Anti-Tumor and Radiosensitization Effects of N-Butylidenephthalide on Human Breast Cancer Cells.

N-Butylidenephthalide (BP), which is extracted from a traditional Chinese medicine, Radix Angelica Sinensis (danggui), displays antitumor activity against various cancer cell lines. The purpose of this study was to investigate the cytotoxic and radiosensitizing effect of BP and the underlying mechanism of action in human breast cancer cells. BP induces apoptosis in breast cancer cells, which was revealed by the TUNEL assay; the activation of caspase-9 and PARP was detected by western blot. In addition, BP-induced G2/M arrest was examined by flow cytometry and the expression levels of the G2/M regulatory protein were detected by western blot. BP also suppresses the migration and invasion of breast cancer cells, which was tested by wound healing and the matrigel invasion assay; the involvement of EMT-related gene expressions was detected by real-time PCR. Furthermore, BP enhanced the radiosensitivity of breast cancer cells, which was measured by the colony formation assay and comet assay, where the foci of γ-H2AX after radiation significantly increased in BP pretreated cells and was evidenced by immunocytochemistry staining and western blot. The homologous recombination (HR) repair protein Rad51 was down-regulated after BP pretreatment. These results indicate that BP might be a potential chemotherapeutic and radiosensitizing agent for breast cancer therapy.

A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.

We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.

The biological effects of hsa-miR-1908 in human adipocytes.

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of gene expression. MiR-1908 is a recently identified miRNA that is highly expressed in human adipocytes. However, it is not known what role of miR-1908 is involved in the regulation of human adipocytes. In this study, we demonstrate that the level of miR-1908 increases during the adipogenesis of human multipotent adipose-derived stem (hMADS) cells and human preadipocytes-visceral. Overexpression of miR-1908 in hMADS cells inhibited adipogenic differentiation and increased cell proliferation, suggesting that miR-1908 is involved in the regulation of adipocyte cell differentiation and metabolism, and, thus, may have an effect on human obesity.

A Novel pro-adipogenesis factor abundant in adipose tissues and over-expressed in obesity acts upstream of PPARγ and C/EBPα.

An important question about adipogenesis is how master adipogenesis factors (defined as being able to initiate adipogenesis when expressed alone) peroxisome proliferator-activated receptor (PPAR) initiate adipogenesis only in differentiating preadipocytes. The objective of our research was to find previously unidentified factors that are unique or highly enriched in cells of the adipocyte lineage during adipogenesis that may provide functional tissue specificity to preadipocytes. We reasoned that such factors may alter expression profile specifically in obese individuals. Omental adipose tissues were obtained from obese and non-obese male patients undergoing emergency abdominal surgery. mRNAs extracted from either group were used for suppression subtraction hybridization (SSH). Genes corresponding to mRNAs enriched in obese versus non-obese patients were identified through sequencing and further analyzed for tissue distribution. Out of ~20 genes, we found several that showed clear fat cell specific expression patterns. In this study, we functionally studied one of these genes, previously designated as open reading frame C10orf116. Our data demonstrated that C10orf116 is highly expressed in adipose tissue and is localized primarily within the nucleus. Over-expression studies in 3T3-L1 cells indicated that it up-regulates the levels of CCAAT/enhancer binding protein α (C/EBPα) and PPARγ and promotes adipogenic differentiation starting from the early stage of adipogenesis. Over-expressed in omental tissues from obese patients, C10orf16 manifested the characteristics of an adipocyte lineage-specific nuclear factor that can modulate the master adipogenesis transcription factors early during differentiation. Further studies of this factor should help reveal tissue-specific events leading to fat cell development at the transcriptional level.

Effects of Lyrm1 knockdown on mitochondrial function in 3 T3-L1 murine adipocytes.

To explore the effects of Lyrm1 knockdown on the mitochondrial function of 3 T3-L1 adipocytes using small interfering RNA (siRNA). 3 T3-L1 preadipocytes were infected with either a negative control (NC) expression lentivirus or a Lyrm1-shRNA expression lentivirus and induced to differentiate. The knockdown efficiency of Lrym1-specific shRNA in 3 T3-L1 cells was evaluated by real-time PCR. The ultrastructure of the mitochondria in adipocytes was visualized using transmission electron microscopy after differentiation. The levels of mitochondrial DNA copy numbers and Ucp2 mRNA were detected by real-time quantitative PCR. The levels of ATP production was detected using a photon-counting luminometer. The mitochondrial membrane potential and ROS levels of cells were analyzed with a FACScan flow cytometer using Cell Quest software. Cells transfected with lentiviral-Lyrm1-shRNA showed a significantly reduced transcription of Lyrm1 mRNA compared with NC cells. The size and ultrastructure of mitochondria in Lyrm1 knockdown adipocytes was similar to those of the NC cells. There was no significant difference in mtDNA copy number between the two groups. The total level of ATP production, mitochondrial membrane potential and Ucp2 mRNA expression levels were dramatically increased in adipocytes transfected with Lyrm1 RNAi. Furthermore, the level of ROS was dramatically decreased in Lyrm1 knockdown adipocytes. Knockdown of the Lyrm1 gene in adipocytes resulted in dramatically increased cellular ATP production, mitochondrial membrane potentials and levels Ucp2 mRNA, while ROS levels were significantly decreased. These results imply that mitochondrial function is improved in adipocytes after the knockdown of Lyrm1.

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway.

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.

Monoclonal antibody to six transmembrane epithelial antigen of prostate-4 influences insulin sensitivity by attenuating phosphorylation of P13K (P85) and Akt: possible mitochondrial mechanism.

We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002 mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.

UCP4 overexpression improves fatty acid oxidation and insulin sensitivity in L6 myocytes.

Obesity, which is caused by energy uptake being greater than energy expenditure, is widely prevalent today. Currently, only a limited number of efficient interventional strategies are available for the prevention of obesity. Previous studies have shown that UCP4 transcription occurs at a considerable level in mouse skeletal muscle; however, the exact functions of UCP4 remain unclear. In this study, we investigated the effect of UCP4 on mitochondrial function and insulin sensitivity in mature L6 myocytes. UCP4 overexpression in L6 myocytes induced increased mitochondrial carnitine palmitoyltransferase 1A (CPT1A) and decreased citrate synthase (CS) mRNA in the basal condition (i.e., in the absence of insulin). UCP4 overexpression significantly improved insulin sensitivity, increased tyrosine phosphorylation of IRS-1 in the presence of insulin, and significantly reduced intracellular triglyceride (TG). Additionally, intracellular ATP content and mitochondrial membrane potential were downregulated. We also observed that intracellular ROS, mitochondrial morphology, and mitochondrial mtDNA copy number were maintained upon UCP4 expression, with no change in mitochondrial fusion and fission. In summary, our findings provide evidence to show that UCP4 overexpression reduced the insulin sensitivity and mitochondrial fatty acid oxidation of L6 myocytes. These findings support the notion that UCPs are ideal targets for treatment of insulin resistance.

The mutation of SPI51, a protease inhibitor of silkworm, resulted in the change of antifungal activity during domestication.

Domestication of silkworm has led to alterations in various gene expression patterns. For instance, many protease inhibitors were significantly downregulated in the domestic silkworm cocoon compared to its wild progenitor. Considering that SPI51 is the most abundant protease inhibitor in silkworm cocoons, herein, we compared the gene structures and sequences of SPI51 from B. mori (BmoSPI51) and B. mandarina (BmaSPI51). Comparing to the "RGGFR" active site in BmaSPI51, that of BmoPI51 is "KGSFP" and the C-terminal "YNTCECSCP" tail sequence is lost in the latter. To investigate the effect elicited by the active site and tail sequences on the function of SPI51, we expressed two mutated forms of BmoSPI51, namely, BmoSPI51 + tail and BmoSPI51M. BmoSPI51, BmoSPI51 + tail and BmoSPI51M were compared and found to have similar levels of inhibitory activity against trypsin. However, the BmoSPI51 + tail and BmoSPI51M proteins exhibited significantly stronger capacities to inhibit fungi growth, compared to BmoSPI51. We concluded that the specific amino acid sequence of the active site, as well as its the disulfide bond formed by C-terminal sequence in the BmaSPI51, represent the key factors responsible for its higher antifungal activity. This study provided new insights into the antifungal mechanisms elicited by protease inhibitors in the cocoons of silkworms.

Diterpenoid anthraquinones as chemopreventive agents altered microRNA and transcriptome expressions in cancer cells.

OBJECTIVE: Cryptotanshinone (CPT) and dihydrotanshinone (DHT) are diterpenoid anthraquinone compounds extracted from traditional Chinese herbal medicine (TCM). Recent studies have shown that CPT regulates the signal transduction pathways via microRNA (miRNA) alterations. However, few studies have investigated the role of DHT in miRNA alterations affecting cell-signaling pathways. This study aimed to investigate the miRNA alterations and post-transcriptional regulation activities of DHT in comparison to CPT. METHODS: HepG2 and HT-29 cells were treated with DHT or CPT for 72 h. MiRNA, transcription factor encoding mRNA, and downstream gene expression were determined using real-time quantitative PCR. Protein expression was analyzed using western blotting. RESULTS: The results revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via miR-15a-5p, miR-27a-5p, miR-100-5p, and miR-200a-5p alterations.In silico target predictions showed that downregulation of epidermal growth factor receptor (EGFR) mRNA expression by DHT might also suppress the expression of STAT family proteins and lead to anti-proliferation effects. We also found that, compared to CPT, DHT might possess higher potency in cell growth regulation via multi-miRNA and transcription factor alterations. CONCLUSION: This study revealed that CPT and DHT targeted cell proliferation and apoptosis signaling pathways via alterations in miRNAs and transcription factors. In addition, the findings of this study suggest that DHT is more potent than CPT in cancer chemopreventive activities. Therefore, DHT at a low dose is a TCM compound with less toxic side effects and may contribute to the development of natural medicine as a potential cancer chemopreventive agent.

Human-Induced Pluripotent Stem Cell Culture Methods Under cGMP Conditions.

The discovery of induced pluripotent stem cells (iPSCs) revolutionized the approach to cell therapy in regenerative medicine. Reprogramming of somatic cells into an embryonic-like pluripotent state provides an invaluable resource of patient-specific cells of any lineage. Implementation of procedures and protocols adapted to current good manufacturing practice (cGMP) requirements is critical to ensure robust and consistent high-quality iPSC manufacturing. The technology developed at Allele Biotechnology for iPSC generation under cGMP conditions is a powerful platform for derivation of pluripotent stem cells through a footprint-free, feeder-free, and xeno-free reprogramming method. The cGMP process established by Allele Biotechnology entails fully cGMP compliant iPSC lines where the entire manufacturing process, from tissue collection, cell reprogramming, cell expansion, cell banking and quality control testing are adopted. Previously, we described in this series of publications how to create iPSCs using mRNA only, and how to do so under cGMP conditions. In this article, we describe in detail how to culture, examine and storage cGMP-iPSCs using reagents, materials and equipment compliant with cGMP standards. © 2020 The Authors. Basic Protocol 1: iPSC Dissociation Support Protocol 1: Stem cell media Support Protocol 2: ROCK inhibitor preparation Support Protocol 3: Vitronectin coating Basic Protocol 2: iPSC Cryopreservation Basic Protocol 3: iPSC Thawing.

Ultrafine and High-Strength Silk Fibers Secreted by Bimolter Silkworms.

Ultrafine fibers are widely employed because of their lightness, softness, and warmth retention. Although silkworm silk is one of the most applied natural silks, it is coarse and difficult to transform into ultrafine fibers. Thus, to obtain ultrafine high-performance silk fibers, we employed anti-juvenile hormones in this study to induce bimolter silkworms. We found that the bimolter cocoons were composed of densely packed thin fibers and small apertures, wherein the silk diameter was 54.9% less than that of trimolter silk. Further analysis revealed that the bimolter silk was cleaner and lighter than the control silk. In addition, it was stronger (739 MPa versus 497 MPa) and more stiffness (i.e., a higher Young's modulus) than the trimolter silk. FTIR and X-ray diffraction results revealed that the excellent mechanical properties of bimolter silk can be attributed to the higher ß-sheet content and crystallinity. Chitin staining of the anterior silk gland suggested that the lumen is narrower in bimolters, which may lead to the formation of greater numbers of ß-sheet structures in the silk. Therefore, this study reveals the relationship between the structures and mechanical properties of bimolter silk and provides a valuable reference for producing high-strength and ultrafine silk fibers.

Over-expression of NYGGF4 inhibits glucose transport in 3T3-L1 adipocytes via attenuated phosphorylation of IRS-1 and Akt.

AIM: NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese patients. The purpose of this study was to investigate the effects of NYGGF4 on basal and insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes and to understand the underlying mechanisms. METHODS: 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. Glucose uptake was determined by measuring 2-deoxy-D-[3H]glucose uptake into the adipocytes. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting also was used to measure the phosphorylation and total protein contents of insulin signaling proteins such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt, ERK1/2, p38, and JNK. RESULTS: NYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, and JNK. CONCLUSION: NYGGF4 regulates the functions of IRS-1 and Akt, decreases GLUT4 translocation and reduces glucose uptake in response to insulin. These observations highlight the potential role of NYGGF4 in glucose homeostasis and possibly in the pathogenesis of obesity.

Gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides by cDNA microarrays.

To investigate the gene expression profiles of adipose tissue of obese rats after central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs), Y5 receptor antisense, mismatched ODNs or vehicle was intracerebroventricularly injected and cDNA microarrays were undertaken. Central administration of NPY-Y5 receptor antisense ODNs decreased food intake, body weight and serum insulin compared with both vehicle and mismatched ODNs. The average area of adipocytes both at retroperitoneal and epididymal adipose tissue were fall in antisense group while only the weight of the retroperitoneal fat pats was reduced in antisense group. cDNA microarrays containing 18,000 genes/Ests were used to investigate gene expression of adipose tissue. Autoradiographic analysis showed that 404, 81, and 34 genes were differently expressed over twofold, threefold, and fivefold, respectively. The analysis of gene expression profiles indicated that 332 genes were up-regulated and 187 genes were down-regulated in response to Y5 receptor antisense ODNs treatment. Different clusters of genes associated with apoptosis, signal transduction, energy metabolism, lipid metabolism, etc., such as FXR1, PHLDA1, MAEA, PIK3R1, ICAM2, PITPN, CALM2, CAMK2D, PKIA, DRD2, SLC25A14, CKB, AADAC, LIPA, ACOX3, FADS1, were concerned. Analysis of differentially expressed genes will help to understand the effects of Y5 receptor antisense ODNs therapy.

Downregulation of STEAP4, a highly-expressed TNF-alpha-inducible gene in adipose tissue, is associated with obesity in humans.

AIM: To determine the relationship between six-transmembrane epithelial antigen of the prostate 4 (STEAP4) expression and obesity. METHODS: RT-PCR and immunoblot analyses were performed to determine the differential expressions of STEAP4 mRNA and protein, respectively, in human omental adipose tissue from obese patients and normal weight controls. The expression pattern of STEAP4 mRNA in various human tissues was determined by RT-PCR. The subcellular localization of the STEAP4 protein in human adipose tissue was confirmed by immunohistochemistry. Finally, we confirmed that cultured human omental adipose tissue undergoes TNF-alpha-mediated regulation of the STEAP4 expression. RESULTS: STEAP4 mRNA and protein levels were downregulated in omental adipose tissue from obese patients relative to normal controls. The STEAP4 expression was most abundant in human adipose tissue. An immunohistochemical analysis confirmed that STEAP4 was associated with the plasma membrane of adipocytes. The STEAP4 expression was induced by TNF-alpha in a dose-dependent manner in human adipose tissue. CONCLUSION: STEAP4 was abundantly expressed in human adipose tissue, and the STEAP4 expression was significantly downregulated in obese patients. STEAP4 localized to the plasma membrane of adipocytes, and the STEAP4 expression was induced by TNF-alpha in adipose tissue. These data suggest that STEAP4 may play a significant role in the development of human obesity.

[Overexpression of resistin affect 3T3-L1 adipocyte lipid metabolism].

OBJECTIVE: To investigate the effect of resistin overexpression on 3T3-L1 adipocyte lipid and glucose metabolism. METHODS: Expression vector for rat resistin gene was constructed and transfected into 3T3-L1 adipocytes. Cell differentiation and lipid accumulation was determined by Oil Red O staining. Differentiation marker genes (pref-1, C/EBPalpha, FAS) and glucose transporter 4 (GLUT4) gene mRNA expressions were evaluated by reverse transcription-PCR (RT-PCR). Triglyceride (TG) and free fatty acids (FFAs) in adipocytes were measured by colorimetric kit. RESULTS: (1) In resistin-overexpressed adipocytes, the lipid droplets presented at the second day which was earlier than the control cells. (2) The expression of C/EBPalpha and FAS genes in resistin-overexpressed adipocytes were up-regulated and the pref-1 was down-regulated compared with that of the control cells. (3) In resistin-overexpressed adipocytes, cellular TG and FFAs levels were significantly increased (P<0.05). (4) There was no difference in the expression of GLUT4 gene between 3T3-L1 adipocytes and resistin-overexpressed adipocytes (P> 0.05). CONCLUSION: Overexpression of resistin can affect 3T3-L1 adipocyte lipid metabolism and thereby result in obesity and insulin resistance, but have no effect on GLUT4 gene expression.

Identification and characterization of NYGGF4, a novel gene containing a phosphotyrosine-binding (PTB) domain that stimulates 3T3-L1 preadipocytes proliferation.

A novel gene named NYGGF4, which was expressed at a higher level in obese subjects, was isolated and characterized. It is a 1527-bp cDNA, containing 753 nucleotides of an ORF (open reading frame) predicting 250 amino acids with a molecular mass of 28.27 kDa. Amino acid sequence analysis revealed NYGGF4 has a phosphotyrosine-binding (PTB) domain. Northern blot analysis revealed NYGGF4 is expressed primarily in adipose tissue, heart, and skeletal muscle but not in any other tissue examined. Confocal imagery analyses with green fluorescent protein-tagged protein transiently expressed in 3T3-L1 preadipocytes and 293-T cells show that NYGGF4 localizes in the cytoplasm. Furthermore, ectopic expression of NYGGF4 dramatically increases the proliferation of 3T3-L1 peadipocytes without affecting adipocytic differentiation. And the NYGGF4 expression 3T3-L1 cells had a greater number of cells in S-phase. Our data suggest that NYGGF4 might be a signaling molecule and could play a role in cell growth and adipogenesis process.

Overexpression of uncoupling protein 4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes.

Uncoupling proteins are a family of mitochondrial proteins involved in energy metabolism. We previously showed that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. However, the effect of UCP4 on adipocytes is unclear. In this work, we established a stable preadipocyte cell line overexpressing UCP4 to observe the direct effect of UCP4 on adipocytes. Cells overexpressing UCP4 showed significantly attenuated differentiation of preadipocytes into adipocytes. During differentiation, expression of adipogenesis-associated markers such as fatty acid synthetase, peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, adipocyte lipid binding protein and lipoprotein lipase were downregulated. Preadipoctes expressing UCP4 grew faster and more of them stayed in S phase compared to control cells. In addition, UCP4 overexpression protected preadipocytes from apoptosis induced by serum deprivation. Our results demonstrate that overexpression of UCP4 can promote proliferation and inhibit apoptosis and differentiation of preadipocytes.

A resistin binding peptide selected by phage display inhibits 3T3-L1 preadipocyte differentiation.

BACKGROUND: Resistin, a newly discovered cysteine-rich hormone secreted mainly by adipose tissues, has been proposed to form a biochemical link between obesity and type 2 diabetes. However, the resistin receptor has not yet been identified. This study aimed to identify resistin binding proteins/receptor. METHODS: Three cDNA fragments with the same 11 bp 5' sequence were found by screening a cDNA phage display library of rat multiple tissues. As the reading frames of the same 11 bp 5' sequence were interrupted by a TGA stop codon, plaque lift assay was consequently used to prove the readthrough phenomenon. The stop codon in the same 11 bp 5' sequence was replaced by tryptophan, and the binding activity of the coded peptide [AWIL, which was designated as resistin binding peptide (RBP)] with resistin was identified by the confocal microscopy technique and the affinity chromatography experiment. pDual GC-resistin and pDual GC-resistin binding peptide were co-transfected into 3T3-L1 cells to confirm the function of resistin binding peptide. RESULTS: Three cDNA fragments with the same 11 bp 5' sequence were found. The TGA stop codon in reading frames of the same 11 bp 5' sequence was proved to be readthroughed. The binding activity of RBP with resistin was consequently identified. The expression of the resistin binding peptide in 3T3-L1 preadipocytes expressing pDual GC-resistin significantly inhibited the adipogenic differentiation. CONCLUSION: RBP could effectively rescue the promoted differentiation of resistin overexpressed 3T3-L1 preadipocyte.

cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA.

Reprogramming somatic cells to generate induced pluripotent stem cells (iPSCs) has presented the biomedical community with a powerful platform to develop new models for human disease. To fully realize the promise of this technology in cell therapy and regenerative medicine, creating iPSCs under current Good Manufacture Practice (cGMP) conditions is paramount. Some reports have described efforts in this regard, resulting in iPSC lines that are cGMP compliant. The technology developed at Allele Biotechnology for footprint-free, feeder-free, and xeno-free reprogramming using only mRNA is very suitable for creating iPSC lines through an established cGMP process. This technology has resulted in a licensing agreement between Allele Biotechnology and Ocata (formerly ACT, now a wholly owned division of Astellas) for clinical applications. All reagents and vessels are certified as cGMP-produced, all equipment and software are certifiable, and all procedures are carried out in Industry ISO 7 or Class 10,000-grade cleanrooms. In this revised version of the unit, we describe the core improvements to implement steps toward cGMP-compliant generation of iPSCs. Recreating a process close to cGMP production in academic research will make these findings more applicable to translational research. © 2016 by John Wiley & Sons, Inc.