Drug effects are usually evaluated in animals housed under maximally standardized conditions. However, it is assumed that an enriched environment (EE) more closely resembles human conditions as compared to maximally standardized laboratory conditions. In the present study, we examined the acute cognition enhancing effects of vardenafil, a PDE5 inhibitor, which stimulates protein kinase G/CREB signaling in cells, in three different groups of male Wistar rats tested in an object recognition task (ORT). Rats were either housed solitarily (SOL) or socially (SOC) under standard conditions, or socially in an EE. Although EE animals remembered object information longer in the vehicle condition, vardenafil only improved object memory in SOL and SOC animals. While EE animals had a heavier dorsal hippocampus, we found no differences between experimental groups in total cell numbers in the dentate gyrus, CA2–3 or CA1. Neither were there any differences in markers for pre- and postsynaptic density. No changes in PDE5 mRNA- and protein expression levels were observed. Basal pCREB levels were increased in EE rats only, whereas β-catenin was not affected, suggesting specific activation of the MAP kinase signaling pathway and not the AKT pathway. A possible explanation for the inefficacy of vardenafil could be that CREB signaling is already optimally stimulated in the hippocampus of EE rats. Since previous data has shown that acute PDE5 inhibition does not improve memory performance in humans, the use of EE animals could be considered as a more valid model for testing cognition enhancing drugs. Many pharmacological animal studies have shown the beneficial effects that drugs can have on memory performance. However, the effects of these drugs are generally only modest, or even absent, when tested in humans. Many factors could explain this possible discrepancy in the effectiveness of drugs on memory performance From the early studies of Rosenzweig, it is known that environmental enrichment in rats has clear effects on brain development. The ‘EE’ brain is heavier and has more neuronal connections compared to brains of standard housed animals In the present study, we examined the efficacy of vardenafil in EE rats and rats that were kept either solitary (SOL) or socially (SOC) in a standard environment. First, we used the object recognition task (ORT) to investigate the effects of the 3 different housing conditions on memory performance, cf. Simpson and Kelly 2), and 22 were individually housed in standard cages (SOL). The 3 different housing conditions are presented in This study was approved by the local animal experimental committee of Maastricht University, the Dier Experimenten Commissie (DEC) Maastricht. Fifty-eight male Wistar rats were obtained from Harlan  just after weaning (four weeks of age). Eighteen were housed in an enriched environment , 18 were socially housed  in standard polycarbonate cages  and a test-trial (T2), separated by a retention interval. In T1, two identical objects were placed in the arena and in T2 the arena contained a novel (different) object and a copy from T1. In both trials, the animals were allowed to freely explore the objects and the arena for 3 min, during which the exploration of each object was scored manually on a personal computer. The relative discrimination index (d2) was used to assess memory performance. The d2 measure is calculated from the exploration of the objects in the second trial ((time novel object–time familiar object)/(time novel object+time familiar object)). For an elaborate description of the ORT setup, objects and procedures, see Akkerman et al. Before drug testing started, we examined the performance of the different groups at a 1 h and a 24 h retention interval, which typically relates to a good and poor performance, respectively Vardenafil  was freshly suspended in 1% tylose (methyl-cellulose) in milli-Q, on each experimental day. Vardenafil was tested at different doses: 0 (vehicle), 0.03, 0.1, 0.3 and 1 mg/kg. Administrations were always p.o. (injection volume 2 ml/kg) immediately after the first trial.Animals were sacrificed 2 weeks after the behavioral testing. Half of the animals in each housing group were killed by decapitation and the dorsal hippocampus was dissected (on ice), weighed, and immediately frozen in liquid nitrogen. Samples were then stored at −80°C and later used for qPCR and Western Blotting. We focused on the dorsal hippocampus as it is preferentially involved in memory processes, in contrast to the ventral hippocampus which is preferentially involved in emotional processes 2 gas and cut into coronal sections  at −25°C using a CM3050 cryostat . Sections were stored at −80°C until further processing for stereological measurements.The other half of the animals was killed by intracardial perfusion under lethal pentobarbital anesthesia . Ice cooled tyrode solution was used during the first minute of perfusion, followed by a fixative containing 4% paraformaldehyde, 15% picric acid, and 0.05% glutaraldehyde in phosphate buffer (0.1 M) Nissl staining was performed on one series of the coronal sections. Sections were put in a 1x TBS solution and mounted on coated glass slides. After mounting, the glass slides were left to air-dry overnight at room temperature. Sections were immersed in a 1% acetic acid, 0.04 M sodium acetate solution (solution A) for 20 min, followed by immersion in a 75% ethanol, 1.25% triton x-100 (Solution B) solution for 20 min, after which the sections were immersed in solution A again, for 20 min. Subsequently, sections were stained by immersion into solution A, containing 0.1% cresyl violet, for 10 min. After staining, sections were washed three times by immersion into solution A for 1 min and dehydrated via immersion into 100% ethanol for 1 min, thrice. Next, the sections were immersed into isopropanol, 2×5 min, followed by immersion in xylene, 2×5 min. Finally, Depex was used to close the sections and fix the cover glasses.The sections were investigated using design-based stereology Pde5 was performed to compare the levels of PDE5 mRNA between the different housing conditions. RNA isolation was performed by TRIzol reagent mediated extraction, according to the manufacturer’s instructions . 500 ng of total RNA was converted into cDNA using iScript Advanced cDNA Synthesis Kit for RT-qPCR . Quantitative PCR of Pde5 and the housekeeping gene Hprt were performed with the IQ SYBR Green Supermix Kit , 0.2 µl primer (10 µM) was added to 4.6 µl H2O and 10 µl SYBR Green per 5 µl cDNA sample (2 ng/µl). The following sequences  were used: Hprt, 5′TTGCTGGTGAAAAGGACCTC3’; Hprt-1, 5′ TCCACTTTCGCTGATGACAC 3′; Pde5, 5′ TGGTGACGTTAGAGGTCCTG3’; Pde5, 5′CGCTGTTTCCAGATCAGACA3’. Analysis of the data was performed via the delta-delta CT method and expression of Pde5 was normalized against Hprt.RNA in the dorsal hippocampus was isolated from half the animals of each housing group. Quantitative PCR (qPCR) of Western Blots were used to assess protein levels of BDNF, PDE5, pCREB, β-catenin, pGSK-3β, synaptophysin, PSD95, and ERK2 in the dorsal hippocampus of half of the animals of each housing condition. GAPDH and β-actin were used for normalization.One hundred to 130 mg dorsal hippocampus tissue was homogenized using a Mini-Bead Beater three times for 30 s in 1 ml ice-cold homogenizing buffer  and one protease inhibitor tablet/20 ml  were added to the homogenizing buffer ). Samples were centrifuged for 20 min at 4°C, 16000 g and the supernatant was stored at −80°C. Protein concentrations were determined using Bio-Rad Lowry Protein Assay .http://rsbweb.nih.gov/ij/), corrected for background signal and GAPDH signal.Brain homogenates in sample buffer were boiled for five minutes and then separated on a 10% or 14% (mBDNF and proBDNF) SDS–PAGE gel (30 µg per sample). Following electrophoresis, proteins were transferred to a nitrocellulose membrane , which was subsequently blocked with blocking buffer  for one hour at room temperature. Next, the membranes were incubated overnight at 4°C with the primary antibodies in blocking buffer: 1∶600 rabbit anti-BDNF ; 1∶500 rabbit anti-PDE5A1 , 1∶100 rabbit anti-pCREB , 1∶2000 mouse anti- β -catenin , 1∶1000 rabbit anti-pGSK-3β , 1∶1000 mouse anti-synaptophysin , 1∶2000 mouse anti-PSD95 , 1∶1000 rabbit anti-ERK2 . For normalization, 1∶2.000.000 mouse anti-Gapdh  or 1∶1000 mouse anti-β-actin  was used. After washing with phosphate-buffered saline-0.1%Tween (PBS-T), membranes were incubated for 1 hour at room temperature with the following secondary antibodies in blocking buffer: 1∶5000 goat anti-rabbit IRDye 800 (Li-Cor #926-32211) and 1∶5000 donkey anti-mouse IRDye 680 (Li-Cor #926-32222). Membranes were washed in PBS-T and fluorescent bands were visualized using an Odyssey Infrared Imaging System (Li-Cor). Intensities of specific bands were quantified using ImageJ  and proBDNF (16 kDa), as previously described The basic readout parameter of the ORT is the time spent exploring each object during a trial. Trials in which animals had explored both objects for less than 6 s in T1 or 9 s in T2 were excluded from analysis, since these data may not be reliable t-tests.The effects of retention delay on object discrimination performance (d2) were examined using a two way ANOVA  on all vehicle treated conditions. Housing effects on d2 were further evaluated for each retention interval separately, using one-way ANOVA followed by Tukey HSD post-hoc tests. Effects of vardenafil on object discrimination were assessed by performing one-way ANOVA’s on each housing condition separately, within the 24 h and 48 h retention intervals. Significant effects were further investigated with Tukey HSD post-hoc tests, comparing each different dose with the vehicle condition. In order to examine whether the animals actually showed object discrimination, the d2 of each individual condition was also compared to 0 using one-sample Differences in hippocampal weight, stereological- and biochemical measures were also analyzed using a one-way ANOVA and post-hoc Tukey HSD tests.F’s >10.17, p’s<0.001) and e2  = 17.94, p’s<0.001), as well as significant Interval by Housing interaction effects  >16.16, p’s<0.001). Therefore, one-way ANOVAs were performed on each separate retention interval.The exploration data of the animals is shown in F = 22.59, p<0.001) and e2  = 23.36, p<0.001). Tukey HSD post-hoc tests showed that SOL animals had a lower e1 and e2 compared to the SOC and EE animals. At the 24 h retention interval one-way ANOVA also revealed significant differences on e1  = 8.72, p<0.01) and e2  = 18.85, p<0.001) between the vehicle treated animals. Post-hoc analysis demonstrated that exploration levels (e1 and e2) were lower in vehicle treated EE animals as compared to the two other housing conditions. At the 48 h retention interval, no significant differences between the housing conditions were found  <1.38, n.s.).At the 1 h interval, one-way ANOVA revealed significant differences between vehicle treated housing conditions on e1  = 0.31 and F = 2.18, both n.s.). There were, on the other hand, significant overall effects of Housing on exploration  = 16.31, e2: F = 19.39, both p<0.001) and Treatment by Housing interaction effects  <1.71, n.s.). Post-hoc analysis with Tukey HSD tests showed that, on the 24 h retention interval, the overall exploration (both e1 and e2) of EE animals was lower compared to SOL and SOC animals.Housing×Treatment ANOVA at the 24 h retention interval showed that there were no overall Treatment effects on e1 or e2  = 6.30, p<0.05) and that no differences were present on e2  = 0.84, n.s.). In the EE animals, no effect was found of Treatment on e1  = 1.85, n.s.), however, there was an effect of Treatment on e2  = 4.54, p<0.01). Post-hoc analysis showed that EE animals treated with 0.03 and 0.1 mg/kg vardenafil had a lower e2 compared to vehicle treated EE animals. Finally, in SOC animals, no effects of vardenafil treatment on exploration levels were found .In the SOL animals, one-way ANOVA showed that animals treated with 1 mg/kg vardenafil had a higher e1 compared to vehicle treated animals  = 21.33, p<0.001). Post-hoc analysis revealed that the overall d2 value was higher at the 1 h interval compared to the 24 h and 48 h intervals. There was also a significant main effect of Housing on d2  = 3.05, p<0.05). Post-hoc analysis revealed that EE animals showed better overall object discrimination (d2 was higher) compared to SOC animals. No significant Interval by Housing interaction effects were found for d2  = 0.50, n.s).The discrimination performance of the animals is shown in F = 0.19, n.s.) and 48 h  <0.63, n.s.) retention intervals. However, there were significant differences between the d2 measures at the 24 h interval  = 3.49, p<0.05). Post-hoc analysis revealed that the d2 value of EE animals was higher at the 24 h retention interval, compared to SOC animals.One-way ANOVA’s were performed on the vehicle conditions of each separate retention interval. No significant d2 differences were found between the housing conditions at the 1 h , with the exception of the SOL animals  = 4.35, p<0.01). Post-hoc analysis showed that the d2 of SOL animals treated with 1 mg/kg vardenafil was increased compared to vehicle treated animals.In the 24 h interval, one-way ANOVAs within each separate Housing condition revealed no significant differences between d2 values of the different vardenafil treatments  = 12.13, p<0.001) and the SOC animals  = 7.92, p<0.01). Thus, after 48 h hours, object discrimination was still improved by the vardenafil doses of 1 mg/kg and 0.3 mg/kg in SOL and SOC animals, respectively. However, in EE animals, one-way ANOVA revealed no significant Treatment effect on discrimination performance (d2)  = 0.58, n.s.).In SOL and SOC animals, only the most effective vardenafil dose from the 24 h interval was re-tested in the 48 h retention interval. Multiple vardenafil doses were tested in EE animals. We analyzed the effects of vardenafil treatment within each housing condition using one-way ANOVA. This revealed an effect of Treatment on the discrimination (d2) performance in the SOL animals  = 4.36, p = 0.023). Post-hoc analysis revealed that EE animals had a heavier dorsal hippocampus, though only compared to SOC animals. However, there was also a strong trend (p = 0.052) towards a significant difference between EE and SOL animals  <1.50, n.s.) and cell density  <2.13, n.s.). The only significant difference found was on the volume of the DG region  = 19,26, p<0.001) which was larger in SOL animals, compared to both other groups.The DG, CA1 and CA2–3 of the dorsal hippocampus were compared between the different housing conditions . One-wayF = 1.41, n.s.; F = 0.21, n.s.; One-way ANOVA revealed no differences in PDE5 mRNA expression between the housing conditions in the dorsal hippocampus  = 0.74, n.s.; F = 1.99, n.s.; F  = 0.72, n.s.; F = 1.78, n.s.; F = 0.46, n.s.; F = 1.32, n.s.; F = 17.62, p<0.001; F = 3.79, p<0.05; Differences between the different housing conditions in dorsal hippocampal protein levels were investigated using Western Blots . One-wayBy analyzing the vehicle conditions, we examined how the different housing conditions affected baseline memory performance of rats in an ORT. One hour after learning, object memory appeared to be equal for all housing groups, as all groups discriminated equally well between the novel and familiar objects. However, after 24 h EE animals outperformed both other groups, which no longer showed any sign of object memory after this retention interval. These findings are in accordance with previous studies Multiple studies have produced evidence which suggests that vardenafil has memory enhancing properties in SOL and SOC rats After the 48 h retention interval, there was no more object discrimination in any of the vehicle treated housing conditions. Even after 48 h, memory performance in SOL (1 mg/kg) and SOC (0.3 mg/kg) animals was still fully enhanced by vardenafil. However, like in the 24 h retention interval, none of the tested vardenafil doses caused memory improvement in EE rats, ruling out the possibility of a ceiling effect. The lowered effective dose of vardenafil in SOC animals compared to SOL animals suggests that the effective dose in EE animals might be even lower and the lowest tested dose of 0.03 mg/kg may still have been excessive. On the other hand, considering the low brain-plasma ratio and IC50 of vardenafil, it is questionable as to whether there would still be enough vardenafil available in the brain to be biologically effective with a further lowering of the dose When animals display a statistically significant preference for the novel objects, it can be safely inferred that they were able to remember/recognize the sample objects. On the other hand, if animals fail to display a preference for the novel object, this may not necessarily indicate an absence of object memory as perceptual, attentional and motivational mechanisms also play an important role in the manifestation of a preference for the novel object The insensitivity of EE animals to vardenafil treatment may be related to changes in brain plasticity, influencing their memory performance. To assess brain plasticity, we analyzed several morphological and neurochemical signaling markers. Based on the enhanced ORT performance and the increased dorsal hippocampal weight in the EE rats, we argue that an effective EE procedure was used in the present study. However, we did not find any effect of Housing in the dorsal hippocampus on absolute cell numbers nor on the synaptic markers synaptophysin and PSD95. Neither did we observe a change in pro- or mature BDNF levels, a neurotrophic protein typically implicated in plastic changes. This appears to be in contrast with the findings of most studies on EE We also assessed two BDNF-related signaling pathways, the MAP kinase/CREB signaling pathway and the AKT/β-catenin pathway. ERK is a kinase, activated by BDNF signaling, which phosphorylates the transcription factor CREB. CREB is involved in the expression of genes important for plasticity (e.g. neurogenesis or synaptogenesis) as well as memory processes The additional exercise in EE has been shown to be the major stimulant of BDNF increases and neurogenic effects The hippocampus has been shown to contain PDE5 mRNA and protein, though at low levels and in different types of cells It has been suggested that the cognition-enhancing effects of vardenafil are related to activation of cellular cGMP/protein kinase G (PKG)/CREB pathways implicated in glutamatergic signaling Taken together, our study showed that baseline memory performance in the ORT was better in EE rats compared to SOL and SOC animals, i.e. they were able to remember object information over a longer period of time. However, no effects were found in the dorsal hippocampus on BDNF levels, number of cells and markers for synapses. Furthermore, the effectiveness of vardenafil diminished with the level of enrichment, yet the target of vardenafil, PDE5, was unaffected by EE. Therefore, the effect of EE on vardenafil efficacy is probably due to a more general mechanism, which means that other classes of drugs are likely to be affected as well. If EE reduces the efficacy of drugs via widespread, general molecular memory mechanisms, this might partly explain the discrepancy in success rate between pre-clinical drug studies in animals and studies performed in human subjects. EE may provide a more valid and predictive animal model for pre-clinical drug screening compared to highly impoverished standard housing conditions. Additionally, it would significantly increase animal welfare Figure S1Dorsal hippocampal weights. Dorsal hippocampal weights of the three experimental groups in grams (mean + SEM). Significant differences between groups are indicated with asterisks : p = 0.052; *: p<0.05).(TIF)Click here for additional data file.Figure S2Stereology. The volume (mm3) total number of cells and cell density (number of cells/mm3) in the DG, CA1 and CA2–3 sub-regions of dorsal hippocampus of solitary- (SOL), social- (SOC) and environmentally enriched (EE) animals. One-way ANOVA only revealed significant differences between the housing conditions in the DG, these are indicated with asterisks .(TIF)Click here for additional data file.Figure S3PDE5 mRNA and protein concentration in the dorsal hippocampus. (A) qPCR analysis showing mRNA levels of PDE5 of the three experimental conditions. Data was normalized relative to the housekeeping gene HGPRT. Analysis with one-way ANOVA revealed no significant differences between groups. (B) Western Blot analysis showing relative protein expression of PDE5, corrected for β-actin, of the three experimental groups in the dorsal hippocampus. Analysis with one-way ANOVA revealed no significant differences between groups. Bars represent the mean value (+ SEM) per group.(TIF)Click here for additional data file.Figure S4Protein levels of synaptophysin and PSD95 in the dorsal hippocampus. Western Blot analysis showing protein levels of synaptophysin (A) and PSD95 (B) of the three experimental conditions. Data was normalized relative to the housekeeping protein β-actin. Bars indicate mean (+ SEM) per group. One-way ANOVA revealed no statistical differences between the experimental groups.(TIF)Click here for additional data file.Figure S5Protein levels of proBDNF and mBDNF in the dorsal hippocampus. Western Blot analysis showing protein levels of proBDNF (A) and mBDNF (B) of the three experimental conditions. Data was normalized relative to the housekeeping protein β-actin. Bars indicate mean (+ SEM) per group. One-way ANOVA revealed no statistical differences between the experimental groups.(TIF)Click here for additional data file.Figure S6Protein levels of pGSK-3β and β-catenin in the dorsal hippocampus. Western Blot analysis showing protein levels of pGSK-3β (A), and β-catenin (B) of the three experimental groups. Data was normalized relative to the housekeeping protein β-actin. Bars indicate mean (+ SEM) per group. One-way ANOVA revealed no statistical differences between the experimental groups.(TIF)Click here for additional data file.Figure S7Protein levels of ERK2 and pCREB in the dorsal hippocampus. Western Blot analysis showing protein levels of ERK2 (A) and pCREB (B) of the three experimental conditions. Data was normalized relative to the housekeeping protein GAPDH. Bars indicate mean (+ SEM) per group. Significant differences between housing conditions are indicated with asterisks .(TIF)Click here for additional data file.


Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomal pairs. The phenotypic anomalies in such cases are attributed to gene disruption, superimposed cryptic imbalances in the genome, and/or position effects. We report a 14-year-old girl who presented with multiple congenital anomalies and developmental delay. Chromosome and FISH analysis indicated a highly complex chromosomal rearrangement involving three chromosomes , seven breakpoints as a result of one inversion, two insertions, and two translocations forming three derivative chromosomes. Additionally, chromosomal microarray study (CMA) revealed two submicroscopic deletions at 3p12.3 (467 kb) and 12q13.12 (442 kb). We postulate that microdeletion within the ROBO1 gene at 3p12.3 may have played a role in the patient’s developmental delay, since it has potential activity-dependent role in neurons. Additionally, factors other than genomic deletions such as loss of function or position effects may also contribute to the abnormal phenotype in our patient. Complex chromosomal rearrangements (CCRs) are balanced or unbalanced structural rearrangements involving three or more cytogenetic breakpoints on two or more chromosomes -5. The aChromosomal rearrangements may occur via several mechanisms , includiBalanced and unbalanced CCRs are associated with a significant risk of mental retardation and phenotypic anomalies attributable to gene disruption, cryptic imbalances and/or from position effects -18. FluoWe report a patient with multiple congenital anomalies and developmental delay who presented with a CCR involving three chromosomes 3, 7 and 12. G-banding, chromosomal microarray (CMA), and FISH were performed to clarify the nature of this complex abnormality.The patient was a 14-year-old female who presented clinically with developmental delay and multiple congenital anomalies including abnormal teeth and abnormal faces. No further clinical information was available regarding this patient.http://genome.ucsc.edu/; hg19 release). Similarly, the deletion at 12q13.12 is likely resulted from the translocation between chromosomes 3 and 12. This microdeletion extended from 49,988,357 to 50,429,906 bp and encompassed thirteen genes. CMA testing of the mother detected no dosage abnormality (gain or loss). The father was not available for follow up.Peripheral blood sample from the patient was referred to our laboratory for chromosome analysis. Metaphase chromosomes were prepared according to standard procedures -27. AnalThe combination of cytogenetic, FISH, and array analysis revealed a complex rearrangement with nine breakpoints:46,××, der (3) (3pter → 3p12::7q11.2 → 7q22::3q27 → 3p12::12q13 → 12q24.3::7p22 → 7pter), der (7) (12qter → 2q24.3::7p22 → 7q11.2::7q22 → 7qter), der (12) (12pter → 12q13::3q27 → 3qter).arr [hg19] 3p12.3 ×1, 12q13.12 ×1.http://www.ncbi.nlm.nih.gov/mapview/), the 3p12.3 deletion was within the ROBO1 gene, while the deletion at 12q13.12 involved thirteen genes . It is not clear if these copy number losses are de novo or paternally inherited, since the patient’s father was not available for follow up studies. The ROBO1 gene encodes a receptor that is a member of the neural cell adhesion molecule  family of receptors, acting as an axon guidance receptor. ROBO1 may play a role in neuronal development, and its disruption may predispose humans to developmental dyslexia [NCKAP5L (OMIM 615104), AQP2 (OMIM 107777) and AQP5 (OMIM 600442). NCKAP5L gene encodes a protein involved in proteolysis, GTPase-mediated signaling, cytoskeletal organization, and other pathways. Furthermore, neuronal depolarization regulates the transcription of these genes, suggesting potential activity-dependent roles in neurons [The disease-associated CCRs are frequently used to establish the genotype-phenotype relationship ,29. A codyslexia ,37. Amon neurons . MutatioDYX5 gene (OMIM 606896) at 3p12, which is associated with neurofunctional disorder, developmental dyslexia [MASP1 (OMIM 257920) located at the distal end (3q27) of the inversion 3 may have resulted in 3MC syndrome, which encompasses four rare autosomal recessive disorders previously designated as the Carnevale, Mingarelli, Malpuech, and Michels syndromes, respectively. The main features of these syndromes are facial dysmorphism, cleft lip and palate, postnatal growth deficiency, cognitive impairment, and craniosynostosis [Since the exact genomic location of at least five out of seven breakpoints in our patient is unknown, we can only speculate as to the disruption of genes resulting in loss of function ,39 or podyslexia , or speedyslexia . Furthernostosis . The othnostosis . Alternanostosis . AdditioROBO1 gene with potential activity-dependent roles in neurons may have played a role in our patient’s developmental delay. Furthermore, gene disruptions or position effects altering gene regulation by chromosomal rearrangements due to interference with some gene regulatory elements, may have also contributed to our patient’s abnormal phenotype.Two sub-microscopic deletions resulted from this apparently balanced CCR. Microdeletion within the These studies were performed on anonymized samples received in the clinical laboratory and thus were exempted from the requirement for consent by an opinion for the Western Institutional review Board.CCR: Complex chromosomal rearrangement; SNP-microarray: Single nucleotide polymorphism.The authors declare that they have no competing interests.MH, First authors; performed analysis, interpretation of the results, drafting and finalizing the manuscript. XY, participated in writing the FISH results. PC, RM and LR Performed the analysis and literature review. FZB reviewed the manuscript. All authors read and approved the final manuscript.


According to the results, root-zone heat stress inhibited photosynthesis mainly through decreasing Rubisco activity, while aerial heat stress mainly through inhibiting PSII acceptor side. The imbalance in light absorption and utilization resulted in accumulation of reactive oxygen species that caused damage to photosynthetic apparatus, forming a vicious cycle. On the contrary, grafting cucumber onto heat-tolerant luffa rootstock alleviated heat-induced photosynthetic inhibition and oxidative stress by maintaining higher root vitality, HSP70 accumulation, and antioxidant potential.Photosynthesis is one of the most thermo-sensitive processes in plants. Although the severity of heat stress could be attenuated by grafting approach, the primary damaged site of photosynthesis system under heat stress and the regulatory mechanism of rootstock-mediated heat tolerance are poorly understood. In the current study, cucumber plants grafted onto their own roots and heat-tolerant luffa roots were exposed to root-zone heat (25/40°C) and aerial heat (40/25°C) individually and in combination (40/40°C) to understand the response of photosynthetic process by investigating energy absorption and distribution, electron transport in photosystem (PS) II and I, and CO As sessile organisms, plants perceive various environmental stimuli that often appear as stress such as extreme temperature, drought, and salinity in their natural ecosystems. The episodes of heat, especially extremity and severity of heat events such as heat wave and/or hot days will become more prevalent in the future because of global climate change . Heat stA, PQ)  , carbon A, PQ) , and thyA, PQ) . However , H2O2, and ⋅OH. As strong oxidant, ROS could lead to the degradation of chlorophyll and decrease of photochemical efficiency of photosystem II (PSII)  . MoreoveI (PSII)  and inhimembrane . To defemembrane .2+, H2O2, phosphatidylinositol-4,5-bisphosphate (PIP2), and then activate different transcriptional regulators such as heat shock factor (HSF) via activation of several calcium-dependent protein kinases (CDPKs) and multiple mitogen-activated protein kinases (MAPKs)  . Finally (MAPKs) . Likewis (MAPKs) .Plant photosynthetic capacity is largely dependent on the vitality of roots that maintain the mineral and water states in leaves through proper acquisition of ion and water from root zone area. Importantly, uptake and translocation of water and nutrients, and related physiological and molecular metabolism in leaves are regulated by the root-sourced signals such as abscisic acid . When th2 assimilation, and root vitality, ROS-generating and -scavenging, and the expression of HSP70 under different aerial and root-zone temperature regimes were investigated. This study adds further evidence concerning primary limiting sites due to heat stress and also provides novel insights into the mechanism of luffa rootstock-mediated heat stress mitigation in cucumber.Cucumber is one of the important horticultural crops, highly sensitive to heat stress. Previously, we observed that the photosynthetic rate of cucumber was significantly decreased under heat stress; however, such inhibition could be alleviated by using luffa as rootstock ,b. In thCucumis sativus L., cv. Jinyan No. 4, Cs) grafted onto cucumber and luffa , were used. For rootstocks, seeds of cucumber and luffa were sown directly into trays filled with a mixture of peat/vermiculite , and for scion, cucumber seeds were sown 7 days later. When the cotyledon of the cucumber sown for scion expanded, a ‘top approach grafting’ was performed  and PPFD of 600 μmol m-2 s-1. The plants were watered daily and fertilized with Hoagland’s nutrient solution every 2 days. Upon the appearance of the first true fully expanded leaves, a group of eight seedlings was transplanted into a container (40 cm × 25 cm × 15 cm) filled with Hoagland’s nutrient solution.Two different grafted plants, cucumber , aerial heat (40/25°C), and the combined heat (40/40°C) as described previously , inhibitor of HSP70 gene expression, for 12 h before imposition of heat treatment (40/40°C). KNK437 and Quercetin were dissolved in DMSO to prepare a 100 mM stock solution and diluted to 200 μM with Milli-Q water. The corresponding amount of DMSO in the same buffer was used as control. After exposure to a 48 h heat treatment, the chlorophyll fluorescence was measured in the third leaf from the bottom.To study the role of HSP70 in luffa-induced heat tolerance, -1 and an ambient CO2 concentration of 380 μmol mol-1. A PPFD of 1000 μmol m-2s-1 was provided by a mixture of red and blue LEDs.The gas exchange of attached leaves was measured using an infrared gas analyser, Li-Cor-6400 , at a temperature of 25°C, a relative humidity of 85%, a cuvette air flow rate of 500 ml minLeaf chlorophyll (chl a and chl b) were extracted in 80% acetone and their contents were determined according to the method of F0, the minimum fluorescence, by a measuring light at low intensity (<0.1 μmol m-2 s-1). A saturating pulse  was then applied to detect Fm (the maximum fluorescence after dark adaptation). The maximal change of P700 signal (Pm) was measured through application of a saturation pulse  after illumination of far-red light for 10 s. A saturating pulse with duration of 300 ms was applied every 20 s after the onset of the actinic light to determine the maximum fluorescence signal (Fm′) and maximum P700+ signal (Pm′) under the actinic light (27 μmol photons m-2 s-1). The slow induction curve was recorded for 300 s to achieve the steady state of the photosynthetic apparatus, and then the actinic light was turned off. Data  derived after the final saturating pulse (Table 1), were recorded and used for analysis of activities of PSI and PSII. According to Pm′-P)/Pm, Y(NA) = (Pm-Pm′)/Pm, Y(ND) = (P-Po)/Pm, ETR(I) = 0.5×Y(I)×PAR×0.84μmol×m-2×s-1. According to Fm′-F)/Fm′, ETR(II) = 0.5×Y(II)×PAR×0.84 μmol×m-2×s-1, qP = (Fm′/-F)/(Fm′-Fo′), qL = qP(Fo′/F′), NPQ = (Fm-Fm′)/Fm′, Y(NO) = 1/NPQ + 1 + qL ((Fm/Fo)-1), Y(NPQ) = 1-Y(II)-Y(NO).Quantum yield of PSI [Y(I)] and PSII [Y(II)] of grafted plants was measured simultaneously with a Dual-PAM-100 system  on the measure mode of Fluo + P700 . All seeF0  was measured at 20 μs, when all PSII RCs are open; F300μs is the fluorescence at 300 μs; Fj and Fi are the fluorescence intensity at step J (2 ms) and at step I (30 ms), respectively; Fm  is the peak of fluorescence at the step P when all RCs are closed; Area is total complementary area between fluorescence induction curve. Selected parameters quantifying PSII behavior were calculated from the original data following the formulae as shown in Supplementary Table S1 . All samples were adapted to the dark for 15 min. The chlorophyll fluorescence transients were recorded up to 1 s on a logarithmic time-scale. Data were acquired every 20 μs. The polyphasic fluorescence induction kinetics was analyzed according to the JIP test that reflected valuable information on photosystem II (PSII) function . In the Table S1 .2, 12.5% (v/v) glycerin, 10% polyvinylpyrrolidone (PVP), and 10 mM β-mercaptoethanol. The homogenates were centrifuged at 15,000 g for 15 min at 4°C.The frozen leaf samples were homogenized using a chilled pestle and mortar with cooled extraction buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM MgCl2, and 10 mM NaHCO3. The initial and total Rubisco activity measurements were then carried out in a 0.1 ml reaction medium containing 5 mM HEPES-NaOH (pH 8.0), 1 mM NaHCO3, 2 mM MgCl2, 0.25 mM dithiothreitol (DTT), 0.1 mM EDTA, 1 U creatine phosphokinase, 1 U 3-phosphoglyceric phosphokinase, 1 U glyceraldehyde 3-phosphate dehydrogenase, 0.5 mM ATP, 0.015 mM NADH2, 0.5 mM phosphocreatine, 0.06 mM RuBP, and 10 μl of extract. The change in absorbance at 340 nm was monitored for 90 s.Rubisco activity was measured spectrophotometrically by coupling 3-phosphoglyceric acid formation with NADH oxidation at 25°C according to + determination. Each homogenate was heated in a boiling water bath for 5 min, cooled in an ice bath, then centrifuged at 10,000 g at 4°C for 10 min. Supernatants were neutralized with respectively 0.2 M NaOH or HCI and centrifuged at 10,000 g at 4°C for 10 min. Final supernatants were transferred to separate tubes and kept on ice for coenzyme assay.The fresh leaf samples (0.3 g) were homogenized using a chilled pestle and mortar with 3.0 ml of 0.2 M HCI for NADPH determination or 3.0 ml of 0.2 M NaOH for NADPg at 4°C for 5 min, the supernatants were carefully removed and the pellet was solubilized in 1 ml 96% ethanol. Finally, absorbance at 570 nm was determined.Enzyme cycling assays of NADP(H) was performed in low light with MTT as the terminal electron acceptor according to the method described by 2HPO4–KH2PO4 at 37°C for 3 h and 0.05% (v/v) Tween 20 were added and the samples were vacuum-infiltered for 15 min. After incubation, the root pieces were washed twice with 5 ml of distilled water. Then, the samples were extracted in 95% (v/v) ethanol at 80°C for 15 min. Absorption at 520 nm was measured by Multimode Plate Reader Label-free System .Root vitality was determined by triphenyltetrazolium chloride (TTC) method according to leaf) was determined on intact excised leaves from plants with a dew point potential meter .Leaf water potential  staining. Leaf disks (1.5 cm in diameter) were directly infiltrated with 0.1 mg ml-1 NBT in 25 mM K-HEPES buffer (pH7.8) and incubated at 25°C in the dark for 4 h. Then, the leaf disks were rinsed in 95% (v/v) ethanol for 10 min at 95°C and photographed with a digital camera .The 2O2, leaves were detached and placed in a solution containing 1 mg ml-1 3,3′-diaminobenzidine  for 6 h after a light vacuum infiltration. Leaf disks were boiled in 95% (v/v) ethanol for 10 min, stored in 50% glycerol, and then photographed with a digital camera  or an Olympus motorized system microscope  at 400 magnifications  contents were determined according to For the measurement of GSH and GSSG, plant leaf tissue (0.3 g) was homogenized in 2 mL of 5% metaphosphoric acid containing 2 mM EDTA and centrifuged at 4°C for 15 min at 12,000 2O2 decomposition at A240 according to Antioxidant enzyme activities in leaves were assayed with spectrophotometric methods. The protein content was determined according to the 3VO4, 10 mM NaF, 50 mM b-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 5 mg mL-1 antipain, 5 mg mL-1 aprotinin, 5 mg mL-1 leupeptin, 10% glycerol and 7.5% polyvinylpolypyrrolidone (PVP)]. After centrifugation at 13,000 g for 20 min, the supernatants were transferred into clean tubes. The protein concentrations of the extracts were determined using the Bio-Rad protein assay kit using bovine serum albumin (BSA) as the standard. Denatured protein extracts were separated by 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and the proteins were then transferred to a nitrocellulose membrane (Bio-Rad) by semidry electroblotting. The membrane was blocked for 2 h in TBS buffer  with 5% BSA at room temperature and then incubated for 1 h in TBS buffer (with BSA) containing the Rabbit polyclonal antibody HSP70 . After incubation with HRP (horseradish peroxidase)-linked antibody , the complexes on the blot were visualized using an enhanced chemiluminescence kit (Perkin Elmer) following the manufacturer’s instructions. The quantitation of HSP70 was performed by ImageJ.Heat Shock Protein 70 was extracted as described by RBCL , RBCS , RCA , and the actin gene  was used as an internal control. The qRT-PCR assays were performed using an iCycleriQTM Real-time PCR Detection System . PCRs were performed using the SYBR Green PCR Master Mix . The PCR conditions consisted of denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s and extension at 72°C for 30 s. The quantification of mRNA levels was based on the method of Total RNA was extracted from grafted cucumber leaves using an RNA extraction kit  according to the supplier’s instructions. DNA contamination was removed using a purifying column. One microgram of total RNA was reverse-transcribed using the ReverTra Ace qPCR RT Kit  following the supplier’s recommendations. The gene-specific primers for qRT-PCR were designed based on their cDNA sequences, as follows: P < 0.05) between treatments were determined using Tukey’s test (Table 2).The experiment was a completely randomized design with four replications. Each replicate contained at least 10 plants. Two-way analysis of variance (ANOVA) was used to test for significance, and significant differences  and aerial heat (40/25°C) individually and in combination  significantly decreased the satA and PIABS in self-grafted (Cs/Cs) cucumber plants. Most pronounced decrease was observed in the 40/40°C treatment, followed by 25/40°C and 40/25°C. Significantly, the leaves of reciprocally grafted cucumber (Cs/Lc) plants where luffa was used as rootstock showed much higher satA and PIABS values under heat stress compared to the Cs/Cs plants. For example, heat stress decreased the satA by 47.0, 33.4, and 90.9%, and PIABS by 66.9, 86.7, and 93.4% following exposure of Cs/Cs plants to 25/40, 40/25, and 40/40°C for 48 h, respectively. On the other hand, decreases in the Cs/Lc plants for the satA were only 24.4%, 14.1% and 54.5%, while for PIABS were 36.3, 37.9, and 76.4%, respectively under the same treatments.The satA and PIABS following heat stress, we firstly analyzed the light energy absorption in Cs/Cs and Cs/Lc plants. Unlike satA and PIABS, the ABS/CSm and chlorophyll contents in Cs/Cs and Cs/Lc plants remained almost unchanged following 25/40°C, 40/25°C, and 40/40°C temperature regimes (Table 1). It is well known that part of the absorbed energy is used to drive photosynthesis (photochemistry). However, the flux channelled to the RC reducing QA to QA-  and the flux transported beyond QA- which is re-oxidized to QA  were significantly decreased by root-zone or/and aerial heat stress in both grafted plants, resulting in the decreases of φP0 and φE0. Lower TR/CSm, ET/CSm, φP0, φE0, Y(II), ETRII, Y(I), and ETR(I) were observed in Cs/Cs plants after exposure to aerial heat than those in Cs/Cs plants after exposure to root-zone heat. Lc as rootstock significantly alleviated the decreases in most of these parameters. For example, following 25/40°C, 40/25°C and 40/40°C treatments, ET/CSm and Y(II) in Cs/Cs plants were decreased by 33.3, 50.0, 63.0 and 25.6, 51.2%, 67.4, respectively, while those in Cs/Lc plants were decreased by 22.8, 26.3, 43.9 and 18.2, 27.3, 50.0%, respectively.To explore the reason of drastic reduction in Cs/Lc plants. Furthermore, Y(NO) in PSII and Y(NA) in PSI represent the extent of optical damage  by root-zone heat]. However, Lc as rootstock inhibited such increases in Y(NO) and Y(NA). Over all, heat stress caused minor damage and consequently lower decrease in electron transport of PSI than those of PSII due to the higher light protection capacity of PSI to a certain degree (Table 1). Moreover, the protection of PSII and PSI by Lc rootstock was partially associated with Lc-induced light protection capacity.The Y(NPQ) in PSII and Y(ND) in PSI represent heat dissipation related to light protection capacity of plants. After all three heat treatments, Y(NPQ) in both grafted plants remained almost unchanged, while Y(ND) was increased significantly, and relatively greater increase was noticed in l damage . Both Y is a proxy for the redox state of QA which is the primary electron acceptor after PSII, Sm reflects the size of PQ pools, and oxidized nicotin amide adenine (NADP+) is the terminal electron acceptor (m and Y(II), OEC, RC/CSm, qP, and Sm in Cs/Cs plants were significantly decreased after heat stress, while root-zone heat caused drastic decreases than aerial heat . However, Lc as rootstock alleviated heat-induced decrease of these parameters. NADP+ was significantly decreased in Cs/Cs plants, while the reduced nicotinamide adenine (NADPH) was increased by root-zone heat . However, the sum of NADPH + NADP+ was not altered by root zone or/and aerial heat treatments. Accordingly, the ratio of NADP+/NADPH was decreased significantly in Cs/Cs plants after root-zone heat stress. Lc as rootstock reversed the decrease in NADP+ content and ratio of NADP+/NADPH under root zone heat stress. Notably, the content of NADP+ or/and NADPH and the ratio of NADP+/NADPH were almost unchanged after 40/25°C and 40/40°C heat treatments in both Cs/Cs and Cs/Lc plants.Oxygen-evolving complex is the primary electron donor, RC/CSacceptor . Like ET+ is partly dependent on the rate of Calvin–Benson Cycle. Therefore, we analyzed the activity and transcript levels of Rubisco, a key rate-limiting enzyme in Calvin-Benson Cycle. At the optimal growth temperature, i.e., 25/25°C, luffa rootstock didn’t affect RBCL and RCA expression significantly . However, heat stress upregulated RBCL, RBCS, and RCA expression in Cs/Lc plants, except for RCBS in 25/40°C and RCA in 25/40°C and 40/40°C treatments. Notably, Cs/Lc plants showed relatively higher RBCL, RBCS, and RCA transcripts abundance than Cs/Cs plants after 25/40°C, 40/25°C, and 40/40 °C heat treatments for 48 h. Total and initial Rubisco activities and Rubisco activation rate in Cs/Cs plants were decreased significantly after exposure to heat stress. However, luffa rootstock alleviated heat stress-induced decreases of Rubisco activities and activation rate. Ultimately, total and initial Rubisco activities and Rubisco activation rate in Cs/Lc plants were higher than those in Cs/Cs plants after exposure to root-zone or/and aerial heat.The regeneration rate of NADP2 concentration is an important factor that impacts Rubisco activity and photosynthetic rate. Ci in Cs/Cs plants was not affected by aerial heat, but was significantly decreased by root-zone heat and combined heat treatments . Ci is dependent on the balance between the CO2 assimilation and CO2 intake through stomata. Similarly, Gs was significantly decreased by root-zone as well as combined heat. As stomatal closure is closely associated with the leaf water potential (Ψleaf) which is basically affected by water supply from root, we further analyzed changes in Ψleaf and root vitality. Ψleaf and root vitality showed similar change trends as like as Gs and Ci. However, root-zone heat- and combined heat-induced decreases in Ci, Gs, Ψleaf, and root vitality were alleviated when cucumber was grafted onto Lc rootstock. In both grafted plants, Ci, Ψleaf, and root vitality were almost unchanged, but Gs was significantly increased after exposure to aerial heat.Intercellular CO) and hydrogen peroxide (H2O2). After in situ leaf staining with NBT and 3,3′-diaminobenzidine(DAB), we found intensive accumulation of  and H2O2 in Cs/Cs plants following heat treatments, where highest ROS accumulation was observed under combined heat treatment followed by root-zone heat and aerial heat . However, Lc as rootstock significantly reduced heat-induced accumulation of  and H2O2. Similarly, relative electrical conductivity (REC) which reflected damage of cell membrane was obviously increased by heat-induced ROS in Cs/Cs plants, and this increase was attenuated by Lc rootstock.Inhibition of photosynthesis could induce generation of ROS such as superoxide . However, luffa rootstock alleviated the decreases or improved the increases of most enzyme activities by heat stress. As a result, the activities of most of these enzymes in Cs/Lc plants were significantly higher than those in Cs/Cs plants after different heat treatments. The content of GSH in Cs/Cs plants was increased by aerial heat but unchanged by root-zone and overall heat, while the GSSG content was increased by overall heat but unchanged by root-zone or aerial heat. Finally, the ratio of GSH/GSSG in Cs/Cs plants was increased by aerial heat, decreased by overall heat, but unchanged by oot-zone heat. With few exception, the contents GSH and GSSG in Cs/Lc plants were significantly higher and lower, respectively than those in Cs/Cs plants, after exposure to root-zone or/and aerial heat treatment. Thus, Cs/Lc plants had higher ratio of GSH/GSSG than Cs/Cs plants after heat treatments.Through evolution, plants have developed a set of antioxidant systems to remove excessive ROS that are generated by environmental stresses. The activities of major antioxidant enzymes such as SOD, CAT, GR, and APX in Cs/Cs plants was almost unchanged by 25/40°C and 40/25°C, but was increased by 40/40°C. HSP70 in Cs/Lc plants was increased by all heat treatments to a higher level, compared to that in Cs/Cs plants . Finally, the levels of HSP70 in Cs/Lc plants were increased by 1.27, 1.39, and 1.38 fold compared with those in Cs/Cs control, after 25/40°C, 40/25°C, and 40/40°C heat treatment for 48h, respectively. However, pretreatment with Q and K which are inhibitors of HSP70 expression, aggravated heat-induced damages as evident by drastic reduction in the Fv/Fm and PSII in Cs/Cs and Cs/Lc, indicating that inhibition of HSP70 reduced the tolerance of grafted plants to heat stress and thus HSP70 might play a crucial role in Lc rootstock-mediated heat stress tolerance .HSP70, an important molecular chaperone in heat shock proteins family, plays important roles in protecting proteins from heat-induced damage . HSP70 psatA and photosynthetic performance (PIABS) in Cs/Cs plants were significantly decreased by root-zone heat (25/40°C), aerial heat (40/25°C), and combined heat (40/40°C) treatments. However, use of luffa, a heat-tolerant species, as rootstock alleviated such photosynthetic inhibition . Consistently, grafted tomato plants with heat tolerant tomato or eggplant cultivar as rootsstock showed higher photosynthetic performance based on the higher values of chlorophyll fluorescence under heat stress conditions .Photon flux is absorbed by the antenna pigments that excite chlorophyll, and part of the excitation energy (the other is dissipated as heat and fluorescence emission) is converted to redox energy via electron transport and leads to ultimate COfixation . A numbefixation . Both phfixation . In the A fixation and stability of PSII , and these indicated severe depression in electron transport beyond QA. Thus, other than PSII donor side, acceptor side maybe the primary limitation site of photosynthesis system by aerial heat stress. The underlying reason is that aerial heat stress was carried out under growth light not in dark, and the block of electron transport from PSII may be a self-protection way by reducing the possibility of PSI photoinhibition , which inhibited Calvin cycle capacity by depressing Rubisco  activation state and activity and then reduced the rate of NADP+ regeneration. It has been well documented that activation state of Rubisco is a crucial limiting factor for photosynthesis, and Rubisco activase is highly susceptible to high temperature . It is worth mentioning that limited supply of CO2 accelerates photodamage to PSII via the excessive reduction of QA , inhibits the repair of photodamaged PSII , which are potentially dangerous for plants . Excessive ROS block the electron transport by affecting the repair process of PSII and inducing cleavage aggregation of RC proteins, which eventually form a vicious cycle , and the increase of REC indicated excess ROS caused membrane damage . The antioxidant system including antioxidant enzymes and non-enzymatic antioxidants is responsible to remove excessive ROS under moderate stress and suppress cell death under severe stress . According to our previous results, the induction of HSP70 by luffa rootstock was closely related to luffa rootstock-induced increase of ABA content  are inhibitors of HSP70 synthesis in plant and animal cells  . As a tyeatments , and thi content . Quercetal cells . More imLc rootstock alleviated heat-induced photosynthetic inhibition by maintaining higher root vitality and by inducing accumulation of HSP70. HSP70 was potentially involved in photoprotection and repair of PSII, as well as activation of antioxidant enzymes.Root-zone heat stress inhibited photosynthesis mainly through decreasing Rubisco activity, while aerial heat stress mainly through inhibiting PSII acceptor side in cucumber plants. Restriction of electron transport resulted in accumulation of ROS which caused damage to photosynthetic apparatus, forming a vicious cycle. In the field condition, aerial temperature fluctuates frequently, and thus PSII acceptor side is to be considered as the primary limiting site of photosynthesis under heat stress. Grafting onto HL and YZ designed research; HL, GA, and GZ performed research; HL, XX, JZ, KS, JY, and YZ analyzed data; HL, GJ, and YZ wrote and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer AS-S and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Apis mellifera. Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts.There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones),  This Advances in agrochemical research highlight a lack of knowledge of the sublethal effects of insecticides on their target insect pests , as wellApis mellifera) has served as a model insect to investigate the effects of various anthropogenic and environmental stressors [Historically, the honeybee  and sub-lethal  effects compared with drones from control colonies based on previous studies demonstrating strong sublethal effects of neonicotinoids on female insect reproduction ,18,30,402.A. mellifera L. honeybee colonies that were established at the beginning of the experimental period using the shook swarm method [The study was performed in Bern, Switzerland, between April and September 2015 using 20 m method  to sourcm method .(a)et al. [et al. [In early May 2015, colonies were randomly assigned to one of two treatments (insecticide or control). Each colony was provided daily with 100 g pollen paste  according to Sandrock et al.  and Will [et al. . Pollen  [et al. ; applied [et al. ,53. Duri [et al. .(b)Thirty-eight days post-initial pollen paste feeding, queens of each colony were first caged for approximately 48 h to a drone brood frame, and then 1 day later to a worker brood frame for an additional approximately 48 h to obtain sufficient numbers of drones and workers of the same known age cohort. Both experimental brood frames remained within their corresponding colonies until approximately 24 h prior to simultaneous drone and worker emergence; frames were then transferred to a laboratory incubator maintained in complete darkness at 34.5°C and 60% relative humidity .(c)Varroa destructor. For each colony, the first 30 drones to emerge, which were free of V. destructor infestation and abnormalities, were weighed to the nearest 0.1 mg using an analytic scale (Mettler Toledo AT400). These drones, plus the next 30 of similar status  to emerge per colony, were then placed in standard hoarding cages (250 cm3) [ad libitum to provide a carbohydrate energy source and ample proteins for organ and tissue development [Upon emergence, each experimental drone and worker was visually examined to assess for physical abnormalities and the presence of the parasitic mite 250 cm3)  correspo250 cm3) –57. Cage250 cm3) , and givelopment ,59, respelopment . The ass(d)et al. [® tube containing 500 µl Kiev+ buffer, and crushed to form a diluted stock sperm solution.Three cages per colony were randomly selected to assess drone sperm quantity and viability at 14 days post-cage assay initiation, the typical age drones reach sexual maturity ,61. Dronet al.  the test® tube for analyses of sperm viability  solution (1 mg ml−1) and 1 µl of Hoechst 33342 (0.5 mg ml−1) [Immediately, a 50 µl aliquot of the stock sperm solution was set aside in a separate 1.5 ml Eppendorfrm alive ). Sperm rm alive  and Stür [et al. . In briemg ml−1)  (both Simg ml−1) . Ten vismg ml−1) .+ buffer (1 : 5 dilution) in a 1.5 ml Eppendorf® tube to perform sperm counts. Sperm densities were measured using a Neubauer counting chamber under light microscopy . The final density of sperm was quantified using the following calculation [In addition, 20 µl of each stock sperm solution were diluted with 80 µl Kievculation : total s(e)Three-level generalized regression mixed models with random intercepts were fitted using STATA14 , whereinp = 0.44), so a general linear model was fitted using the meglm function. Total sperm quantity and the total living sperm quantity are count data, and were not normally distributed  so were therefore fitted to negative binomial models using the menbreg function. Sperm viability is a score ranging from 0 to 100% and was also not normally distributed  so an ordered logistic model was employed [Drone teneral body mass was normally distributed  between two groups .3.(a)p = 0.80; p < 0.001; median ± 95% CI; figure 2a). Furthermore, insecticide drone survival was significantly reduced compared with controls for up to 14 days ; mortality was 16.82 ± 0.02% and 32.08 ± 0.03% for controls and insecticides, respectively, which represents an approximately 50% difference . By contrast, no significant difference in worker median longevity was observed between controls (23 ± 22–24 days) and insecticides (26 ± 25–29 days) .No significant difference was observed between treatments for drone teneral body mass ((b)p = 0.14; median ± 95% CI; figure 3a). By contrast, sperm viability was significantly different between the two treatment groups, with insecticide drones having 8 ± 4.6–11.3% (median difference ± 95% CI) lower sperm viability than controls . Sperm viability was 92 ± 90–94% and 83.5 ± 80–86% in the controls and insecticides, respectively (median ± 95% CI). No correlation was observed between sperm quantity and sperm viability . In addition, a significant difference was observed between control (1.98 ± 1.72–2.18 million) and insecticide (1.2 ± 0.20–1.6 million) treatments for total living sperm quantity , which represents on average approximately 39% less living sperm in insecticides compared with controls. The median difference and its 95% CI was 0.61 ± 0.32–0.90 million less living sperm in insecticides compared with controls.No evidence of treatment effect was found between control (2.19 ± 1.93–2.55 million) and insecticide (1.55 ± 1.33–2.05 million) drone sperm quantity 14 days post-cage assay initiation (4.Factors governing reproductive success have a profound influence on shaping populations by affecting fitness ,75. BounFor eusocial insects such as honeybees, polyandry conveys several fitness benefits, such as reducing parasitism ,82, buffHoneybee teneral body mass immediately succeeding pupation is often used as an index for an individual's overall condition ,96; bothThe successful transfer of male sperm is the primary goal of copulation . TherefoAlthough only a small proportion of transferred sperm is stored by the queen , any decAs the primary egg layer and an important source of colony cohesion, the queen is intimately connected to colony performance . IncreasBy demonstrating the effects of neonicotinoid insecticides on male insect reproduction, our study provides a possible mechanism, in addition to introduced parasites and other land-use practices, for honeybee queen failure ,31 and a


Botrytis cinerea is a major threat to grapevine cultivation worldwide. A screen of 41 Vitis genotypes for leaf resistance to B. cinerea suggested species independent variation and revealed 18 resistant Chinese wild Vitis genotypes, while most investigated V. vinifera, or its hybrids, were susceptible. A particularly resistant Chinese wild Vitis, “Pingli-5” (V. sp. [Qinling grape]) and a very susceptible V. vinifera cultivar, “Red Globe” were selected for further study. Microscopic analysis demonstrated that B. cinerea growth was limited during early infection on “Pingli-5” before 24 h post-inoculation (hpi) but not on Red Globe. It was found that reactive oxygen species (ROS) and antioxidative system were associated with fungal growth. OB. cinerea 4 hpi on both Vitis genotypes. Lower levels of O2O2) were detected 4 hpi and ROS (H2O2 and OB. cinerea triggered sustained ROS production in “Red Globe” but not in “Pingli-5” with subsequent infection progresses. Red Globe displayed little change in antioxidative activities in response to B. cinerea infection, instead, antioxidative activities were highly and timely elevated in resistant “Pingli-5” which correlated with its minimal ROS increases and its high resistance. These findings not only enhance our understanding of the resistance of Chinese wild Vitis species to B. cinerea, but also lay the foundation for breeding B. cinerea resistant grapes in the future.The necrotrophic fungus  Botrytis cinerea causes gray mold disease in a broad range of plant species, including grape. Grape production, of great economic importance in China, relies almost exclusively on European grapevine varieties  plants overexpressing the transcription factor SlSHINE3, which regulates cuticle production, were observed to be more resistant to B. cinerea with lower levels of ROS production and more cuticles than wild-type plants  synthesis) effectively blocked infection by the pathogen  leaves, it was shown that the secondary oxidative burst was much stronger following challenge by a non-aggressive B. cinerea strain than by an aggressive strain, indicating that ROS-mediated responses have the capacity to block infection by the pathogen  to V. vinifera was reported to improve resistance to B. cinerea by inducing ROS production and the expression of genes involved in defense through different signal pathways  in the greenhouse and was maintained on Potato Glucose Agar medium in the dark at 22°C. After 21 days, conidia were washed down with distilled water, counted, and added to the inoculation solution at concentrations detailed in the following sections. Conidia were pre-germinated for 2 h at 22°C before inoculations were performed  and inoculation treatments were incubated in the dark for the first 24 h and then in a light/dark (12/12 h) regime at 22°C  selected randomly from vines. Detached leaves were washed carefully, first under tap water and then distilled water, and were then quickly transferred to a bed of 0.8% agar in trays and then uniformly sprayed with B. cinerea were determined. The conidia germination in solutions with different glucose (Glc) and phosphate concentrations was determined under a light microscope after 6 and 24 h. The four solutions tested in this study were: (i) sterile; (ii) 1 × 106 spores mL−1, 0.1 M Glc, 67 mM KH2PO4; (iii) 1 × 106 spores mL−1, 0.05 M Glc, 33 mM KH2PO4; and (iv) 1 × 106 spores mL−1, 0.01 M Glc, 6.7 mM KH2PO4 , “Hunan-1” (V. pseudoreticulata), “Taishan-2” (V. adstrica), “Baihe-41” (V. sp. [Maihuang grape]) were evaluated after infection with conidia suspensions of different concentrations .To evaluate detached leaves , at least 18 leaves from three biological replicates of each genotype were tested. Four days after inoculation, the infection was evaluated by counting the percentage of spreading lesions on each leaf. Before evaluation, the optimal inoculation solution and conidia concentration of For time series experiments, single inoculated, and control leaves were sampled 4, 8, 12, 18, 24, 36, 48, 72, and 96 hpi (hours post-inoculation) in a randomized manner from each of three biological replicates, except in the case of samples used for DAB (diaminobenzidine) staining.Disease severity was evaluated from 2011 to 2013 and scored as previously described  containing 0.15% (w/v) trichloroacetic acid, before clearing in saturated chloral hydrate, and were then stored in 20% glycerol. Samples were subsequently stained with aniline blue solution  and examined with an Olympus BX-51 microscope . For each sample, fungal germination, and infection percentages were examined. For scanning electron microscopy (SEM), leaf tissues were cut into small pieces (0.5–1 cm2), fixed in 4% (v/v) glutaraldehyde in phosphate buffer  for 12 h at 4°C, and rinsed in the same buffer four times for 10–15 min. After dehydration in a graded ethanol series , the samples were then critical-point dried, coated with gold in a sputter coater, and examined with a JEOL FESEM S-4800 scanning electron microscope at 15 kV  and “Red Globe”  by 2O2 and O2 leaf segments were immersed under direct light in a DAB solution (1 mg/mL with HCl acidifying to pH 3.8) 8 h before sample collection except for that samples 4 hpi were directly immersed in DAB solution once inoculated. The leaves were prepared for observation as described above. Leaf segments of the same size were collected directly into 0.1% (w/v) NBT solution in 10 mM phosphate buffer (pH 7.8) prior to a vacuum infiltration for 30 min and an exposure to direct light for 20 min. The NBT stained samples were then observed as above, except for the omission of aniline blue staining. The percentages of conidia, germ tubes, and infection sites exhibiting O2O2 accumulation were evaluated.H2O2 was measured at 470 nm.Crude protein extracts to assess superoxide dismutase (SOD)   and significant differences and Duncan LSD analysis by a completely random design and correlation analyses of resistance evaluation data from 2011 to 2013 were performed using SPSS Statistics , “Hunan-1” (V. pseudoreticulata) and “Baihe-41” (V. sp. [Maihuang grape]), were also randomly selected for the comparative assay with different concentrations of spores in sterile water  were counted 4 days post-inoculation, Chinese wild Vitis species generally exhibited a greater degree of variation in their resistance to B. cinerea than other species did (Table P ≤ 0.05) among the different genotypes through completely random Duncan LSD analysis  in which only 10 belonged to Chinese wild Vitis species (Table Among the 20 genotypes that were classified as resistant at least (scores between 0 and 3.50), 18 were Chinese wild Vitis to B. cinerea is shown in Figure V. vinifera cultivars. Indeed, five of the eight HS genotypes were cultivars of V. vinifera or its hybrids. Four V. romanetii genotypes and three V. davidii genotypes but “Tangwei” were classified as susceptible at least, while four of five V. amurensis genotypes were resistant at least. Furthermore, all three V. piasezkii, three V. adstricta and two V. sp. genotypes were classified as R (scores between 1.51 and 3.5) or HR, as were V. sp. (Qinling grape) and V. yenshanensis, although there was only one representative. It is noteworthy that all six genotypes identified as HR were Chinese wild Vitis: “Pingli-5” (V. sp. [Qinling grape]); “Yanshan-1” (V. yenshanensis); “Taishan-2” (V. adstricta); and three V. amurensis genotypes .Variation in the resistance levels of Chinese wild V. vinifera or Chinese wild Vitis showed macroscopic mycelium 4 days after infection (Table V. quinquangularis) was 42.4% and spreading lesions reached 42.9%, the infection rate was only 14.1% that was even lower than the R genotype “Gansu-91” (V. piasezkii)  Table , the latB. cinerea 4 days after inoculation  “Pingli-5” , and one V. adstricta  is resistant to anthracnose, powdery and downy mildew  and thus experienced less ROS-induced stress. Since substantial ROS was induced in “Red Globe” but not in “Pingli-5,” the precise coordination of ROS production and associated scavenging mechanisms by antioxidative system during combined biotic and abiotic stress . This accumulation began 4 hpi, at the earliest stage of the infection  responses and antioxidative system changes between the highly susceptible Vitis vinifera “Red Globe” and the highly resistant Chinese wild Vitis “Pingli-5” [V. sp. (Qinling grape)] after the infection with this pathogen. Our results demonstrated that minimal fungal development as well as minimal production of ROS and a timely elevation in antioxidative capacity were correlated with a high level of resistance in “Pingli-5,” while highly suscepitble “Red Globe” suffered massive infection and sustained ROS production due to relatively unchanged antioxidative activities. Moreover, we speculated O2O2 production, may be among the first consequences of an interaction between B. cinerea with grapevines, suggesting a potential ROS response responsible for the timely recognition and defense of Chinese wild Vitis “Pingli-5” to B. cinerea. However, this remains to be resolved through futher experiments on spatiotemporal relationship of ROS and molecular mechanism.In conclusion, we explored germplasm resources from Chinese wild XPW and RW designed the study. RW, XH, and XHW contributed to the experiments. RW, XH, and JQ performed data analysis. RW, XH, and SS assisted with the interpretation of the results. XPW and YW provided guidance throughout the study. RW, XH, SS, and XPW wrote and revised the manuscript. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dipyrithione (PTS2) is widely used as a bactericide and fungicide. Here, we investigated whether PTS2 has broad-spectrum antitumor activity by studying its cytotoxicity and proapoptotic effects in four cancer cell lines.t-test was used for statistical analysis.We used MTT assays and trypan blue staining to test the viability of cancer cell lines. Hoechst 33258 and DAPI staining were used to observe cell apoptosis. Cell-cycle percentages were analyzed by flow cytometry. Apoptosis was assayed using caspase-3 and poly (ADP-ribose) polymerase (PARP) combined with Western blotting. Student’s PTS2 inhibited proliferation in four cancer cell lines in a dose-dependent manner. Treated cells showed shrinkage, irregular fragments, condensed and dispersed blue fluorescent particles compared with control cells. PTS2 induced cycle-arrest and death. Cleavage of caspase-9, caspase-3, and PARP were detected in PTS2-treated cells. Antitumor activity of PTS2 was more effective against widely used cancer drugs and its precursor.PTS2 appears to have novel cytotoxicity and potent broad-spectrum antitumor activity, which suggests its potential as the basis of an anticancer drug. Male ICR mice  purchased from Shanghai Laboratory Animal Center, Chinese Academy Sciences, were kept in groups of five animals per cage in a temperature-controlled room at 20 ± 2°C. They were fed a standard pellet diet and water ribed in, two grot test for other data. P < 0.05 was considered statistically significant.Statistical analysis used SPSS 12.0 . Results are expressed as means ± S.D. Differences between means were determined by one-way ANOVA, followed by Student–Newman–Keuls tests for multiple comparisons and Student’s To assess effects of PTS2 on cancer cell growth or proliferation, 4 cancer cell lines, including KB, 231, U937 and K562, were assayed using MTT. After 36 h treatment with various concentrations of PTS2, cell viability was determined for all 4 cell lines. PTS2 (0.25–5 μg/ml) was found to decrease cell viability in a dose-dependent manner  inhibits the cell cycle through its interaction with cyclin–CDK complexes[1 growth arrest[1 arrest in cancer cells could be mediated via modulation of p53, p21, CyclinD1 and CyclinE1 levels.As p21omplexes, and is h arrest. We furth arrest,18, and Morphology alteration and chromatin condensation are two indicators of cell apoptosis. To verify whether the growth inhibitory effect of PTS2 was due to apoptosis, KB, 231, U937 and K562 cells were treated with PTS2 at 2.5 μg/ml for 36 h, and then observed under a light microscope or under a fluorescence microscope after Hoechst33258 and DAPI staining. We found the PTS2-treated cells were shrunken, with irregular fragments and apoptotic bodies, in contrast to control cells Figure A. The tyCaspase family members, including caspase-9 and caspase-3, as well as downstream substrates such as PARP, are crucial mediators of the apoptotic process. To see whether PTS2-induced cell death involved activation of caspases and PARP, we analyzed cleavage of caspase-9, caspase-3 and PARP in PTS2-treated cancer cells. Cells were incubated with 2.5 μg/ml of PTS2 for 36 h. As expected, Western blot results showed that caspase-9, caspase-3 and PARP were cleaved  and cisplatin (DDP) are widely used cancer drugs. To further evaluate the efficacy of PTS2 in decreasing cancer cell viability, we compared its effects on KB cells to those of ADM and DDP. KB cells were exposed to 2.5 μg/ml of PTS2, ADM or DDP and assayed with MTT. At 3–36 h after treatment, PTS2 also showed faster results than did ADM or DDP  were transplanted subcutaneously into the right groins of mice; 24 h after tumor implantation, the mice were injected intraperitoneally with PTS2, DDP or saline and observed for 10 days. PTS2 at 2.5 mg/kg/day reduced the weight of H22 tumors; DDP had a similar effect at 25 mg/kg/day  cells  and cisplatin (a platinum compound), which suggests PTS2’s potential as an anticancer drug Figure . AlthougHere, we report novel toxicity of PTS2 on various cancers, and show PTS2 to inhibit proliferation of four cancer cell lines and induce apoptosis involving activation of caspase-9, caspase-3 and PARP. These results suggest that PTS2 has broad-spectrum antitumor activity and could be the basis of an anticancer drug.The authors declare that they have no competing interests.YF, CL and SW carried out the experimental studies. YF and YZ drafted and completed the manuscript. YH performed the statistical analysis. JZ and LC cultured cells. ZY proofread the manuscript. XD and ZY refined the manuscript. FY and XD conceived of and designed the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/2050-6511/14/54/prepub


Thermotogae bacteria: Kosmotoga sp. strain DU53, isolated from a continental oil reservoir, and Kosmotoga arenicorallina, isolated from hydrothermal sediments. The sequences will provide further insight into evolution of the Kosmotogales.Here, we announce the draft genome sequences of two thermophilic  Kosmotoga are anaerobic thermophilic bacteria isolated from oil reservoirs and hydrothermal environments . Kosmotoga sp. DU53 was isolated from free-water-knockout (FWKO) water collected from oil field D  in Alberta, Canada  using a 316 D Chip and 500 flows. The K. arenicorallina S304 library was constructed using the Nextera XT kit and sequenced as one of 10 pooled barcoded libraries on a MiSeq  using 500 cycles generating 2 × 250-bp paired-end reads.DNA was extracted from 50-ml cultures of Forterre . The purK. arenicorallina S304 was assembled de novo by CLC Genomics Workbench 7.0.4, using trimming settings, automatic word size, a bubble size corresponding to the average length of the input reads, a minimum contig length of 1,000 bp, and reads mapped back to the contigs. Four contigs containing parts of two nonidentical 16S genes were located and manually resolved using its published 16S gene sequences (accession numbers AB530678 and AB530679). This resulted in 40 contigs totaling 2,113,627 bp, with an N50 of 109,886 bp, a longest contig size of 350,318 bp, and a G+C content of 41.0%.The genome of De novo assembly of the Kosmotoga sp. DU53 genome was done using MIRA 3, with Ion Torrent settings (http://sourceforge.net/projects/mira-assembler/), resulting in 97 contigs totaling 2,375,260 bp, with an N50 of 66,806 bp, a longest contig size of 221,738 bp, and a G+C content of 41.4%.settings  , which JFHK00000000 and JGCK00000000 for Kosmotoga arenicorallina S304 and Kosmotoga sp. DU53, respectively. The versions described in this paper are the first versions, JFHK01000000 and JGCK01000000.Both whole-genome shotgun projects have been deposited at DDBJ/EMBL/GenBank under the accession numbers


The predicted amino acid sequences for the yellow catfish grp78, crt, perk, ire-1α, and atf-6α revealed that the proteins contained all of the structural features that were characteristic of the five genes in other species, including the KDEL motif, signal peptide, sensor domain, and effector domain. mRNAs of the five genes mentioned above were expressed in various tissues, but their mRNA levels varied among tissues. Dietary Cu excess, but not Cu deficiency, activated the chaperones (grp78 and crt) and folding sensors in ER, and the UPR signaling pathways  in a tissue-specific manner. For the first time, our study cloned grp78, crt, perk, ire-1α, and atf-6α genes in yellow catfish and demonstrated their differential expression among tissues. Moreover, the present study also indicated differential regulation of these ER stress–related genes by dietary Cu deficiency and excess, which will be beneficial for us to evaluate effects of dietary Cu levels in fish at the molecular level, based on the upstream pathway of lipid metabolism (the ER) and thus provide novel insights regarding the nutrition of Cu in fish.Two endoplasmic reticulum (ER) molecular chaperones [glucose-regulated protein 78 ( Grp78) (Crt) (Perk), inositol requiring enzyme (ire)-1α, and activating transcription factor (Atf)-6α  is an important intracellular organelle responsible for protein and lipid synthesis, xenobiotic detoxification, and cellular calcium storage . UPR is Grp78, crt, Perk, Ire-1α, and Atf-6α have been previously reported in human, mouse, and rat species  were only determined in very limited fish species, such as grass carp Ctenopharyngodon idella (grp78) (Oncorhynchus mykiss (crt)  (Oryzias latipes (atf6)  . Althoug−1 feed, which depends on the species, feeding regime, and life stage  is an essential micronutrient for vertebrate animals including fish  and three ER stress sensors  genes were first cloned and their tissue-specific expressions were determined. Meanwhile, the effect of dietary Cu deficiency and excess on their mRNA levels was explored. The results will be beneficial for us to elucidate the molecular basis of ER stress, and to further evaluate effects of dietary Cu levels in fish at a molecular level, based on the upstream pathway of lipid metabolism, which will greatly extend our understanding of the nutrition of Cu in fish.Yellow catfish grp78, crt, perk, ire-1α, and atf-6α transcripts. The second experiment was designed to evaluate a possible transcriptional regulation of grp78, crt, perk, eif2α, ire-1α, xbp-1, and atf-6α by dietary Cu deficiency and excess. We ensured that the experiments performed on animals followed the ethical guidelines of Huazhong Agricultural University and Animal Care and Use Committee (IACUC) in Huazhong Agricultural University specifically approved this study.Here, two experiments were conducted. The first experiment was involved in the cloning and tissue expression profile analysis of grp78, crt, perk, ire-1α, and atf-6α genes were obtained from a local fish pond  and transferred to indoor fiberglass tanks (400 liters in water volume) for 2-wk acclimation. During the acclimation period, the fish were fed to apparent satiation twice daily with a commercial pellet diet. The experiment was conducted at ambient temperature and subjected to natural photoperiod (approximately 14 hr light/10 hr darkness). Water quality parameters were monitored twice per week in the morning. The following parameters were used: water temperature, 27.9 ± 1.2°; pH, 7.66 ± 0.11; dissolved oxygen, 6.34 ± 0.15 mg liter−1; and total hardness, 151.8 ± 1.1 mg liter−1 as CaCO3. Fish were anesthetized with 3-aminobenzoic acid ethyl ester methanesulfonate (MS-222 at 10 mg liter−1) and killed by decapitation. Skeletal muscle, gill, head kidney, mid-intestine, brain, spleen, liver, heart, testis, and ovary from six fish were dissected and immediately prepared for RNA isolation (n = 6).Yellow catfish  for cloning the 260/OD280 of total RNA were determined using a Nanodrop ND-2000 spectrophotometer . Total RNA (1 μg) was reverse-transcribed to cDNA with oligo-dT primers and a cDNA Synthesis Kit  according to manufacturer’s instructions.Each sample was pulverized with its own mortar and pestle, and the mortars and pestles were heated at 230° for 8 hr to inactivate RNases. Total RNA was extracted using TRIzol RNA reagent  based on the acid guanidinium thiocyanate-phenol-chloroform extraction method, according to the manufacturer’s instructions. The quality of total RNA was checked by agarose gel electrophoresis. The concentration and the ratio (between 1.8 and 2.0) of ODgrp78, crt, perk, ire-1α, and atf-6α sequences available in the GenBank and Ensembl databases were used to amplify partial cDNA fragments of grp78, crt, perk, ire-1α, and atf-6α. The PCR program consisted of initial denaturing for 4 min at 94° followed by 30 cycles of 94° for 30 sec, 55° for 30 sec, 72° for 1 min, and a final elongation step at 72° for 5 min, with 500 ng cDNA template, 0.2 μM primer, 5 μl 10 × Ex Taq Buffer, 2.5 mM dNTP, and 1.25 units Ex Taq polymerase . The target fragments were purified using the EZNA gel extraction kit  according to manufacturer’s instructions, and then subcloned into the pMD 19-T cloning vector . Positive clones containing inserts of an expected size were sequenced with sanger dideoxy mediated chain termination method .In the present study, we used the liver of yellow catfish to clone the cDNA sequences of the five genes. Degenerate primers  designedgrp78, crt, perk, ire-1α, and atf-6α, nested 3′ and 5′ RACE PCR were performed with a SMART RACE cDNA Amplification Kit  using the DNase treated total RNA, with 1 μl 10 × Reaction Buffer, 1 units DNase I for 1 μg RNA incubated at 37° for 15 min, then with 1 μl 25 mM EDTA incubated at 65° for 15 min to inactivate the DNase I. In the first PCR, the cDNA was amplified with two outer primer sets . In the second PCR, inner primer sets . The PCR parameters were 30 cycles at 94° for 30 sec, 55° for 30 sec, and 72° for 1 min, with an additional initial 3-min denaturation at 94° and a 10-min final extension at 72°. The RACE products were sequenced as described above.To obtain the 3′ and 5′ end sequences of mer sets  and Univmer sets  and Nestgrp78, crt, perk, ire-1α, and atf-6α cDNAs. The sequences were edited and analyzed using the program EDITSEQ of DNASTAR package to search for the open reading frame (ORF) and then translated into amino acid sequences using standard genetic codes. The nucleotide sequences were compared with DNA sequences present in the GenBank database using BLAST network service at the NCBI (http://blast.ncbi.nlm.nih.gov/). Sequence alignments and percentage of amino acid conservation were assessed with the Clustal-W multiple alignment algorithm. For phylogenetic analysis, multiple sequence alignments were made with MAFFT (http://guidance.tau.ac.il/)  and subjected to natural photoperiod (approximately 14 hr light/10 hr darkness). Each diet was assigned to three tanks in a completely randomized design, with nine tanks for the experiment. Water in each tank was renewed 80% twice daily, before feeding. They were provided with continuous aeration to maintain the dissolved oxygen level near saturation. The fish were fed to apparent satiation twice daily at two equal meals (09:00 and 16:30 hr) during the week. Care was taken to ensure that no noneaten food remained in the tanks during feeding, thus leaching of CuSO4 into water was very low and negligible. Cu concentrations in water samples collected 10 min before and after feeding remained low throughout the experiment (1.7 ± 0.2 μg 1−1). The experiment continued for 8 wk. At the end of the 8-wk period, 24 hr after the last feeding, all fish were killed with MS-222 (10 mg l−1). Three fish per tank were randomly selected. After the skin was rapidly removed from muscle, the liver and skeletal muscle above the lateral line were removed immediately using sterile forceps, frozen in liquid nitrogen, and stored at −80° (not longer than 2 wk) for total RNA extraction .Experimental procedures are in agreement with these in our parallel study  method. Primers used for qPCR are shown in 2O. Reactions were based on a three-step method as followed: at 95° for 30 sec, 40 cycles of 5 sec at 95°, 10 sec at 57°, and 30 sec at 72°. All reactions were performed in duplicate and each reaction was verified to contain a single product of the correct size by agarose gel electrophoresis. A nontemplate control and dissociation curve were performed to ensure that only one PCR product was amplified and that stock solutions were not contaminated. A 10-fold dilution series was created from a random pool of cDNA from our sample groups ranging from ×1 dilution to ×100,000 dilutions. The PCR efficiency and correlation coefficients of each primer pair were generated using the slopes of the standard curves. The amplification efficiencies of all genes were approximately equal and ranged from 98 to 103%. Because housekeeping gene sequences were not available in P. fulvidraco, a set of eight housekeeping genes  were selected from the literature  as suggested by geNorm .Quantitative data were expressed as means ± SEM. Prior to statistical analysis, all data were tested for normality of distribution using the Kolmogorov–Smirnov test. The homogeneity of variances among the different tissues was tested using the Barlett’s test. Data were then subjected to one-way ANOVA and Tukey’s multiple range tests. Differences between control (adequate Cu) and individual Cu-treated groups (Cu deficiency and Cu excess) were analyzed by student’s File S1 contains detailed data about tissue expression mRNA of grp78, crt, perk, ire-1α, and atf-6α. File S2 contains detailed about effect of dietary Cu deficiency and excess on epression of grp78, crt, perk, eif2α, ire-1α, xbp-1α, and atf-6α.grp78, crt, perk, ire-1α, and atf-6α genes, which were shown to be 2544 bp, 1678 bp, 3776 bp, 3440 bp, and 2785 bp, respectively  formed an independent cluster, whereas amphibian and mammalian corresponding proteins formed another cluster, clustering the yellow catfish most closely with Astyanax mexicanus and Danio rerio, and more distantly from Oryzias latipes  (Oncorhynchus mykiss (crt)  (Oryzias latipes (atf6)  revealed that the peptide contained all of the structural features that are characteristic of ER molecular chaperones in other species, containing 16- and 18-residue signal peptide sequences and the KDEL motif   in all tested tissues, with the highest expression in liver and the lowest in skeletal muscle and gill. At present, mRNA expression profiles of these genes in fish were very scarce. Studies pointed out that the expression level of ER stress proteins was expected to be high in secretory cells, as these cells are rich in ER  and enzyme activities  in yellow catfish  involved in ER stress response from yellow catfish and investigated their molecular characterization, which will facilitate further studies on the regulation of ER stress response at the molecular level. mRNA expression patterns of these genes in different tissues also provided a basis for further exploring the differential regulatory patterns of ER stress response in different tissues for the fish species. Dietary Cu excess activated ER stress and the UPR signaling in a tissue-specific manner in yellow catfish, which will help us to understand the effects of dietary Cu levels on the upstream pathway of lipid metabolism (the ER) and thus provide some novel insights on the nutrition of Cu in fish.In summary, we cloned the full-length sequences of five key genes (