Association of TGF-ß1 and IL-10 Gene Polymorphisms with Osteoporosis in a Study of Taiwanese Osteoporotic Patients.

Osteoporosis is a rising health threat in the increasingly aging world population. It is a common skeletal disease strongly linked to genetic predisposition. We aim to identify the effects of the anti-inflammatory TGF-ß1- and IL-10-specific single-nucleotide polymorphism (SNP) combination on the risk for osteoporosis. We investigated and analyzed the relationships between three TGF-ß1 SNPs (-509C/T, +869 T/C and +29T/C), one IL-10 SNP (+1927A/C) and the level of bone mineral density (BMD), as well as the risk of osteoporosis in Taiwanese osteoporotic patients. A total of 217 subjects were recruited, including 88 osteoporotic patients and 129 healthy controls, for SNPs, BMD and clinical characteristics statistical analyses. Females with TGF-ß1 SNP (-509 C/C) and IL-10 SNP (+1927 C/C) genotypes showed a great benefit for femoral neck T-scores. However, the combination of TGF-ß1 SNP (-509 T/T) and IL-10 SNP (+1927 A/A) genotypes in all subjects showed a significant decrease in total hip BMD T-scores. The TGF-ß1 SNP (-509 C/T) genotype in all subjects and TGF-ß1 SNP (-509 T/T) and IL-10 SNP (+1927 A/C) genotypes in males showed positive effects on body height. The combination of the many SNPs in the anti-inflammatory TGF-ß1 and IL-10 genes may be cooperatively involved in the development of osteoporosis. Our data suggested that the specific SNP combination of TGF-ß1 (-509) and IL-10 (+1927) may act as a predictive factor for postmenopausal osteoporosis in Taiwanese women.

Phosphoproteomics and Bioinformatics Analyses Reveal Key Roles of GSK-3 and AKAP4 in Mouse Sperm Capacitation.

Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.

A novel osteoporosis model with ascorbic acid deficiency in Akr1A1 gene knockout mice.

The AKR1A1 protein is a member of the aldo-keto reductase superfamily that is responsible for the conversion of D-glucuronate to L-gulonate in the ascorbic acid (vitamin C) synthesis pathway. In a pCAG-eGFP transgenic mouse line that was produced by pronuclear microinjection, the integration of the transgene resulted in a 30-kb genomic DNA deletion, including the Akr1A1 gene, and thus caused the knockout (KO) of the Akr1A1 gene and targeting of the eGFP gene. The Akr1A1 KO mice (Akr1A1eGFP/eGFP) exhibited insufficient serum ascorbic acid levels, abnormal bone development and osteoporosis. Using micro-CT analysis, the results showed that the microarchitecture of the 12-week-old Akr1A1eGFP/eGFP mouse femur was shorter in length and exhibited less cortical bone thickness, enlargement of the bone marrow cavity and a complete loss of the trabecular bone in the distal femur. The femoral head and neck of the proximal femur also showed a severe loss of bone mass. Based on the decreased levels of serum osteocalcin and osteoblast activity in the Akr1A1eGFP/eGFP mice, the osteoporosis might be caused by impaired bone formation. In addition, administration of ascorbic acid to the Akr1A1eGFP/eGFP mice significantly prevented the condition of osteoporotic femurs and increased bone formation. Therefore, through ascorbic acid administration, the Akr1A1 KO mice exhibited controllable osteoporosis and may serve as a novel model for osteoporotic research.

Using Dual Fluorescence Reporting Genes to Establish an In Vivo Imaging Model of Orthotopic Lung Adenocarcinoma in Mice.

PURPOSE: Lung adenocarcinoma is characterized by a poor prognosis and high mortality worldwide. In this study, we purposed to use the live imaging techniques and a reporter gene that generates highly penetrative near-infrared (NIR) fluorescence to establish a preclinical animal model that allows in vivo monitoring of lung cancer development and provides a non-invasive tool for the research on lung cancer pathogenesis and therapeutic efficacy. PROCEDURES: A human lung adenocarcinoma cell line (A549), which stably expressed the dual fluorescence reporting gene (pCAG-iRFP-2A-Venus), was used to generate subcutaneous or orthotopic lung cancer in nude mice. Cancer development was evaluated by live imaging via the NIR fluorescent signals from iRFP, and the signals were verified ex vivo by the green fluorescence of Venus from the gross lung. The tumor-bearing mice received miR-16 nucleic acid therapy by intranasal administration to demonstrate therapeutic efficacy in this live imaging system. RESULTS: For the subcutaneous xenografts, the detection of iRFP fluorescent signals revealed delicate changes occurring during tumor growth that are not distinguishable by conventional methods of tumor measurement. For the orthotopic xenografts, the positive correlation between the in vivo iRFP signal from mice chests and the ex vivo green fluorescent signal from gross lung tumors and the results of the suppressed tumorigenesis by miR-16 treatment indicated that lung tumor size can be accurately quantified by the emission of NIR fluorescence. In addition, orthotopic lung tumor localization can be accurately visualized using iRFP fluorescence tomography in vivo, thus revealing the trafficking of lung tumor cells. CONCLUSIONS: We introduced a novel dual fluorescence lung cancer model that provides a non-invasive option for preclinical research via the use of NIR fluorescence in live imaging of lung.

Sexually Dimorphic Expression of eGFP Transgene in the Akr1A1 Locus of Mouse Liver Regulated by Sex Hormone-Related Epigenetic Remodeling.

Sexually dimorphic gene expression is commonly found in the liver, and many of these genes are linked to different incidences of liver diseases between sexes. However, the mechanism of sexually dimorphic expression is still not fully understood. In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty. The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation. Through this de novo sexually dimorphic expression of the transgene, the Akr1A1(eGFP) mouse provides a useful model to study the mechanisms and the dynamic changes of sexually dimorphic gene expression during either development or pathogenesis of the liver.

Silymarin and protein kinase A inhibitor modulate glucose-mediated mouse sperm motility: An in vitro study.

Glucose is suggested to play a key role in motility hyperactivation of mammalian spermatozoa. The current study aimed to investigate the modulatory effects of silymarin and/or a protein kinase A (PKA) inhibitor (H-89) on glucose-mediated motility parameters of mouse spermatozoa. Spermatozoa were incubated in HEPES medium containing normal (NG; 5.5mM) or high (HG; 25 mM) glucose concentration. The results of computer-assisted analysis showed that samples incubated in HG resulted in a larger (p < 0.05) percentage of motile spermatozoa at 120 min (59.5 ± 14.8% vs. 34.0 ± 4.4%) compared to those incubated in NG. The average pathway velocity (VAP), curvilinear velocity (VCL), and straight-line velocity (VSL) exhibited similar patterns at 60 and 120 min when incubated in HG (p < 0.05). Treatments with silymarin (5, 10, 20 µg/mL) did not significantly affect sperm motility under NG conditions, but decreased the HG-enhanced motility, VAP, and VCL at 120 min (p < 0.05). H-89 (30 µM) reduced (p < 0.05) motility at 30 min examined in the NG or HG medium. At 90 min, H-89 also reduced (p < 0.05) the HG-enhanced motility of spermatozoa incubated with or without 20 µg/mL silymarin by 49% or 32%, respectively. In conclusion, the H-89-inhibition of glucose-mediated mouse sperm motility and certain types of velocity suggests that the glycolysis-PKA pathway is involved in the regulation of sperm motility. Silymarin may maintain sperm motility under NG conditions, but it inhibits glucose-activated sperm motility.

Simultaneous Determination of Plasma Deferasirox and Deferasirox-Iron Complex Using an HPLC-UV System and Pharmacokinetics of Deferasirox in Patients With ß-Thalassemia Major: Once-daily Versus Twice-daily Administration.

PURPOSE: Deferasirox (DEFR), when administered BID, improves iron overload and decreases DEFR-related adverse effects in patients with ß-thalassemia major. However, the pharmacokinetic (PK) disposition of DEFR and the iron-DEFR complex (Fe-[DEFR]2) in this dosing strategy is unclear. METHODS: Chromatographic analysis was performed using a solvent delivery system coupled to an HPLC-UV detector to determine the steady-state concentrations of DEFR (CDEFR) and Fe-(DEFR)2 (CFe-[DEFR]2) in ß-thalassemia major patients (n = 8) following either once-daily or BID dosing, during which the PK parameters of the 2 dosing schedules were compared. FINDINGS: An HPLC-UV system for the analysis of blood samples following solid-phase extraction was validated. Patients who received 40 mg/kg of DEFR had higher mean CDEFR and CFe-[DEFR]2 values at all sampling times. However, concentrations of iron-DEFR complex were similar in patients who received 30 or 40 mg/kg of DEFR in the once-daily group at the 6- to 24-hour sampling times. There was no significant difference in any of the PK parameters; however, DEFR administration BID increased the mean trough levels of DEFR (183.8 [157.5] µmol/L) compared with once daily (87.7 [56.8] µmol/L), whereas all the patients had increased peak levels per individual DEFR dose when they were switched from once daily to BID (139.0 [59.8] µmol/L vs 289.2 [145.8] µmol/L, respectively). IMPLICATIONS: Splitting the dose increased the peak levels of DEFR per unit dose in all patients and tends to increase drug exposures, but there were no significant differences in DEFR PK parameter estimates. Switching from once daily to BID may be considered for patients with an inadequate response to chelation therapy to achieve optimal drug levels. Further research is needed with a larger sample size to determine the clinical importance of the significant results due to the interindividual variability of DEFR.

Lung tumorigenesis induced by human vascular endothelial growth factor (hVEGF)-A165 overexpression in transgenic mice and amelioration of tumor formation by miR-16.

Many studies have shown that vascular endothelial growth factor (VEGF), especially the human VEGF-A165 (hVEGF-A165) isoform, is a key proangiogenic factor that is overexpressed in lung cancer. We generated transgenic mice that overexpresses hVEGF-A165 in lung-specific Clara cells to investigate the development of pulmonary adenocarcinoma. In this study, three transgenic mouse strains were produced by pronuclear microinjection, and Southern blot analysis indicated similar patterns of the foreign gene within the genomes of the transgenic founder mice and their offspring. Accordingly, hVegf-A165 mRNA was expressed specifically in the lung tissue of the transgenic mice. Histopathological examination of the lung tissues of the transgenic mice showed that hVEGF-A165 overexpression induced bronchial inflammation, fibrosis, cysts, and adenoma. Pathological section and magnetic resonance imaging (MRI) analyses demonstrated a positive correlation between the development of pulmonary cancer and hVEGF expression levels, which were determined by immunohistochemistry, qRT-PCR, and western blot analyses. Gene expression profiling by cDNA microarray revealed a set of up-regulated genes (hvegf-A165, cyclin b1, cdc2, egfr, mmp9, nrp-1, and kdr) in VEGF tumors compared with wild-type lung tissues. In addition, overexpressing hVEGF-A165 in Clara cells increases CD105, fibrogenic genes (collagen α1, α-SMA, TGF-ß1, and TIMP1), and inflammatory cytokines (IL-1, IL-6, and TNF-α) in the lungs of hVEGF-A165-overexpressing transgenic mice as compared to wild-type mice. We further demonstrated that the intranasal administration of microRNA-16 (miR-16) inhibited lung tumor growth by suppressing VEGF expression via the intrinsic and extrinsic apoptotic pathways. In conclusion, hVEGF-A165 transgenic mice exhibited complex alterations in gene expression and tumorigenesis and may be a relevant model for studying VEGF-targeted therapies in lung adenocarcinoma.

Recombinant Derp5 allergen with αS1-casein signal peptide secreted in murine milk protects against dust mite allergen-induced airway inflammation.

Recent advances in recombinant technology make transgenic animals that produce pharmaceutical proteins in their milk more feasible. The group 5 allergen isolated from Dermatophagoides pteronyssinus (Derp5) is one of the most important dust mite allergens in humans. The aims of this study were to develop transgenic mice that could secrete recombinant Derp5-containing milk and to demonstrate that ingesting recombinant milk protects against allergic airway inflammation. Two transgenes were constructed separately. The α-LA-Derp5f transgene consisted of the bovine α-lactalbumin (α-LA) promoter and full-length Derp5 cDNA. The α-LA-CN-Derp5t transgene included the α-LA promoter, a leader sequence of αS1-casein (CN), and signal peptide-truncated Derp5 cDNA. Both species of transgenic mice were confirmed to have successful transgene integration and stable germline transmission. Western blot analysis of the milk obtained from the offspring of transgenic mice demonstrated that recombinant Derp5 was secreted successfully in the milk of αLA-CN-Derp5t transgenic mice but not in that of αLA-Derp5f transgenic mice. This study provides new evidence that transgenic mice can secrete recombinant Derp5 efficiently in milk by adding a signal peptide of αS1-casein. The antigenic activity of recombinant Derp5 milk was demonstrated to have a protective effect against allergic airway inflammation in a murine model in which the ingestion of recombinant Derp5-containing milk was used as pretreatment.

FTSJ2, a heat shock-inducible mitochondrial protein, suppresses cell invasion and migration.

Ribosomal RNA large subunit methyltransferase J (RrmJ), an Escherichia coli heat shock protein, is responsible for 2'-O-ribose methylation in 23S rRNA. In mammals, three close homologs of RrmJ have been identified and have been designated as FTSJ1, FTSJ2 and FTSJ3; however, little is known about these genes. In this study, we characterized the mammalian FTSJ2, which was the most related protein to RrmJ in a phylogenetic analysis that had similar amino acid sequence features and tertiary protein structures of RrmJ. FTSJ2 was first identified in this study as a nucleus encoded mitochondrial protein that preserves the heat shock protein character in mammals in which the mRNA expressions was increased in porcine lung tissues and A549 cells after heat shock treatment. In addition, a recent study in non-small cell lung cancer (NSCLC) suggested that the FTSJ2 gene is located in a novel oncogenic locus. However, our results demonstrate that the expression of FTSJ2 mRNA was decreased in the more invasive subline (CL1-5) of the lung adenocarcinoma cells (CL1) compared with the less invasive subline (CL1-0), and overexpression of FTSJ2 resulted in the inhibition of cell invasion and migration in the rhabdomyosarcoma cell (TE671). In conclusion, our findings indicate that mammalian FTSJ2 is a mitochondrial ortholog of E. coli RrmJ and conserves the heat shock protein properties. Moreover, FTSJ2 possesses suppressive effects on the invasion and migration of cancer cells.

Amniotic fluid stem cells from EGFP transgenic mice attenuate hyperoxia-induced acute lung injury.

High concentrations of oxygen aggravate the severity of lung injury in patients requiring mechanical ventilation. Although mesenchymal stem cells have been shown to effectively attenuate various injured tissues, there is limited information regarding a role for amniotic fluid stem cells (AFSCs) in treating acute lung injury. We hypothesized that intravenous delivery of AFSCs would attenuate lung injury in an experimental model of hyperoxia-induced lung injury. AFSCs were isolated from EGFP transgenic mice. The in vitro differentiation, surface markers, and migration of the AFSCs were assessed by specific staining, flow cytometry, and a co-culture system, respectively. The in vivo therapeutic potential of AFSCs was evaluated in a model of acute hyperoxia-induced lung injury in mice. The administration of AFSCs significantly reduced the hyperoxia-induced pulmonary inflammation, as reflected by significant reductions in lung wet/dry ratio, neutrophil counts, and the level of apoptosis, as well as reducing the levels of inflammatory cytokine (IL-1ß, IL-6, and TNF-α) and early-stage fibrosis in lung tissues. Moreover, EGFP-expressing AFSCs were detected and engrafted into a peripheral lung epithelial cell lineage by fluorescence microscopy and DAPI stain. Intravenous administration of AFSCs may offer a new therapeutic strategy for acute lung injury (ALI), for which efficient treatments are currently unavailable.

Aerosolized human extracellular superoxide dismutase prevents hyperoxia-induced lung injury.

An important issue in critical care medicine is the identification of ways to protect the lungs from oxygen toxicity and reduce systemic oxidative stress in conditions requiring mechanical ventilation and high levels of oxygen. One way to prevent oxygen toxicity is to augment antioxidant enzyme activity in the respiratory system. The current study investigated the ability of aerosolized extracellular superoxide dismutase (EC-SOD) to protect the lungs from hyperoxic injury. Recombinant human EC-SOD (rhEC-SOD) was produced from a synthetic cassette constructed in the methylotrophic yeast Pichia pastoris. Female CD-1 mice were exposed in hyperoxia (FiO2>95%) to induce lung injury. The therapeutic effects of EC-SOD and copper-zinc SOD (CuZn-SOD) via an aerosol delivery system for lung injury and systemic oxidative stress at 24, 48, 72 and 96 h of hyperoxia were measured by bronchoalveolar lavage, wet/dry ratio, lung histology, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) in lung and liver tissues. After exposure to hyperoxia, the wet/dry weight ratio remained stable before day 2 but increased significantly after day 3. The levels of oxidative biomarker 8-oxo-dG in the lung and liver were significantly decreased on day 2 (P<0.01) but the marker in the liver increased abruptly after day 3 of hyperoxia when the mortality increased. Treatment with aerosolized rhEC-SOD increased the survival rate at day 3 under hyperoxia to 95.8%, which was significantly higher than that of the control group (57.1%), albumin treated group (33.3%), and CuZn-SOD treated group (75%). The protective effects of EC-SOD against hyperoxia were further confirmed by reduced lung edema and systemic oxidative stress. Aerosolized EC-SOD protected mice against oxygen toxicity and reduced mortality in a hyperoxic model. The results encourage the use of an aerosol therapy with EC-SOD in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure, including acute respiratory distress syndrome (ARDS).

Curcumin reduces pulmonary tumorigenesis in vascular endothelial growth factor (VEGF)-overexpressing transgenic mice.

SCOPE: We investigated the inhibition of pulmonary tumor formation through treatment with curcumin in transgenic mice. METHODS AND RESULTS: In this study, a strain of transgenic mice carrying human vascular endothelial growth factor A165 (hVEGF-A165) gene to induce pulmonary tumor was used as an in vivo cancer therapy model. We found that curcumin significantly reduced hVEGF-A165 overexpression to normal, specifically in Clara cells of the lungs of transgenic mice, and suppressed the formation of tumors. In addition, we demonstrated a relationship between curcumin treatment and the expression of VEGF, EGFR, ERK2, and Cyclin A at the transcriptional and translational levels. We also noticed a reduction of Cyclin A and Cyclin B after curcumin treatment that had an effect on the cell cycle. Curcumin-induced inhibition of Cyclin A and Cyclin B likely results in decreased progression through S and G2/M phases. These results demonstrated that the expression of proteins involved in the S to M phase transition in transgenic mice is suppressed by curcumin. CONCLUSION: A Data suggest that a blockade of the cell cycle may be a critical mechanism for the observed effects on vasculogenesis and angiogenesis following treatment with curcumin.

Porcine lactoferrin administration enhances peripheral lymphocyte proliferation and assists infectious bursal disease vaccination in native chickens.

The purpose of this study was to investigate the effects of dietary supplementation with recombinant porcine lactoferrin (rPLF) produced by yeast culture on peripheral lymphocyte proliferation and serum antibody titers in chickens vaccinated against the infectious bursal disease (IBD) virus. Treatment groups were fed with rPLF powder in their diet (2.0%, w/w), and the IBD vaccine was administrated at 1 and 3 weeks of age. At 8, 12, and 16 weeks after vaccination, serum IBD antibody titers were measured via the micro-method and T cell proliferation rates were evaluated. In gene expression analyses, rPLF-treated chicken peripheral T lymphocytes were stimulated with concanavalin A (ConA) for 24h. The mRNA expression levels of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and interleukin-12 (IL-12) were determined using a semi-quantitative RT-PCR assay. The results revealed that the rPLF additive led to significant increases in serum IgG and IBD-specific antibody titers (P<0.05). The rPLF administration significantly increased chicken intestinal villous lengths and also enhanced the expression of IFN-gamma and IL-12 in chicken T lymphocytes. These data suggest that rPLF enhances cell-mediated immunity and augment the ability of IBD vaccination to benefit chicken industry in disease resistance.

Application of porcine lipase secreted by pichia pastoris to improve fat digestion and growth performance of postweaning piglets.

The aim of the study was to use Pichia pastoris to express a recombinant porcine lipase gene (pLip). The expression-secretion cassette was constructed using the P. pastoris GAPDH (glyceraldehyde-3-phosphate-dehydrogenase) promoter and an 89-residue prepro-alpha-factor secretion signal fused to the AOX1 terminator (the pGAPZalphaA vector). A total of 1,408 bp of pancreatic lipase cDNA was produced, which was located from the position of 4-nt upstream of ATG to 1408-nt inside the intact coding region of the pLip sequence. In an animal trial, three concentrations of recombinant lipase activity (0, 5,000 and 10,000 U/kg) were blended with the basal diet and fed to weaned piglets for six weeks. During the experimental period, the growth performance (bodyweight, feed intake, and feed efficiency) of the test groups was superior to that of the control group (p < 0.05). Furthermore, the group fed the diet blended with 10,000 U/kg of recombinant lipase showed significant (p < 0.05) increases in blood triglyceride (TG) concentration on the seventh day postweaning. These results suggested that the porcine lipase protein yielded by transformed yeast cells may improve fat digestibility and enhance the growth performance in postweaning piglets.