Reversibly immortalization establishes a hair follicle stem cell line with hair follicle reconstruction ability.

Hair follicle stem cells (HFSCs) play critical roles in the periodic regeneration of hair follicles. HFSCs are also a good model for stem cell biology research. However, no stable mouse HFSC cell line has been reported, which restricts the research and application of HFSCs. We isolated HFSCs from mouse hair follicles and immortalized them by inducing a reversible SV40 large T antigen. Through monoclonal screening, we identified a reversibly immortalized cell line, immortalized HFSC (iHFSC2). RNA sequencing, fluorescence-activated cell sorting, western blotting and immunofluorescence experiments revealed that the expression patterns of iHFSC2 and HFSC were similar at the protein and mRNA levels. After that, iHFSC2s were passaged and morphologically monitored for up to 40 times to detect their long-term culture potential. The long-term cultured iHFSC2 could regenerate hair follicles with complete hair follicle structure and HFSCs in the bulge area. This work successfully established an HFSC cell line with the ability of hair follicle reconstruction.

Profiling the oral microbiomes in patients with Alzheimer's disease.

OBJECTIVES: To analyse the characteristics of the oral microbiomes and expected to find biomarkers about Alzheimer's disease (AD). SUBJECTS AND METHODS: AD patients (n = 26) and cognitive intact people (n = 26) were examined for cognition, depression, oral health and collected saliva and gingival crevicular fluid (GCF) in the morning. Full-length 16S rRNA gene was amplified and sequencing was performed using the PacBio platform. RESULTS: The predominant bacterium of salivary microbiome and periodontal microbiome from AD patients was Streptococcus oralis and Porphyromonas gingivalis, respectively. With respect to ß diversity analysis, there was a significance difference in periodontal microbiome between AD patients and cognitively intact subjects. The relative abundance of Veillonella parvula significantly increased in oral microbiomes from AD patients. Interestingly, the dominant species were different between early-onset AD and late-onset AD patients. Moreover, the predominant species were changed as the clinical severity of AD. Furthermore, the correlation analysis revealed that V. parvula was associated with AD in both saliva and GCF and that P. gingivalis was associated with AD only in GCF. CONCLUSIONS: In this study, the microbiome community of oral microbes was altered in AD patients and periodontal microbiome was sensitive to cognition changes. Moreover, V. parvula and P. gingivalis were associated with AD.

Influence of power generation period on the oily sludge bio-electrical system.

The single-chamber bio-electrical systems can degrade oily sludge in sediments while generating electricity from the microbial fuel cells (MFCs) and their characteristics in energy and environmental effects have attracted wide international attention in recent years. To explore the influence of the power generation period on the oily sludge bio-electrical system, an oily sludge bio-electrical system was constructed. The output voltage, polarization curve, power density curve, crude oil removal rate and microflora were detected during different power generation periods, respectively. The results of this study showed that under the stable power generation period, the power generation and oily sludge degradation performance of MFC are higher than the voltage rise period and voltage attenuation period. Besides, the oily sludge bio-electrical system during the stable period contained more electricity-producing bacteria than the other two periods. The voltage in the stable period of oily sludge bio-electrical system is about 280 mV, the electromotive force is 493.1 mV and the power density is 134.93 mW·m-3. It lays a foundation for the improvement of degradation of crude oil and power generation performance in oily sludge bio-electrical system.

Screening and Demulsification Mechanism of Fluorinated Demulsifier Based on Molecular Dynamics Simulation.

In order to solve the problem of demulsification difficulties in Liaohe Oilfield, 24 kinds of demulsifiers were screened by using the interface generation energy (IFE) module in the molecular dynamics simulation software Materials Studio to determine the ability of demulsifier molecules to reduce the total energy of the oil-water interface after entering the oil-water interface. Neural network analysis (NNA) and genetic function approximation (GFA) were used as technical means to predict the demulsification effect of the Liaohe crude oil demulsifier. The simulation results show that the SDJ9927 demulsifier with ethylene oxide (EO) and propylene oxide (PO) values of 21 (EO) and 44 (PO) reduced the total energy and interfacial tension of the oil-water interface to the greatest extent, and the interfacial formation energy reached -640.48 Kcal/mol. NNA predicted that the water removal amount of the SDJ9927 demulsifier was 7.21 mL, with an overall error of less than 1.83. GFA predicted that the water removal amount of the SDJ9927 demulsifier was 7.41mL, with an overall error of less than 0.9. The predicted results are consistent with the experimental screening results. SDJ9927 had the highest water removal rate and the best demulsification effect. NNA and GFA had high correlation coefficients, and their R2s were 0.802 and 0.861, respectively. The higher R2 was, the more accurate the prediction accuracy was. Finally, the demulsification mechanism of the interfacial film breaking due to the collision of fluorinated polyether demulsifiers was studied. It was found that the carbon-fluorine chain had high surface activity and high stability, which could protect the carbon-carbon bond in the demulsifier molecules to ensure that there was no re-emulsion due to the stirring external force.

Correction to: The balance of Bmp6 and Wnt10b regulates the telogen-anagen transition of hair follicles.

Following publication of the original article [1], the authors reported that they would like to correct the second last sentence of "Authors' information" section as PW is an undergraduate, but was incorrectly described as a Ph.D. in the sentence. The sentence should read "PW is an undergraduate. YZ, YX, WX, HG, FD and YL are Ph.D.". The authors sincerely apologize for having this unintentional error in the article, and apologize for any inconvenience caused.

A Meta-Analysis of Oral Health Status of Children with Autism.

Purpose: To present a meta-analysis whether the risks of caries and periodontal problems in autistic children are higher than those in healthy children. Study design: A literature search that included PubMed, Embase, Web of Science, Cochrane, China National Knowledge Infrastructure (CNKI), Wan fang, and Chinese Scientific and Technological Journal (VIP) databases was conducted. The primary outcomes of interest included the DMFT index, Plaque index (PI), Gingival index (GI), and Salivary pH. Quality assessment was performed in accordance with the Newcastle-Ottawa Scale (NOS). Dichotomous variables are presented as relative risk (RR), and continuous variables are presented as weighted mean difference (WMD). Results: Eight studies were included in this meta-analysis. Among these 8 studies, six studies compared the DMFT index, three studies compared PI, three studies compared GI, and three studies compared salivary pH. Meta-analysis showed that the mean DMFT index in autistic children was higher than that in healthy children, and the difference was statistically significant {MD = 0.50, 95% CI [0.04-0.96], P<0.00001}. Similarly, PI and GI in autistic children were higher than those in healthy children, and the difference between PI was statistically significant {MD = 0.59, 95%CI [0.36-0.82], P=0.02}, while the difference between GI was not statistically significant {MD = 0.52, 95%CI [0.30-0.75], P=0.08}. But the salivary pH in autistic children was lower than that in healthy children {MD = -0.28, 95%CI [-0.54--0.02], P = 0.02}, and the difference was statistically significant. Conclusion: The present analysis suggests that children with autism have poorer oral hygiene, higher risk of caries, and a lower salivary pH than healthy children.

The balance of Bmp6 and Wnt10b regulates the telogen-anagen transition of hair follicles.

BACKGROUND: The periodic growth of hair follicles is regulated by the balance of activators and inhibitors. The BMP signaling pathway plays an important role during hair follicle regeneration, but the exact BMP protein that controls this process has not been revealed. METHODS: The expression of BMP6 was determined via in situ hybridization and immunofluorescence. The in vivo effect of BMP6 overexpression was studied by using a previously established adenovirus injection model. The hair follicle regeneration was assessed by gross observation, H&E staining and 5-bromo-2-deoxyuridine (BrdU) tracing. The expression patterns of BMP6 signaling and Wnt10b signaling in both AdBMP6-treated and AdWnt10b-treated skins were determined by in situ hybridization and immunofluorescence. RESULTS: BMP6 was expressed differently in the stages of hair follicle cycle. The telogen-anagen transition of hair follicles was inhibited by adenovirus-mediated overexpression of BMP6. In the in vivo model, the BMP6 signaling was inhibited by Wnt10b and the Wnt10b signaling was inhibited by BMP6. The activation of hair follicle stem cells (HFSCs) was also competitively regulated by Wnt10b and BMP6. CONCLUSIONS: Combined with previously reported data of Wnt10b, our findings indicate that BMP6 and Wnt10b are major inhibitors and activators respectively and their balance regulates the telogen-anagen transition of hair follicles. To the best of our knowledge, our data provide previously unreported insights into the regulation of hair follicle cycling and provide new clues for the diagnosis and therapies of hair loss.

Differential expression of miR-17-92 cluster among varying histological stages of minor salivary gland in patients with primary Sjögren's syndrome.

OBJECTIVES: To investigate the differential expression of miR-17-92 cluster, which encodes 6 microRNAs (miR-NAs) including miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a, among varying histological stages of labial minor salivary gland (MSG) tis- sues in patients with primary Sjögren's syndrome (pSS). METHODS: Fifty-seven pSS patients and 13 healthy volunteers were enrolled in this study. The pSS patients were allocated to 3 subgroups of advanced clinical stages according to the histological findings of the MSG biopsies. Salivary flow rate, Schirmer test and some laboratory indexes were also tested. The expression levels of the 6 miRNAs in MSG were evaluated using quantitative real-time polymerase chain reaction with TaqMan miRNA assay. RESULTS: The differences between the healthy individuals and the 3 pSS subgroups were statistically significant for positive findings of salivary flow rate, Schirmer test and laboratory indexes. In the labial MSG tissues, we observed that the expression level of miR-18a was significantly up-regulated in patients of the 3 pSS subgroups compared to healthy individuals, while the expression level of miR-92a was significantly down-regulated. We also observed that there was no notable difference in the expression levels of miR-17, miR-19a, miR-19b, and miR-20a. Furthermore, we distinguished that miR-18a was progressively up-regulated along the advanced histological stages of the 3 pSS subgroups, while the miR-92a was progressively down-regulated. CONCLUDIONS: This is the first study on the expression of the miR-17-92 cluster in MSG of pSS patients. The association of increased expression levels of miR-18a and reduced expression levels of miR-92a with advanced clinical stages of pSS could significantly reduce the substantial subjectivity of scoring inflammatory infiltrates and may aid in the diagnosis of pSS.

Gsdma3 is required for mammary gland development in mice.

AE (alopecia and excoriation) is a mouse mutant phenotype that harbours a mutation in Gsdma3. Gsdma3 has been reported to regulate the development of skin and hair follicles. However, its role in the mammary glands has not been reported. In this study, we found that descendants bred from an AE mother died within 12 days after birth. Then, we found that the expression of Gsdma3 varied among the developmental stages of mammary glands. Subsequently, we systematically studied the phenotype of the mammary glands of AE and wild-type mice, revealing that the mammary glands were smaller in AE mice. The mammary glands of AE mice exhibited shorter ductal extension and less bifurcation. Immunohistochemistry staining indicated that the mammary glands of AE mice displayed more proliferating cells during puberty while secreting less ß-casein during pregnancy and lactation. The lymph nodes in the mammary glands of the AE mice were larger and showed some pigmentation, suggesting that the immune reaction in the mammary glands was up-regulated. Under a transmission electron microscope, residual bodies were observed in the lymph nodes in the mammary glands of AE mice. Thus, we report a new function of Gsdma3 in regulating the development of mammary glands, and we demonstrate that the Gsdma3 gene may act as a suppressor of the immune reaction.

Effectiveness and safety of Chinese massage therapy (Tui Na) on post-stroke spasticity: a prospective multicenter randomized controlled trial.

OBJECTIVE: To evaluate the effectiveness and safety of Chinese massage therapy (Tui Na) for patients with post-stroke spasticity. DESIGN: A prospective, multicenter, blinded, randomized, placebo-controlled intervention trial. SUBJECT: A total of 90 patients with post-stroke spasticity were randomly assigned to the experimental (Tui Na therapy) group ( n = 45) or control (placebo Tui Na therapy) group ( n = 45). INTERVENTION: Participants in the experimental group received Tui Na therapy, while those in the control group received placebo-Tai Na (gentle rubbing) for 20-25 minutes per limb, once per day, five days per week for a total of four weeks. All participants in both groups received conventional rehabilitation. MAIN MEASURE: The Modified Ashworth Scale, the Fugl-Meyer Assessment and the Modified Barthel Index were used to assess the severity of spasticity, motor function of limbs and activities of daily living, respectively. Assessments were performed at baseline, at four weeks and at three months. RESULTS: Tui Na group had a significantly greater reduction in Modified Ashworth Scale in only four muscle groups than the control did (elbow flexors, P = 0.026; wrist flexors, P = 0.005; knee flexors, P = 0.023; knee extensors, P = 0.017). Improvements were sustained at three months follow-up. There was no significant difference between the two groups in Fugl-Meyer Assessment ( P = 0.503) and Modified Barthel Index ( P = 0.544). No adverse reaction was recorded in any of the cases mentioned at all study sites. CONCLUSIONS: Tui Na might be a safe and effective treatment to reduce post-stroke spasticity of several muscle groups.

Cloning and sequence analysis of Wild Argali short palate, lung and nasal epithelium clone 1 cDNA.

OBJECTIVE: Experiments were conducted to clone the sequence of Wild Argali short palate, lung and nasal epithelium clone 1 (SPLUNC1) cDNA, and to lay the foundation for further study the biological function of Wild Argali SPLUNC1. METHODS: The complete sequence of Wild Argali SPLUNC1 cDNA was generated by rapid amplification of cDNA ends. The entire coding sequence was inserted into the pPIC9K vector and expressed in Pichia pastoris (P. pastoris) GS115. The recombinant SPLUNC1 protein was detected by Western blot and purified by Ni2+ chelate affinity chromatography. The test of effect of the protein on Mycoplasma ovipneumoniae (MO) was performed with real-time polymerase chain reaction. RESULTS: The Wild Argali SPLUNC1 cDNA was 1,076 bp with an open reading frame of 768 bp, which encoded a 26.49 kDa protein composed of 255 amino acids. Its amino acid sequence shared 98.4%, 96.9%, 94.5%, 90.2%, 80.8%, 78.4%, 78.3%, 72.5%, 72.3%, 68.8% identity with those of SPLUNC1 cDNA from Ovis aries (accession no. NP_001288334.1), Capra hircus (accession no. XP_005688516.1), Pantholops hodgsonii (accession no. XP_005979709.1), Bos taurus (accession no. NP_776851.1), Felis catus (accession no. XP_006929910.1), Homo sapiens (accession no. NP_001230122.1), Sus scrofa (accession no. NP_001005727.1), Chinchilla lanigera (accession no. NP_001269294.1), Mus musculus (accession no. NP_035256.2), and Rattus norvegicus (accession no. NP_742028.1), respectively. The recombinant protein corresponded to the expected molecular mass of 25.47 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was detected in the supernatant of P. pastoris, and it could be purified. The results from the test of inhibition effect of argali recombinant SPLUNC1 protein on MO showed that the product could inhibit MO very well (p<0.01). CONCLUSION: The amino acid sequence of Wild Argali SPLUNC1 was different from other organisms. The recombinant SPLUNC1 protein has good biological activity.

12-O-tetradecanoylphorbol-13-acetate activates hair follicle melanocytes for hair pigmentation via Wnt/ß-catenin signaling.

Melanocyte stem cells (McSCs) undergo cyclical activation and quiescence together with hair follicle stem cells (HFSCs). This process is strictly controlled by the autonomous and extrinsic signaling environment. However, the modulation of factors important for the activation of McSCs for hair pigmentation remains unclear. 12-O-tetradecanoylphorbol-13-acetate (TPA) mimics vital signaling pathways involved in melanocyte growth and melanogenesis in vitro. To investigate whether TPA regulates quiescent McSCs for hair pigmentation, we topically smeared TPA on 7-week-old mouse dorsal skin and found that TPA stimulated hair growth and hair matrix pigmentation. These changes were associated with a significant increase in the number of hair bulb melanocytes. Moreover, in the TPA-treated group, hair bulge McSCs and hair bulb melanoblasts actively proliferated. At the molecular level, nuclear ß-catenin, a key factor of Wnt/ß-catenin signaling, was highly synthesized in melanocytes and keratinocytes in TPA-induced hair bulbs. Inhibition of Wnt/ß-catenin signaling by injecting Dickkopf1 plasmids into TPA-treated skin decreased hair matrix pigmentation and inhibited the proliferation and differentiation of McSCs. Our findings suggest that the topical application of TPA stimulates the proliferation and differentiation of McSCs and their progeny for hair matrix pigmentation by activating Wnt/ß-catenin signaling. This might provide a useful experimental model for the study of signals controlling the activation of McSCs.

Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b.

The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.

Wnt5a Suppresses ß-catenin Signaling during Hair Follicle Regeneration.

Hair follicles display periodic growth. Wnt signaling is a critical regulator for hair follicle regeneration. Previously, we reported that Wnt5a inhibits the telogen-to-anagen transition of hair follicles, but the mechanism by which this process occurs has not yet been reported. Here, we determined the expression patterns of Wnt signaling pathway molecules by quantitative reverse transcription polymerase chain reaction, western blot, and immunohistochemistry and found that ß-catenin signaling was suppressed by Wnt5a. We then compared the phenotypes and expression patterns following ß-catenin knockdown and Wnt5a overexpression during hair follicle regeneration induced by hair depilation and observed similar patterns. In addition, we performed a rescue experiment in the JB6 cell line and found that the inhibitory effect of Wnt5a on cell proliferation could be rescued by the addition of Wnt3a. Our data reveal that Wnt5a suppresses the activation of ß-catenin signaling during hair follicle regeneration.

Modulating hair follicle size with Wnt10b/DKK1 during hair regeneration.

Hair follicles have characteristic sizes corresponding to their cycle-specific stage. However, how the anagen hair follicle specifies its size remains elusive. Here, we showed that in response to prolonged ectopic Wnt10b-mediated ß-catenin activation, regenerating anagen hair follicles grew larger in size. In particular, the hair bulb, dermal papilla and hair shaft became enlarged, while the formation of different hair types (Guard, Awl, Auchene and Zigzag) was unaffected. Interestingly, we found that the effect of exogenous WNT10b was mainly on Zigzag and less on the other kinds of hairs. We observed dramatically enhanced proliferation within the matrix, DP and hair shaft of the enlarged AdWnt10b-treated hair follicles compared with those of normal hair follicles at P98. Furthermore, expression of CD34, a specific hair stem cell marker, was increased in its number to the bulge region after AdWnt10b treatment. Ectopic expression of CD34 throughout the ORS region was also observed. Many CD34-positive hair stem cells were actively proliferating in AdWnt10b-induced hair follicles. Importantly, subsequent co-treatment with the Wnt inhibitor, DKK1, reduced hair follicle enlargement and decreased proliferation and ectopic localization of hair stem cells. Moreover, injection of DKK1 during early anagen significantly reduced the width of prospective hairs. Together, these findings strongly suggest that Wnt10b/DKK1 can modulate hair follicle size during hair regeneration.

Recent progress in hair follicle stem cell markers and their regulatory roles.

Hair follicle stem cells (HFSCs) in the bulge are a multipotent adult stem cell population. They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing. An increasing number of biomarkers have been used to isolate, label, and trace HFSCs in recent years. Considering more detailed data from single-cell transcriptomics technology, we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.

Codonopsis pilosula polysaccharides (CPP) intervention alleviates sterigmatocystin (STC)-induced liver injury and gut microbiota dysbiosis.

Codonopsis pilosula polysaccharides (CPP), the main active ingredient of Codonopsis pilosula, has gained significant attention as a liver-protective agent. Previous studies have demonstrated that CPP could alleviate gut microbiota dysbiosis in colitis or obese mice. However, the effects of CPP on mycotoxin-induced liver injury and gut microbiota dysbiosis are still poorly understood. In this study, we aimed to investigate the protective effects of CPP on sterigmatocystin (STC)-induced liver injury, as well as its regulatory effects on gut microbiota. Our results revealed that CPP intervention significantly alleviated STC-induced liver injury, as evidenced by decreased liver index, reduced liver histopathological changes, and modulation of related molecular markers. Additionally, we found that CPP could alleviate liver injury by reducing liver inflammation and oxidative stress, inhibiting hepatocyte apoptosis, and regulating lipid metabolism. Notably, we also observed that CPP could alleviate STC-induced gut microbiota dysbiosis by modulating the diversity and richness of gut microbiota, suggesting that gut microbiota modulation may also serve as a mechanism for CPP-mediated remission of liver injury. In summary, our study not only provided a new theoretical basis for understanding the hepatotoxicity of STC and the protective effects of CPP against STC-induced liver injury, but also provided new perspectives for the application of CPP in the fields of food, healthcare products, and medicine.

Wnt5a inhibits the proliferation and melanogenesis of melanocytes.

Wnt5a, which is a noncanonical Wnt molecule, has been shown to be involved in a variety of developmental processes and cellular functions. In this study, we used "melan-a" cells as a cell model to investigate the effects of Wnt5a on melanocyte proliferation and melanogenesis, and to elucidate the possible mechanisms involved. We infected melan-a cells with recombinant Wnt5a adenoviruses to express Wnt5a protein and to simulate the Wnt5a processing environment. MTT assay and BrdU incorporation assay revealed that Wnt5a significantly inhibited the proliferation of melan-a cells. Melanin content and tyrosinase activity assays showed that Wnt5a was an inhibitor of melanin synthesis. Furthermore, RT-PCR and Western blot showed that this suppressive effect depended on noncanonical Wnt/Ror2 pathway activation and accessed the inhibition of the canonical Wnt pathway. The above results provided a novel insight into the role of Wnt5a and its related signaling in melanocyte homeostasis.

Wnt10b promotes differentiation of mouse hair follicle melanocytes.

Previous research has revealed that Wnt10b activates canonical Wnt signaling, which is integral to melanocyte differentiation in hair follicles (HFs). However, the function of Wnt10b in HF melanocytes remains poorly understood. We determined using Dct-LacZ transgenic mice that Wnt10b is mainly expressed near and within melanocytes of the hair bulbs during the anagen stage of the hair cycle. We also found that Wnt10b promotes an increase in melanocyte maturation and pigmentation in the hair bulbs of the mouse HF. To further explore the potential functions of Wnt10b in mouse HF melanocytes, we infected iMC23 cells with Ad-Wnt10b to overexpress Wnt10b. We demonstrated that Wnt10b promotes the differentiation of melanocytes by activating canonical Wnt signaling in melanocytes.

Adenovirus-mediated Wnt5a expression inhibits the telogen-to-anagen transition of hair follicles in mice.

The canonical Wnt/ß-catenin pathway plays an important role in hair cycle induction. Wnt5a is a non-canonical Wnt family member that generally antagonizes canonical Wnt signaling in other systems. In hair follicles, Wnt5a and canonical Wnt are both expressed in cells in the telogen stage. Wnt5a has been shown to be critical for controlling hair cell fate. However, the role that Wnt5a plays in the transition from the telogen to anagen stage is unknown. In this study, using whole-mount in situ hybridization, we show that Wnt5a is produced by several other cell types, excluding dermal papilla cells, throughout the hair cycle. For example, Wnt5a is expressed in bulge and secondary hair germ cells in the telogen stage. Our studies focused on the depilated 8-week-old mouse as a synchronized model of hair growth. Interestingly, overexpression of adenovirus Wnt5a in the dorsal skin of mice led to the elongation of the telogen stage and inhibition of the initiation of the anagen stage. However, following an extended period of time, four pelage hair types grew from hairless skin that was induced by Wnt5a, and the structure of these new hair shafts was normal. Using microarray analysis and quantitative arrays, we showed that the expression of ß-catenin and some target genes of canonical Wnt signaling decreased after Wnt5a treatment. These data demonstrate that Wnt5a may inhibit the telogen stage to maintain a quiescent state of the hair follicle.