[Regulation of autophagy in the treatment of prostate cancer: Progress in research].

Prostate cancer (PCa) is one of the most common genital malignancies in males, with gradually increasing morbidity and mortality in recent years. Improving the treatment of PCa has an important clinical significance for the patients. Lots of recent studies show that autophagy is closely related to this malignancy. More and more emphasis is being attached to the investigation of the role of autophagy in the development and progression of PCa as well as to the treatment of the disease by regulating the level of autophagy. This review summarizes the advances in the studies of the role of autophagy in the treatment of PCa.

Copy number variant hotspots in Han Taiwanese population induced pluripotent stem cell lines - lessons from establishing the Taiwan human disease iPSC Consortium Bank.

BACKGROUND: The Taiwan Human Disease iPSC Service Consortium was established to accelerate Taiwan's growing stem cell research initiatives and provide a platform for researchers interested in utilizing induced pluripotent stem cell (iPSC) technology. The consortium has generated and characterized 83 iPSC lines: 11 normal and 72 disease iPSC lines covering 21 different diseases, several of which are of high incidence in Taiwan. Whether there are any reprogramming-induced recurrent copy number variant (CNV) hotspots in iPSCs is still largely unknown. METHODS: We performed genome-wide copy number variant screening of 83 Han Taiwanese iPSC lines and compared them with 1093 control subjects using an Affymetrix genome-wide human SNP array. RESULTS: In the iPSCs, we identified ten specific CNV loci and seven "polymorphic" CNV regions that are associated with the reprogramming process. Additionally, we established several differentiation protocols for our iPSC lines. We demonstrated that our iPSC-derived cardiomyocytes respond to pharmacological agents and were successfully engrafted into the mouse myocardium demonstrating their potential application in cell therapy. CONCLUSIONS: The CNV hotspots induced by cell reprogramming have successfully been identified in the current study. This finding may be used as a reference index for evaluating iPSC quality for future clinical applications. Our aim was to establish a national iPSC resource center generating iPSCs, made available to researchers, to benefit the stem cell community in Taiwan and throughout the world.

Recovery of CD45(-)/Lin(-)/SSEA-4(+) very small embryonic-like stem cells by cord blood bank standard operating procedures.

BACKGROUND AIMS: Very small embryonic-like (VSEL) stem cells are a rare cell population present in bone marrow, cord blood and other tissues that displays a distinct small cell size and the ability to give rise to cells of the three germ layers. VSEL stem cells were reported to be discarded in the red blood cell fraction by Ficoll-Paque density gradient centrifugation during the processing of bone marrow and cord blood specimens. However, most cord blood banks do not include density gradient centrifugation in their procedures while red blood cells are removed by Hespan sedimentation following the Cord Blood Transplantation Study cord blood bank standard operating procedures (COBLT SOP). To clarify the retention of VSEL stem cells, we investigated the recovery of VSEL stem cells following COBLT SOP guidelines. METHODS: The recovery of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells of umbilical cord blood was examined by flow cytometry before and after COBLT SOP processing, and relative expression of pluripotent genes was analyzed by quantitative polymerase chain reaction. RESULTS: CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells were mostly recovered in the final products following COBLT SOP guidelines. The expression of pluripotent genes could be maintained at >80% in products after hetastarch (Hespan; B. Braun Medical Inc., Irvine, CA, USA) processing. CONCLUSIONS: The rare sub-population of CD45(-)/Lin(-)/SSEA-4(+) VSEL stem cells survived after Hespan sedimentation. This finding suggests that umbilical cord blood units cryopreserved by COBLT SOP in cord blood banks should retain most VSEL stem cells present in the un-processed specimens.

Lysophosphatidic acid induces erythropoiesis through activating lysophosphatidic acid receptor 3.

Lysophosphatidic acid (LPA), an extracellular lipid mediator, exerts multiple bioactivities through activating G protein-coupled receptors. LPA receptor 3 (LPA(3)) is a member of the endothelial differentiation gene family, which regulates differentiation and development of the circulation system. However, the relationship among the LPA receptors (LPARs) and erythropoiesis is still not clear. In this study, we found that erythroblasts expressed both LPA(1) and LPA(3), and erythropoietic defects were observed in zLPA(3) antisense morpholino oligonucleotide-injected zebrafish embryos. In human model, our results showed that LPA enhanced the erythropoiesis in the cord blood-derived human hematopoietic stem cells (hHSCs) with erythropoietin (EPO) addition in the plasma-free culture. When hHSCs were treated with Ki16425, an antagonist of LPA(1) and LPA(3), erythropoietic process of hHSCs was also blocked, as detected by mRNA and protein expressions of CD71 and GlyA. In the knockdown study, we further demonstrated that specific knockdown of LPA(3), not LPA(1), blocked the erythropoiesis. The translocation of ß-catenin into the nucleus, a downstream response of LPAR activation, was blocked by Ki16425 treatment. In addition, upregulation of erythropoiesis by LPA was also blocked by quercetin, an inhibitor of the ß-catenin/T-cell factor pathway. Furthermore, the enhancement of LPA on erythropoiesis was diminished by blocking c-Jun-activated kinase/signal transducer and activator of transcription and phosphatidylinositol 3-kinase/AKT activation, the downstream signaling pathways of EPO receptor, suggested that LPA might play a synergistic role with EPO to regulate erythropoietic process. In conclusion, we first reported that LPA participates in EPO-dependent erythropoiesis through activating LPA(3).

Microlens Array Fabrication by Using a Microshaper.

A simple, easy, inexpensive, and quick nonsilicon-based micromachining method was developed to manufacture a microlens array. The spherical surface of the microlens was machined using a microshaper mounted on a three-axis vertical computer numerical control (CNC) machine with cutter-path-planning. The results show the machined profiles of microlens agree well with designed profiles. The focus ability of the machined microlens array was verified. The designed and measured focal lengths have average 1.5% error. The results revealed that the focal lengths of micro lens agreed with the designed values. A moderate roughness of microlens surface is obtained by simply polishing. The roughness of the lens surface is 43 nm in feed direction (x-direction) and 56 nm in path interval direction (y-direction). It shows the simple, scalable, and reproducible method to manufacture microlenses by microshaper with cutter-path-planning is feasible.

Generation of induced pluripotent stem cells FIRDIi001-A from a Taiwanese subject carrying ALDH2 pE487K mutation.

The ALDH2 mutation (ALDH2*2) is caused by an amino acid substitution ALDH2 rs671 G>A (pE487K) which reduces ALDH2 enzyme activity. When individuals with the ALDH2 mutation consume alcohol, accumulating acetaldehyde in the blood can cause reddened face, headache, nausea, and palpitations; symptoms referred to as Alcohol Flushing Reaction. We report the production of an induced pluripotent stem cell (iPSC) line, FIRDIi001-A, developed from peripheral blood mononuclear cells of a 39-year-old male subject with the ALDH2*2 mutation. The ALDH2-pE487K iPSCs will be valuable in investigating pathogenic mechanisms involved in the link between the ALDH2 polymorphism and alcohol-related diseases.

Generation of IBMS-iPSC-021, -022, -023 human induced pluripotent stem cells (IBMSi016-A, IBMSi017-A, and IBMSi018-A) derived from patients with the ALDH2 rs671 polymorphism.

ALDH2 gene is coded for the aldehyde dehydrogenase (ALDH), which is an enzyme involved in alcohol metabolism. Compared to normal aldehyde dehydrogenases, a homozygous point mutation on exon 12 from G to A significantly reduces its efficiency. In this study, we have reported the generation of IBMS-iPSC-021-04, IBMS-iPSC-022-01, and IBMS-iPSC-023-03 as induced pluripotent stem cell (iPSC) lines carrying the homozygous form of ALDH2 with the rs671 genetic polymorphism (E487K mutation). These cell lines were characterized in terms of pluripotency and differentiation potential. They serve as useful platforms to study alcohol metabolism and other chronic diseases associated with alcohol consumption.

Exploring the molecular mechanisms of OSU-03012 on vascular smooth muscle cell proliferation.

Restenosis is resulted from the proliferation and migration of vascular smooth muscle cells (VSMCs) from the arterial media into the intima within the vessel lumen following percutaneous transluminal coronary angioplasty (PTCA). OSU-03012, a synthetic compound (2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl} acetamide) acting as a PDK-1 inhibitor, is used as an apoptosis-promoting anticancer drug. However, whether OSU-03012 can inhibit VSMC proliferation and migration following PTCA remains unclear. In this study, we used A10 smooth muscle cells cultured in 10% FBS for stimulating proliferation and evaluated the inhibitory effects of OSU-03012 on cell proliferation and migration. The data demonstrated that OSU-03012 dose-dependently inhibited A10 cell proliferation examined by Trypan blue, MTT and morphological alteration assays, and inhibited the levels of proliferation-related proteins, proliferating cell nuclear antigen (PCNA), phosphorylated ERK examined by western blotting. Additionally, 10 µM OSU-03012 also enhanced apoptosis examined using DAPI assay by regulating apoptosis-related proteins. Furthermore, compared with the control group, A10 cells treated with 10 µM OSU-03012 showed a lower number of migrating cells examined by Boyden Chamber assay, and a dose-dependently reduced NFκB-dependent and interferon-stimulated response element (ISRE) promoter luciferase activities, implying the anti-migration and anti-inflammation effects of OSU03012. Taken together, this study provides insights into the pharmacological mechanisms of OSU-03012 in preventing smooth muscle cell proliferation, migration, and inflammation supporting the novel discovery of OSU-03012 as an adjuvant therapy for balloon injury-induced restenosis.

Matrix metalloproteinases (MMP) 1 and MMP10 but not MMP12 are potential oral cancer markers.

The aim of this study was to investigate the mRNA performance of matrix metalloproteinases (MMP) 1, MMP10 and MMP12 as oral cancer markers. With gingiva as the control, the areas under the receiver- operating characteristic curves (AUCs) of the relative gene expressions for MMP1, MMP10 and MMP12 were 0.715, 0.727 and 0.513, respectively. With the margins or neck platysma muscles as controls, the AUCs of MMP1, MMP10 and MMP12 were 0.746 vs 0.626, 0.712 vs 0.683 and 0.697 vs 0.630, respectively. MMP10 displayed the best sensitivity for oral cancer detection with any controls. MMP1 and MMP10 were suitable markers for cancer detection with gingiva and margin as controls. Using neck tissue as the control, only MMP10 was suitable for cancer detection. With margin and neck controls, there were no significant differences for MMP1, MMP10 and MMP12 in different stages, invasion and locations or different habits. Therefore, MMP1 and MMP10 but not MMP12 are potential oral cancer markers.

A novel growth hormone 1 gene-derived probe for Oncorhynchus masou formosanus distinguished from the Oncorhynchus subspecies.

Traditional mitochondrial 16S rRNA is commonly used in many species identification studies. However, it is difficult to apply to the phylogenetic studies among the Oncorhynchus subspecies, which is a crucial need for management purposes for Oncorhynchus masou formosanus, Taiwan salmon. In this study, we have developed an improved species identification method for Taiwan salmon distinguished with other Oncorhynchus subspecies tested by exploiting PCR for growth hormone (GH) 1 gene. By comparing DNA sequences for GH1 from 11 species of Oncorhynchus subspecies we designed novel PCR primers that exploit differences between Taiwan salmon and other Oncorhynchus subspecies. Therefore, the technique is an important tool in the management of populations of the endangered land-locked Taiwan salmon preventing from their possible hybrids with other Oncorhynchus subspecies once tested.

Generation of induced pluripotent stem cells (IBMSi011-A) from a patient with Parkinson's disease carrying LRRK2 p.I1371V mutation.

Leucine rich repeat kinase 2 (LRRK2) is the causative gene for autosomal-dominant familial forms of Parkinson's disease (PD). Here, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a female patient with LRRK2 c.4111A > G (p.I1371V) mutation by using the Sendai-virus delivery system. The resulting iPSCs had a normal karyotype. The iPSCs also showed pluripotency confirmed by immunofluorescent staining and differentiated into the three germ layers in vivo. This cellular model will provide a platform for studying the role of LRRK2 in the disease process.

Upregulation of matrix metalloproteinase-9 and tissue inhibitors of metalloproteinases in rapid atrial pacing-induced atrial fibrillation.

Remodeling of atrial extracellular matrix (ECM) in atrial fibrillation (AF) involves changes in the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). The contributions of MMPs and TIMPs to the pathogenesis of AF development have not been clearly defined. This study evaluated the in situ activity and expression of gelatinases (MMP-2 and MMP-9) and their relationship with TIMP-1 or TIMP-3 in atria undergoing rapid atrial pacing for the induction of AF (4 weeks' pacing followed by 2 weeks of maintained AF) in pigs. In AF atria, in situ gelatinase activity was mainly localized in the interstitium of atrial myocardium, and was significantly larger than that of sinus rhythm control (i.e., sham control). The significant increase of MMP-9 in its pro-form and mRNA level, but not MMP-2, was shown to be responsible for the increased gelatinase activity in atria with AF. The inhibitory activities of glycosylated TIMP-1 and TIMP-3, but not TIMP-2, in AF tissues were markedly elevated and also localized in the atrial interstitium. TIMP-1 was found to be mostly colocalized with gelatinase activity over the AF tissues, implying the coexistence of gelatinase activity and TIMP-1, but TIMP-3 appeared only partially colocalized and discontinued the gelatinase activity surrounding the cardiomyocytes. TIMP-1 and TIMP-3 may play differential roles in the inhibition of gelatinase activity in vivo. Together with the survey of several MMPs transcripts and the level of transforming growth factor-beta1 (TGF-beta1), we proposed that the increased activity of gelatinase (i.e., MMP-9), TIMP-1 and TIMP-3 and their interaction may contribute to atrial ECM remodeling of AF.

High-throughput avian molecular sexing by SYBR green-based real-time PCR combined with melting curve analysis.

BACKGROUND: Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved. RESULTS: Here, we are the first to introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Spilornis cheela hoya (S. c. hoya) and Pycnonotus sinensis (P. sinensis) were used to illustrate this novel molecular sexing technique. The difference in the length of CHD genes in S. c. hoya and P. sinensis is 13-, and 52-bp, respectively. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis of S. c. hoya and in PCR/MCA of S. c. hoya and P. sinensis. In contrast, we redesigned sex-specific primers to yield 185- and 112-bp PCR products for the CHD-Z and CHD-W genes of S. c. hoya, respectively, using PCR/MCA. Using this specific primer set, at least 13 samples of S. c. hoya were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were distinguished. CONCLUSION: In this study, we introduced a high-throughput avian molecular sexing technique and successfully applied it to two species. This new method holds a great potential for use in high throughput sexing of other avian species, as well.

Restriction enzyme mining for SNPs in genomes.

Many different single nucleotide polymorphisms (SNPs) genotyping methods have been developed recently. However, most of them are expensive. Using restriction enzymes for SNP genotyping is a cost-effective method. However, restriction enzyme mining for SNPs in a genome sequence is still challenging for researchers who do not have a background in genomics and bioinformatics. In this review, the basic bioinformatics tools used for restriction enzyme mining for SNP genotyping are summarized and described. The objectives of this paper include: i) the introduction of SNPs, genotyping and PCR-restriction fragment length polymorphism (RFLP); ii) a review of components for genotyping software, including tools for primer design only or restriction enzyme mining only; iii) a review of software providing the flanking sequence for primer design; iv) recent advances in PCR-RFLP tools and natural and mutagenic PCR-RFLP; v) highlighting the strategy for restriction enzyme mining for SNP genotyping; vi) a discussion of potential problems for multiple PCR-RFLP. The different implications for restriction enzymes on sense and antisense strands are also discussed. Our PCR-RFLP freeware, SNP-RFLPing, is included in this review to illustrate many characteristics of PCR-RFLP software design. Future developments will include further sophistication of PCR-RFLP software in order to provide better visualization and a more interactive environment for SNP genotyping and to integrate the software with other tools used in association studies.

Combinational polymorphisms of four DNA repair genes XRCC1, XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan.

BACKGROUND: Many single nucleotide polymorphisms (SNPs) have been found to be associated with oral cancer but the biological interactions through SNPs are seldom addressed. In this study, we focused on the joint effect for SNP combinations of four DNA repair genes, X-ray repair cross-complementing groups (XRCCs) 1-4, involved in major cancer-related pathways. METHODS: Single nucleotide polymorphism genotyping was determined using by polymerase chain reaction-restriction fragment length polymorphism in this study (case = 103, control = 98). Different numbers of combinational SNPs with genotypes called the pseudo-haplotypes from these chromosome-wide genes were used to evaluate their joint effect on oral cancer risk. RESULTS: Except for XRCC2 rs2040639-AG, none of these SNPs was found to individually contribute to oral cancer risk. However, for two combined SNPs, the proportion of subjects with oral cancer was significantly higher in the pseudo-haplotype with AG-CC genotypes in rs2040639-rs861539 (XRCC2-XRCC3) compared with those with non-AG-CC genotypes. Similarly, the pseudo-haplotype of rs2040639-rs861539-rs2075685 (XRCC2-XRCC3-XRCC4) and rs2040639-rs861539-rs2075685-rs1799782 (XRCCs 1-4) with specific genotype pattern (AG-CC-TG and CT-AG-CC-TG) among three and four combinational SNPs were significantly associated with oral cancer. After controlling for age, gender, smoking, drinking, and betel nut chewing, the estimated odds ratio of oral cancer were 2.45, 5.03, and 10.10 for two, three and four specific SNP combinations, respectively, comparing these specific pseudo-haplotypes to their corresponding non-pseudo-haplotypes. CONCLUSION: We have identified the potential combined XRCCs 1-4 SNPs with genotypes that were associated with oral cancer risk and may have an impact on identification of a high-risk population.

SNP combinations in chromosome-wide genes are associated with bone mineral density in Taiwanese women.

Osteoporosis is a major public health problem, mainly quantified by low BMD. Eleven polymorphisms were investigated in this study; TNFalpha-857 (rs1799724), TGFbeta1-509 (rs1800469), osteocalcin (rs1800247), TNFalpha-308 (rs1800629), PTH BstB I (rs6254), PTH Dra II (rs6256), IL-1ra (VNTR), HSP70 hom (rs2227956), HSP 70-2 (rs1061581), CTR (rs1801197), and BMP-4 (rs17563). The relationship between the combined polymorphisms in different genomic regions and BMD variation was investigated. Among the female subjects, the proportion of subjects with low BMD in low BMI group (< or = 18.50) was significantly higher than that of the middle (18.51-22.99) and high (> or = 23.00) BMI groups (P < 0.05). In post-menopausal women, there was a significant association between low BMD and genotypes ranging from 2 to approximately 7 SNPs. For two combined SNPs, the portion of subjects with low BMD was significantly higher in those with CC-AA genotypes in rs1799724-rs1800629, compared to those with non-CC-AA genotypes in post-menopausal women and the combination of all women. Similarly, part of the combined SNPs with rs1799724-rs1800629-rs6254-rs6256-IL-1ra-rs2227956-rs1801197 was significantly associated with reduced BMD. After controlling for age and BMI, post-menopausal women with certain specific SNP combination had a 3.54- to 4.68-fold increased risk for low BMD, comparing to other SNP combinations. In conclusion, our data suggest that several gene polymorphisms may be cooperatively involved in the development of osteoporosis.

Generation of novel induced pluripotent stem cell (iPSC) line from a 16-year-old sialidosis patient with NEU-1 gene mutation.

Sialidosis is a rare autosomal recessive disorder that affects the intralysosomal catabolism of sialylated glycoconjugates and is involved in cellular immune response. Mutations in NEU1, which encodes the sialidase enzyme, result in sialidosis. Sialidosis is characterized by the progressive lysosomal storage of sialylated glycopeptides and oligosaccharides. In this study, we used Sendai virus reprogramming to generate an induced pluripotent stem cell (iPSC) line carrying the A544G mutation combined with the 667-679 deletion of the NEU1 gene from a sialidosis patient. The patient-specific iPSCs expressed pluripotent markers, possessed a normal karyotype, and displayed the capability to differentiate into three germ layers.

Generation of an induced pluripotent stem cell (iPSC) line from a 40-year-old patient with the A8344G mutation of mitochondrial DNA and MERRF (myoclonic epilepsy with ragged red fibers) syndrome.

Mitochondrial defects are associated with clinical manifestations from common diseases to rare genetic disorders. Myoclonus epilepsy associated with ragged-red fibers (MERRF) syndrome results from an A to G transition at nucleotide position 8344 in the tRNALys gene of mitochondrial DNA (mtDNA) and is characterized by myoclonus, myopathy and severe neurological symptoms. In this study, Sendai reprogramming method was used to generate an iPS cell line carrying the A8344G mutation of mtDNA from a MERRF patient. This patient-specific iPSC line expressed pluripotent stem cell markers, possessed normal karyotype, and displayed the capability to differentiate into mature cells in three germ layers.

Generation of induced pluripotent stem cells from a patient with spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited neurodegenerative disease caused by a trinucleotide repeat (CAG) expansion in the coding region of ATXN3 gene resulting in production of ataxin-3 with an elongated polyglutamine tract. Here, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a male patient with SCA3 by using the Sendai-virus delivery system. The resulting iPSCs had a normal karyotype, retained the disease-causing ATXN3 mutation, expressed pluripotent markers and could differentiate into the three germ layers. Potentially, the iPSCs could be a useful tool for the investigation of disease mechanisms of SCA3.

Generation of induced pluripotent stem cells from a patient with Parkinson's disease carrying LRRK2 p.I2012T mutation.

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by interactions between genetic and environmental factors. Leucine rich repeat kinase (LRRK2) is the most prevalent mutation in autosomal-dominant inheritance of PD. Here, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a female patient with p.I2012T mutation in LRRK2 gene by using the Sendai-virus delivery system. The resulting iPSCs had a normal karyotype. The iPSCs also showed pluripotency confirmed by immunofluorescent staining and differentiated into the 3 germ layers in vivo. This cellular model will provide a useful platform for further pathophysiological studies of PD.