From genetics to epigenetics: new insights into keloid scarring

Keloid scarring is a dermal fibroproliferative response characterized by excessive and progressive deposition of collagen; aetiology and molecular pathology underlying keloid formation and progression remain unclear. Genetic predisposition is important in the pathogenic processes of keloid formation, however, environmental factors and epigenetic mechanisms may also play pivotal roles. Epigenetic modification is a recent area of investigation in understanding the molecular pathogenesis of keloid scarring and there is increasing evidence that epigenetic changes may play a role in induction and persistent activation of fibroblasts in keloid scars. Here we have reviewed three epigenetic mechanisms: DNA methylation, histone modification and the role of non‐coding RNAs. We also review the evidence that these mechanisms may play a role in keloid formation ‐ in future, it may be possible that epigenetic markers may be used instead of prognostic or diagnostic markers here. However, there is a significant amount of work required to increase our current understanding of the role of epigenetic modification in keloid disease.     

Discovery and identification of anti-U1-A snRNP antibody in lung cancer

There are multiple reports of autoimmune response in patients with lung cancer. To investigate whether a novel autoantibody is present in patients with lung cancer and evaluate its clinical diagnostic and prognostic value, sera from 10 patients with lung cancer and 10 normal individuals were analyzed using immunofluorescence and Western blotting. It was found that one serum sample from the patients with squamous carcinoma gave a fine speckled pattern staining in nucleus and had a high titer antinuclear autoantibody which could recognize 31 kD of nuclear protein isolated from both cancer cells and normal cells. The same patient’s serum was further used to immunoprecipitate the target antigen. The protein bands were excised from the SDS-PAGE gels and were analyzed with a Qstar Pulser I Quadrupole time-flight mass spectrometer, and the 31 kD target antigen was identified as U1-AsnRNP. To test the prevalence of anti-U1-AsnRNP antibody, sera from 93 patients including 36 squmaous carcinomas (SCC), 26 adenocarcinomas (Ad), and 31 small cell carcinomas (SCLC) were screened by Western blotting. The results demonstrated that anti-U1-A snRNP antibody was present in 50% of SCC sera, 26.9% of Ad sera and 54.8% of SCLC sera. In this paper, we report for the first time that anti-U1-AsnRNP antibody could be detected in the patients with lung cancer.     

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals

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Nucleophilic participation of reduced flavin coenzyme in mechanism of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) requires reduced FAD (FAD(red)) to catalyze the reversible interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf). Recent structural and mechanistic studies of UGM have provided evidence for the existence of an FAD-Galf/p adduct as an intermediate in the catalytic cycle. These findings are consistent with Lewis acid/base chemistry involving nucleophilic attack by N5 of FAD(red) at C1 of UDP-Galf/p. In this study, we employed a variety of FAD analogues to characterize the role of FAD(red) in the UGM catalytic cycle using positional isotope exchange (PIX) and linear free energy relationship studies. PIX studies indicated that UGM reconstituted with 5-deaza-FAD(red) is unable to catalyze PIX of the bridging C1-OP(β) oxygen of UDP-Galp, suggesting a direct role for the FAD(red) N5 atom in this process. In addition, analysis of kinetic linear free energy relationships of k(cat) versus the nucleophilicity of N5 of FAD(red) gave a slope of ρ = -2.4 ± 0.4. Together, these findings are most consistent with a chemical mechanism for UGM involving an S(N)2-type displacement of UDP from UDP-Galf/p by N5 of FAD(red).     

Treatment with BX471, a CC chemokine receptor 1 antagonist, attenuates systemic inflammatory response during sepsis

Sepsis is a complex clinical syndrome resulting from a harmful host inflammatory response to infection. Chemokines and their receptors play a key role in the pathogenesis of sepsis. BX471 is a potent nonpeptide CC chemokine receptor-1 (CCR1) antagonist in both human and mouse. The aim of the present study was to evaluate the effect of prophylactic and therapeutic treatment with BX471 on cecal ligation and puncture-induced sepsis in the mouse and to investigate the underlying mechanisms. In sepsis induced by cecal ligation and puncture, treatment with BX471 significantly protected mice against lung and liver injury by attenuating MPO activity, an indicator of neutrophil recruitment in lungs and livers and attenuating lung and liver morphological changes in histological sections. Blocking CCR1 by BX471 also downregulated ICAM-1, P-selectin, and E-selectin expression at mRNA and protein levels in lungs and livers compared with placebo-treated groups. These findings suggest that blockage of CCR1 by specific antagonist may represent a promising strategy to prevent disease progression in sepsis.     

New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

Intratumoral expression of IL-17 and its prognostic role in gastric adenocarcinoma patients

In this study, we characterized the intratumoral expression of IL-17 and CD8(+) TILs in gastric adenocarcinoma patients after resection and determined the correlation between the survival probability of gastric adenocarcinoma patients and the expression of IL-17 in tumor. Expression of IL-17 and CD8 was assessed by immunohistochemistry, and the prognostic effects of intratumoral IL-17 expression and CD8(+) TILs were evaluated by Cox regression and Kaplan-Meier analysis. Immunohistochemical detection revealed the presence of IL-17 and CD8(+) cells in gastric adenocarcinoma tissue samples (90.6%, 174 out of 192 patients and 96.9%, 186 out of 192 patients, respectively). We have also found that intratumoral IL-17 expression was significantly correlated with age (p=0.004) and that the number of CD8(+)TILs was significantly correlated with UICC staging (p=0.012) and the depth of tumor invasion (p=0.022). The five-year overall survival probability among patients intratumorally expressing higher levels of IL-17 was significantly better than those expressing lower levels of IL-17 (p=0.036). Multivariate Cox proportional hazard analyses revealed that intratumoral IL-17 expression (HR: 0.521; 95% CI: 0.329-0.823; p=0.005) was an independent factor affecting the five-year overall survival probability. We conclude that low levels of intratumoral IL-17 expression may indicate poor prognosis in gastric adenocarcinoma patients.     

Region-specific enhancement of basal extracellular and cocaine-evoked dopamine levels following constitutive deletion of the Serotonin(1B) receptor

The behavioral effects of cocaine are enhanced following constitutive deletion of the serotonin(1B) receptor. The neural substrates mediating the enhanced response to cocaine are unknown. The present studies determined whether basal dopamine dynamics or cocaine-evoked dopamine levels are altered in projection areas of mesostriatal or mesoaccumbens dopamine neurons following serotonin(1B) receptor deletion. Male wild-type and serotonin(1B) knockout mice were implanted with microdialysis guide cannulas aimed at the dorsal striatum or nucleus accumbens. The zero net flux method of quantitative microdialysis was used to quantify basal extracellular dopamine concentrations (DA(ext)) and the extraction fraction of dopamine (E(d)), which provides an index of dopamine uptake. Conventional microdialysis techniques were used to quantify cocaine (0, 5.0, and 20.0 mg/kg)-evoked dopamine overflow. Basal DA(ext) and E(d) did not differ in striatum of wild-type and knockout mice. Similarly, cocaine-stimulated dopamine overflow did not differ between genotype. The basal E(d) did not differ in the nucleus accumbens of wild-type and knockout mice. However, DA(ext) was significantly elevated in the nucleus accumbens of knockout mice. Cocaine-evoked dopamine overflow (nM) was also enhanced in the nucleus accumbens of knockout mice. However, the cocaine-induced increase in dopamine levels, relative to basal values, did not differ between genotype. These data demonstrate that deletion of the serotonin(1B) receptor is associated with increases in basal DA(ext) in the nucleus accumbens. This increase is not associated with an alteration in E(d), suggesting increased basal dopamine release in these animals. It is hypothesized that these alterations in presynaptic neuronal activity are a compensatory response to constitutive deletion of the serotonin(1B) receptor and may contribute to the enhanced behavioral effects of psychostimulants observed in knockout mice.     

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer¹. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells^2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/α-ketoglutarate-dependent dioxygenases^{2, 3}. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development^{2, 5-10}. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC¹¹. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically¹².Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by β-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.