[High-intensity interval training (HIIT) induces hepatic ketone body production possibly through altering expression of mTORC1, PPARα and FGF21 in mice].

The present study aims to investigate the production of ketone body in the liver of mice after 6 weeks of high-intensity interval training (HIIT) intervention and explore the possible mechanisms. Male C57BL/6J mice (7-week-old) were randomly divided into control and HIIT groups. The control group did not engage in exercise, while the HIIT group underwent a 6-week HIIT (10° slope treadmill exercise). Changes in weight and body composition were recorded, and blood ketone body levels were measured before, immediately after, and 1 h after each HIIT exercise. After 6-week HIIT, the levels of free fatty acids in the liver and serum were detected using reagent kits, and expression levels of regulatory factors and key enzymes of ketone body production in the mouse liver were detected by Western blot and qPCR. The results showed that, the blood ketone body levels in the HIIT group significantly increased immediately after a single HIIT and 1 h after HIIT, compared with that before HIIT. The body weight of the control group gradually increased within 6 weeks, while the HIIT group mice did not show significant weight gain. After 6-week HIIT, compared with the control group, the HIIT group showed decreased body fat ratio, increased lean body weight ratio, and increased free fatty acid levels in liver and serum. Liver carnitine palmitoyl transferase-I (CPT-I), peroxisome proliferator activated receptor α (PPARα), and fibroblast growth factor 21 (FGF21) protein expression levels were up-regulated, whereas mammalian target of rapamycin complex 1 (mTORC1) protein expression level was significantly down-regulated in the HIIT group, compared with those in the control group. These results suggest that HIIT induces hepatic ketone body production through altering mTORC1, PPARα and FGF21 expression in mice.

Functional reconstruction of the impaired cortex and motor function by hMGEOs transplantation in stroke.

Stroke is the leading cause of death and long-term disability worldwide. But treatments are not available to promote functional recovery, and efficient therapies need to be investigated. Stem cell-based therapies hold great promise as potential technologies to restore function in brain disorders. Loss of GABAergic interneurons after stroke may result in sensorimotor defects. Here, by transplanting human brain organoids resembling the MGE domain (human MGE organoids, hMGEOs) derived from human induced pluripotent stem cells (hiPSCs) into the infarcted cortex of stroke mice, we found that grafted hMGEOs survived well and primarily differentiated into GABAergic interneurons and significantly restored the sensorimotor deficits of stroke mice for a long time. Our study offers the feasibility of stem cell replacement therapeutics strategy for stroke.

Integrating GWAS and eQTL to predict genes and pathways for non-syndromic cleft lip with or without palate.

OBJECTIVE: To explore susceptibility genes and pathways for non-syndromic cleft lip with or without cleft palate (NSCL/P). MATERIALS AND METHODS: Two genome-wide association studies (GWAS) datasets, including 858 NSCL/P cases and 1,248 controls, were integrated with expression quantitative trait loci (eQTL) dataset identified by Genotype-Tissue Expression (GTEx) project in whole-blood samples. The expression of the candidate genes in mouse orofacial development was inquired from FaceBase. Protein-protein interaction (PPI) network was visualized to identify protein functions. Go and KEGG pathway analyses were performed to explore the underlying risk pathways. RESULTS: A total of 233 eQTL single-nucleotide polymorphisms (SNPs) in 432 candidate genes were identified to be associated with the risk of NSCL/P. One hundred and eighty-three susceptible genes were expressed in mouse orofacial development according to FaceBase. PPI network analysis highlighted that these genes involved in ubiquitin-mediated proteolysis (KCTD7, ASB1, UBOX5, ANAPC4) and DNA synthesis (XRCC3, RFC3, KAT5, RHNO1) were associated with the risk of NSCL/P. GO and KEGG pathway analyses revealed that the fatty acid metabolism pathway (ACADL, HSD17B12, ACSL5, PPT1, MCAT) played an important role in the development of NSCL/P. CONCLUSIONS: Our results identified novel susceptibility genes and pathways associated with the development of NSCL/P.

[Research advances on high-intensity interval training and cognitive function].

High-intensity interval training (HIIT) has proven to be a time-saving and efficient exercise strategy. Compared with traditional aerobic exercise, it can provide similar or even better health benefits. In recent years, a number of studies have suggested that HIIT could be used as a potential exercise rehabilitation therapy to improve cognitive impairment caused by obesity, diabetes, stroke, dementia and other diseases. HIIT may be superior to regular aerobic exercise. This article reviews the recent research progress on HIIT with a focus on its beneficial effect on brain cognitive function and the underlying mechanisms. HIIT may become an effective exercise for the prevention and/or improvement of brain cognitive disorder.

Identification of novel susceptibility loci for non-syndromic cleft lip with or without cleft palate.

Although several genome-wide association studies (GWAS) of non-syndromic cleft lip with or without cleft palate (NSCL/P) have been reported, more novel association signals are remained to be exploited. Here, we performed an in-depth analysis of our previously published Chinese GWAS cohort study with replication in an extra dbGaP case-parent trios and another in-house Nanjing cohort, and finally identified five novel significant association signals (rs11119445: 3' of SERTAD4, P = 6.44 × 10-14 ; rs227227 and rs12561877: intron of SYT14, P = 5.02 × 10-13 and 2.80 × 10-11 , respectively; rs643118: intron of TRAF3IP3, P = 4.45 × 10-6 ; rs2095293: intron of NR6A1, P = 2.98 × 10-5 ). The mean (standard deviation) of the weighted genetic risk score (wGRS) from these SNPs was 1.83 (0.65) for NSCL/P cases and 1.58 (0.68) for controls, respectively (P = 2.67 × 10-16 ). Rs643118 was identified as a shared susceptible factor of NSCL/P among Asians and Europeans, while rs227227 may contribute to the risk of NSCL/P as well as NSCPO. In addition, sertad4 knockdown zebrafish models resulted in down-regulation of sox2 and caused oedema around the heart and mandibular deficiency, compared with control embryos. Taken together, this study has improved our understanding of the genetic susceptibility to NSCL/P and provided further clues to its aetiology in the Chinese population.

[Aerobic exercise reduces the expression of pyroptosis-related proteins and inflammatory factors in hippocampus of mice with insulin resistance].

The aim of the present study was to observe the expression of pyroptosis- and inflammation-related proteins in the hippocampus of mice with insulin resistance (IR) after aerobic exercise, and to explore the possible mechanism of exercise to improve IR. C57BL/6J male mice of 6 weeks old were randomly fed with normal diet (n = 12) and high-fat diet (HFD) (n = 26) for 12 weeks respectively. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to determine whether IR occurred in HFD mice. Then the mice were randomly divided into control group (n = 12), IR group (n = 10) and IR + aerobic exercise group (AE, n = 10). Mice in AE group performed a 12-week progressive speed treadmill training after being adapted to the treadmill for one week. After the intervention, the expression of pyroptosis- and inflammation-related proteins in hippocampus was detected by Western blot. The results showed that compared with control group, NFκB, Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), pyroptosis-related proteins like pro-Caspase-1, gasdermin D (GSDMD), GSDMD-N, and inflammatory factors IL-1ß, IL-18 were significantly increased. The inflammasome-related protein NIMA-related kinase 7 (NEK7) and pyroptosis-related protein Caspase-1 showed an increasing trend, but there was no significant difference. Compared with the IR group, progressive speed treadmill training significantly reduced the expression of NFκB, NLRP3, NEK7, ASC, pro-Caspase-1, GSDMD, GSDMD-N, IL-1ß, and IL-18 in the hippocampus of mice with IR. These results suggested 12-week progressive speed treadmill training can significantly reduce the expression of pyroptosis-related proteins and inflammatory factors in the hippocampus of mice with IR, and inhibit pyroptosis.

Non-syndromic cleft lip with or without palate-susceptible SNPs is associated with hyperdontia.

BACKGROUND: Non-syndromic supernumerary teeth (NSST) or hyperdontia may share common genetic determinants with non-syndromic cleft lip with or without palate (NSCL/P). The aim of this study was to test the associations between five genome-wide-associated NSCL/P-susceptible single nucleotide polymorphisms (SNPs) (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) and the occurrence of NSST. MATERIALS AND METHODS: A total of 163 cases and 326 controls were recruited and their genomic DNA was extracted from blood samples. Five NSCL/P-susceptible SNPs (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) were genotyped by TaqMan method. Odds ratio (OR) and 95% confidence interval (CI) were used to estimate the associations between the SNPs and the risk of NSST by PLINK software. RESULTS: Rs4791774 (A > G) and rs13041247 (T > C) were associated with risk of NSST (rs4791774: Padd  = 0.011, OR, 95% CI = 0.62, 0.43-0.90; rs13041247: Phomo  = 0.031, OR, 95% CI = 1.79, 1.05-3.05) and one supernumerary tooth (rs4791774: Pdom  = 0.009, OR, 95% CI = 0.56, 0.36-0.87; rs13041247: Phomo  = 0.034, OR, 95% CI = 1.82, 1.05-3.15). Rs4791774 (A > G) was also showed association with risk of upper arch supernumerary teeth only (Padd  = 0.010, OR, 95% CI = 0.60, 0.41-0.89). CONCLUSION: Non-syndromic cleft lip with or without palate-susceptible loci rs4791774 (A > G) and rs13041247 (T > C) were associated with the risk of supernumerary teeth.

Non-syndromic cleft lip with or without palate susceptible loci is associated with tooth agenesis.

OBJECTIVE: Non-syndromic tooth agenesis (NSTA) may share common genetic factors with non-syndromic cleft lip with or without cleft palate (NSCL/P). Single-nucleotide polymorphisms (SNPs) were associated with individual's susceptibility to these anomalies. We selected five NSCL/P-associated SNPs from our previous genome-wide association study (GWAS) to test for the associations with NSTA. MATERIALS AND METHODS: A total of 677 NSTA cases and 1,144 healthy controls were recruited in this case-control study. Five genome-wide NSCL/P-associated SNPs (rs2235371, rs7078160, rs8049367, rs4791774, and rs13041247) were genotyped by TaqMan platform and evaluated for the associations with NSTA using plink software. RESULTS: No significant associations between these SNPs and risk of NSTA were observed in the overall analysis and subgroup analysis with the number of missing teeth. However, in the subgroup analysis by tooth position, rs8049367 was nominally associated with mandibular premolar agenesis (Dominant model: ORdom  = 0.66, 95% CIdom  = 0.47-0.93, pdom  = 0.016; Heterozygote model: ORhet  = 0.60, 95% CIhet  = 0.41-0.88, Phet  = 0.008). Rs4791774 showed a nominal association with congenitally missing maxillary canine (Dominant model: ORdom  = 0.53, 95% CIdom  = 0.28-0.98, pdom  = 0.041; Heterozygote model: ORhet  = 0.50, 95% CIhet  = 0.26-0.97, Phet  = 0.041) and premolar (Additive model: OR = 0.59, 95% CI = 0.36-0.96, p = 0.035). CONCLUSION: This study showed that NSCL/P susceptible loci rs8049367 and rs4791774 were probably associated with the risk of NSTA.

HSP70 plays a role in the defense of acute and chronic heat stress in Mongolian gerbils (Meriones unguiculatus).

Mongolian gerbils (Meriones unguiculatus) show a wide thermal neutral zone (TNZ, 26.5-38.9 °C). Whether heat shock proteins (HSPs) are involved in thermal tolerance for gerbils has still been unknown. We investigated the effects of acute and chronic high temperature within and above TNZ on the expressions of HSP70 and HSP90 and oxidative status in Mongolian gerbils, to test the hypothesis that the gerbils need increase the expression in HSPs to defense the acute and chronic heat stress. In experiment I, 50 Mongolian gerbils were exposed to 23 °C, 27 °C, 37 °C, 40 °C and 43.5 °C for 80 min respectively, and then sacrificed 12 h after treatment. HSP70 expression in the liver increased at 40 °C compared to that at 23 °C, but did not change after 27 °C, 37 °C or 43.5 °C exposure. There were no differences in HSP90 expression, oxidative stress parameters such as malonaldehyde (MDA) and hydrogen peroxide (H2O2), or antioxidant parameters such as superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in the liver. HSP70 and HSP90 expression both in the heart and brain showed no differences among groups. In experiment II, another set of 30 gerbils were acclimated to 23 °C, 27 °C and 37 °C for 21 days, respectively. During chronic acclimation, HSP70 expression increased and H2O2 level decreased in the liver in 37 °C group compared to other two groups. Both H2O2 and SOD in the brain decreased in 37 °C group, but there were no differences in HSP70, MDA or T-AOC in the brain. These data indicate that Mongolian gerbils can maintain basal levels of HSPs after acute exposure to temperatures within the wide TNZ, but rely on increased HSP70 in the liver to protect from heat damage at temperatures above TNZ and during chronic heat acclimation. The increased HSP70 expression in the liver may contribute to keeping from heat damage in desert rodents.

A functional polymorphism in the pre-miR-146a gene is associated with the risk of nonsyndromic orofacial cleft.

microRNAs (miRNAs) are widely involved in craniofacial development, and genetic variants of miRNAs may be associated with the risk of nonsyndromic orofacial cleft (NSOC). Here, we systematically selected five single nucleotide polymorphisms (SNPs) of miRNAs and investigated the associations between these variants and NSOC susceptibility in a two-stage case-control study including 1,406 NSOC patients and 1,578 controls from the Chinese population. We found that compared with the C allele, the rs2910164 G allele of pre-miR-146a was associated with an increased risk of NSOC (additive model: odds ratio [OR] = 1.17, 95% confidence interval [CI]: 1.06-1.30, P = 0.002), including both cleft lip with or without cleft palate (CL/P) and cleft palate only (CPO). Bioinformatic prediction and functional assays revealed that the C allele of rs2910164 was significantly associated with inhibited HEK-293 and HEPM cell proliferation and decreased abundance of TRAF6. Both miR-146a and TRAF6 were expressed in the lip tissue samples of NSOC patients, and a moderate inverse correlation was observed between them. Taken together, these results demonstrated that miR-146a/rs2910164 is associated with susceptibility to NSOC, providing novel insights into the genetic etiology and underlying biology of NSOC.

[Research advances on anti-inflammatory, antioxidant and anti-apoptotic biological effects of methane].

Methane (CH4) is the simplest hydrocarbons and endogenous CH4 has been thought only to be generated by methanogens in the oral cavity and gastrointestinal tract of the mammals. However, recent animal studies have shown that endogenous CH4 can also be generated from choline and its metabolites in the mammals to protect the plasma membrane from reactive oxygen species attack and repair the membrane. In addition, exogenous CH4 can ameliorate the oxidative stress injury of multiple tissues and organs through its anti-inflammatory, antioxidant and anti-apoptosis effects. This paper reviews the recent researches about CH4 synthetic metabolism and biological functions, and highlights its potential of wide application in the prevention and treatment of oxidative stress related diseases and the significance for the development of gas medicine.

The signaling mechanisms of hippocampal endoplasmic reticulum stress affecting neuronal plasticity-related protein levels in high fat diet-induced obese rats and the regulation of aerobic exercise.

High fat diet (HFD)-induced obesity has been shown to reduce the levels of neuronal plasticity-related proteins, specifically brain-derived neurotrophic factor (BDNF) and synaptophysin (SYN), in the hippocampus. However, the underlying mechanisms are not fully clear. Endoplasmic reticulum stress (ERS) has been reported to play a key role in regulating gene expression and protein production by affecting stress signaling pathways and ER functions of protein folding and post-translational modification in peripheral tissues of obese rodent models. Additionally, HFD that is associated with hyperglycemia could induce hippocampal ERS, thus impairing insulin signaling and cognitive health in HFD mice. One goal of this study was to determine whether hyperglycemia and hyperlipidemia could cause hippocampal ERS in HFD-induced obese SD rats, and explore the potential mechanisms of ERS regulating hippocampal BDNF and SYN proteins production. Additionally, although regular aerobic exercise could reduce central inflammation and elevate hippocampal BDNF and SYN levels in obese rats, the regulated mechanisms are poorly understood. Nrf2-HO-1 pathways play roles in anti-ERS, anti-inflammation and anti-apoptosis in peripheral tissues. Therefore, the other goal of this study was to determine whether aerobic exercise could activate Nrf2-HO-1 in hippocampus to alleviate obesity-induced hippocampal ERS, which would lead to increased BDNF and SYN levels. Male SD rats were fed on HFD for 8weeks to establish the obese model. Then, 8weeks of aerobic exercise treadmill intervention was arranged for the obese rats. Results showed that HFD-induced obesity caused hyperglycemia and hyperlipidemia, and significantly promoted hippocampal glucose transporter 3 (GLUT3) and fatty acid transport protein 1 (FATP1) protein expression. These results were associated with the activation of hippocampal ERS and ERS-mediated apoptosis. At the same time, we found that excessive hippocampal ERS not only significantly decreased proBDNF-the precursor of mature BDNF, but also attenuated p38/ERK-CREB signaling pathways and activated NLRP3-IL-1ß pathways in obese rats. These results were associated with reduced BDNF and SYN protein production. However, these adverse changes were obviously reversed by aerobic exercise intervention through activating the Nrf2-HO-1 pathways. These results suggest that dietary obesity could induce hippocampal ERS in male SD rats, and excessive hippocampal ERS plays a critical role in decreasing the levels of BDNF and SYN. Moreover, aerobic exercise could activate hippocampal Nrf2 and HO-1 to relieve ERS and heighten BDNF and SYN production in obese rats.

WNK1 is involved in Nogo66 inhibition of OPC differentiation.

LINGO-1 is a transmembrane receptor expressed primarily in the central nervous system (CNS) and plays an important role in myelination. Recent studies have indicated that it is also involved in oligodendrocyte precursor cell (OPC) survival and differentiation; however, the downstream signaling pathway underlying OPC development is unknown. In our previous study, we found that LINGO-1 is associated with WNK1 in mediating Nogo-induced neurite extension inhibition by RhoA activation. In an effort to identify the role of LINGO-1-WNK1 in OPCs, we first confirmed that WNK1 is also expressed in OPCs and co-localized with LINGO-1, which suppresses WNK1 expression by RNA interference-attenuated Nogo66-induced inhibition of OPC differentiation. Furthermore, we mapped the WNK1 kinase domain using several fragmented peptides to identify the key region of interaction with LINGO-1. We found that a sequence corresponding to the D6 peptide is necessary for the interaction. Finally, we found that using the TAT-D6 peptide to introduce D6 peptide into primary cultured OPC inhibits the association between LINGO-1 and WNK1 and significantly attenuates Nogo66-induced inhibition of OPC differentiation. Taken together, our results show that WNK1, via a specific region on WNK1 kinase domain, interacts with LINGO-1, thus mediating Nogo66-inhibited OPC differentiation.

Effect of Anterior Ankle-Foot Orthoses on Weight Shift in Persons With Stroke.

OBJECTIVE: To examine the effects of an anterior ankle-foot orthosis (AAFO) on the speed and accuracy of weight shift in persons with stroke. DESIGN: Cross sectional, repeated measures. SETTING: Neurologic rehabilitation department. PARTICIPANTS: People with stroke (N=24) who were unable to voluntarily dorsiflex the foot against gravity. INTERVENTION: The weight-shift performance was measured with and without the AAFO. MAIN OUTCOME MEASURES: The speed and accuracy of sustained and cyclic bilateral weight shift were measured using the computerized dynamic posturography. The movement velocity, maximum excursion, and directional control of sustained weight shift were calculated using the limits of stability test. The on-axis velocity gap, directional control, and stability of cyclic bilateral weight shift were calculated using the rhythmic weight shift test. RESULTS: For sustained weight shift, the maximum excursion of weight shift to the affected side was greater with the AAFO (P=.002). For cyclic bilateral weight shift, the on-axis velocity gap in the mediolateral (ML) direction was smaller at a fast speed (P=.004). The stability of the ML and anteroposterior weight shift was higher at slow (P=.002 and P<.001, respectively) and fast (P=.001 and P<.001, respectively) speeds when wearing the AAFO. CONCLUSIONS: The findings demonstrated that persons with stroke who wear an AAFO might improve the excursion of the sustained weight shift to the affected side and the speed and stability of cyclic bilateral weight shift in the ML direction.

Effects of an anterior ankle-foot orthosis on walking mobility in stroke patients: get up and go and stair walking.

OBJECTIVE: To examine the effects of an anterior ankle-foot orthosis (AAFO) on walking mobility in stroke patients. DESIGN: Cross-sectional and repeated-measures study design. SETTING: A university's neurologic rehabilitation department. PARTICIPANTS: Ambulant stroke patients (N=21). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Walking mobility was measured by the Timed Up and Go (TUG) test and the Timed Up and Down Stairs (TUDS) test. The paired t test was used to determine the difference between the mobility performances measured with and without the AAFO. RESULTS: There were significant differences between mobility performances with and without an AAFO in the TUG test (P=.038) and the TUDS test (P=.000). CONCLUSIONS: This study supports the effect of an AAFO on walking mobility in stroke patients. The findings demonstrate that stroke patients wearing an AAFO may ambulate with greater speed and safety on level surfaces and stairs.

Effects of the type and direction of support surface perturbation on postural responses.

BACKGROUND: Postural control is organized around a task goal. The two most frequently used types of tasks for postural control research are translational (translation along the anterior-posterior axis) and rotational (rotation in sagittal plane) surface perturbations. These types of perturbations rotate the ankle joint, causing different magnitudes and directions of body sway. The purpose of this study was to investigate the effects of the type (translation vs. rotation) and direction (forward/toe up vs. backward/toe down) of the perturbation on postural responses. METHOD: Nineteen healthy subjects were tested with four perturbations, i.e., forward and backward translation and toe up and toe down rotation. The onset latency and magnitude of muscle activations, angular changes, and COM displacements were measured. In addition, the kinematic data were divided into two phases. The initial phase reflected the balance disturbance induced by the platform movement, and the reversal phase reflected the balance reaction. RESULTS: The results showed that, in the initial phase, rotational perturbation induced earlier ankle movement and faster and larger vertical COM displacement, while translational and forward/toe up perturbations induced larger head and trunk angular change and faster and larger horizontal COM displacement. In the reversal phase, balance reaction was attained by multi-joint movements. Translational and forward/toe up perturbations that induced larger upper body instability evoked faster muscle activation as well as faster and larger hip or knee joint movements. CONCLUSIONS: These findings provide insights into an appropriate support surface perturbation for the evaluation and training of balance.

Integrative Multi-omics Analysis Identifies Genetic Variants Contributing to Non-syndromic Cleft Lip with or without Cleft Palate.

OBJECTIVE: To provide novel insights into the aetiology of non-syndromic cleft lip with or without cleft palate (NSCL/P) by integrating multi-omics data and exploring susceptibility genes associated with NSCL/P. METHODS: A two-stage genome-wide association study (GWAS) of NSCL/P was performed, involving a total of 1,069 cases and 1,724 controls. Using promoter capture Hi-C (pCHi-C) datasets in human embryonic stem cells (hESC) and chromatin immunoprecipitation sequencing (ChIP-seq) in craniofacial tissues, we filtered out single nucleotide polymorphisms (SNPs) with active cis-regulation and their target genes. Additionally, we employed expression quantitative trait loci (eQTL) analysis to identify candidate genes. RESULTS: Thirteen SNPs were identified as cis-regulation units associated with the risk of NSCL/P. Five of these were proven to be active in chromatin states in early human craniofacial development (rs7218002: odds ratio [OR] 1.50, P = 8.14E-08; rs835367: OR 0.78, P = 3.48E- 05; rs77022994: OR 0.55, P = 1.05E-04; rs961470: OR 0.73, P = 1.38E-04; rs17314727: OR 0.73, P = 1.85E-04). Additionally, pCHi-C and eQTL analysis prioritised three candidate genes (rs7218002: NTN1, rs835367: FGGY, LINC01135). NTN1 and FGGY were expressed in mouse orofacial development. Deficiencies in NTN1, FGGY and LINC01135 were associated with cleft palate and cleft lip, abnormal facial shape and bifid uvula, and abnormality of the face, respectively. CONCLUSION: Our study identified five SNPs (rs7218002, rs835367, rs77022994, rs961470 and rs17314727) and three susceptibility genes (NTN1, FGGY and LINC01135) associated with NSCL/P. These findings contribute to a better understanding of the genetic factors involved.

Genetic Variants in METTL16 Affect the Risk of Non-Syndromic Orofacial Clefts.

OBJECTIVE: N6-methyladenosine (m6A) is the most prevalent modification of RNA in eukaryotes which is associated with many cellular processes and diseases. Here, our objective is to explore whether genetic variants in m6A modification genes are associated with the risk of non-syndrome orofacial clefts (NSOCs). METHODS: The transmission disequilibrium test (TDT) was performed to calculate the association between single nucleotide polymorphisms (SNPs) in m6A modification genes and NSOCs risk in 944 case-parent trios. The function of SNP was predicted by HaploReg, RegulomeDB and histone enrichment data. The expression quantitative trait locus (eQTL) analysis was examined using Genotype-Tissue Expression (GTEx) and eQTLGen. The role of gene in the development of NSOCs was assessed with correlation and enrichment analysis based on gene expression data in mice craniofacial tissue and zebrafish embryo. RESULTS: We identified that rs8078195 (A > C) in METTL16 was suggestively associated with the increased risk of NSOCs (OR = 1.32, p = 1.80E - 03). The region surrounding rs8078195 was subjected to deoxyribonuclease hypersensitivity and enriched with multiple histone modifications. In addition, it had a significant eQTL effect with METTL16 in skin tissue and human peripheral blood, which played an important role in NSOCs development. Bioinformatic analysis indicated that METTL16 contributed to the development of NSOCs probably by regulating cell cycle process. CONCLUSIONS: Rs8078195 in METTL16 was associated with the occurrence of NSOCs.

Genetic variants in BCL-2 family genes influence the risk of non-syndromic cleft lip with or without cleft palate.

BACKGROUND: The BCL-2 family is crucial for cell death regulation and is involved in development, tissue homeostasis, and immunity. This study aimed to investigate the association between genetic variants in BCL-2 family genes and non-syndromic cleft lip with or without cleft palate (NSCL/P) risk. METHODS: A two-stage case-control study was conducted in this association study. Gene-based analysis using Multi-marker Analysis of GenoMic Annotation was performed in the first stage cohort, which included 565 cases and 1269 controls. A logistic regression model was employed to assess the effect of single nucleotide polymorphisms (SNPs) on susceptibility to NSCL/P. Candidate SNPs were replicated by extra dbGaP case-parent trios. Haploreg, RegulomeDB, and UCSC Genome Browser were used to identify enhancer effects of promising SNPs. Bulk RNA sequencing data obtained from the Gene Expression Omnibus was used to identify co-expressed genes. Single-cell RNA sequencing dataset was used to infer the cell population of the candidate gene. The "Monocle" package was used to analyze the pseudotime cell trajectories. RESULTS: Rs3943258 located in the enhancer region was associated with the risk of NSCL/P (Pmeta = 5.66 × 10-04 ) and exhibited an eQTL effect for BCL2 (P = 3.96 × 10-02 ). Co-expression and pathway enrichment analysis revealed that genes related to Bcl2 were significantly enriched in the PI3K-Akt signaling pathway, MAPK signaling pathway, and Wnt signaling pathway. Five cell clusters were identified in single-cell RNA sequencing, and Bcl2 was mainly located in the mesenchyme. CONCLUSION: The rs3943258 located within BCL2 was probably related to NSCL/P susceptibility.

METTL3-dependent m6A modification of PSEN1 mRNA regulates craniofacial development through the Wnt/ß-catenin signaling pathway.

Craniofacial malformations, often associated with syndromes, are prevalent birth defects. Emerging evidence underscores the importance of m6A modifications in various bioprocesses such as stem cell differentiation, tissue development, and tumorigenesis. Here, in vivo, experiments with zebrafish models revealed that mettl3-knockdown embryos at 144 h postfertilization exhibited aberrant craniofacial features, including altered mouth opening, jaw dimensions, ethmoid plate, tooth formation and hypoactive behavior. Similarly, low METTL3 expression inhibited the proliferation and migration of BMSCs, HEPM cells, and DPSCs. Loss of METTL3 led to reduced mRNA m6A methylation and PSEN1 expression, impacting craniofacial phenotypes. Co-injection of mettl3 or psen1 mRNA rescued the level of Sox10 fusion protein, promoted voluntary movement, and mitigated abnormal craniofacial phenotypes induced by mettl3 knockdown in zebrafish. Mechanistically, YTHDF1 enhanced the mRNA stability of m6A-modified PSEN1, while decreased METTL3-mediated m6A methylation hindered ß-catenin binding to PSEN1, suppressing Wnt/ß-catenin signaling. Pharmacological activation of the Wnt/ß-catenin pathway partially alleviated the phenotypes of mettl3 morphant and reversed the decreases in cell proliferation and migration induced by METTL3 silencing. This study elucidates the pivotal role of METTL3 in craniofacial development via the METTL3/YTHDF1/PSEN1/ß-catenin signaling axis.