Upregulation of SMAD4 inhibits thyroid cancer cell growth via MAPK/JNK pathway repression

Purpose: To investigate whether the effect of mothers against decapentaplegic homolog 4 (SMAD4) on thyroid cancer cell survival was via the MAPK/JNK pathway. Methods: Papillary thyroid cancer (TPC)-1 cells were cultured and transfected with SMAD4 overexpression plasmid or siRNA to achieve SMAD4 overexpression or knockdown, respectively. In TPC-1 cells, the mRNA and protein expression levels of SMAD4, mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) were quantified using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Cell viability and apoptosis were measured using MTT assay and flow cytometry, respectively. MAPK and JNK inhibitors (U0126 and SP600125) were used for rescue experiments. The sensitivity of TPC-1 cells to chemotherapeutic drugs, cisplatin and doxorubicin, was also assessed. Results: A reduction in viability and an enhancement in apoptosis (p < 0.01) were found when SMAD4 was overexpressed in TPC-1 cells. Knockdown of SMAD4 elicited opposite results (p < 0.01). Overexpression of SMAD4 caused a decrease in the activation of MAPK and JNK, as evidenced by lower levels of phosphorylated MAPK and phosphorylated JNK (p < 0.05). Results from rescue experiments indicate that the increase in cell viability after SMAD4 knockdown was reversed by MAPK/JNK inhibitors (p < 0.05 and p < 0.01). Finally, overexpression of SMAD4 increased cytotoxic susceptibility of thyroid cancer cells to cisplatin/doxorubicin. Conclusion: These results indicate that SMAD4 inhibits thyroid cancer cell growth via inactivation of MAPK/JNK pathway. Overexpression of SMAD4 also increased thyroid cancer cell sensitivity to cisplatin/doxorubicin

Resveratrol enhances brown adipocyte formation and function by activating AMP-activated protein kinase (AMPK) α1 in mice fed high-fat diet

© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Scope: Enhancing the formation and function of brown adipose tissue (BAT) increases thermogenesis and hence reduces obesity. Thus, we investigate the effects of resveratrol (Resv) on brown adipocyte formation and function in mouse interscapular BAT (iBAT). Methods and results: CD1 mice and stromal vascular cells (SVCs) isolated from iBAT were treated with Resv. Expression of brown adipogenic and thermogenic markers, and involvement of AMP-activated protein kinase (AMPK)α1 were assessed. In vivo, Resv-enhanced expression of brown adipogenic markers, PR domain-containing 16 (PRDM16) and thermogenic genes, uncoupling protein 1 (UCP1) and cytochrome C in iBAT, along with smaller lipid droplets, elevated AMPKα activity and increased oxygen consumption. Meanwhile, Resv promoted expression of PRDM16, UCP1, PGC1α, cytochrome C and pyruvate dehydrogenase (PDH) in differentiated iBAT SVCs, suggesting that Resv enhanced brown adipocyte formation and function in vitro. In addition, Resv stimulated AMPKα and oxygen consumption in differentiated iBAT SVCs. However, the promotional effects of Resv were diminished by AMPK inhibition or AMPKα1 knockout, implying the involvement of AMPKα1 in this process. Conclusion: Resv enhanced brown adipocyte formation and thermogenic function in mouse iBAT by promoting the expression of brown adipogenic markers via activating AMPKα1, which contributed to the anti-obesity effects of Resv

Calcium Supplementation Enhanced Adipogenesis and Improved Glucose Homeostasis Through Activation of Camkii and PI3K/Akt Signaling Pathway in Porcine Bone Marrow Mesenchymal Stem Cells (pBMSCs) and Mice Fed High Fat Diet (HFD)

Background/Aims: It has been implicated that calcium supplementation is involved in reducing body weight/fat and improving glucose homeostasis. However, the underlying mechanisms are still not fully understood. Here, we investigated the effects of calcium supplementation on adipogenesis and glucose homeostasis in porcine bone marrow mesenchymal stem cells (pBMSCs) and high fat diet (HFD)-fed mice and explored the involved signaling pathways. Methods: In vitro, pBMSCs were treated with 4 mM extracellular calcium ([Ca2+]o) and/or 1 μM nifedipine, 0.1 μM BAPTA-AM, 1 μM KN-93, 50 nM wortmannin for 10 days. The intracellular calcium ([Ca2+]i) levels were measured using Fluo 3-AM by flow cytometry. The adipogenic differentiation of pBMSCs was determined by Oil Red-O staining and triglyceride assay. The expression of marker genes involved in adipogenesis (peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα)) and glucose uptake (glucose transporter 4 (GLUT4)), as well as the activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and PI3K/Akt-FoxO1/AS160 signaling pathways were determined by Western blotting. Glucose uptake and utilization were examined using 2-NBDG assay and glucose content assay, respectively. In vivo, C57BL/6J male mice were fed a HFD (containing 1.2% calcium) without or with 0.6% (w/w) calcium chloride in drinking water for 13 weeks. The adipogenesis, glucose homeostasis and the involvement of CaMKII and PI3K/Akt signaling pathway were also assessed. Results: In vitro, [Ca2+]o stimulated pBMSCs adipogenesis by increasing [Ca2+]i level and activating CaMKII and PI3K/Akt-FoxO1 pathways. In addition, [Ca2+]o promoted glucose uptake/utilization by enhancing AS160 phosphorylation, GLUT4 expression and translocation. However, the stimulating effects of [Ca2+]o on pBMSCs adipogenesis and glucose uptake/utilization were abolished by L-VGCC blocker Nifedipine, [Ca2+]i chelator BAPTA-AM, CaMKII inhibitor KN-93, or PI3K inhibitor Wortmannin. In vivo, calcium supplementation decreased body weight and fat content, increased adipocyte number, and improved glucose homeostasis, with elevated PPARγ and GLUT4 expression and PI3K/Akt activation in iWAT. Conclusion: calcium supplementation enhanced adipogenesis and glucose uptake in pBMSCs, which was coincident with the increased adipocyte number and improved glucose homeostasis in HFD-fed mice, and was associated with activation of CaMKII and PI3K/Akt-FoxO1/AS160 pathways. These data provided a broader understanding of the mechanisms underlying calcium-induced body weight/fat loss and glycemic control

Production of Transgenic Pigs Mediated by Pseudotyped Lentivirus and Sperm

Sperm-mediated gene transfer can be a very efficient method to produce transgenic pigs, however, the results from different laboratories had not been widely repeated. Genomic integration of transgene by injection of pseudotyped lentivirus to the perivitelline space has been proved to be a reliable route to generate transgenic animals. To test whether transgene in the lentivirus can be delivered by sperm, we studied incubation of pseudotyped lentiviruses and sperm before insemination. After incubation with pig spermatozoa, 62±3 lentiviral particles were detected per 100 sperm cells using quantitative real-time RT-PCR. The association of lentivirus with sperm was further confirmed by electron microscopy. The sperm incubated with lentiviral particles were artificially inseminated into pigs. Of the 59 piglets born from inseminated 5 sows, 6 piglets (10.17%) carried the transgene based on the PCR identification. Foreign gene and EGFP was successfully detected in ear tissue biopsies from two PCR-positive pigs, revealed via in situ hybridization and immunohistochemistry. Offspring of one PCR-positive boar with normal sows showed PCR-positive. Two PCR-positive founders and offsprings of PCR-positive boar were further identified by Southern-blot analysis, out of which the two founders and two offsprings were positive in Southern blotting, strongly indicating integration of foreign gene into genome. The results indicate that incubation of sperm with pseudotyped lentiviruses can incorporated with sperm-mediated gene transfer to produce transgenic pigs with improved efficiency

A Comprehensive Expression Profile of MicroRNAs in Porcine Pituitary

MicroRNAs (miRNAs) are an abundant class of small RNAs that regulate expressions of most genes. miRNAs play important roles in the pituitary, the “master” endocrine organ.However, we still don't know which role miRNAs play in the development of pituitary tissue or how much they contribute to the pituitary function. By applying a combination of microarray analysis and Solexa sequencing, we detected a total of 450 miRNAs in the porcine pituitary. Verification with RT-PCR showed a high degree of confidence for the obtained data. According to the current miRBase release17.0, the detected miRNAs included 169 known porcine miRNAs, 163 conserved miRNAs not yet identified in the pig, and 12 potentially new miRNAs not yet identified in any species, three of which were revealed using Northern blot. The pituitary might contain about 80.17% miRNA types belonging to the animal. Analysis of 10 highly expressed miRNAs with the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that the enriched miRNAs were involved not only in the development of the organ but also in a variety of inter-cell and inner cell processes or pathways that are involved in the function of the organ

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Peritoneal dialysis related eosinophilic peritonitis: a case report and review of the literature

Abstract Background Overt eosinophilic peritonitis (EP) is a relatively uncommon complication of peritoneal dialysis (PD), although not rare. Here we reported a case of EP relieved after changing dialysate.  Case presentation A 28-year old male patient developed cloudy PD effluents within the first month after PD started. Cytological study of PD effluents showed elevated white blood cells and polynuclear cells. Bacteria culture of PD effluents repeated for several times were all negative, and no pathogen was found by metagenomics next generation sequencing (mNGS). Antibiotic therapy for 28-day was ineffective. Based on these and increased eosinophils in peritoneal fluid, he was finally diagnosed as EP. PD dialysate was changed (consists of the same buffer agent and electrolytes, but is packed in bags that do not contain PVC), and the patient’s PD effluent became clear. Of note, EP did not relapse 5 months later when the patient started to use the former PD solution again. Conclusion Although PD effluent turbidity almost always represents infectious peritonitis, there are other differential diagnoses including EP. For patients with cloudy fluid accompanied by mild symptoms who do not response to antibiotic therapy, it is reasonable to consider the possibility of this disease. EP tends to heal spontaneously, however, antihistamines or glucocorticoids are required sometimes to avoid catheter obstruction. For patients with no obvious incentives, replacement of dialysate may be useful