Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

Zebrafish Mbd5 binds to RNA m5C and regulates histone deubiquitylation and gene expression in development metabolism and behavior.

Complex biological processes are regulated by both genetic and epigenetic programs. One class of epigenetic modifications is methylation. Evolutionarily conserved methyl-CpG-binding domain (MBD)-containing proteins are known as readers of DNA methylation. MBD5 is linked to multiple human diseases but its mechanism of action remains unclear. Here we report that the zebrafish Mbd5 does not bind to methylated DNA; but rather, it directly binds to 5-methylcytosine (m5C)-modified mRNAs and regulates embryonic development, erythrocyte differentiation, iron metabolism, and behavior. We further show that Mbd5 facilitates removal of the monoubiquitin mark at histone H2A-K119 through an interaction with the Polycomb repressive deubiquitinase (PR-DUB) complex in vivo. The direct target genes of Mbd5 are enriched with both RNA m5C and H2A-K119 ubiquitylation signals. Together, we propose that zebrafish MBD5 is an RNA m5C reader that potentially links RNA methylation to histone modification and in turn transcription regulation in vivo.

Systematical Evaluation of the Structure-Cardiotoxicity Relationship of 7-Azaindazole-based PI3K Inhibitors Designed by Bioisosteric Approach.

A growing concern of cardiotoxicity induced by PI3K inhibitors has raised the requirements to evaluate the structure-cardiotoxicity relationship (SCR) in the development process of novel inhibitors. Based on three bioisosteric 7-azaindazole-based candidate inhibitors namely FD269, FD268 and FD274 that give same order of inhibitory concentration 50% (IC50) magnitude against PI3Ks, in this work, we proposed to systematically evaluate the SCR of 7-azaindazole-based PI3K inhibitors designed by bioisosteric approach. The 24-h lethal concentrations 50% (LC50) of FD269, FD268 and FD274 against zebrafish embryos were 0.35, 4.82 and above 50 µM (not detected), respectively. Determination of the heart rate, pericardial and yolk-sac areas and vascular malformation confirmed the remarkable reduction in the cardiotoxicity of from FD269 to FD268 and to FD274. The IC50s of all three compounds against the hERG channel were tested on the CHO cell line that constitutively expressing hERG channel, which were all higher than 20 µM. The transcriptomic analysis revealed that FD269 and FD268 induced the up-regulation of noxo1b, which encodes a subunit of an NADPH oxidase evoking the oxidative stress. Furthermore, immunohistochemistry tests confirmed the structure-dependent attenuation of the overproduction of ROS and cardiac apoptosis. Our results verified the feasibility of bioisosteric replacement to attenuate the cardiotoxicity of 7-azaindazole-based PI3K inhibitors, suggesting that the screening for PI3K inhibitors with both high potency and low cardiotoxicity from bioisosteres would be a beneficial trial.

Quartet RNA reference materials improve the quality of transcriptomic data through ratio-based profiling.

Certified RNA reference materials are indispensable for assessing the reliability of RNA sequencing to detect intrinsically small biological differences in clinical settings, such as molecular subtyping of diseases. As part of the Quartet Project for quality control and data integration of multi-omics profiling, we established four RNA reference materials derived from immortalized B-lymphoblastoid cell lines from four members of a monozygotic twin family. Additionally, we constructed ratio-based transcriptome-wide reference datasets between two samples, providing cross-platform and cross-laboratory 'ground truth'. Investigation of the intrinsically subtle biological differences among the Quartet samples enables sensitive assessment of cross-batch integration of transcriptomic measurements at the ratio level. The Quartet RNA reference materials, combined with the ratio-based reference datasets, can serve as unique resources for assessing and improving the quality of transcriptomic data in clinical and biological settings.

Application of modern computer technology in classical genetics lab course--Development of a mobile, lightweight and high-precision batch identification system for genetic traits of Drosophila.

Drosophila is a crucial biological experimental teaching material extensively utilized in experimental teaching. In this experimental teaching, each student typically needs to manually identify hundreds of fruit flies and record multiple of each fly. This task involves substantial workload, and the classification standards can be inconsistent. To address this issue, we introduce a deep convolutional neural network that classifies the traits of every fruit fly, using a two-stage consisting of an object detector and a trait classifier. We propose a keypoint-assisted classification model with tailored training session for the trait classification task and significantly enhanced the model interpretability. Additionally, we've enhanced the RandAugment method to better fit the features of our task. The model is trained with progressive learning and adaptive regularization under limited computational resources. The final classification model, which utilizes MobileNetV3 as backbone, achieves an accuracy of 97.5%, 97.5% and 98% for the eyes, wings, gender tasks, respectively. After optimization, the model is highly lightweight, classifying 600 fruit fly traits from raw images in 10 seconds and having a size less than 5 MB. It can be easily deployed on any android device. The development of this system is conducive to promoting the experimental teaching, such as verifying genetic laws with Drosophila as the research object. It can also be used for scientific research involving a large number of Drosophila classifications, statistics and analyses.

Non-invasive brain stimulation for limb motor function and daily living activity improvement in acute stroke: A meta-analysis.

OBJECTIVE: To evaluate the effect of non-invasive brain stimulation (NIBS) in improving limb motor dysfunction and daily living activity during at the phase of acute stroke. MATERIALS AND METHODS: Randomized controlled trials about the effect of NIBS on hemiparesis in acute stroke were retrieved from databases of China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP), Wanfang Data, CBM, PubMed, Embase, Cochrane Library, and Web of Science from inception until January 3rd 2022. The quality of the trials was assessed, and the data were extracted according to the Cochrane Handbook for Systematic Reviews of Interventions. A statistical analysis was carried out using Review Manager 5.3 and STATA 14. The effect size was evaluated by using the weighed mean difference (WMD) and a 95% confidence interval (CI). The stability and sensitivity of the results and sources of heterogeneity were also analyzed. RESULTS: 12 studies involving 639 patients were included. Our meta-analysis showed that NIBS could improve the Fugl-Meyer Assessment (weighed mean difference = 3.96, 95% confidence interval = 3.45 to 4.48) and Barthel Index (weighed mean difference = 12.29, 95% confidence interval = 4.93 to 19.66), while reducing the National Institutes of Health Stroke Scale (weighed mean difference = -2.37, 95% confidence interval = -3.43 to -1.31). CONCLUSION: NIBS is effective in improving paretic limb motor function and activities of daily living in patients during at the phase of acute stroke.

Novel DNA nanoflower biosensing technologies towards next-generation molecular diagnostics.

DNA nanoflowers (DNFs) are topological flower-like nanostructures based on ultralong-strand DNA and inorganic metal-ion frameworks. Because of their programmability, biocompatibility, and controllable assembly size for specific responses to molecular recognition stimuli, DNFs are powerful biosensing tools for detecting biomolecules. Here, we review the current state of DNF-based biosensing strategies for in vivo and in vitro detection, with a view of how the field has evolved towards molecular diagnostics. We also provide a detailed classification of DNF-based biosensing strategies and propose their future utility. Particularly as transduction elements, DNFs can accelerate biosensing engineering by signal amplification. Finally, we discuss the key challenges and further prospects of DNF-based biosensing technologies in developing applications of a broader scope.

Autism-Risk Gene necab2 Regulates Psychomotor and Social Behavior as a Neuronal Modulator of mGluR1 Signaling.

Background: De novo deletion of the neuronal calcium-binding protein 2 (NECAB2) locus is associated with idiopathic autism spectrum disorders (ASDs). The in vivo function of NECAB2 in the brain remains largely elusive. Methods: We investigated the morphological and behavioral profiles of both necab2 knock-out and overexpression zebrafish models. The expression pattern and molecular role of necab2 were probed through a combination of in vitro and in vivo assays. Results: We show that Necab2 is a neuronal specific, cytoplasmic, and membrane-associated protein, abundantly expressed in the telencephalon, habenula, and cerebellum. Necab2 is distributed peri-synaptically in subsets of glutamatergic and GABAergic neurons. CRISPR/Cas9-generated necab2 knock-out zebrafish display normal morphology but exhibit a decrease in locomotor activity and thigmotaxis with impaired social interaction only in males. Conversely, necab2 overexpression yields behavioral phenotypes opposite to the loss-of-function. Proteomic profiling uncovers a role of Necab2 in modulating signal transduction of G-protein coupled receptors. Specifically, co-immunoprecipitation, immunofluorescence, and confocal live-cell imaging suggest a complex containing NECAB2 and the metabotropic glutamate receptor 1 (mGluR1). In vivo measurement of phosphatidylinositol 4,5-bisphosphate further substantiates that Necab2 promotes mGluR1 signaling. Conclusions: Necab2 regulates psychomotor and social behavior via modulating a signaling cascade downstream of mGluR1.

A Course-Based Undergraduate Research Experience Improves Outcomes in Mentored Research.

Infusing undergraduate curricula with authentic research training is an important contemporary challenge. Such exposure typically occurs through mentored research (MR) or course-based undergraduate research experiences (CUREs). In Asian contexts, CURE implementation is rare, while MR is often a graduation requirement. In this study, mentor interviews and mentee focus groups were used to characterize the learning challenges associated with this requirement at a Chinese university. An intensive 6-week CURE was then implemented as an MR preparatory program to help mitigate the identified challenges. This program contained seven site-specific features not typically included in other CUREs, each designed to improve different aspects of student readiness for MR. Post-CURE surveys, focus groups, and interviews demonstrated CURE enrollment significantly improved subsequent MR outcomes. Almost 90% of all enrollees, for example, began their first MR experience in their second year, more than twice the rate of non-enrollees. Enrollees also reported greater confidence in their research skills and more frequent experiences working in multiple labs. This study reports both immediate CURE and downstream MR outcomes, using the former to help explain the latter. A comprehensive CURE implementation process is described, offering a potential model for the design of other programs with similar research enhancement goals.

Anorectal dysfunction in patients with mid-low rectal cancer after surgery: A pilot study with three-dimensional high-resolution manometry.

BACKGROUND: The quality of life in patients who develop low anterior resection syndrome (LARS) after surgery for mid-low rectal cancer is seriously impaired. The underlying pathophysiological mechanism of LARS has not been fully investigated. AIM: To assess anorectal function of mid-low rectal cancer patients developing LARS perioperatively. METHODS: Patients diagnosed with mid-low rectal cancer were included. The LARS score was used to evaluate defecation symptoms 3 and 6 mo after anterior resection or a stoma reversal procedure. Anorectal functions were assessed by three-dimensional high resolution anorectal manometry preoperatively and 3-6 mo after surgery. RESULTS: The study population consisted of 24 patients. The total LARS score was decreased at 6 mo compared with 3 mo after surgery (P < 0.05), but 58.3% (14/24) lasted as major LARS at 6 mo after surgery. The length of the high-pressure zone of the anal sphincter was significantly shorter, the mean resting pressure and maximal squeeze pressure of the anus were significantly lower than those before surgery in all patients (P < 0.05), especially in the neoadjuvant therapy group after surgery (n = 18). The focal pressure defects of the anal canal were detected in 70.8% of patients, and those patients had higher LARS scores at 3 mo postoperatively than those without focal pressure defects (P < 0.05). Spastic peristaltic contractions from the new rectum to anus were detected in 45.8% of patients, which were associated with a higher LARS score at 3 mo postoperatively (P < 0.05). CONCLUSION: The LARS score decreases over time after surgery in the majority of patients with mid-low rectal cancer. Anorectal dysfunctions, especially focal pressure defects of the anal canal and spastic peristaltic contractions from the new rectum to anus postoperatively, might be the major pathophysiological mechanisms of LARS.

Selective effects of exercise on reactive and proactive inhibition in Parkinson's disease.

Objective: Patients with Parkinson's disease (PD) have an obvious motor inhibition disorder, which is closely related to their motor symptoms. Although previous studies have shown that exercise can improve their inhibition deficits, the effect of exercise on different types of inhibition (proactive and reactive inhibition) has not been addressed. Methods: We used a behavioral paradigm combined with a series of questionnaires to explore the effect of long-term exercise on different types of motor inhibition in 59 patients with PD aged 55-75 years. According to the intensity and frequency of exercise, the participants were divided into regular-exercise and no-exercise groups. To obtain the average reference value for inhibition ability at the same age, we also recruited 30 healthy elderly people as controls. Results: The main defect in the motor inhibition of PD is reactive inhibition, while proactive inhibition has no obvious differences compared with healthy controls. Additionally, compared with the non-exercise group, PD in the exercise group showed significantly better reaction speeds and reactive control ability, fewer motor symptoms and negative emotions. Conclusions: Taken together, the motor inhibition defects of patients with PD affect only reactive inhibition. In addition, PD with exercise reported fewer negative emotions than that of the non-exercise group, indicating that exercise can relieve negative emotions and improve behavioral symptoms and quality of life in PD to a certain extent. We demonstrate for the first time that exercise has and can improve reactive inhibition in PD patients and has no effect on proactive inhibition.

Screening models combining maternal characteristics and multiple markers for the early prediction of preeclampsia in pregnancy: a nested case-control study.

To identify maternal laboratory markers to predict the risk of preeclampsia (PE) in different stages of pregnancy, we analysed 67, 25, and 73, pregnancies developing PE at 11-13, 16-20, and 24-28 wks, respectively. Routine laboratory markers were measured in whole blood or serum and binary logistic regression analysis was used to identify predictive models. At 11-13 wks of gestation, patients who went on to develop PE showed significantly higher concentrations of alanine aminotransferase, aspartate aminotransferase, α-L-fucosidase, 5'-nucleotidase, glutamyl transpeptidase, cholinesterase, and uric acid; plateletcrit was also higher. At 16-20 wks, inhibin A concentration and plateletcrit were significantly elevated. At 24-28 wks, platelets, plateletcrit, and glucose concentration were significantly elevated. Logistic regression analysis showed that an elevation in 5'-nucleotidase was independently associated with PE at 11-13 wks. The combination of inhibin A, diastolic blood pressure, and body mass index was a significant predictor for PE at 16-20 wks, while the combination of glucose and systolic blood pressure was a significant predictor for PE at 24-28 wks. In conclusion, when combined with maternal characteristics, the measurement of 5'-nucleotidase, inhibin A, and glucose levels, represents a potentially valuable risk assessment for PE.Impact statementWhat is already known on this subject? Preeclampsia (PE) may be viewed as a spectrum of disorders with a severity that is reflected in the levels of specific biomarkers. Consequently, there is a clear need for additional biomarkers that can be used to stratify pregnancies as high or low risk soon after conception.What do the results of this study add? At 11-13 wks of gestation, maternal assays for platelets, plateletcrit, alanine aminotransferase, aspartate aminotransferase, α-L-fucosidase, 5'-nucleotidase, glutamyl transpeptidase, cholinesterase, and uric acid, demonstrated significantly higher values in patients with PE when compared with normal controls. Furthermore, assay results for inhibin A and platelets showed increased values at 16-20 wks of gestation. Assays performed at 24-28 wks of gestation revealed elevated levels of platelets, plateletcrit, and glucose. Our analysis indicated that increases in the levels of 5'-nucleotidase, inhibin A, and glucose, are effective and significant biomarkers that could be used in combination with maternal characteristics to screen for PE at 11-13, 16-20, and 24-28 wks of gestation, respectively. These findings provide a new basis for our understanding of the aetiology underlying PE.What are the implications of these findings for clinical practice and/or further research? Further studies that consider the entire population are now needed and should include the investigation of laboratory markers across different stages of pregnancy. Long-term follow up would also be necessary if we are to explore the full role of laboratory markers in the pathophysiology of PE.

Knowledge Discovery-Based Analysis of Health Factors of Urinary Infections in Elderly Cardiology Inpatients.

A set of semantic similarity calculation methods combining full-text text and domain knowledge topics is proposed for the current study of entity association relations such as disease-gene in medical texts combined with topics in knowledge discovery, which is insufficient to reveal the deep semantic association relations of medical domain knowledge at topic level. Taking urinary infections in elderly inpatients as the research subject, word embedding representation of word vectors and topic vectors is performed by the TWE model, and similarity calculation is performed by combining text and domain knowledge topics based on Siamese Network framework. The urinary microbiological culture results of both groups were dominated by Escherichia coli, accounting for 34.65% and 47.92%, respectively; the use of antimicrobial drugs in the symptomatic urinary infection group was 94.19% higher than that in the asymptomatic bacteriuria group, 77.27% (x 2 = 8.158, P=0.004).

The Feasibility and Positive Effects of Wuqinxi Exercise on the Cognitive and Motor Functions of Patients with Parkinson's Disease: A Pilot Study.

INTRODUCTION: Parkinson's disease (PD) is a chronic degenerative disease of the central nervous system common in middle-aged and elderly people, which has a serious impact on patients' cognitive and motor functions. Exercise can improve the nonmotor symptoms of PD patients, but the optimal type of exercise for the cognitive function of patients is unclear. Therefore, the purpose of this study is the impact of 12 weeks of Wuqinxi exercise on the cognitive and motor function in PD patients. METHODS: Thirty PD patients participated in the study and were randomly assigned to two groups: Wuqinxi group (n = 15) or stretching group (n = 15). All the participants performed a 12-week exercise program twice a week, 90 min/session. The assessments were conducted before and after exercise intervention, included cognitive function (frontal assessment battery (FAB); Stroop test I and II), motor functions (Unified Parkinson's Disease Rating Scale Part III (UPDRS-III); timed up and go (TUG)). RESULTS: We found the FAB and Stroop I scores were significantly higher in the Wuqinxi group than in the stretching group. Participants in the Wuqinxi group significantly improved their UPDRS-III (17.73 ± 9.88) and TUG (10.50 ± 1.79) score after 12 weeks of training intervention. CONCLUSION: The results show that the use of Wuqinxi for rehabilitation therapy for cognition is feasible, widely accepted, and effective in patients with Parkinson's disease. This study provides preliminary evidence for further large-scale and controlled studies.

Anti-IgLON5 disease with distinctive brain MRI findings responding to immunotherapy: A case report.

RATIONALE: Anti-IgLON5 disease was first described as a progressive antibody-associated encephalopathy, with multiple non-specific clinical symptoms including sleep dysfunction, bulbar symptoms, progressive supranuclear palsy-like syndrome, cognitive impairment, and a variety of movement disorders. This newly discovered disease presents with unremarkable or unspecific brain magnetic resonance imagings (MRI), and have poor responsiveness to immunotherapy. PATIENT CONCERNS: In this case, a 37-year-old man presented with 4-day history of gait instability, dysarthria, and oculomotor abnormalities. The initial neurologic examination revealed mild unsteady gait, subtle dysarthria, and left abducent paralysis. DIAGNOSIS: The patient was diagnosed with anti-IgLON5 disease, based on clinical features and positive anti-IgLON5 antibodies in serum. INTERVENTIONS: Initially, the patient was treated with high dosages of methylprednisolone and immunoglobulins.Outcomes: The symptoms of patient rapidly improved after high-dose intravenous methylprednisolone and immunoglobulins. CONCLUSIONS: In this paper, we report a new case of anti-IgLON5 disease with major symptoms of gait instability, dysarthria, and oculomotor abnormalities, with distinctive brain MRI findings, and responsive to immunotherapy.

An optimized procedure for detection of genetically modified DNA in refined vegetable oils.

In this study, the amplifiable DNA from refined vegetable oils was isolated by using commercial DNA extraction kits based on the CTAB method in combination with nucleic acid enrichment, and then the presence of genetically modified (GM) soybean and maize DNA in the oils was traced by PCR. The results showed that the duration and intensity of heating had no significant effect on the DNA stability and concentration in oils for a short period, suggesting that DNA in oils could be stably reserved for a certain time, thus making it possible to trace down refined vegetable oils reliably and effectively. The results provided a set of primers suitable for systematic GM oil detection. More importantly, this study made an important contribution to the economical and reliable detection of GM vegetable oils regarding food authenticity issues.

Characteristics and predictors of muscle strength deficit in mechanical ankle instability.

PURPOSE: Muscle strength training is a common strategy for treating chronic ankle instability (CAI), but the effectiveness decreases for mechanical ankle instability (MAI) patients with initial severe ligament injuries. The purpose of this study was to investigate the characteristics and the potential predictors of muscle strength deficit in MAI patients, with a view to proposing a more targeted muscle strength training strategy. METHODS: A total of 220 MAI patients with confirmed initial lateral ankle ligament rupture and a postinjury duration of more than 6 months were included. All patients underwent a Biodex isokinetic examination of the ankle joints of both the affected and unaffected sides. Then, the associations between the limb symmetry index (LSI) (mean peak torque of the injury side divided by that of the healthy side) and the patients' sex, body mass index, postinjury duration, presence of intra-articular osteochondral lesions, presence of osteophytes and ligament injury pattern (i.e., isolated anterior talofibular ligament (ATFL) injury or combined with calcaneofibular ligament injury) were analysed. RESULTS: There was significantly weaker muscle strength on the affected side than on the unaffected side in all directions (p < 0.05). The LSI in plantar flexion was significantly lower than that in dorsiflexion at 60°/s (0.87 vs 0.98, p < 0.001). A lower LSI in eversion was significantly correlated with female sex (0.82 vs 0.94, p = 0.016) and isolated ATFL injury (0.86 vs 0.95, p = 0.012). No other factors were found to be associated with muscle strength deficits. CONCLUSION: MAI patients showed significant muscle strength deficits on the affected side, especially in plantar flexion. There were greater strength deficits in eversion in females and individuals with an isolated ATFL injury. Thus, a muscle strength training programme for MAI patients was proposed that focused more on plantar flexion training and eversion training for females and those with an isolated ATFL injury.

Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development.

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development.

A novel role of FKN/CX3CR1 in promoting osteogenic transformation of VSMCs and atherosclerotic calcification.

Fractalkine (FKN) and its specific receptor CX3CR1 play a critical role in the pathogenesis of atherosclerosis including recruitment of vascular cells and the development of inflammation. However, its contribution to regulating the development of atherosclerotic calcification has not been well documented. Osteogenic transformation of vascular smooth muscle cells (VSMCs) is critical in the development of calcification in atherosclerotic lesions. In this study, for the first time, we evaluated the effect of FKN/CX3CR1 on the progression of VSMCs calcification and defined molecular signaling that is operative in the FKN/CX3CR1-induced osteogenic transformation of VSMCs. We found that high-fat diet induced atherosclerotic calcification in vivo was markedly inhibited in the Apolipoprotein E (ApoE) and CX3CR1 deficient (ApoE-/-/CX3CR1-/-) mice compared with their control littermates. FKN and CX3CR1 were both expressed in VSMCs and up-regulated by oxidized low-density lipoprotein (ox-LDL). FKN/CX3CR1 promoted the expression of osteogenic markers, including osteopontin (OPN), bone morphogenetic protein (BMP)-2 and alkaline phosphatase (ALP) and decreased VSMCs markers, including smooth muscle (SM) α-actin and SM22-α in a dose-dependent manner. The essential role of FKN/CX3CR1 in VSMCs calcification was further confirmed by lentivirus-mediated knockdown or overexpression of CX3CR1 blocked or accelerated osteogenic transformation of VSMCs. This response was associated with reciprocal up- and down-regulation of osteogenic factor, runt-related transcription factor 2 (RUNX2), transcription factors in osteoclast differentiation, receptor activator of nuclear factor-κB (RANK), RANK ligand (RNAKL) and osteoprotegerin (OPG), respectively. Inhibition of FKN/CX3CR1-activated Jak2/Stat3 signaling by the Jak/Stat inhibitor AG490 blocked osteogenic transformation of VSMCs and RUNX2 induction concurrently. Taken together, our data uncovered novel roles of FKN/CX3CR1 in promoting VSMC osteogenic transformation and atherosclerotic calcification by activating RUNX2 through Jak2/Stat3 signaling pathway and suppressing OPG. Our findings suggest that targeting FKN/CX3CR1 may provide new strategies for the prevention and treatment of atherosclerotic calcification.