The small leucine-rich proteoglycans (SLRPs) are a group belonging to the leucine-rich repeat (LRR) superfamily of proteins.

This includes decorin and biglycan (Figure 1C), which have a central region of 10 leucine residues flanked by cysteine residues [73]. Decorin is the best characterized SLRP member and is traditionally associated with ‘decorating’ collagen fibrils. The core protein is 40 kDa and has a single GAG chain attached to a serine residue near the N-terminus. Biglycan is structurally similar, www.selleckchem.com/products/BMS-777607.html with a core protein of 45 kDa and two GAG chains. SLRPs evoke a number of signalling pathways and are implicated in multiple interactions including modulation of collagen I and II fibrillogenesis [74]. Decorin expression may have positive effects on repair. It is known to inhibit activity of TGFβ [75] and EGFR [76,77], which have click here regulatory effects on synthesis of inhibitory CSPGs [78,79]. Biglycan also binds TGFβ, and soluble glycosylated biglycan acts as an endogenous ligand of the innate immunity

receptors TLR4 and TLR2 in macrophages (reviewed in [80]). Thus, the CSPGs comprise a complex family of molecules that are key components of the ECM. The multiple interactions of CSPGs with other ECM molecules as well as their binding affinity for a diverse array of growth factors, cytokines and receptors all suggest that they are crucial players in the CNS response to injury and that ECM modification will be an important therapeutic target. In addition to specific targeting of individual CSPGs (such as the function blocking NG2 antibody), global targeting of CSPGs has been a widely used strategy in experimental studies, for example by enzymatic digestion of CS-GAG chains to reduce the growth inhibitory properties of CSPGs. These approaches will be discussed

in detail later in this review. Many of the above ECM molecules have been targeted in repair strategies, often in an attempt to recapitulate developmental processes, where they play an important role in cell proliferation, migration, axon guidance and plasticity. Below we will discuss some of these heptaminol processes. Correct wiring of the nervous system requires the precise distribution and connectivity of millions of cells during development. The ECM plays a key role, conferring many of the properties required to form intricate networks with specificity and reliability. During embryogenesis, neural induction and neural tube formation are followed by rapid cell proliferation, migration and differentiation of cells to neurones and glia to form the CNS. Subsequent to regionalization of neurones, connections form between them. Connections form when a differentiated neurone sends out an axon, tipped by a growth cone which responds to multiple sources of extracellular cues to reach its target.

The endophytic fungus was grown on PDA at 30 °C for 7–9 days, and the formation of conidia was examined under a microscope. A slide culture technique was also used to observe the morphology of the fungus. The isolated endophytic fungus was identified at the Centre for Advanced

Studies in Botany, University of Madras, Tamilnadu, India. The identification of endophytic fungal strain C. gloeosporioides was confirmed by 18S rRNA gene sequence comparisons (Altschul et al., 1990). The 18S rRNA gene sequencing was done at Synergy Scientific Services, Chennai, India. The sequence alignment was done at a blast server. click here The radial growth of the fungus was studied on different solid media: Czapek Dox agar, malt extract agar, glucose peptone yeast agar, potato carrot agar and PDA. The mycelial agar plugs (5 mm in diameter) were inoculated at the centre of each Petri plate containing the respective medium and incubated for 7 days at 30 °C. The diameter of mycelial growth was measured at 24-h intervals. The fungus was grown in potato dextrose PD broth with the initial pH adjusted to 4.0,

4.5 5.0, 5.5, 6.0, 6.5 and 7.0. The culture was incubated for 21 days at 30 °C under selleck chemicals llc static conditions. After the incubation, the fungal mycelium was removed by cheesecloth and dried in a hot air oven at 70 °C. The growth of the fungus was estimated by determining the dry weight of the mycelium. Disks Rebamipide were cut from the edge of an actively growing colony on PDA with a flamed cork borer (5 mm diameter) and transferred

aseptically into 500-mL Erlenmeyer flasks containing 100 mL PD broth. The culture was incubated for 21 days at 30 °C under static conditions. After the incubation period, fungal mycelium was separated from the culture filtrate by cheesecloth. The filtrate and dried mycelium were extracted three times with hexane followed by ethyl acetate. The culture filtrate was dried at 70 °C in a hot air oven. The dried culture filtrate and mycelium were extracted with methanol and the solvent was removed by evaporation under reduced pressure at 35 °C using a rotary vacuum evaporator. After evaporation, the dried fungal extract was dissolved in 50% dimethyl sulphoxide (DMSO) and used to determine antibacterial activity. Staphylococcus aureus (MTCC 3160), Bacillus subtilis (MTCC 619), Escherichia coli (MTCC 4296), Pseudomonas aeruginosa (MTCC 2488) and Candida albicans (MTCC 3018) were purchased from the Microbial Type Culture Collection (Chandigarh, India). The clinical strains of S. aureus (1–10) were obtained from Bose Clinical Laboratory and X-ray (Madurai, Tamilnadu, India). Staphylococcus aureus strains were identified by standard biochemical methods (Essers & Radebold, 1980; Pourshadi & Klaas, 1984). The Kirby–Bauer disk diffusion test was used to determine the antibiotic resistance of S. aureus strains (1–10).

Ejarque-Ortiz et al. [9] have also shown that the restoration of C/EBP-α levels may be a strategy for attenuating neurotoxic effects. Moreover, LPS can induce C/EBP-β expression by astrocytes and microglia in primary mouse

glial cultures. It has been demonstrated by Straccia et al. [8] that C/EBP-β-null glial culture in activated microglia abrogates neurotoxicity, implying that C/EBP-β is a possible therapeutic Stem Cells inhibitor target for ameliorating neuronal damage due to neuroinflammation. However, the relationships between the response of microglial cells to environmental damage or inflammatory processes and the profound changes of gene expression associated with ER stress-related signaling have not been clearly established [10, 11]. This study hypothesizes that enhancement of calpain-II-regulated C/EBP-β downregulation by IL-13 through the induction of ER stress-related signaling in activated microglia may exacerbate microglial cell death and lead to the inhibition of proinflammatory cytokines release from deteriorated microglia. Neuronal cells will no longer be exposed to toxic damage. Thus, this change may reduce neuronal damage due to neuroinflammation. The present study also shows that IL-13-enhanced ER stress-related calpain activation plays an important role in the downregulation of C/EBP-β-regulated PPAR-γ/HO-1 expression in activated

microglia. In activated microglia, IL-13 may potentially many confer functional and therapeutic benefits in neurologic disorders by abrogating neurodegeneration. Previously, PGE2 production was reportedly involved in activated microglial death [6]. Here, find more the role of C/EBP-α and C/EBP-β was analyzed using specific small interfering RNA (siRNA) to elucidate whether IL-13-enhanced activated microglia PGE2 expression using ELISA. IL-13 increased PGE2 expressions in LPS-induced primary and BV-2 microglial cells (Fig. 1A). C/EBP is thought to play a crucial role in the activation of microglia following brain injury. Moreover, transfection of siRNA targeting C/EBP-α significantly decreased PGE2 production, whereas

silencing C/EBP-β alone resulted in minor effects. To more directly assess IL-13 enhancement on NO induction in activated microglia, NO production was examined by Griess reagents. NO production was detected in LPS-treated cells (Fig. 1B). The combination of IL-13 in LPS showed no effects. These suggested that C/EBP-α could be a factor mediating IL-13-induced PGE2 production and death of activated microglia. IL-13-enhanced apoptotic cell death in activated microglia has been shown to be involved in neurodegenerative disorders [5-7, 12, 13]. Related genes in activated microglia were analyzed to determine whether they were regulated by C/EBP-α and C/EBP-β. LPS significantly increased C/EBP-α and C/EBP-β in primary microglia cells and BV-2 microglia (Fig. 2).

Surprisingly, we found that Tregs produce high amounts of CXCL8 (IL-8), a potent neutrophil chemoattractant. Tregs also produced other CC and CXC family chemokines, including CCL2-5, CCL7, and CXCL10. Whereas ectopic expression of FOXP3 suppressed cytokine production, it

significantly induced CXCL8. Moreover, supernatants from Tregs attracted neutrophils via a CXCL8-dependent mechanism. These data provide the first evidence that selleckchem although classical Tregs are defined by their lack of proinflammatory cytokine production, they secrete significant quantities of chemokines and thus may have an unappreciated role in directing the recruitment of immune cells. A notable characteristic of classically defined FOXP3+ Tregs is their inability to secrete T-cell-derived inflammatory cytokines such as IFN-γ and TNF-α 1. Although it is generally accepted that Tregs express a variety of chemokine receptors 2–5, very little is known about their capacity to produce chemokines and thereby direct trafficking of immune cells. Tregs reside in both lymphoid and non-lymphoid tissues 4, 6, and are present during the initiation of inflammatory responses. We speculated that, in addition to their known capacity to suppress immune cells upon arrival into inflammatory tissues, Tregs might regulate the recruitment of additional

immune cells by directly secreting chemokines themselves. We therefore investigated Selleck Dabrafenib the chemokine expression profile of human FOXP3+ Tregs and surprisingly found that they produce substantial amounts of CXCL8 in addition to other chemokines. Evidence that Tregs also stimulated the migration of neutrophils

suggests that these immunoregulatory cells may have an unappreciated role in recruitment of innate immune cells. As Tregs Glycogen branching enzyme are present in the early stages of an immune response, we investigated whether they may have the capacity to influence the recruitment of innate immune cells such as neutrophils via production of chemokines. We initially focused on CXCL8, which is made by a variety of leukocytes and signals through CXCR1 and CXCR2, since this is a strong chemoattractant for neutrophils 7, 8. CD4+CD25− and CD4+CD25+ T cells were isolated using magnetic separation, stimulated with αCD3/αCD28-coated beads and levels of secreted CXCL8 in supernatants were determined. As shown in Fig. 1A, CD4+CD25+ T cells produced similar levels of CXCL8 compared to CD4+CD25− T cells, with an average of 2.3±2.1 ng/mL of CXCL8 and 0.7±0.8 ng/mL of CXCL8, respectively. Recent studies have demonstrated that a significant proportion of Tregs have the capacity to produce IL-17 9–12 and Th17 cells are known to produce CXCL8 13, 14.

Given the body of evidence now available, it is now widely accepted that MCs have a role in the immune response of fish (16,18,26,27). MCs are motile and their distribution and abundance change in response to the pathogen that is attempting to infect the host (8,17,23,28). At the site of parasitic infection, these cells release selleckchem their contents that include various tryptases, lysosyme, piscidin and antimicrobial peptides (6,25); their degranulation

in response to the presence of parasites having been reported in several recent studies (29,30). It has been suggested that the secretions produced by MCs may have a role in attracting other types of granulocytes such as neutrophils, which are among the first cell types to arrive at the sites of inflammation and are a critical component of the teleost innate immune defence system (31). Neutrophils are involved in the inflammatory process, especially during the period of initial pathogen challenge (22,32), migrating to

and accumulating at the site of parasitic infection or injury (5), their number increasing in response to the parasitic infection (33,34). Fish neutrophils have been shown to phagocytize small foreign particles (8) and to degranulate in close RO4929097 supplier proximity to parasites, releasing the contents (11,34, current study). Rodlet cells (RCs) are a type of an inflammatory cell that are closely linked to other piscine inflammatory cells, such as MCs (23), mesothelial and epithelioid cells (23). RCs are commonly associated with epithelia, for example intestine, and the general consensus among researchers is that they have an important role in host defence (23,35). Interestingly, in infected tench, RCs have been frequently observed distributed among MCs and neutrophils within the submucosal layer of the intestine (4). Cestodes possess a diverse range of glands within www.selleck.co.jp/products/Gefitinib.html their scolices, the secretions of which have an array of different functions and effects on their hosts (36,37). Many

of these secretions are histolytic in nature (38), protecting the tapeworm from the host’s immune response (37). The noted increase in the number of host neutrophils and MCs at the site of M. wageneri infection in T. tinca (4) and the intense degranulation of both cell types in close proximity to the cestode’s tegument prompted a further study and comparative survey of un- and infected hosts. Findings from this study provide evidence for the role of the immune system of T. tinca in the modulation of the inflammatory response to a M. wageneri infection. Twenty-three tench from Lake Piediluco (Province of Terni, Central Italy 42° 31′ 01″ N; 12° 45′ 00″ E) were caught by professional fishermen belonging to the Piediluco Fish Consortium using a gill net that was deployed on two occasions (April and July 2011).

Representatives of five ixodid tick genera were compared, both metastriate species (D. reticulatus, R. appendiculatus, H. excavatum and A. variegatum) and a prostriate species (I. ricinus). The D. reticulatus and I. ricinus ticks were collected by flagging the vegetation in selected locations of western Slovakia previously used for tick collecting; R. appendiculatus, H. excavatum and A. variegatum were obtained from colonies maintained at the Institute of Zoology (Bratislava). Hyalomma excavatum was the kind gift of Dr Michael Samish, Kimron Veterinary Institute,

Bait Dagan, Israel. It is both a two-host ditropic tick, with larvae and nymphs feeding on the same selleckchem individual host animal while adults feed on entirely different host species, and a three-host tick with larvae, nymphs and adults each feeding on a different animal. To maintain our H. excavatum colony, the ticks were fed on rabbits: 70–80% followed a two-host strategy while the remainder were three-host. Larvae fed for 6–9 days, nymphs for 7–10 days and adult females fed for 8–12 days to complete engorgement; larvae + nymphs (two-host strategy) completed engorgement as nymphs in 11–28 days. see more SGE was prepared by modifying the method of [13]. Briefly, at given times, ticks were gently removed from the laboratory animals and their

salivary glands dissected out in ice-cold sterile 0.15 m NaCl (0.9%) and washed three times in the same

solution. Salivary gland tissues were then homogenized and centrifuged at 10 000 g for 30 min at 4°C. Supernatant fluids were dried using a Speed-Vac, stored at 4°C and reconstituted in PBS before use. Pooled SGE was prepared from ticks feeding on laboratory rabbits for two time periods: 3 days representing the early (slow) period and 7 days representing Osimertinib molecular weight the late (rapid) phase of engorgement (Table 1). Before testing, the pooled dried SGE was diluted such that 10 μL contained SGE from a single tick. The hypostome of ticks is sclerotized and does not change size or shape once the tick has moulted [14]. Live ticks were immobilized on double-sided tape, and the tube-shaped hypostome from the apex to the base of the cheliceral shaft (dorsal aspect) was measured by means of an eyepiece and lens micrometre using a binocular microscope (Nikon SMZ 645; Optoteam S.R.O., Bratislava, Slovak Republic) at magnification, ×50. Antigrowth factor activities were measured using commercial ELISA kits and recombinant growth factors obtained from R&D Systems (Abingdon, UK): human fibroblast growth factor, FGF-2 (basic; DFB50); human hepatocyte growth factor, HGF (DHG00); human IL-6 (D6050); human keratinocyte growth factor, KGF (DKG00); human/mouse platelet-derived growth factor, PDGF-AA (DAA00); human stromal cell-derived factor, SDF-1α (DSA00); and human-transforming growth factor, TGF-β1 (DY240).

Most studies on this topic were retrospective and used questionnaires to survey donors and potential donors. The majority of donors were satisfied with the donation process and did not regret their decision. However, several concerns frequently reported by donors related to surgical pain, recipient wellbeing (complications and side-effects), uncertainty about donor health, assessment

of donor eligibility, poor follow-up care, lifestyle restrictions, financial impact and inadequate information. Kidney Disease Outcomes Quality Initiative: No recommendation. UK Renal Association: The doctor looking after the donor has a responsibility to inform donors of psychosocial Cell Cycle inhibitor issues around transplantation. Canadian Society of Nephrology: No recommendation. European Best Practice Guidelines: No recommendation.

Organ Procurement and Transplantation Network (OPTN): The program has a responsibility to have available to the potential donor a donor team that consists of at least the following: physician/surgeon, transplant coordinator/nurse clinician, medical social worker, psychiatrist or psychologist, ethicist/clergy. The donor team’s function is to: 1 Educate buy AZD6244 the potential donor regarding the potential risks and benefits Psychiatric and social screening: the dedicated mental health professional familiar with transplantation and living donation should evaluate the potential donor for: 1 Psychosocial history The Canadian Council for Donation and Transplantation:22 Pre-donation psychosocial evaluation should be conducted by a clinical social worker (with the appropriate knowledge and skill set) who is independent of the intended recipient’s Thiamet G care team. A psychosocial evaluation should be based on a semi-structured tool.

This tool should guide discussion while enabling the latitude necessary for individual variation. The timing of the psychosocial evaluation should be left to the discretion of the living donor coordinator on the basis of the initial interview. Suggested components of the evaluation include: An exploration of the motivation for organ donation (how the decision was made, evidence of coercion or inducement, expectations and ambivalence) 1 Renal units could conduct a standard comprehensive psychosocial assessment, using a semi-structured questionnaire, during the postoperative clinical check up. The questionnaire should be evaluated. Emma van Hardeveld and Allison Tong have no relevant financial affiliations that would cause a conflict of interest according to the conflict of interest statement set down by CARI. We would like to acknowledge Karen Penberthy who helped to analyze the data. “
“Allograft thrombosis is a devastating early complication of renal transplantation that ultimately leads to allograft loss.

After 6 hr the medium was replaced with basal medium and the transfected cells were incubated for 24 hr. After 24 hr of incubation, the transfected cells were harvested and the cell lysates were prepared with 1 × lysis buffer (Promega, selleck chemical Madison, WI) containing 10 μg/ml aprotinin and 0·5 μm PMSF. Twenty microlitres of luciferase assay reagent (Promega)

was added to each 50-μg protein sample, and the luciferase activities were evaluated at least in triplicate. The assay results were expressed in relative luciferase activity units. The results are expressed as the average of three independent experiments ± SD. A total of 5 μg RNAs were isolated from SiHa and CaSki cells transfected with mock, E7AS, IL-32, COX-2, siCONTROL and siIL-32 using an easy-BLUE total RNA extraction

kit (iNtRon Biotechnology, Sungnam, South Korea), and the cDNA products were prepared with Moloney murine leukaemia virus reverse transcriptase (New England Biolabs, Beverly, MA). Reverse transcription–PCR (RT-PCR) analysis was performed using a Dice PCR thermal cycler (TaKaRa, Shiga, Japan) with the following primer sets: HPV E7: 5′-ATGCATGGAGATACACCTACATTGC-3′ (forward), 5′-TTATGGTTTCTGAGAACAGATGGGGC-3′ (reverse); IL-32: 5′-ATGTGCTTCCCGAAGGTCCTC-3′ (forward), 5′-TCATTTTGAGGAT TGGGGTTC-3′ (reverse); COX-2: 5′-GAAACCCACTCCAAACACAG-3′ (forward), 5′- CCCTCGCTTATGATCTGTCT-3′ (reverse); IL-1β: 5′-ATGGCAGAAGTACCTAAGCTCGC-3′ (forward), 5′-TTGACTGAAGTGGTACGTTAAACACA-3′ BAY 57-1293 in vivo (reverse); TNF-α: 5′-GTCAGATCATCTTC TCGAACC-3′ (forward), 5′-AAAGTAGACCTGCCCAGACTC-3′

(reverse); IL-18: 5′-ATAGGATCCATGGCTGCTGAACCAGTA-3′ (forward), 5′-GACAGATCTGTCTTCGTTTTGAACAG T-3′ (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. Expression of proteins was analysed using Western blotting with Ribonucleotide reductase specific antibodies. The cell lysates were prepared by treating cells with a lysis buffer [0·1% SDS, 0·1% sodium deoxycholate, 1% Triton-X-100, 1 mm EDTA, 0·5 mm EGTA, 140 mm NaCl, 10 mm Tris–HCl (pH 8·0), 10 μg/ml aprotinin and 0·5 mm PMSF] on ice and centrifuged for 30 min at 11 269 g. The protein concentration of the supernatant was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA) and 50 μg proteins were resolved on 12% SDS–PAGE. The proteins were then transferred onto PVDF membranes (Millipore, Billerica, MA) and blocked overnight with 5% skimmed milk. The antibodies used were specific to COX-2, GAPDH, p21 (Santa Cruz Biotechnology, Santa Cruz, CA), poly-ADP-ribose-polymerase (PARP; Cell Signaling Technology, Beverly, MA), cyclin E and cyclin A (BD Biosciences Pharmingen, San Diego, CA), and IL-32 (KU32-52).30 The blots were probed with enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK) or WEST-ZOL Plus (iNtRon Biotechnology) Western blot detection systems according to the respective manufacturers’ instructions. Culture media were collected after incubating the transfected cells for 24 hr.

“
“Autologous microvascular breast reconstruction is an increasingly common procedure. While arterial

anastomoses are traditionally being hand-sewn, venous anastomoses are often completed with a coupler device. The largest coupler size possible should be used, as determined by the smaller of either the donor or recipient vein. While its efficacy Ibrutinib has been shown using 3.0-mm size and greater couplers, little is known about the consequences of using coupler sizes less than or equal to 2.5 mm. Methods: A retrospective chart review of patients undergoing autologous breast reconstruction was conducted at NYU Medical Center between November 2007 and November 2011. Flaps were divided into cohorts based on coupler size used: 2.0 mm, 2.5 mm, and 3.0 mm. Outcomes included selleck inhibitor incidence of arterial or venous insufficiency, hematoma, fat necrosis, partial flap loss, full flap loss, and need for future fat grafting. Results: One-hundred ninety-seven patients underwent 392 flaps during the study period. Patients were similar in age, type of flap, smoking status, and radiation history. Coupler size less than or equal to 2.0 mm was found to be a significant risk factor for venous insufficiency (P = 0.038), as well as for development of fat necrosis (P = 0.041) and future need for fat grafting (P = 0.050). In multivariate analysis, body mass index was found

to be an independent risk factor for skin flap necrosis (P = 0.010) and full flap loss (P = 0.035). Conclusions: FER Complications were significantly increased in patients where couplers of 2.0 mm or less were used, therefore to be avoided whenever possible. When needed, more aggressive vessel exposure through rib harvest, the use

of thoracodorsal vessels or hand-sewing the anastomosis should be considered in cases of internal mammary vein caliber of 2.0 mm or less. Therapeutic Level III. © 2013 Wiley Periodicals, Inc. Microsurgery 33:514–518, 2013. “
“Intraoperative near-infrared indocyanine-green (ICG) angiography enables the visualization of microvascular perfusion and may help in the early detection of complications. The purpose of the present study was to examine whether the effect of microvascular stenoses can be quantitatively assessed by analysis of ICG-angiography in a microvascular model. Graded stenoses and total vessel occlusion of the carotid, aorta, and femoral arteries were created in 25 Wistar rats. Stenoses were graded to reduce arterial flow by 25%, 50%, 75%, and 100% of baseline flow as measured by transit-time flowmeter analyzing the emission signal of the ICG detected and investigated by the mathematical software tool (FLOW 800). ICG angiography was performed to assess vessel perfusion and flow curves were analyzed and correlated with the stenosis rate. A total of 576 investigations were performed. The area under the curve (P < 0.001), first and second maximum (P < 0.001), and the maximum slope to the first maximum (P < 0.

To disrupt each sample of tissue we added 400 μl of cell disruption buffer and then homogenized the sample with a motorized rotorstator. Total RNA was isolated from tissue samples using the mirVanaTMParisTM kit (Ambion/Applied Biosystems). The RNA obtained from each sample was then quantified by NanoDrop. Pools of three tissue samples in each were analysed using a final concentration of 50 ng/μl. A total of 3 μl of the small 5-Fluoracil nmr RNA fraction were reverse-transcribed using the miRNA Megaplex reverse

transcription primers (for pools A and B) and the TaqMan® microRNA reverse transcription kit (both from Applied Biosystems). The cDNA obtained was amplified using TaqMan® PreAmp Master Mix and Megaplex PreAmp Primers (for pools A and B). For the reverse transcription of cel-miR-39, we prepared a reaction with RT master mix using the TaqMan® microRNA reverse transcription kit, cel-miR-39 RT primer (TaqMan MicroRNA assay) and total RNA. The reaction was incubated at 16°C for 30 min, followed by 42°C for 30 min and then 85°C for 5 min. An initial reverse check details transcription–quantitative polymerase chain

reaction (RT–qPCR) was performed to test the quality of cDNA before the definitive analysis. At this point, three types of quality control were used. Cel-miR-39 was used as a spiked-in control in serum samples. RNU48 was used to test the quality and integrity of the obtained cDNA tissue. Mammalian U6 (U6) was used in both types of samples (serum and tissue).

Ct values of 16–19 in serum samples and 15–18 in tissue samples were considered as valid. Each RT reaction was performed using TaqMan® 2× Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems). Up to 700 miRNAs were evaluated by the TaqMan® human miRNA array. A TaqMan® human microRNA array card is a high-throughput PCR-based miRNA array that enables analysis of more than 700 miRNA assays on a microfluidic card. Simultaneous synthesis of cDNA for mature miRNAs was performed using Megaplex reverse transcription human pool A and B (Applied Biosystems). Each of these, DCLK1 A and B, is a set of predefined pools of 384 stem-looped reverse transcription primers. RT–qPCR was performed using the Applied Biosystems 7900HT fast real-time PCR system and default thermal-cycling conditions. Data analysis was performed using Expression Suite software (Applied Biosystems) and the HTqPCR library in r [27]. The ΔCt values were obtained using the mean expression value of all expressed miRNAs in a given sample as a normalization factor for miRNA RT–qPCR data, according to the procedure described by Mestdagh et al. [28]. The results were expressed as log2 fold change from ΔCt values. We discarded fold change values between −2 and 2 in absolute terms, with mean values between −1 and 1 expressed as log2 fold change.