SURFACE MARKERS AND GENE EXPRESSION TO CHARACTERIZE THE DIFFERENTIATION OF MONOLAYER EXPANDED HUMAN ARTICULAR CHONDROCYTES

Autologous chondrocyte implantation (ACI) is a method of cartilage repair. To improve the quality of regenerated tissue by ACI, it is essential to identify surface marker expression correlated with the differentiation status of monolayer expanded human articular chondrocytes and to define the index for discriminating dedifferentiated cells from monolayer expanded human articular chondrocytes. Normal human articular chondrocytes were cultured in monolayer until passage 4. At each passage, mRNA expression of collagen type I, II, and X and aggrecan was analyzed by real-time quantitative PCR, and the surface marker expression of CD14, CD26, CD44, CD49a, CD49c, CD54, and CD151 was analyzed by fluorescence-activated cell sorting (FACS). The ratios of mRNA levels of collagen type II to I (Col II/Col I) represented the differentiation status of chondrocytes more appropriately during monolayer culture. The surface marker expression of CD44, CD49c, and CD151 was upregulated according to the dedifferentiation status, whereas that of CD14, CD49a, and CD54 was downregulated. The most appropriate combination of the ratio of Col II/Col I was CD54 and CD44. Cell sorting was performed using a magnetic cell sorting system (MACS) according to CD54 and CD44, and real-time quantitative PCR was performed for the cell subpopulations before and after cell sorting. The expression of collagen type II and aggrecan of the chondrocytes after MACS was higher than that before sorting, but not significantly. The mean fluorescence intensity (MFI) ratio of CD54 to CD44 could be an adequate candidate as the index of the differentiation status

MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes

INTRODUCTION: Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA. METHODS: MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA). RESULTS: In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125. CONCLUSIONS: Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA

An integrated RH map of porcine chromosome 10

BACKGROUND: Whole genome radiation hybrid (WG-RH) maps serve as "scaffolds" to significantly improve the orientation of small bacterial artificial chromosome (BAC) contigs, order genes within the contigs and assist assembly of a sequence-ready map for virtually any species. Here, we report the construction of a porcine: human comparative map for pig (Sus scrofa) chromosome 10 (SSC10) using the IMNpRH2(12,000-rad )porcine WG-RH panel, integrated with the IMpRH(7000-rad )WG-RH, genetic and BAC fingerprinted contig (FPC) maps. RESULTS: Map vectors from the IMNpRH2(12,000-rad )and IMpRH(7,000-rad )panels were merged to construct parallel framework (FW) maps, within which FW markers common to both panels have an identical order. This strategy reduced map discrepancies between the two panels and significantly improved map accuracy. A total of 216 markers, including 50 microsatellites (MSs), 97 genes and ESTs, and 69 BAC end sequences (BESs), were ordered within two linkage groups at two point (2 pt) LOD score of 8. One linkage group covers SSC10p with accumulated map distances of 738.2 cR(7,000 )and 1814.5 cR(12,000), respectively. The second group covers SSC10q at map distances of 1336.9 cR(7,000 )and 3353.6 cR(12,000), yielding an overall average map resolution of 16.4 kb/cR(12,000 )or 393.5 kb per marker on SSC10. This represents a ~2.5-fold increase in map resolution over the IMpRH(7,000-rad )panel. Based on 127 porcine markers that have homologous sequences in the human genome, a detailed comparative map between SSC10 and human (Homo sapiens) chromosome (HSA) 1, 9 and 10 was built. CONCLUSION: This initial comparative RH map of SSC10 refines the syntenic regions between SSC10 and HSA1, 9 and 10. It integrates the IMNpRH2(12,000-rad )and IMpRH(7,000-rad), genetic and BAC FPC maps and provides a scaffold to close potential gaps between contigs prior to genome sequencing and assembly. This map is also useful in fine mapping of QTLs on SSC10

An Autopsy Case of Anaplastic Pancreatic Ductal Carcinoma (Spindle Cell Type) Multiple Onset in the Pancreas

This is a case of a 75-year-old man who was diagnosed with anaplastic pancreatic ductal carcinoma (spindle cell type). His image findings showed pancreatic head cysts and pancreatic head, body, and tail tumors respectively. EUS-FNA was performed to the pancreatic head and pancreatic body tumors, and the same high atypical type cells suspected of cancer were obtained from either specimen, and finally total pancreatectomy was performed. On the specimen, there were 4 lesions in the pancreas; histology showed that the same anaplastic pancreatic ductal carcinoma (spindle cell type) was obtained from the pancreatic head cyst and the pancreatic tumors

Difference between far-infrared photoconductivity spectroscopy and absorption spectroscopy: theoretical evidence of the electron reservoir mechanism

The intriguing difference between far-infrared photoconductivity spectroscopy and absorption spectroscopy in the measurement of the magnetoplasmon frequency in GaAs quantum wells reported by Holland et al. [Phys. Rev. Lett. 93, 186804 (2004)] remains unexplained to date. This Letter provides a consistent mechanism to solve this puzzle. The mechanism is based on the electron reservoir model for the integer quantum Hall effect in graphene [Phys. Lett. A 376, 616 (2012)]. We predict sharp kinks to appear in the magnetic induction dependence of the magnetoplasmon frequency at very low temperatures such as 14 mK in the same GaAs quantum well sample used by Holland et al.