Fast method for skeletal tissue gene expression analysis

open9Several chronic diseases have been associated with bone alteration in the last few years. Despite the wealth of information provided by the analysis of the transcriptome in affected tissues, only a limited number of studies evaluated gene expression in bone tissue due to the difficulty to obtain high quality RNA. Therefore, skeletal pathologies have been often associated to a defective maturation process that occurs during recruitment of progenitor stem cells. In order to explore the possibility of analysing the gene expression during osteogenic differentiation in skeletal tissue, a single-step method to extract well-preserved RNA from bone specimens was performed. A comparison between this technique and a traditional method was made by analysing the quality and yield of RNA obtained. In addition, RNAs were assayed by reverse transcription-quantitative polymerase chain reaction to analyse the expression levels of the bone genes associated with the differentiation process in a mouse model. The present data showed that good quality RNA can be obtained from bone tissue by a simple single-step method allowing the expression analysis of the genes encoded by skeletal tissue. In conclusion, the present study allows the possibility to easily obtain good quality RNA from bone tissue that is suitable for gene expression studies of bone diseases.openDalle Carbonare, Luca; Vilei, Maria Teresa; Stranieri, Chiara; Innamorati, Giulio; Rosato, Antonio; Boldrin, Elisa; Sella, Stefania; Giannini, Sandro; Valenti, Maria TeresaDALLE CARBONARE, LUCA GIUSEPPE; Vilei, MARIA TERESA; Stranieri, Chiara; Innamorati, Giulio; Rosato, Antonio; Boldrin, Elisa; Sella, Stefania; Giannini, Sandro; Valenti, Maria Teres

Effects of time culture and prototypical CYP3A inducers on CYP2B22, CYP2C, CYP3A28 and nuclear receptors mRNAs in cryopreserved pig hepatocytes

Introduction. The constitutive expression of major drug metabolizing enzymes like cytochromes P450 (CYPs) and related nuclear receptors (NRs) is considered of fundamental importance in drug metabolism studies made in whole-cell systems like hepatocyte primary cultures (HPCs). In fresh and cryopreserved pig HPCs, time-dependent variations of CYP gene expression have been investigated by and large at the post-translational level; furthermore, few data have been published about the effect of time and known CYP3A inducers on NRs mRNAs. In the present study, the transcriptional effects of time and prototypical CYP3A inducers upon CYP2B22, 2C, 3A28 and NR1I2, NR1I3, NR2B1 and NR3C1 were investigated by using cryopreserved pig HPCs. Materials and Methods. To measure time-dependent changes in aforementioned target genes, HPCs were stopped 24, 48, 72 and 96 hrs after plating. As regards the HPCs response to known CYP3A inducers, media containing phenobarbital (PB, 2 mM), pregnenolone 16\u3b1-carbonitrile (PCN, 10 \u3bcM), rifampicin (RIF, 10 \u3bcM), dexamethasone (DEX, 10 \u3bcM) and dimethyl sulfoxide (control) were daily added to monolayers from 24 hrs after plating and up to 72 hrs. Additionally, specific reference genes were identified by using geNormPLUS and Normfinder algorithms. Transcriptional effects were measured by using specific qPCR assays. Results. The geometric mean of three reference genes, namely ribosomal protein large P0 (RPLP0), cyclophilin A (PPIA), glyceraldheyde 3-phosphte dehydrogenase and RPLP0, PPIA and \u3b2-actin was used to normalize time-course and induction qPCR data, respectively. CYP gene expression was strongly down-regulated as a function of time culture; at 48 hrs, CYP2B22 and CYP3A accounted (%) for 7.14\ub11.08 and 19.37\ub111.21 of the RQ value measured at 24 hrs. A low and constant constitutive expression of CYP2C, NR1I2 and NR1I3 was noticed during the whole culturing time, while NR2B1 and NR3C1 mRNA levels were increased (p<0.001 for NR2B1at 96 hrs). Hepatocytes were responsive to model CYP3A inducers; PB significantly increased CYP2B22 (p<0.05), 2C (p<0.01) and 3A (p<0.001) gene expression, while DEX induced CYP3A, NR1I2 (p<0.001) and NR1I3 (p<0.05). Finally, RIF up-regulated only CYP3A mRNA (p<0.05). Conclusions. Data obtained agree with previous published comparative results about time-course and induction in fresh and cryopreserved HPCs. Furthermore, present results would confirm the role played by NRs in CYP expression and regulation phenomena as well as the presence of species-differences in CYP3A drug metabolism. Confirmatory immunoblotting investigations are actually running

Is there a place for medical treatment in children with gallstones?

BACKGROUND: Medical treatment of gallstones with ursodeoxycholic acid (UDCA) or chenodeoxycholic acid (CDCA) has not been evaluated in children. AIM: The purpose of this study was to assess the effectiveness of UDCA in the treatment of gallstones in children. METHODS: UDCA was used to treat 15 patients, (7 boys and 8 girls; mean age, 7.8 years; range, 3 months to 15 years) for 1 year. All had radiolucent stones with a maximum diameter of 10 mm and a normally contractile gallbladder. RESULTS: The stones disappeared completely in two children but returned later. All symptomatic patients became symptom free. CONCLUSION: UDCA is ineffective in the treatment of gallstones in children except in terms of relieving symptoms while on treatment

Declining trends in the incidence of hip fractures in people aged 65years or over in years 2000-2011

The aim of this study was to explore hip fracture (HFx) incidence in the Veneto Region of Italy, looking at potential differences with the national data

The relationship between the Spine Deformity Index, biochemical parameters of bone metabolism and vascular calcifications: results from the Epidemiological VERtebral FRACtures iTalian Study (EVERFRACT) in dialysis patients.

Abstract Background: The Spine Deformity Index (SDI) is a measure of vertebral fractures (VFs), providing information on both their number and severity. Methods: We evaluated the relationships between SDI and clinical, biochemical and arterial calcification parameters in 387 hemodialysis (HD) patients. VFs, assessed by quantitative vertebral morphometry, and vascular calcifications were identified in the same lateral spinal X-ray. To improve the detection of fracture severity, we created a corrected SDI (c-SDI), by dividing SDI for the number of VFs. We assessed routine biochemistry, bone-Gla-protein (BGP), undercaboxylated BGP (ucBGP), and matrix-Gla-protein (MGP). Results: VFs prevalence was 55.3%. HD patients with a SDI >1 were more frequently males (p1 had higher LDL-cholesterol (p1 (p1 (p1. Age (OR 1.05, CI 1.03-1.07), LDL-cholesterol (OR 1.74, CI 1.04-2.92) and ucBGP (OR 0.35, CI 0.18-0.70) were associated with c-SDI >1. Conclusions: We conclude that the severity of VFs was associated with age, atherogenic factors and bone metabolism markers