Forkhead Research Articles

Cardiomyocyte Foxp1‐Specific Deletion Promotes Post‐injury Heart Regeneration via Targeting Usp20‐HIF1ɑ‐Hand1 Signaling Pathway

AbstractThe adult mammalian heart has limited regenerative capacity to replace lost tissue after a major injury. Forkhead box P1 (Foxp1) regulates embryonic cardiomyocyte proliferation and heart development. However, whether Foxp1 participates in postnatal‐injury cardiomyocyte proliferation and heart regeneration remains unclear. This study demonstrates that Foxp1 is downregulated at border zone cardiomyocytes of both neonatal apical resection and adult myocardial infarction. Analysis of the Single‐cell transcriptome database reveals reduced Foxp1 expression in the cardiomyocyte population with high regenerative capacity. Cardiomyocyte‐Foxp1 loss‐of‐function significantly promotes, whereas cardiomyocytes‐Foxp1 gain‐of‐function suppresses cardiomyocyte proliferation. Mechanistically, Foxp1 directly regulates ubiquitin specific peptidase 20 (USP20), a de‐ubiquitinase that prevents hypoxia inducible factor 1ɑ (HIF1α) degradation. Thus, Foxp1 regulates HIF1α and downstream heart and neural crest derivatives expressed 1 (Hand1) to control the cardiomyocyte proliferation via metabolic transition from fatty acid oxidation to glycolysis. Finally, cardiac type troponin T2 (cTnT)‐promoter‐driven adeno‐associated virus 9 (AAV9) for Hand1 induction in cardiomyocytes significantly promoted cardiac regeneration and functional recovery. These findings may provide novel molecular strategies to promote heart regeneration and therapeutic interventions for heart failure.

Fraxetin attenuates DNA damage and inflammation in cisplatin-induced nephrotoxicity via FoxO1 activation.

Cisplatin-induced acute kidney injury (CKI) represents a severe renal dysfunction characterized by DNA damage and tubular injury. Fraxetin, derived from the Chinese herb Qinpi (Fraxinus bungeana A.DOC), is recognized for its neuroprotective effects and has been used for the prevention of various diseases. This study investigated the renoprotective effects and molecular mechanisms of fraxetin in CKI. A mouse CKI model and a cisplatin-induced tubule epithelial cell (TEC) injury model were established to evaluate fraxetin's effects by measuring diverse parameters associated with kidney injury, focusing on DNA damage and inflammation. Additionally, network pharmacology and cellular sequencing analysis were employed to identify altered pathways or targets after fraxetin treatment. Subsequent experiments involved siRNA and pharmacological regulation to identify fraxetin targets, alongside molecular docking to unravel binding mechanisms. Fraxetin pretreatment significantly ameliorated CKI, with a 45% reduction in in tubular damage compared to the cisplatin-only group. Additionally, fraxetin notably enhanced DNA repair. Fraxetin pretreatment reduced cisplatin-induced DNA damage in HK-2 cells by 42.8% in comet assays. Fraxetin also mitigated inflammation, with pro-inflammatory cytokine levels decreasing by approximately 20-30% in both mouse and cell models. Notable changes were observed in the FoxO pathway. Specifically, manipulating Forkhead box O1 (FoxO1), a transcription factor involved in stress responses and longevity, influenced fraxetin's protective effect. Molecular docking revealed that fraxetin binds to the Forkhead (FH) domain of FoxO1, promoting its nuclear localization. Fraxetin protects against CKI by activating FoxO1, providing a foundation for novel therapeutic strategies and underscoring fraxetin's potential in treating kidney injury.

Klotho supplementation decreases blood pressure and albuminuria in mice with lupus nephritis.

Klotho deficiency is prevalent in various chronic kidney diseases. Although klotho is known to bind transforming growth factor β (TGFβ) receptor 1 to antagonize renal fibrosis, TGFβ also maintains regulatory T cells with inducing forkhead box protein P3 (FOXP3). Female New Zealand Black/White F1 (NZBWF1) mice were divided into two groups (n=10 for each): one group was treated with daily subcutaneous injection of klotho protein (30μg/kg/day) for 8 weeks, and the other only received vehicle. Klotho supplementation suppressed blood pressure, 8-epi-prostaglandin F2α excretion, albuminuria, and renal angiotensin II levels (p<0.05 for all) without affecting the glomerular filtration rate (GFR) in NZBWF1 mice. Klotho protein supplementation reduced the number of cluster of differentiation (CD)4+FOXP3+ T cells (p<0.05) without altering the anti-DNA antibody levels. Klotho supplementation augmented glomerular cellularity, but decreased glomerular crescent formation and interstitial fibrosis in NZBWF1 mice (p<0.05). Klotho protein supplementation inactivated renal renin-angiotensin system, ameliorating blood pressure and albuminuria in NZBWF1 mice. Klotho supplementation hampered regulatory T cells without altering autoantibodies, exerting dual effects on glomerular pathology in NZBWF1 mice without changes in GFR.

Inhibition of DNA Methyltransferase DNMT1 Reverses Th2 Response Polarisation and Alleviates Allergic Rhinitis.

Type 2 T helper (Th2) cells-mediated immune response plays vital roles in allergic rhinitis (AR), and DNA methylation is previously found to be closely related to AR development. Our study aims to reveal the detail mechanism of DNA methylation affecting Th2 response in AR. Mice were stimulated with ovalbumin (OVA) to induce AR symptoms, and CD4+ T cells were subjected to Th2 induction culture. Real-time quantitative PCR, western blot, flow cytometry, and enzyme-linked immunosorbent assay were performed to analyse the activation of Th2 response. DNA methyltransferase 1 (DNMT1) was significantly upregulated in OVA-induced AR model mice, and DNMT1 knockdown alleviated AR symptoms and pathological changes of nasal mucosa tissues in the model mice. DNMT1 knockdown obviously reduced the expression of GATA binding protein 3 (GATA3), the ratio of interleukin (IL)-4+CD4+ cells and the release of Th2 cytokines, but elevated the expression of T-box expressed in T cells (T-bet), the ratio of interferon (IFN)-γ+CD4+ cells and the levels of Th1 cytokines to improve Th1/Th2 imbalance in the model mice and Th2-induced CD4+T cells. Mechanistically, DNMT1 promoted promoter methylation of forkhead box O3 (FOXO3), inhibited FOXO3 expression and activated the nuclear factor kappa-B (NF-κB)/GATA3 signalling. FOXO3 overexpression remarkably inactivated the NF-κB/GATA3 pathway and mitigated Th2 polarisation in DNMT1-deficient and Th2-conditined CD4+T cells, which was reversed by a NF-κB inhibitor. Altogether, DNMT1 downregulated FOXO3 expression to activate the NF-κB/GATA3 pathway and promote Th2 response in AR.

Association analysis between forkhead box E1 gene and non-syndromic cleft lip with or without cleft palate in Han Chinese population.

This study aims to explore the association between single nucleotide polymorphisms (SNPs) loci near the haplotype region hg19 chr9:100560865-100660865 of the forkhead box E1 (FOXE1) gene and the occurrence of non-syndromic cleft lip with or without cleft palate (NSCL/P) in western Han Chinese population. In the first stage, our study recruited 159 NSCL/P patients and performed targeted region sequencing to screen SNPs loci near the haplotype region of the FOXE1 gene associated with NSCL/P. In the second stage, we selected 21 common SNPs and re-enrolled 1 000 non-syndromic cleft lip only (NSCLO) patients, 1 000 non-syndromic cleft palate only (NSCPO) patients, and 1 000 normal controls to verify the association. PLINK software was used to perform Hardy-Weinberg equilibrium (HWE) test. Association analysis for common variants, gene burden analysis for rare mutations, and function prediction of SNPs with non-synonymous mutations were performed using Mutation Taster and other software programs. In the first stage, 126 variants, including 76 single nucleotide variants and 50 insertion-deletions were identified. All the included SNPs confirmed to HWE, and the results of gene burden analysis and prediction of functional harmfulness for rare variants were not statistically significant. Association analysis showed that rs13292899 of the FOXE1 gene was significantly associated with NSCL/P (P=1.85E-27) and was also correlated with NSCLO (P=6.41E-23) and non-syndromic cleft lip with cleft palate (NSCLP) (P=2.36E-15) subtypes. In the validation phase, rs79268293 (P=0.013, P=0.022), rs10983951 (P=0.009 2, P=0.007 6), rs117227387 (P=0.009 2, P=0.007 6), rs3758250 (P=0.009 2, P=0.007 6), and rs116899397 (P=0.009 2, P=0.007 6) were significantly associated with NSCLO and NSCPO; rs13292899 (P=0.008 5), rs74606599 (P=0.008 3), rs143226042 (P=0.008 3), and rs117236550 (P=0.01) were associated with the occurrence of NSCLO; and rs12343182 (P=0.008 7), rs10119760 (P=0.012), rs10113907 (P=0.012), and rs13299924 (P=0.012) were associated with the occurrence of NSCPO. This study found a new susceptible SNP rs13292899 of the FOXE1 gene that is closely associated with NSCL/P and NSCLO subtype and 13 other SNPs associated with NSCLO or NSCPO.

Structural profile and diversity of immunoglobulin genes in the Arctic Fox.

Immunoglobulins are important components of humoral immunity and play a crucial role in protecting the body from external antigens. The Arctic fox is an important member of furbearer farming, but due to the lack of research on the immune system of the Arctic fox, animal welfare regarding Arctic fox farming has still not received enough attention. In this study, we used the Arctic fox as a research subject, described the gene locus structure of the Arctic fox immunoglobulin germline by genome comparison, and analysed the mechanism of expression diversity of the antibody pool of the Arctic fox by rapid amplification of cDNA 5' ends and high-throughput sequencing. The results showed that the Arctic fox IgH, Igκ and Igλ loci were located on chromosome 6, chromosome 5 and chromosome 14, respectively. The number of variable (V) genes identified were 18, 11 and 10, and the number of joining (J) were 3, 4 and 13 and six diversity (D) genes in the heavy chain, respectively. Among them, the D genes, J genes and constant region genes of the heavy chain were arranged in the middle of the two variable heavy gene clusters; Arctic fox had a strong preference for the use of V genes, D genes and J genes, which resulted in a low level of V(D)J recombination diversity; linkage diversity analyses showed that random deletion of the V and J genes and insertion of the N and P nucleotides of the immunoglobulins of the Arctic foxes had a large impact on the linkage diversity of the IgH, whereas the light chain The linkage diversity was mainly contributed by the random deletion of V and J genes, and the insertion of N and P nucleotides had a smaller effect; somatic hypermutation (SHM) analysis showed that the mutation types of SHM of the heavy and light chains of the Arctic fox had a strong bias towards G>A and A>G, and the bias exhibited by the three chains was basically the same. By analysing the structure and expression diversity analysis of the Arctic fox gene loci, this study could provide a theoretical basis for antibody design and vaccine development in the Arctic fox, and provide new insights to further improve the animal welfare level of Arctic fox farming.

Histone Demethylase PHF8 Confers Protection against Oxidative Stress and Cardiomyocyte Apoptosis in Heart Failure by Upregulating FOXA2

Oxidative stress and cardiomyocyte apoptosis are hallmarks of heart failure (HF) development. Plant homeodomain finger protein 8 (PHF8) is a histone demethylase downregulated in failing human hearts. Nevertheless, the potential role of PHF8 in HF remains unclear. Therefore, this study aimed to explore the biological action and molecular mechanism of PHF8 in HF.A rat model of left anterior descending coronary artery (LAD) ligation-induced HF and a cardiomyocyte model of oxygen-glucose deprivation/reperfusion (OGD/R) were developed after gain- or loss-of-function experiments in rats and cardiomyocytes, respectively. Heart function indexes, such as left ventricular end-diastolic diameter, left ventricular end-systolic diameter, left ventricular ejection fraction, and left ventricular fractional shortening, were detected. Changes in myocardial tissues were examined by pathological staining. Cardiomyocyte apoptosis and oxidative stress markers, such as malondialdehyde, reactive oxygen species, superoxide dismutase, and catalase, were examined. The relationship between PHF8 and forkhead box A2 (FOXA2) was analyzed by luciferase and chromatin immunoprecipitation-quantitative polymerase chain reaction assays.PHF8 was downregulated in LAD-ligated rats and OGD/R-exposed cardiomyocytes. Following PHF8 upregulation, pathological changes in myocardial tissues and heart dysfunction were improved in LAD-ligated rats. Importantly, cardiomyocyte apoptosis and oxidative stress were diminished in vivo and in vitro upon PHF8 upregulation. Mechanistically, PHF8 increased FOXA2 expression in a histone demethylase-dependent manner. FOXA2 silencing abrogated the protective effect of PHF8 upregulation on cardiomyocytes against OGD/R-induced apoptosis and oxidative stress.PHF8 exerts protective functions against cardiomyocyte apoptosis, oxidative stress, and heart dysfunction in HF, in correlation with FOXA2 upregulation. These results suggest that the PHF8/FOXA2 axis may be a promising therapeutic target to prevent HF.

Reference genome provide insights into sex determination of silver aworana (Osteoglossum bicirrhosum)

BackgroundSilver arowana (Osteoglossum bicirrhosum) is a basal fish species with sexual monomorphism, while its sex determination mechanism has been poorly understood, posing a significant challenge to its captive breeding efforts.ResultsWe constructed two high-quality chromosome-level genome assemblies for both female and male silver arowana, with scaffold N50 values over 10 Mb. Combining re-sequencing data of 109 individuals, we identified a female-specific region, which was localized in a non-coding region, i.e., around 26-kb upstream of foxl2 gene (encoding forkhead box L2). Its strong interaction with the neighboring foxl2 on the same chromosome suggests foxl2 as a candidate sex-related gene in silver arowana. We subsequently propose a complex gene network in the sex determination process of silver arowana, with foxl2 acting as the central contributor. Transcriptome sequencing of gonads support our hypothesis that the regulation of foxl2 can be influenced by the spatial proximity of the female-specific fragment, thereby promoting ovarian function or inhibiting testicular function to stimulate gonadal differentiation. Furthermore, we found the sex chromosomes to be homomorphic with a potentially recent origin, as a linkage disequilibrium analysis proved minor recombination suppression.ConclusionsThese results taken together serve as a crucial foundation for conducting extensive investigations on the evolution and differentiation of sex-determining mechanisms, as well as the emergence and development of sex chromosomes in various fishes.

Nucleotide variation in Foxp3 gene and prognosis of bladder cancer: a case-control study

BackgroundNumerous researches have investigated the correlation between single nucleotide polymorphisms (SNPs) in the transcription factor forkhead box protein 3 (Foxp3) gene and the development of various cancers. However, the relationship of Foxp3 polymorphism and bladder cancer (BC) remain unclear.MethodThis hospital-based case-control study enrolled a total of 316 patients diagnosed with BC and 643 healthy controls. Two Foxp3 SNPs (rs3761548 C/A, rs5902434 del/ATT) were selected, and genotyping of the samples was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. SPSS and online SNPstats software were used to determine the disparities between groups.ResultsFor the rs3761548 C/A polymorphism, patients with the CA/AA genotype showed a notable decrease in the case group (22.1% versus 34.8%, P = 0.003, OR = 0.61, 95%CI = 0.44-0.85), and the heterozygous CA genotype presented a distinctly lower risk for BC (P = 0.0003, OR = 0.43, 95%CI = 0.26-0.70). Notably, individuals who were homozygous for the AA genotype demonstrated a markedly lower overall survival (OS) rate compared to those with the CC/CA genotypes (P = 0.03, OR = 5.89, 95%CI = 1.23-28.15), after adjusting for factors such as age, gender, smoking status, tumor grade, metastasis, and clinical stage. For the rs5902434 del/ATT polymorphism, a decreased risk was observed across the codominant and over-dominant models with statistical significance (codominant model: P = 0.01, OR = 0.61, 95%CI = 0.42-0.89; over-dominant model: P = 0.004, OR = 0.60, 95%CI = 0.42-0.85), and no significant association was observed between the rs5902434 polymorphism and patient’s OS rate.ConclusionsOur findings indicate that Foxp3 polymorphisms may be associated with BC susceptibility, and that rs3761548 could potentially serve as an independent risk factor for the OS rate.

Sirtuin 1 Suppresses Hydrogen Peroxide-Induced Senescence and Promotes Viability and Migration in Lens Epithelial Cells by Inhibiting Forkhead Box Protein O1/Toll-Like Receptor 4 Pathway.

Age-related cataracts (ARCs) are associated with increased oxidative stress and cellular senescence. Our objective is to investigate the function of Sirtuin 1 (SIRT1) within ARCs. In ARCs tissues and H2O2-treated lens epithelial cells (LECs), the expression levels of SIRT1 were examined. Senescence-associated β-galactosidase (SA-β-gal) staining was employed to evaluate cellular senescence. The Cell Counting Kit-8 assay was employed to measure viability. A wound healing assay was performed to assess migratory capacity in LECs. Oxidative stress-related indicators were determined by enzyme-linked immunosorbent assay kits. Additionally, the Coxpresdb and GeneCards databases were utilized to identify downstream pathways of SIRT1 in ARCs. The expression levels of protein and mRNA were detected using western blot and real-time quantitative polymerase chain reaction, respectively. The expression of SIRT1 was downregulated in ARCs tissues with an increase in reactive oxygen species. In H2O2-induced LECs, SIRT1 was downregulated and its overexpression inhibited oxidative stress and cellular senescence while promoting viability and migration. Furthermore, FoxO1/TLR4 pathway was screened out as the key pathway of SIRT1, which was activated in H2O2-induced LECs senescence. Overexpression of SIRT1 suppressed FoxO1/TLR4 pathway. Further research demonstrated that the activation of FoxO1/TLR4 pathway reversed the inhibitory role of SIRT1 in oxidative stress-induced cellular senescence and the promotion effect of SIRT1 on viability and migration in H2O2-induced LECs. SIRT1 inhibits oxidative stress-induced cellular senescence and promotes the viability and migration in H2O2-induced LECs via suppressing FoxO1/TLR4 pathway.

Lactiplantibacillus plantarum 22 A-3 ameliorates leaky gut in mice through its anti-inflammatory effects

There are limited studies on the improvement of leaky gut with minor inflammation associated with various diseases. To explore the therapeutic potential of Lactiplantibacillus plantarum 22 A-3, a member of the Lactobacillus species, in addressing a leaky gut. Lactiplantibacillus plantarum 22 A-3 was administered to a leaky gut mice model with low dextran sulfate sodium concentrations. The Lactiplantibacillus plantarum 22 A-3-treated group exhibited amelioration of increased intestinal permeability, as indicated by lower blood fluorescein isothiocyanate-dextran levels compared with that of the control group. Furthermore, the messenger RNA expression of interleukin-10, an anti-inflammatory cytokine, was upregulated in the small intestine of Lactiplantibacillus plantarum 22 A-3-treated mice. Moreover, forkhead box P3 was upregulated in the small intestine and colon following Lactiplantibacillus plantarum 22 A-3 administration. Flow cytometry showed that forkhead box P3-positive regulatory T cells tended to increase in the small intestine and colon; however, this was not significant. Messenger RNA levels for the pro-inflammatory cytokines, interleukin-1 beta, and tumor necrosis factor-alpha showed no significant changes in the small intestine; however, their expressions significantly decreased in the colon. Blood fluorescein isothiocyanate-dextran levels showed that intestinal permeability also decreased in Lactiplantibacillus plantarum 22 A-3-dead bacteria. The bacterial component of Lactiplantibacillus plantarum 22 A-3 ameliorates increased intestinal permeability through its anti-inflammatory effect in the intestinal tract and may be a novel treatment for leaky gut.

Capecitabine enhances sensitivity to oxaliplatin in advanced gastric cancer and the effects on patients’ FOXP1 and GGT levels

ObjectiveTo investigate the effect of capecitabine on the sensitivity of oxaliplatin and on the level of transcription factor forkhead box P1 (FOXP1) and gamma-glutamyl transpeptidase (GGT) in patients with intermediate and advanced gastric cancer.MethodsA total of 152 Patients with advanced gastric cancer who were continuously diagnosed and treated in our hospital were selected as the study objects. The general data were retrospectively analyzed. The patients in the control group received oxaliplatin, while the patients in the study group received capecitabine on the basis of the control group. The FOXP1 expression level was detected using immunohistochemistry. Serum levels of GGT were measured by chemiluminescence. Protein levels were detected by Western blot. The prognostic factors were analyzed by the COX regression model. The Kaplan–Meier survival curve was used to analyze the survival of gastric cancer.ResultsThe effective rates (complete response, partial response, and stability) of the study group and the control group were 94.74% and 76.32%, respectively. Compared with adjacent normal tissues, the expression level of FOXP1 in gastric cancer tissues was lower (P < 0.05). After treatment, the average expression level of FOXP1 in the gastric cancer tissue of the study group was higher than the control group (P < 0.05). Moreover, lower FOXP1 expression was associated with lower overall survival (OS) (1-year survival and 3-year survival were 75.76% and 53.03%, respectively) (P < 0.05). Further analysis showed that capecitabine combined with oxaliplatin down-regulated the expression of DNA repair related-proteins and up-regulated the expression of key molecules of the apoptosis pathway, thus enhancing the killing effect of oxaliplatin on gastric cancer cells (P < 0.05). Both the 1-year and 3-year survival rates of the study group were higher than that in the control group (P < 0.05). The 1-year survival rate of 152 patients with gastric cancer was 84.87% (129/152) and the 3-year survival rate was 63.17% (96/152). Age, tumor-node-metastasis (TNM) stage, lymph node metastasis, chemotherapy regimen, FOXP1, and GGT levels were important factors in determining OS.ConclusionCapecitabine effectively enhanced the sensitivity of intermediate and advanced gastric cancer to oxaliplatin, improved the therapeutic effect and ameliorated the prognosis of patients.Graphical

Adipose Tissue-Derived Mesenchymal Stem Cells Regulate Th17/Treg in a Rat Model of Allergic Rhinitis by Activating IL-2/JAK3/STAT5 Signaling Pathway.

To investigate how adipose-derived mesenchymal stem cells (ADSCs) regulate the balance between regulatory T cells (Treg) and Th17 cells through the IL-2/JAK3/STAT5 signaling pathway in a rat model of allergic rhinitis (AR). Adipose-derived stem cells (ADSCs) were used to treat an ovalbumin (OVA)-induced AR rat model. The pathological changes and nasal symptoms were observed by HE staining and scanning electron microscopy. The concentrations of OVA-sIgE, IL-10, and IL-17 in serum were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expressions of JAK-3, STAT-5, forkhead box P3 (Foxp3), and retinoic acid-associated orphan receptor γt (RORγt) in nasal mucosa were detected by reverse transcription-polymerase chain reaction (RT-PCR). The expression of IL-2, JAK-3, and STAT-5 proteins in nasal mucosa was detected by Western blotting, and the expression patterns of these proteins were analyzed. ADSCs significantly reduced nasal symptoms and histological abnormalities by significantly reducing the levels of OVA-sIgE and IL-10. The expressions of IL-2, JAK-3, STAT-5, IL-2, JAK-3, STAT-5, Foxp3 mRNA, and RORγ t mRNA in the nasal mucosa of AR rats were significantly increased. ADSC can control the development of Treg/Th17 cells by the IL-2/JAK-3/STAT-5 signaling pathway and reduce OVA-induced allergic inflammation.

Dexamethasone-loaded fibroin nanoparticles promote retinal reattachment in rats by regulating the Th17/Treg balance

Retinal detachment (RD) is a common acute blinding eye disease, and dexamethasone (DEX), an adrenocorticosteroid, shows protective effects against RD. However, its poor water solubility and low bioavailability limit its effectiveness. To address this, we developed SF@DEX nanomaterials and investigated their therapeutic potential and mechanisms in RD. The nanomaterials were successfully synthesized and characterized, achieving 90% encapsulation efficiency and releasing 60% of DEX within 12 h.In vitro, phagocytosis was measured by flow cytometry, and enzyme-linked immunosorbent assay determined interleukin-17 (IL-17) and interleukin-10 (IL-10) levels. A rat RD model was established surgically, followed by oral administration of silk fibroin (SF), SF@DEX, and DEX. Polymerase chain reaction (PCR) assessed IL-17A and forkhead box P3 (FOXP3) expression, while Western blot analysed transforming growth factor-β1 (TGF-β1), IL-10, IL-17A, and FOXP3 levels. Apoptosis of retinal ganglion cells was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy detected colocalization of IL-17A and FOXP3. SF@DEX treatment significantly reduced Th17 cells and IL-17A while increasing Tregs, FOXP3, TGF-β1, and IL-10 levels. The severity of RD in rats was notably alleviated by SF@DEX, demonstrating its anti-inflammatory effects through modulation of the Th17/Treg immune balance. These results highlight SF@DEX as a promising nano-based therapy for RD.

Identification of glycogen synthase kinase 3alpha/beta as a host factor required for hepatitis B virus transcription using high-throughput screening.

Hepatitis B virus (HBV) leads to severe liver diseases, such as cirrhosis and hepatocellular carcinoma. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1,827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.

CAMKIIδ Reinforces Lipid Metabolism and Promotes the Development of B Cell Lymphoma.

The most prevalent types of lymphomas are B cell lymphomas (BCL). Newer therapies for BCL have improved the prognosis for many patients. However, approximately 30% with aggressive BCL either remain refractory or ultimately relapse. These patients urgently need other options. This study shows how calcium/calmodulin-dependent protein kinase II delta (CAMKIIδ) is pivotal for BCL development. In BCL cells, ablation of CAMKIIδ inhibits both lipolysis from lipid droplets and oxidative phosphorylation (OXPHOS). With lipolysis blocked, BCL progression is markedly suppressed in two distinct BCL mouse models: MYC-driven EµMyc mice and Myc/Bcl2 double-expressed mice. When CAMKIIδ is present, it destabilizes transcription factor Forkhead Box O3A (FOXO3A) by phosphorylating it at Ser7 and Ser12. This then permits transcription of downstream gene IRF4 - a master transcription factor of lipid metabolism. The CAMKIIδ/FOXO3A axis bolsters lipid metabolism, mitochondrial respiration, and tumor fitness in BCL under metabolic stress. This study also evaluates Tetrandrine (TET), a small molecule compound, as a potent CAMKIIδ inhibitor. TET attenuates metabolic fitness and elicits therapeutic responses both in vitro and in vivo. Collectively, this study highlights how CAMKIIδ is critical in BCL progression. The results also pave the way for innovative therapeutic strategies for treating aggressive BCL.

Cardiac allograft tolerance can be achieved in nonhuman primates by donor bone marrow and kidney cotransplantation.

Long-term, immunosuppression-free allograft survival has been induced in human and nonhuman primate (NHP) kidney recipients after nonmyeloablative conditioning and donor bone marrow transplantation (DBMT), resulting in transient mixed hematopoietic chimerism. However, the same strategy has consistently failed in NHP heart transplant recipients. Here, we investigated whether long-term heart allograft survival could be achieved by cotransplanting kidneys from the same donor. Cynomolgus monkeys were transplanted with heart allografts alone or heart and kidney allografts from the same major histocompatibility complex (MHC)-mismatched donors. All animals except one received DBMT, either at the same time or after a 2- to 4-month delay, plus short-term costimulation blockade and calcineurin inhibitor treatment. Long-term, immunosuppression-free heart allograft survival was consistently achieved in heart/kidney, but not heart-alone, recipients. This was not associated with greater donor/recipient histocompatibility or altered lymphoid cell reconstitution after conditioning. The maintenance of tolerance after heart/kidney transplantation was associated with the presence of forkhead box P3 (Foxp3+) regulatory T cell (Treg)-rich organized lymphoid structures in kidneys but not hearts. Substituting high-dose erythropoietin treatment for kidney transplantation was unsuccessful, suggesting that it was not the sole mechanism of action. RNA sequencing analysis revealed that gene expression in hearts from tolerant recipients closely resembled that in hearts from chronically immunosuppressed recipients but differed markedly from rejecting allografts and naïve hearts. A version of this protocol may be able to induce tolerance in patients with end-stage heart and kidney failure who require combined heart and kidney transplantation.

Feedback loop centered on MAF1 reduces blood–brain barrier damage in sepsis-associated encephalopathy

BackgroundA previous study found that MAF1 homolog, a negative regulator of RNA polymerase III (MAF1), protects the blood–brain barrier (BBB) in sepsis-associated encephalopathy (SAE); however, the related molecular mechanisms remain unclear.Subjects and methodsIn this study, a rat sepsis model was constructed using the cecum ligation and puncture (CLP) method. In vitro, rat brain microvascular endothelial cells and astrocytes were stimulated with serum from the sepsis model rats. The loss of MAF1 protein levels and the molecular mechanisms leading to cell damage were investigated.ResultsIt was shown in the SAE models that MAF1 was expressed at low levels. Knockdown of Cullin 2 (CUL2) stimulated the accumulation of MAF1 protein, attenuated the RNA sensor RIG-I/interferon regulatory factor 3 (IRF3) signaling pathway, and reduced cell apoptosis. Furthermore, it increased phosphatase and tensin homolog (PTEN) expression and inactivated the serine/threonine kinase (AKT)/mechanistic target of the rapamycin kinase (mTOR) signaling pathway. Interference with forkhead box O1 (FOXO1) inhibited MAF1 expression and activated the RIG-I/IRF3 signaling pathway, while MAF1 overexpression promoted PTEN expression, decreased cell apoptosis, and normalized autophagy.ConclusionsThese findings demonstrate that CUL2 promoted MAF1 ubiquitination and caused BBB injury in SAE. Through the regulatory loop of PTEN/AKT/FOXO1/MAF1, CUL2 initiated the gradual downregulation of MAF1, which subsequently regulated polymerase III (Pol III)-dependent transcription and played essential roles in cell apoptosis in SAE.Clinical trial number: not applicable.Graphical

Exploring Integrin α5β1 as a Potential Therapeutic Target for Pulmonary Arterial Hypertension: Insights From Comprehensive Multicenter Preclinical Studies.

Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PAs) and progressive increase in pulmonary vascular resistance leading to right ventricular failure. Although several drugs are approved for the treatment of PAH, mortality rates remain high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. The expression of the RGD (arginylglycylaspartic acid)-binding integrin α5β1 was assessed in PAs, PA smooth muscle cells, and PA endothelial cells from patients with PAH and controls using NanoString, immunoblotting, and Mesoscale Discovery assays. RNA sequencing was conducted to identify gene networks regulated by α5β1 inhibition in PAH PA smooth muscle cells. The therapeutic efficacy of α5β1 inhibition was evaluated using a novel small molecule inhibitor and selective neutralizing antibodies in Sugen/hypoxia and monocrotaline rat models, with validation by an external contract research organization. Comparisons were made against standard-of-care therapies (ie, macitentan, tadalafil) and sotatercept and efficacy was assessed using echocardiographic, hemodynamic, and histological assessments. Ex vivo studies using human precision-cut lung slices were performed to further assess the effects of α5β1 inhibition on pulmonary vascular remodeling. We found that the arginine-glycine-aspartate RGD-binding integrin α5β1 is upregulated in PA endothelial cells and PA smooth muscle cells from patients with PAH and remodeled PAs from animal models. Blockade of the integrin α5β1 or depletion of the α5 subunit downregulated FOXM1 (forkhead box protein M1)-regulated gene networks, resulting in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. We demonstrated that α5β1 integrin blockade safely attenuates pulmonary vascular remodeling and improves hemodynamics and right ventricular function and matched or exceeded the efficacy of standard of care and sotatercept in multiple preclinical models. Ex vivo studies further validated its potential in reversing advanced remodeling in human precision-cut lung slices. These findings establish α5β1 integrin as a pivotal driver of PAH pathology and we propose its inhibition as a novel, safe, and effective therapeutic strategy for PAH.

Evidence for Multiple Independent Expansions of Fox Gene Families Within Flatworms.

Expansion and losses of gene families are important drivers of molecular evolution. A recent survey of Fox genes in flatworms revealed that this superfamily of multifunctional transcription factors, present in all animals, underwent extensive losses and expansions during platyhelminth evolution. In this paper, I analyzed Fox gene complement in four additional species of platyhelminths, that represent early-branching lineages in the flatworm phylogeny: catenulids (Stenostomum brevipharyngium and Stenostomum leucops) and macrostomorphs (Macrostomum hystrix and Macrostomum cliftonense). Phylogenetic analysis of Fox genes from this expanded set of species provided evidence for multiple independent expansions of Fox gene families within flatworms. Notably, FoxG, a panbilaterian brain-patterning gene, appears to be the least susceptible to duplication, while FoxJ1, a conserved ciliogenesis factor, has undergone extensive expansion in various flatworm lineages. Analysis of the single-cell atlas of S. brevipharyngium, combined with RNA in situ hybridization, elucidated the tissue-specific expression of the selected Fox genes: FoxG is expressed in the brain, three of the Fox genes (FoxN2/3-2, FoxO4 and FoxP1) are expressed in the pharyngeal cells of likely glandular function, while one of the FoxQD paralogs is specifically expressed in the protonephridium. Overall, the evolution of Fox genes in flatworms appears to be characterized by an early contraction of the gene complement, followed by lineage-specific expansions that have enabled the co-option of newly evolved paralogs into novel physiological and developmental functions.