Eur J Cancer 2010, 46:1359–64.PubMedCrossRef 17. Bae J, Lim MC, Choi JH: Prognostic

factors of secondary cytoreductive surgery for patients with recurrent epithelial ovarian cancer. J Gynecol Oncol. 2009, 20:101–6.PubMedCrossRef 18. Chi DS, McCaughty K, Diaz JP: Guidelines and selection criteria for secondary cytoreductive surgery in patients with recurrent, platinum-sensitive epithelial ovarian carcinoma. Cancer 2006, 106:1933–9.PubMedCrossRef 19. International INCB018424 concentration Collaborative Ovarian Neoplasm Group: Paclitaxel plus carboplatin versus standard chemotherapy with either single-agent carboplatin or cyclophosphamide, doxorubicin, and cisplatin in women with ovarian cancer: the ICON3 randomised trial. Lancet 2002, 360:505–15.CrossRef Competing interests The authors declare that they have

no competing interests. Authors’ contributions MS participated in the design of the study, collected data, prepared specimens for staining, analyzed the results and drafted CHIR98014 research buy the manuscript. LB participated in the design of the study and performed the statistical analysis. BG and KZ carried out the immunochemistry staining and assessed the slides. RS helped to analyze the data and draft the manuscript. WK helped to analyze the data. CS participated in the study design and coordination, revised the manuscript critically and gave final approval of the version to be published. All authors read and SCH727965 in vitro approved the final manuscript.”
“Introduction Several benzoquinones have been found to be effective in the treatment of some forms of cancer; previous studies demonstrated that these drugs act on cells by numerous mechanisms, such as apoptosis, abrogation of the cell cycle, activation of caspases, stimulation of the production of reactive oxygen species (ROS), inhibition of topoisomerases I and II, activation of intracellular second messengers, and production of free radicals to attack DNA. However, their cumulative heart toxicity limits their use1; therefore, an important goal PLEKHB2 of present and future work is to develop quinoid compounds that display anticancer activity but with less side effects. Among the 1,4 benzoquinones, there are several naturally occurring quinones having

potent anticancer activity. A recent work [1] demonstrated that Ardisianone, a natural benzoquinone derivative, displayed anti-proliferative and apoptotic activities against human hormone-refractory prostate cancer cells (HRPC), PC-3, and DU-145. Previous investigations also showed that Primin isolated from the leaves of Miconia Lepidota present in Suriname forests, exhibited activity towards mutant yeast strains, indicative of their cytotoxicity and potential antitumor activity [2]. Furthermore Kaul and co-workers isolated a known cytotoxic quinine Irisoquin which demonstrated cytotoxic properties [3]. In previous reports Muhammad et al. [4] evaluated cytotoxic and antioxidant activities of alkylated benzoquinones from the leaves of Maesa Lanceolata.

The PM has a decreased expression of 19 and 42 of the 99 genes that encode for cellulosomal components in standard and Vorinostat molecular weight Populus hydrolysate

media, respectively (Additional file 4). The statistically significant decreased expression in cellulosome genes by the PM may be an attempt to conserve energy since the cells were adapted in media containing cellobiose and soluble glucans present from the hydrolysate. It has been hypothesized that the downregulation of the cellulosome on soluble substrate such as cellobiose occurs via Dibutyryl-cAMP datasheet catabolite repression [42]. The PM has a synonymous SNP at codon 415 in RsgI6 (Cthe_2119) which is an anti-σI factor involved in regulating the expression of cellulosomal genes in the presence of xylans and cellulose [17]. It is possible that this mutation changes the specificity of the anti-σI factor and reduces the expression of the cellulosomal genes over and above the reduction that would be achieved by catabolite repression alone. The PM has lower expression than the WT of 31 and 54 genes that encode for cell envelope

proteins in standard and Populus hydrolysate medium (Additional file 4). The PM also downregulated 21 and 50 genes that encode for cell motility in standard and Populus hydrolysate media compared to the WT. It has been proposed that the σD in B. PX-478 mw subtilis controls flagellin production and possibly has a role in the expression of the methyl-accepting chemotaxis proteins [31]. Sigma factor σD (Cthe_0495) is downregulated in the PM compared to the WT in standard and Populus hydrolysate media by 3-fold and 10-fold at the mid-log time point (Table 1) and may cause the decrease in cell motility genes. The PM also downregulated 12 genes that encode Megestrol Acetate for various inorganic ion transport and metabolism proteins compared to the WT in standard medium and upregulates 17 genes in 10% v/v Populus hydrolysate medium.

However, the downregulated genes do not belong to any specific pathway. The change in expression may be due to the downregulation of inorganic ion transport and metabolism genes in the standard versus Populus hydrolysate media comparison below. The PM also downregulated 26 genes in the miscellaneous category compared to the WT in standard medium. Beyond a simple conservation of cellular resources, the benefits of reducing the expression level of genes in these categories are unclear. Hydrolysate comparison The Populus hydrolysate concentration comparison represents the difference in gene expression for various hydrolysate concentrations within a given strain.

The lowest dilution that allowed detection of the gene within the linear working range was chosen as the dilution

to be used for the analysis of the genes of interest. To control for contaminating DNA in the reaction, tubes with template from control 1 (see above) and tubes with water instead of template were included in the analysis. The controls gave Ct values (Ct is the threshold cycle) below detection level or at least 8 cycles later than the corresponding cDNA. Relative copy numbers (RCN) of selected genes were expressed in relation to the expression of the housekeeping gene tul4 [24] and calculated according PCI-32765 ic50 to the following equation: RCN = 2- ΔCt × 100 where ΔCt is Ct (target) – Ct(tul4) [25]. Thus, the copy number of a given gene is related to the copy number of tul4. Normalized Ct-values were used for statistical evaluation of the data. Chromazurol-S (CAS) plate assay Chrome-azurol sulfonate-C-CDM agar plates (CAS plates) were prepared essentially as described [13]. Briefly, 40 ml of CAS/Fe(III)-hexadecyltrimethylammonium solution was mixed with 50 ml of a 4% (wt/vol)

solution of GC II Agar Elacridar in vitro Base (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and 110 ml of C-CDM. The resulting CAS-C-CDM agar solution (1% agar) was poured into 20 ml Petri dishes. All components of the CAS-solution were purchased from Sigma-Aldrich, Buchs, Switzerland. Bacteria were cultivated overnight in C-CDM and thereafter washed three times in C-CDM before dilution in C-CDM to 1.0 OD600. The suspension was added as a droplet of 2.5 μl to the center of the CAS plate. The plates were incubated at 37°C in 5% CO2 and the size and appearance of the halo formed around the bacterial colony was scored at 72 h. Ferrozine assay A ferrozine-based method was used to measure the total amount of iron in the bacterial samples and in culture medium [26]. Ferrozine forms a complex with Fe2+ that absorbs light at 562 nm.

To determine the iron content of bacteria, a volume corresponding to 1.0 OD600 was withdrawn from the culture and bacteria collected by centrifugation for 5 min at 13,000 rpm. The bacteria were resuspended in PBS and collected Thiamine-diphosphate kinase by centrifugation. The resulting bacterial pellet was lysed with 100 μl of 50 mM NaOH. The solution was mixed selleck screening library thoroughly to ensure complete lysis of the bacteria. One hundred μl of 10 mM HCl was added to the lysate. To release protein-bound iron, the samples were treated with 100 μl of a freshly prepared solution of 0.7 M HCl and 2.25% (w/v) KMnO4 in H2O and incubated for 2 h at 60°C. All chemicals used were from Sigma-Aldrich. Thereafter, the samples were mixed with 100 μl of the iron detection reagent composed of 6.5 mM ferrozine, 6.5 mM neocuproine, 2.5 M ammonium acetate, and 1.0 M ascorbic acid dissolved in water. For determination of iron in medium, 30 μl of iron detection reagent was mixed with 170 μl of bacterial-free culture medium.

Procter & Gamble: speaking, consulting, research support (through the university). sanofi-aventis: speaking, consulting.. Frederick A Anderson: Research grant: sanofi-aventis: GRACE, GLOW, ENDORSE; The Medicines Company: STAT; Scios: Orthopedic Registry; Consultant/Advisory Board: sanofi-aventis, Scios,

GlaxoSmithKline, The Medicines Company, Millennium Pharmaceuticals. Pierre Delmas: None Open Access This article www.selleckchem.com/products/epz-5676.html is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References 1. Hays J, Hunt JR, Hubbell FA, Anderson GL, Limacher M, Allen C, Rossouw JE (2003) The Women’s Health Initiative recruitment methods and results. Ann Epidemiol 13:S18–S77PubMedCrossRef 2. Cummings SR, Nevitt MC, Browner WS, Stone K, Fox KM, Ensrud KE, Cauley J, Black

D, Vogt TM (1995) Risk factors for hip fracture in white women. Study of osteoporotic fractures research MLN2238 nmr group. N Engl J Med 332:767–773PubMedCrossRef 3. Tanko LB, Bagger YZ, Nielsen SB, Christiansen C (2003) Does serum cholesterol contribute to vertebral bone loss in postmenopausal women? Bone 32:8–14PubMedCrossRef 4. European Prospective Osteoporosis Study Group (2002) Incidence of vertebral fracture in Europe: results from the European Prospective Osteoporosis Study (EPOS). J Bone Miner Res 17:716–724CrossRef 5. Hofman A, Grobbee DE, de Jong PT, van den Ouweland FA (1991) Determinants of disease and disability in the elderly: the Rotterdam Elderly Study. Eur J Epidemiol 7:403–422PubMedCrossRef 6. O’Neill TW, Felsenberg D, Varlow J, Cooper C, Kanis JA, Silman AJ (1996) The prevalence of vertebral deformity in European men and women: the European Vertebral Osteoporosis Study. J Bone Miner Res 11:1010–1018PubMedCrossRef 7. Ismail AA, Pye SR, Cockerill WC, Lunt Terminal deoxynucleotidyl transferase M, Silman AJ, Reeve J, Banzer D, Benevolenskaya LI, Bhalla A, Bruges Armas J, Cannata JB, Cooper C, Delmas PD, Dequeker J, Dilsen G, Falch JA, Felsch B, Felsenberg D, Finn JD, Gennari C, Hoszowski K, Jajic I, Janott J, Johnell O, Kanis JA, Kragl

G, Lopez Vaz A, Lorenc R, Lyritis G, Marchand F, Masaryk P, Matthis C, Miazgowski T, Naves-Diaz M, Pols HA, Poor G, Rapado A, Raspe HH, Reid DM, Reisinger W, Scheidt-Nave C, Stepan J, Todd C, Weber K, Woolf AD, O’Neill TW (2002) Incidence of limb fracture across Europe: results from the European Prospective Osteoporosis Study (EPOS). Osteoporos Int 13:565–571PubMedCrossRef 8. Adachi JD, Loannidis G, Berger C, Joseph L, Papaioannou A, Pickard L, Papadimitropoulos EA, Hopman W, Poliquin S, Prior JC, Hanley DA, Olszynski WP, Anastassiades T, Brown JP, Murray T, Jackson SA, DAPT price Tenenhouse A (2001) The influence of osteoporotic fractures on health-related quality of life in community-dwelling men and women across Canada. Osteoporos Int 12:903–908PubMedCrossRef 9.

Real-time polymerase chain reaction Total RNA was isolated from HeLa-S3 cells by Trizol® Reagent (Life Technologies), and reverse transcription was carried out using the

Applied Biosystems High Capacity cDNA Reverse Transcription Kit (Life Technologies) Selleck OSI 906 according to the manufacturer’s instructions. The cDNA was diluted to a final concentration of approximately 1 ng/μl and reacted with gene-specific primer pairs and Applied Biosystems SYBR® Green PCR Master Mix (Life Technologies) according to the manufacturer’s protocol. The primer sequences for GAPDH (NM_002046) and β-actin (NM_001101) were designed by Origene (Rockville, MD, USA). Primer specificity was confirmed by Primer-BLAST developed at NCBI, and primer PCR efficiency was validated to be close to 100%. Genes of interest were this website detected and amplified by Applied Biosystems 7300 Real-Time PCR System (Life Technologies) with the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min, followed by melting curve analysis. Amplicons were visualized with electrophoresis on a 1.4% agarose gel to ensure the presence of a single product. The mRNA level of each gene was analyzed by the Applied Biosystems Sequence Detection Software

V1.2 (Life Technologies) and normalized to that of GAPDH. Relative gene expression was calculated by the comparative Ct (2−ΔΔct) method [31] and expressed as fold changes (x-fold) relative to the control. Statistical analysis Statistical analysis was performed on data from at least three independent experiments. find more Significant difference relative to the control was tested using Student’s t test. Levels of significance of p < 0.05 and 0.01 were accepted

as significant and highly significant, respectively. Results and discussion Results PEI-NH-CNT suspensions PEI functionalization remarkably increased the degree of dispersibility of SWNTs and MWNTs. After being dispersed in ddH2O at 1 mg/ml and sonicated for 15 min, PEI-NH-MWNTs and PEI-NH-SWNTs can be solubilized in water and maintained in suspension form for over 6 months without further sonication (left Dapagliflozin images, Figure 1A, B). Because agglomeration of carbon nanotubes as a result of van der Waals’ interaction tends to increase cytotoxicity [32, 33], PEI-NH-CNTs were subjected to centrifugation to remove large aggregates, and the supernatant gave a more homogeneous solution of PEI-NH-CNTs for the following studies (right images, Figure 1A, B). Figure 1 Suspension of PEI-NH-SWNTs and PEI-NH-MWNTs in water. PEI-NH-SWNTs (A) and PEI-NH-MWNTs (B) were solubilized in ddH2O at a concentration of 1 mg/ml and sonicated for 15 min (left images). Large aggregates were removed by centrifugation at 3,000 rpm for 30 min to obtain a more homogeneous suspension (right images). Morphology of PEI-NH-CNTs The morphology of PEI-NH-CNTs compared to pristine CNTs was studied by SEM and TEM.

The rgg 0182 gene (864 bp) potentially encodes a protein of 288 amino acids with a predicted molecular mass of 35.6 kDa. This protein exhibited an identity of about 30% with other streptococcal proteins belonging to the Rgg family of transcriptional regulators and 35% identity (e-value = 8e-48) with Rgg1358 from S. thermophilus LMD-9 which was recently https://www.selleckchem.com/products/incb28060.html shown to be involved in a quorum sensing (QS) mechanism [9]. Rgg0182 contained a HTH-XRE motif from amino acid 11 to 67 typical of Rgg regulators and a Rgg-C-terminal motif from amino acid 70 to 288 (Figure 1). Therefore, the rgg 0182 gene was predicted to encode a transcriptional

regulator. Figure 1 Schematic representation of the rgg 0182 and rgg 1358 loci (A) and of the corresponding proteins (B). Although the rgg 0182 and rgg 1358 loci present selleckchem analogies (A), they

encoded distinct proteins (B). Numbers in panel A indicate the position of nucleotides, with the +1 position being that of the first nucleotide of the rgg 0182 gene. The “”deletion fragment”" corresponds to the deleted portion of the rgg 0182 gene in the Δrgg 0182 mutant. The broken arrows indicate the promoters. Mocetinostat ic50 Pshp 0182 and Ppep 0182 materialized the position of the 126 bp and 165 bp PCR fragment respectively used in EMSA. In panel B, amino acids sequence identities are indicated in percent. HTH indicated the Helix-Turn-Helix-XRE motif. The gene rgg 0182 was surrounded by two ORFs (Figure 1), not annotated in the genome of the strain LMG18311, but revealed using the software bactgeneSHOW designed for small-gene detection [29]. Indeed,

upstream of the rgg 0182 gene was the shp 0182 gene (63 nucleotides long), potentially encoding a small hydrophobic peptide belonging to the group I of the SHP family [9]. Downstream of rgg 0182 was the pep 0182 gene (42 nucleotides long), encoding a small peptide with no similarity with peptides found in databases. Although, the genetic organization of the rgg Vildagliptin 0182 locus was similar to that of the rgg 1358 of the LMD-9 strain from S. thermophilus, these two loci were distinct as illustrated by the low sequence identity between the proteins encoded by them (Figure 1). The two shp genes were classified in two distinct groups from the SHP family [9]. Finally, the rgg 0182 locus and its flanking genes were also found in the genome of CNRZ1066 strain but missing in the genome of ND03 and LMD-9 strains. Transcription analysis of the rgg 0182 gene In the literature, studies of rgg genes transcription are scarce. Indeed, only the ropB transcription from Streptococcus pyogenes has been studied [10]. Thus, it was of interest to determine whether transcription of rgg was constitutive or not.

Cervical epithelial (HeLa) cells were plated in triplicate on microscopic slides and infected with M. genitalium G37 and TIM207 strains at an MOI of 1:50. Cells were observed with confocal laser microscope with 20X objective after 2–3 h incubation at 37°C. PBS indicates un-infected cells; G37, TIM207, TIM262 and HKG37 indicate infection of cells with M. genitalium wild type G37 strain, TIM207 mutant strain, TIM262 control strain and heat killed G37 bacteria, respectively. Figure 6 Hydrogen peroxide (H 2 O 2 ) production by M. genitalium cells. Cells of

M. genitalium strains (G37 wild type, TIM207 mutant and TIM262 mutant control) were sonicated in PBS and the presence of H2O2 in each sample was determined by FOX assay at 560 nm. The amount of hydrogen peroxide in each sample was determined using standard curve generated with H2O2 and the 10058-F4 values expressed as μmoles

H2O2/ μg protein. * indicate significant difference from G37 and TIM262 (p≤0.05). TIM207 strain fails to differentiate THP-1 cells THP-1 cell line is an undifferentiated monocyte cell line from human and its differentiation into macrophages requires incubation with 100 nM of Phorbol-12-myristate-13-acetate (PMA) for 48 to 72 h. Usually, differentiated THP-1 cells adhere to the selleck products surface of culture flask, while undifferentiated THP-1 cells only float in the culture PF-6463922 medium. We have recently discovered that M. genitalium can induce the differentiation of monocytic THP-1 cells into macrophages, similar to that of PMA, and this ability of M. genitalium may be affected by the absence of protein like MsrA [54]. To test whether absence of MG207 Tacrolimus (FK506) had any effect on the differentiation of THP-1 cells by M. genitalium, we labeled THP-1 cells with CFSE and infected with TIM207 strain and control strains of M. genitalium. Figure 7A shows confocal microscopy observation of THP-1 cells adhered to surface of the culture slides due to differentiation induced by M. genitalium strains. Although THP-1 cells infected with G37 and TIM262 exhibited higher number of adhered cells, relatively less number of cells

adhered with THP-1 cells infected with TIM207 and heat killed bacteria of G37 strain (Figure 7B). This suggested that the product of MG_207 plays an important role in the induction of differentiation of THP-1 cells by M. genitalium. Figure 7 Differentiation of THP-1 cells by M. genitalium strains. A. Adherent THP-1 cells showing fluorescence. Images of adherent cells were acquired using confocal laser scanning microscope with 10X objective and 488 nm laser. G37, TIM207 and TIM262 are wild type, TIM207 mutant and TIM262 control M. genitalium strains, respectively. HKG37 represents heat killed bacteria of wild type M. genitalium. Flour, Fluorescence; DIC, Differential interference contrast. B. Graph showing the amount of adherent cells for each infection. The numbers of labeled cells in each image were counted using the particle plugin of Image J software.

Densely packed alkyl chains with hydrophilic head groups can have both a hydrophobic interaction and a hydrophilic one with guest substrates. This specific environment provided by lipid membranes is essential for molecular recognition in biological membranes and has been studied in the areas of chemical sensing and separation [1, 2]. Proteins or other biologically active substances, especially those produced by recombinant microorganisms, are usually contaminated with lipopolysaccharide (LPS) [3]. LPS, which originates from an outer membrane

of Gram-negative bacteria, consists HKI-272 solubility dmso of a polysaccharide and a terminal lipid A moiety. Lipid A is composed of a diglucosamine that is highly substituted with amide- and ester-linked long-chain fatty acids and negatively charged with phosphate groups. For pharmaceutical uses of those active substances, LPS has to be removed to not higher than 0.1 ng mL-1 because of its strong pyrogenicity [4]. Intensive studies

have been done on the removal of LPS from protein solutions [5]. For example, LPS was selectively removed by ion-exchange chromatography using DEAE-Sepharose CL-6B [6] and affinity chromatography using adsorbents bearing histidine [7], polymyxin B [8], and polycation selleck compound [9]. However, the removal of LPS is suggested to be extremely difficult when LPS is associated with protein to be purified [5] and has been an issue in pharmaceutical technology and science. We have reported the covalent immobilization of polymeric lipid membranes of N-octadecylchitosan consisting

of 2-deoxy-2-octadecylamino-d-glucopyranose (GlcNC18; Figure 1), 2-amino-2-deoxy-d-glucopyranose Montelukast Sodium (GlcN), and 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) to carboxylated porous supports composed of chitosan [10], and the selective removal of LPS from bovine serum albumin (BSA) solution using the resulting porous materials [11]. In this paper, we would like to report further data of a successful LPS removal to as low as 0.02 ng mL-1 from human serum albumin (HSA) solution with a quantitative recovery of protein showing the possibility of their practical use. PF-02341066 datasheet Figure 1 Monosaccharide components of N -octadecylchitosan. Methods Materials and general methods Chitosan was purchased from Dai-ichi Kogyo Seiyaku Co., Ltd. (Kyoto, Japan). The degree of deacetylation was determined as 87 mol% by colloidal titration. Intrinsic viscosity was 1.42 dL g-1 (0.2 M CH3COOH/0.1 M CH3COONa, 30°C) which corresponded to 2.67 × 104 of molecular weight relative to poly(ethylene glycol). A cross-linked porous chitosan having a particle size of 45 to 420 μm and an average pore diameter of 2 μm, a product of Kurita Water Industries, Ltd. (Tokyo, Japan), was used as obtained. 1-Bromooctadecane, succinic anhydride (Kishida Chemical Co., Ltd.

The patients, ranging in age from 21 to 78 years (mean, 51.3 years) Erastin cell line and having adequate liver function reserve, had survived for at least 2 months after hepatectomy, and none received treatment prior to surgery such as transarterial chemoembolization or radiofrequency ablation. Clinicopathologic features of the 120 HCCs in this study are described in Table 1. Surgically resected specimens were partly embedded in paraffin after fixation in 10% formalin for histological processing and

partly immediately frozen in liquid nitrogen and stored at -80°C. All available hematoxylin and eosin YAP-TEAD Inhibitor 1 order stained slides were reviewed. The tumor grading was based on the criteria proposed by Edmondson and Steiner (I, well differentiated; II, moderately differentiated; III, poorly

differentiated; IV, undifferentiated) [16]. The conventional TNM system outlined in the cancer staging manual (6th ed.) by the American Joint Committee on Cancer (AJCC) was used in tumor staging. Table 1 Relations between NNMT mRNA levels and clinicopathologic features in HCC   All patients (n = 120)   Clinicopathologic parameters High NNMT (n = 48) Copy number ratio ≥ 4.40 Low NNMT (n = 72) Copy number ratio < 4.40 P value Age     0.730 < 55 years 31 43   ≥ 55 years 17 29   Gender     0.758 Male 38 54   Female 10 VX-689 clinical trial 18   HbsAg     0.885 Absent 8 14   Present 40 58   HCV     0.823 Absent 45 67   Present 3 5   Liver cirrhosis     0.852 Absent 25 40   Present 23 32   Tumor stage     0.010 I 23 23   II 9 33   III & IV 16 16   AFP level     0.314 < 100 ng/ml 28 34   ≥ 100 ng/ml 20 38   Tumor size     0.733 < 5 cm 27 44   ≥ 5 cm 21 28   Edmondson grade     0.368 I 13 15   II 30 43   III & IV 5 Ribonucleotide reductase 14   RNA extraction and cDNA synthesis Total RNA was extracted from cancerous and surrounding non-cancerous frozen tissues using an RNeasy minikit (Qiagen, Germany) according to the manufacturer’s instructions. The integrity

of all tested total RNA samples was verified using a Bioanalyzer 2100 (Agilent Technologies, United States). DNase I treatment was routinely included in the extraction step. Residual genomic DNA contamination was assayed by a quantitative real-time PCR assay for GAPDH DNA and samples with contaminating DNA were re-subjected to DNase I treatment and assayed again. Samples containing 4 μg of total RNA were incubated with 2 μl of 1 μM oligo d(T)18 primer (Genotech, Korea) at 70°C for 7 min and cooled on ice for 5 min. The enzyme mix was separately prepared in a total volume of 11 μl by adding 2 μl of 0.1 M DTT (Duchefa, Netherlands), 2 μl of 10× reverse-transcription buffer, 5 μl of 2 mM dNTP, 1 μl of 200 U/μl MMLV reverse-transcriptase, and 1 μl of 40 U/μl RNase inhibitor (Enzynomics, Korea). After adding the enzyme mix to the annealed total RNA sample, the reaction was incubated for 90 min at 42°C prior to heat inactivation of reverse-transcriptase at 80°C for 10 min.