At least within the crown measures this is not surprising, since,

At least within the crown measures this is not surprising, since, in contrary to the 2-dimensional crown projection area in the crown surface area the crown length, as additional information of the third dimension, is EX 527 chemical structure included. Obviously, crown surface area shows a more realistic model of the actual crown shape. Furthermore, the coefficients of the log-linear relationship with leaf area did not differ significantly between the stands, and the

common coefficient of this relationship was nearest to one. Thus, within stands, crown surface area can be assumed to be proportional to leaf area. Some other authors who also worked on non-destructive methods for estimating leaf area found their models also improved by adding crown parameters. But, in contrary to our study, they used crown length (Pereira et al., 1997 and Kenefic and Seymore, 1999) or crown ratio (Valentine et al., 1994). Like crown surface area, their influential crown parameters also contained selleck chemical information about the third dimension of the crown. Hence, the importance to consider crown variables describing the length of the crown to find models of high quality for the estimation of leaf area seems to be crucial. Our test to improve the leaf area estimation through additional variables showed that for all stands together, the common relationship with crown surface area and dbh was better than the one with

crown surface area alone. However, this relationship with both variables had significantly different coefficients between the

stands, and therefore Gemcitabine research buy it would have to be parameterized separately in every stand. Thus, the advantages of crown surface area as a measure for leaf area within stands are (i) its high correlation with leaf area, even better than that for sapwood area at breast height (see Table 3 and Table 4), (ii) its property of having a relationship with leaf area with a coefficient not different between stands, and (iii) a coefficient very near 1, so that it can be assumed being proportional to leaf area. All together makes the crown surface area an applicable measure for the leaf area within stands. Because of this strong relationship the crown surface area could also be used to distribute a given stand’s leaf area appropriately to individual trees within this stand. In some studies regarding crown damage and tree growth the crown surface area was used as a kind of substitute for dry needle mass without testing the relationship between these two parameters (Kramer, 1986 and Halmschlager et al., 2007). Given that the leaf area is highly correlated with the dry needle mass (Hager and Sterba, 1985) – in our study leaf area is actually calculated out of the dry needle mass – the results of these studies are justified retrospectively by our results. So far, only the within-stand relationships between leaf area and its surrogates have been discussed.

These results alongside with those previously obtained by other a

These results alongside with those previously obtained by other authors suggest that this group of natural compounds might be promising for future antiviral

drug design. This study was supported by CNPq/MCT/Brazil (grant number 470235/2009-8). J.W. Bertol, C.M.O. Simões, F.C. Braga, R.M. Pádua and C.R.M. Barardi are grateful to CNPq for their research fellowships, as well as C. Rigotto thanks to CAPES/MEC/Brazil for her postdoc fellowship. “
“Herpes DNA Damage inhibitor Simplex Virus types 1 and 2 (HSV-1 and HSV-2) are human neurotropic viruses usually associated with infections of the skin and mucosae of different locations, most commonly the oral and genital regions. Although infections are often subclinical, HSV can cause mild to severe diseases, especially in neonates and immunocompromised individuals. Currently, there is no cure for PI3K inhibitor the persistent infection, and prolonged therapy with the available antiherpes drugs has induced the emergence of drug-resistant virus strains.

Moreover, HSV has been described as a risk factor for HIV infection (Roizman et al., 2007). This scenario has triggered the search for new antiherpetic agents, especially those with mechanisms of action different from that of nucleoside analogs, the major class of antiviral agents used for the management of HSV infections. Besides, a treatment based on the combination of different antiviral agents can be considered a promising approach to increase antiviral selectivity while simultaneously enabling the reduction of the Ergoloid active concentrations of the drugs (Chou, 2006). Many synthetic or naturally occurring sulfated polysaccharides from different species of marine algae, bacteria, fungi, and animals have been previously shown to have antiviral activity against human and animal viruses (Ghosh et al., 2009). In the case of fungi, cell wall polysaccharides have been chemically modified to increase their solubility and enhance their biological activities (Liu et al., 2010), including their antiviral action (Zhang et al., 2004). The pharmacological effects of Agaricus brasiliensis,

a Basidiomycete fungus native to the Brazilian Atlantic forest region, have been mainly related to the presence of polysaccharides and protein–polysaccharide complexes ( Firenzuoli et al., 2008). Concerning its previous antiviral evaluation, Sorimachi et al. (2001) showed that the ethanolic fractions of A. brasiliensis mycelium and fruiting bodies inhibited HSV, poliovirus, and Western equine encephalitis virus replication. The inhibition of HSV-1 and herpes bovine virus by an aqueous extract of A. brasiliensis fruiting bodies was also demonstrated by Bruggemann et al. (2006). Additionally, both aqueous and ethanolic fruiting bodies extracts and an isolated polysaccharide from this species displayed antiviral activity against poliovirus 1, as reported by Faccin et al. (2007).

16 showing a 1 2 log10 reduction at this concentration (Fig 4A)

16 showing a 1.2 log10 reduction at this concentration (Fig. 4A). In previous studies 226/8.1 showed a higher neutralizing activity than 133/3.16 (Takada et al., 2003), clearly corresponding to our results. For testing of the DsiRNA, 293 cells were pretransfected with a DsiRNA directed against L (targeting the same region as the siRNA EK1, that has been successfully used to protect both guinea pigs and NHPs against lethal EBOV challenge (Geisbert et al., 2006 and Geisbert et al., 2010)) check details or control DsiRNAs, and then infected with 100

TCID50 (equivalent to an MOI of 0.005) of rgEBOV-luc2. After two days, reporter activity was measured, and as expected we observed a clear drop in reporter activity of about 2 log10 at the highest amount of DsiRNA used, and smaller reductions of reporter activity at lower amounts of DsiRNA (Fig. 4B). No effects of the control DsiRNAs were observed, indicating that the reduction in reporter activity was due to a sequence specific effect of the DsiRNA on virus replication. Antiviral screening of EBOV poses unique challenges. While reporter-expressing recombinant EBOVs have enabled rapid detection of infection, the need for a BSL4 laboratory when working with live virus remains, making fully automated high-throughput screenings for

these viruses challenging. However, screening of libraries containing several thousand compounds in a 96-well format is feasible, as was recently demonstrated

(Panchal et al., 2012), and rgEBOV-luc2 is highly amenable to be used in such a screen. rgEBOV-luc2 also has several advantages over eGFP-expressing EBOVs, including its ease of use (no requirement for removal of samples from BSL4, very little labor intensive), low equipment costs and the ability to use either a much lower infectious dose, or alternatively the much faster readout times when using higher infectious doses. These faster readout times, in addition to obvious practical advantages, also mean that compounds with a low stability in culture medium can be more reliably screened. However, too short readout times also have to be avoided, since otherwise the virus does not have time to complete a full life cycle, which CYTH4 would results in inhibitors of late stages of the virus life cycle (e.g. budding inhibitors) not being recognized in the screen. In contrast, high-content screening, which so far is the most extensively used screening approach that has been performed with EBOV-GFP, requires extensive and costly automated imaging equipment. Until now this kind of screening has relied on a multistep approach in which cells are first infected in a BSL4 laboratory for several days, and then fixed for several days in formalin before they are analyzed under BSL2 conditions (of course we cannot exclude the possibility that despite the very complex technology high-content imaging will in future become available under BSL4 conditions).

An influential theory in this field is “scanpath theory” (Norton

An influential theory in this field is “scanpath theory” (Norton & Stark, 1971), which proposed that reinstatement of the sequence of eye-movements made during encoding of a visual stimulus plays a causal role in its subsequent successful recognition. A hard interpretation of this theory entails that recapitulation of eye-movements made during encoding of visual scenes facilitates successful recall. However, a recent study Wortmannin in vitro by Martarelli and Mast (2013) manipulated eye-position during pictorial recall and found that there was no increase in memory accuracy when participants looked at areas where stimuli had previously appeared, in comparison to when

they looked at non-corresponding areas of screen. Similarly, Foulsham and Kingstone (2013) have recently reported a series of experiments in which participants’ fixations were constrained during click here encoding and recognition of images in order to manipulate scanpath similarity. Although scanpath similarity was a predictor of recognition accuracy, there was no recognition advantage when participants re-viewed their own fixations of a

scene versus someone else’s, or for retaining serial order of fixations between encoding and recognition. Foulsham and Kingston conclude that while congruency in eye-movements between encoding and retrieval is beneficial for scene recognition, there is no evidence to suggest recapitulation of the exact scanpath at encoding is necessary for accurate recall. Our own results are broadly in line with these recent findings, as there is no evidence from Experiment 3 in the present study that the ability to engage in saccade preparation to memorized locations

is necessary for their accurate recall. Thus, while the rehearsal of directly salient locations in the oculomotor system allows for optimal spatial memory at recall, we regard this as a contributing mnemonic mechanism that operates in conjunction with visually-based strategies such as mental path construction or visual imagery (Parmentier et al., 2005 and Rudkin et al., 2007). Critically, we have previously shown Staurosporine that eye-abduction only reduces, rather than abolishes, spatial memory even when applied across all encoding, maintenance, and retrieval stages of a trial (Ball et al., 2013). Therefore, clearly the involvement of oculomotor encoding and rehearsal enhances spatial memory for a sequence of visually-salient locations rather than critically enables it. However, this position is not dissimilar to that observed when articulatory suppression is used to prevent subvocal rehearsal of words and digits during verbal working memory ( Baddeley et al., 1975 and Murray, 1967), where verbal memory span is significantly reduced but not abolished ( Baddeley, 2003). Both the findings of Ball et al.

For multivariate analysis, data were z-score standardized and Euc

For multivariate analysis, data were z-score standardized and Euclidean distance matrices produced for each

parameter group. Permutational Multivariate Analysis of Variance (MANOVA) was used with GC# and site location as factors to determine if each category differed by stream and up and downstream of golf course facilities. Significant multivariate interactions were examined by trajectory analysis where the magnitude and direction of change for each stream and site location pair was explored ( Collyer and Adams, 2007). When interactions between stream and site location were not significant, multivariate post hoc tests LBH589 manufacturer were run to determine which streams differed. Multivariate categories for each sampling location were visualized with principle components analysis as biplots of components 1 and 2. Mantel and partial mantel tests and two block partial least squares were used to examine multivariate correlation between parameter groups. All statistical analyses were carried out in R 2.14.1 with the assistance of vegan and geomoph packages. Watershed area ranged for each sampling point from 10 to 93 km2. Anthropogenic land use (e.g., agriculture, development, tree plantations, etc.) ranged 48–78% among stream riparian zones (Table

1). The multivariate landscape group was PCI-32765 similar up and downstream of golf course facilities (Pillai’s Trace = 0.2, p = 0.914; Table 1; Fig. 2A). The landscape group significantly differed by stream (Pillai’s T = 16.9, p = 0.001). Post hoc comparison indicated that GC1 was only similar

to GC2 and GC5. The landscape of GC6 was Reverse transcriptase significantly different from GC2. The landscapes of GC2, GC3, and GC4 were similar ( Fig. 2A). Water quality among streams ranged from oligotrophic to eutrophic (Table 2). DOC ranged from 1.3 to 16.9 mg-C l−1 and was significantly lower downstream of golf courses (Wilcoxon’s paired test, p = 0.002; Fig. 3). SpCond, TDN, BACT, and BP were variable among sites but did not differ up and downstream of golf course facilities. TDP ranged from 4.1 to 44.1 μg-P l−1 and was significantly higher downstream of golf course facilities (Wilcoxon’s paired test, p = 0.023; Fig. 3). All together, the water quality group up and downstream of golf course facilities was similar (Pillai’s T = 0.2, p = 0.913), but significantly differed in water quality among streams (Pillai’s T = 14.3, p = 0.001; Fig. 2B). Post hoc comparison indicated that GC1 and GC2 were similar but significantly differed from the other streams, except between GC1 and GC5 which did not differ (p = 0.064). GC3, GC4, GC5, and GC6 had similar water quality. DOM ranged from strongly humic-like with features of terrestrial inputs (e.g., higher aromaticity (SUVA) and contributions of C2 and C3) to humic-like with features of microbial inputs (e.g.

KRG protects aflatoxin B1- [20] and acetaminophen-induced hepatot

KRG protects aflatoxin B1- [20] and acetaminophen-induced hepatotoxicity [21] and increases liver regeneration after partial hepatectomy [22] in animal models. We recently reported that KRG effectively protects against liver fibrosis induced by chronic CCl4 treatment [23]. However, the effects of KRG on alcohol-induced liver damage and the expression of lipogenic genes have not yet been fully established. In the present study, we examined the effect of KRG in mice after chronic EtOH treatment and in EtOH-treated hepatocytes. Histopathology and biochemical analysis verified the ability of KRG extract (RGE) to protect against EtOH-induced

fat accumulation and oxidative stress, and to restore liver function. Moreover, CB-839 cost RGE recovered the activity of AMPK and Sirt1 in alcohol-fed mice. In agreement with the in vivo data, RGE and its major ginsenosides possess the ability to recover homeostatic lipid metabolism in hepatocytes. These results demonstrate that KRG inhibits alcohol-induced steatosis through the AMPK/Sirt1 signaling pathway in vivo and in vitro, suggesting that KRG may have a potential to treat ALD. Lieber–DeCarli liquid diet was purchased from Dyets, Inc. (Bethlehem, PA, USA). Antibodies directed against CYP2E1, 4-hydroxynonenal

(4-HNE), PPARα, and SREBP-1 were supplied by Abcam (Cambridge, UK). Antibodies that specifically recognize phosphorylated AMPK, AMPK, phosphorylated ACC, and Sirt1 were obtained from Cell Signaling (Beverly, MA, USA). The nitrotyrosine polyclonal antibody was purchased Pexidartinib nmr from Millipore Corporation (Billerica, MA, USA). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G and goat anti-mouse immunoglobulin G were provided by Zymed Laboratories Inc. (San Francisco, CA, USA). RGE was kindly provided by KT&G Central Research Institute (Daejeon, Korea). Briefly, RGE was obtained from PIK-5 6-year-old roots of P. ginseng Meyer. The ginseng was steamed at 90–100°C for 3 h and dried at 50–80°C. The red ginseng was extracted six

times with water at 87°C for 12 h. The water content of the pooled extract was 36% of the total weight. Ginsenosides (Rb1, Rb2, and Rd) were obtained from Sigma-Aldrich Corporation (St Louis, MO, USA). Animal studies were conducted under the guidelines of the Institutional Animal Use and Care Committee at Chosun University, Gwangju, South Korea. C57BL6 mice were obtained from Oriental Bio (Sungnam, Korea) and acclimatized for 1 week. Mice (n = 8/group) were given free access to either the control diet or the Lieber–DeCarli liquid diet containing EtOH with or without RGE. The body weight and general condition of the animals were monitored at least once a week. The diet was kept refrigerated in the dark. EtOH was incorporated into the diet just before it was supplied to the animals. We used two animal models to evaluate the effect of RGE on alcohol-induced fatty liver and liver injury as previously reported [24], [25] and [26].

9% of patients with CCCs, may be secondary to differences in the

9% of patients with CCCs, may be secondary to differences in the characteristics between the study samples and those used in the score development. Murphy-Filkins et al. reported that changes in the demographic characteristics of patients and in the prevalence of diseases alter the mix of cases, which may influence score performance.20 Perhaps, with the recently observed increase in the prevalence of CCCs among patients admitted to the PICU and the observation that these patients have a higher mortality rate than the general population, it will be necessary that future scores for outcome prediction use this condition when their models are being constructed. The

SMR of 1.65 TGF-beta inhibitor (95% CI: 1.26 to 2.04) for the general population and of 1.75 (95% CI: 1.31 to 2.19) for the subgroup of

patients with CCCs indicated that the quality of health care offered at the PICU for these patients during the study period was worse than the quality of health care offered by the PICUs that participated in the PIM2 development study in the period between 1997 and 1999. However, the SMR of 1.14 (0.29 to 1.99) for patients without CCCs indicated that the quality of health care offered at the PICU for this subgroup in the study period was similar to the quality of health care offered by the PICUs that participated in PIM2 development study between 1997 and Buparlisib price 1999. The SMR is the main indicator of health care quality in the PICU, and is part of the first set of quality indicators that were submitted to the Joint Commission on Accreditation of Healthcare Organizations, published in 2005.21 However, the accuracy of this indicator depends on the capacity of the score in predicting mortality in the studied population;8 thus, in addition

to the hypothesis of inadequate quality as the cause of the high SMR in the overall study population and in the subgroup of patients with CCCs, the hypothesis that the PIM2 Reverse transcriptase may not be adequate for the studied population cannot be ruled out. This study has some limitations. The principal limitation is the fact that the study was conducted in a single PICU, making it difficult to prove the hypothesis and subsequently, the generalizability of the findings. Operating conditions (equipment, medications, and staff) in the studied PICU may not be similar to those of PICUs in developed countries, which may have influenced the results. Finally, the statistical analysis may have been influenced by the relatively small sample size. The PIM2 showed poor performance in the subgroup of patients with CCCs and the overall study population, which had 83.9% of patients with CCCs. Although the poor performance of the score may be secondary to the quality of health care offered by the PICU, the hypothesis that the difference in characteristics between the study sample and the sample used for the score development is responsible for inadequate performance of PIM2 cannot be ruled out.

Weight was measured using a digital scale, appropriately calibrat

Weight was measured using a digital scale, appropriately calibrated and suitable for every age group. Length measurement Autophagy inhibitor in children younger than 2 years was performed using an infantometer; a stadiometer was used for the older children. The z-scores of the weight/height (W/H), height/age (H/A),

and weight/age (W/A) indicators were calculated using the Anthro program, available from the WHO. The nutritional diagnosis was made following the WHO criteria.18 Before the intervention, all meals served to the children (four per day) were calculated using food composition tables19 and 20 and product labels, in order to quantify the macro and micronutrients present in a serving of 100 g, followed by estimates of daily consumption. To quantify the consumption, the preparations were weighed before and after ingestion of each meal using a scale accurate to 1 g. All records of consumption and anthropometry were made by the study dietitians. During 90 days, the subjects in group A received one sachet of sprinkles with added zinc and micronutrients daily, whereas group B received

the same micronutrients, without zinc (Table 1). The supplements were mixed in a small portion of the meal, always at the same time. Due to the nutritional composition and day-care routine, the most adequate meal for addition of supplements and the one best accepted by the children was the afternoon snack (silver banana). The supplements were only opened and added to food immediately before serving, by dietitians not blinded to the study, as the sachets were identified for the presence of zinc. The snacks were monitored by the team members (physicians SPTLC1 and dietitians blinded to the study) to prevent exchange of plates or loss of the food serving that contained the supplements, as well as to identify those who did not ingest it. Based on the assessment of what had been eaten at the afternoon snack, it was recorded in a form whether the child had ingested all the supplement (the entire snack), at least half of it (half the snack), or none of it. Based on this information, acceptance of the supplement was assessed. On weekends, holidays,

and planned absences, to ensure the continuity of the intervention process, parents or guardians were given enough supplements for consumption during the period. They were instructed to offer them once per day, in a meal similar to that served at the daycare. DD was defined as the presence of three or more liquid or semi-liquid stools in 24 hours, lasting less than 14 days. The Brazilian Ministry of Health criteria was used for the diagnosis of ARI.21 Daily, prior to the start of the day at the daycare, the parents/guardians were questioned by the nursing staff, which were properly trained, as to the health status of the children. If any child was identified as having one of the outcomes (DD and/or ARI), he or she was referred for evaluation by the physicians of this project.


which suggested that the


which suggested that the Palbociclib chemical structure Dox localized in the endosomes [12]. Thus, these results suggested that AG73–Dox could enhance the intracellular uptake of Dox compared with Dox–PEG or AG73T–Dox. To examine the therapeutic efficiency of AG73–Dox, the cytotoxicity of Dox-encapsulating liposomes against cancer cells was evaluated using a WST assay. The cytotoxicity studies were initially performed with empty liposomes on the cancer cells to determine whether the liposomes themselves contributed to the cytotoxicity. The concentration of lipids tested was matched to the amount of lipids used in the drug-encapsulating formulations. At the concentration of lipids used, the cell viability was more than 80% that of the control (data not shown). These data indicated that the empty liposomes alone BLU9931 cost did not have a significant cytotoxicity. As shown in Fig. 5, the cell viability was dependent on the concentration of Dox. Furthermore, the cytotoxicity of AG73–Dox was more sensitive than that of Dox–PEG or AG73T–Dox against both types of cancer cells. In particular, the cytotoxicity of AG73–Dox against 293T-Syn2 was notably higher in comparison with that against colon26. This result may be due to the fact that the ability and

sensitivity of the intracellular uptake of free Dox into the cancer cells were distinct from those of 293T-Syn2 and colon26, as shown by confocal microscopy (Fig. 4), as it has been reported that colon26 exhibits a constitutive expression of mdr1a and mdr1b that encodes the drug efflux transporter P-glycoprotein [16]. To understand the drug delivery and release behaviors of AG73–Dox, the cells were washed after the treatment of AG73–Dox

and placed into fresh medium for further incubation at 37 °C. They were imaged using confocal microscopy at various time points after washing. As shown in Fig. 6, Dox was diffused through the 293T-Syn2 in a time-dependent manner and then was colocalized with the nuclei. This result could be due to the release Fossariinae of Dox from AG73–Dox inside endosomes with a lower pH. In addition, at 24 h post-incubation, we observed fragmentation of the nuclei, which is characteristic of apoptosis (indicated by white arrows in Fig. 6). Moreover, at 48 h post-incubation, we observed increased fragmentation of the nuclei. Therefore, these results suggested that after the cellular uptake of AG73–Dox, Dox was slowly released from AG73–Dox and was subsequently transferred to nuclei, which led to cytotoxicity (Fig. 5). Next, as AG73–Dox showed higher cellular uptake and cytotoxicity against cancer cells compared to Dox–PEG, the antitumor efficacy of AG73–Dox in vivo was evaluated in colon26 tumor-bearing mice at a dose of 2 mg Dox/kg body weight. As shown in Fig. 7, the Dox–PEG group had suppressed tumor growth compared with the free Dox group.

The bound crystallin was eluted with binding buffer containing im

The bound crystallin was eluted with binding buffer containing imidazole Y-27632 mw in a step-gradient manner (100–500 mM). The crystallin protein peaks eluted with 250–350 mM imidazole were combined and dialyzed against buffer A (50 mM Tris–HCl, pH 8.0,

1 mM dithiothreitol, 50 mM NaCl, 5 mM MgCl2, 10% glycerol), followed by freezing at −70°C in a minimal aliquot. Protein concentrations were determined using a Bio-Rad protein assay kit with a bovine serum albumin (BSA) standard. To obtain anti-grouper crystallin rabbit polyclonal antibody, the (His)6-tagged crystallin were used to immunize two New Zealand White rabbits with a primary injection emulsified in Freund’s incomplete adjuvant at 1 mg/mL, and 1 mL was injected subcutaneously into two rabbits. The rabbits were boosted after 4, 8, and 12 weeks with the same amount of antigen in the adjuvant. The crystallin antibody was obtained after clotting overnight at 4°C

followed by centrifugation at 1200 rpm [26]. Healthy grouper eyes were crushed in liquid nitrogen and homogenized SCR7 price with 10% trichloroacetic acid and 0.07% β-mercaptoethanol in cold acetone. After centrifugation, each pellet was washed twice with cold acetone. Supernatants were discarded and the pellets were vacuum-dried to a protein powder. The powder was solubilized in 1 mL of lysis buffer [9.5 M urea, 2% (w/v) CHAPS, 0.8% (w/v) Pharmalyte pH 3–10, and 1% (w/v) dithiothreitol]. For isoelectric focusing, 50 μL of each sample was mixed with 300 μL of a rehydration solution Verteporfin [8 M urea, 2% (w/v) CHAPS, 0.8% (w/v), 15 mM dithiothreitol, and 0.5% (v/v) (Immobilized pH Gradient (IPG)-buffer pH 3–10)] to produce a final protein amount of 150 μg per sample. Isoelectric

focusing was performed using immobilized pH gradient strips. The IPG strips (13 cm, pH 3–10 NL) were rehydrated overnight at 50 V and focused for 3 h at 8000 V at 20 °C under mineral oil. Strips were then incubated for 10 min in equilibration buffer I [6 M urea, 30% (w/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) dithiothreitol in 0.05 M Tris–HCl buffer pH 8.8] following by incubation in equilibration buffer II [6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS, and 4% (w/v) iodacetamide in 0.05 M Tris/HCl buffer pH 8.8]. After the equilibration steps the strips were transferred to a 22 cm×22 cm 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) system. Electrophoresis in the second dimension was performed at 150 V and 150 mA at 20 °C for approximately 18 h [27]. Total RNA was isolated from post-hatch day 40–45 orange-spotted grouper, Epinephelus coioides, following the single-step acid guanidinium thiocyanate-phenol-chlorofrom extraction method [28]. Extracted cellular total RNA (5 μg) as template was incubated at 42 °C for 60 min in 20 μL of 1X reaction buffer containing 2 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega), 0.25 mM dNTP and 4 μM oligo(dT)15 primer, and 0.