Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.

Genetic properties of endemic Chinese porcine epidemic diarrhea virus strains isolated since 2010.

Acute diarrhea outbreaks caused by porcine epidemic diarrhea virus (PEDV) have been observed in various pig-breeding provinces of China since December 2010. Endemic strains of PEDV were isolated from different areas, and the complete genome sequences of 10 isolates were determined. Our objective in this study was to genetically characterize current Chinese field isolates of PEDV to better understand their epidemiology and genetic diversity. Sequence analysis showed that 10 post-2010 isolates shared high homology with each other and were always clustered together with the virulent DR13 strains (South Korea) and/or one earlier Chinese strain, CH-S, in phylogenetic analysis. All post-2010 isolates possessed common sequence changes in each gene. Our results suggest that current Chinese PEDV isolates originated from either South Korean and/or Chinese ancestors that underwent some genetic variation, thereby forming a new PEDV genotype in China.

Antiviral effect of lithium chloride on porcine epidemic diarrhea virus in vitro.

Porcine epidemic diarrhea virus (PEDV), a member of the Coronaviridae family, causes acute diarrhea, vomiting, dehydration, and high mortality rates in neonatal piglets. Severe outbreaks of PEDV variants have re-emerged in Asia and North America since 2010, causing tremendous economic losses to the swine industry. The lack of effective therapeutic treatment promotes the research for new antivirals. Lithium chloride (LiCl) has been reported as a potential antiviral drug for certain viruses. In this study, the antiviral effect of LiCl on PEDV in Vero cells was evaluated. Real-time quantitative PCR and indirect immunofluorescence assay indicated that LiCl effectively inhibited the entry and replication of PEDV in Vero cells. The expression of viral RNA and protein of PEDV in Vero cells was suppressed in a dose-dependent manner by LiCl. Moreover, addition of LiCl inhibited both early and late cell apoptosis induced by PEDV. Our data implied that LiCl could be a potential antiviral drug against PEDV infection. Further studies are required to explore the antiviral effect of lithium chloride on PEDV infection in vivo.

QT hysteresis in long-QT syndrome children with exercise testing.

BACKGROUND: Congenital long QT syndrome (LQTS) is an inherited ion channel disorder resulting in abnormal cardiac repolarization that can cause syncope and sudden death associated with a prolonged rate-corrected QT interval and polymorphic ventricular tachycardia. Several studies in adults showed that LQTS patients have altered QT adaptation to heart rate changes compared with normal subjects which forming a "hysteresis loop" in the QT-circle length plot. This study was to observe the QT interval changing during exercise testing in children long QT syndrome (LQTS) patients, explore the new diagnosis methods of LQTS. METHODS: The subjects were divided into 3 groups according to 1993 LQTS diagnostic criteria. Group 1: LQTS group (n = 17) who scored > or = 4 points indicating definite LQTS. Group 2: Middle group (n = 16), patients who have prolonged QT interval but scored 1.5 to 3.5. Group 3: Normal control group (n = 18). The average age of all study population is (12.3 +/- 5.8) years. No case had beta-adrenergic antagonists administration before exercise testing. All subjects were underwent tread mill exercise testing and electrocardiograph in whole exercise testing and recovery were recorded. QT and heart rate changing during whole exercise testing period were recorded. DeltaQT, the QT interval at 1, 2, 4, 6 minutes into recovery subtract from the QT interval at a similar heart rate during exercise, were calculated. RESULTS: In all three groups, QT intervals were shortening with the increasing of heart rate, but QTc had no significant change. DeltaQT at 1 minute ((45 +/- 11) ms), 2 minutes ((37 +/- 15) ms), 4 minutes ((23 +/- 12) ms) into recovery in LQTS group were significantly greater than that of the other two groups (P < 0.05, P < 0.01, P < 0.01, respectively). There was no DeltaQT significant difference between middle group and normal control group at recovery time. During the recovery phase in LQTS group, the QT interval remained shortened despite a decelerating heart rate, forming a hysteresis "loop" in the curve relating the QT interval to the cycle length. CONCLUSIONS: In children LQTS patients, there is significant QT hysteresis loop in the relation of QT interval with heart rate during recovery of exercise testing, which could be useful to the early diagnosis for LQTS.

Effect of exogenously applied molybdenum on its absorption and nitrate metabolism in strawberry seedlings.

Molybdenum (Mo)-an essential element of plants-is involved in nitrogen (N) metabolism. Plants tend to accumulate more nitrate and show lower nitrogen use efficiency (NUE) under Mo-deficient conditions. Improving NUE in fruits reduces the negative effect of large applications of chemical fertilizer, but the mechanisms underlying how Mo enhances NUE remain unclear. We cultivated strawberry seedlings sprayed with 0, 67.5, 135, 168.75, or 202.5 g Mo·ha-1 in a non-soil culture system. The Mo concentration in every plant tissue analyzed increased gradually as Mo application level rose. Mo application affected iron, copper, and selenium adsorption in roots. Seedlings sprayed with 135 g Mo·ha-1 had a higher [15N] shoot:root (S:R) ratio, and 15NUE, and produced higher molybdate transporter type 1 (MOT1) expression levels in the roots and leaves. Seedlings sprayed with 135 g Mo·ha-1 also had relatively high nitrogen metabolic enzyme activities and up-regulated transcript levels of nitrate uptake genes (NRT1.1; NRT2.1) and nitrate-responsive genes. Furthermore, there was a significantly lower NO3- concentration in the leaves and roots, a higher NH4+ concentration in leaves, and a higher glutamine/glutamate (Gln/Glu) concentration at 135 g Mo·ha-1. Seedlings sprayed with 202.5 g Mo·ha-1 showed the opposite trend. Taken together, these results suggest that a 135 g Mo·ha-1 application was optimal because it enhanced NO3- transport from the roots to the shoots and increased NUE by mediating nitrogen metabolic enzyme activities, nitrate transport, and nitrate assimilation gene activities.

[The relation between en-dormancy and changes in two main electron transport pathways of nectarine (Prunus persica var. nectariana) buds].

Respiration rate of buds of Prunus persica var. nectariana Maxim. cv. Shuguang and Mayfire was studied during dormancy. The results showed that the bud respiration rate changed with the stage of dormancy for both cultivars. Respiration rate of buds of the controls decreased steadily before dormancy, while increased slowly from November 5th for Shuguang and October 29th for Mayfire, and then decreased sharply on December 17th at which dormancy was terminated. Cyanide (KCN) only partially inhibited respiration rate of dormant buds of nectarine during the whole period, and the trend of the buds respiration rate followed the same pattern as the control samples during the entire period. Cyanide inhibited the respiration rate of dormant nectarine buds to roughly the same degree. The effect of salicylhydroxamic acid (SHAM) on bud respiration rate varied with time. SHAM decreased bud respiration rate before dormancy and after the release of dormancy, but increased bud respiration rate during dormancy. SHAM increased bud respiration rate more than three times at the beginning of dormancy for all buds and the degree decreased gradually along with the dormant period until turn to decrease the respiration rate again at the end of dormancy. KCN plus SHAM had the same effect on bud respiration rate as SHAM acting alone. The time KCN plus SHAM began to increase bud respiration rate or terminated was about 7 d earlier than SHAM did, which was closely associated with the different stage of dormancy.

[Responses of chilling resistance to photoperiodic dormancy induction in peach leaf.] / æ¡ƒå¶ç‰‡æŠ—å†·æ€§å¯¹å ‰å‘¨æœŸè¯±å¯¼ä¼‘çœ çš„å“åº”.

Taking 6-year-old "Chunjie" peach as test material, and the peach in natural condition as the control, this paper investigated induced effects of long-day and short-day photoperiod on dormancy and responses of chilling resistance to photoperiodic induction during dormancy induction process. The results showed that the trees of long-day and short-day treatments could both enter dormancy induction under the gradually decreasing temperature. The long-day treatment was 1 week later than the control, while the short-day treatment was 1 week earlier. The total water content and free water content both decreased, and the bound water content and the ratio of bound water/total water increased with the development of dormancy. SOD and CAT activities changed as unimodal curve during dormancy induction, and the peak values appeared at the late stage of dormancy induction, POD activity decreased rapidly after the start of dormancy induction, and rebounded to form a small peak at the late stage of dormancy induction. The soluble protein content declined, proline and malonaldehyde (MDA) increased continuously, and the injury rate increased. Long-day could increase SOD and CAT activities and proline content, alleviate the decline of POD activity and soluble protein content, and reduce the growth rate of MDA and injury rate, which indicated leaf damage was lighter in long-day treatment than in the control. However, they changed differently under short-day treatment, especially the leaf injury rate was higher than the control, exhibiting a lower chilling resistance. Prolonging illumination was suggested to improve leaf chilling resistance in practical production if environmental temperature permitted.

The flavor and nutritional characteristic of four strawberry varieties cultured in soilless system.

Strawberry fruits (cv. Benihoppe, Tochiotome, Sachinoka, and Guimeiren) were harvested and evaluated the flavor and nutritional parameters. By principal component analysis and hierarchical clustering analysis, differences were observed based on the volatile compounds composition, sugar and acid concentration, sweetness, and total soluble sugars/total organic acids of the four varieties. A total of 37, 48, 65, and 74 volatile compounds were identified and determined in cv. Benihoppe, Tochiotome, Sachinoka, and Guimeiren strawberry fruits extracted by head-space solid-phase microextraction (HS-SPME), respectively. Esters significantly dominated the chemical composition of the four varieties. Furaneol was detected in cultivars of Sachinoka and Guimeiren, but mesifuran was only found in cv. Tochiotome. Tochiotome and Sachinoka showed higher content of linalool and (E)-nerolidol. Sachinoka showed the highest content of total sugars and total acids. Guimeiren showed higher sweetness index than the other three cultivars. Firmness of Tochiotome was highest among all the varieties. The highest total soluble solids TSS value was found in cv. Sachinoka, followed by the Guimeiren and Tochiotome varieties. Sachinoka had the highest titratable acidity TA value. The content of ascorbic acid (AsA) of cv. Tochiotome was higher than the others, but there was no significant difference in cultivars of Benihoppe, Tochiotome, and Sachinoka. Fructose and glucose were the major sugars in all cultivars. Citric acid was the major organic acid in cv. Tochiotome, cv. Sachinoka, and cv. Guimeiren. Tochiotome had higher ratios of TSS/TA and total sugars/total organic acids than others, arising from its lower acid content. The order of the comprehensive evaluation score was Sachinoka>Guimeiren>Tochiotome>Benihoppe.

Analysis of basic leucine zipper genes and their expression during bud dormancy in peach (Prunus persica).

Dormancy is a biological characteristic developed to resist the cold conditions in winter. The bZIP transcription factors are present exclusively in eukaryotes and have been identified and classified in many species. bZIP proteins are known to regulate numerous biological processes, however, the role of bZIP in bud dodormancy has not been studied extensively. In total, 50 PpbZIP transcription factor-encoding genes were identified and categorized them into 10 groups (A-I and S). Similar intron/exon structures, additional conserved motifs, and DNA-binding site specificity supported our classification scheme. Additionally, chromosomal distribution and collinearity analyses suggested that expansion of the PpbZIP transcription factor family was due to segment/chromosomal duplications. We also predicted the dimerization properties based on characteristic features of the leucine zipper and classified PpbZIP proteins into 23 subfamilies. Furthermore, qRT-PCR results indicated that PpbZIPs genes may be involved in regulating dormancy. The same gene of different species might participate in different regulating networks through interactions with specific partners. Our expression profiling results complemented the microarray data, suggesting that co-expression patterns of bZIP transcription factors during dormancy differed among deciduous fruit trees. Our findings further clarify the molecular characteristics of the PpbZIP transcription factor family, including potential gene functions during dormancy. This information may facilitate further research on the evolutionary history and biological functions of bZIP proteins in peach and other rosaceae plants.

Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry.

[Effect of Astragalus injection on plasma levels of apoptosis-related factors in aged patients with chronic heart failure].

OBJECTIVE: To investigate the effect of Astragalus injection (AI) on plasma levels of apoptosis-related factors in aged patients with chronic heart failure (CHF). METHODS: Seventy-two CHF patients were randomly divided into the AI group (36 cases) treated with AI and the control group (36 cases) treated with conventional treatment. Plasma levels of soluble Fas (sFas), soluble Fas ligand (sFasL), tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) were measured by enzyme-linked immunosorbent assays (ELISA) with monoclonal anti-human antibodies. Besides, New York Heart Association (NYHA) grading was assessed according to improved symptoms and left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV) and left ventricular ejection fraction (LVEF) were assessed by echocardiogram after 4 weeks of treatment. RESULTS: After 4 weeks of treatment, NYHA grading was markedly improved in the two groups, but it was significantly better in AI group than that in the control group (P < 0.05). As compared with the control group, sFas, sFasL, TNF-alpha and IL-6 in the AI group were obviously lower, the difference between the two groups and between before and after treatment were significant (P < 0.05 or P < 0.01). Moreover, in AI group, LVESV and LVEDV decreased, LVEF increased, which was significantly different than that before treatment (P < 0.05), respectively. CONCLUSION: AI could lower plasma levels of apoptosis-related factors, and is one of the effective drugs in improving cardiac function in the aged patients with CHF.

[Dual chamber pacing for vasovagal syncope].

OBJECTIVE: To investigate the effect of DDD pacing on vasovagal syncope (VVS). METHODS: Eleven patients diagnosed VVS (7 with heart inhibiton type, 4 with combination type)by tilt table test (TTT) in Shanghai Chest Hospital were selected into the study. They all were implanted DDD pacemaker in whom 9 with rate-drop response (RDR) function. All of them were prescribed beta-receptor blocker. The patients were followed-up at 1, 3, 6, 12, 24 months after being discharged. RESULTS: All the 11 patients with pacemaker implantation were free from syncope, in whom 4 still had pre-syncope discomfort, but the symptoms were slightly. Six patients took TTT again after pacemaker implantation. It was observed that pacing at an elevated rate started immediately when heart rate dropping to the lower threshold, and as a result syncope were prevented. CONCLUSION: In the VVS patients with recurrent syncope and severe heart inhibition, DDD pacing with RDR function could prevent from syncope efficiently.

[Relationships between H2O2 metabolism and Ca2+ transport in dormancy-breaking process of nectarine floral buds].

In order to explore regulatory function of H2O2 in bud dormancy release, main effects of three dormancy-breaking treatments (high temperature, hydrogen cyanamide and TDZ) on H2O2 metabolism were determined, and impacts of H2O2 on Ca2+ transport were tested using non-invasive micro-test technique. The results showed that both high temperature and hydrogen cyanamide induced H2O2 accumulation and CAT inhibition were efficient in breaking dormancy during deep dormancy period. However, TDZ showed little impacts on H2O2 metabolism and was much less effective in breaking dormancy. Dormant floral primordium was absorbing state to exogenous Ca2+ due to active calcium channels. The Ca2+ transport could be changed by exogenous H2O2. H2O2 of low concentration reduced the absorption rate of Ca2+, and at high concentration, it changed the Ca2+ transport direction from absorption to release. The results indicated that H2O2 signals were related with Ca2+ signals in dormant buds. Ca2+ signal regulated by H2O2 accumulation might be important in the dormancy-breaking signal transduction process induced by high temperature and hydrogen cyanamide.

[Clinical study on effect of Astragalus injection on left ventricular remodeling and left ventricular function in patients with acute myocardial infarction].

OBJECTIVE: To observe the effect of Astragalus injection (AI) on left ventricular remodeling and left ventricular function in patients with acute myocardial infarction (AMI). METHODS: AMI patients were randomly divided into the AI group (54 cases) treated with AI and the control group (54 cases) treated with conventional treatment. Left ventricular end-diastolic volume index (LVEDVI), left ventricular end-systolic volume index (LVESVI), anterior endocardial segmental length (ASL), posterior endocardial segmental length (PSL) were assessed by echocardiogram at the 1st and the 4th week of treatment; and the cardiac systolic and diastolic functions were detected by nuclide gating cardiac blood pool imaging on the 4th week. Besides, the plasmic levels of lipid peroxide (MDA), count of endothelial cell (CEC) and superoxide dismutase (SOD) were determined before and after treatment. RESULTS: At the 4th week, changes of LVEDVI, LVESVI and ASL in the AI group were not obvious, but increased significantly in the control group, the significant difference in comparison between the two groups was shown (P < 0.05). As compared with the control group, in the AI group, the left ventricular ejection fraction, left ventricular peak ejecting rate and left ventricular peak filling rate were higher, and the left ventricular time for peak filling rate was shorter, moreover, MDA and CEC were lower and SOD was higher. The difference between groups and between before and after treatment were significant (P < 0.01 or P < 0.05). CONCLUSION: AI is one of the effective drugs in reversal of left ventricular remodeling and improving left ventricular function in patients with AMI.

Endoplasmic reticulum proteins quality control and the unfolded protein response: the regulative mechanism of organisms against stress injuries.

The endoplasmic reticulum is the cellular compartment in which secretory proteins are synthesized and folded. Perturbations of endoplasmic reticulum homeostasis lead to the accumulation of unfolded proteins. The activation of the unfolded protein response during endoplasmic reticulum stress transmits information about the status of protein folding to the cytosol and nucleus. The unfolded protein response leads to the upregulation of genes encoding endoplasmic reticulum chaperones, attenuation of translation, and initiation of the endoplasmic reticulum quality control system to restore endoplasmic reticulum homeostasis. When the unfolded protein response is insufficient to rebuild the steady state in endoplasmic reticulum, the programmed cell death or apoptosis would be initiated, by triggering cell injuries, even to cell death through apoptosis signals. In this review, we briefly outline research on the chaperones and foldases conserved in eukaryotes and plants, and describe the general principles and mechanisms of the endoplasmic reticulum quality control and the unfolded protein response. We describe the current models for the molecular mechanism of the unfolded protein response in plants, and emphasize the role of inositol requiring enzyme-1-dependent network in the unfolded protein response. Finally, we give a general overview of the directions for future research on the unfolded protein response in plants and its role in the response to environmental stresses.

[Effects of photoperiod on photosynthesis and PSII performance in peach during dormancy induction].

Long-day and short-day photoperiods were set artificially, with natural condition as the control, to examine photosynthetic parameters and chlorophyll fluorescence transient kinetics of 6-year-old 'Chunjie' peach cultivar (Prunus persica cv. Chunjie), and to investigate the effects of photoperiod on photosynthesis of the northern deciduous fruit trees. The tree advanced into the dormancy induction period under the short-day condition, and delayed under the long-day condition. In the dormancy induction period, the leaf net photosynthetic rate (Pn) and stomatal conductance (g(s)) decreased, and the intercellular CO2 concentration (Ci) increased, suggesting that the Pn decreased because of non-stomatal limitation. Maximum quantum yield for primary photochemistry (PhiPo, or Fv/Fm), potential activity (Fv/Fo), probability that a trapped exciton moved an electron into the electron transport chain beyond Q(A)-(Psi(o)) and performance index on absorption basis (Pl(ABS)) decreased in the dormancy induction period, suggesting that the electron transport capacity of photosynthetic electron transport chain was inhibited, possibly due to the damage to downstream electron transport chain (after Q(A)- acceptor) of PS II reaction center. Long-day photoperiod improved Pn in the dormancy induction period, and reduced the range of decline in PI(ABS) and the injured degree of photosystem. Short-day photoperiod deepened and accelerated the damage to photosynthetic apparatus significantly. The induced effect of photoperiod was associated with the dormancy processes.

Roles of endoplasmic reticulum stress and unfolded protein response associated genes in seed stratification and bud endodormancy during chilling accumulation in Prunus persica.

Dormancy mechanisms in seeds and buds arrest growth until environmental conditions are optimal for development. A genotype-specific period of chilling is usually required to release dormancy, but the underlying molecular mechanisms are still not fully understood. To discover transcriptional pathways associated with dormancy release common to seed stratification and bud endodormancy, we explored the chilling-dependent expression of 11 genes involved in endoplasmic reticulum stress and the unfolded protein response signal pathways. We propose that endoplasmic reticulum stress and the unfolded protein response impact on seed as well as bud germination and development by chilling-dependent mechanisms. The emerging discovery of similarities between seed stratification and bud endodormancy status indicate that these two processes are probably regulated by common endoplasmic reticulum stress and unfolded protein response signalling pathways. Clarification of regulatory pathways common to both seed and bud dormancy may enhance understanding of the mechanisms underlying dormancy and breeding programs may benefit from earlier prediction of chilling requirements for uniform blooming of novel genotypes of deciduous fruit tree species.

Complete genome sequence of a variant porcine epidemic diarrhea virus strain isolated in central china.

We report here the complete genome sequence of the porcine epidemic diarrhea virus strain CH/ZMDZY/11 isolated from central China. Our data, together with sequence data of porcine epidemic diarrhea virus (PEDV) isolates from other parts in China, will help to understand better the epidemiology and genetic diversity of PEDV field isolates in China.

[Effects of high temperature and hydrogen cyanamide on dormant nectarine's floral bud respiratory metabolism].

Taking 3-year old potted 'Shuguang' nectarine (Prunus persica var. nectariana cv. Shuguang) as test material, this paper studied the effects of high temperature (50 degrees C, HT) and hydrogen cyanamide (HC) on the floral bud respiratory metabolism of the tree during its natural dormancy. Both HT and HC could break the natural dormancy of the tree, and lead to a significant decrease in the respiratory metabolism of floral buds for several hours. The main respiratory pathways, tricarboxylic acid cycle (TCA) and pentose phosphate pathway (PPP), were affected. For the buds not received dormancy-breaking treatments, both the TCA and the PPP decreased, while treating with HT and HC induced a rapid recovery of PPP after the early respiratory attenuation. HT also induced the recovery of TCA, but HC did not show this effect in 96 hours. Therefore, respiratory attenuation and the following PPP activation could be the important part in the floral bud respiratory mechanism of HT- and HC-induced dormancy release.