SMAD Family Member Research Articles

Clinicopathological relevance of SMAD4 and RUNX3 in patients with resected pancreatic cancer

Abstract Objectives Treating pancreatic ductal adenocarcinoma (PDAC) is a major clinical challenge, owing to its poor prognosis. Understanding the underlying molecular interactions may be crucial in determining PDAC intractability. We elucidated the combinatory role of SMAD family member 4 (SMAD4) and Runt-related transcription factor 3 (RUNX3) in PDAC. Methods Formalin-fixed and paraffin-embedded tissues were obtained from 101 patients who underwent surgical resection of PDAC. SMAD4 and RUNX3 expression in the tissues was evaluated using immunohistochemistry. SMAD4 and KRAS mutations were evaluated using Sanger sequencing. SMAD4 copy number variations were evaluated using quantitative real-time PCR. These molecular characteristics were compared with clinicopathological features of patients. Results Retained SMAD4 expression in PDAC tissues was associated with higher tumor, node, metastasis (TNM) stage, and metastatic recurrence. Defective RUNX3 expression was associated with higher TNM stages. Further analysis to determine significance of retained SMAD4 indicated that all these cases had defective RUNX3 expression; therefore, the combined expression status of retained SMAD4 and defective RUNX3 expression in tissues was determined to be associated with higher TNM stage and metastatic recurrence than defective SMAD4 expression with defective or retained RUNX3 expression. Survival analysis showed that patients with tumors with retained SMAD4 and defective RUNX3 expression had relatively worse overall survival (OS) rates. KRAS mutations were not associated with SMAD4/RUNX3 expression or OS. Conclusions Despite inherent limitations of this retrospective study and small sample size, our findings highlight the potential combined role of SMAD4 and RUNX3 in node metastasis and metastatic recurrence in patients with resected pancreatic cancer.

Increased susceptibility to diet-induced obesity in female mice impairs ovarian steroidogenesis: the role of elevated leptin signalling on Nodal activity inhibition in theca cells

ObjectiveSusceptibility to obesity in humans is driven by the intricate interplay of genetic, environmental and behavioural factors. Moreover, the mechanisms linking maternal obesity to infertility remain largely understudied. In this study, we investigated how variable susceptibility to obesity in mice affects ovarian steroidogenesis, with a particular focus on the leptin-mediated dysregulation of Nodal signalling pathway in theca cells (TC). MethodsC56BL/6J (B6) and 129S1/SvlmJ (129) mice, models of maternal obesity (MO), were fed a chow diet (CD) and a high fat diet (HFD) for 16 weeks. To investigate the contrasting effects of leptin on ovarian steroidogenesis, B6 mice pharmacologically treated with leptin for 16 days on CD were used to model hyperleptinemia, while homozygous ob/ob (-/-) mice with genetic leptin deficiency, also on a CD, were used to examine the effects of obesity in the absence of leptin. Following the characterisation of the mouse phenotype, gonadal fat (GON), whole ovaries (WO), ovarian TC and granulosa cell (GC) fractions were collected for mRNA transcription and protein expression analysis. Finally, in vitro treated ovarian explants obtained from B6 mice were used to further elucidate the effects of Nodal on steroidogenesis. ResultsThe significant gain in body weight (BW) and fat mass (FM) in HFD-fed B6 mice (p<0.05), was associated with increased mRNA transcription of the adipose tissue expansion genes Polymerase I and transcript release factor (Cavin), Secreted frizzled- related protein 5 (Sfrp5) and Mesoderm specific transcript (Mest) in GON (p<0.05). Furthermore, the HFD-fed B6 mice presented also impaired glucose metabolism and insulin sensitivity (p<0.05). In contrast, the HFD-fed 129 mice exhibited no changes in BW and FM, maintaining glucose and insulin metabolism. At the ovarian level, decreased protein expression of Steroidogenic Acute Regulatory Protein (StAR) in WO obtained from HFD-fed B6 mice (p=0.05), was followed by reduced transcription of key steroidogenic genes like Star and Cytochrome P450 17a1 (Cyp17a) in TC (p<0.05). Furthermore, the transcription of Nodal and its receptors was downregulated (p<0.05), whereas mRNA levels of Suppressor of cytokine signalling 3 (Socs3) and SMAD family member 7 (Smad7) were upregulated in TC obtained from HFD-fed B6 mice (p<0.05). No changes were seen in the genes regulating steroidogenesis, Nodal signalling, or Socs3 and Smad7 activity in the ovaries of HFD-fed 129 mice. Importantly, the pharmacological treatment of lean mice with leptin, upregulated the ovarian transcription of Socs3 and Smad7, while downregulating Nodal and its receptors (p<0.05). Finally, in vitro pharmacological inhibition of Nodal signalling pathway in ovarian explants isolated from CD-fed B6 mice decreased the transcription of Star and Cyp17a in TC (p<0.05), whereas Nodal treatment of explants obtained from HFD-fed B6 mice restored the transcription of both genes (p<0.05). ConclusionsIncreased susceptibility to obesity in MO is associated with systemic hyperleptinemia and hypoestrogenism due to compromised ovarian steroidogenesis, largely driven by the inhibitory effects of leptin-Smad7 pathway on Nodal signalling activity in the TC compartment of ovarian follicles.

Circ_0008035 Promotes Gastric Cancer Development via the miR-429/SMAD2 Cascade.

The vital roles of circular RNAs (circRNAs) in human tumorigenesis have attracted more attention. Circ_0008035 is one of the most up-regulated circRNAs in gastric cancer (GC). Herein, we explored the associated mechanism of circ_0008035 in GC. EdU incorporation experiments were performed to monitor cell proliferation ability. Cell cycle progression, apoptosis, angiogenesis, migration, and invasion were analyzed using flow cytometry, Tube formation, and Transwell assays respectively. Protein expression was detected by Western blot. Dual-luciferase reporter experiments were applied to demonstrate the relationship between circ_0008035 or SMAD family member 2 (SMAD2) and microRNA-429 (miR-429). Mouse xenograft assays were conducted for evaluation of the role of circ_0008035 in vivo. Circ_0008035 content was elevated in GC tissues (P < .0001) and cell lines (P < .001), and its deficiency hindered GC cell proliferation (P < .01), HUVEC angiogenesis (P < .05), and GC cell metastasis (P < .01) and triggered apoptosis (P < .01). Circ_0008035 could sponge miR-429 to up-regulate SMAD2 expression (P < .0001). Circ_0008035 absence restrained tumor growth in vivo (P < .01). MiR429 was a mediator of circ_0008035 function, and miR-429 hindered GC cell malignant phenotypes by SMAD2. Circ_0008035 aggravates GC cell malignant progression partially by targeting the miR-429/SMAD2 axis. Considering the inhibitory effect of circ_0008035 deficiency on GC progression, targeting circ_0008035 may be a potential approach to prevent or treat GC.

FTO-mediated SMAD2 m6A modification protects cartilage against Osteoarthritis

N6-methyladenosine (m6A) modification is one of the most prevalent forms of epigenetic modification and plays an important role in the development of degenerative diseases such as osteoarthritis (OA). However, the evidence concerning the role of m6A modification in OA is insufficient. Here, m6A modification was increased in human OA cartilage and degenerated chondrocytes. Among all of the m6A enzymes, the expression of the demethylase fat mass and obesity-associated protein (FTO) decreased dramatically. Conditional knockout of FTO in chondrocytes accelerates OA progression. FTO transcription is regulated by runt-related transcription factor-1 (RUNX1). Reduced FTO elevates m6A modification at the adenosine N6 position in SMAD family member 2 (SMAD2) mRNA, whose stability is subsequently modulated by the recruited m6A reader protein YTH N6-methyladenosine RNA binding protein F2 (YTHDF2). Collectively, these findings reveal the function and mechanism of the m6A family member FTO in OA progression. Therefore, reducing m6A modification to increase SMAD2 stability by activating FTO might be a potential therapeutic strategy for OA treatment.

Tinglu Yixin granule inhibited fibroblast-myofibroblast transdifferentiation to ameliorate myocardial fibrosis in diabetic mice

Ethnopharmacological relevanceMyocardial fibrosis is one of the pathological characteristics of advanced diabetic cardiomyopathy (DCM) and serves as the strong evidence of poor prognosis. Among them, the transdifferentiation of cardiac fibroblasts (CFs) may play a crucial role in the development of myocardial fibrosis in DCM. Tinglu Yixin granule (TLYXG) has been clinically used for many years and can significantly improve cardiac function of patients with DCM. However, the effect of TLYXG on myocardial fibrosis in DCM remains unknown, and the underlying mechanisms of its efficacy have yet to be fully understood. Aim of the studyThis study aimed to investigate the impact and underlying mechanism of TLYXG on myocardial fibrosis in diabetes mice. Materials and methodsThe bioactive compounds in TLYXG were identified using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). The potential mechanism of TLYXG in treating DCM was predicted using network pharmacology combined with molecular docking and protein-protein docking. The mice model of type 2 diabetes were established by intraperitoneal injection of streptozotocin (STZ) and the high-fat diet (HFD). Indicators of pancreatic islet function, lipids, oxidative stress, and inflammatory factors were tested using kits. Cardiac function was assessed in diabetic mice using echocardiography. Histologic staining was performed to evaluate myocardial hypertrophy and fibrosis. Mechanistically, the hypothesis was tested through rescue experiments. The expression levels of transient receptor potential channel 6 (TRPC6), transforming growth factor-β1 (TGF-β1), collagen I (COL-I) and alpha-smooth muscle actin (α-SMA), along with the mRNA and phosphorylation levels of SMAD family member 3 (Smad3) and protein 38 mitogen-activated protein kinase (p38 MAPK), were assessed using quantitative RT-qPCR, western blot, immunohistochemistry, and immunofluorescence. Neonatal lactating mice were used to extract primary CFs for vitro experiments. Scratch and transwell assays were conducted to assess CFs migration and invasion abilities. Western blot and immunofluorescence were used to evaluate the expression levels of CFs transdifferentiation markers COL-I and α-SMA. ResultsA total of 168 active ingredients were detected in TLYXG based on UPLC-MS and databases. Network pharmacology indicated that TLYXG could improve DCM through inflammatory mediator regulation of TRP channels, TGF-beta signaling pathway, and MAPK signaling pathway. ELISA results showed that TLYXG could ameliorate metabolic levels, inflammation, and oxidative stress in diabetic mice. Echocardiography suggested that TLYXG improved cardiac systolic and diastolic dysfunction in diabetic mice. Histological analysis revealed that TLYXG alleviated myocardial fibrosis in diabetes mice. Additionally, molecular docking analysis indicated strong binding activity between the main active ingredients of TLYXG and TRPC6 of the TRP family. At the molecular level, TLYXG reduced the mRNA and protein expression levels of TRPC6 and TGF-β1 and inhibited the mRNA and phosphorylation levels of Smad3 and p38 MAPK. Furthermore, TLYXG inhibited CFs migration and invasion, and reduced the expression levels of the CFs transdifferentiation markers COL-I and α-SMA. ConclusionTLYXG inhibited the proliferation, migration, invasion and transdifferentiation of CFs by suppressing TGF-β1/Smad3/p38 MAPK signaling through down-regulation of TRPC6, thereby ameliorating myocardial fibrosis in diabetes mice.

CircSMAD3 represses VSMC phenotype switching and neointima formation via promoting hnRNPA1 ubiquitination degradation.

Circular RNAs (circRNAs) are novel regulatory RNAs with high evolutionary conservation and stability, which makes them effective therapeutic agents for various vascular diseases. The SMAD family is a downstream mediator of the canonical transforming growth factor beta (TGF-β) signalling pathway and has been considered as a critical regulator in vascular injury. However, the role of circRNAs derived from the SMAD family members in vascular physiology remains unclear. In this study, we initially identified potential functional circRNAs originating from the SMAD family using integrated transcriptome screening. circSMAD3, derived from the SMAD3 gene, was identified to be significantly downregulated in vascular injury and atherosclerosis. Transcriptome analysis was conducted to comprehensively illustrate the pathways modulated by circRNAs. Functionally, circSMAD3 repressed vascular smooth muscle cell (VSMC) proliferation and phenotype switching in vitro evidenced by morphological assays, and ameliorated arterial injury-induced neointima formation in vivo. Mechanistically, circSMAD3 interacted with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) within the nucleus, enhanced its interaction with E3 ligase WD repeat domain 76 to promote hnRNPA1 ubiquitination degradation, facilitated p53 pre-RNA splicing, activated the p53γ signalling pathway, and finally suppressed VSMC proliferation and phenotype switching. Our study identifies circSMAD3 as a novel epigenetic regulator that suppresses VSMC proliferation and phenotype switching, thereby attenuating vascular remodelling and providing a new circRNA-based therapeutic strategy for cardiovascular diseases.

Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity

Obesity is a global issue that warrants the identification of more effective therapeutic targets and a better understanding of the pivotal molecular pathogenesis. Annexin A1 (ANXA1) is known to inhibit phospholipase A2, exhibiting anti-inflammatory activity. However, the specific effects of ANXA1 in obesity and the underlying mechanisms of action remain unclear. Our study reveals that ANXA1 levels are elevated in the adipose tissue of individuals with obesity. Whole-body or adipocyte-specific ANXA1 deletion aggravates obesity and metabolic disorders. ANXA1 levels are higher in stromal vascular fractions (SVFs) than in mature adipocytes. Further investigation into the role of ANXA1 in SVFs reveals that ANXA1 overexpression induces lower numbers of mature adipocytes, while ANXA1-knockout SVFs exhibit the opposite effect. This suggests that ANXA1 plays an important role in adipogenesis. Mechanistically, ANXA1 competes with MYC binding protein 2 (MYCBP2) for interaction with PDZ and LIM domain 7 (PDLIM7). This exposes the MYCBP2-binding site, allowing it to bind more readily to the SMAD family member 4 (SMAD4) and promoting its ubiquitination and degradation. SMAD4 degradation downregulates peroxisome proliferator-activated receptor gamma (PPARγ) transcription and reduces adipogenesis. Treatment with Ac2-26, an active peptide derived from ANXA1, inhibits both adipogenesis and obesity through the mechanism. In conclusion, the molecular mechanism of ANXA1 inhibiting adipogenesis was first uncovered in our study, which is a potential target for obesity prevention and treatment.

The phosphorylation of Smad3 by CaMKIIγ leads to the hepatocyte pyroptosis under perfluorooctane sulfonate exposure

Perfluorooctane sulfonate (PFOS) is a persistent organic pollutant and accumulated in the liver of mammals. PFOS exposure is closely associated with the development of pyroptosis. Nevertheless, the underlying mechanism is unclear. We found here that PFOS induced pyroptosis in the mice liver and L-02 cells as demonstrated by activation of the NOD-like receptor protein 3 inflammasome, gasdermin D cleavage and increased release of interleukin-1β and interleukin-18. The level of cytoplasmic calcium was accelerated in hepatocytes upon exposure to PFOS. The phosphorylated/activated form of calcium/calmodulin-dependent protein kinase II (CaMKII) was augmented by PFOS in vivo and in vitro. PFOS-induced pyroptosis was relieved by CaMKII inhibitor. Among various CaMKII subtypes, we identified that CaMKIIγ was activated specifically by PFOS. CaMKIIγ interacted with Smad family member 3 (Smad3) under PFOS exposure. PFOS increased the phosphorylation of Smad3, and CaMKII inhibitor or CaMKIIγ siRNA alleviated PFOS-caused phosphorylation of Smad3. Inhibiting Smad3 activity was found to alleviate PFOS-induced hepatocyte pyroptosis. This study puts forward that CaMKIIγ-Smad3 is the linkage between calcium homeostasis disturbance and pyroptosis, providing a mechanistic explanation for PFOS-induced pyroptosis.

Efficacy and Safety of KRASG12C Inhibitor IBI351 Monotherapy in Patients With Advanced NSCLC: Results From a Phase 2 Pivotal Study

IntroductionKRAS glycine-to-cysteine substitution at codon 12 (G12C) mutation is a well-recognized and increasingly promising therapeutic target with huge unmet clinical needs in NSCLC patients. IBI351 is a potent covalent and irreversible inhibitor of KRAS G12C. Here, we present the efficacy and safety of IBI351 from an open-label, single-arm, phase 2 pivotal study. MethodsEligible patients with NSCLC with KRAS G12C who failed standard therapy were enrolled. IBI351 was orally administered at a dose of 600 mg twice daily. The primary endpoint was confirmed objective response rate assessed by an independent radiological review committee (IRRC) as per Response Evaluation Criteria in Solid Tumors v1.1. Other endpoints were safety, IRRC-confirmed disease control rate, duration of response, progression-free survival (PFS), and overall survival. ResultsAs of December 13, 2023, 116 patients were enrolled (Eastern Cooperative Oncology Group Performance Status 1: 91.4%; brain metastasis: 30.2%; prior treatments with both anti-PD-1 or anti-PD-L1 inhibitors and platinum-based chemotherapy: 84.5%). As per the IRRC assessment, the confirmed objective response rate was 49.1% (95% confidence interval [CI]: 39.7–58.6), and the disease control rate was 90.5% (95% CI: 83.7–95.2). The median duration of response was not reached whereas disease progression or death events occurred in 22 patients (38.6%), and the median PFS was 9.7 months (95% CI: 5.6–11.0). overall survival data was immature. Treatment-related adverse events (TRAEs) occurred in 107 patients (92.2%) whereas 48 patients (41.4%) had equal to or higher than grade three TRAEs. Common TRAEs were anemia (44.8%), increased alanine aminotransferase (28.4%), increased aspartate aminotransferase (27.6%), asthenia (26.7%) and presence of protein in urine (25.0%). TRAEs leading to treatment discontinuation occurred in nine patients (7.8%). In biomarker evaluable patients (n = 95), all patients had positive KRAS G12C in tissue whereas 72 patients were blood-positive and 23 were blood-negative for KRAS G12C. Patients with KRAS G12C in both blood and tissue had higher tumor burden at baseline (p < 0.05) and worse PFS (p < 0.05). Tumor mutation profiling identified tumor protein p53 (45.3%), serine/threonine kinase 11 (STK11) (30.5%), and kelch-like ECH-associated protein 1 (21.1%) as the most common genes co-mutated with KRAS G12C. Among 13 genes with mutation frequency equal to or higher than 5%, mutations of six genes (STK11, kelch-like ECH-associated protein 1, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma, DNA polymerase epsilon, SMAD family member 4, and BMP/retinoic acid-inducible neural-specific protein 3) were significantly associated with worse PFS (p < 0.05). Mutation in STK11 was also found to have a significant association with higher tumor burden at baseline and lower response rate (p < 0.05). ConclusionsIBI351 monotherapy demonstrated promising and sustained efficacy with manageable safety, supporting its potential as a new treatment option for KRAS G12C-mutant NSCLC.

Suppression of NUPR1 in fibroblast-like synoviocytes reduces synovial fibrosis via the Smad3 pathway

BackgroundSynovial fibrosis is a common complication of knee osteoarthritis (KOA), a pathological process characterized by myofibroblast activation and excessive extracellular matrix (ECM) deposition. Fibroblast-like synoviocytes (FLSs) are implicated in KOA pathogenesis, contributing to synovial fibrosis through diverse mechanisms. Nuclear protein 1 (NUPR1) is a recently identified transcription factor with crucial roles in various fibrotic diseases. However, its molecular determinants in KOA synovial fibrosis remain unknown. This study aims to investigate the role of NUPR1 in KOA synovial fibrosis through in vivo and in vitro experiments.MethodsWe examined NUPR1 expression in the murine synovium and determined the impact of NUPR1 on synovial fibrosis by knockdown models in the destabilization of the medial meniscus (DMM)-induced KOA mouse model. TGF-β was employed to induce fibrotic response and myofibroblast activation in mouse FLSs, and the role and molecular mechanisms in synovial fibrosis were evaluated under conditions of NUPR1 downexpression. Additionally, the pharmacological effect of NUPR1 inhibitor in synovial fibrosis was assessed using a surgically induced mouse KOA model.ResultsWe found that NUPR1 expression increased in the murine synovium after DMM surgical operation. The adeno-associated virus (AAV)-NUPR1 shRNA promoted NUPR1 deficiency, attenuating synovial fibrosis, inhibiting synovial hyperplasia, and significantly reducing the expression of pro-fibrotic molecules. Moreover, the lentivirus-mediated NUPR1 deficiency alleviated synoviocyte proliferation and inhibited fibroblast to myofibroblast transition. It also decreased the expression of fibrosis markers α-SMA, COL1A1, CTGF, Vimentin and promoted the activation of the SMAD family member 3 (SMAD3) pathway. Importantly, trifluoperazine (TFP), a NUPR1 inhibitor, attenuated synovial fibrosis in DMM mice.ConclusionsThese findings indicate that NUPR1 is an antifibrotic modulator in KOA, and its effect on anti-synovial fibrosis is partially mediated by SMAD3 signaling. This study reveals a promising target for developing novel antifibrotic treatment.

Primary cilia mediate skeletogenic BMP and Hedgehog signaling in heterotopic ossification.

Heterotopic ossification (HO), defined as the formation of extraskeletal bone in muscle and soft tissues, is a diverse pathological process caused by either genetic mutations or inciting trauma. Fibrodysplasia ossificans progressiva (FOP) is a genetic form of HO caused by mutations in the bone morphogenetic protein (BMP) type I receptor gene activin A receptor type 1 (ACVR1). These mutations make ACVR1 hypersensitive to BMP and responsive to activin A. Hedgehog (Hh) signaling also contributes to HO development. However, the exact pathophysiology of how skeletogenic cells contribute to endochondral ossification in FOP remains unknown. Here, we showed that the wild-type or FOP-mutant ACVR1 localized in the cilia of stem cells from human exfoliated deciduous teeth with key FOP signaling components, including activin A receptor type 2A/2B, SMAD family member 1/5, and FK506-binding protein 12kD. Cilia suppression by deletion of intraflagellar transport 88 or ADP ribosylation factor like GTPase 3 effectively inhibited pathological BMP and Hh signaling, subdued aberrant chondro-osteogenic differentiation in primary mouse or human FOP cells, and diminished in vivo extraskeletal ossification in Acvr1Q207D, Sox2-Cre; Acvr1R206H/+ FOP mice and in burn tenotomy-treated wild-type mice. Our results provide a rationale for early and localized suppression of cilia in affected tissues after injury as a therapeutic strategy against either genetic or acquired HO.

Novel Circular RNA CircSLC2A13 Regulates Chicken Muscle Development by Sponging MiR-34a-3p.

The skeletal muscle is the major muscle tissue in animals, and its production is subject to a complex and strict regulation. The proliferation and differentiation of myoblasts are important factors determining chicken muscle development. Circular RNAs (circRNAs) are endogenous RNAs that are widely present in various tissues of organisms. Recent studies have shown that circRNA plays key roles in the development of skeletal muscles. The solute carrier (SLC) family functions in the transport of metabolites such as amino acids, glucose, nucleotides, and essential nutrients and is widely involved in various basic physiological metabolic processes within the body. In this study, we have cloned a novel chicken circular RNA circSLC2A13 generated from the solute carrier family 2 member 13 gene (SLC2A13). Also, circSLC2A1 was confirmed by sequencing verification, RNase R treatment, and reverse transcription analysis. Currently, our results show that circSLC2A13 promoted the proliferation and differentiation of chicken myoblasts. The double luciferase reporter system revealed that circSLC2A13 regulated the proliferation and differentiation of myoblasts by competitive binding with miR-34a-3p. In addition, results indicated that circSLC2A13 acts as a miR-34a-3p sponge to relieve its inhibitory effect on the target SMAD3 gene. In summary, this study found that chicken circSLC2A13 can bind to miR-34a-3p and weaken its inhibitory effect on the SMAD family member 3 gene (SMAD3), thereby promoting the proliferation and differentiation of myoblasts. This study laid foundations for broiler industry and muscle development research.

O-037 An update on the genetics of dizygotic twinning

Abstract Study question Which genes are involved in female fertility defined as giving birth to naturally conceived dizygotic (DZ) twins? Summary answer In 2016 we identified FSHB and SMAD3 as first loci for having DZ twins. We now report four additional loci: GNRH1, FSHR, ZFPM1 and IPO8. What is known already The propensity to give birth to DZ twins differs around the globe, is positively associated with body size and smoking, and runs in families. Heritability estimates tend to be low. Study design, size, duration We conducted a genome-wide association meta-analysis (GWAMA) of mothers of naturally conceived dizygotic (DZ) twins (8,265 cases, 264,567 controls) and of independent DZ twin offspring (26,252 cases, 417,433 controls). Participants were carefully screened to exclude twins born after use of Medically Assisted Reproduction (MAR) technologies. Participants/materials, setting, methods Over 700.000 participants from Australia, New Zealand, Europe and USA were included. Genetic association analyses by cohort for being a mother of DZ twins and being a DZ twin were combined by fixed-effects inverse variance methods as implemented in METAL software meta-analysis. To predict the most likely effector gene in a locus, we applied the new FLAMES framework which integrates machine learning with biological data linking SNPs to genes, and GWAS-wide convergence of gene interactions. Main results and the role of chance By enlarging sample size from previous efforts, both by including new cohorts and by combining through meta-analyses with independent DZ offspring, we identified 4 genome-wide significant loci, in addition to FSHB (follicle stimulating hormone subunit beta ) and SMAD3 (SMAD family member 3). Two novel loci GNRH1 (gonadotropin releasing hormone 1) and FSHR (follicle stimulating hormone receptor) have well-established roles in female reproduction. The 2 further novel genes were implicated by gene level enrichment analyses. Both ZFPM1 (Zinc Finger Protein, FOG Family Member 1) and IPO8 (Importin 8) have not previously been implicated. Significant genetic correlations were found with multiple aspects of female reproduction and body size as well as with a recent GWAMA of female infertility. The 26 top SNPs from our GWAMA in European-origin participants predicted the crude twinning rates in 47 non-European populations (r = 0.23 between risk score and population prevalence, p=.058) indicating that GWAS are needed in African and Asian populations to explore the causes of their respectively high and low DZ twinning rates. A Phenome-wide association study (PheWAS) based on the GWAMA, linked SNPs predisposing to DZT with reduced risk of both ovarian dysfunction and polycystic ovaries. Limitations, reasons for caution So far, our studies only included European ancestry cohorts. Inclusion of data from Africa (the highest world-wide DZ twining rates are observed in Western Africa) and Asia (lowest DZ twinning rate) will further illuminate the etiology of twinning and female fertility. Wider implications of the findings About one in 40 babies born in the world is a twin and there is still much unknown why they run in families. We hope our results will inform investigations of female fertility and infertility. Trial registration number not applicable

SIRT6 Inhibits the Proliferation and Collagen Synthesis of Keloid Fibroblasts through MAPK/ERK Pathway.

Keloid, a fibroproliferative disorder, significantly impacts patients' quality of life, yet effective therapies remain elusive. This study explored the role of silent information regulator 6 (SIRT6) in modulating the proliferation, invasion, and collagen synthesis of keloid fibroblasts. Keloid and normal skin specimens were collected, and fibroblasts were isolated from the keloid tissue. SIRT6 recombinant adenovirus (Ad) was constructed to infect keloid fibroblasts to overexpress SIRT6. This study entails three groups: Control group, adenovirus-Negative Control (Ad-NC) group, and Ad-SIRT6 group. SIRT6 protein and mRNA levels were measured via Western blotting and Quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. Cell viability was determined using 5-ethynyl-2'-deoxyuridine (EdU) assay. Flow cytometry was exploited to measure cell apoptosis. To investigate cell migration, wound healing assay and Transwell assay were employed. Western blotting was also utilized to study the expression levels of apoptotic proteins, collagen deposition-related proteins, and Mitogen-Activated Protein Kinases (MAPK)/extracellular regulated protein kinases (ERK) pathway-related proteins. Compared to the control and Ad-NC groups, the Ad-SIRT6 group exhibited significantly elevated SIRT6 level; diminished cell proliferation, migration and invasion; reduced protein levels of α-smooth muscle actin (α-SMA), collagen I, collagen III, phospho SMAD Family Member 3 (p-Smad3), transforming growth factor-β 1 (TGF-β1), and MAPK/ERK pathway proteins (phospho extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phospho MAP kinase-ERK kinase (p-MEK) and phospho-c-Raf (p-c-Raf)). Treatment with epidermal growth factor (EGF), an MAPK/ERK pathway agonists, reversed the inhibitory effect of SIRT6 on cell activity and inhibited apoptosis in keloid fibroblasts. SIRT6 overexpression in keloid fibroblasts attenuates proliferation, invasion, and collagen synthesis, while fostering apoptosis, likely through the suppression of MAPK/ERK pathway activity. This suggests a potential therapeutic target for keloid treatment.

Naringin inhibits cisplatin resistance of ovarian cancer cells by inhibiting autophagy mediated by the TGF-β2/smad2 pathway.

Resistance to cisplatin (DDP) in patients with ovarian cancer (OC) poses a great challenge to improving the quality of life of patients. Past reports have revealed that naringin can induce apoptosis of OC cells and delay the occurrence of drug resistance in OC cells. However, the molecular role by which naringin inhibits DDP resistance in OC has not been definitively proven by researchers. The objective of this study is to investigate the effect of naringin on DDP resistance in OC cells and the specific mechanism of naringin mediating autophagy. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were selected to evaluate the role of naringin or DDP on the proliferation and apoptosis of human OC cells (SKOV3/A2780). The protein levels of Sequestosome 1 (SQSTM1/p62, hereinafter referred to as p62), microtubule-associated protein 1 light chain 3 (LC3), transforming growth factor-β2 (TGF-β2) and SMAD family member 2 (smad2) were detected with Western blotting assay. Immunofluorescence assay was also used to evaluate the level of LC3 in different groups of cells. Besides, functional analyses were performed in vivo. Naringin was shown to promote DDP sensitivity and apoptosis of human OC DDP-resistant cell line (SKOV3/A2780-DDP cells). Significantly increased p62 expression and reduced LC3 expression were found in naringin-treated cells. The autophagy agonist, rapamycin, reversed the effect of naringin on the resistance of SKOV3-DDP cells to DDP. Naringin inhibited levels of TGF-β2/smad2 pathway-related proteins, and regulated autophagy in SKOV3-DDP cells. In vivo experiments demonstrated that injection of naringin inhibited DDP resistance and autophagy in mice xenograft model. In summary, naringin inhibits DDP resistance in OC cells by inhibiting autophagy mediated by the TGF-β2/smad2 pathway.

MiR-21/SMAD2 Is Involved in the Decrease in Progesterone Synthesis Caused by Lipopolysaccharide Exposure in Follicular Granulosa Cells of Laying Goose.

Lipopolysaccharide (LPS) is one of the important pathogenic substances of E. coli and Salmonella, which causes injury to the reproductive system. Ovarian dysfunction due to Gram-negative bacterial infections is a major cause of reduced reproductive performance in geese. However, the specific molecular mechanisms of LPS-induced impairment of sex steroid hormone synthesis have not been determined. The regulatory mechanism of miRNA has been proposed in many physiological and pathogenic mechanisms. Therefore, the role of miRNA in breeding geese exposed to LPS during the peak laying period was investigated. In this study, twenty Yangzhou geese at peak laying period were injected with LPS for 0 h, 24 h, and 36 h. The follicular granulosa layer was taken for RNA-seq and analyzed for differentially expressed miRNAs. It was observed that LPS changed the appearance of hierarchical follicles. miRNA sequencing analysis was applied, and miR-21 and SMAD2 (SMAD family member 2) were selected from 51 differentially expressed miRNAs through bioinformatics prediction. The results showed that miR-21 down-regulated SMAD2 expression and progesterone (P4) production in LPS-treated goose granulosa cells (GCs). It also determined that overexpression of miR-21 or silence of SMAD2 suppressed the sex steroid biosynthesis pathway by decreasing STAR and CYP11A1 expression. Down-regulation of miR-21 exacerbates the LPS-induced decline in P4 synthesis and vice versa. The findings indicated that miR-21 was involved in LPS regulation of P4 synthesis in goose granulosa cells by down-regulating SMAD2. This study provides theoretical support for the prevention of LPS-induced ovarian dysfunction in geese.

The human SMAD9 (GCC) repeat links to natural selection and late-onset neurocognitive disorders.

Whereas (GCC)-repeats are overrepresented in genic regions, and mutation hotspots, they are largely unexplored with regard to their link with natural selection. Across numerous primate species and tissues, SMAD9 (SMAD Family Member 9) reaches highest level of expression in the human brain. This gene contains a (GCC)-repeat in the interval between + 1 and + 60 of the transcription start site, which is in the high-ranking (GCC)-repeats with respect to length. Here we sequenced this (GCC)-repeat in 396 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N = 181) and controls (N = 215). We detected two predominantly abundant alleles of 7 and 9 repeats, forming 96.2% of the allele pool. The (GCC)7/(GCC)9 ratio was in the reverse order in the NCD group versus controls (p = 0.005), resulting from excess of (GCC)7 in the NCD group (p = 0.003) and (GCC)9 in the controls (p = 0.01). Five genotypes, predominantly consisting of (GCC)7 and lacking (GCC)9 were detected in the NCD group only (p = 0.008). The patients harboring those genotypes received the diagnoses of Alzheimer's disease (AD) and vascular dementia (VD). Five genotypes consisting of (GCC)9 and lacking (GCC)7 were detected in the control group only (p = 0.002). The group-specific genotypes formed approximately 4% of the genotype pool in the human samples studied. We propose natural selection and a novel locus for late-onset AD and VD at the SMAD9 (GCC)-repeat in humans.

Characteristics and Functions of IL-37

Interleukin-37 (IL-37), previously known as IL-1F7, is a member of the IL-1 family of cytokines. There are five basic subtypes of IL-37, including IL-37a, IL-37b, IL-37c, IL-37d, and IL-37e. Like other members of the IL-1 family, IL-37 is initially expressed as an immature precursor protein that needs to be processed enzymatically by caspase-1 to generate the bioactive protein. However, unlike most other members of the IL-1 family, IL-37 induces anti-inflammatory activities in IL-37 receptor-positive target cells. IL-37 functions as an extracellular protein by binding to the IL-18 receptor, IL-18R, and an intracellular protein via its interaction with SMAD family member-3 (SMAD-3). This article reviews recent findings regarding the IL-37 protein maturation process and the biological functions mediated by this cytokine.

Evaluation of the maternal systemic immune system during frozen euploid embryo transfer according to cycle outcome

Infertility affects 15 % of couples in the US, and many turn to assisted reproductive technologies, including in vitro fertilization and subsequent frozen embryo transfer (FET) to become pregnant. This study aimed to perform a broad assessment of the maternal immune system to determine if there are systemic differences on the day of FET in cycles that result in a live birth compared to those that do not. Women undergoing FET of euploid embryos were recruited and blood was collected on the day of FET as well as at early timepoints in pregnancy. Sixty immune and angiogenic proteins were measured in plasma, and gene expression of 92 immune-response related genes were evaluated in peripheral blood mononuclear cells (PBMCs). We found plasma concentrations of interleukin-13 (IL-13) and macrophage derived chemokine (MDC) were significantly lower on the day of FET in cycles that resulted in a live birth. We also found genes encoding C-C chemokine receptor type 5 (CCR5), CD8 subunit alpha (CD8A) and SMAD family member 3 (SMAD3) were upregulated in PBMCs on the day of FET in cycles that resulted in live birth. Measurements of immune mediators from maternal blood could serve as prognostic markers during FET to guide clinical decision making and further our understanding of implantation failure.

Qiangxinyin formula protects against isoproterenol-induced cardiac hypertrophy

Heart failure is a life-threatening cardiovascular disease and characterized by cardiac hypertrophy, inflammation and fibrosis. The traditional Chinese medicine formula Qiangxinyin (QXY) is effective for the treatment of heart failure while the underlying mechanism is not clear. This study aims to identify the active ingredients of QXY and explore its mechanisms protecting against cardiac hypertrophy. We found that QXY significantly protected against isoproterenol (ISO)-induced cardiac hypertrophy and dysfunction in zebrafish. Eight compounds, including benzoylmesaconine (BMA), atractylenolide I (ATL I), icariin (ICA), quercitrin (QUE), psoralen (PRN), kaempferol (KMP), ferulic acid (FA) and protocatechuic acid (PCA) were identified from QXY. PRN, KMP and icaritin (ICT), an active pharmaceutical ingredient of ICA, prevented ISO-induced cardiac hypertrophy and dysfunction in zebrafish. In H9c2 cardiomyocyte treated with ISO, QXY significantly blocked the calcium influx, reduced intracellular lipid peroxidative product MDA, stimulated ATP production and increased mitochondrial membrane potential. QXY also inhibited ISO-induced cardiomyocyte hypertrophy and cytoskeleton reorganization. Mechanistically, QXY enhanced the phosphorylation of Smad family member 2 (SMAD2) and myosin phosphatase target subunit-1 (MYPT1), and suppressed the phosphorylation of myosin light chain (MLC). In conclusion, PRN, KMP and ICA are the main active ingredients of QXY that protect against ISO-induced cardiac hypertrophy and dysfunction largely via the blockage of calcium influx and inhibition of mitochondrial dysfunction as well as cytoskeleton reorganization.