Alteration of Histone Acetylation Pattern during Long-Term Serum-Free Culture Conditions of Human Fetal Placental Mesenchymal Stem Cells

Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.     

The Role of Toll-Like Receptor 9 in Chronic Stress-Induced Apoptosis in Macrophage

Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.     

Region-Oriented and Staged Treatment Strategy in Reconstruction of Severe Cervical Contracture

Introduction

Severe cervical contracture after burns causes obvious impairment of neck movement and the aesthetic silhouette. Although various surgical techniques for treatment have been described, there is not a definitive strategy to guide treatment. Over the past 6 years, we have been utilizing a region-oriented and staged treatment strategy to guide reconstruction of severe cervical contracture. Satisfactory results have been achieved with this strategy.

Methods

The first stage of treatment focuses on the anterior cervical region and submental region. Procedures include cicatrix resection, contracture release, division and elevation of the platysma to form two platysma flaps, and skin grafting. Three to six months later, the second stage treatment is performed, which localize to the mental region. This includes scar resection, correction of the lower lip eversion, and reconstruction with free (para)scapular skin flap. Three subtypes of cervicomental angle that we proposed were measured as quantitative tool for evaluation of the reconstruction.

Results

24 patients who completed the treatment were reviewed. By the 3rd postoperative month, their CM angles changed significantly: the soft tissue CM angle was reduced from 135.0° ± 17.3° to 111.1° ± 11.3°, the osseous CM angle increased from 67.1° ± 9.0° to 90.5° ± 11.6° and the dynamic CM angle increased from 21.9° ± 8.7° to 67.4° ± 13.1°. 22 in 24 (91.7%) of these patients gained notable improvement of cervical motion and aesthetic contour.

Conclusions

Our results suggest that the region-oriented and staged treatment strategy can achieve satisfactory functional and aesthetic results, combining usage of both skin graft and skin flap while minimizing the donor site morbidity.     

Determinants for Achieving the LDL-C Target of Lipid Control for Secondary Prevention of Cardiovascular Events in Taiwan

Background

Epidemiological and clinical studies have clearly established the link between low-density lipoprotein cholesterol (LDL-C) and atherosclerosis-related cardiovascular consequences. Although it has been a common practice for physicians to prescribe lipid-lowering therapy for patients with dyslipidemia, the achievement rate is still not satisfied in Taiwan. Therefore, the determinants for achieving the LDL-C target needed to be clarified for better healthcare of the patients with dyslipidemia.

Method

This registry-type prospective observational study enrolled the patients with cardiovascular diseases (coronary artery disease (CAD) and cerebrovascular disease (CVD)) from 18 medical centers across Taiwan, and clinically followed them for five years. At every clinical visit, vital signs, clinical endpoints, adverse events, concurrent medications and laboratory specimens were obtained as thoroughly as possible. The lipid profile (total cholesterol, high-density lipoprotein cholesterol, LDL-C, triglyceride), liver enzymes, and creatinine phosphokinase were evaluated at baseline, and every year thereafter. The cross sectional observational data was analyzed for this report.

Result

Among the 3,486 registered patients, 54% had their LDL-C < 100 mg/dL. By univariate analysis, the patients achieving the LDL-C target were associated with older age, more male sex, taller height, lower blood pressure, more under lipid-lowering therapy, more smoking cessation, more history of CAD, DM, physical activity, but less history of CVD. The multivariate analysis showed statin therapy was the most significant independent determinant for achieving the treatment target, followed by age, history of CAD, diabetes, blood pressure, and sex. However, most patients were on regimens of very-low to low equipotent doses of statins.

Conclusion

Although the lipid treatment guideline adherence is improving in recent years, only 54% of the patients with cardiovascular diseases have achieved their LDL-C target in Taiwan, and the most significant determinant for this was statin therapy.     

Sevoflurane Inhibits Nuclear Factor-κB Activation in Lipopolysaccharide-Induced Acute Inflammatory Lung Injury via Toll-Like Receptor 4 Signaling

Background

Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR₄) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms.

Methods

In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR₄ and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR₄ and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards.

Results

The mRNA and protein levels of NF-κB and TLR₄ in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR₄ and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol.

Conclusions

This study demonstrates the crucial role of TLR₄ activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo and in vitro via inhibition of TLR₄/NF-κB pathway.     

Macrophage Migration Inhibitory Factor Mediates PAR-Induced Bladder Pain

IntroductionMacrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine, is constitutively expressed in urothelial cells that also express protease-activated receptors (PAR). Urothelial PAR1 receptors were shown to mediate bladder inflammation. We showed that PAR1 and PAR4 activator, thrombin, also mediates urothelial MIF release. We hypothesized that stimulation of urothelial PAR1 or PAR4 receptors elicits release of urothelial MIF that acts on MIF receptors in the urothelium to mediate bladder inflammation and pain. Thus, we examined the effect of activation of specific bladder PAR receptors on MIF release, bladder pain, micturition and histological changes.MethodsMIF release was measured in vitro after exposing immortalized human urothelial cells (UROtsa) to PAR1 or PAR4 activating peptides (AP). Female C57BL/6 mice received intravesical PAR1- or PAR4-AP for one hour to determine: 1) bladder MIF release in vivo within one hour; 2) abdominal hypersensitivity (allodynia) to von Frey filament stimulation 24 hours after treatment; 3) micturition parameters 24 hours after treatment; 4) histological changes in the bladder as a result of treatment; 5) changes in expression of bladder MIF and MIF receptors using real-time RT-PCR; 6) changes in urothelial MIF and MIF receptor, CXCR4, protein levels using quantitative immunofluorescence; 7) effect of MIF or CXCR4 antagonism.ResultsPAR1- or PAR4-AP triggered MIF release from both human urothelial cells in vitro and mouse urothelium in vivo. Twenty-four hours after intravesical PAR1- or PAR4-AP, we observed abdominal hypersensitivity in mice without changes in micturition or bladder histology. PAR4-AP was more effective and also increased expression of bladder MIF and urothelium MIF receptor, CXCR4. Bladder CXCR4 localized to the urothelium. Antagonizing MIF with ISO-1 eliminated PAR4- and reduced PAR1-induced hypersensitivity, while antagonizing CXCR4 with AMD3100 only partially prevented PAR4-induced hypersensitivity.ConclusionsBladder PAR activation elicits urothelial MIF release and urothelial MIF receptor signaling at least partly through CXCR4 to result in abdominal hypersensitivity without overt bladder inflammation. PAR-induced bladder pain may represent an interesting pre-clinical model of Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS) where pain occurs without apparent bladder injury or pathology. MIF is potentially a novel therapeutic target for bladder pain in IC/PBS patients.     

Cloning and Functional Characterization of SAD Genes in Potato

Stearoyl-acyl carrier protein desaturase (SAD), locating in the plastid stroma, is an important fatty acid biosynthetic enzyme in higher plants. SAD catalyzes desaturation of stearoyl-ACP to oleyl-ACP and plays a key role in determining the homeostasis between saturated fatty acids and unsaturated fatty acids, which is an important player in cold acclimation in plants. Here, four new full-length cDNA of SADs (ScoSAD, SaSAD, ScaSAD and StSAD) were cloned from four Solanum species, Solanum commersonii, S. acaule, S. cardiophyllum and S. tuberosum, respectively. The ORF of the four SADs were 1182 bp in length, encoding 393 amino acids. A sequence alignment indicated 13 amino acids varied among the SADs of three wild species. Further analysis showed that the freezing tolerance and cold acclimation capacity of S. commersonii are similar to S. acaule and their SAD amino acid sequences were identical but differed from that of S. cardiophyllum, which is sensitive to freezing. Furthermore, the sequence alignments between StSAD and ScoSAD indicated that only 7 different amino acids at residues were found in SAD of S. tuberosum (Zhongshu8) against the protein sequence of ScoSAD. A phylogenetic analysis showed the three wild potato species had the closest genetic relationship with the SAD of S. lycopersicum and Nicotiana tomentosiformis but not S. tuberosum. The SAD gene from S. commersonii (ScoSAD) was cloned into multiple sites of the pBI121 plant binary vector and transformed into the cultivated potato variety Zhongshu 8. A freeze tolerance analysis showed overexpression of the ScoSAD gene in transgenic plants significantly enhanced freeze tolerance in cv. Zhongshu 8 and increased their linoleic acid content, suggesting that linoleic acid likely plays a key role in improving freeze tolerance in potato plants. This study provided some new insights into how SAD regulates in the freezing tolerance and cold acclimation in potato.     

Thermal Modelling Analysis of Spiral Wound Supercapacitor under Constant-Current Cycling

A three-dimensional modelling approach is used to study the effects of operating and ambient conditions on the thermal behaviour of the spiral wound supercapacitor. The transient temperature distribution during cycling is obtained by using the finite element method with an implicit predictor-multicorrector algorithm. At the constant current of 2A, the results show that the maximum temperature appears in core area. After 5 cycles, the maximum temperature is 34.5°C, while in steady state, it’s up to 42.5°C. This paper further studies the relationship between the maximum temperature and charge-discharge current. The maximum temperature will be more than 60°C after 5 cycles at the current of 4A, and cooling measurements should be taken at that time. It can provide thoughts on inner temperature field distribution and structure design of the spiral wound supercapacitor in working process.     

Knowledge of HIV and Willingness to Conduct Oral Rapid HIV Testing among Dentists in Xi’an China

Introduction

China is considered a country of low HIV prevalence (780,000 people living with HIV), however, HIV infections among high-risk populations continue to grow at alarming rates. Voluntary Counseling and Testing services were first implemented in 2003, and oral rapid HIV testing (ORHT) began in 2012. Dentists, as oral health experts, would be well placed to conduct ORHT. We assessed willingness of dentists to undertake ORHT in their clinical practice.

Methods

A cross-sectional, paper-based survey of dentists from the Xi’an region of China was conducted from April to June 2013. Dentists were recruited from Shaanxi Stomatological Association using a stratified sampling methodology. A 40-item survey was used to measure knowledge of HIV, attitudes toward people living with HIV and willingness to conduct ORHT.

Results

477 dentists completed the survey with a mean HIV knowledge test score of 13.2/18 (SD 1.9). If made available in the dental setting, 276 (57.9%) preferred to use blood to diagnose HIV, only 190 (39.8%) preferred saliva or both. Four hundred and thirty-five (91.2%) thought that ORHT was needed in dental clinics. Female dentists felt more accepting of ORHT than males (93.8% vs. 87.8%; χ²=5.145; p<0.05). 42.6% of the participants who responded thought that lack of education on ORHT for dentists was the most urgent problem to solve for ORHT, 144 (31.3%) thought that lack of support for ORHT from patients was the most urgent problem. There was statistically significant difference among dental hospital, dentistry and department of dentistry (χ²=24.176; p<0.05).

Conclusions

The majority of Chinese dentists thought that ORHT was needed in the dental setting. Providing opportunities for dentists and dental students to learn about HIV testing guidelines and practices is needed as well as feasibility and implementation science research.     

Adverse Effects of High Concentrations of Fluoride on Characteristics of the Ovary and Mature Oocyte of Mouse

Reproductive toxicity has been an exciting topic of research in reproductive biology in recent years. Soluble fluoride salts are toxic at high concentrations; their reproductive toxicity was assessed in this study by administering different fluoride salt concentrations to mice. Continuous feeding for five weeks resulted in damage to the histological architecture of ovaries. The expression of genes, including Dazl, Stra8, Nobox, Sohlh1, and ZP3 gene, associated with oocyte formation were much lower in the experimental group as compared with the control group. The number of in vitro fertilization of mature oocytes were also much lower in the experimental group as compared with control. Moreover, the fertility of female mice, as assessed by mating with normal male mice, was also lower in experimental compared with control groups. The expression of the oocyte-specific genes: Bmp15, Gdf9, H1oo, and ZP2, which are involved in oocyte growth and the induction of the acrosome reaction, decreased with the fluoride administration. DNA methylation and histone acetylation (H3K18ac and H3K9ac) are indispensable for germline development and genomic imprinting in mammals, and fluoride administration resulted in reduced levels of H3K9ac and H3K18ac in the experimental group as compared with the control group, as detected by immunostaining. Our results indicate that the administration of high concentrations of fluoride to female mice significantly reduced the number of mature oocytes and hampered their development and fertilization. Thus, this study lays a foundation for future studies on fluoride-induced reproductive disorders in women.