Norepinephrine protects against apoptosis of mesenchymal stem cells induced by high glucose.

In diabetes, the number of bone mesenchymal stem cells (MSCs) decreases and their differentiation is impaired. However, the exact mechanism is unclear. Patients with diabetes often experience sympathetic nerve injury. Norepinephrine (NE), a major mediator of the sympathetic nervous system, influences rat MSC migration in culture and in vivo. The present study aimed to investigate the effect of NE on MSCs under high glucose conditions; therefore MSCs were treated with high glucose and NE. High glucose-induced MSCs apoptosis, which was reversed by NE. To verify the effect of NE, mice underwent sympathectomy and were used to establish a diabetic model. Diabetic mice with sympathectomy had a higher apoptosis rate and higher levels of reactive oxygen species in their bone marrow-derived cells than diabetic mice without sympathectomy. High glucose inhibited p-AKT production and B-Cell CLL/Lymphoma 2 expression, and promoted BAX and caspase-3 expression. NE reversed these effects of high glucose. An AKT inhibitor enhanced the effects of high glucose. Thus, NE had a protective effect on MSC apoptosis induced by high glucose, possibly via the AKT/BCL-2 pathway.

Identification of circular RNA-associated competing endogenous RNA network in the development of cleft palate.

Circular RNAs (circRNAs) serve as competing endogenous RNAs (ceRNAs) and indirectly regulate gene expression through shared microRNAs (miRNAs). However, the regulatory mechanisms of circRNA as ceRNA associated with the fusion of palatal shelves in palatogenesis are yet unclear. This study aimed to explore the potential mechanism underlying the role of circRNA as ceRNA in cleft palate (CP). First, we systematically analyzed RNA-seq and miRNA-seq data after high-throughput sequencing for embryonic palatal shelf tissues from a mouse CP model induced by maternal exposure to all-trans retinoic acid on embryonic gestation day 14.5 (E14.5). Thirty-nine circRNAs, 18 miRNAs, and 936 messenger RNAs (mRNAs) were significantly dysregulated (log2 [fold change {FC}] > 1; P < 0.05). Thereafter, we constructed a circRNA-associated ceRNA network. Finally, we determined the circRNA_0954-miRNA-881-3p-PRKAR1α ceRNA network as a hub involved in palatogenesis. Gene Ontology analysis revealed that ceRNA-related genes were associated with facial morphogenesis and developmental gene silencing. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that ceRNA-related genes are involved in apoptosis (P < 0.05, fold enrichment >1). Quantitative reverse transcription polymerase chain reaction was performed to verify the results of ceRNA analysis. We found that the circRNA-miRNA-mRNA ceRNA network is involved in palatogenesis. The present results imply that circRNA_0954-miRNA-881-3p-PRKAR1α ceRNA network may cause dysfunctional palatal fusion and might facilitate the development of novel epigenetic biomarkers to treat CP in the future.

Application of standardized platelet-rich plasma in elderly patients with complex wounds.

In recent years, autologous platelet-rich plasma (PRP) derivatives have been used widely in the regeneration and repair of tissue, but a standard definition and preparation method for PRP are lacking. We developed a standardized method using platelet indices as quality-control indicators for PRP preparation. Twenty-one elderly patients (9 males, 12 females) with complex wounds were treated with standardized platelet-rich plasma (S-PRP). The platelet count in PRP after the second centrifugation was 1,069-1,436 × 109 /L. We adjusted the platelet concentration in PRP after a second centrifugation to 1,000 × 109 /L according to a formula using platelet-poor plasma (PPP). The standardized preparation method that we developed gave S-PRP with a relatively uniform platelet concentration. The wounds of 21 patients showed accelerated healing after S-PRP treatment, and there were no obvious side effects during treatment. These data suggest that our preparation method of S-PRP, using platelet indices as quality-control indicators with platelet count of 1,000 × 109 /L could be used for the treatment of complex wounds in the elderly. The preparation method of S-PRP proposed in the present study may be a simple and effective method of PRP quality control.

Position-dependent correlation between TBX22 exon 5 methylation and palatal shelf fusion in the development of cleft palate.

DNA methylation is essential for spatiotemporally-regulated gene expression in embryonic development. TBX22 (Chr X: 107667964-107688978) functioning as a transcriptional repressor affects DNA binding, sumoylation, and transcriptional repression associated with X-linked cleft palate. This study aimed to explore the relationship and potential mechanism between TBX22 exon 5 methylation and palatal shelf fusion induced by all-trans retinoic acid (ATRA). We performed DNA methylation profiling, using MethylRAD-seq, after high throughput sequencing of mouse embryos from control (n=9) and ATRA-treated (to induce cleft palate, n=9) C57BL/6J mice at embryonic gestation days(E) 13.5, 14.5 and 16.5. TBX22 exon 5 was hyper-methylated at the CpG site at E13.5 (P=0.025, log2FC=1.5) and E14.5 (P=0.011, log2FC:1.5) in ATRA-treated, whereas methylation TBX22 exon 5 at the CpG site was not significantly different at E16.5 (P=0.808, log2FC=-0.2) between control and ATRA-treated. MSP results showed a similar trend consistent with the MethylRAD-seq results. qPCR showed the change in TBX22 exon 5 expression level negatively correlated with its TBX22 exon 5 methylation level. These results indicate that changes in TBX22 exon 5 methylation might play an important regulatory role during palatal shelf fusion, and may enlighten the development of novel epigenetic biomarkers in the treatment of CP in the future.

Investigating the effect of age on platelet ultrastructure using transmission electron microscopy.

In the present study, the age- and sex-related differences in platelet ultrastructure were investigated using transmission electron microscopy (TEM). A total of 15 healthy volunteers were grouped according to age, with 5 people in each of the following groups: young group (25-45 years), middle-aged group (46-65 years), and old-aged group (> 65 years). In the TEM micrographs, the internal components, specifically the α-granules, dense granules, and lysosomal granules, of 20 platelets were counted for each group. Two-way analysis of variance of age and sex variance was used to compare the results. The ultrastructure of the platelets in the old-aged group was observed to be quite different from those of the young and middle-aged groups. Specifically, with ageing, the platelet membrane becomes more irregular in shape and non-smooth, and multiple platelet membrane ruptures are observed. Furthermore, the pseudopodia and protuberances become more numerous and slender, and the number of α-granules is significantly reduced. These morphological changes indicate that ageing may affect the function of platelets, which in turn affects the efficacy of platelet concentrates. Thus, the effects of age should be considered when using platelet concentrates prepared from elderly autologous blood.

A Modified Hyaluronic Acid-Based Dissolving Microneedle Loaded With Daphnetin Improved the Treatment of Psoriasis.

Psoriasis is a common chronic immune-inflammatory disease. Challenges exist in the present treatment of psoriasis, such as difficulties in transdermal drug administration and severe side effects. We hope to achieve a better therapeutic outcome for psoriasis treatment. By using modified soluble microneedles (MNs) loaded with daphnetin, the psoriasis symptoms of mice, the abnormal proliferation of keratinocytes, and the secretion of inflammatory factors were significantly reduced. In vitro, daphnetin is proven to inhibit the NF-κB signaling pathway and to inhibit the proliferation of HaCaT cells and the release of inflammatory factors, especially CCL20. This research showed that the modified microneedle loaded with daphnetin optimized transdermal drug delivery and relieved the symptoms of psoriasis more effectively. The novel route of Daph administration provides a future research direction for the treatment of psoriasis.

Intense Pulsed Light Treatment for Itch Associated with Allergic Keratoconjunctivitis: A Retrospective Study of 35 Cases.

Objective: To conduct a preliminary assessment of intense pulsed light (IPL) treatment for allergic keratoconjunctivitis (AKC)-associated ocular itch. Background: Current control measures for AKC rely primarily on drugs. IPL is effective for dry eye disease (DED). Furthermore, phototherapy is effective for managing skin inflammation and pruritus, suggesting that eye itching could decrease in some patients having AKC complicated with DED following IPL treatment to control dry eye symptoms. Methods: Thirty-five patients having DED complicated with mid-to-severe AKC were administered three IPL treatments to the periorbital skin. The eye scores of subjective symptoms and signs of AKC and tear film breakup time (TBUT) were retrospectively assessed before and after each treatment. Results: The scores for AKC-related symptoms and signs were determined four times: on Day 1 (time 0), Day 15 (time 1), Day 45 (time 2), and Day 75 (time 3) before each treatment. The average symptom score significantly decreased with treatments (time 0: 30.97, time 1: 15.03, time 3: 10). The average sign score for both eyes decreased after the first IPL treatment (left eye: 7.97 vs. 11.38; right eye: 8.1 vs. 11.1). There were no further improvements in the signs after the last treatment. The TBUT value in the right eye increased from times 0 to 3 (2.31 vs. 4.66 vs. 7.71 vs. 7.74). The TBUT value in the left eye increased from times 0 to 3 (2.50 vs. 6.97 vs. 7.57 vs. 8.24). Conclusions: Symptoms and signs improved after IPL treatment in patients with AKC. Eye itching was gradually controlled and rarely recurred. IPL may be effective for AKC treatment.

Co-administration of platelet-rich plasma and small intestinal submucosa is more beneficial than their individual use in promoting acute skin wound healing.

Background: Acute skin wounds may compromise the skin barrier, posing a risk of infection. Small intestinal submucosa (SIS) is widely used to treat acute and chronic wounds. However, the efficacy of SIS to accelerate wound healing still needs to be improved to meet clinical demands. To tackle this problem, platelet-rich plasma (PRP) is used due to its potency to promote proliferation, migration and adhesion of target cells. In this study, we applied PRP and SIS to skin wounds to explore their effects on wound healing by evaluating re-epithelialization, collagen production, angiogenesis and the inflammatory response. Methods: A 1 × 1-cm full-thickness skin defect was established in mice. Sixty mice were divided into four treatment groups: PRP + SIS, PRP, SIS and control. On days 3, 5, 7, 10 and 14 post-surgery, tissue specimens were harvested. Haematoxylin and eosin, Masson's trichrome, immunohistochemical and immunofluorescence double staining were used to visualize epidermal thickness, collagen and vascular regeneration and inflammation. Results: Wound contraction in the PRP and PRP + SIS groups was significantly greater, compared with the other groups, on days 3 and 5 post-surgery. A histological analysis showed higher collagen expression in the PRP and PRP + SIS groups on day 7, which was associated with a thicker epidermal layer on day 14. In addition, immunohistochemical staining showed that CD31-positive blood vessels and vascular endothelial growth factor expression in the PRP + SIS and PRP groups were significantly higher, compared with the control group. Furthermore, immunofluorescence double staining showed that the number of M1 and M2 macrophages in the PRP + SIS and PRP groups was higher, compared with the control and SIS groups alone, on day 3. However, on day 7, the number of M1 macrophages dramatically decreased in the PRP + SIS and PRP groups. The ratio of M2 to M1 macrophages in the PRP + SIS and PRP groups was 3.97 and 2.93 times that of the control group and 4.56 and 3.37 times that of the SIS group, respectively. Conclusion: Co-administration of SIS and PRP has a better effect on promoting angiogenesis, re-epithelialization and collagen regeneration in managing acute wound healing than either agent alone.

Histatin 1 enhanced the speed and quality of wound healing through regulating the behaviour of fibroblast.

OBJECTIVES: Histatin 1(Hst 1) has been proved to promote wound healing. However, there was no specific study on the regulation made by Hst 1 of fibroblasts in the process of wound healing. This research comprehensively studied the regulation of Hst 1 on the function of fibroblasts in the process of wound healing and preliminary mechanism about it. MATERIALS AND METHODS: The full-thickness skin wound model was made on the back of C57/BL6 mice. The wound healing, collagen deposition and fibroblast distribution were detected on days 3, 5 and 7 after injury. Fibroblast was cultured in vitro and stimulated with Hst 1, and then, their biological characteristics and functions were detected. RESULTS: Histatin 1 can effectively promote wound healing, improve collagen deposition during and after healing and increase the number and function of fibroblasts. After healing, the mechanical properties of the skin also improved. In vitro, the migration ability of fibroblasts stimulated by Hst 1 was significantly improved, and the fibroblasts transformed more into myofibroblasts, which improved the function of contraction and collagen secretion. In fibroblasts, mTOR signalling pathway can be activated by Hst 1. CONCLUSIONS: Histatin 1 can accelerate wound healing and improve the mechanical properties of healed skin by promoting the function of fibroblasts. The intermolecular mechanisms need to be further studied, and this study provides a direction about mTOR signalling pathway.

Erbium fractional laser irradiation combined with autologous platelet-rich plasma and platelet-poor plasma application for facial rejuvenation.

BACKGROUND: Erbium fractional laser treatment has immense skin rejuvenation effects, but it is associated with side effects such as erythema and pigmentation. Platelet-rich plasma (PRP) can enhance the restorative effects of Erbium fractional laser, but there are few studies on this combined treatment in the Asian population, and no study has examined the effects of adding platelet-poor plasma (PPP) to the combination. OBJECTIVE: This study aims to investigate the effects and safety of Erbium fractional laser irradiation combined with autologous PRP and PPP therapy for facial rejuvenation. METHODS: Between January 2010 and June 2016, 158 patients with facial skin aging were treated by Erbium fractional laser irradiation combined with autologous PRP and PPP. After three sessions, patients and experienced physicians evaluated the effectiveness of the treatment. RESULTS: The symptoms of skin aging, especially skin color, pore expansion, and skin texture, showed obvious improvement after the treatment, according to the evaluation of the patients and the physicians, who reported a total treatment effectiveness rate of 90.51% and 88.61%, respectively. The treatment was well-tolerated by all the participants, and no hyperpigmentation or depigmentation was observed in any of the cases. The reported side effects were edema (1-3 days), erythema (2-4 days), and crusting (3-10 days). CONCLUSION: Erbium fractional laser irradiation combined with PRP and PPP application is an effective and safe approach for improving facial skin aging and has minimal side effects. Future investigations on a bigger sample with a longer follow-up period should focus on optimizing the treatment protocol and settings.

Human Salivary Histatin-1 Is More Efficacious in Promoting Acute Skin Wound Healing Than Acellular Dermal Matrix Paste.

A rapid wound healing is beneficial for not only recovering esthetics but also reducing pain, complications and healthcare burdens. For such a purpose, continuous efforts have been taken to develop viable dressing material. Acellular dermal matrix (ADM) paste has been used to repair burn wounds and is shown to promote angiogenesis as well as fibroblast attachment and migration. However, its efficacy still needs to be significantly improved to meet clinical demands for accelerating acute skin wound healing. To approach this problem, we studied the added value of a human salivary peptide - Histatin 1 (Hst1). Hst1 was chosen because of its potency to promote the adhesion, spreading, migration, metabolic activity and cell-cell junction of major skin cells and endothelial cells. In this study, we hypothesized that ADM paste and Hst1 showed a better effect on the healing of surgically created acute skin wounds in mice since ADM paste may act as a slow release system for Hst1. Our results showed that the healing efficacy of 10 µM topically administrated Hst1 was significantly higher compared to the control (no Hst1, no ADM) from day 3 to day 10 post-surgery. In contrast, ADM alone failed in our system at all time points. Also, the combination of ADM paste and Hst1 did not show a better effect on percentage of wound healing. Histological analysis showed that 10 µM Hst1 was associated with maximal thickness of newly formed epidermal layer on day 7 as well as the largest collagen area on day 14. In addition, immunohistochemical staining showed that the number of CD31-positive blood vessels in the group of 10 µM Hst1 was 2.3 times compared to the control. The vascular endothelial growth factor (VEGF) expression in the groups of 10 µM Hst1 group and ADM + 10 µM Hst1 group was significantly higher compared with the control group. Furthermore, 10 µM Hst1 group was associated with significantly lower levels of CD68-positive macrophage number, interleukin-1ß (IL-1ß) expression and C-reactive protein (CRP) expression than those of the other groups (control, ADM alone and ADM + 10 µM Hst1). In contrast, ADM was only associated with significantly lower CD68-positive macrophage number and IL-1ß expression in comparison with the control. The co-administration of Hst1 and ADM paste did not yield more beneficial effects than Hst1 alone. In conclusion, the topically administrated of 10 µM Hst1 could be a promising alternative dressing in managing acute wound healing.

TNF-α suppresses sweat gland differentiation of MSCs by reducing FTO-mediated m6A-demethylation of Nanog mRNA.

An effect of inhibition of tumor necrosis factor-α (TNF-α) on differentiation of mesenchymal stromal cells (MSCs) has been demonstrated, but the exact mechanisms that govern MSCs differentiation remain to be further elucidated. Here, we show that TNF-α inhibits the differentiation of MSCs to sweat glands in a specific sweat gland-inducing environment, accompanied with reduced expression of Nanog, a core pluripotency factor. We elucidated that fat mass and obesity-associated protein (FTO)-mediated m6A demethylation is involved in the regulation of MSCs differentiation potential. Exposure of MSCs to TNF-α reduced expression of FTO, which demethylated Nanog mRNA. Reduced expression of FTO increased Nanog mRNA methylation, decreased Nanog mRNA and protein expression, and significantly inhibited MSCs capacity for differentiation to sweat gland cells. Our finding is the first to elucidate the functional importance of m6A modification in MSCs, providing new insights that the microenvironment can regulate the multipotency of MSCs at the post-transcriptional level. Moreover, to maintain differentiation capacity of MSCs by regulating m6A modification suggested a novel potential therapeutic target for stem cell-mediated regenerative medicine.

DNA hypermethylation of Fgf16 and Tbx22 associated with cleft palate during palatal fusion.

OBJECTIVE: Cleft palate (CP) is a congenital birth defect caused by the failure of palatal fusion. Little is known about the potential role of DNA methylation in the pathogenesis of CP. This study aimed to explore the potential role of DNA methylation in the mechanism of CP. METHODOLOGY: We established an all-trans retinoic acid (ATRA)-induced CP model in C57BL/6J mice and used methylation-dependent restriction enzymes (MethylRAD, FspEI) combined with high-throughput sequencing (HiSeq X Ten) to compare genome-wide DNA methylation profiles of embryonic mouse palatal tissues, between embryos from ATRA-treated vs. untreated mice, at embryonic gestation day 14.5 (E14.5) (n=3 per group). To confirm differentially methylated levels of susceptible genes, real-time quantitative PCR (qPCR) was used to correlate expression of differentially methylated genes related to CP. RESULTS: We identified 196 differentially methylated genes, including 17,298 differentially methylated CCGG sites between ATRA-treated vs. untreated embryonic mouse palatal tissues (P<0.05, log2FC>1). The CP-related genes Fgf16 (P=0.008, log2FC=1.13) and Tbx22 (P=0.011, log2FC=1.64,) were hypermethylated. Analysis of Fgf16 and Tbx22, using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), identified 3 GO terms and 1 KEGG pathway functionally related to palatal fusion. The qPCR showed that changes in expression level negatively correlated with methylation levels. CONCLUSIONS: Taken together, these results suggest that hypermethylation of Fgf16 and Tbx22 is associated with decreased gene expression, which might be responsible for developmental failure of palatal fusion, eventually resulting in the formation of CP.

Properties of an alginate-gelatin-based bioink and its potential impact on cell migration, proliferation, and differentiation.

Three-dimensional (3D) bioprinting allows embedding of cells within a bioink, creating cell-based 3D structures to promote tissue regeneration and repair. The bioink should be biocompatible with the cells and its effect on cell behavior should be determined. Alginate-gelatin (Alg-Gel) blends with mouse planta dermis (PD) induced epidermal progenitors for sweat gland regeneration, confirming the role of 3D support during the process. The present study aimed to investigate the chemical and physical properties of the Alg-Gel-PD bioink, and its effect on embedded mouse mesenchymal stem cells (MSCs). MSCs showed increased proliferation and migration in 2D culture with an Alg-Gel-based bioink extract. Gene expression analysis confirmed MSC differentiation towards sweat gland cells. The extract had no effect on protein expression in differentiated cells. Mixing MSCs with the Alg-Gel-based bioink for 3D bioprinting resulted in gene and protein expression characteristic of differentiation, including YAP1 activation. The mechanical strength of the bioink was similar to that of mouse dermal tissue and scanning electron microscopy showed that PD induced a more regular pore structure, suggesting advantages for the physical properties of the embedded cells. This study determined the influence of bioink on cellular behavior, thereby promoting therapeutic stem cell function via 3D cell printing processes.

Redirecting differentiation of mammary progenitor cells by 3D bioprinted sweat gland microenvironment.

BACKGROUND: Mammary progenitor cells (MPCs) maintain their reproductive potency through life, and their specific microenvironments exert a deterministic control over these cells. MPCs provides one kind of ideal tools for studying engineered microenvironmental influence because of its accessibility and continually undergoes postnatal developmental changes. The aim of our study is to explore the critical role of the engineered sweat gland (SG) microenvironment in reprogramming MPCs into functional SG cells. METHODS: We have utilized a three-dimensional (3D) SG microenvironment composed of gelatin-alginate hydrogels and components from mouse SG extracellular matrix (SG-ECM) proteins to reroute the differentiation of MPCs to study the functions of this microenvironment. MPCs were encapsulated into the artificial SG microenvironment and were printed into a 3D cell-laden construct. The expression of specific markers at the protein and gene levels was detected after cultured 14 days. RESULTS: Compared with the control group, immunofluorescence and gene expression assay demonstrated that MPCs encapsulated in the bioprinted 3D-SG microenvironment could significantly express the functional marker of mouse SG, sodium/potassium channel protein ATP1a1, and tend to express the specific marker of luminal epithelial cells, keratin-8. When the Shh pathway is inhibited, the expression of SG-associated proteins in MPCs under the same induction environment is significantly reduced. CONCLUSIONS: Our evidence proved the ability of differentiated mouse MPCs to regenerate SG cells by engineered SG microenvironment in vitro and Shh pathway was found to be correlated with the changes in the differentiation. These results provide insights into regeneration of damaged SG by MPCs and the role of the engineered microenvironment in reprogramming cell fate.

Expression and localization of Forkhead transcription factor A1 in the three-dimensional reconstructed eccrine sweat glands.

Previously studies showed that Forkhead transcription factor A1 (FoxA1) was associated with sweat secretion. To investigate the expression and localization of FoxA1 in the three-dimensional (3D) reconstructed eccrine sweat glands, eccrine sweat gland cells were transplanted subcutaneously into nude mice with Matrigel, and at 2, 3, 4, 5, 6, 8, 10 and 12 weeks post-transplantation, the reconstructed eccrine sweat glands were removed and immunostained for FoxA1 and co-immunostained for FoxA1 and eccrine sweat markers, K7, carbonic anhydrase II (CA Ⅱ), gross cystic disease fluid protein-15 (GCDFP-15) and α-smooth muscle actin (α-SMA), and FoxA1 and sweat secretion-related proteins, Na+-K+-ATPase α and Na+-K+-2Cl- cotransporter 1 (NKCC1). The results showed that FoxA1-positive cells weren't detected until 3 weeks post-implantation, a time point of the differntiation of secretory coil-like structures. From the fourth week on, the number of FoxA1-positive cells increased and thereafter maintained at a high number. Double immunofluorescence staining showed that FoxA1-positive cells co-expressed dark cell marker GCDFP-15 and myoepithelial cell marker α-SMA, as well as secretion-related proteins, Na+-K+-ATPase α and NKCC1 in both the native and reconstructed eccrine sweat glands. In conclusion, FoxA1 might be related to the development and differentiation of secretory coil-like structures, as well as the secretory function of the 3D reconstructed eccrine sweat glands.

Differential Innervation of Secretory Coils and Ducts in Human Eccrine Sweat Glands.

BACKGROUND: Previous studies demonstrate that eccrine sweat glands are innervated by both cholinergic and adrenergic nerves. However, it is still unknown whether the secretory coils and ducts of eccrine sweat glands are equally innervated by the sympathetic nerve fibers. To well understand the mechanisms on sweat secretion and reabsorption, the differential innervation of secretory coils and ducts in human eccrine sweat glands was investigated in the study. METHODS: From June 2016 to June 2017, six human skins were fixed, paraffin-embedded, and cut into 5 µm-thick sections, followed by costaining for nerve fiber markers protein gene product 9.5 (PGP 9.5), tyrosine hydroxylase (TH) and vasoactive intestinal peptide (VIP), and eccrine sweat gland markers K7, S100P, and K14 by combining standard immunofluorescence with tyramide signal amplification (IF-TSA). Stained sections were observed under the microscope, photographed, and analyzed. RESULTS: The fluorescent signals of PGP 9.5, TH, and VIP were easily visualized, by IF-TSA, as circular patterns surrounding eccrine sweat glands, but only PGP 9.5 could be observed by standard IF. The IF-TSA method is more sensitivity than standard IF in detecting antigens expressed at low levels. PGP 9.5, TH, and VIP appeared primarily surrounding the secretory coils and sparsely surrounding the sweat ducts. CONCLUSION: Sweat secretion is mainly controlled by autonomic nerves whereas sweat reabsorption is less affected by nerve activity.

Genome-Wide DNA Methylation Analysis During Palatal Fusion Reveals the Potential Mechanism of Enhancer Methylation Regulating Epithelial Mesenchyme Transformation.

Epithelial mesenchyme transformation (EMT) of the medial edge epithelium (MEE) is the crucial process during palatal fusion. This work is aimed to elucidate the enhancer regulatory mechanism by genome-wide DNA methylation analysis of EMT during palatal fusion. Over 800 million clean reads, 325 million enzyme reads, and 234 million mapping reads were generated. The mapping rate was 68.85-74.32%, which included differentially methylated 17299 CCGG sites and 2363 CCWGG sites (p < 0.05, log2FC >1). Methylated sites in intron and intergenic regions were more compared to other regions of all DNA elements. GO and KEGG analysis indicated that differential methylation sites related to embryonic palatal fusion genes (HDAC4, TCF7L2, and PDGFRB) at the enhancer were located on CCWGG region of non-CpG islands. In addition, the results showed that the enhancer for HDAC4 was hypermethylated, whereas the enhancers for TCF7L2 and PDGFRB were hypomethylated. The methylation status of enhancer regions of HDAC4, PDGFRB, and TCF7L2, involved in the regulation of the EMT during palatal fusion, may enlighten the development of novel epigenetic biomarkers in the treatment of cleft palate.

DNA hypermethylation of Fgf16 and Tbx22 associated with cleft palate during palatal fusion

Abstract Objective: Cleft palate (CP) is a congenital birth defect caused by the failure of palatal fusion. Little is known about the potential role of DNA methylation in the pathogenesis of CP. This study aimed to explore the potential role of DNA methylation in the mechanism of CP. Methodology: We established an all-trans retinoic acid (ATRA)-induced CP model in C57BL/6J mice and used methylation-dependent restriction enzymes (MethylRAD, FspEI) combined with high-throughput sequencing (HiSeq X Ten) to compare genome-wide DNA methylation profiles of embryonic mouse palatal tissues, between embryos from ATRA-treated vs. untreated mice, at embryonic gestation day 14.5 (E14.5) (n=3 per group). To confirm differentially methylated levels of susceptible genes, real-time quantitative PCR (qPCR) was used to correlate expression of differentially methylated genes related to CP. Results: We identified 196 differentially methylated genes, including 17,298 differentially methylated CCGG sites between ATRA-treated vs. untreated embryonic mouse palatal tissues (P<0.05, log2FC>1). The CP-related genes Fgf16 (P=0.008, log2FC=1.13) and Tbx22 (P=0.011, log2FC=1.64,) were hypermethylated. Analysis of Fgf16 and Tbx22, using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG), identified 3 GO terms and 1 KEGG pathway functionally related to palatal fusion. The qPCR showed that changes in expression level negatively correlated with methylation levels. Conclusions: Taken together, these results suggest that hypermethylation of Fgf16 and Tbx22 is associated with decreased gene expression, which might be responsible for developmental failure of palatal fusion, eventually resulting in the formation of CP.