The aim of this study was to examine the feasibility of expanding and regulating mesenchymal stem cells (MSCs) from isolated adult human bone marrow mononuclear cells, seeded on gelatin-hyaluronic acid biomatrices, and then to quantitatively compare the gene expression in three different culture systems. Individual and interactive effects of model system parameters on construct structure, function, and molecular properties were evaluated. The results showed that these adult human MSCs even at old age not only expressed primitive mesenchymal cell markers but also maintained a high level of colony-forming efficiency and were capable of differentiating into osteoblasts, chondrocytes, and adipocytes upon appropriate inductions. After 21 days of culture, we found that the osteoblastic and chondrocytic lineage gene expression were earlier and higher expressed in spinner flask bioreactor culture group when compared with the static culture and rotating wall vessel reactor culture. The osteogenic lineage proteins type I collagen, alkaline phosphatase, and osteocalcin were strongly stained in histological sections of spinner flask bioreactor culture, whereas these were less detected in the other two groups, especially in rotating wall vessel reactor culture. As for the markers associated with the chondrogenic lineage differentiation proteins, type II collagen was apparently expressed in spinner flask culture group, while the expression of proteoglycans (aggreacan, decorin) in three culture conditions took the lead of each other. We conclude that the spinner flask bioreactor with appropriate induction medium reported in this study may be used to rapidly expand adult MSCs and is likely to possess better induction results toward osteoblastic and chondrocytic lineages.

The objective was to assess effectiveness of apraxia treatments using a systematic review. In contrast to previous reviews, each study was rated as to its applicability to occupational therapy practice and its focus on occupational performance using the FAME rating system (defined by four categories: Feasibility, Appropriateness, Meaningfulness, Effectiveness). This systematic review included eight studies: four randomized controlled trials (level 1 evidence) and four pre-post designs (level 3 evidence). Three treatment approaches were reported: errorless learning with training of details; gesture training; and strategy training. FAME scores ranged from A to C. All studies reported significant treatment effects, but only one demonstrated an impact on observed occupational performance that transferred from clinic to home.

This study used DNA methyltransferase 3b (DNMT3b) knockout cells and the functional loss of DNMT3b mutation in immunodeficiency-centromeric instability-facial anomalies syndrome (ICF) cells to understand how DNMT3b dysfunction causes genome instability. We demonstrated that R-loops contribute to DNA damages in DNMT3b knockout and ICF cells. More prominent DNA damage signal in DNMT3b knockout cells was due to the loss of DNMT3b expression and the acquirement of p53 mutation. Genome-wide ChIP-sequencing mapped DNA damage sites at satellite repetitive DNA sequences including (peri-)centromere regions. However, the steady-state levels of (peri-)centromeric R-loops were reduced in DNMT3b knockout and ICF cells. Our analysis indicates that XPG and XPF endonucleases-mediated cleavages remove (peri-)centromeric R-loops to generate DNA beaks, causing chromosome instability. DNMT3b dysfunctions clearly increase R-loops susceptibility to the cleavage process. Finally, we showed that DNA double-strand breaks (DSBs) in centromere are probably repaired by error-prone end-joining pathway in ICF cells. Thus, DNMT3 dysfunctions undermine the integrity of centromere by R-loop-mediated DNA damages and repair.

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Measurement of four dNTP pools is important for investigating metabolism, genome stability, and drug action. In this report, we developed a two-step method for quantitating dNTPs by the combination of rolling circle amplification (RCA) and quantitative polymerase chain reaction (qPCR). We used CircLigase to generate a single-strand DNA in circular monomeric configuration, which was then used for the first step of RCA reaction that contained three dNTPs in excess for quantification of one dNTP at limiting levels. The second step is the amplification of RCA products by qPCR, in which one primer was designed to be completely annealed with the polymeric ssDNA product but not the monomeric template DNA. Using 1 amol of the template in the assay, each dNTP from 0.02 to 2.5 pmol gave a linearity with r 2 > 0.99, and the quantification was not affected by the presence of rNTPs. We further found that the preparation of biological samples for the RCA reaction required methanol and chloroform extraction. The method was so sensitive that 1 × 10 4 cells were sufficient for dNTP quantification with the results similar to those determined by a radio-isotope method using 2 × 10 5 cells. Thus, the RCA/qPCR method is convenient, cost-effective, and highly sensitive for dNTP quantification.