Mediating effect of cumulative lipid profile burden on the effect of diet and obesity on hypertension incidence: a cohort study of people aged 35-65 in rural China.

BACKGROUND AND OBJECTIVES: Cumulative lipid profile burden is designed to dynamically measure lipid accumulation, and its effect on hypertension has been poorly studied. Our main purpose was to investigate the effect of cumulative lipid profile burden on the incidence of essential hypertension (EH) and to investigate whether cumulative lipid burden mediates the pathogenesis of the effects of diet and obesity on EH. SUBJECTS AND METHODS: A total of 1295 participants were included in the study, which started in 2017. The average follow-up time was 2.98 years. A total of 240 EH patients occurred during the follow-up period. RESULTS: The HR (95% CI) of the highest quartile in cumulative Total cholesterol (TC), triglyceride (TG) and high density lipoprotein (HDL) burden were 1.747 (1.145 - 2.664), 1.502 (1.038 - 2.173), 0.615 (0.413 - 0.917) for incidence of EH respectively, compared to the respective reference groups. Participants with EH consumed more red meat and refined grains, and red meat was positively associated with cumulative TC burden. BMI and Waist-To-Height Ratio (WHtR) increased the incidence of EH, and obesity was positively correlated with cumulative TG burden. Mediating analysis showed that cumulative TG had a partial mediating effect in the causal relationship between obesity and EH, and Mendelian randomization (MR) also proved this result. Diet was not found to influence EHn through cumulative lipid profile burden. CONCLUSIONS: The cumulative TG burden partially mediates the effect of obesity on EH.

Surgical Treatment for Chest "Lock" Keloid Using Autologous Split-Thickness Skin Grafting and Postoperative Radiotherapy.

BACKGROUND: The treatment of chest "lock" keloids is challenging due to skin defects and a high recurrence rate. OBJECTIVE: Evaluation of the effectiveness of autologous split-thickness skin graft with local radiotherapy for treating chest "lock" keloids. METHODSAND MATERIALS: Fifty-seven patients with chest "lock" keloids were treated from July 2018 to September 2020. The skin defects were closed with an autologous split-thickness skin graft (STSG) and vacuum sealing drainage. The donor and the recipient sites received the first session of radiotherapy 72 hours postoperation for 3 consecutive days. Patients underwent follow-up examinations 12 months after surgery. The Patient and Observer Scar Assessment Scale (POSAS) was used to assess the treatment outcome. RESULTS: Except for the complaints of pain, which did not improve in the patients' assessments (p = .368), POSAS improved significantly after treatment (p < .0001). The cure rate (including cured and partially cured scars) was 100%. No keloid recurrence was observed during the follow-up period. CONCLUSION: The procedure of treating chest "lock" keloid by keloid debulking and autologous STSG followed by postoperational radiotherapy is a novel combined methodology for treating keloids.

Comparative efficacy of targeted structural patterns of electroencephalography neurofeedback in children with inattentive or combined attention deficit hyperactivity disorder.

OBJECTIVE: To evaluate and compare the effects of three courses of different structural patterns of electroencephalography neurofeedback on predominantly inattentive attention deficit hyperactivity disorder (ADHD-PI) and combined ADHD (ADHD-CT). METHODS: Thirty-eight ADHD-PI and ADHD-CT children were selected and completed three courses of different structural patterns of electroencephalography neurofeedback according to their ADHD type. Before and after each course, relative power value of electroencephalography, including θ, ß, α, SMR and their ratios (θ/ß, θ/α), and eighteen integrated visual and auditory continuous performance test (IVA/CPT) quotients were obtained and compared. Data were analyzed by SPSS software, and p < .05 was considered statistically significant. RESULTS: After one course, θ, three IVA/CPT quotients in both types and two comprehensive quotients in ADHD-CT changed significantly (all p < .05). After two courses, θ/α, θ/ß and five IVA/CPT quotients in both types, θ and α in ADHD-PI, four comprehensive quotients, and four respond control quotients in ADHD-CT varied significantly compared to before treatment and after one course (all p < .05). After three courses, α, ß, θ, θ/α, θ/ß and ten IVA/CPT quotients in both types changed significantly compared to before treatment and after one course (all p < .05). In addition, six IVA/CPT quotients in both types after three courses were significantly higher than those after two courses (all p < .05). CONCLUSION: Different structural patterns of electroencephalography neurofeedback targeted for ADHD-CT and ADHD-PI were both effective and feasible. Three courses of EEG neurofeedback were most effective.

Inflammation mediated the effect of dietary fiber on depressive symptoms.

Background and aims: Previous studies showed that inflammation affects depressive symptoms. Dietary fiber may be associated with inflammation and depressive symptoms. We aimed to investigate the relationship between inflammation and depressive symptoms at different levels of dietary fiber intake and to explore whether dietary fiber affects depression through inflammation. Methods: A total of 8,430 National Health and Nutrition Examination Survey (NHANES) samples were collected between 2015 and 2018. Factor analysis was used to determine dietary patterns. Linear regression and logistic regression analysis were used to explore the relationship between nutrients, inflammation, and depressive symptoms, and the mediation analysis was conducted using the bootstrap method. Results: Factor 3 (dietary fiber and vitamins) was inversely associated with depressive symptoms and inflammation. The upper quartile scores of the dietary inflammatory index (DII) and C-reactive protein (CRP) were associated with depressive symptoms compared with controls (DII: OR = 1.851, 95% CI: 1.267-2.705; CRP: OR = 1.737, 95% CI: 1.136-2.656). The DII score and CRP were associated with depressive symptoms in the group with low dietary fiber intake (DII: OR = 2.736, 95% CI: 1.628-4.598; CRP: OR = 2.092, 95% CI: 1.196-3.658) but not in the high dietary fiber intake group. Mediating analysis showed that CRP partially mediated the effect of dietary fiber intake on depressive symptoms (ßindirect = -0.0025, 95% CI: -0.0038 to -0.0013), and the mediated proportion was 10.5%. Conclusion: In this study, we found that DII scores and CRP were not associated with depressive symptoms in participants with high dietary fiber intake, and inflammation partially mediates the effect of dietary fiber on depressive symptoms.

[Effects of aerobic exercise combined with Lycium ruthenicum on some indicators of myocardial lipid metabolism in rats with high-fat diet].

OBJECTIVE: To study the effects of aerobic exercise combined with Lycium ruthenicumon on some indicators of myocardial lipid metabolism in rats with high-fat diet. METHODS: Fifty-five male Wistar rats were subjected to adaptive feeding for 4 days and weight-free swimming training for 3 days, 20 min/d. After eliminating 5 rats that were not suitable for swimming training, the others were randomly divided into 5 groups according to their weight: regular diet + quiet control group (RDC), high fat diet + quiet control group (HDC), high-fat diet + Lycium ruthenicum quiet control group (HDLC), high fat diet + aerobic exercise group (HDM), high fat diet + Lycium ruthenicum + aerobic exercise group (HDLM), 10 in each group. Group HDM and HDLM did 60 min/d swimming training for 6 weeks with no-bearing. Group C were fed regular diet; The other groups were fed with high-fat diet; Group HDLC and HDLM were intragastrically treated with Lycium ruthenicum at the dose of 4.48 g/(kg·d), and the volume was 5 mL/kg, and the other groups were given equivalent distilled water. The Lee's index, serum and myocardial biochemical indexes were measured after 6 weeks. RESULTS: Compared with group RDC, Lee's index, serum free fatty acids (FFA), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), myocardial FFA and intercellular adhesion molecule-1 (ICAM-1) increased significantly (P＜0.01), serum level of high-density lipoprotein cholesterol (HDL-C) decreased significantly (P＜0.01) in group HDC. Compared with group HDC, Lee's index, serum FFA, IL-6, TNF-α, TC, TG, LDL-C, myocardial FFA and ICAM-1 decreased significantly (P＜0.05 or P＜0.01), serum HDL-C levels increased significantly (P＜0.05 or P＜0.01) in group HDLC, HDM and HDLM. Compared with group HDLC and HDM, Lee's index, serum FFA, IL-6, TNF-α, TC, TG, LDL-C, myocardial FFA and ICAM-1 decreased significantly (P＜0.05), serum HDL-C level increased significantly (P＜0.05) in group HDLM. CONCLUSION: Aerobic exercise and/or Lycium ruthenicum can improve lipid metabolism in rats with high-fat diet, reduce lipotoxicity caused by obesity. Combined intervention is more effective.

Generic and rapid determination of veterinary drug residues and other contaminants in raw milk by ultra performance liquid chromatography-tandem mass spectrometry.

A generic, rapid and simple analytical method able to identify 255 veterinary drug residues and other contaminants in raw milk had been developed. The method was based on two-step simple precipitation and ultra performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (UPLC-ESI-MS/MS) operating both in positive and negative multiple reaction mode (MRM). For most of the target analytes, the optimized pretreatment processes led to no significant interference on analysis from complicated sample matrix. For quantification, matrix-fortified calibration curves were performed to compensate for the matrix effect and loss in sample preparation. Competent linearity was found for over 90% of target compounds with linear regression coefficients (R) higher than 0.99. Detection limits ranged from 0.05 to 10µg/kg. Average recoveries spiked into raw milk were in the range from 63% to 141% with associated RSD values from 1% to 29% under the selected conditions. The method had been validated for its extraction sensitivity, linearity, recoveries and precision. The results clearly demonstrated the feasibility of the approach proposed. Application of this method, which improved efficiency and coverage of residues, would imply a drastic reduction of both effort and time in routine monitoring programs.

[Influence of electroacupuncture of meridian acupoints on the related hormones of the hypothalamus-pituitary-adrenal axis in rats with cerebral ischemia reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the related hormones of the hypothalamus-pituitary-adrenal (HPA) axis in rats with cerebral ischemia-reperfusion injury (CI-RI). METHODS: Ninety Wistar rats were randomly divided into normal control, sham-operation (sham), model, EA acupoint and EA non-acupoint groups. CI-RI model was established by using modified middle cerebral artery occlusion (MCAO) and reperfusion. These rats were further divided into 1 day (d), 3 d and 7 d subgroups, with 6 cases in each. EA (1 mA, 2 Hz/30 Hz) was applied to acupoint "Zusanli" (ST 36) and "Quchi" (LI 11), and non-acupoints (5 mm lateral to ST 36 and 5 mm apart from LI 11 on the radial side respectively) for 30 min, once daily for 1 d, 3 d and 7 d separately. Serum cortisole (CORT) content was assayed by enzyme linked immunosorbent assay (ELISA), and the expression of hypothalamic adrenocorticotropic releasing factor (CRF) mRNA and glucocorticoid receptor (GR) mRNA, and pituitary adrenocorticotrophic hormone (ACTH) mRNA was assayed by reverse transcription polymerase chain reaction (RT-PCR) method. RESULTS: Compared with normal and sham groups, serum CORT levels of model and EA non-acupoint groups on the 1st, 3rd and 7th day increased significantly (P < 0.05), while compared with model groups, serum CORT level of EA acupoint groups decreased apparently (P < 0.05). Compared with normal and sham groups, hypothalamic CRF mRNA and pituitary ACTH mRNA expression of model groups were upregulated significantly on the 1st, 3rd and 7th d after CI-RI (P < 0.01, P < 0.05), and hypothalamic GR mRNA expression of model groups were obviously downregulated (P < 0.01, P < 0.05). In comparison with model groups, hypothalamic CRF mRNA and pituitary ACTH mRNA expression of EA acupoint groups at the 3 time-points, and their expression of EA non-acupoint groups on the 1st and 3rd day were remarkably downregulated (P < 0.01, P < 0.05), and hypothalamic GR mRNA expression of EA acupoint groups was upregulated significantly at the 3 time-points (P < 0.01, P < 0.05). Comparison between EA acupoint and EA non-acupoint groups showed that the effect of EA acupoint groups was obviously superior to that of EA non-acupoint groups in downregulating CRF mRNA and ACTH mRNA expression and in upregulating GR mRNA expression at the 3 time-points (P < 0.01, P < 0.05). CONCLUSION: EA of meridian-acupoints can effectively downregulate serum CORT content, hypothalamic CRF mRNA and pituitary ACTH mRNA expression and upregulate hypothalamic GR mRNA expression in CI-RI rats, which may contribute to its effect in relieving CI-RI.

Development of an automated on-line pre-column derivatization procedure for sensitive determination of histamine in food with high-performance liquid chromatography-fluorescence detection.

An improved sensitive method was developed and validated for the determination of histamine in food samples by using automated on-line pre-column derivatization coupled with high performance liquid chromatography and fluorescence detection (HPLC-FLD). o-Phthaldialdehyde (OPA) was adopted as derivatization reagent, and a "sandwich" (OPA+histamine+OPA) aspiration mode for the automated on-line pre-column derivatization was found to efficiently enhance the method sensitivity and precision. Histamine in food samples was efficiently extracted with a methanol-phosphate buffer solution (50:50, v/v) at 60 degrees C for 30 min, and purified with Waters Oasis MCX solid-phase extraction (SPE) cartridge. The limit of quantification for this method is 0.2 mg/kg, which is very sensitive for histamine determination. With the "sandwich" injection program, 3.7% of relative standard deviation (RSD) was achieved by five replicative determinations of a sample blank spiked with 0.25 mg/kg histamine standard. Histamine in food samples such as fumitory skipjack and mackerel was analyzed with relative recoveries over 95% at spiking level of 150 mg/kg, as well as canned tuna fish and cheese with relative recoveries up to 98% at spiking levels of 0.50 and 5.0 mg/kg, respectively. The proposed method was validated with a sample from the Food Analysis Performance Assessment Scheme (FAPAS) as a standard certified material; and the results (140+/-6 mg/kg) agreed well with the assigned value (139 mg/kg).

[Clinical observation on acupuncture with pushing manipulation for treatment of finger flexion in the patient of poststroke].

OBJECTIVE: To compare therapeutic effects of acupuncture with pushing manipulation and routine acupuncture on finger flexion in the patient of poststroke. METHODS: Eighty cases were randomly divided into a group of acupuncture with pushing manipulation and a routine acupuncture group. Hegu (LI 4), Houxi (SI 3), Waiguan (TE 5) were selected in the both groups. For the group of acupuncture with pushing manipulation (n=42), after arrival of qi acupuncture with pushing manipulation was given, the needle was inserted forcedly downwards, heavily thrust and lightly lifted with the thumb forward and the index-finger backward, and the needle was retained for 30 min. For the routine acupuncture group, after arrival of qi the needle was retained for 30 min. The therapeutic effects were assessed by modified Ashworth spastic rating and activity of metacarpophalangeal articulations after treatment of 30 days and 60 days in the two groups. RESULTS: The total effective rate of 81.0% in the group of acupuncture with pushing manipulation was significantly better than 57.9% in the routine acupuncture group (P<0.05); there was a significant or a very significant difference between the two groups in changes of the spastic degree and the activity of metacarpophalangeal articulations after treatment (P<0.05, P<0.01). CONCLUSION: Acupuncture with pushing manipulation has a definite therapeutic effect on finger flexion in the patient of poststroke.

[Clinical observation on abdominal electroacupuncture for treatment of poststroke constipation].

OBJECTIVE: To compare therapeutic effects of abdominal electroacupuncture (EA) and western medicine on poststroke constipation. METHODS: Eighty cases were randomly divided into an EA group and a medication group, 40 cases in each group. The EA group were treated with EA at Daheng (SP 15), Fujie (SP 14), Tianshu (ST 25), Shuidao (ST 28), etc., once a day, 30 min each session, and the medication group with oral administration of 10 mg Cisapride, thrice each day. Seven days constituted one course. After 2 courses, clinical therapeutic effects were evaluated by cumulative scores of symptoms. RESULTS: The total effective rate of 92.5% in the EA group was significantly better than 72.5% in the medication group (P < 0.05). After treatment, the cumulative scores of clinical symptoms significantly decreased in the two groups (P < 0.05) and the improving degrees of symptoms in the EA group was significantly better than that in the medication group (P < 0.05). CONCLUSION: Abdominal electroacupuncture has a definite therapeutic effect on poststroke constipation, accelerating gastrointestinal movement.

Functional role of transforming growth factor-beta type III receptor during palatal fusion.

The molecular regulation of palatogenesis continues to be an active area of investigation to provide a foundation for understanding the molecular etiology of cleft palate. Transforming growth factor (TGF) -beta type III receptor (TbetaR-III) has been shown to be specifically expressed in the medial edge epithelium at critical stages of palatal shelf adherence during palatogenesis. The aim of this study was to examine TbetaR-III mRNA localization and expression levels in vivo and to determine the requirement for TbetaR-III expression during palatal fusion in vitro. TbetaR-III gene expression was analyzed by in situ hybridization in tissue specimens and real-time reverse transcriptase-polymerase chain reaction using specific cells in the palatal shelf isolated by laser capture microdissection. TbetaR-III was knocked down in embryonic day (E) 13 palatal shelves in organ culture. Palatal shelf organ cultures were treated with small interfering RNA (siRNA) at final concentrations of 300, 400, and 500 nM, respectively. The treatment with siRNA specific for TbetaR-III decreased the amount of protein by approximately 75%. The reduction in TbetaR-III resulted in a delay in the process of palatal fusion compared with control. The protein expression of phospho-Smad2 was decreased in the TbetaR-III siRNA group. In addition, palatal organ cultures treated with TbetaR-III siRNA + rhTGF-beta3 completely fused by 72 hr in vitro. These results support our hypothesis that TbetaR-III has a critical role in the process of palatal fusion.

Inhibition of SMAD2 expression prevents murine palatal fusion.

Transforming growth factor (TGF)-beta 3 is known to regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Our previous studies showed that SMAD2, a TGF-beta signaling mediator, was expressed and phosphorylated primarily in the MEE and that SMAD2 phosphorylation in the MEE was temporospatially regulated by TGF-beta 3. The goal of this study was to examine the requirement for SMAD2 to complete the developmental events necessary for palatal fusion. SMAD2 expression was inhibited with Smad2 siRNA transfection into palatal tissues in vitro. The results showed that Smad2 siRNA transfection resulted in the maintenance of MEE cells in the palatal midline. Western blot and immunofluorescence analyses confirmed that the endogenous SMAD2 and phospho-SMAD2 levels were reduced following siRNA transfection. The SMAD3 level was not altered by the Smad2 siRNA transfection. The persistence of the MEE and the decreased SMAD2/phospho-SMAD2 levels were coincident with increased MEE cell proliferation. Addition of exogenous TGF-beta 3 increased p-SMAD2 level but not the total SMAD2 level. Therefore, exogenous TGF-beta 3 was not able to induce p-SMAD2 enough to rescue the palatal phenotype in the Smad2 siRNA group. The results indicated that the endogenous SMAD2 level is crucial in the regulation of disappearance of MEE during palatal fusion.

Overexpression of Smad2 in Tgf-beta3-null mutant mice rescues cleft palate.

Transforming growth factor (TGF)-beta3 is an important contributor to the regulation of medial edge epithelium (MEE) disappearance during palatal fusion. SMAD2 phosphorylation in the MEE has been shown to be directly regulated by TGF-beta3. No phospho-SMAD2 was identified in the MEE in Tgf-beta3-null mutant mice (Tgf-beta3-/-), which was correlated with the persistence of the MEE and failure of palatal fusion. In the present study, the cleft palate phenotype in Tgf-beta3-/- mice was rescued by overexpression of a Smad2 transgene in Keratin 14-synthesizing MEE cells following mating Tgf-beta3 heterozygous mice with Keratin 14 promoter directed Smad2 transgenic mice (K14-Smad2). Success of the rescue could be attributed to the elevated phospho-SMAD2 level in the MEE, demonstrated by two indirect evidences. The rescued palatal fusion in Tgf-beta3-/-/K14-Smad2 mice, however, never proceeded to the junction of primary and secondary palates and the most posterior border of the soft palate, despite phospho-SMAD2 expression in these regions at the same level as in the middle portion of the secondary palate. The K14-Smad2 transgene was unable to restore all the functional outcomes of TGF-beta3. This may indicate an anterior-posterior patterning in the palatal shelves with respect to TGF-beta3 signaling and the mechanism of secondary palatal fusion.

Effect of N'-nitrosonornicotine (NNN) on murine palatal fusion in vitro.

Maternal smoking has been linked to an increased risk for orofacial clefts. N'-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines that has been shown to be linked to the deleterious effects of tobacco and could be linked to the formation of cleft palate birth defects. The effect of NNN on palatal fusion was examined using an in vitro organ culture model of palatal development. The organ cultures were exposed to NNN (0.01, 0.1, 1, 10 and 100 mM) and the effects on palatal development characterized at defined points. Palatal fusion was evaluated at embryonic day 13 (E13)+72 h by characterizing the remaining medial edge epithelium (MEE) and determining the extent of fusion compared to controls. The NNN-treated group (1 mM) had more MEE remaining in the palatal midline than the untreated group at E13+72 h (P<0.05). Changes in cell proliferation in the MEE resulting from NNN exposure were examined by BrdU incorporation in replicating DNA. Changes in the pattern of MEE cell death were examined by TUNEL. BrdU incorporation and TUNEL staining showed that the NNN (1 mM)-treated palates had more MEE cell proliferation and less apoptosis than the untreated-palates at E13+24 h (P<0.05). The mechanism altered by NNN was further evaluated by characterizations of extracellular signal-regulated kinase (ERK) 1/2, p38 and c-jun amino-terminal kinase (JNK). NNN at 1 mM induced ERK1/2 phosphorylation, but reduced p38 phosphorylation (P<0.05, P<0.01, respectively) in the MEE. The results suggest that NNN inhibited palatal fusion by effects on cell proliferation and MEE cell death.

Role of ERK1/2 signaling during EGF-induced inhibition of palatal fusion.

During mammalian palatal fusion, the medial edge epithelial (MEE) cells must stop DNA synthesis prior to the initial contact of opposing palatal shelves and thereafter selectively disappear from the midline. Exogenous EGF has been shown to inhibit the cessation of DNA synthesis and induce cleft palate; however, the precise intracellular mechanism has not been determined. We hypothesized that EGF signaling acting via ERK1/2 would maintain MEE DNA synthesis and cell proliferation and consequently inhibit the process of palatal fusion. Palatal shelves from E13 mouse embryos were maintained in organ cultures and stimulated with EGF. EGF-treated palates failed to fuse with intact MEE and had significant ERK1/2 phosphorylation. Both EGF-induced ERK1/2 phosphorylation and BrdU-incorporation were localized in the nucleus of MEE cells. Subsequent inhibition assays using U0126, a specific inhibitor of ERK1/2 phosphorylation, were conducted. U0126 inhibited EGF-induced ERK1/2 phosphorylation in a dose-dependent manner and consequently MEE cells stopped proliferation. The threshold of ERK1/2 inactivation to stop MEE DNA synthesis coincides with the level required to rescue the EGF-induced cleft palate phenotype. These results indicate that EGF-induced inhibition of palatal fusion is dependent on nuclear ERK1/2 activation and that this mechanism must be tightly regulated during normal palatal fusion.

TGF-beta3-dependent SMAD2 phosphorylation and inhibition of MEE proliferation during palatal fusion.

Transforming growth factor (TGF) -beta3 is known to selectively regulate the disappearance of murine medial edge epithelium (MEE) during palatal fusion. Previous studies suggested that the selective function of TGF-beta3 in MEE was conducted by TGF-beta receptors. Further studies were needed to demonstrate that the TGF-beta signaling mediators were indeed expressed and phosphorylated in the MEE cells. SMAD2 and SMAD3 were both present in the MEE, whereas SMAD2 was the only one phosphorylated during palatal fusion. SMAD2 phosphorylation was temporospatially restricted to the MEE and correlated with the disappearance of the MEE. No phosphorylated SMAD2 was found in MEE in TGF-beta3(-/-) mice, although nonphosphorylated SMAD2 was present. The results suggest that TGF-beta3 is required for initiating and maintaining SMAD2 phosphorylation in MEE. Phospho-SMAD3 was not detectable in palate during normal palatal fusion. Previous results suggested TGF-beta-induced cessation of DNA synthesis in MEE cells during palatal fusion in vitro. The present results provide evidence that inhibition of MEE proliferation in vivo was controlled by endogenous TGF-beta3. The number of 5-bromo-2'-deoxyuridine (BrdU) -labeled MEE cells was significantly reduced in TGF-beta3(+/+) compared with TGF-beta3(-/-) mice when the MEE seam formed (t-test, P < 0.05). This finding suggests that TGF-beta3 is required for inhibiting MEE proliferation during palatal fusion. The inhibition of MEE proliferation may be mediated by TGF-beta3-dependent phosphorylation of SMAD2.