A Reversible Neural Stem Cell Quiescence and Activation Culture System for Metabolic Study.

Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.

Artemisinin potentiates apoptosis and triggers cell cycle arrest to attenuate malignant growth of salivary gland tumor cells.

One of the rare malignant tumors developing within the glands of the buccal cavity in human beings is salivary gland tumors (SGTs). The hallmark of SGTs is the fusion of nuclear factor IB (NFIB) and myeloblastosis (MYB) genes developed after the translocation of q22-23; p23-24. Although the aetiology of SGTs is not clear, however, the therapeutic modalities are surgical resection followed by the combination of chemotherapy and radiotherapy if a chance of recurrence seems to develop. Owing to have numerous side effects of chemotherapy, the drug development has been shifted to natural products with minimal side effects. One of the key phytochemical artemisinin derived from wormwood Artemisia annua exhibits various pharmacological activities against various in-vivo and in-vitro cellular models. Here, we evaluated the cytotoxic potential of artemisinin against A-253 cells with possible underlying cell death mechanisms. Our results showed that artemisinin reduces the proliferation of cells in a concentration-dependent manner and displays IC50 value in a range of 10.23, 14.21 µM, and 203.18 µM against A-253/HTB-41 and transformed salivary gland SMIE cells, respectively. Flow cytometry analysis demonstrated that artemisinin promotes a significant amount of apoptotic cellular population and triggers G0/G1 arrest of A-253 cells in a concentration-dependent manner. To verify the mechanism of cell death induced by artemisinin in A-253 cells, we found an increased level of Bax, Bim, Bad, Bak and reduced level of antiapoptotic protein Bcl-2, Bcl-XL with concomitant release of mitochondrial resident protein cytochrome c into the cytoplasm. Additionally, we found that artemisinin augments the production of reactive oxygen species which further leads to the activation of proapoptotic proteins PARP1, and caspase-3, in a concentration-dependent manner thereby triggering apoptosis. In conclusion, artemisinin exhibits promising anticancer therapeutic potential against A-253 cells and needs further validation of in-vitro results in preclinical models.

Direct in vivo application of induced pluripotent stem cells is feasible and can be safe.

Increasing evidence suggests the consensus that direct in vivo application of induced pluripotent stem cells (iPSCs) is infeasible may not be true. Methods: Teratoma formation and fate were examined in 53 normal and disease conditions involving brain, lung, liver, kidney, islet, skin, hind limb, and arteries. Results: Using classic teratoma generation assays, which require iPSCs to be congregated and confined, all mouse, human, and individualized autologous monkey iPSCs tested formed teratoma, while iPSC-derived cells did not. Intravenously or topically-disseminated iPSCs did not form teratomas with doses up to 2.5×108 iPSCs/kg and observation times up to 18 months, regardless of host tissue type; autologous, syngeneic, or immune-deficient host animals; presence or absence of disease; disease type; iPSC induction method; commercial or self-induced iPSCs; mouse, human, or monkey iPSCs; frequency of delivery; and sex. Matrigel-confined, but not PBS-suspended, syngeneic iPSCs delivered into the peritoneal cavity or renal capsule formed teratomas. Intravenously administered iPSCs were therapeutic with a dose as low as 5×106/kg and some iPSCs differentiated into somatic cells in injured organs. Disseminated iPSCs trafficked into injured tissue and survived significantly longer in injured than uninjured organs. In disease-free animals, no intravenously administered cell differentiated into an unwanted long-lasting cell or survived as a quiescent stem cell. In coculture, the stem cell medium and dominant cell-type status were critical for iPSCs to form cell masses. Conclusion: Teratoma can be easily and completely avoided by disseminating the cells. Direct in vivo iPSC application is feasible and can be safe.

Postnatal Vitamin D Intake Modulates Hippocampal Learning and Memory in Adult Mice.

Vitamin D (VD) is a neuroactive steroid crucial for brain development, function and homeostasis. Its deficiency is associated with numerous brain conditions. As such, VD and its variants are routinely taken by a broad of groups with/without known VD deficiency. In contrast, the harmful effects of VD overdose have been poorly studied. Similarly, the developmental stage-specific VD deficiency and overdose have been rarely explored. In the present work, we showed that postnatal VD supplementation enhanced the motor function transiently in the young adult, but not in the older one. Postnatal VD intake abnormality did not impact the anxiety and depressive behavior but was detrimental to spatial learning and hippocampus-dependent memory. At the molecular level we failed to observe an obvious and constant change with the neural development and activity-related genes examined. However, disrupted developmental expression dynamics were observed for most of the genes, suggesting that the altered neural development dynamics and therefore aberrant adult plasticity might underlie the functional deficits. Our work highlights the essence of VD homeostasis in neural development and adult brain function. Further studies are needed to determine the short- and long-term effects VD intake status may have on brain development, homeostasis, and diseases.

Autophagy mediates glucose starvation-induced glioblastoma cell quiescence and chemoresistance through coordinating cell metabolism, cell cycle, and survival.

Metabolic reprogramming is pivotal to sustain cancer growth and progression. As such dietary restriction therapy represents a promising approach to starve and treat cancers. Nonetheless, tumors are dynamic and heterogeneous populations of cells with metabolic activities modulated by spatial and temporal contexts. Autophagy is a major pathway controlling cell metabolism. It can downregulate cell metabolism, leading to cancer cell quiescence, survival, and chemoresistance. To understand treatment dynamics and provide rationales for better future therapeutic strategies, we investigated whether and how autophagy is involved in the chemo-cytotoxicity and -resistance using two commonly used human glioblastoma (GBM) cell lines U87 and U251 together with primary cancer cells from the GBM patients. Our results suggest that autophagy mediates chemoresistance through reprogramming cancer cell metabolism and promoting quiescence and survival. Further unbiased transcriptome profiling identified a number of clinically relevant pathways and genes, strongly correlated with TCGA data. Our analyses have not only reported many well-known tumor players, but also uncovered a number of genes that were not previously implicated in cancers and/or GBM. The known functions of these genes are highly suggestive. It would be of high interest to investigate their potential involvement in GBM tumorigenesis, progression, and/or drug resistance. Taken together, our results suggest that autophagy inhibition could be a viable approach to aid GBM chemotherapy and combat drug resistance.

Clinical value of serum markers of type 2 diabetes mellitus complicated by chronic periodontitis / 口腔疾病防治

Objective@#To investigate the relation between serum markers, the degree of lesions and the active period of chronic periodontitis in patients with type 2 diabetes mellitus.@*Methods@#A total of 595 patients with type 2 diabetes mellitus were selected, and oral examinations and laboratory tests were conducted. The patients were divided into a periodontally healthy group mild, moderate and severe periodontitis groups depending on the diagnostic criteria for chronic periodontitis. The patients were also divided into periodontally healthy, resting and active groups depending on the diagnostic criteria of the active period. The relationships between serum biochemical indices, the degree of lesions and chronic periodontitis activity were analyzed. @*Results@#The prevalence of chronic periodontitis in patients with type 2 diabetes was 74.6%, and the proportions of patients with mild, moderate, and severe chronic periodontitis were 44.9%, 16.1% and 13.6%, respectively. The composite ratio of active periodontal chronic periodontitis was 33.1%. Compared with the patients in the control group, the fasting plasma glucose and HbA1c levels were more poorly controlled in the type 2 diabetes mellitus groups with chronic periodontitis (P < 0.05). There were significant differences in fasting blood glucose levels between mild periodontitis group and moderate, severe periodontitis group (P < 0.05). There was no significant difference in HbA1c levels among the mild, moderate and severe groups (P>0.05). However, there were significant differences in fasting blood glucose levels between the groups with various degrees of progression in the following order: active (11.24 mmol /L) > resting (9.64 mmol/L) > control (8.82 mmol/L) (P < 0.05).@*Conclusion@#The severity of chronic periodontitis plays no role in the level of HbA1c, instead, the level of fasting plasma glucose changes with the severity and progression of chronic periodontitis.

Study on application of natural medicine epigallocatechin gallate for primary enamel caries remineralization / 口腔疾病防治

Objective@#This in vitro study aimed to investigate the remineralization effect of the natural medicine epigallocatechin gallate on artificial enamel caries in primary human teeth. @*Methods @#We divided 30 sound primary upper anterior teeth into 3 groups according to a random number table, including experimental group (epigallocatechin gallate group), positive control group (NaF group) and blank control group (artificial saliva group), with 10 teeth in each group. After test in vitro, Micro Hardness Tester was applied to measure hardness of samples before and after demineralization. Scanning electron microscopy (SEM) was used to observe the result of primary enamel surface remineralization. @*Results@# A significant increase in enamel surface microhardness between the three groups after remineralization (F=1 199.975, P < 0.05). The difference between 2 groups was compared with each other among 3 groups. Statistical significance was found (vs experimental group q=41.986, P < 0.05; vs positive control group q=68.174, P < 0.05), suggesting that both positive control group and experimental group could promote the remineralization of primary enamel, and the effect of epigallocatechin gallate was weaker than NaF (q=26.188, P < 0.05 ). The results from SEM indicated that there was large amount of sediment on the surface of primary enamel surface of incisors in the experimental group and positive control group, while primary enamel surface of incisors in the blank control group was honeycomb and uneven, with less sediment. @*Conclusion@#Based on this in vitro study, epigallocatechin gallate can promote the remineralization of demineralized enamel of primary teeth, indicating its potential use as a natural remineralization medicine.

Transduction of interleukin-10 through renal artery attenuates vascular neointimal proliferation and infiltration of immune cells in rat renal allograft.

Renal transplantation is the treatment of choice for end-stage renal failure. Although acute rejection is not a major issue anymore, chronic rejection, especially vascular rejection, is still a major factor that might lead to allograft dysfunction on the long term. The role of the local immune-regulating cytokine interleukin-10 (IL-10) in chronic renal allograft is unclear. Many clinical observations showed that local IL-10 level was negatively related to kidney allograft function. It is unknown this negative relationship was the result of immunostimulatory property or insufficient immunosuppression property of local IL-10. We performed ex vivo transduction before transplantation through artery of the renal allograft using adeno-associated viral vectors carrying IL-10 gene. Twelve weeks after transplantation, we found intrarenal IL-10 gene transduction significantly inhibited arterial neointimal proliferation, the number of occluded intrarenal artery, interstitial fibrosis, peritubular capillary congestion and glomerular inflammation in renal allografts compared to control allografts receiving PBS or vectors carrying YFP. IL-10 transduction increased serum IL-10 level at 4 weeks but not at 8 and 12 weeks. Renal IL-10 level increased while serum creatinine decreased significantly in IL-10 group at 12 weeks compared to PBS or YFP controls. Immunohistochemical staining showed unchanged total T cells (CD3) and B cells (CD45R/B220), decreased cytotoxic T cells (CD8), macrophages (CD68) and increased CD4+ and FoxP3+ cells in IL-10 group. In summary, intrarenal IL-10 inhibited the allograft rejection while modulated immune response.

Pre-existing interleukin 10 in cerebral arteries attenuates subsequent brain injury caused by ischemia/reperfusion.

Recurrent stroke is difficult to treat and life threatening. Transfer of anti-inflammatory gene is a potential gene therapy strategy for ischemic stroke. Using recombinant adeno-associated viral vector 1 (rAAV1)-mediated interleukin 10 (IL-10), we investigated whether transfer of beneficial gene into the rat cerebral vessels during interventional treatment for initial stroke could attenuate brain injury caused by recurrent stroke. Male Wistar rats were administered rAAV1-IL-10, rAAV1-YFP, or saline into the left cerebral artery. Three weeks after gene transfer, rats were subjected to occlusion of the left middle cerebral artery (MCAO) for 45 min followed by reperfusion for 24 h. IL-10 levels in serum were significantly elevated 3 weeks after rAAV1-IL-10 injection, and virus in the cerebral vessels was confirmed by in situ hybridization. Pre-existing IL-10 but not YFP decreased the neurological dysfunction scores, brain infarction volume, and the number of injured neuronal cells. AAV1-IL-10 transduction increased heme oxygenase (HO-1) mRNA and protein levels in the infarct boundary zone of the brain. Thus, transduction of the IL-10 gene in the cerebral artery prior to ischemia attenuates brain injury caused by ischemia/reperfusion in rats. This preventive approach for recurrent stroke can be achieved during interventional treatment for initial stroke.

HSF1 is a transcriptional activator of IL-10 gene expression in RAW264.7 macrophages.

The heat shock transcription factor (HSF) is an important transactivator of the heat shock genes. Recent studies have shown that HSF1 acts as a repressor of non-heat shock genes to protect against endotoxemia. In this study, we found that heat shock treatment and HSF1 over-expression augmented the induction of interleukin (IL)-10 mRNA. Computational analysis of the mouse IL-10 promoter region showed that three potential heat shock elements (HSEs) were located at mouse IL-10 gene promoter, among which only the -387/-360 probe formed a complex with HSF1. The lack of binding of the other two HSEs to HSF1 suggested the critical role of the flanking sequences in the binding specificity of HSE to HSF1. Moreover, we showed that HSF1 overexpression transactivated mouse IL-10 gene promoter and this transcriptional activation was inhibited by the mutation of HSE in the -387/-360 region of IL-10 gene promoter using luciferase reporter assay. These findings indicate that HSF1 is a transcriptional activator of anti-inflammatory mediator IL-10 gene in RAW264.7 macrophages.

[Quality evaluation of randomized controlled trials on treatment of cognitive-behavioral therapy on temporomandibular disorders].

OBJECTIVE: To evaluate methodological and reporting quality of the randomized controlled trials on cognitive-behavioral therapy(CBT) on temporomandibular disorders(TMD). METHODS: The electronic databases of Medline via Ovid, EMBASE, Cochrane Central Register of Controlled Trial, CBM and CNKI, and five Chinese stomatological journals were included to collect randomized controlled trial(RCT) and quasi-RCT(qRCT) on CBT on TMD. Data were assessed using the quality assessment criteria recommended by the Cochrane Collaboration, and the reporting quality was assessed using the consolidated standards of reporting trials (CONSORT) checklist. RESULTS: 232 articles were collected by the search strategy, of which 5 (3 RCTs and 2 qRCTs) met the inclusion criteria. The methodological quality varied among the studies with 2 scored as B and 3 as C. The highest score evaluated by the CONSORT checklist was 24. CONCLUSION: The methodological qualities of included studies on CBT on TMD are generally low, and its reporting quality which is checked by CONSORT is also unsatisfactory yet.

HSF1 regulates expression of G-CSF through the binding element for NF-IL6/CCAAT enhancer binding protein beta.

Heat shock factor 1 (HSF1) is the major heat shock transcription factor and plays an essential role in mediating the cellular response to physiological and environmental stress. We found that LPS-induced expression of the granulocyte-colony stimulating factor (G-CSF) gene was upregulated in HSF1 knock-out (HSF1(-/-)) mice using a gene array. In order to determine whether and how HSF1 regulates the induced expression of G-CSF, mRNA, and protein levels of G-CSF were detected by Northern blotting and ELISA, the promoter of G-CSF was analyzed with an online transcription element search system and the transcriptional activity of the G-CSF promoter was analyzed by EMSA and a reporter gene assay. The results showed that transcription and protein secretion of G-CSF induced by LPS are both inhibited by HSF1. Three high affinity binding sites for NF-IL6/CCAAT enhancer binding protein beta, but no heat shock element, were identified in the core promoter of G-CSF. The DNA-binding capability of NF-IL6 to the G-CSF promoter was reinforced by LPS but not influenced by heat shock or HSF1. However, HSF1 was observed to bind to the binding sites of NF-IL6 in the G-CSF promoter. The transcriptional activity of the G-CSF promoter was enhanced by LPS or NF-IL6 and inhibited by HSF1 in a dose dependent manner. We conclude that HSF1 regulates expression of G-CSF through binding to the NF-IL6-binding element.

[Screen of inflammatory genes regulated by heat shock factor 1 and corroboration with SOCS3 gene].

OBJECTIVE: To screen the inflammatory mediators genes regulated by HSF1, and explore the mechanism of downstream genes regulated by HSF1. METHODS: HSF-/- and HSF1+/+ mice were injected with 15 mg/kg LPS intraperitoneally (ip), respectively, and were treated as previous after HSR. The total RNA of lung tissues were extracted and filtrated by SuperArray gene Microarry. The promoter of candidate genes were analyzed by transcription element search software to search for heat shock element (HSE). Select the suppressor of cytokine signaling 3 (SOCS3) with HSE. Macrophage cells were stimulated with 400 ng/mL LPS, and were treated as previous after HSR, then the total RNA was extracted respectively. RT-PCR and northern blot assay were performed to detect the expression levels of SOCS3 mRNA. RESULTS: Fifteen genes were repressed by HSF1, including 9 genes with complete HSE. Eleven genes were accelerated by HSF1 possibly, including 8 genes with complete HSE. The promoter of SOCS3 gene contained one complete HSE. LPS stimulation obviously increased the levels of SOCS3 mRNA in macrophages of RAW264.7 mice, which was inhibited by HSR and over-expression of HSF1. CONCLUSION: HSR or HSF1 inhibits LPS induced expression of SOCS3 mRNA; HSF1 might inhibit LPS-induced expression of SOCS3 mRNA by binding to HSE in the promoter of SOCS3 gene.