There, along 4 radii, the sapwood border was recorded in order to

There, along 4 radii, the sapwood border was recorded in order to calculate the sapwood area. In a first step we compared the predictive power of crown surface area (CSA), crown projection area (CPA), and basal area (BA) with that of other often used substitutes for leaf area, e.g., sapwood area at crown base (SAPcb), at breast height (SAPdbh), and at three tenth of the tree height (SAP03), for each stand separately by using log-linear regression models of the following form: equation(11) ln LA=a+b⋅ln Xln LA=a+b⋅ln Xwith LA the leaf area, a the intercept and b the coefficient for the respective

substitute variable X. The coefficients were estimated by log-linear regression in order to avoid heteroscedasticity. Further on, analysis of covariance was used to test Small molecule library solubility dmso if (i) the assumption of a common slope for all stands was justified, (ii) the relation between LA and X was proportional (b = 1), and (iii) the intercepts did not differ between the stands. Here should be mentioned that, if b = 1 the intercept a represents the proportionality factor of LA to X in the delogarithmized form of Eq. (11). In a next step the same procedures were used to test if the estimation of leaf area within the stands can be improved by including more variables into the above equation (11). Finally, we investigated if the leaf area models can be generalized by using tree and stand variables in the

mixed model equation (12). equation(12) ln(LA)=a+b⋅ln(X)+cT⋅STANDVAR+u+eln(LA)=a+b⋅ln(X)+cT⋅STANDVAR+u+eAdditionally N-acetylglucosamine-1-phosphate transferase to the variables and Doxorubicin in vivo coefficients of Eq. (11) following variables

were included: cT a vector of the coefficients of STANDVAR which is a vector of the stand variables ( Table 2) and a dummy variable for the thinning treatment. In the models the natural logarithm of each variable in Table 2 has been used. Finally, u, and e are the random effects of the stands and the trees, respectively. All statistical analysis were performed with Microsoft® Office Excel 2003 (2003) and the statistical software package SPSS for Windows – Rel. 13.0 (2004). The mixed models were analysed and parameterized with the procedure “MIXED” of SPSS for Windows. In all models only variables with significant coefficients (p ≤ 0.05) were included. For comparing the models and finding the final ones, following goodness of fit criteria were used: R2 for log-linear regression models with the same number of predictor variables, adjusted R2 for log-linear regression models with a different number of predictor variables, and the Akaike Information Criterion (AIC) for mixed models according to Demidenko (2004). Judged from the average R2 and the standard error of estimate of the natural logarithm of leaf area, the sapwood areas at crown base and at three tenth of the tree height are the best predictors for leaf area ( Table 3).

The same results were

The same results were this website obtained when

the treatments were performed at 6 h p.i. (data not shown). Nevertheless, Su et al. (2008) demonstrated that UL52 (β) and UL13 (γ) mRNA levels were inhibited by digitoxin. The β-actin (Fig. 5D), was used as an internal standard, and the expression level of its mRNA was not affected. Since the glucoevatromonoside did not inhibit mRNA expression, the next step of HSV replication to be evaluated was the protein synthesis. Likewise glucoevatromonoside, acyclovir, furosemide + potassium chloride (KCl) as well as their combinations were also tested. The relevance of intracellular K+ to the viral replication has already been reported (Hartley et al., 2006, Hartley et al., 1993 and Nagai et al., 1972). Furosemide is a loop diuretic also known as an inhibitor of Na+K+Cl− cotransport activity (NKCC), which prevents the entry of K+ into the cells (Russel, 2000), and has also shown antiherpes activity (Hartley et al., 2006). Thus, furosemide was investigated in order to check if it was able to reduce the viral protein levels. Likewise, the supplementation of Enzalutamide molecular weight K+ by adding KCl to the culture medium was also tested to confirm if this ion was important for the viral inhibition caused by glucoevatromonoside. Fig. 6 shows

the effects of these treatments on some HSV-1 proteins and on β-actin that is use as an internal standard. As shown in Fig. 6, β-actin cell protein was expressed in all treatments; consequently, the tested drugs were not cytotoxic. The individual treatments with glucoevatromonoside (lane 5) and acyclovir (lane 3) reduced the levels of all tested viral proteins, when compared to virus control. The glucoevatromonoside completely inhibited all viral protein synthesis, whereas acyclovir was able to inhibit completely only the UL42 and the gB proteins expression. The treatment with furosemide (lane 8) did not reduce the levels of any viral protein, when compared to viral control indicating that this drug Loperamide could not affect

this stage of HSV-1 replication or that the tested concentration was insufficient to induce protein synthesis inhibition. When the treatment was performed with glucoevatromonoside + acyclovir (lane 4) or glucoevatromonoside + furosemide (lane 9), a complete inhibition of protein levels was also detected, as well as when glucoevatromonoside (lane 5) was tested alone. Therefore, it was not possible to verify synergistic effects between glucoevatromonoside and acyclovir or glucoevatromonoside and furosemide. However, the inhibition caused by glucoevatromonoside on HSV protein levels could indicate that the Na+K+ATPase has been inhibited for this compound, as it is a cardenolide and its inhibition ability is well established. This inhibition could reduce the K+ concentration, and the HSV replication will not occur as usual.

This would provide an advantage since CPE-based TCID50 assays req

This would provide an advantage since CPE-based TCID50 assays require a relatively Ceritinib molecular weight long incubation to allow a clear distinction between infected and uninfected wells, particularly at higher dilutions. To this end, we performed TCID50 assays on a virus stock with known concentration, measured luciferase activity after 1, 2, 3, 4, 7 and 10 days to determine infected vs. uninfected wells, and then calculated a TCID50 titer based on these data (Fig. 2A). While at 1 and 2 days after infection

the calculated titer did not concur with the actual titer, after 3 days and at all later time points the luminescence-based TCID50 matched the actual titer as previously determined by CPE-based TCID50 analysis, indicating that this assay reliably allows rapid titration of rgEBOV-luc2 within 3 days, and is able to detect single infectious particles (as determined by conventional TCID50) with the same sensitivity as conventional TCID50 assays.

When analyzing the data from the TCID50 assay, we observed that the reporter signal declined about 1 log10 for each of the 10-fold dilution steps (data not shown), which lead us to explore the possibility of a linear relationship between reporter activity and input virus titer. To this end, we performed a 0.5 log10 dilution series of our virus stock, and determined reporter activity Enzalutamide cost for each Org 27569 sample 2 days post-infection (Fig. 2B). Our data show that there is a clear linear relation between the input titer and luciferase activity in the range between 102.7 TCID50/ml and 105.2 TCID50/ml. At higher titers we no longer observed an equivalent increase in reporter activity, most likely due to the fact that these

signals exceeded the linear dynamic range of the luminometer, whereas at lower titers we observed occasional samples that showed only background activity, suggesting that at these low concentration stochastic effects (i.e. an increasing probability that a sample of a highly diluted virus contains no infectious particles) start to significantly influence the outcome of the assay. Based on these findings, we developed a luminescence-based direct titration assay, in which the luminescence of an unknown sample is compared to a known standard dilution series. In order to increase the linear range of this assay, we measured both undiluted and 1000-fold diluted samples, to circumvent the fact that higher titers exceeded the linear dynamic range of the luminometer. To evaluate this assay, unknown samples were titered both using luminescence-based TCID50 assays and LBT assays, and both titration methods showed good concurrence (Fig. 2C), indicating that the LTB assay can be used to accurately titer rgEBOV-luc2 samples within 2 days. One obvious application for the rgEBOV-luc2 virus is in the screening for antivirals.

In Experiment 2 (eye abduction during retention and retrieval) th

In Experiment 2 (eye abduction during retention and retrieval) the only significant reduction in spatial span was observed when memoranda were presented in the Temporal 40° Abducted condition, with no comparable drop or trend in the 20° Abducted condition. Considering the further absence of any effect of abduction in Experiment 3 (abduction during retrieval only), we argue these results offer strong support for oculomotor involvement during the maintenance of directly-indicated spatial locations in working memory. As outlined in the introduction,

previous studies have struggled to reliably decouple attentional processes from oculomotor control processes in VSWM. We propose the present study is the first to unambiguously demonstrate Akt inhibitor that the oculomotor system contributes to the maintenance of spatial locations in working memory independently from any involvement of covert attention. This claim rests on the decoupling of oculomotor processes and attention that occurs when participants are placed in a 40° Abducted position and spatial memoranda are presented wholly in the temporal hemifield. Critically, participants can still see everything in the display and can covertly shift their attention within the

abducted hemifield, but are they physically unable find more to make any further eye-movements. It is only in this condition that spatial memory span is significantly reduced. This reduction cannot be explained by differences in the quality of sensory information between conditions, as previous studies have shown that eye-abduction does not reduce visual acuity (Ball et al., 2013 and Craighero et al., 2004). Given that our interpretation of these data rests on the decoupling of endogenous attention and saccade control, it is worth noting that there is substantial behavioral and neuropsychological evidence for this dissociation. For example, neuropsychological evidence supporting separation between the oculomotor system and attentional control comes from reported cases of patients with defective oculomotor control who are still Baricitinib able to covertly orient their attention (Gabay et al., 2010,

Rafal et al., 1988 and Smith et al., 2004). Smith et al. (2012) have also previously shown using an eye-abduction paradigm that numeric cues elicit covert endogenous shifts of attention to locations in the temporal hemispace even when they cannot become the goal of saccadic eye movements. In healthy participants, a series of studies by Klein and colleagues have shown that covert shifts of attention triggered by symbolic cues do not facilitate subsequent saccadic eye-movements (Hunt and Kingstone, 2003, Klein, 1980 and Klein and Pontefract, 1992). Furthermore, Belopolsky and Theeuwes, 2009b and Belopolsky and Theeuwes, 2012 have argued that endogenous attention associated with maintaining attention at a spatial location is independent from the preparation of an eye-movement to the same location.

One eye of each patient was selected randomly when both eyes were

One eye of each patient was selected randomly when both eyes were eligible. Glaucomatous

eyes were defined by a glaucoma specialist based on a glaucomatous visual field (VF) defect confirmed by two reliable VF tests and typical appearance of a glaucomatous optic nerve head including cup-to-disc ratio > 0.7, intereye cup asymmetry > 0.2, or neuroretinal rim notching, focal thinning, disc hemorrhage, or vertical elongation of the optic cup. Exclusion criteria included a history of any ocular surgery, evidence of acute or chronic infections, an inflammatory condition of the eye, a history Selleck Raf inhibitor of intolerance or hypersensitivity to any component of the study medications, women of childbearing age, and the presence of current punctal occlusion. Patients with media opacity or other diseases affecting the VF were also excluded. All participants were provided with the same artificial tears (1 mg sodium hyaluronate) to use as required during the study period, whereas individuals who were on medications for dry eye treatment other than artificial tears were excluded.

Participants were randomized to receive one of two treatment regimens for 8 weeks. The treatments were 1 g of KRG administered as two 500-mg powder capsules or placebo administered BKM120 cell line as two identically appearing capsules, taken three times daily in both groups. KRG powder was manufactured by the Korea Ginseng Corporation (Seoul, Republic of Korea) from roots of a 6-year-old KRG, Panax ginseng, harvested in the Republic of Korea. KRG was made by steaming fresh ginseng at 90–100°C for 3 hours and then drying at 50–80°C. KRG powder prepared from grinded red ginseng, and a capsule contained 500 mg of powder. KRG was analyzed by high-performance

liquid chromatography. KRG extract contained major ginsenoside-Rb1: 5.61 mg/g, -Rb2: 2.03 mg/g, -Rc: 2.20 mg/g, -Rd: 0.39 mg/g, -Re: 1.88 mg/g, -Rf: 0.89 mg/g, -Rg1: 3.06 mg/g, -Rg2s: 0.15 mg/g, -Rg3s: 0.17 mg/g, -Rg3r: 0.08 mg/g, and other minor ginsenosides. Dichloromethane dehalogenase Placebo capsules were also provided by the Korea Ginseng Corporation, and they were identical in size, weight, color, and taste. The participants were instructed to avoid taking other forms of KRG or any type of ginseng for the duration of the study. Group assignment of the participants was determined prior to the initiation of the study. Block randomization, which was generated by our institutional biostatistics department using a computer-generated random sequence, was used to randomize the participants. Study investigators, participants, and their caregivers were blinded through the provision of the medication as identically appearing capsules in boxes, with neither the investigator providing the medication nor the participants aware of the allocated treatment.

g , avalanches, debris flows, rock-falls, causing problems of par

g., avalanches, debris flows, rock-falls, causing problems of particular relevance for protection forests services ( Brang et al., 2006 and Beghin et al., 2010), including water supply. Moreover, large fires at the rural–urban interface involve civil protection issues ( Höchtl et al., 2005 and Ascoli and Bovio, 2010) and increasing costs due to post-fire restoration ( Beghin et al.,

2010, Wohlgemuth et al., 2010 and Ascoli et al., 2013a). On the contrary, the second generation of large fires, e.g., in the south-western Alps in 1989–90, characterized by mixed severity effects, i.e., a mosaic of low, intermediate and high severity stand replacing phases, might promote structural and species diversity in formerly exploited forests (e.g., chestnut and beech coppice woodlands, conifer

plantations) that are now no more managed, thus accelerating Smad inhibition the transition to alternative ecosystem states dominated by semi-natural ecological processes, e.g., Moretti et al. (2006), Maringer et al. (2012), Ascoli et al. (2013a), Fernandes et al. (2013), which is the aim of forest management in most unproductive forested areas of the Alps. Concerns about the long-term consequences of uncharacteristic fire regimes, and expected benefits from planning fire use, recently gave rise to a discussion about the suitability of implementing prescribed burning programmes in the Alpine environment (Lemonnier-Darcemont, 2003, Bernard-Laurent and Weber, 2007, Lyet et al., 2009, Valese et al., 2011b and Ascoli et al., 2013b). In particular, prescribed see more burning has been applied since the beginning of the 1980s over relatively large areas in the French Alps (e.g., ∼2000 ha yr−1 in the Department of Alpes Maritimes) both to regulate pastoral fire use (Fig. 8) and to abate fire risk by periodically reducing hazardous fuels in fuel Chlormezanone breaks strategically placed in the landscape (Fernandes et al., 2013). Long-term results (>20

yrs) of prescribed burning programmes in the French Alps have shown a shift from a fire regime characterized by uncontrolled fires, usually on high fire danger days, with a high inter-annual variability in overall burnt area, to a prescribed burning regime of lower severity and on a yearly planned area (Réseau Brûlage Dirigé, 2012). Experimental prescribed burning for similar objectives has also been carried out in the Italian Alps (Ascoli and Bovio, 2013), both to prevent the surreptitious use of fire by shepherds and to preserve habitats of interest included in the Habitat Directive (HD) 92/43/EEC, such as Calluna heathlands (cod. HD: 4030) in the western Alps ( Ascoli et al., 2013b), eastern sub-Mediterranean dry grasslands (Scorzoneretalia villosae – cod. HD: 62A0) and lowland hay meadows (Alopecurus pratensis, Sanguisorba officinalis – cod. HD: 6510) in the eastern Alps ( Valese et al., 2011b).

9% vs 5 6%, p = 0 5) The score discrimination assessed by the a

9% vs. 5.6%, p = 0.5). The score discrimination assessed by the area under the ROC curve was considered adequate both in the general population and in the subgroups of patients with and without CCC (Table 2). The score calibration assessed by the Hosmer-Lemeshow goodness-of-fit test was considered inadequate in the general population and in the subgroup of patients with CCC, but it was considered appropriate in the subgroup of patients without CCC (Table 2). The PIM2 score was chosen for evaluation, as it

is considered to be user-friendly and efficient, and it is public domain.10 The PIM2 is the updated version of the Pediatric Index of Mortality that was published in 1997, and has been used extensively since then.11 and 12 Since its publication in 2003, several studies have been published evaluating the score performance in populations and scenarios that were different Selleck ZVADFMK from those used in the score development study, with most studies showing

adequate score performance.10 In the present study, 83.9% selleck inhibitor of patients admitted to the PICU had one CCC, higher values than those found in a recent study that found a prevalence of 53% in a cohort that included patients from 54 PICUs in the United States.2 The high proportion of patients with CCCs that was observed in the present study and in the recent literature may be related to advances in medical care in recent years, especially in pediatric and neonatal intensive care, which have resulted in improvement in survival rates of patients with previously unmanageable diseases, leading to an increase of patients with CCCs, who have higher risk of hospitalization enough in the PICU than the general population.13 and 14 The overall mortality of the study population was 9.7%, consistent with the currently observed rates in the PICUs, which

range from 5-10%.15 There was a trend toward higher mortality in patients with CCCs, when compared with patients without CCCs, but without statistical significance. The observation that patients with chronic diseases have higher mortality rates when admitted to the PICU has been reported in recent literature. Wölfler et al., in a study evaluating the PIM2, observed a high mortality in patients with chronic diseases compared with the general population (15.6% vs. 5.2%). 16 Odetola et al. observed a significantly higher mortality in patients with comorbidities when compared to patients without comorbidities admitted to the PICUs in the United States in 1997 (12.5% vs. 8.6%) and in 2006 (10.8% vs. 7.8%). 17 Edwards et al. also observed a higher mortality rate among patients with CCCs, when compared to patients without CCCs admitted to PICUs in the United States in 2008 (3.9% vs. 2.2%). 2 However, this trend of increased mortality in patients with CCCs in the present study was not verified by the probability of death estimated by PIM2, which was similar in patients with and without CCCs (5.9% vs. 5.6%, p = 0.5) and may indicate poor score performance.

Several studies have identified the benefits of zinc are not limi

Several studies have identified the benefits of zinc are not limited to specific groups, and that intervention should include all children at risk, mainly those living in developing countries with high rates of

morbidity and mortality from infectious diseases.14In Canada, in the 1990s, with the objective of preventing micronutrient deficiencies PR171 in children, sprinkles were developed as a strategy for home fortification of foods. The sprinkles are sachets containing dried and predetermined micronutrients encapsulated by a thin lipid layer, which prevents interaction with other nutrients and confers an almost imperceptible level of food modification regarding color, flavor, and texture, facilitating Anti-infection Compound Library clinical trial their acceptance by children.15, 16 and 17 This study aimed to evaluate the incidence of DD and ARI in children receiving zinc supplementation combined with other micronutrients through the use of sprinkles, as well as their acceptance. Between August and November of 2009, a randomized, controlled, double-blinded study was conducted in a non-profit day care center located in a lower socioeconomic class neighborhood of Salvador, capital of the state of Bahia, Brazil. Data were collected by the staff of Centro de Pesquisa Fima Lifishitz – Universidade Federal da Bahia. The sprinkles were donated by Emory

University, Atlanta, USA. A sample was calculated to detect a 20% reduction in the occurrence of diarrheal episodes and respiratory infections compared to commonly observed rates over a period of three months in the children attending the studied day care center. Considering a beta error of 0.80 and an alpha error of 0.05, it was necessary to recruit at least 60 subjects

per group. The inclusion criteria were: almost healthy children aged 6 to 48 months, of both genders, whose parents or legal guardians consented to participation by signing an informed consent, who agreed not to offer any vitamin and/or mineral supplement during the study period, except for the sprinkles, which were sent home on weekends and holidays. The exclusion criteria were: participants with severe malnutrition (z-score W/H < -3), severe anemia (Hb < 9.0 mg/dL); any active severe illness requiring hospitalization, including DD or ARI; and history of underlying disease that could eventually interfere in the evaluation. Children enrolled in the institution considered eligible for the study were randomized into two groups: group A (test) and group B (control). Randomization was performed by rooms and nurseries, according to a computer-generated sequence. The study’s medical team was responsible for identifying eligible subjects, collecting the medical history, and performing the physical examination. Finally, blood was collected for complete blood count analysis, to eliminate subjects with severe anemia from the analysis.

For immunohistochemistry, the tumor section was blocked with 10%

For immunohistochemistry, the tumor section was blocked with 10% lapine serum in TBS for 30 min at 4 °C and incubated with anti-mouse CD31 antibody (BD Pharmingen) overnight at 4 °C. Subsequently, the section was incubated with Alexa Fluor 488 rabbit anti-rat IgG (Molecular Probes) for 1 h at room temperature. Each tumor and organ section was then mounted with VECTASHIELD Hard Set Medium with DAPI (VECTOR LABORATORIES, INC, USA) and fluorescently observed with BZ-8100 (KEYENCE, Japan). First, we sought to prepare Dox-encapsulating AG73 peptide-modified liposomes (AG73–Dox). As shown in Table 1, the mean particle diameter

of Dox–PEG, AG73–Dox, or AG73T–Dox ranged from 130 to 170 nm with a relatively narrow distribution (Fig. 1). The zeta potential of the Dox-encapsulating liposomes was slightly negative selleck compound in value. The efficiency for the remote loading of Dox into the liposomes was 90–95% with a drug/lipid ratio of 1:5 (molar ratio). The encapsulated efficiency also remained unchanged even in the case of peptide modification.

After one month of storage at 4 °C, the encapsulated efficiency was less than 5%. To examine the selective cellular uptake of Dox transfected by liposomes via the syndecan-2 receptor, the amount of Dox uptake into cancer cells including syndecan-2 overexpressing ZD1839 price cells was evaluated by flow cytometry analysis. As shown in Fig. 2, the cellular uptake of AG73–Dox on both 293T-Syn2 and colon26 was higher

than that of Dox–PEG or AG73T–Dox. The laminin-derived AG73 peptide is known as a ligand for syndecans, and it has been reported that syndecan-2 is highly expressed in various cancer cells [3], [5], [8], [24] and [26]. In addition, the AG73 peptide has been shown to bind to the heparin sulfate side chains of syndecan [8]. Therefore, to verify that AG73–Dox can bind to syndecan-2 on the surface of cancer cells, the cancer cells were treated with AG73–Dox and heparin (Fig. 3). Our data showed that the cellular uptake of AG73–Dox was down-regulated by the treatment with heparin. However, the cellular uptake of Dox–PEG or AG73T–Dox was not down-regulated by the treatment (data not shown). Therefore, these results suggested that AG73–Dox could effectively target cancer cells via the syndecan-2 receptor. To further elucidate the intracellular uptake of Dox, the intracellular localization of Dox VAV2 was evaluated after the treatment of cells with Dox–PEG, AG73–Dox, AG73T–Dox, or free Dox using confocal microscopy. As shown in Fig. 4, the cells treated with AG73–Dox showed a strong red fluorescence, whereas cells treated with Dox–PEG or AG73T–Dox showed a faint red fluorescence. The intracellular localization of Dox in the cells treated with AG73–Dox could be observed on the surface of the cell membrane and in the cytoplasm with a slight colocalization in the nuclei. However, these localizations after the treatment with AG73–Dox were blocked by heparin.

The dityrosine-conjugated Keyhole Limpet Hemocyanin (KLH; 500 μL,

The dityrosine-conjugated Keyhole Limpet Hemocyanin (KLH; 500 μL, 1 mg mL−1) was emulsified with an equal volume of complete Freund’s adjuvant to a final concentration of 0.5 mg mL−1,

and the solution was then intramuscularly injected into New Zealand White rabbits. The rabbits were boosted after 4, 8, and 12 weeks with the same amount of antigen in Freund’s incomplete adjuvant until learn more an adequate antibody generation was achieved. Immunohistochemical studies were performed as previously described [25]. Briefly, grouper at post-hatch day 40–45 were fixed in 10% formalin and embedded in paraffin following a routine procedure. Each 5μm-thick section was mounted on a polylysine-coated slide, deparaffinized in xylene, and rehydrated in descending grades (100–70%) of ethanol. Endogenous Tanespimycin cost peroxidase activity was blocked by 10 min incubation at room temperature with absolute methanol containing 3% H2O2. The sections were sequentially blocked with a power block solution (BioGenex, San Ramon, CA, USA), washed with phosphate-buffered saline (PBS), and incubated with polyclonal rabbit anti-grouper crystallin, dityrosine, or polyclonal rabbit anti-coat protein antibody (1:500 dilution) at 4 °C overnight. The sections were washed twice with PBS, incubated with secondary

antibody (Super Sensitive Polymer-HRP IHC; BioGenex, San Ramon, CA, USA) for 30 min at room temperature. Peroxidase activity was ascertained with 3,3′-diaminobenzidine (used as chromogen) for 10 min. The sections were counterstained with Harris hematoxylin for nuclei, dehydrated, and mounted. Negative controls were performed with Epothilone B (EPO906, Patupilone) preimmune rabbit serum and incubation with PBS instead of anti-grouper crystallin antibodies.

The sections were observed using an Axiovert 40 microscope (Carl Zeiss, Jena, Germany). The images were obtained with an SPOT RT3 camera (Diagnostic, Sterling Heights, MI, USA). cDNA from amino acids 1–231 for the mature crystallin of an orange-spotted grouper was cloned into a pGEM-T vector (Promega, Madison, WI, USA) and subcloned into the pET29a vector (Novagen, San Diego, CA, USA) between the EcoRI and XhoI sites to obtain pET29a-crystallin. The resulting expression vector encoded crystallin with a (His)6 and several extra amino acids at the N-terminus. This vector was transformed into the bacterial host, Escherichia coli BL21 (DE3), for expression driven by T7 polymerase. Induction by 0.5 mM isopropyl-β-thiogalactopyranoside was carried out at 37 °C for 3 h. After undergoing freezing and thawing once, cells were sonicated on ice, and the cleared lysate was obtained by centrifugation at 12,000 rpm for 15 min.