Probe sets of unknown identification/function are listed under “Unknown”. Based on these categories, a higher percentage of NOD altered genes (∼22%) were involved in apoptosis/cellular proliferation at each of 2 or 3 weeks than at 4 weeks (12%). Conversely, a higher percentage

of genes (22%) were involved in immune response at 4 weeks than at 2 or 3 weeks (4.1% and 12%, respectively). Overall, a high percentage of genes at all 3 ages were involved in enzymatic activity, including several kinases/phosphatases at 3 weeks. Moreover, a large number of the CD4 T-cell NOD altered genes lie within known Idds, 52%, RO4929097 47% and 38% at 2, 3, and 4 weeks, respectively ( Table 1, Table 2, Table 3 and Table 4). A total of 14 (25%),

20 (∼19%) and 10 (∼17%) genes at 2, 3, and 4 weeks, respectively, lie within Idd13, a region located on chromosome (Chr) 2 and previously identified to confer resistance to diabetes in NOR mice [ 4, [15], [16] and [17], 30]. These genes included 6 that were common to all 3 ages: Bloc1s6, Venetoclax order Trp53bp1 (transformation related protein 53 binding protein 1), Tmem87a, Ctdspl2 (CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase like 2), Gatm and Raly (hnRNP-associated with lethal yellow). Two genes (Khdrbs1 − KH domain containing, RNA binding, signal transduction associated 1 and Ptp4a2, both altered at 2 and 3 weeks, but only Khdrbs1 at 4 weeks) lie within Idd9/11, a region on Chr4 that also confers resistance to diabetes in NOR. This region has also been demonstrated previously by several groups Exoribonuclease to regulate the diabetogenic activity of CD4 T-cells [ 5, 10, 18, 19]. To gain further insights into the biological processes associated with the NOD altered genes, we performed Gene ontology (GO) and KEGG pathway analyses. Because GO analysis is better suited for larger gene lists, we also analyzed gene lists generated

at a slightly lower statistical stringency. Analysis at p < 0.05, Benjamini–Hochberg (FDR of 5%) identified 134, 252, and 185 NOD altered genes at 2, 3, and 4 weeks, respectively ( Tables S2, S3 and S4, respectively). The topmost ranked major biological process of these gene lists was metabolism ( Table 5). Although common to all 3 ages, this functional category was most significantly enriched at 2 weeks. Several biological processes unique to 3 weeks were also identified, including biopolymer modification, localization and transport, and T cell activation. Under “molecular function”, hydrolase activity and binding were the common top ranked categories. “Intracellular” was the most significantly enriched cellular component for all the 3 lists. Nucleus and cytoplasm/endoplasmic reticulum were uniquely enriched at 2 and 3 weeks, respectively.

Thus, multiple neurotrophins are probably necessary for the survival of proprioceptive neurons in the trigeminal nervous system. This suggestion can be partly supported by our previous finding that the exogenous BDNF prevents cell death of Mes5 neurons after neonatal masseteric nerve transection [45]. It is likely that the neurotrophin dependency of proprioceptive neurons is different in the spinal and trigeminal nervous systems. Brn-3a/Brn-3.0 is a member of the POU family of transcription factors expressed by neurons [46], [47], [48] and [49]. This factor protects neurons from their cell death and stimulates outgrowth of neuronal processes [50], [51] and [52]. Targeted deletion of the

Brn-3a gene in mice has little or no effect on the number of sensory neurons in the DRG [53]. However, loss of Brn-3a causes a marked reduction of sensory neurons in the TG; only 30% of the normal see more GSK1210151A concentration complement of neurons survive until birth [53] and [54].

By cell size analysis, the proportion of small neurons markedly increases while that of medium-sized and large neurons significantly decreases in the TG of Brn-3a knockout mice [55]. Thus, it is likely that Brn-3a deficiency predominantly causes the cell death of medium-sized and large TG neurons. Our immunohistochemical studies have indicated that Brn-3a is necessary for the development of nociceptors and low threshold-mechanoreceptors Bay 11-7085 in the TG (Table 2) [55] and [56]. The loss of Brn-3a reduces

the number of medium-sized CGRP-containing neurons [56]. In Brn-3a knockout mouse, medium-sized and large TRPV2-containing neurons disappear whereas small TRPV1-containing neurons remain unchanged (Table 2) [55] and [57]. In addition, a 63% decrease of parvalbumin-containing TG neurons is detected (Table 2) [56]. In the Brn-3a knockoutvibrissa, parvalbumin-containing endings are lost and Merkel endings are reduced. Thus, it is suggested that primary nociceptors and mechanoreceptors with medium-sized and large cell bodies in the TG require Brn-3a for their survival. On the other hand, the number of small TG neurons which contain CGRP, calbindin D-28k or calretinin is increased by the loss of Brn-3a [56]. Brn-3a may also have a function to suppress the expression of these neurochemical substances in small TG neurons. The trigeminal motor nucleus contains abundant motoneurons in wild-type and Brn-3a knockout mice [58]. In the Mes5 of wild-type mice, many proprioceptors that contain parvalbumin are also observed. In Brn-3a knockout mice, however, parvalbumin-containing Mes5 neurons cannot be detected (Table 2) [58]. Therefore, Brn-3a is probably required for the survival of proprioceptors but not motoneurons in the trigeminal nervous system. Dystoninis known as Bullous Pemphigoid Antigen 1 (Bpag1) which is a member of the plakin family of high molecular weight cytoskeletal linker proteins [59].

Myoblasts, including C2C12

cells, are maintained in vitro in the presence of highly concentrated serum to sustain their self-renewal ability and to inhibit their differentiation. Over-expression of Smad7 in C2C12 cells markedly enhanced their myogenic differentiation even in the presence of concentrated serum, suggesting that the TGF-β family is involved in the inhibition of myogenesis by serum [55]. This result also indicated that serum contains a BMP-like activity because serum induced the expression of the Id1 gene and ALP activity in C2C12 cells, which were inhibited by adding a soluble form of ALK3/BMPR-IA learn more or noggin [55]. Bovine BMP-4 was identified in the highly purified BMP-like fraction from calf serum [55]. BMP-9 is also found in the circulating blood of humans, suggesting that BMPs are not only local factors but also systemic factors [56]. The phosphorylation of Smad1/5/8 is induced within 1 h after BMP stimulation, and the transcription of several genes is also induced by BMPs within the same period. Id1 was identified as a dominant-negative regulator of MyoD activity and an inhibitor of myogenesis [57] and [58]. The expression of Id1 mRNA

is up-regulated within 1 h in C2C12 cells following BMP-2 stimulation, suggesting that the induction Src inhibitor of Id1 expression would inhibit myogenesis [37]. A GC-rich 29-bp region was identified as a BMP-responsive element (BRE) in the Id1 gene, and this BRE is conserved 100% in mice and humans [59]. Smad1 and Smad4 form a DNA–protein complex with the BRE in response to BMP-2 stimulation [59]. Smad4 is an essential co-activator of Id1 expression because deletion of the Smad4 gene prevented its transcription in response to BMP stimulation [48] and [59]. In addition to Id1, Id2 and Id3 are also early-responsive genes of BMP signaling, and GC-rich

Smad-binding elements were identified in these genes [60], [61] and [62]. A novel non-coding RNA, called BMP-inducible transcript-1 (BIT-1), was identified by searching databases for the conserved Montelukast Sodium BRE sequence [62]. The expression of BIT-1 mRNA is increased in C2C12 cells in response to BMP-4 stimulation [62]. Although BIT-1 mRNA is highly expressed in cartilage, the physiological role of BIT-1 remains unknown. Among the genes induced by BMP stimulation, osterix was shown to play an important role in bone formation [63]. Osterix (also called SP7) was identified as a transcription factor that is expressed in C2C12 cells treated with BMP-2 but not in the parental C2C12 cells [63]. The level of Osterix mRNA is increased up to 24 h after the BMP-2-stimulation of C2C12 cells and is specifically expressed in bone tissue in vivo [63].

, 2012 and Wang et al., 2001): (BE = fresh weight of mushrooms/dry weight of substrate) × 100 Subsequently, the mushrooms were dried in an oven at 45 °C for the determination of their dry

weight. To determine the content of minerals, crude proteins and to evaluate the accessibility of Li, the dried mushrooms were ground using a knife mill and passed through a 2-mm sieve. Samples of 100 mg of dry mushrooms were milled and submitted to digestion with a mixture of nitric acid and perchloric acid (3:1, v:v) at 200 °C for 2 h (Tedesco, Gianello, Crizotinib concentration Bissani, Bohnen, & Volkweis, 1995). The levels of Li were determined using a flame photometer. The standard curve was prepared with the following concentrations of this element: 0.00; 0.09; 0.36; 0.72; 1.44; 1.80; 2.88; 3.60 and 9.00 mg L−1. The percentage of Li was calculated according to the formula: ConcentrationofLiindrymass(μgg-1)=[M]×DFDM×1000where, [M] = mineral concentration in mg L−1, DF = dilution

factor = 0.025, DM = dry mass of sample. The content of Fe, Zn, Cu, potassium (K), calcium (Ca), phosphorus (P), sulphur (S), lead (Pb), chromium (Cr), magnesium (Mg), aluminium (Al),, cadmium (Cd) and nickel (Ni) contained in the mushrooms were measured by inductively-coupled plasma optical emission spectrometry (Optima 3300 DV; Perkin Elmer, Waltham, MA), using specific standards for each mineral. The crude MEK inhibitor protein content was determined using the semimicro-Kjeldahl method (AOAC, 1996). The nitrogen content was multiplied by a factor of 4.38 to calculate

the percentage of crude protein (Kalac, 2009). The sequential extraction and in vitro methods were used to evaluate the accessibility of Li. We compared mushrooms grown in substrate enriched or not with LiCl (0 and 500 mg kg−1) and a psychiatric drug containing lithium carbonate (140.9 mg of Li per g of pill, as reported by the manufacturer). To evaluate the solubility of Li, 1 g of dried mushroom and also 1 g of the psychiatric drug pill were processed according to sequential extraction CHIR-99021 nmr methodology described by Ramos, Hernandez, and Gonzalez (1994) and modified by Ma and Rao (1997). After each successive extraction, the extracts were separated by centrifugation at 1500g for 10 min, and the supernatant was collected. The sediment obtained after each extraction was resuspended and again subjected to extraction to collect a new supernatant. This procedure was repeated until six fractions were obtained. We then conducted the analysis of dissolved Li using a flame photometer. The second method was the in vitro simulation of gastrointestinal digestion, with the purpose of predicting the accessibility of Li in the digestive tract ( Elless et al., 2000 and Glahn et al., 1998). For this, 250 mg of samples of both dried mushrooms and of the psychiatric drug were crushed. Next, the samples were centrifuged at 1500g for 10 min and filtered to obtain soluble extracts.

, 2001 and Lin and Harnly,

2007) and for the standard analysed under the same conditions. Peaks 8, 9, 10 and Lapatinib cell line 11 were identified as myricetin glucoside, myricetin pentoside, myricetin rhamnoside and myricetin acetyl-rhamnoside, respectively. The following elution order is expected on reversed phase for the same aglycone: hexoside < pentoside < deoxyhexoside, and acylated derivatives elute after their non-acylated flavonoids (Lin and Harnly, 2007 and Wu and Prior, 2005). In addition, the λmax values at 349–355 nm, about 20 nm lower than the λmax of myricetin (371 nm), indicate the typical hypsochromic effect of flavonol glycosides in relation to its aglycone ( Lin & Harnly, 2007). The mass spectra indicated the presence of the aglycone at m/z 319 (ESI+) and at m/z 317 (ESI-), which corresponds to myricetin. In addition, the MS/MS fragmentation pattern obtained from these ions (m/z at 319 and m/z at 317) showed the same fragments at m/z 301, 273, 245, 165 and 153 as those found for myricetin. In the case of myricetin glucoside (peak 8), the loss of 162 u, both in positive and negative modes, indicated the presence of an hexose in the molecule, whereas the loss of 132 u indicated

the presence of a pentose in peak 9 (myricetin pentoside). However, the analysis by MS itself does not allow distinguishing whether the sugar Romidepsin is xylose or arabinose, which are the most commonly pentoses found in fruits. For myricetin rhamnoside (peak 10), the loss of 146 u from [M−H]− (m/z at 463) is characteristic of a deoxyhexose

unit, and rhamnose is the only deoxyhexose found in fruit flavonoids. Finally, the MS/MS spectrum of myricetin acetyl-rhamnoside (peak 11) showed a loss of 188 u, corresponding to an acetylated rhamnose unit (146 + 42 u) ( Cuyckens and Claeys, 2004 and Mahmoud et al., 2001). The C3 position is the most likely location for all these glycosides ( Cuyckens & Claeys, medroxyprogesterone 2004). For flavanonols, considering the biosynthetic flavonoid pathway (proposed in Fig. S4 from Supplementary data), the aglycones at m/z 321 (ESI+, peak 3) and at m/z 305 (ESI+, peak 5) were identified as dihydromyricetin and dihydroquercetin, respectively, since these flavanonols are precursors of myricetin (peak 12). Considering that simple flavonoids with a hydroxyl in ring B may be modified during biosynthesis through hydroxylation and methylation reactions ( Heller & Forkmann, 1994), the aglycones at m/z 335 (ESI+, peak 6) and at m/z 349 (ESI+, peak 7) were identified as methyl and dimethyl derivatives of dihydromyricetin diglucoside (peak 3) ( Fig. S3 from Supplementary data).

, 2004, Hammond et al., 2006a, Hammond et al., 2006b, Healy et al., 2008,

Qi et al., 2012 and Zhu et al., 2004) simply stated that a pathologist or veterinary pathologist performed the analysis, RGFP966 supplier but no mention was given as to what these analyses entailed, for example what pathological parameters were used or what was measured and why. The exception appears to be a study by Teshima et al. (2000) who stated that the morphology of the small intestine mucosa was assessed, in particular the composition of goblet cells and intraepithelial lymphocytes. According to the authors, the analysis was based on a chapter in an immunotoxicology textbook (Kawabata, 1996). However, that chapter did not mention the purpose or even how the investigation of the OTX015 order small intestine should appear. In particular, it did not include the definition of what constitutes abnormal or diseased, such as, what changes in goblet cell population would indicate a pathology. A paper that appears to be well-structured and thorough was the Tutel’ian et al. (2008) study published in Russian. The methods section clearly stated that the morphometric analysis of the internal organs was conducted according to textbook guidelines (Avtandilov, 1982 and Avtandilov, 1990) and results were compared according to guidelines set out by Stefanov (1985). The two Russian textbooks (Avtandilov, 1982 and Avtandilov, 1990) are manuals on how to conduct

quantitative research to obtain a meaningful assessment of morphological PTK6 changes. In other words, the Tutel’ian et al. (2008) study appears to be thorough and well set out, especially since detailed results are provided for the analyses. However, the publication lacks basic information. It does not specify the number of rats used in the study and it does not list which organs were collected for the histopathological analyses. Results

seem to imply that the ileum was the only section of the GI tract to be analysed. A more thorough study would have investigated other sections of the GI tract to more accurately ensure that the GM crop did not have any adverse effects. Another Russian study (Tutel’ian et al., 2010) also appears to be properly conducted. Its safety assessment is based on the Tutel’ian et al. (2008) study, which implies that the same rigorous morphometric analysis was also utilised. However, even this publication lacks key information. For example, the paper indicated that the morphometric analysis was conducted on the small intestine and colon, but results were only reported for the small intestine. In addition, the publication does not specify which section of the small intestine these results pertain to. This lack of detail in both Russian papers makes it difficult to determine the veracity of the results. It also makes it difficult to reproduce and further the study or to compare these studies to others.

, 2009). A similar observation was made with a group of adult homesigners from Nicaragua (Spaepen et al., 2011): In a series of set-reproduction tasks, homesigners used one-to-one correspondence strategies only rarely, and when Dorsomorphin research buy they did so, they used it by mapping their fingers (the constituents of their number signs) to objects, never by mapping

two sets directly onto each other, a seemingly simpler strategy. Understanding how words (or, in the case of homesigners, fingers) stand in one-to-one correspondence with objects while counting may be the first step that leads to a more general understanding of one-to-one correspondence relations, and in particular of how one-to-one correspondence warrants exact numerical equality. Our findings shed light both on the extent and the limits of children’s numerical knowledge, before they master the meanings of all the number words they use in counting. Children who have not mastered the exact numerical meanings of “five” and “six” are able to use one-to-one correspondence cues to reconstruct a set of exactly five or six objects, even when the sets are moved around, rearranged in space, and kept out of view for some time, and even if one individual is first subtracted and then added back to the set, as long as the identity INCB024360 manufacturer of the items

forming the sets is not modified. However, children do not know how set transformations that change the individual members affect the way sets can be measured by one-to-one correspondence. Hence, before children acquire symbols for exact number, one-to-one correspondence defines a relation of identity between sets: a relation that is not limited to approximate numerical equality but falls short of exact numerical Thymidylate synthase equality. Furthermore, children do not understand how one-to-one mappings interact with the addition of one, i.e. the successor function. At 3 years of age, the child’s state of knowledge for number thus corresponds to the initial stage of Russell–Frege’s formal definition of cardinal integers:

they have a relation of set identity, but yet have not figured out how this notion interacts with basic operations, and how the numbers can be ordered in a list structured by a successor function. This research was supported by grants from NIH (HD 23103) and NSF (0633955) to E.S.S., and by a postdoctoral grant from the Fyssen Foundation and a Starting Grant from the European Research Council (MathConstruction 263179) to V.I. We thank Ariel Grace, Kate Ellison and Konika Banerjee for help in data collection; Amy Heberle and LeeAnn Saw for help in offline data recoding; Renée Baillargeon, David Barner, Susan Carey, Lola de Hevia, Lisa Feigenson, Justin Halberda, Mathieu Le Corre, Peggy Li, T.R. Virgil, and one anonymous reviewer for helpful discussions and comments throughout the course of the project; and all the parents and children who kindly participated in the research.

, 1993 and Gilmore et al., 1996). Furthermore, recently conducted studies show that climate can also influences the hydraulic architecture and therefore the ratio of leaf area to sapwood area (Poyatos et al., 2007 and Martínez-Vilalta et al., 2009). And even within trees Adriamycin the relationship between leaf area and sapwood area can vary with the position within

the tree (Mencuccini and Bonosi, 2001). Thus, for studies regarding the development of leaf area over time, other indirect methods for estimating leaf area should be found, preferably ones which are based on tree characteristics which can be collected easily and in a non-destructive way. The use of other crown characteristics to estimate leaf area, such as crown ratio, crown length, crown projection area, and crown surface area is rarely investigated (Pereira et al., 1997 and Kenefic and Seymore, 1999). Badoux (1945) and Assmann

(1970) used crown surface area as substitute for leaf area with the evident assumption that most of the growth influencing photosynthetically active leaves are at the crown surface. Assmann (1970) also described that therefore differences in efficiency Z-VAD-FMK clinical trial (there: growth per crown projection area) should lead to differences in the ratio of crown surface area to crown projection area and further, that trees with large crowns are less efficient than trees with smaller crowns due to their large inner crown volume (cubic content) bearing no leaves or needles. Therefore, this study aimed at the question if traditional forest crown measures, particularly

crown surface area (CSA) and crown projection area (CPA) are good measures for leaf area (LA), and if not, whether they can be improved by corrections through additional tree measures or stand measures. The study area was located near Bärnkopf, Lower Austria (15°00′20″ E, 48°23′24″ N) in the Bohemian Massif. Cetuximab ic50 On similar sites 8 even-aged Norway spruce (Picea abies L. Karst.) stands were investigated. The stands represented three different age classes and two thinning variants. We selected four pole stage stands, two premature, and two mature stands (ages of about 40, 80, and 125 years); two of the pole stage stands, and one of the premature and mature stands, respectively, were thinned 5 years ago (subsequently named “thinned”) and the other ones were not thinned for more than 10 years (subsequently named “un-thinned”). No other management, e.g., pruning or fertilization was performed in any of the investigated stands. Because of the relatively small size of the pole stage stands, for each thinning treatment two stands were selected. The fieldwork was conducted between April and September 2008. At first in each stand, the diameter at breast height (dbh), the tree height, the height to the crown base, and the coordinates of each tree were assessed.

These orifices containing the files were filled with epoxy resin. After 24 hours of resin curing, the resin cylinders were removed from the device and sectioned by using a diamond disk (Arotec, São Paulo, Brazil) mounted in an Isomet cutting machine (Buehler, Lake Bluff, IL) under constant irrigation with alcohol. The cut positions were selected in such a way to correspond

to the D14, D6, and D3 of the files. Consequently, flat surfaces containing the whole cross section of each file were obtained (Fig. 1). The metallic this website surface was then polished by using sandpapers of different granulations: 220, 400, 600, and 1200 (3M, São Paulo, Brazil). After polishing, the surface of each fragment was abraded with a steel fine tip by using ultrasonic vibration. This method was adopted to promote the mechanical removal of any protector layer resultant from the previous surface polishing. http://www.selleckchem.com/products/sotrastaurin-aeb071.html Consequently, 3 types of file fragments

were created, generating 3 experimental groups: group D14, group D6, and group D3 composed of fragments with exposed cross section correspondent to the D14, D6, and D3, respectively. Current register tests were used to evaluate the dissolution process of the embedded fragments. An electrochemical cell was used that contained a saturated calomel electrode as reference, platinum as counter-electrode, and a platinum wire with diameter equal to 0.1 mm as the working electrode. The working electrode was used in contact with the file fragment. A polymeric tube with internal diameter equal to 0.15 mm was used to support the platinum wire. Cyanoacrylate ester was used to attach the platinum wire to the polymeric tube. Another polymeric tube with internal diameter equal to 0.40 mm was used as an overlay of the first tube to increase the mechanical

resistance of the described setup. The attachment aminophylline between the tubes containing the working electrode was made by using the same cyanoacrylate ester. The electrodes were immersed in a [NaF 5 g/L + NaCl 1 g/L] solution with pH 5.0 and connected to a digital potentiostat (Metrohm Autolab, Herisau, Switzerland). The fragment of file was immersed in the same solution in such position that the center of the file cross-section surface could be in contact with the working electrode. An anodic potential equal to 700 mVsce was applied to the fragment during 50 minutes, and the potentiostat registered the anodic current generated. The total electrical charge of each test was obtained by integrating the current value over the time by using the program of data analysis Origin 6.0 (Microcal Software Inc, Northampton, MA). Three fragments of each group were tested in these conditions. Analysis of variance (ANOVA) (P < .05) was used to compare the total electrical charge among the fragments of the 3 groups.

Recombinant adenoviral vectors expressing Ad5-directed amiRNAs were amplified in T-REx-293 cells. All other adenoviral vectors and wt Ad5 (ATCC VR-5) were amplified in HEK 293 cells. Titers of infectious adenoviruses expressing amiRNAs were determined on T-REx-293 cells by 50% tissue culture infective dose (TCID50) assays. Titers of wt Ad5 present in mixed virus suspensions containing both wt and recombinant virus as obtained in combined transduction/infection experiments were determined on A549 cells using

the same method. All other TCID50 assays were performed with HEK 293 cells. The vectors employed in dual-luciferase assays for the screening of Ad5-directed amiRNAs have been described elsewhere (Kneidinger et al., 2012). The dual-luciferase target vector used for the Protein Tyrosine Kinase inhibitor determination of Renilla luciferase gene silencing in Ad5-infected cells was constructed as follows: a part of the modified coding region of the firefly (Photinus pyralis) luciferase open reading frame (ORF) representing the target sequence for the corresponding amiRNA was amplified selleck inhibitor by PCR with primers Fluc-f2 (5′-ATAAGGCTATCTCGAGATACGCCCTGGTTCC-3′) and Fluc-r2 (5′-AATGTCGTTCGCGGCCGCAACTGCAACTCCGAT-3′) from vector pGL3 (Promega, Mannheim, Germany). This fragment was restricted with XhoI and NotI and inserted into the corresponding sites located

within the 3′UTR of the Renilla luciferase gene present on plasmid psiCHECK-2 (Promega, Mannheim, Germany). From the resulting vector (psiCHECK-FLuc2), a BglII-BamHI fragment comprising both the firefly and Renilla luciferase expression cassettes was transferred into pENTR4 (Life Technologies Austria, Vienna, Austria) that had been restricted with XmnI and EcoRV. From the resulting vector (pENTR-Luc), the entire expression Sitaxentan cassette was eventually moved into the deleted E1 region of the adenoviral vector pAd/PL-DEST (Life Technologies Austria, Vienna, Austria) giving rise to vector Ad-Luc-as ( Fig. 1). This final transfer was mediated

by employing Life Technologies’ Gateway technology, i.e., by site-specific recombination between sequences flanking the expression cassette on pENTR-Luc and the corresponding sequences located on the adenoviral vector. The recombination reaction was performed according to the instructions of the manufacturer (Life Technologies Austria, Vienna, Austria). The adenoviral vector expressing the amiRNA directed against the target sequence present in the 3′UTR of the Renilla gene on Ad-Luc-as was constructed in a similar way by transferring the enhanced green fluorescence protein (EGFP)/amiRNA expression cassette of plasmid pcDNA6.2-GW/EmGFP-miR-luc (Life Technologies Austria, Vienna, Austria) into pAd/CMV/V5-DEST™ via site-specific recombination as before.