterioration ,4. ImpaiIn this study, first, we found that Pin1 protein was dramatically upregulated in the colons of dextran sodium sulfate (DSS)-induced colitis model mice, and therefore, investigated Pin1 involvement in the pathogenesis of IBD. Pin1 is a unique peptidyl-propyl isomerase, which specifically recognizes phosphorylated serine or threonine residues located at the immediate N-terminus to a proline, followed by isomerization of the bound peptide and catalyzation of the isomerization of prolyl peptide bonds from trans to cis. These conformational changes stabilize proteins, protein–protein interactions, cellular localizations and catalytic activities, responses which vary according to the individual protein. As a consequence, Pin1-catalyzed conformational changes have a profound impact on many key proteins playing roles in the regulation of cell growth, immune responses, differentiation and survival ,8,9,10. Pin1 involvement has been identified in numerous biological processes related to pathological conditions, including cancer, inflammation, Alzheimer’s disease, aging, asthma and microbial infection ,11,12,13Pin1 fl/fl mice on a C57BL/6 background was reported previously and is, thus, not repeated in detail [Pin1 flox mice were crossed with CAG-Cre mice. The genotyping primers used were P1 (tct cct tcc act ggg caa ctt cct g) and P2 (tgc tgt ccc cat cgg gac ct) for both the wild and the floxed type allele, and P1 and P3 (ctg tgg tgc tct tgg ggg tg) for the knockout (KO) allele, yielding 180 bp, 375 bp, and 419 bp products, respectively. Animal studies were conducted using age-matched controls for each experiment. Eight- and 10-week-old mice were used for the experiments. Colitis was induced in these mice by administering 3% DSS  in drinking water for 7 days.The generation of n detail . To creaFor treatment with Pin1 inhibitor, juglone was dissolved in dimethyl sulfoxide (DMSO) and orally administered to the mice (5 mg/Kg BW).These experiments were approved by Hiroshima University Animal Research Committee. The animals were handled in an accordance with the animal experimentation guidelines of Hiroshima University .Anti-Pin1 , anti-α tubulin  and anti-actin  antibodies were purchased from Santa Cruz Biotechnology . Anti-active caspase-3  antibody was purchased from Cell Signaling .Flow cytometry analysis was performed using LSRFortessa X-20 . Anti-PE-F4/80 , anti-APC-CD11c and anti-Brilliant Violet 421-CD206  were used for the flow cytometric analyses.Gapdh. Primer sequences are presented in Real time PCR was performed, as previously reported ,15. BrieFormalin-fixed and paraffin-embedded murine colon tissue was sectioned, followed by standard hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) staining. Briefly, deparaffinized sections were stained with hematoxylin. After being washed, they were also incubated with eosin solutions. For PAS staining, deparaffinized slides were reacted with periodic acid. After being washed, they were incubated with Schiff’s reagent and then reacted with hematoxylin. Finally, the slides were dehydrated and embedded.For immunofluorescence staining, the entire colon was fixed in 10% formalin and embedded in paraffin. Paraffin-embedded samples were sectioned. The sections were incubated in 0.1% Triton solution. After washing with phosphate buffered saline (PBS), the sections were boiled in citrate buffer (pH = 6.0) followed by a 30-min microwave exposure for antigen retrieval. The slides were then reacted with Pin1 antibody (1:200) at 4 °C overnight. After being washed with PBS, the slides were stained by the avidin–biotin complex staining method, according to the manufacturer’s protocol (Santa Cruz). The samples were examined using a BZ 9000 All-in-one Fluorescence Microscope .3VO4, and 1 mM phenylmethylsulfonyl fluoride. The lysates were incubated on ice for 30 min and then centrifuged at 15,000 rpm for 10 min and subsequently for 30 min at 4 °C. After adjusting the protein concentrations, the supernatants were mixed and boiled with sample buffer. The same amounts of samples (40 µg) were electrophoresed with SDS (sodium dodecyl sulfate)-polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 3% bovine serum albumin for 1 h at room temperature, and then incubated with primary antibody (1:3000). After being washed with PBS containing 0.1% Tween 20 three times, the membranes were reacted with HRP-conjugated secondary antibody (1:5000) for 1 hr at room temperature. After washing, proteins on the membranes were subjected to immunoblotting using Supersignal West Pico Substrate .Western blotting was conducted as reported previously ,15. Colop values. The p values ≤0.05 are considered to indicate a statistically significant difference.Data were analyzed in GraphPad Prism . For multiple group comparisons, we used a one-way ANOVA with a Tukey multiple comparison posttest to calculate It is reported that mice given drinking water supplemented with 3% DSS for 7 days show symptoms including diarrhea, rectal bleeding, reduction in colon length, body weight loss and decreased activity, which mimic the clinical and histological features of IBDs. Microscopic observation indicated severe damage in the epithelial layer. Interestingly, Pin1 protein expression levels in the colons of mice treated with DSS for 7 days were dramatically increased, i.e., by approximately 45 folds, as compared with those of control mice, while Pin1 mRNA levels were not significantly altered A,B. NextSince Pin1 protein was markedly increased in the colonic tissues of DSS-treated mice, we speculated that Pin1 is involved in the development of colitis. To address this possibility, first, global Pin1 KO mice, generated by crossing Pin1 flox and CAG-Cre mice, were subjected to experiments resulting in the development of DSS-induced colitis. As expected, Pin1 expressions were fully deleted in the colons of KO mice . After 7Tnfα, Il6 and Il1b and the chemokine Mcp1 were markedly elevated, and also showed significant normalization in the Pin1 KO mice  administration A. Howevereatment C,D. Juglpression D. These To date, many physiological as well as pathogenic roles of Pin1 have been elucidated. For example, Pin1 interacts with and promotes the degradation of tau, advantageously contributing to protection from Alzheimer disease . As an uPin1 KO mice showed resistance to DSS-induced colitis development characterized by body weight loss, colon length reduction and epithelial tissue disruption.This is the first study demonstrating the pathological role of Pin1 in the development of DSS-induced colitis. Pin1 expression was shown to be markedly increased in the DSS-treated mouse colons. In addition, global Interestingly, ablation of the Pin1 gene reduced expressions of pro-inflammatory cytokines including Th17-related cytokines and increased the M2 macrophage population in the colons of DSS-treated mice. Many functions of Pin1 in the regulation of immune or inflammatory responses were previously described in the literature ,24. T heGmcsf and Tgfβ by preventing exosome-mediated translation. Pin1 reportedly induces AUF1 isomerization, leading to the loss of its binding ability with Gmcsf mRNA, and thereby stabilizing Gmcsf mRNA [Besides the involvement of Th17 cells, multiple mechanisms underlying the contribution of hematopoietic Pin1 to the pathogenesis of colitis are possible. First, Pin1 interacts with and controls the ARE mRNA-binding activity of AUF1 in T lymphocytes and eosinophils. AUF1 induces the catabolization of several mRNAs including those of csf mRNA ,30. In addition, Pin1 binds to the p65 subunit of NF-κB and promotes its transcriptional activity . NF-κB rTaken together, these findings show that orally-administered Pin1 inhibitor exerts a significant protective effect against DSS-induced colitis, raising the possibility of Pin1 inhibitors serving as novel therapeutic agents for treating IBDs. We conclude that Pin1 plays a key role in the pathogenesis of DSS-induced colitis in mice. Although further studies are necessary to fully elucidate the roles of Pin1 in colitis development, the development of new agents with high specificity for inhibiting Pin1 activity are eagerly awaited.


Deletions and duplications at the 22q11.2 locus are associated with significant neurodevelopmental and psychiatric morbidity. Previous diffusion-weighted magnetic resonance imaging (MRI) studies in 22q11.2 deletion carriers (22q-del) found nonspecific white matter (WM) abnormalities, characterized by higher fractional anisotropy. Here, utilizing novel imaging and processing methods that allow separation of signal contribution from different tissue properties, we investigate whether higher anisotropy is driven by (1) extracellular changes, (2) selective degeneration of secondary fibers, or (3) volumetric differences. We further, for the first time, investigate WM microstructure in 22q11.2 duplication carriers (22q-dup). Multi-shell diffusion-weighted images were acquired from 26 22q-del, 19 22q-dup, and 18 healthy individuals (HC). Images were fitted with the free-water model to estimate anisotropy following extracellular free-water elimination and with the novel BedpostX model to estimate fractional volumes of primary and secondary fiber populations. Outcome measures were compared between groups, with and without correction for WM and cerebrospinal fluid (CSF) volumes. In 22q-del, anisotropy following free-water elimination remained significantly higher compared with controls. BedpostX did not identify selective secondary fiber degeneration. Higher anisotropy diminished when correcting for the higher CSF and lower WM volumes. In contrast, 22q-dup had lower anisotropy and greater extracellular space than HC, not influenced by macrostructural volumes. Our findings demonstrate opposing effects of reciprocal 22q11.2 copy-number variation on WM, which may arise from distinct pathologies. In 22q-del, microstructural abnormalities may be secondary to enlarged CSF space and more densely packed WM. In 22q-dup, we see evidence for demyelination similar to what is commonly observed in neuropsychiatric disorders. Copy number variation (CNV) at the 22q11.2 locus is associated with significant neurodevelopmental and psychiatric morbidity. Deletion of the 22q11.2 locus occurs in approximately one in 4000 live births , and immGiven the importance of white matter (WM) microstructure for cognitive functioning , its disLower FA has been implicated in multiple idiopathic psychiatric disorders , as well as neurodegenerative disorders, and is often interpreted as a proxy for abnormal WM organization , 32. Howhigher FA in most tracts in 22q-del [While there are some earlier studies that reported decreased FA in single WM tracts in 22q-del , 34, 35, 22q-del . Studies 22q-del .In the present study, we compared diffusion MRI data between reciprocal 22q11.2 CNVs and tested whether opposing effects of gene dosage, similar to the effects reported for GM, also occur in WM. To determine the potential mechanisms that might drive FA alterations, we applied advanced dMRI analysis approaches which allowed considering: (1) extracellular and cellular changes, as quantified by free-water (FW) imaging ; (2) theThe sample consisted of 26 22q-del, 19 22q-dup, and 18 HC that passed visual quality control. Patients have been recruited from either (1) Clinical Genetics, Allergy/Immunology, or Craniofacial Clinics from medical centers in the Southern California area, or (2) through local support groups and websites. HC were recruited from the community via web-based advertisements and local schools, pediatric clinics, and community sites. Exclusion criteria for all participants were significant neurological or medical conditions (unrelated to 22q11.2 deletion/duplication), history of head injury with loss of consciousness, insufficient fluency in English, and/or substance abuse or dependency within the past six months. Further exclusion criteria for HC were current or past major mental disorders (except for ADHD or past episodes of depression) and/or intellectual disability (IQ below 70). HC were screened for mental disorders via the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders Version 4  or CompuAll participants or parents, if participants were under the age of 18, provided written informed consent, and the University of California at Los Angeles Institutional Review Board approved all study procedures.Participants underwent a comprehensive clinical and cognitive test battery administered by trained clinical psychology students and supervised by PhD-level clinicians.Given the previously reported high rates of neuropsychiatric disorders in 22q-del and 22q-dup , 47–49, Furthermore, we were interested in characterizing global and cognitive functioning. Functional status was, therefore, determined by the Global Functioning Scales (GAF) . In addib = 1500 s/mm2, 46 gradient directions with b = 3000 s/mm2, 3 gradient directions with b = 200 s/mm2, 6 gradient directions with b = 500 s/mm2, and 7 volumes with b = 0 s/mm2.Images were acquired on a 3 T whole-body scanner (Siemens Magnetom Prisma) with a 32-channel head coil. Scanning protocols were derived from the Lifespan Human Connectome Project (HCP) study, including multi-shell diffusion-weighted images that were acquired with the following protocol: AP and PA sequence each with voxel size = 2.0 × 2.0 × 2.0 mm, TR = 8000 ms, TE = 66 ms, flip angle = 90°, FOV = 208 mm, slice thickness = 2.00 mm, slices = 72, and 108 volumes, including 46 gradient directions with All data underwent a visual quality check for movement artifacts, echo-planar imaging (EPI) distortions, and structural abnormalities by investigators blinded to group allocation. We excluded individuals who did not have complete data for the AP and PA sequence and individuals who presented with severe motion artifacts . We preprocessed the remaining images utilizing the HCP Minimal Preprocessing Pipeline v4.0.0 . First, We first investigated group differences in FA to determine the comparability of our findings to previous findings of increased FA in 22q-del. From the preprocessed diffusion-weighted images, we, therefore, computed diffusion tensors utilizing a least-squares fit and calculated FA maps from these tensors .T) is derived [T represents anisotropy following the elimination of FW contribution, and therefore FAT more specifically reflects changes in the WM tissue  than FA [To identify if the FA abnormalities originate from the cellular or extracellular domains, we fitted a two-compartment FW model to the multi-shell diffusion data using a regularized non-linear fit . One com derived , 64, 65. than FA , 66. FW  than FA , 68, moo than FA , 70, eat than FA , traumat than FA , dementi than FA , 74, and than FA , 76) hav than FA .iso). Degeneration of secondary fibers is expected to be indicated by a decrease of F2 but not F1. Previous studies have demonstrated that this selective degeneration of secondary fibers yields less partial volume in crossing fibers areas, resulting in an increase of FA values [We used the crossing fiber BedpostX model  to obtaiA values .https://pnlbwh.github.io/TBSS/TUTORIAL.html) that performed all registrations using the Advanced Normalization Tools (ANTs) toolbox [T, and FW maps were projected onto the ENIGMA skeleton [To facilitate group comparisons, we employed the tract-based spatial statistics (TBSS) pipeline –82 by no toolbox , 84. FA,skeleton  to perfoiso maps onto the skeleton provided by ENIGMA to perform statistical analyses. .To test if macrostructural volume measures interact with the microstructural measures, we utilized a novel segmentation algorithm  that allp < 0.05 and included age, sex, and motion as covariates.For all TBSS analyses, non-parametric voxel-wise permutation tests for each voxel on the WM skeleton were performed in FSL’s Randomize . We testT, FW, F1, F2, and Fiso), we conducted voxel-wise F-tests and post hoc t-tests to compare the three groups .For each of the maps .We performed all subsequent analyses using SPSS Version 26 and GraphPad Prism 8.2.0. First, we correlated FA with IQ , 59, glo with IQ , and the with IQ  for 22q-p-value < 0.013 was considered significant (Bonferroni corrected for four tests). In the case of a significant group effect in the ANCOVA, we then calculated post hoc t-tests between the single groups .Next, to investigate group differences in macrostructural volume, we carried out four ANCOVAs with ICV, CSF, WM, or GM volume as the independent variable, group as the dependent variable , and age, sex, and motion as covariates. A F-test and post-hoc t-tests comparing FAT, FW, F1, F2, and Fiso, between groups with the volumes that were different between groups as an additional covariate.Last, we included the volumes that demonstrated group differences in our previous analyses to identify the effect of macrostructural volume on microstructure. We, therefore, repeated the voxel-wise Demographic information is shown in Table F-test revealed a significant group effect on FA in 54% of the WM skeleton. Post hoc t tests demonstrated that 22q-del had significantly higher FA than HC in 41% of the WM skeleton, and 22q-dup had significantly lower FA than HC in 37% of the WM skeleton. Further, 22q-del displayed significantly higher FA than 22q-dup in 66% of the WM skeleton  and lower FW (in 49% of the WM skeleton). 22q-dup presented with the opposite pattern: lower FAT (in 8% of the WM skeleton) and higher FW (in 59% of the WM skeleton) than HC. In addition, 22q-del demonstrated higher FAT (54% of the WM skeleton) and lower FW (76% of the WM skeleton) than 22q-dup  and secondary (F2 in 40% of the WM skeleton) fibers, as well as the partial volume fraction of the isotropic compartment (Fiso in 66% of the WM skeleton). Post-hoc t-tests revealed that 22q-del had higher primary (F1 in 8% of the WM skeleton) and secondary (F2 in 28% of the WM skeleton) fiber fractions and a lower isotropic fraction (Fiso in 49% of the WM skeleton) than HC, which is not consistent with a pattern of degeneration of secondary fibers [lower primary (26% of the WM skeleton) and secondary (21% of the WM skeleton) fiber fractions than HC and a higher isotropic fraction (in 48% of the WM skeleton) than HC  or GM volume . However, there was a significant group effect on WM volume  and CSF volume . Post hoc t-tests revealed that 22q-del had lower WM volume than both 22q-dup (p = 0.062) and HC (p = 0.050). In addition, 22q-del had higher CSF volume than both 22q-dup (p = 0.002) and HC (p < 0.001)  extracellular changes, (2) selective degeneration of secondary fibers, or (3) macrostructural volumetric effects. Utilizing the FW imaging approach , 64 to culations , 79. InsWhile our current analysis cannot deduce causality, we speculate that enlarged CSF volume  in 22q-del might contribute to smaller WM volume, which then leads to reduced extracellular space within the WM and therefore more densely packed cellular structures. Several previous studies –99 have In line with our proposed interpretation of more densely packed WM in 22q-del, other clinical populations that observed higher FA  have speculated that the increased FA could be reflective of greater WM packing density –103. FurAlong the same lines, future longitudinal studies observing macro- and microstructural relationships are needed to explore the role of brain pathologies underlying psychiatric symptoms in 22q-del. Similar to our finding of enlarged CSF in 22q-del, previous studies have highlighted the role of increased CSF in various neurological and psychiatric disorders, including dementia  and moodT, lower primary and secondary fiber fractions (F1 and F2), and higher isotropic space (FW and Fiso) than HC. Interestingly, while we observed a high spatial overlap between abnormalities in 22q-del and 22q-dup, in 22q-dup, microstructural WM abnormalities were independent of macrostructural volume. Similar WM abnormalities  occur in healthy aging, in neurodegenerative disorders such as dementia, as well as psychiatric disorders including depression and schizophrenia [As the first study to investigate WM architecture in 22q-dup, our findings indicate that the 22q11.2 duplication is associated with an opposing pattern of WM alterations to those that characterize 22q-del. Specifically, 22q-dup demonstrated lower FA and FAophrenia , 110–112The opposing WM architecture findings in 22q-del and 22q-dup are consistent with imaging studies of other reciprocal CNVs, including 15q11.2 and 16p11.2, in which opposite effects on WM structure have also been observed , 114. Auincreased myelination, given previous postmortem studies [While future studies are needed to further explore the interaction between genes within the 22q11.2 locus and WM structure, our results suggest that the pathologies underlying opposing patterns of WM abnormalities in 22q-del and 22q-dup might differ from one another. Specifically, while lower FA in 22q-dup could be related to less myelin, the higher FA observed in 22q-del is less likely to result from abnormally  studies , 26 and  studies , 119 ass studies , 121.The limited sample size did not allow us to investigate subgroups of 22q11.2 CNV carriers with particular psychiatric phenotypes or to explore the association between brain structure and clinical and cognitive impairments in depth. Future investigations in larger cohorts are warranted to compare 22q-del with and without psychotic symptoms, given the increased risk for psychosis in 22q-del . SpecifiAs highlighted above, longitudinal studies are also of interest to establish trajectories of neuroanatomical abnormalities and to further elucidate the complex interaction between microstructural and macrostructural abnormalities. Last, while we applied advanced models to investigate WM pathology in 22q-del and 22q-dup, we could not include all factors that might influence the diffusion signal. For example, we could not account for potential vascular abnormalities, which may be present in 22q11.2 CNV carriers, affecting diffusion MRI measurements .Here, we conducted the first study to apply advanced dMRI analysis methods to elucidate WM microstructure abnormalities in reciprocal 22q11.2 CNVs. Findings revealed opposing effects of the 22q11.2 deletion and duplication on WM architecture. In 22q-del, we identified an association between WM microstructure findings and macrostructural volumetric measures, suggesting microstructural abnormalities are directly related to macrostructural features. WM alterations in 22q-dup, however, appear similar to those observed in other neuropsychiatric disorders such as schizophrenia, bipolar disorder, and dementia, suggesting reduced myelination of WM fibers. Hence, the pathology underlying these opposing effects may differ between 22q-del and 22q-dup and thus does not suggest a direct gene dosage effect on WM. Further longitudinal and translational studies are needed to elucidate the mechanisms underlying WM microstructure and macrostructure alterations resulting from CNV at the 22q11.2 gene locus.Supplementary Material


Transcription factor-7-like-2 (TCF7L2), a vital member of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family, plays an important role in normal human physiological and pathological processes. TCF7L2 exhibits multiple anti-atherosclerotic effects through the activation of specific molecular mechanisms, including regulation of metabolic homeostasis, macrophage polarization, and neointimal hyperplasia. A single-nucleotide substitution of TCF7L2, rs7903146, is a genetic high-risk factor for type 2 diabetes and indicates susceptibility to cardiovascular disease as a link between metabolic disorders and atherosclerosis. In this review, we summarize the anti-atherosclerosis effect and novel mechanisms underlying the function of TCF7L2 to elucidate its potential as an anti-atherosclerosis biomarker and provide a novel therapeutic target for cardiovascular diseases. Atherosclerosis precedes and forecasts the pathological process of cardiovascular disease (CVD), the leading cause of mortality worldwide , 2. Athevia the hybridization screening of TCF7 cDNA for the first time and described TCF7L2 as an important transcription factor of Wnt signaling. During embryogenesis, TCF7L2 promotes embryonic organ development as a key player in Wnt signaling (TCF7L2 is well characterized as a member of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family, whose remaining members are LEF1, TCF7, and TCF7L1 in humans. It was also previously called T-cell factor 4 (TCF4), not to be mistaken for transcription factor 4 . Korinekignaling . In adulignaling –13. Currignaling , 15. Stuignaling –18. In cignaling , 20. Thiignaling –23. ThesIn humans, TCF7L2 is located on chromosome 10q25.2-q25.3; it spans over 217,432 bp of DNA and consists of 17 exons. The full-length TCF7L2 protein contains 596 amino-acid residues in a certain subsequence of the N terminal, β catenin-binding domain, Groucho-binding sequence, HMG box-DNA-binding domain (HGM-DBD), cysteine clamp (C clamp), and C terminal , 25. Witvia a series of processes, including glucolipid metabolism, macrophage polarization, and neointimal hyperplasia , high low-density lipoprotein (LDL)-C, and low high-density lipoprotein (HDL)-C levels , 55. SomIn summary, TCF7L2 rs7903146 has a great impact on metabolic balance, linking dyslipidemia and hyperglycemia, and results in greater risk of CVD in T-allele carriers, especially in T2D patients . DecadesAs mentioned previously, TCF7L2 rs7903146 is a genetic risk factor for CVD, which cause a reduction in TCF7L2 protein. However, TCF7L2 is a functional transcription factor with pleiotropic anti-atherosclerotic effects on glucose and lipid metabolism. Metabolic syndrome comprises a complex group of pathological circumstances associated with metabolic and proinflammatory states, which play an important role in the atherosclerotic process by gathering atherogenic risk factors  65). It. It65). in vivo and in vitro experiments  activity. To enhance insulin synthesis, TCF7L2 significantly promotes the expression of the proinsulin genes Ins1 and Ins2. Subsequently, TCF7L2 can upregulate PSCK1 and PSCK2 to cleave proinsulin into mature insulin and the C peptide , high LDL, and low HDL levels –94; partG levels , 98. Anoic state . A recenic state . In concA levels .In addition to metabolic disorders, inflammatory infiltrates and neointimal hyperplasia aggravate the progression of atherosclerosis. Previously, we have illustrated TCF7L2 as a regulatory element in metabolism, but recent research has revealed other anti-atherosclerotic effects of TCF7L2, such as those against inflammation and neointimal hyperplasia .In vitro, curcumin can upregulate TCF7L2 to suppress M1 macrophage-derived inflammation  and long noncoding RNAs (lncRNAs), are master regulators of gene expression, and many are involved in the development of atherosclerosis . Serum m2+ channel CAV1.2, which promotes insulin efflux through exocytosis , Natural Science Foundation of Hunan Province, China (No. 2019JJ40249), Outstanding Young Aid Program for Education Department of Human Province (No. 18B274), the Major Project of social science achievement review committee in Hunan Province (XSP20ZDI013), Key Project of Hunan Provincial Department of Education (20A427), and Graduate Research innovation Project of School of Nursing, University of South China (2021CX22).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


GASA) gene family is critical for plant growth, development, and stress response. GASA gene family has been studied in various plant species, however, the GASA gene family in tobacco (Nicotiana tabacum) have not been characterized in detail. In this study, we identified 18 GASA genes in the tobacco genome, which were distributed to 13 chromosomes. All the proteins contained a conserved GASA domain and highly specific 12-cysteine residues at the C-terminus. Phylogenetic analysis divided the NtGASA genes into three well-conserved subfamilies. Synteny analysis suggested that tandem and segmental duplications played an important role in the expansion of the NtGASA gene family. Cis-elements analysis showed that NtGASA genes might influence different phytohormone and stress responses. Tissue expression analysis revealed that NtGASA genes displayed unique or distinct expression patterns in different tissues, suggesting their potential roles in plant growth and development. We also found that the expression of NtGASA genes were mostly regulated by abscisic and gibberellic acid, signifying their roles in the two phytohormone signaling pathways. Overall, these findings improve our understanding of NtGASA genes and provided useful information for further studies on their molecular functions.The gibberellic acid stimulated Arabidopsis ( GASA) gene family is widespread in monocotyledonous and dicotyledonous plant species -deficient (gib1) mutant gene , rice (Oryza sativa), wheat (Triticum aestivum), grapevine (Vitis vinifera L.), and tomato (Solanum lycopersicum)  . GASA gen of FLC . In petuongation .GASA genes are involved in GA signaling pathways. In soybean (Glycine max), GmGASA32 is upregulated by GA and interacts with GmCDC25 to control plant height , FaGAST genes are upregulated by the exogenous application of GA and affect fruit ripening (GASA genes is increased by other phytohormones such as brassinosteroid (BR), salicylic acid (SA), abscisic acid (ABA), naphthalene acetic acid (NAA), and indole-3-acetic acid (IAA) , the expression of MdGASA are upregulated by GA and ABA applications during the flowering stage (Most t height . In Gerbxpansion . In straripening . Besidesid (IAA) . In riceDIM/DWF1 . In Arabongation . In applng stage .GASA gene family also involved in plant response to abiotic and biotic stresses. In Arabidopsis, overexpression of AtGASA4 suppresses the accumulation of reactive oxygen species (ROS) and nitric oxide in wounded leaves  improves tolerance to salt, ROS, and heat stress  and fungi  by regulating the redox levels (Hevea brasiliensis), HbGASA genes are upregulated upon inoculation with Colletotrichum gloeosporioides and are involved in innate immunity by regulating ROS accumulation  is widely cultivated and has been used as a model plant for biological research. GASA genes are important in plant growth and development, however, the tobacco GASA gene family were not characterized previously. In this study, we identified GASA gene family in the tobacco genome with bioinformatics methods, and characterized their gene structure, phylogenetic relationships, protein motifs, chromosomal locations, syntenic regions, cis-acting elements, and expression patterns in different tissues. Our findings provide useful clues for further studies of GASA gene family in tobacco.Tobacco  were collected at the flowering stage to analyze NtGASA expression. For phytohormone treatments, 3-week-old seedlings were transferred to plates containing 10 μM ABA, 10 μM GA, 10 μM IAA, 10 μM SA, 50 μM methyl-jasmonate (MeJA), or 1% (v/v) dimethyl sulfoxide (control) and incubated for 5 h under the same photoperiod, temperature, and humidity conditions  and used as queries for BLAST search against the Solanaceae Genomics Network (https://solgenomics.net/). Subsequently, the Hidden Markov Model-based profile of the GASA domain PFAM 02704 was used to verify the presence of the complete GASA domain in NtGASA sequences. The non-redundant putative NtGASA sequences with a conserved GASA domain were used for further bioinformatics  and expression analysis.For http://web.expasy.org/protparam/). The sequences of GASA from Arabidopsis (AtGASA), rice (OsGASA), grapevine , and tobacco (NtGASA) were used to construct a phylogenetic tree using MEGA 7.0 with the neighbor-joining (NJ) method and a bootstrap test of 1,000 replicates (NtGASA genes was illustrated using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn). The conserved motifs of NtGASA proteins were analyzed using MEME 5.1.1 (http://meme-suite. org/tools/meme)  .NtGASA genes, the DNA sequence of each gene was mapped using MG2C 2.0 (http://mg2c.iask.in/mg2c_v2.0/). Segmental and tandem duplicated gene pairs within the tobacco genome, as well as collinear gene pairs among the Arabidopsis, rice, grapevine, and tobacco genomes, were identified using MCScanX , and cDNA synthesis was performed using the M-MLV reverse transcriptase Kit , according to the manufacturer’s instructions. Quantitative reverse-transcription (qRT)-PCR was carried out using the Bio-Rad CFX96 real-time system  with SYBR Green Master Mix (Bio-Rad). The t method . The priNtGASA genes was examined for the presence of cis-regulatory elements. Cis-regulatory elements in the promoters of each NtGASA gene were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and classified according to their regulatory functions.The 3 kb DNA sequence upstream of the start codon of NtGASA genes were 186 to3,715 bp and 186 to 444 bp, respectively. The deduced NtGASA proteins varied from 61 to 147 amino acids with a molecular weight of 6.6–16.17 kDa, and the isoelectric point ranged from 6.66 to 9.75. Apart from these, the instability index for most of the proteins (77.8%) were more than 35. According to the Grand average of Hydropathicity (GRAVY), the NtGASA proteins were hydrophilic except for NtGASA3, NtGASA4, and NtGASA9. The amino acid content of NtGASA was conserved, cysteine, lysine, and leucine were predominant amino residues. Most NtGASA proteins were localized in the extracellular membrane, chloroplasts, and mitochondria. Detailed information about NtGASA physicochemical characteristics is presented in To identify the GASA genes in tobacco, we used 15 AtGASA sequences as queries for BLAST search, and identified 18 putative NtGASA based on amino acid similarities. As shown in GASA genes from Arabidopsis, rice, grapevine and tobacco, an unrooted NJ tree was constructed aligning 15 AtGASA, 10 OsGASA, 14 VvGASA, and 18 NtGASA. According to the phylogenetic tree, GASA genes could be classified into three subfamilies: subfamily I included six AtGASA genes (AtGASA1/2/3/9/11/13), three OsGASA genes (OsGASA3/5/7), six VvGASA genes (VvGASA1/5/8/10/11/12), and nine NtGASA genes (NtGASA1–9). Subfamily II included five AtGASA genes (AtGASA4/5/6/12/15), four OsGASA genes (OsGASA4/6/9/10), five VvGASA genes (VvGASA4/6/7/13/14), and four NtGASA genes (NtGASA10–13). Subfamily III included four AtGASA genes (AtGASA7/8/10/14), three OsGASA genes (OsGASA1/2/8), three VvGASA genes (VvGASA2/3/9), and five NtGASA genes (NtGASA14–18)  . TherefoNtGASA genes in the chromosomes of tobacco were further determined. Using a simplified physical map, we found that the 18 NtGASA genes were unevenly distributed in 11 chromosomes in the tobacco genome. Chromosome (Chr.) 1, 4, 6, 8, and 21 contained two copies each, whereas Chr. 2, 10, 12, 14, 15, 16, 17, and 18 contained one copy each  were tandemly duplicated on Chr.1. In addition, five pairs  were segmental duplicated /AtGASA1, /OsGASA2, /VvGASA10 and /VvGASA11. There were also one-to-many matches, such as NtGASA9/, NtGASA15,NtGASA16/ and NtGASA18/. The one-to-one matches were NtGASA16/AtGASA7,NtGASA13/VvGASA4 and NtGASA17/VvGASA3  compared with vegetative parts   .cis-elements could provide clues about regulatory pathways of gene expression, then we analyzed the 3,000-bp upstream promoter sequences of NtGASA genes. The largest number of cis-elements observed across the NtGASA genes was associated with light-responsiveness. In addition, cis-elements involved in phytohormone  and stress  responses were also identified in the promoter sequences of NtGASA genes  showed ubiquitous expression, whereas most NtGASA genes were upregulated only in specific tissues   . Previoud cotton . Here, 1cis-elements assists in its functional characterization , indicating that unidentified cis-elements might regulate their expression (NtGASA genes under phytohormone applications highlighted their potential integral roles in various physiological processes.The promoter region of a gene is related to its function, and thus, the analysis of rization . Our resesponses , suggestpression . The comGASA genes in tobacco. We identified 18 NtGASA genes and analyzed their physicochemical characteristics, phylogenetic relationships, gene structure, conserved motifs, chromosomal locations, synteny, and cis-elements in the promoters, which showed a clear evolutionary history for this family in tobacco. We also studied the expression patterns of NtGASA genes in various tissues and under different phytohormone applications. Overall, our results provided insights into the role of NtGASA genes in several physiological and biological pathways and laid a solid foundation for further exploring the underlying molecular and biochemical mechanisms.To our knowledge, this is the first report on the identification and characterization of


Scutellaria baicalensis, induces the entire autophagy that proceeds through the autolysosome maturation stage in human hepatoma cells. In addition, baicalein-induced autophagy is demonstrated to target mitochondria for degradation. Further studies show that baicalein triggers the translocation of Parkin and TBK1 to mitochondria to induce mitophagy. Moreover, the phosphorylation of TBK1 at Ser172 and ubiquitin at Ser65 is shown to trigger mitophagy in baicalein-treated cells. Furthermore, two specific autophagy cargo receptors, NDP52 and OPTN, that function in baicalein-activated mitophagy are identified. Taken together, these findings not only delineate the molecular process of Parkin-dependent mitophagy in liver cells, but also reveal baicalein as a novel inducer of hepatic mitophagy.The elimination of intracellular components by autophagy maintains metabolic homeostasis and is a quality-control pathway that enables organelle regeneration. Mitophagy is a type of selective autophagy that regulates mitochondrial turnover, and the dysregulation of mitophagy has been implicated in the pathogenesis of liver diseases. However, the detailed molecular mechanism underlying mitophagy regulation in liver cells remains unclear, and the small molecules that may potentially modulate hepatic mitophagy are still unavailable. Here, we report that baicalein, a flavonoid extracted from  Autophagy is an evolutionarily conserved process that catabolizes intracellular components via lysosomal degradation ,2. TargeIn addition to bulk and nonselective degradation, autophagy selectively eliminates aggregated proteins and damaged organelles through selective autophagy ,11,12,13Mitophagy is a form of organellophagy that specifically degrades deformed mitochondria to promote mitochondrial turnover ,17,18. DScutellaria baicalensis  (P8131) and ethanol (1.00983.2500) used in sample preparation for electron microscopy (EM) were purchased from Sigma–Aldrich and Merck Millipore, respectively. The poly-D-lysine (A3890401) used for correlative light and electron microscopy was purchased from Thermo Fisher Scientific. The nutrient starvation medium containing 20 mM Hepes, pH 7.4, 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM glucose, and 1% bovine serum albumin (BSA) was prepared as previously described ) at RT for 1 h, followed by two washes with 0.1 M CAB buffer and three washes with double-distilled water (ddH2O). Then, the cells were incubated with 0.4% uranyl acetate at 4 °C for 1 h. After extensive washing with ddH2O, the cells were dehydrated in a graded series of ethanol and embedded in Spurr’s resin according to the manufacturer’s instructions. The embedded samples were trimmed and sectioned to 70 nm thickness by an EM UC7 ultramicrotome . The cell sections were stained with 0.4% uranyl acetate at RT for 20 min and then incubated with 4% lead citrate solution for 6 min. Electron micrographs were obtained with a JEM 1230 transmission electron microscope .For conventional TEM analysis, the cells were harvested and washed with PBS three times, followed by 0.1 M CAB buffer, pH 7.2 twice. Subsequently, the cells were treated with fixative I  at RT for 2 h. After washing with 0.1 M CAB buffer three times (each time for 5 min), the cells were incubated with fixative II  12 h prior to baicalein treatment. The baicalein-treated cells were washed once with PBS and once with 0.1 M CAB buffer, pH 7.2; then, the nuclei were stained with Hoechst 33342. After three washes with 0.1 M CAB buffer, pH 7.2, the cells were incubated with fixative III  as described above at 37 °C for 30 min. Then, the cells were washed twice with 0.1 M CAB buffer, pH 7.2, and stored in 2 mL 0.1 M CAB buffer, pH 7.2. The images of fluorescence-tagged reporters in cells were obtained by a Plan-Apochromat 63×/1.4 Oil DIC M27 objective equipped with confocal microscopy. The cell of interest was first marked by grids on dishes; then, the images of different Z-positions  of a defined cell were captured to further correlate fluorescence images with electron micrographs. Subsequently, the cells were fixed with fixative II, washed, incubated with 0.4% uranyl acetate, dehydrated, and embedded in Spurr’s resin as described above. The embedded samples were then trimmed and sectioned by the EM UC7 ultramicrotome. Serial sections  were collected and stained. Images of each section were captured by a JEM 1230 electron microscope. The EM images were assembled into one montage and used to relocate the position of the defined cell on confocal fluorescence Z-stack images. Finally, the confocal fluorescence images were aligned with the montage of EM images by Adobe Photoshop CS6 software.CLEM was performed according to previously described procedures ,54,55. TThe generation of gene knockout cells was performed as previously described ,57,58. TAn increasing number of studies have suggested that baicalein may regulate autophagy in liver cancer cells to promote apoptosis and enhance chemosensitivity ,47. In a+/GFP+ puncta of RFP-GFP-LC3) and autolysosomes (RFP+/GFP− puncta of RFP-GFP-LC3) were dramatically increased in baicalein-treated cells, whereas no significant puncta were detected in untreated cells , ATG5KO, ATG7KO, and ATG16KO cells were successfully generated , treatedKO cells . These r+/GFP− autolysosomes in the RFP-GFP-LC3 reporter and HSP60 was detected in baicalein-treated cells  and RFP+/GFP− autolysosomes (indicated by white arrows) in baicalein-treated cells  in human cytochrome c subunit VIII oxidase (COX8A) fused with GFP, we found that baicalein triggered the fragmentation of mitochondria, as demonstrated by the large population of fission mitochondria in baicalein-treated cells . In conted cells . TherefoFurthermore, we used CLEM to analyze mitochondrial engulfment at the ultrastructural level during baicalein-induced autophagy. In baicalein-treated RFP-LC3/Mito-GFP cells, confocal microscopy images taken at different Z-positions A were as+/GFP− dot-like structures; indicated by white arrowheads in magnified field-1), but the dominant RFP+/GFP+ signal of Mito-QC remained dominant in the untreated cells  in human fission 1 (FIS1), was used to assess mitochondrial turnover in baicalein-treated cells. When mitochondria are targeted to autolysosomes for degradation, the GFP signal emitted by the Mito-QC reporter is quenched by the low lysosomal pH, whereas the RFP signal emitted by Mito-QC remains stable. This reporter has been used to quantitatively assess mitophagy in vivo and in vitro ,65, and ed cells A. In addnd LAMP2 . In addind LAMP2 . In contnd LAMP2 . Notablynd LAMP2 . In addind LAMP2 , suggestnd LAMP2 C. In conNext, we tested whether baicalein treatment triggers the translocation of Parkin to mitochondria, indicating the initiation of PINK1/Parkin-dependent mitophagy . As showThe translocation of TBK1 to mitochondria and the TBK1-mediated phosphorylation of p62/SQSTM1 and OPTN have been implicated in PINK1/Parkin-dependent mitophagy ,25,26. WSeveral studies have identified active cargo receptors in PINK1/Parkin-dependent mitophagy ,28. TherWe then employed CLEM analysis of the cellular ultrastructure to determine whether NDP52 and OPTN participate in the elimination of mitochondria through baicalein-activated mitophagy. In baicalein-treated cells, deformed mitochondria were located inside RFP-LC3-labeled autophagic vacuoles with overlapping GFP-NDP52 signals . SimilarIn the past decade, accumulating evidence has shown that diverse mechanisms underlie the regulation of mitophagy ,17. AddiOn the other hand, interference with autophagy in genetic mouse models and human patients has been demonstrated in the pathogeneses of liver diseases, such as liver cancer ,74,75,76S. baicalensis that has hepatoprotective activity and has been used for a long time as a traditional Chinese medicine [Baicalein is a flavonoid isolated from the Chinese herb medicine ,34,37,38medicine ,23. Fina4-induced damage [Although the findings presented herein convincingly underscore that baicalein induces hepatic autophagy, the early events regulating the biogenesis of autophagosomes in baicalein-induced autophagy remain unclear. Previous studies have noted that baicalein represses the phosphorylation of mTOR and activates ULK1 signaling ,80, suggd damage . In accod damage .Previous studies have indicated that the regulators of the autophagy initiation complex, including ULK1, DFCP1, WIPI, and FIP200, are translocated to damaged mitochondria, thereby proximately triggering autophagosome maturation in mitophagy ,81,82. ICan baicalein-induced autophagy and mitophagy be applied to treat liver diseases? The answer remains unclear because whether baicalein induces autophagy and mitophagy in normal liver cells and in vivo remains to be determined. Further investigations are urgently needed to verify the autophagy- and mitophagy-induction ability of baicalein in primary human hepatocytes (PHHs) and experimental mouse models of liver diseases. Intriguingly, recent studies have implied that baicalein suppresses the accumulation of hepatic fat and prevents the formation of fatty liver in mice ,85. Aski


In the present study, a transcriptome analysis of intramuscular preadipocytes from Chinese Guizhou Congjiang pigs, including analyses of mRNAs, lncRNAs, and circRNAs at days 0 (D0), 4 (D4), and 8 (D8) was performed. A total of 1,538, 639, and 445 differentially expressed (DE) mRNAs, 479, 192, and 126 DE lncRNAs, and 360, 439, and 304 DE circRNAs were detected between D4 and D0, D8 and D0, and D8 and D4, respectively. Functional analyses identified many significantly enriched RNAs related to lipid deposition, cell differentiation, metabolism processes, and obesity-related diseases, biological processes, and pathways. We identified two lncRNAs (TCONS_00012086 and TCONS_00007245) closely related to fat deposition according to their target genes and tissue expression profiles. Subcellular distribution analysis using quantitative real-time PCR (qRT-PCR) revealed that both TCONS_00012086 and TCONS_00007245 are cytoplasmic lncRNAs. These data provide a genome-wide resource for mRNAs, lncRNAs, and circRNAs potentially involved in Chinese Guizhou Congjiang pig fat metabolism, thus improving our understanding of their function in adipogenesis.Intramuscular fat content is an important determinant of meat quality, and preadipocyte differentiation plays a critical role in intramuscular fat deposition in pigs. However, many types of RNA differentiation, including messenger RNA (mRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) remain unreported despite their crucial roles in regulating adipogenesis. Chinese Guizhou Congjiang pigs are raised in the Guizhou province of China for their high-quality meat. Therefore, it is important for breeders to explore the mechanisms of proliferation and differentiation of intramuscular adipocytes from the  Intramuscular fat content is positively correlated with flavor, tenderness, and juiciness in pork and is closely related to pork quality ,2. Thereet al. [et al. [In recent years scientists have identified thousands of non-coding RNAs in humans, plants, fish, insects, and mammals due to advances in sequencing technologies and bioinformatic approaches . Two newet al.  identifi [et al.  analyzed [et al. . CircRNA [et al.  and have [et al. . Several [et al. , mammals [et al. , and chi [et al. . HoweverChinese Guizhou Congjiang pigs are a heritage breed that is largely restricted to the Guizhou Province and is raised primarily for meat. Their meat is high-quality, fragrant, and tender due to its high intramuscular fat content. However, RNA expression profiles of these pigs during intramuscular preadipocyte differentiation have not yet been reported. Therefore, in the present study, mRNA, lncRNA, and circRNA expression were detected using RNA sequencing (RNA-Seq) during different stages of intramuscular preadipocyte differentiation (days 0 (D0), 4 (D4), and 8 (D8)). This research can help us to understand the involvement of RNAs in intramuscular fat.Three 3-day-old Chinese Guizhou Congjiang piglets were obtained from a local livestock farm. All animal care and experimental procedures were approved by the Guizhou University Animal Care and Use Committee, Guizhou, China. The ethics approval document number was EAE-GZU-2021-P004.longissimus dorsi muscle. The tissue was then washed with phosphate-buffered saline (PBS) three times and cut into 1–2 cm pieces. The pieces were digested with 2 mg/mL collagenase type Ⅰ at 37°C for 65 min and shaken well every 10 min. The digested tissue was diluted with an equal volume of DMEM/F12 growth medium and filtered with gauze to remove any undigested tissues. The filtered solution was then strained using 200 and 400 μm cell strainers and centrifuged at 1500 r/min for 10 min to collect the progenitor cells. The cells were cultured in DMEM/F12 growth medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in an atmosphere of 5% CO2. Because preadipocytes attach much earlier than myoblasts, the cultured cells were washed with PBS three times in order to remove unadherent cells and insoluble myofibrillar proteins after culturing for two hours [Three piglets were killed via venous administration of sodium pentobarbital (30 mg/kg of body weight) and sterilized with 75% ethanol to obtain samples of the wo hours . After tThe cultured cells were washed three times with PBS prior to Oil Red O staining, and then fixed in 4% paraformaldehyde for 30 min. The stained cells were again washed three times with PBS and observed under an inverted microscope . Triglyceride levels were measured using a commercial assay kit .Total RNA was isolated on D0, D4, and D8 using the Trizol reagent . RNA integrity was assessed using an RNA Nano 6000 Assay Kit in a Bioanalyzer 2100 system  and 1% agarose gel electrophoresis. The RNA integrity numbers (RINs) for all samples were > 9.8. RNA concentration was measured using a Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer .Ribosomal RNA was removed using a Epicentre Ribo-Zero™ rRNA Removal Kit , and sequencing libraries were generated using an rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®  following manufacturers’ recommendations. Briefly, fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. dNTPs with dTTP were replaced by dUTP in the reaction buffer and any remaining overhangs were converted into blunt ends via exonuclease/polymerase activity. After adenylation of 3’ends of DNA fragments, NEBNext Adaptors with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments 150–200 bp in length, the library fragments were purified using an AMPure XP system . Next, 3μl USER Enzyme  was added to size-selected, adaptor-ligated cDNA and processed at 37°C for 15 min and at 95°Cfor 5 min. PCR was then performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Nine strand-specific libraries were sequenced on an Illumina Hiseq 4000 platform at Novogene Corporation . The flowchart of library construction is shown in Sus scrofa 11.1) using HISAT2 [Clean data (clean reads) used in downstream analyses were obtained by removing reads containing adapter, reads containing ploy-N, and low quality reads from the raw data. At the same time, the Q20, Q30, and GC content of the clean data were calculated. All of the downstream analyses were based on the clean data with high quality. The index of the reference genome was built and paired-end clean reads were aligned to the pig reference genome (g HISAT2 .All of the transcripts were merged using Cuffmerge software. LncRNAs were then identified from the assembled transcripts in four steps: (1) removal of lowly-expressed transcripts with FPKM < 0.5; (2) removal of transcripts with < 200 bp and < 2 exons; (3) removal of transcripts with protein-coding capability using the coding-non-coding index (CNCI), protein folding domain database (Pfam), coding potential calculator 2 database (CPC2), and phylogenetic codon substitution frequency (PhyloCSF); (4) removal of transcripts mapped within the 1 kb flanking regions of an annotated gene using Cuffcompare. Novel lncRNAs were named according to HGNC rules . The floAfter the clean reads were aligned to the porcine reference genome, the junctions of the unmapped reads were identified using Findcirc software . In brieCandidate circRNAs were then identified using the CIRI (circRNA identifier) algorithm . In brieDue to the high false positive rate in circRNA identification, the circRNA identified by the Findcirc software and CIRI were combined, and their intersection was retained for use.DESeq2 R package, which identified DE genes as padj ≤ 0.05 and |log2(FoldChange)| > 1. The function of DE lncRNAs was predicted using Gene Ontology (GO) analysis of their co-location and co-expression target mRNAs. These were screened based on their genomic positional relation 100 kilobase pairs (kb) upstream and 100 kb downstream for co-location mRNAs and based on the Pearson correlation coefficient of lncRNA-RNA pairs (≥ 0.95) for co-expression mRNAs. The functions of DE circRNAs were analyzed using GO analysis of their parental genes. GO terms were performed for differentially expressed genes using GOseq [The expression of mRNA and lncRNA was evaluated using fragments per kilobase of transcript sequenced per million base pairs sequenced (FPKM), and circRNA expression was estimated using TPM (transcripts per million). Differential expression of mRNAs, lncRNAs, and circRNAs was analyzed using the ng GOseq , and p <ng GOseq .−△△Ct method and the data were expressed as values relative to the D0 group.The primer pairs used in quantitative real-time RT-PCR (qRT-PCR) are listed in Porcine intramuscular preadipocytes were used for subcellular localization of lncRNA. Porcine intramuscular preadipocytes were lysed in cold lysis buffer and placed on ice for 10 min. Then, cells were centrifuged  and the supernatant retained as the cytoplasmic fraction and immediately frozen (80°C) for subsequent analysis. The nuclear pellet was resuspended with nuclear extraction buffer and placed on ice for 30 min, and then centrifuged . The supernatant was removed and the remainder (nuclear fraction) was frozen (80°C) for subsequent analysis.p<0.05.Data were analyzed using one-way ANOVAs in SPSS 18.0 statistical software. Differences were considered significant at Our sequencing data have been submitted to the Sequence Read Archive database with accession number PRJNA718643.Preadipocytes changed from fibrous during the initial phase (D0) to spherical, and lipid droplets were visible on D4. The number of lipid droplets increased through D8 . The triA total of 962,969,676 clean reads were obtained from all samples containing nine complementary libraries . The perThe total numbers of mRNAs, lncRNAs, and circRNAs obtained from the three differentiation stages were 25,880, 4,278, and 11,439 respectively. A total of 9,940 mRNAs, 397 lncRNAs, and 1,284 cirRNAs  were detected as reliably expressed genes during intramuscular preadipocyte differentiation . Three cBetween D4 and D0, we found significant GO terms for DE mRNA related to metabolic processes, primary metabolic processes, cellular component organization, intracellular processes, cytoplasmic parts, catalytic activity, and protein binding . Based oBetween D4 and D0, significantly enriched KEGG terms for DE mRNAs were primarily involved in ribosomes, proteoglycans in cancer, PPAR signaling pathways, Parkinson’s disease, oxidative phosphorylation, non-alcoholic fatty liver disease, Huntington’s disease, focal adhesion, ECM-receptor interaction, Alzheimer’s disease, glycerolipid metabolism, and citrate cycles . SignifiFor DE mRNAs between D8 and D0, significant GO terms were found related to lipid metabolic processes, lipid biosynthetic processes, cardiovascular system development, growth factor binding, receptor binding, insulin-like growth factor binding, and protein binding . BetweenBetween D8 and D0, significantly enriched KEGG terms for DE mRNAs were primarily involved in terpenoid backbone biosynthesis, systemic lupus erythematosus, steroid biosynthesis, PI3K-Akt signaling pathways, focal adhesion, ECM-receptor interaction, and alcoholism . SignifiFor DE mRNAs between D8 and D4, significant GO terms were found related to biological processes, cell adhesion, tissue development, regulation of responses to stimulus, regulation of signaling, and cytosolic parts . SignifiBetween D8 and D4, significantly enriched KEGG terms for DE mRNAs were primarily involved in ribosomes, ECM-receptor interactions, the phosphatidylinositol signaling system, and fatty acid degradation . SignifiThrough comparison to the initial phase of differentiation (D0), 33 mRNAs, 4 lncRNAs, and 11 circRNAs were identified as common DE genes during the intramuscular preadipocyte differentiation process. Significant GO terms for DE mRNAs related to lipid metabolism and cell proliferation/differentiation were commonly significantly enriched based on comparisons between D4 and D0, D8 and D0, and D8 and D4 . SignifiAPOE, DGAT2, MAOA, TSC22D3, CEBPA, KLF9, LPL, COL15A1, COL14A1, SEMA3C, CCND1, FOS, CCNG1, and COL3A1), 9 lncRNAs , and 5 circRNAs  to validate. The nine cell samples used in RNA-Seq were also used for qRT-PCR validation. Relative gene expression levels were calculated based on the mean value from nine cell samples using the comparative Ct method. After comparison with the RNA-Seq data, similar expression trends for qRT-PCR were discovered , consistent with results described in other research [CEBPA can impact growth arrest on D4 through downregulation of CCND1 expression. APOE regulates lipid homeostasis by mediating lipid transport from one tissue or cell type to another, and positively correlates with triglyceride content [LPL also plays a key role in lipid metabolism by catalyzing triglyceride hydrolysis of lipoproteins such as chylomicron and very-low-density lipoprotein (VLDL) into fatty acids [COL14A1 and COL15A1 was significantly lower during the early stage of differentiation (D4 vs D0). COL14A1 and COL15A1 have an anti-proliferative role in reducing de novo DNA synthesis in 3T3-L1 preadipocytes [COL14A1 and COL15A1 may be associated with the observed changes in intramuscular preadipocyte morphology.The intramuscular preadipocyte differentiation process consists of three critical stages, including growth arrest (D0), mitotic clonal expansion (D4), and late events and terminal differentiation (D8). DE mRNAs were enriched during lipid metabolism, cell proliferation, cell differentiation, cell development, and cell growth in the early stage of differentiation (D0 vs D4). At the same time, triglyceride content on D4 was significantly higher than on D0. The pathway of lipid metabolism and cell development/proliferation/growth was essential during the early stages of intramuscular preadipocyte differentiation, and this was consistent with other research . Accordiipocytes , and canipocytes . The expresearch . Our dat content . LPL alsty acids . The expipocytes . Therefolongissimus dorsi muscle of different porcine breeds [Throughout the last decade, many studies have revealed the important roles that lncRNAs and circRNAs play in regulating adipocyte development, metabolic processes, and physiological processes ,31. There breeds ,34. Chane breeds , wherease breeds . These re breeds .GO and KEGG pathway analyses were performed in order to explore differential expression of mRNAs during preadipocyte differentiation. As expected, many classical functional categories were found to be significantly enriched, including metabolic processes, intracellular parts, protein binding, biological processing, and regulation of responses to stimuli. Genes within these functional groups may play essential roles in the conversion of intramuscular preadipocytes to adipocytes .The PPARThe function of DE lncRNAs can be predicted from their cis- or trans-acting mRNAs, both of which may be important in the regulation of transcription and posttranscriptional modification. Trans target gene predictions were based on correlations between lncRNA and mRNA expression levels . The maiGO and KEGG pathway analyses were performed to explore the function of DE circRNAs based on their genomic locations. As expected, many lipid metabolism-related functional categories were significantly enriched, including metabolic processes, enzyme regulator activities, and the regulation of biological processes. The MAPK signaling pathway was enriched on D4 relative to D0. The MAPK signaling pathway plays an important role in regulating adipocyte proliferation and differentiation in mammals because of the function of signal systems in mediating cell responses to external stimuli . The celXLOC_046142, XLOC_004398, and XLOC_015408 play main regulatory roles in adipogenesis and lipid accumulation by targeting MAPKAPK2, NR1D2, and AKR1C4, respectively [PDE7B) is the target gene of TCONS_00012086. Cyclic nucleotide phosphodiesterases (PDEs) are enzymes regulating cellular cAMP (or cGMP) concentration by controlling the rate of hydrolysis [XLOC_005242, first found in this study, is the target gene of TCONS_00007245, which was expressed at high levels in the liver. We speculate that TCONS_00007245 is related to lipid metabolism due to the important function of the liver in regulating lipid metabolism [In pig intramuscular preadipocytes, tabolism . HoweverDGAT2, MAOA, TSC22D3, CEBPA, KLF9, LPL, COL15A1, COL14A1, SEMA3C, CCND1, FOS, CCNG1, and COL3A1), nine lncRNAs  and five circRNAs  were validated using qRT-PCR, and these were consistent with our RNA-seq results. Two lncRNAs (TCONS_00012086 and TCONS_00007245) that are closely related to fat deposition according to their target genes were identified, and their tissue expression profiles were detected. However, further study is necessary to identify their functions in pig preadipocytes. These findings provide a reliable foundation for future studies investigating the molecular mechanisms underlying preadipocyte differentiation in pigs.In conclusion, a genome-wide investigation of the expression of mRNAs, lncRNAs, and circRNAs during porcine intramuscular preadipocyte differentiation was undertaken. A large number of DE genes, which may contribute to phenotypic changes in adipocytes at different stages of differentiation, were identified. Our study provides a comprehensive record of the expression levels of various RNAs during adipocyte differentiation, and in turn provides a tool for better understanding the mechanisms underlying molecular regulation of intramuscular preadipocyte differentiation in pigs. Fourteen mRNAs Click here for additional data file.S2 Fig(DOC)Click here for additional data file.S3 FigSchematic diagram for circRNA discovery for find CIRC (a) and CIRI (b).(DOC)Click here for additional data file.S4 FigVenn diagram of DE mRNAs (a), lncRNAs (b), and circRNAs (c) at three time-points.(DOC)Click here for additional data file.S5 Fig(a) GO analysis of differentially expressed mRNAs. (b) GO analysis of differentially expressed lncRNAs with their trans-acting mRNA. (c) GO analysis of differentially expressed lncRNAs with their cis-acting mRNA. (d) GO analysis of differentially expressed circRNAs.(DOC)Click here for additional data file.S6 Fig(a) GO analysis of differentially expressed mRNAs. (b) GO analysis of differentially expressed lncRNAs with their trans-acting mRNA. (c) GO analysis of differentially expressed lncRNAs with their cis-acting mRNA between. (d) GO analysis of differentially expressed circRNAs.(DOC)Click here for additional data file.S7 Fig(a) GO analysis of differentially expressed mRNAs. (b) GO analysis of differentially expressed lncRNAs with their trans-acting mRNA. (c) GO analysis of differentially expressed lncRNAs with their cis-acting mRNA between. (d) GO analysis of differentially expressed circRNAs.(DOC)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Tabled0, d4 and d8 refer to three different time points during intramuscular preadipocyte differentiation . 1, 2, and 3 refer to the three replicates.(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.