[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.

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.

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.

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.

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.

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 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.

Induced pluripotent stem cells derived from an autosomal dominant polycystic kidney disease patient carrying a PKD1 Q533X mutation.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most prevalent monogenic kidney disorder leading to kidney failure. We generated induced pluripotent stem cells (iPSCs) from a 37-year-old man carrying a PKD1 Q533X mutation who suffered from kidney failure and a myocardial infarction. The iPSCs were reprogrammed from the patient's peripheral blood mononuclear cells using the Sendai virus system, and were confirmed to possess the specific PKD1 Q533X mutation and normal karyotype. Pluripotency was confirmed using in vitro and in vivo assays. This iPSC line will be useful for studying the mechanisms driving the complicated pathophysiology of ADPKD.

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.

Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis.

Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH < 7.2) were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine, n = 20) and control (normal saline, n = 20) groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition, cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5) or to culture medium (control). Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group (p < 0.05). In addition, in pH 6.5 or pH 7.3 HBSS buffer, the expression levels of cell stress (p53) and apoptosis (caspase-3, Bcl-xL) markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR). L-glutamine also increased the beating function of cardiomyocytes, especially at the lower pH level (6.5). More importantly, glutamine decreased cardiomyocyte apoptosis and increased these cells' beating function at a low pH level.

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.

Single nucleotide polymorphism barcoding to evaluate oral cancer risk using odds ratio-based genetic algorithms.

Cancers often involve the synergistic effects of gene-gene interactions, but identifying these interactions remains challenging. Here, we present an odds ratio-based genetic algorithm (OR-GA) that is able to solve the problems associated with the simultaneous analysis of multiple independent single nucleotide polymorphisms (SNPs) that are associated with oral cancer. The SNP interactions between four SNPs-namely rs1799782, rs2040639, rs861539, rs2075685, and belonging to four genes (XRCC1, XRCC2, XRCC3, and XRCC4)-were tested in this study, respectively. The GA decomposes the SNPs sets into different SNP combinations with their corresponding genotypes (called SNP barcodes). The GA can effectively identify a specific SNP barcode that has an optimized fitness value and uses this to calculate the difference between the case and control groups. The SNP barcodes with a low fitness value are naturally removed from the population. Using two to four SNPs, the best SNP barcodes with maximum differences in occurrence between the case and control groups were generated by GA algorithm. Subsequently, the OR provides a quantitative measure of the multiple SNP synergies between the oral cancer and control groups by calculating the risk related to the best SNP barcodes and others. When these were compared to their corresponding non-SNP barcodes, the estimated ORs for oral cancer were found to be great than 1 [approx. 1.72-2.23; confidence intervals (CIs): 0.94-5.30, p < 0.03-0.07] for various specific SNP barcodes with two to four SNPs. In conclusion, the proposed OR-GA method successfully generates SNP barcodes, which allow oral cancer risk to be evaluated and in the process the OR-GA method identifies possible SNP-SNP interactions.

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).

Identifying the regulatory element for human angiotensin-converting enzyme 2 (ACE2) expression in human cardiofibroblasts.

Angiotensin-converting enzyme 2 (ACE2) has been proposed as a potential target for cardioprotection in regulating cardiovascular functions, owing to its key role in the formation of the vasoprotective peptides angiotensin-(1-7) from angiotensin II (Ang II). The regulatory mechanism of ace2 expression, however, remains to be explored. In this study, we investigated the regulatory element within the upstream of ace2. The human ace2 promoter region, from position -2069 to +20, was cloned and a series of upstream deletion mutants were constructed and cloned into a luciferase reporter vector. The reporter luciferase activity was analyzed by transient transfection of the constructs into human cardiofibroblasts (HCFs) and an activating domain was identified in the -516/-481 region. Deletion or reversal of this domain within ace2 resulted in a significant decrease in promoter activity. The nuclear proteins isolated from the HCFs formed a DNA-protein complex with double stranded oligonucleotides of the -516/-481 domain, as detected by electrophoretic mobility shift assay. Site-directed mutagenesis of this region identified a putative protein binding domain and a potential binding site, ATTTGGA, homologous to that of an Ikaros binding domain. This regulatory element was responsible for Ang II stimulation via the Ang II-Ang II type-1 receptor (AT1R) signaling pathway, but was not responsible for pro-inflammatory cytokines TGF-ß1 and TNF-α. Our results suggest that the nucleotide sequences -516/-481 of human ace2 may be a binding domain for an as yet unidentified regulatory factor(s) that regulates ace2 expression and is associated with Ang II stimulation.

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.

Regulation of angiotensin converting enzyme II by angiotensin peptides in human cardiofibroblasts.

Numerous studies have suggested that angiotensin peptides modulate the expression of angiotensin converting enzyme II (ACE2) in the cardiovascular system, but the molecular mechanisms remain poorly understood. In the present study, human cardiac fibroblasts (HCF) were used to test the regulatory effects of angiotensin II (Ang II) and angiotensin-(1-7) [Ang-(1-7)] on ACE2 expression. The results show that Ang II upregulates ACE2 expression. This action is modulated through activation of Ang II type 1 receptor (AT1R). Ang II-mediated ACE2 upregulation was blocked by antagonists of AT1R and ERK-MAPK signaling pathways. Additionally, Ang-(1-7) increased ACE2 expression, and this upregulation was inhibited by Ang-(1-7) Mas receptor blockade. Our results further reveal that the activation of p-ERK1/2 proteins plays a critical role in upregulating ACE2 in Ang-(1-7)-stimulated HCF cells. This effect occurs independently of the Ang II-AT1R signaling pathway. In conclusion, we propose that Ang II-upregulated ACE2 may increase Ang-(1-7) formation from Ang II, and that ACE2 expression is further enhanced by Ang-(1-7) in a positive feedback loop.

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.

Dynamic programming for single nucleotide polymorphism ID identification in systematic association studies.

Single nucleotide polymorphisms (SNPs) play an important role in personalized medicine. However, the SNP data reported in many association studies provide only the SNP nucleotide/amino acid position, without providing the SNP ID recorded in National Center for Biotechnology Information databases. A tool with the ability to provide SNP ID identification, with a user-friendly interface, is needed. In this paper, a dynamic programming algorithm was used to compare homologs when the processed input sequence is aligned with the SNP FASTA database. Our novel system provides a web-based tool that uses the National Center for Biotechnology Information dbSNP database, which provides SNP sequence identification and SNP FASTA formats. Freely selectable sequence formats for alignment can be used, including general sequence formats (ACGT, [dNTP1/dNTP2] or IUPAC formats) and orientation with bidirectional sequence matching. In contrast to the National Center for Biotechnology Information SNP-BLAST, the proposed system always provides the correct targeted SNP ID (SNP hit), as well as nearby SNPs (flanking hits), arranged in their chromosomal order and contig positions. The system also solves problems inherent in SNP-BLAST, which cannot always provide the correct SNP ID for a given input sequence. Therefore, this system constitutes a novel application which uses dynamic programming to identify SNP IDs from the literature and keyed-in sequences for systematic association studies. It is freely available at http://bio.kuas.edu.tw/SNPosition/.