Association of microRNA gene polymorphisms with recurrent spontaneous abortion: An updated meta­analysis.

Numerous studies have reported single nucleotide polymorphisms (SNPs) in microRNAs (miRNAs) associated with unexplained recurrent spontaneous abortion (URSA). The present study aimed to conduct an updated meta-analysis to confirm a pooled effect size of the association between miRNA SNPs and URSA. The relevant literature was searched on PubMed, EMBASE, Web of Science and Cochrane Library before July 2022 to identify case-control studies. The pooled odds ratio and confidence intervals at 95% of the eligible studies were extracted and evaluated under five genetic models. A total of 18 studies involving 3,850 cases and 4,312 controls were included. miR499a rs3746444 A>G, miR-149 rs2292832 T>C, miR-125a rs41275794 G>A and miR-10a rs3809783 A>T may enhance the risk of recurrent spontaneous abortion (RSA) under various genetic models. Although no separate association was found between the miR-125a rs12976445 C>T and miR-27a rs895819 A>G polymorphisms and RSA, statistical significance was found in certain ethnic groups only. The current analysis suggests a high significance of an up-to-date meta-analysis for screening out and preventing URSA among high-risk women by testing miRNA SNPs and RSA susceptibility.

A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells.

Light is a highly attractive actuator that allows spatiotemporal control of diverse cellular activities. In this study, we developed a single-component light-switchable gene expression system for yeast cells, termed yLightOn system. The yLightOn system is independent of exogenous cofactors, and exhibits more than a 500-fold ON/OFF ratio, extremely low leakage, fast expression kinetics, and high spatial resolution. We demonstrated the usefulness of the yLightOn system in regulating cell growth and cell cycle by stringently controlling the expression of His3 and ΔN Sic1 genes, respectively. Furthermore, we engineered a bidirectional expression module that allows the simultaneous control of the expression of two genes by light. With ClpX and ClpP as the reporters, the fast, quantitative, and spatially specific degradation of ssrA-tagged protein was observed. We suggest that this single-component optogenetic system will be immensely helpful in understanding cellular gene regulatory networks and in the design of robust genetic circuits for synthetic biology.

Protein palmitoylation activate zygotic gene expression during the maternal-to-zygotic transition.

Upon fertilization, maternal factors direct development and trigger zygotic genome activation at the maternal-to-zygotic transition (MZT). However, the factors that activate the zygotic program in vertebrates are not well defined. Here, we found that protein palmitoylation played an important role in acquiring transcriptional competency and orchestrating the clearance of the maternal program in zebrafish. After inhibition of protein palmitoylation, zebrafish embryos developed normally before the Mid-Blastula Transition (MBT); however, they did not initiate epiboly. Moreover, our results showed that protein palmitoylation is required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

Melatonin inhibits tumorigenicity of glioblastoma stem-like cells via the AKT-EZH2-STAT3 signaling axis.

Glioblastoma stem-like cells (GSCs) displaying self-renewing and tumor-propagating capacity play a particularly important role in maintaining tumor growth, therapeutic resistance, and tumor recurrence. Therefore, new therapeutic strategies focusing on impairing GSC maintenance are urgently needed. Here, we used GSCs isolated from surgical specimens from patients with glioblastoma multiforme (GBM) to study the roles and underlying mechanisms associated with melatonin in GSC biology. The results showed that melatonin directly targeted glioma tumor cells by altering GSC biology and inhibiting GSC proliferation. Additionally, melatonin altered profile of transcription factors to inhibit tumor initiation and propagation. Furthermore, EZH2 S21 phosphorylation and EZH2-STAT3 interaction in GSCs were impaired following melatonin treatment. These results suggested that melatonin attenuated multiple key signals involved in GSC self-renewal and survival, and further supported melatonin as a promising GBM therapeutic.

Protein Palmitoylation Regulates Neural Stem Cell Differentiation by Modulation of EID1 Activity.

The functional significance of palmitoylation in the switch between self-renewal and differentiation of neural stem cells (NSCs) is not well defined, and the underlying mechanisms of protein palmitoylation are not well understood. Here, mouse NSCs were used as a model system and cell behavior was monitored in the presence of the protein palmitoylation inhibitor 2-bromopalmitate (2BRO). Our data show that 2BRO impaired the differentiation of NSCs into both neurons and glia and impaired NSC cell cycle exit. Moreover, the results show that palmitoylation modified E1A-like inhibitor of differentiation one (EID1) and this modification regulated EID1 degradation and CREB-binding protein (CBP)/p300 histone acetyltransferase activity at the switch between self-renewal and differentiation of NSCs. Our results extended the cellular role of palmitoylation, suggesting that it acts as a regulator in the acetylation-dependent gene expression network, and established the epigenetic regulatory function of palmitoylation in the switch between maintenance of multipotency and differentiation in NSCs.

ZDHHC16 modulates FGF/ERK dependent proliferation of neural stem/progenitor cells in the zebrafish telencephalon.

In vertebrates, neural stem/progenitor cells (NSPCs) maintenance is critical for nervous system development and homeostasis. However, the molecular mechanisms underlying the maintenance of NSPCs have not been fully elucidated. Here, we demonstrated that zebrafish ZDHHC16, a DHHC encoding protein, which was related to protein palmitoylation after translation, was expressed in the developing forebrain, and especially in the telencephalon. Loss- and gain-of-function studies showed that ZDHHC16 played a crucial role in the regualtion of NSPCs proliferation during zebrafish telencephalic development, via a mechanism dependent on its palmitoyltransferase activity. Further analyses showed that the inhibition of ZDHHC16 led to inactivation of the FGF/ERK signaling pathway during telencephalic NSPCs proliferation and maintenance. Taken together, our results suggest that ZDHHC16 activity is essential for early NSPCs proliferation where it acts to activate the FGF/ERK network, allowing for the initiation of proliferation -regulated gene expression programs. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1014-1028, 2016.

ZDHHC17 promotes axon outgrowth by regulating TrkA-tubulin complex formation.

Correct axonal growth during nervous system development is critical for synaptic transduction and nervous system function. Proper axon outgrowth relies on a suitable growing environment and the expression of a series of endogenous neuronal factors. However, the mechanisms of these neuronal proteins involved in neuronal development remain unknown. ZDHHC17 is a member of the DHHC (Asp-His-His-Cys)-containing family, a family of highly homologous proteins. Here, we show that loss of function of ZDHHC17 in zebrafish leads to motor dysfunction in 3-day post-fertilization (dpf) larvae. We performed immunolabeling analysis to reveal that mobility dysfunction was due to a significant defect in the axonal outgrowth of spinal motor neurons (SMNs) without affecting neuron generation. In addition, we found a similar phenotype in zdhhc17 siRNA-treated neural stem cells (NSCs) and PC12 cells. Inhibition of zdhhc17 limited neurite outgrowth and branching in both NSCs and PC12. Furthermore, we discovered that the level of phosphorylation of extracellular-regulated kinase (ERK) 1/2, a major downstream effector of tyrosine kinase (TrkA), was largely upregulated in ZDHHC17 overexpressing PC12 cells by a mechanism independent on its palmitoyltransferase (PAT) activity. Specifically, ZDHHC17 is necessary for proper TrkA-tubulin module formation in PC12 cells. These results strongly indicate that ZDHHC17 is essential for correct axon outgrowth in vivo and in vitro. Our findings identify ZDHHC17 as an important upstream factor of ERK1/2 to regulate the interaction between TrkA and tubulin during neuronal development.

2-Bromopalmitate impairs neural stem/progenitor cell proliferation, promotes cell apoptosis and induces malformation in zebrafish embryonic brain.

2-Bromopalmitate (2BP) is a widely used palmitoylation inhibitor. Besides, it has been reported that 2BP can inhibit T-cell activation, making it a potential immunosuppressor for the treatment of autoimmune diseases. Although the important roles of palmitoylation in a neural system have been noted during the past decades, the effect of 2BP on neural development is still not very clear. In this study, we demonstrated that 25 µM-100 µM 2BP exposure caused apparent neural malformation in the presumptive brains of zebrafish embryos at 14 hpf. Further studies implied that the mRNA quantities and distributions of neural stem/progenitor cell (NSPC) markers (neurog1, sox2, and sox3) in the affected regions of 50 µM 2BP treated embryos significantly decreased. In addition, we found that 2BP impaired the NSPC proliferation at 10 hpf and 14 hpf as well as promoted cell apoptosis at 14 hpf, consistent with which the interference with FGF/ERK signaling pathway was also detected. For the first time, this study provided information about the toxicity and teratogenicity of 2BP for neural development in vivo.

Cytoprotective effect of melatonin against hypoxia/serum deprivation-induced cell death of bone marrow mesenchymal stem cells in vitro.

Bone marrow mesenchymal stem cells (MSCs) have been shown great potential for cardiac regeneration. However the therapeutic efficiency has become a major obstacle due to the poor survival of transplanted MSCs in ischemic cardiac tissue. Previous studies reported that melatonin could protect many different types of cells from apoptosis under various pathological conditions. In the present study, we demonstrated that melatonin, an endogenously secreted indoleamine had cytoprotection from hypoxia/serum deprivation (Hy/SD)-induced cell death in MSCs. We further investigated the possible mechanism and found out that melatonin attenuated (Hy/SD)-induced cell death could be via effectively reducing the generation of intracellular reactive oxygen species, an increase in the ratio of Bax/Bcl-2, loss of mitochondrial membrane potential and then activation of caspase-3 in MSCs in response to Hy/SD exposure. Furthermore, melatonin pretreatment significantly modulated the expression of phospho-P38MAPK and phospho-ERK1/2 in Hy/SD-induced MSCs and the protective effects of melatonin were partially reversed by ERK1/2 inhibitor but not p38 inhibitor, suggesting that melatonin inhibited Hy/SD-induced MSCs cell death through the MAPK signaling pathway in part. Taken together, the findings imply that melatonin could improve the survival of engrafted MSCs under hypoxia and serum deprivation condition. Our findings indicate that combination therapy with melatonin may provide therapeutic benefit for improving myocardial function after infarction.

Melatonin promotes the acquisition of neural identity through extracellular-signal-regulated kinases 1/2 activation.

Melatonin, a major pineal secretory product, exerts a range of physiological and neuroprotective effects. However, the functional significance of melatonin in determining neural identity, and the mechanisms by which this may occur, is unknown. In this study, P19 cells were used as a model system and cell behavior was monitored. Our data show that melatonin plays an important role in determining cell fate during neural commitment and promoting the differentiation of pluripotent P19 cells (Oct4(+) Sox2(+) ) into neural stem cells (Oct4(-) Sox2(+) ). This promotion appears to coincide with the activation of the MT1 receptor and phosphorylation of extracellular-signal-regulated kinases 1/2 (ERK1/2). Furthermore, our results show that melatonin regulates neural fate specification of P19 cells through two distinct mechanisms: the promotion of nuclear localization of ERK1/2 and upregulation of Sox2 transcription, and suppression of Smad1-induced expression of mesodermal-specific genes, such as Bra.

Zinc finger DHHC-type containing 13 regulates fate specification of ectoderm and mesoderm cell lineages by modulating Smad6 activity.

Neither the roles of Asp-His-His-Cys (DHHC)-containing proteins in embryonic cell fate specification are well defined, nor the underlying mechanisms of their activity are well understood. Here, we compared the embryonic function of zinc finger DHHC-type containing (Zdhhc13) in zebrafish embryos and in an in vitro cell model. Zdhhc13, a critical regulator of bone morphogenetic protein (BMP) signaling, specifically bound to Smad6 to induce its perinuclear accumulation and degradation through a mechanism independent of its palmitoyltransferase activity. We showed that Zdhhc13 played a crucial role during zebrafish embryogenesis in the control of germ layer specification, particularly in ectoderm and mesoderm differentiation homeostasis. Depletion of Zdhhc13 led to the neuralization of ectoderm and dorsalization of mesoderm in zebrafish embryos. Moreover, Zdhhc13 antagonized Smad6 during BMP-dependent signaling and early lineage decisions in our in vitro cell model. Our results extended the cellular role of Zdhhc13, suggesting that it acts as a regulator in BMP signaling, and established that the embryonic function of Zdhhc13 is in lineage specification.

2-Bromopalmitate modulates neuronal differentiation through the regulation of histone acetylation.

In order to evaluate the functional significance of palmitoylation during multi-potent neural stem/progenitor cell proliferation and differentiation, retinoic acid-induced P19 cells were used in this study as a model system. Cell behaviour was monitored in the presence of the protein palmitoylation inhibitor 2-bromopalmitate (2BP). Here, we observed a significant reduction in neuronal differentiation in the 2BP-treated cell model. We further explored the underlying mechanisms and found that 2BP resulted in the decreased acetylation of histones H3 and H4 and interfered with cell cycle withdrawal and neural stem/progenitor cells' renewal. Our results established a direct link between palmitoylation and the regulation of neural cell fate specification and revealed the epigenetic regulatory mechanisms that are involved in the effects of palmitoylation during neural development.

Palmitic acid affects proliferation and differentiation of neural stem cells in vitro.

High-lipid diet composed of saturated fatty acids (SFAs) has significant detrimental effects on brain homeostasis, and deleterious effects of SFAs on various cells have been well documented. However, the effects of SFAs on neural stem Cells (NSCs) function have not been fully explored. The aim of this study was to determine whether palmitic acid (PA) affected the proliferation and differentiation of murine-derived NSCs. The results showed that PA dose dependently suppressed viability of NSCs and was cytotoxic at high concentrations. The toxic levels of PA inhibited the proliferation of NSCs as shown by reduced bromodeoxyuridine labeling of NSCs, which is correlated with reactive oxygen species generation. Pretreatment of the cells with the antioxidant N-acetyl-L-cysteine inhibitor significantly attenuated the effects of PA on the proliferation of NSCs. Furthermore, nontoxic levels of PA promoted astrocytogenesis in the differentiated NSCs, associated with Stat3 activation and altered expression of serial of basic helix-loop-helix transcription factor genes. Altogether, our data have demonstrated that PA has a significant impact on proliferation and differentiation of NSCs in vitro and may be useful for elucidating the role of SFAs in regulating NSCs fate in physiological and pathological settings.

Protective effect of melatonin on bone marrow mesenchymal stem cells against hydrogen peroxide-induced apoptosis in vitro.

Bone marrow mesenchymal stem cells (MSCs) transplantation has shown great promises for treating various central nervous system (CNS) diseases. However, poor viability of transplanted MSCs in injured CNS has limited the therapeutic efficiency. Oxidative stress is one of major mechanisms underlying the pathogenesis of CNS diseases and has a negative impact on the survival of transplanted MSCs. Melatonin has recently been reported to have the antioxidant and anti-apoptotic properties in serial of cells. This study was designed to investigate the protective effect and potential mechanisms of melatonin against hydrogen peroxide (H2O2)-induced apoptosis of MSCs. MSCs were pretreated with melatonin (1, 10, and 100 nM, respectively) for 30 min, followed by exposure to 400 µM H2O2 and melatonin together for 12 h. The present study reports that melatonin pretreatment significantly attenuated H2O2-induced MSC apoptosis in a dose-dependent manner. Consistently, melatonin effectively suppressed the generation of intracellular ROS, expression ratio of Bax/Bcl-2, activation of caspase-3 and expression of phospho-P38MAPK in H2O2-induced MSCs. Luzindole, a nonselective melatonin receptor antagonist, significantly counteracted melatonin's promotion effect on cell survival, indicating that melatonin exerts its protective effect on MSCs, at least in part, through the activation of melatonin receptors. The findings suggest that melatonin may be an effectively protective agent against oxidative stress-induced MSC apoptosis.

Palmitic acid increases apoptosis of neural stem cells via activating c-Jun N-terminal kinase.

Elevated plasma free fatty acid (FFA) level is common in many pathological conditions, including neurological disorders, and their deleterious effects on various cells have been well documented. However, it remains to be investigated whether elevated FFAs would have a direct effect on neural stem cells (NSCs). Here, we reported that palmitic acid (PA) impaired cell viability and increased apoptosis of NSCs significantly in a dose- and time-dependent manner. Increased protein levels of Bax and cleaved caspase 3 coupled with decreased expression of Bcl-2 were also observed in NSCs with increasing dose or time of PA treatment, whereas caspase 3 expression remained relatively unaltered. In parallel to this, the expression of phospho-c-Jun N-terminal kinase (p-JNK) in NSCs challenged with PA was increased significantly; however, JNK expression appeared stable. Remarkably, JNK inhibitor effectively reduced the apoptosis of NSCs induced by PA. The expression of phospho-p38 (p-p38), p38, phospho-extracellular regulated protein kinases 1/2 (p-EKR1/2) and EKR1/2 in NSCs was not affected by PA treatment. In consideration of the above, it is suggested that elevated plasma FFA level may induce apoptosis of NSCs in vivo, and that this might be one of possible underlying mechanisms for the cognitive disturbance in neurological disorders.

[Effect of periodontal initial therapy together with Chinese herbal compound for clearing heat and detoxification on interleukin-1 beta and tumor necrosis factor-alpha in gingival crevicular fluid of patients with chronic periodontitis].

OBJECTIVE: To observe the effect of periodontal initial therapy together with Chinese herbal compound for clearing heat and detoxification (Abbr. TCM) on the levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) in gingival crevicular fluid (GCF) of patients with chronic periodontitis (CPD). METHODS: Thirty-four teeth of 24 patients with CPD were randomly assigned to the control group (17 teeth of 11 patients) and the treated group (17 teeth of 13 patients), they were treated for 8 weeks with periodontal initial therapy, but TCM was given additionally to the treated group. The GCF of the observed teeth was collected at the time before initial therapy (datum line) and at 2 and 8 weeks after treatment. At the same time, the clinical parameters, including probing depth (PD), clinical attachment loss (CAL), and bleeding on probing (BOP) were recorded, and the levels of TNF-alpha and IL-1 beta in GCF were measured by radioimmunoassay. RESULTS: The levels of TNF-alpha and IL-1 beta in GCF after treatment were significantly lowered in all patients as compared with those before treatment (P<0.01), while PD, CAL and BOP showed significant reduction (P<0.01). After treatment, TNF-alpha and IL-1 beta levels in the treated group were lower than those in the control group (TNF-alpha: 2.5551 +/- 1.0157 microg/L vs 3.4341 +/- 1.0762 microg/L, IL-1 beta: 0.1335 +/- 0.0216 microg/L vs 0.1471 +/- 0.0169 microg/L; P<0.05). CONCLUSION: TCM could enhance the effect of periodontal initial therapy in lowering TNF-alpha and IL-1 beta levels, which was attributed to reducing the inflammatory reaction of periodontal tissues.

[The experimental research of the influence of cervical settling method on marginal accuracy of wax patter].

OBJECTIVE: To study the influence of cervical settling method on marginal accuracy of wax patters. METHODS: 24 wax patterns were made on the standard cast specimens and round capsules of which twelve patters were made with the method of cervical settling (experiment group), twelve patters were made with the method of wax dripping (control group). Marginal accuracy of patterns were measured before sublation, sublation half an hour and twenty-four hours. RESULTS: The results revealed that marginal accuracy had significant difference between the cervical settling methods and wax dripping methods (P < 0.05). There was significant difference between the cervical settling methods measured after half an hour and twenty-four hours (P < 0.01). There was also significant difference between the wax dripping methods measured after half an hour and twenty-four hours (P < 0.01). CONCLUSION: Wax patterns with the method of cervical settling can improve marginal accuracy of patterns. The marginal accuracy of wax patters are influenced by the existing time.

[The test of metal-ceramic bonding strength among three ceramic alloies].

PURPOSE: To compare the metal-ceramic bonding strength of different ceramic alloies. METHODS: 30 wax sheets were divided into 6 groups at random. Each group included 5 sheets. After being invested conventionally with Bellaves SH,the samples were casted and fused with porcelain.The metal-ceramic bonding strength was evaluated. RESULTS: The highest bonding strength was found in Bio Herador N alloy, and then in TILITE alloy and Heraenium S alloy respectively. Bio Herador N alloy and TILITE alloy all had significant differences of the bonding strength before and after treatment (P<0.05). There was no significant differences in the bonding strength of the Heraenium S alloy after treatment (P>0.05). CONCLUSION: The bonding strength of precious metal-ceramic was higher than that of non-precious metal-ceramic.Pre-oxygen treatment can improve the metal-ceramic bonding strength of Bio Herador N alloy and TILITE alloy,but it had no effect on the metal-ceramic bonding strength of the Heraenium S alloy.