Dgcr8 deletion in the primitive heart uncovered novel microRNA regulating the balance of cardiac-vascular gene program

Primitive mammalian heart transforms from a single tube to a four-chambered muscular organ during a short developmental window. We found that knocking out global microRNA by deleting Dgcr8 microprocessor in Mesp1 cardiovascular progenitor cells lead to the formation of extremely dilated and enlarged heart due to defective cardiomyocyte (CM) differentiation. Transcriptome analysis revealed unusual upregulation of vascular gene expression in Dgcr8 cKO hearts. Single cell RNA sequencing study further confirmed the increase of angiogenesis genes in single Dgcr8 cKO CM. We also performed global microRNA profiling of E9.5 heart for the first time, and identified that miR-541 was transiently highly expressed in E9.5 hearts. Interestingly, introducing miR-541 back into microRNA-free CMs partially rescued their defects, downregulated angiogenesis genes and significantly upregulated cardiac genes. Moreover, miR-541 can target Ctgf and inhibit endothelial function. Our results suggest that microRNAs are required to suppress abnormal angiogenesis gene program to maintain CM differentiation.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Differential regulation of H3S10 phosphorylation, mitosis progression and cell fate by Aurora Kinase B and C in mouse preimplantation embryos

Coordination of cell division and cell fate is crucial for the successful development of mammalian early embryos. Aurora kinases are evolutionarily conserved serine/threonine kinases and key regulators of mitosis. Aurora kinase B (AurkB) is ubiquitously expressed while Aurora kinase C (AurkC) is specifically expressed in gametes and preimplantation embryos. We found that increasing AurkC level in one blastomere of the 2-cell embryo accelerated cell division and decreasing AurkC level slowed down mitosis. Changing AurkB level had the opposite effect. The kinase domains of AurkB and AurkC were responsible for their different ability to phosphorylate Histone H3 Serine 10 (H3S10P) and regulate metaphase timing. Using an Oct4-photoactivatable GFP fusion protein (Oct4-paGFP) and fluorescence decay after photoactivation assay, we found that AurkB overexpression reduced Oct4 retention in the nucleus. Finally, we show that blastomeres with higher AurkC level elevated pluripotency gene expression, which were inclined to enter the inner cell mass lineage and subsequently contributed to the embryo proper. Collectively, our results are the first demonstration that the activity of mitotic kinases can influence cell fate decisions in mammalian preimplantation embryos and have important implications to assisted reproduction.

Analysis of antibacterials drug for department of respiration in old patients / 药学实践杂志

Objective To investigate the utility of antibiotics in elderly patients of respiratory department in No. 210 Hospital of PLA, and to understand the rational use of antibiotics in respiratory department. Methods A total of 534 patients >60 in respiratory department of our hospital from September to December in 2011 were randomly selected. The usage rate of antibiotics, the types that used, dosing routes and so on were recorded and analyzed. Some rational suggestions were dis‐cussed put forward. Results ① The usage rate of antibiotics was 97%. ② A total of 21 kinds of antibiotics had been used. The types of drugs are Amp C, levofloxacin, β‐lactamase inhibitors, nitromidazoles, aminoglycoside, antifungals, MALS, lincomycin. The percentage rate of the top three antibiotics were 59. 97%, 17. 30% and 7. 58%. ③ There were some problems in the usage of antibiotics, such as no indication of medicine, high use rate of antibiotics, improper variety selection, dosing routes and drug intervals, irrational use of drugs combinations. Conclusion The application rate of antibiotics was 97%, there was some illegiti‐mate phenomenon in the use of antibiotics. Some attentions should be paid to the rational use of antibiotics in elderly patients, and to raise the rational use rate of antibiotics.

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.

Efficacy of transvenous embolization of cavernous sinus dural arteriovenous fistula with Onxy or in combination with detachable coils / 中国脑血管病杂志

Objective: To investigate the safety and effectiveness of transvenous embolization of cavernous sinus dural arteriovenous fistula with Onxy or in combination with detachable coils. Methods: Twelve patients with cavernous sinus dural arteriovenous fistula treated by transvenous embolization from February 2007 to December 2010 were analyzed retrospectively. Ten patients were treated via the inferior petrosal sinus approach, 2 were treated via the vein-superior ophthalmic vein approach. Eight patients used Onyx alone, and 4 used Onxy in combination with detachable coils. Results: Circled digit oneAngiography showed complete occlusion in 10 patients immediately after treatment, small amount of residues remained in 2 patients. Six patients were cured with Onyx embolization alone, 2 were improved, 2 were cured with Onyx in combination with detachable coils embolization, and 2 were improved. The total effective rate was 100%, the total cure rate was 66.7%, and none of the patients worsened or died. Circled digit twoTwo patients had intraoperative bradycardia, 3 had postoperative orbital ache. Circled digit threeAll patients received clinical follow-up for 6 to 32 months and no recurrent and worsened were found. Conclusion: The clinical efficacy of transvenous embolization of cavernous sinus dural arteriovenous fistula with Onxy or in combination with detachable coils is effective and safe.

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.

Effect of Huaisanzhen on central analgesic transmitters in the rat of the nerve root pain caused by protrusion of lumbar intervertebra disc / 中国针灸

<p><b>OBJECTIVE</b>To search the mechanism of Huaisanzhen in treating the nerve root pain caused by protrusion of lumbar intervertebra disc.</p><p><b>METHODS</b>Sixty male SD rats were randomly divided into a normal control group, a sham operation control group, a model control group, an aspirin-DL-lysine control group, a routine body acupuncture group, and a Huaisanzhen treatment group. The aspirin-DL-lysine control group, the routine body acupuncture group and the Huaisanzhen treatment group were treated by aspirin-DL-lysine through intramuscular injection, routine acupuncture and Huaisanzhen therapy, respectively. The pain threshold and the content of brain 5-HT in each group were determined.</p><p><b>RESULTS</b>The pain threshold significantly increased in the Huaisanzhen treatment group as compared with that in the model control group at the same hour points (P < 0.05). And at the 30 min, the pain threshold in the aspirin-DL-lysine control group and the routine body acupuncture group was significantly higher than that in the model control group, and the Huaisanzhen treatment group was significantly higher than that in the aspirin-DL-lysine control group and the routine body acupuncture group (all P < 0.05). The 5-HT contents at the same time in the Huaisanzhen treatment group were higher than those of the model control group and the aspirin-DL-lysine control group (P < 0.01). The 5-HT content at 30 min in the Huaisanzhen treatment groups was significantly higher than that of the routine body acupuncture group (P < 0.05).</p><p><b>CONCLUSION</b>The analgesic effect of Huaisanzhen treatment is closely related to increase of 5-HT content in the brain.</p>