BackgroundDifferentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties.MethodsThrough the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers.ResultsThe cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization.ConclusionsMSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.Electronic supplementary materialThe online version of this article (10.1186/s13287-018-0942-x) contains supplementary material, which is available to authorized users.

Microsecond pulsed electric fields (μsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, μsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.

BackgroundHuman mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions.MethodsOne or several electric pulses of 100 μs were used to induce Ca2+ spikes caused by the penetration of Ca2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked.ResultsAccording to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability.ConclusionsAn easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation.

Tax revenue in Indonesia until 2014 contributed approximately 78% of total state revenue. This shows that the tax is important both for the country as a source of income, as well as for the company as a taxpayer. For companies, the tax to be paid is a burden for the company which will reduce net income. Based on earlier research, the management company will tend to act aggressive tax in financial reporting. Tax aggressiveness influenced also by the pattern of ownership of the company in which the role of corporate governance are expected to influence the direction of the management decision-making. The purpose of this study was to examine the effect of financial reporting aggressiveness, family ownership and corporate governance against tax aggressiveness. The samples are all companies listed on the Indonesian Stock Exchange (BEI) except finance, insurance and banks during the period 2010-2014. The method used is multiple regression method. The results showed that the audit committee and institutional ownership has a significant influence on the aggressiveness of the taxes while the aggressiveness of financial reporting and the ownership of the family did not have a significant effect on the aggressiveness of the tax.Keywords: Aggressiveness Financial Reporting, Tax Aggressiveness, Family Ownership, Corporate Governance Abstrak: Penerimaan pajak di Indonesia sampai dengan tahun 2014 memberikan kontribusi sekitar 78% dari total penerimaan negara. Hal ini menunjukkan bahwa pajak adalah penting baik untuk negara sebagai sumber pendapatan, maupun untuk perusahaan sebagai wajib pajak. Bagi perusahaan, pajak yang harus dibayarkan ini merupakan beban bagi perusahaan yang akan mengurangi laba bersih. Berdasarkan penelitian terdahulu, manajemen perusahaan akan cenderung melakukan tindakan pajak agresif dalam pelaporan keuangan. Agresivitas pajak dipengaruhi juga oleh pola kepemilikan perusahaan dimana peran tata kelola perusahaan diharapkan memberikan pengaruh dalam manajemen mengambil keputusan. Tujuan dari penelitian ini adalah untuk menguji pengaruh agresivitas pelaporan keuangan, kepemilikan keluarga dan tata kelola perusahaan terhadap agresivitas pajak. Sampel yang digunakan adalah semua perusahaan yang terdaftar pada Bursa Efek Indonesia (BEI) kecuali perusahaan keuangan, asuransi dan bank selama periode 2010-2014. Metode penelitian yang digunakan adalah metode regresi berganda. Dari hasil penelitian menunjukkan bahwa komite audit, kepemilikan institusional memiliki pengaruh signifikan terhadap agresivitas pajak sedangkan agresivitas pelaporan keuangan dan kepemilikan keluarga tidak memiliki pengaruh yang signifikan terhadap agresivitas pajak.

AbstrakTulisan ini diilhami oleh dua orang pakar bahasa yakni pakar dialektologi Prof. Mahsun, mencoba menawarkan fase pembelajaran bahasa sedangkan pakar linguistik fungsional Prof. Amrin Saragih menawarkan pentingnya linguistik fungsional. Makalah ini menawarkan kepada kita tentang konsep pembelajaran bahasa untuk mewujudkan tujuan pendidikan yakni pembentukan karakter yang terwujud dalam kesatuan esensial si subyek dengan bahasa dan perilaku serta sikap hidup yang dimilikinya dengan mengacu pada teori Foerster. Bagi Foerster, karakter merupakan sesuatu yang mengualifikasi seorang pribadi. Karakter menjadi identitas yang mengatasi pengalaman kontingen yang selalu berubah. Dari kematangan karakter inilah, kualitas seorang pribadi diukur.

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The objectives of this research were three-fold: to explore the types of politeness uttered by the teacher, to analyze the most predominant type of politeness uttered, and to explore the strategies of politeness uttered by the teacher in an English classroom. This research employed a descriptive qualitative analysis. The subject of this research was an English teacher of MAN 1 Kolaka. This research was conducted with two cohorts; they were class XII.IA-2 and class XII.IA-4. The data were collected from three observations and recordings of teacher’s utterances. As many as 117 politeness utterances were identified, encompassing 55 positive politeness utterances, 45 negative politeness utterances, 6 bald on record utterances and 1 off record utterance. There were 17 strategies of politeness used by the English teacher. The most predominant type of politeness was positive politeness, including the strategies of exaggerating sympathy with the Hearer, which was used 3 times, including both the Speaker and the Hearer 6 times, giving gifts to Hearer 26 times, being optimistic 10 times, offering 2 times, notice attend to Hearer 4 times, using group identity mark 3 times, and Intensifying interest to the hearer 1 time.Keywords:Pragmatics, Politeness Strategies, Teacher’s Utterances, English Classroom

Abnormalities of the insulin-like growth factor 2 (IGF2)-H19 locus with the overexpression of IGF2 are frequent findings in adrenocortical carcinoma (ACC). The present study assessed the expression of RNAs and microRNAs (miRNAs/miRs) from the IGF2-H19 locus using PCR-based methods in ACC and adrenocortical adenoma (ACA). The results were associated with proteomics data. IGF2 was overexpressed in ACC, and its expression correlated with that of miR-483-3p and miR-483-5p hosted by IGF2. The downregulated expression of H19 in ACC compared to ACA correlated with miR-675 expression hosted by H19. Several proteins exhibited an inverse correlation in expression and were predicted as targets of miR-483-3p, miR-483-5p or miR-675. Subsets of these proteins were differentially expressed between ACC and ACA. These included several proteins involved in mitochondrial metabolism. Among the mitochondrial respiratory complexes, complex I and IV were significantly decreased in ACC compared to ACA. The protein expression of NADH:ubiquinone oxidoreductase subunit C1 (NDUFC1), a subunit of mitochondrial respiratory complex I, was further validated as being lower in ACC compared to ACA and normal adrenals. The silencing of miR-483-5p increased NDUFC1 protein expression and reduced both oxygen consumption and glycolysis rates. On the whole, the findings of the present study reveal the dysregulation of the IGF2-H19 locus and mitochondrial respiration in ACC. These findings may provide a basis for the further understanding of the pathogenesis of ACC and may have potential values for diagnostics and treatment.