Dicentrarchus labrax). Sea bass embryos, pre-larvae and larvae at specific points of development were exposed to acute stressors and the temporal patterns of cortisol whole body concentrations and the expression of genes involved in corticosteroid biosynthesis, degradation and signaling were determined. Expression of genes  involved into the corticoid response regulation combined with histological data indicated that, although a cortisol stress response is evident for the first time around first feeding, a pattern becomes established in larvae at flexion until the formation of all fins. Moreover, mRNA transcript levels of 11β-hydroxylase and 11β-hsd2 showed a strong correlation with the whole body cortisol concentrations. Concluding, our data reveal the presence of an adaptive mechanism in European sea bass at early ontogeny enabling to cope with external stressful stimuli and provide a better insight into the onset and regulation of the stress response in this species.The cortisol stress response and the molecular programming of the corticoid axis were characterized for the first time during early ontogeny in a Mediterranean marine teleost, the European sea bass ( The teleostean hypothalamic–pituitary–interrenal (HPI) axis is a system comparable with the mammalian stress axis , as a result of convergent evolution113135Exposure to stress can have a profound impact on the physiology and health of an organism later in life79109In teleostean fish, cortisol is the principal corticosteroid and plays an important role in a number of physiological processes including growth, immunoregulation, maintenance of energy balance, and reproduction2161712123Dicentrarchus labrax, and other species showed the presence of maternal cortisol in embryos and that de novo cortisol synthesis starts shortly after hatching but a significant elevation in whole body cortisol in response to a stressor becomes obvious days to weeks later, depending on the species2526272829Studies conducted in European sea bass, gr1, gr2, mr, crf), corticosteroid synthesis (11β-hydroxylase) and cortisol metabolism or inactivation (11β-hsd2) at various stages during early ontogeny in order to assess the ontogenesis of the corticosteroid-signaling pathway. Moreover, we subjected European sea bass embryos, pre-larvae and larvae to an acute stressor in order to determine any differences in the timing or magnitude of the activation of the corticosteroid stress axis and the molecular response at each developmental point/stage.However, our knowledge on the development of the hypothalamic–pituitary– interrenal (HPI) axis of European sea bass and its response to stressors during early ontogeny or the molecular mechanisms involved is scarce. To this end, we examined the temporal patterns of cortisol and genes related to the corticosteroid signaling  that had declined at hatching (0.6 ± 0.3 ng g−1) and subsequently slightly increased at mouth opening (1.5 ± 0.2 ng g−1), but the differences were not statistically significant. The first peak (P < 0.001) was observed at first feeding (6.8 ± 1.3 ng g−1), after which whole-body cortisol mean concentrations dropped gradually from flexion  onwards to the formation of all fins (1.9 ± 0.5 ng g−1)  at hatching and mouth opening, followed by a second increase at first feeding until the formation of all fins (P < 0.001). The mRNA abundance of mr  at first feeding until the formation of all fins. Expression of crf (P < 0.001) and the formation of all fins than at mouth opening (P < 0.05). 11β-hydroxylase mRNA (P < 0.001) in mouth opening and the formation of all fins and a peak at flexion. Finally, transcript levels of 11β-hsd2  . All gen−1) (gr1  showed agr1 (gr2  was highce of mr  showed an of crf  was detease mRNA  showed l11β-hsd2  showed aP < 0.001) increase at 24 h after the application of the stressor, i.e. approximately 48 h following fertilization. An identical pattern of changes was observed after stress at hatching and mouth opening. A statistically significant (P < 0.05) effect of the stressor on whole-body cortisol concentrations soon after the application of the acute stressors was observed for the first time at first feeding, where whole body cortisol increased from the 0 h basal values (6.8 ± 1.3 ng g−1) to a maximum at 0.5 h (10.8 ± 1.0 ng g−1) and 1 h (9.6 ± 0.5 ng g−1) to return to basal values at 24 h (3.5 ± 0.5 ng g−1). At flexion, a prolonged peak was observed with basal cortisol values at 0 h (4.1 ± 1.2 ng g−1) and peak values (P < 0.05) at 0.5 h (17.1 ± 1.7 ng g−1), 1 h (19.9 ± 6.5 ng g−1) and 2 h (23.4 ± 3.1 ng g−1) post stress. In addition, the magnitude of the stress response was higher (P < 0.001) than the respective at first feeding. Finally, at the formation of all fins, cortisol content at 0 h (1.9 ± 0.5 ng g−1) sharply increased at 0.5 h (17.1 ± 1.6 ng g−1) to reach a maximum at 2 h (33.7 ± 2.7 ng g−1) after stress and return to basal values at 24 h (3.3 ± 1.4 ng g−1). The magnitude of the stress response was statistically significant higher (P < 0.001) than the respective in first feeding and flexion. Transcripts of gr1 (P < 0.001) at 24 h, respectively, while at the formation of all fins a significant 1.4-fold downregulation (P < 0.05) compared to controls was observed at 2 h post-stress. A similar pattern of expression was observed in gr2 transcripts (P < 0.05) at 24 h at first feeding and flexion. In the formation of all fins, gr2 reaches a maximum at 0.5 h after stress and then mRNA levels fall down to a minimum at 2 h. Transcript levels of mr  at 2 h and 24 h post-stress. In all fins, crf transcripts peaked, as whole-body cortisol concentrations, at 0.5 h post stress followed by a gradual decrease to the basal levels of control at 24 h  at 24 h (i.e. hatching 100% completed) post-stress, while at mouth opening and first feeding there was a peak at 1 h and 2 h post-stress respectively. In flexion, 11β-hydroxylase abundance remained at high levels from 0 h till 1 h post stress after which there was a statistically significant decrease (P < 0.05) at 2 h. In all fins, the pattern of changes resembled that of cortisol, with low transcripts at 0 h, 1.6 to 1.4-fold upregulation at 0.5 and 1 h followed by a sharp downregulation at 2 h  using routine histology. Similar results were found in cichlid fishes where the head kidney from 12 to 30 days after fertilization is functionally mixed, with the nephron and developing hemopoietic and endocrine The acute stress challenge tests didn't result in a cortisol response in embryos, hatch and mouth opening stages, apart from a maximum at 24 h post stress. However, further research is needed to clarify whether this maximum is a result of a delayed stress response or reflects differences in the developmental point/stage. Histological data showed that the first appearance of a distinct hypothalamo-hypophysial-interrenal axis is observed at first feeding, where a peak in whole body cortisol levels was observed at 0.5 h post stress, followed by a protracted decrease until at 24 h when it reached resting levels. These results, in accordance with the molecular data, imply that as early as at first feeding sea bass individuals are capable of responding to external noxious stimuli. In addition the first peak observed in whole-body cortisol concentrations at first feeding reflects the essential role of cortisol to carbohydrate and protein metabolism towards transition to exogenous feeding. As development proceeds, the magnitude and duration of the response is higher and a pattern seems to be established from flexion until the formation all fins where cortisol values reach a maximum at 2 h. This is further supported by the histological data showing that while the kidney tubules with a distinct morphology were present at hatching, a clear morphological differentiation of the interrenal and chromaffin tissues was possible only at 28days post hatch , stimulates the pituitary corticotropes to secrete adrenocorticotropic hormone (ACTH)43Gr1 expression levels after application of an acute stressor were not altered in any developmental stage apart from all fins, where a down-regulation was observed at 2 h post stress. The same is the case of the mRNA levels of gr2, as the only response to the stress was detected at the stage of all fins, where an increase was observed at 0.5 h post stress followed by a down-regulation at 2 h. The down-regulation of gr1 and gr2 is in accordance with data from other studies carried out in sea bass exposed to very high stocking densitiesgr1 and gr2 observed at 24 h post stress in first feeding may be related, as with the case of cortisol, either to a delayed stress response or to the role of cortisol in metabolism and neural development for the passage of fish to exogenous feeding. The mRNA abundance of mr was not altered at any of the developmental stages examined apart from first feeding where the expression levels decreased from 0.5 h to 2 h post stress. However, as this is not repeated in the later stages and especially at all fins where the HPI axis is expected to be more mature, this alteration may not reflect a response to stress but rather a suppression related to the needs of the developmental stage. Crf expression pattern at all fins follows an increase at 1 h and a gradual decrease until 24 h post stress, which is in accordance with the pattern observed for cortisol at this stage, where the peak of cortisol levels is at 2 h post stress. In flexion, there is a statistically significant increase at 2 h that remains at high values still at 24 h, indicating that at this stage the crf system is being established, but the prolonged expression and the delayed response compared to the pattern of cortisol at the same stage, reveals that it is not mature yet.With the aim to shed light on the molecular mechanisms related to the onset of the cortisol stress response, we carried out qPCR experiments in order to measure the mRNA transcript levels of genes related to the HPI axis, crf and the grs at these stages until only at all fins, might be of critical importance for the survival of the larvae and the normal progress of the development. There are very limited data available about the role of gr and mr in fish development. The major mineralocorticosteroid in mammals and non-mammalian vertebrates is aldosterone. However, in fish, deoxycorticosterone (DOC) is considered to be a mr ligand instead of aldosterone, as the latter is not detected in fish, but also cortisol is a high-affinity ligand for mr. A recent study carried out in zebrafish showed that both GR and MR are present during embryogenesis and suggested that gr plays a more important role after hatching in zebrafish, whereas mr is suggested to be important at the earlier stages of development, and that after hatching a ligand other than cortisol, perhaps DOC, may be responsible for mr signalinggr survive until birth but die shortly thereafter due to impaired lung developmentgr leads to developmental defects in mesoderm formation in zebrafishgr signaling is essential for zebrafish muscle developmentmr expression profiles during the stress response in first feeding seems to give mr a more important role at this stage than that of grs, which at the later stages of development is inverted. This is in accordance with the results obtained from the other studies mentioned above.During fish ontogenesis cortisol is a critical hormone when changes occur at the metabolic demands of the larvae21241611β-hydroxylase that generates cortisol from 11-deoxycortisol11β-hsd2, an enzyme that converts the biologically active cortisol to the inactive cortisone. After the acute stress application the transcript levels of 11β-hydroxylase appear statistically significant altered at hatch and mouth opening but these changes may not reflect a stress response, but these changes could represent the role of this enzyme in gonad differentiation, as 11β-hydroxylase appears to be a key transducer in the mechanism of sex determination in fish11β-hydroxylase transcripts and cortisol increase. This pattern continues also in the later stages of development, where mRNA expression of 11β-hydroxylase is upregulated along with cortisol levels. This is in accordance with data obtained for rainbow trout, where mRNA abundance of 11β-hydroxylase also increases in response to an acute stressor11β-hsd2 mRNA transcripts are at high amounts in the larvae and gradually decline to a minimum at 2 h post stress; if compared with the cortisol pattern, it becomes clear that the mRNA levels of this enzyme are correlated with the amount of cortisol present. At the following developmental stages this becomes more obvious as the maximum mRNA levels of this gene are observed at 1 h post stress, just prior to the cortisol peaks (at 2 h), and at 2 h the transcript levels drop again to the resting values.We tested the hypothesis that the molecular events related to the appearance of the cortisol synthesis pathway are tightly linked to the enzymes which take part in cortisol biosynthesis and degradation. Therefore, we quantified the temporal expression of Dicentrarchus labrax. Sea bass embryos, pre-larvae and larvae were exposed to acute stressors and the temporal patterns of cortisol whole body concentrations and the expression of genes involved in corticosteroid biosynthesis (11β-hydroxylase), degradation (11β-hsd2) and signaling  were determined. Histological data showed that although the kidney was present at hatching, morphological differentiation of the interrenal and chromaffin tissues was difficult at early stages of development using routine histology, and only around flexion the respective endocrine cells were simultaneously evident. Whole body cortisol concentrations showed a decline from embryos to hatching, remained at low levels at mouth opening and peaked at first feeding. In addition, around first feeding an effect of stress was evident for the first time on post-stress cortisol concentrations. As development proceeds, a pattern with a higher magnitude and longer duration of the cortisol stress response was established from flexion until the formation of all fins. Expression data of genes related to the regulation of the corticoid response  indicated that, although a cortisol stress response is evident at first feeding, the HPI axis seems to be fully mature only at the stage of all fins. Moreover, the mRNA transcript levels of 11β-hydroxylase and 11β-hsd2 showed a strong correlation with the whole body cortisol concentrations. Overall, the data indicate that fish do respond to external noxious stimuli as early as at first feeding but the cortisol stress response becomes fully functional and mature until only at the stage of all fins. In addition, several changes occurred in early development  may resemble, apart from an adaptive to stress role of cortisol, its implication in other important aspects of development and metabolism. In conclusion, our data reveal the presence of an adaptive mechanism in European sea bass at early ontogeny enabling to cope with external stressful stimuli and provide a better insight into the onset and regulation of the stress response in this species.The cortisol stress response and the regulation of genes related to the corticoid axis in combination with histological analysis were studied for the first time during early ontogeny in European sea bass, −1 in which both hatching and rearing took place. Tanks were coupled to a biological filter and were initially filled with filtered seawater from a deep well. Aeration was provided by means of a wooden diffuser located in the tank center at a rate of 150–200 ml min−1. Larvae were held during the whole experimental period under mean (±SD) water temperature of 18 (±1.6)°C, dissolved oxygen levels of 7.2 ± 0.8 mg l−1, salinity of 36 and pH of 7.9 ± 0.3. Food was delivered only when inflated swim bladder was observed in more than 80% of the population. Exogenous feeding was based on rotifers (Brachionus sp.) at 5 individuals ml−1 enriched with proteins and PUFA  until 10 days post hatching (dph) while phytoplankton (Chlorella sp.) was supplied until 10 dph. Enriched Artemia nauplii  were delivered since 10 dph until 50 dph at 0.5 to 1.0 individual ml−1. From 30 dph, larvae were offered dry feed  using automatic feeders. On day 50 dph, the type of the dry feed changed to PROTON 3–5 . During larval rearing and pre-weaning a sample of 10 larvae was taken daily to determine the morphological characteristics and record of total length while 2 times per week weight measurements were also performed with a sample of 10 individuals. The trial lasted until individuals completed the formation of their fins on 45 days post hatch (dph).Batches of fertilized European sea bass eggs were obtained from a private fish farm (DIAS S.A.) and transferred to the installations of the Institute of Aquaculture, Hellenic Center for Marine Research . Larval rearing was performed applying the pseudogreen-water technique−1 and then to netting and air exposure for 1 min until distribution to the incubation tanks. Pre-larvae  were exposed to chasing with a net for 20 sec and high aeration  for 90 sec. Larvae  were exposed to high aeration (as above), chasing with a net for 20 sec, confinement (collection in beakers), and air exposure for 5 sec before being transferred to baskets within a 500 L tank. Samples for molecular and endocrine (cortisol) analysis were collected with a net at 0, 0.5 h, 1 h, 2 h and 24 h post-stress, flash frozen in liquid N2 and stored at −80°C. All experiments were performed in accordance with relevant guidelines and regulations. The laboratories of the Hellenic Centre for Marine Research are certified and obtained the codes for breeding animals for scientific purposes (EL-91-BIO-04). Furthermore all procedures involving the handling and treatment of fish used during this study were approved by the HCMR Institutional Animal care and use committee following the Three Rs  guiding principles for more ethical use of animals in testing, in accordance to Greek (PD 56/2013) and EU (Directive 63/2010) legislation on the care and use of experimental animals.Samples were collected at six different embryonic and larvae phases  and fixed in buffered formalin. Samples were dehydrated in a 70–95% ethanol series and embedded in glycol methacrylate resin . Serial sections were obtained at a thickness of 3–5 μm on a microtome  using disposable blades. After drying, slides were stained with methylene blue/azure II/basic fuchsinet al.et al.0. All wells received 90 μl (333 Bq) of 3H-hydrocortisone  solution and plates were incubated at room temperature for 4 hours. The plates were then washed three times with wash buffer and after the final wash step, all wells received 200 μl of ‘Optiphase hisafe-3 scintillation liquid’ . Beta-emission was quantified by a 3 min count per well using a Microbeta Plus .Samples were homogenized according to Stouthart Samples of embryos, pre-larvae and larvae were let to thaw on ice, disrupted and homogenized using the TissueRuptor  for 20 s in 600 μl RLT plus buffer . Total RNA was isolated with the RNeasy Plus Mini Kit . RNA yield and purity was determined by measuring the absorbance at 260 and 280 nm using the Nanodrop® ND-1000 UV–Vis spectrophotometer , and its integrity was tested by electrophoresis in 1% agarose gels. Reverse transcription (RT) was carried out using 1 μg RNA with QuantiTect Reverse transcription kit (Qiagen).gr1), Glucocorticoid Receptor 2 (gr2), Mineralcorticoid Receptor (mr), Corticotropin Releasing Factor (crf), eukaryotic Elongation Factor 1 (eEF1a), 40S Ribosomal protein S30 (Fau) and ribosomal 18S RNA (18S) were obtained by a previous work of our group3011β-hydroxylase (11β-hydroxylase) was based on the available sequence with accession number AF449173.2β-Hydroxysteroid Dehydrogenase type II (11β-hsd2) were designed based on the conserved regions as revealed by multiple sequence alignments of other teleost fish.Primers for Glucocorticoid Receptor 1  was a 21-mer with the sequence 5′ -GGAGGAGGATTGCTGAGAACG- 3′ and the reverse primer (11β_Rev) an 19-mer primer with the sequence 5′ -AGAGGACGACACGCTGAGA- 3′. For 11β-hsd2, the sequence of the forward primer (hsd_Fwd) was 5′ -CACCCAGCCACAGCAGGT- 3′ and the reverse primer (hsd_Rev) had the sequnece 5′-ACCAAGCCCCACAGACC- 3′. The products of each primer pair were further checked with sequencing in order to confirm that they amplify the desired genes.In the case of gr1, gr2, mr, crf, 11β-hydroxylase and 11β-hsd2 was determined with quantitative polymerase chain reaction (qPCR) assays using the KAPA SYBR® FAST qPCR Kit (Kapa Biosystems). Reactions were cycled and the resulting fluorescence was detected with MJ Mini Thermal Cycler (Bio-Rad) under the following cycling parameters: 95°C for 3 min (HotStarTaq DNA Polymerase activation step), 94°C for 15 s (denaturation step), 60°C for 30 s , 72°C for 20 s (extension step), 40 cycles (step 2–step 4). Levels of gr1, gr2, mr, crf, 11β-hydroxylase and 11β-hsd2 mRNA were normalized based on the reference genes 18S, eEF1a and Fau. A relative standard curve was constructed for each gene, using 4 serial dilutions (1:5) of a pool of all cDNA samples. We also performed geNORM analysiseEF1a and 18S.The mRNA expression of genes encoding for P < 0.05.All statistical analyses were performed with SigmaPlot 11.0 (Jandel Scientific). All data are presented as means ± standard error of the mean (SEM). Data were initially screened for normality and homogeneity. Statistical comparisons of temporal patterns of cortisol and gene expression of unstressed specimens (0 h) between the different developmental stages were made using one-way ANOVA. Statistical comparisons of cortisol content and gene expression between the different time points following exposure to a stressor and the various developmental points/stages tested were made using two-way ANOVA. Holm-Sidak's honestly significant difference test for multiple comparisons was used to determine significant differences among groups. The significant level used was A.T. and M. P. wrote the main manuscript. A.T. and N.P. carried out larvae rearing and sampling. M.G. and G.F. made cortisol measurements. P.K. carried out histology experiments and prepared figure 1. A.T. prepared figures 2–7. All authors reviewed the manuscript.


The recovery of precious metals from waste printed circuit boards (PCBs) is an effective recycling process. This paper presents a promising hydrometallurgical process to recover precious metals from waste PCBs. To simplify the metal leaching process, large pieces of PCBs were used instead of a pulverized sample. The chemical coating present on the PCBs was removed by sodium hydroxide (NaOH) treatment prior to the hydrometallurgical treatment. Among the leaching reagents examined, hydrochloric acid (HCl) showed great potential for the recovery of metals. The HCl-mediated leaching of waste PCBs was investigated over a range of conditions. Increasing the acid concentration decreased the time required for complete metal recovery. The shaking speed showed a pronounced positive effect on metal recovery, but the temperature showed an insignificant effect. The results showed that 1 M HCl recovered all of the metals from 4 cm × 4 cm PCBs at room temperature and 150 rpm shaking speed in 22 h. The production of electric and electronic equipment (EEE) is rapidly increasing due to the revolution of informatics technology343), HCl, sulfuric acid (H2SO4) and aqua regia for the recovery of metals from waste PCBs11314Several methods based on pyrometallurgy81117et al.The metals present on the PCB are covered by a chemical coating32033, HCl, and H2SO4 solutions are often used as leaching agents in hydrometallurgical treatments. Hydrometallurgical treatments have more flexibility during the upscaling and control processes23243, H2SO4, acetic acid (C2H4O2) and citric acid (C6H8O7) were used as lixiviants for metal recovery from PCB pieces. It was observed that HCl and HNO3 removed all of the metal present on the PCB pieces , C2H4O2 (96 h) and C6H8O7 (364 h) treatments showed poor metal solubilization compared with HCl and HNO3, with only 8.8, 9.89 and 19.57% recovery of Cu achieved using H2SO4 (96 h), C2H4O2 (96 h) and C6H8O7 (364 h), respectively  showed similar results to those obtained at 150 rpm . Therefo00 rpm shet al.2) when it reacts with HCl. However, Pb can still be leached by applying HCl. According to Zhang et al.2+ complexes with Cl, as per the following reaction:Xiu 2 in the present study) anion under highly acidic conditionsAu has little solubility in HCl. However, Au metal dissolves in the presence of an oxidant (air) and a halide  (data not shown). These results contrast those obtained in previously published reports. Parhi et al.et al.21213+ concentration in the solutions and cause low oxidation. It would also slow down the mass transfer by forming a layer on the surface of the PCB powder−, SO42−), concentration of metal(s), impurities, and economy of the process. However, a very limited number of articles is available on the purification and recovery of individual metals from PCB leach liquor1Another experiment was carried out to study the effect of the size of the PCB piece on metal leaching performance. Here, 8 h was required for complete metal recovery from a 2 cm × 2 cm PCB piece. The time required for complete metal recovery increased with the size of the PCB piece; 22 and 25 h were required for 4 cm × 4 cm and 6 cm × 6 cm PCB pieces, respectively . These rIn the present study, hydrometallurgical leaching was applied to waste PCBs to design a metal recovery process. The results show that the hydrometallurgical recovery of metals from large pieces of waste PCBs is possible. The HCl took less time for the metal recovery and was thus a very effective leachant. The use of large pieces of PCBs for metal recovery will facilitate the recycling of the remaining boards. It will also avoid the problem of precipitate contamination when recovering the metals from the leach liquors. Hence, an economical hydrometallurgical process can be established for the recovery of metals from waste PCBs.All tests were performed in three replicates.3 65%), sulfuric acid (H2SO4 95%), hydrochloric acid (HCl 37%), citric acid (C6H8O7) and acetic acid (C2H4O2) were purchased from Sigma-Aldrich.Nitric acid , and gold-plated terminals of connectors, are attached to the substrate, depending on the specific functions and applications. These components also contain various precious metals1et al.The PCBs have a chemical coating (solder mask), which is commonly made of epoxy. The solder mask covers the metals mounted on the PCBs and does not allow the leaching agent to penetrate through it for the recovery of metals. Adhapure 3, H2SO4, C2H4O2 and C6H8O7. For this study, a 1 M solution (100 ml) of each acid was used. Deionized-distilled water was used in the preparation of the acid solutions. The effect of the incubation time on the leaching performance of each acid was studied. The experiments were carried out by separately immersing the PCB pieces (4 × 4 cm) in 1 M leaching solutions (100 ml) of HCl, HNO3, H2SO4, C2H4O2 and C6H8O7 in 500 ml beakers. The beakers were incubated while covered on an orbital rotary shaker at 150 rpm and room temperature. The PCB pieces were removed after the respective leaching periods, and photographs of the PCB surfaces were taken. Small aliquots of the samples were also taken and sent for ICP analysis to determine the metal content. Further process optimization was carried out using HCl because the best results were obtained using HCl. The effect of the acid concentration on the time required for metal recovery from the PCB piece was studied. Leach solutions (100 ml) were prepared at the required concentration of HCl (1–6 M). The beakers were kept for incubation on an orbital rotary shaker at 150 rpm and room temperature. A study was carried out to find the effect of the rotation speed on the metal recovery. For this study, 100 ml of 1 M HCl solution was used. The PCB pieces were covered with HCl solution in 500 ml beakers, and these beakers were incubated in an orbital rotary shaker at 0–150 rpm at room temperature for 22 h. The effect of temperature on the metal recovery was studied. The PCB pieces were immersed in 100 ml of 1 M HCl solution in 500 ml beakers that were incubated at various temperatures (room temperature-60 °C) and 150 rpm for 22 h. The effect of the size of the PCB on the time required for metal recovery was also studied. PCB pieces with sizes of 2 cm × 2 cm, 4 cm × 4 cm and 6 cm × 6 cm were immersed in 100 ml of 1 M HCl solution in 500 ml beakers at 150 rpm and room temperature.Aqua regia was used to fully leach the metals from the PCB specimen. Following the filtration of the digestion solution, the metal concentrations were determined by inductively coupled plasma resonance spectroscopy (ICP), with the results shown in How to cite this article: Jadhav, U. and Hocheng, H. Hydrometallurgical Recovery of Metals from Large Printed Circuit Board Pieces. Sci. Rep.5, 14574; doi: 10.1038/srep14574 (2015).


These proteins did not display a shift in mobility in native gels when incubated with divalent cations other than Ca2+ – such as Mg2+, Ba2+, and Sr2+, even at 10× concentrations. The data is similar to that obtained with another NCS protein, neurocalcin delta (Viviano et al., 2016, “Electrophoretic Mobility Shift in Native Gels Indicates Calcium-dependent Structural Changes of Neuronal Calcium Sensor Proteins”, In this data article we show the specificity of the Ca Specifications TableValue of the data1.NCS proteins primarily bind and respond to calcium.2.2+However, they have been shown to bind other metal ions, particularly Mg3.2+ was shown to affect the function of GCAP1 Binding of Mg4.2+, Ba2+, and Sr2+, even at 10× concentrations.The data presented reveal that the mobility retardation on native gels is specifically induced only by calcium and not by other tested divalent cations – Mg5.It may be possible to correlate binding of small ligands to structural changes detectable as electrophoretic mobility shift.12+, Mg2+, Ba2+, or Sr2+) and were subjected to electrophoresis in native gels. Representative images are provided in t-tests are also presented for each group against the control group . Mobility retardation is observed with the addition of calcium but not with any of the other divalent cations, even at concentrations ten times that of calcium.Bacterially expressed proteins – Hippocalcin (HPCA), Guanylate Cyclase Activating Proteins 1 and 2 (GCAP1 and GCAP2) – were purified in their myristoylated forms. The proteins were incubated with the divalent cation  to remove any residual calcium. Calcium removal was through the use of Chelex-100 resin  using standard procedures In order to determine if divalent cations such as magnesium, strontium or barium could induce a mobility shift in the same way that calcium can


HSCR) is a congenital disorder caused by the defective function of the embryonic enteric neural crest. The impaired migration of embryonic enteric neural crest plays an important role in the pathogenesis of this disease. Recent studies showed that the ARP2/3 complex and RAC isoforms had effects on actin cytoskeleton remodelling, which contributes to migration. Moreover, some regulatory relationships were identified between ARP2/3 complex and RAC isoforms. Although microRNAs (miRNAs) have been known to modulate target gene expression on the post‐transcriptional level, little is known about the regulation among miRNAs, ARP2/3 complex and RAC isoforms. Here, we report that down‐regulation of ARP2 and ARP3, two main subunits of ARP2/3 complex, suppressed migration and proliferation in 293T and SH‐SY5Y cell lines via the inhibition of RAC1 and RAC2. Meanwhile, as the target genes, ARP2 and ARP3 are reduced by increased miR‐24‐1* and let‐7a*, respectively, in 70 HSCR samples as compared with 74 normal controls. Co‐immunoprecipitation showed that aberrant reduction in ARP2 and ARP3 could weaken the function of ARP2/3 complex. Our study demonstrates that the miR‐24‐1*/let‐7a*‐ARP2/3 complex‐RAC isoforms pathway may represent a novel pathogenic mechanism for HSCR.Hirschsprung disease ( RET, have been identified to be associated with the pathogenesis of HSCR Hirschsprung disease (HSCR) is a congenital abnormality disorder and is caused by the absence of ganglion cells (aganglionosis) in a variable length of the gastrointestinal tract, with an incidence of approximately 1 in 5000 births The actin cytoskeleton has important roles in a wide range of cellular processes, such as motility and pseudopod movement. Various factors contribute to the regulation of actin, including the actin‐related protein 2/3 (ARP2/3) complex. As a branched actin nucleator, it consists of seven subunits, including two main subunits (ARP2 and ARP3) and other five subunits (ARPC1‐5) that provide a framework for positioning the dimer formed by ARP2 and ARP3 MiRNAs are small, non‐coding RNA molecules, which act as repressors in post‐transcriptional control. They are about 19–25 nucleotides long and negatively regulate their target genes by base‐pairing with the complementary sequences in the 3′‐untranslated regions (3′‐UTR) of the target mRNAs This research was approved by the Institutional Ethics Committee of Nanjing Medical University. All research activities involving human cases were carried out in accordance with government policies and the Helsinki Declaration. 70 HSCR patients with a pathological diagnosis and 74 matched controls were selected. Colon tissues were collected from patients undergoing surgical treatment at Nanjing Children's Hospital Affiliated to Nanjing Medical University from October 2009 to April 2013 (the NJMU Birth Cohort). Tissues were collected with informed consent and agreement from patients and were stored at −80°C. All the patients were diagnosed by barium enema and anorectal manometry evaluation before surgical procedures. After surgery, pathological examination was taken to make a definite diagnosis. The control samples were collected after an enterectomy intussusception or inguinal hernia without the ischaemic or necrotic parts.® MicroRNA Assays  were chosen for has‐miR‐24‐1* and has‐let‐7a*. Sn‐RNA U6 was used as a control for normalization. We used 1 μg total RNAs as the template and reverse transcription conditions were as follows: 16°C, 30 min., 42°C, 30 min. and 85°C, 5 min. The cDNAs were 1:40 diluted, and PCR was performed for 5 sec. at 95°C and for 30 sec. at 60°C, 40 cycles.Total RNA was extracted from tissue samples and cell lines using Trizol reagent  according to the manufacturer's instructions. The qRT‐PCR was carried out to measure the expression levels of miRNAs and mRNAs. For miRNAs, TaqManGlyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as an internal control for mRNAs. We extracted total RNAs (500 ng) under 37°C for 15 min. and 85°C for 30 sec. for reverse transcription using the reverse transcription kit . PCR amplification was performed in an ABI 7900 HT  under 5 sec. at 95°C and for 30 sec. at 60°C for 40 cycles. All the primers were shown in Table Total proteins were collected from tissue samples and treated cell lines with RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA‐free, EASYpack . The BCA method was used to determine protein concentrations. Proteins were subjected to 12% SDS‐PAGE and then transferred onto a nitrocellulose membrane. Membrane was blocked using 5% skimmed milk and incubated with antibodies. Primary antibodies of ARP2 (sc15389), ARP3 (sc15390), RAC1 (sc95) and RAC2 (sc96) were from Santa Cruz  and RAC3 (Cat. no.: 5659‐1) was from Epitomics, Inc. . The target proteins were detected using ECL reagent kit . A GAPDH antibody  was used for endogenous control.2. Synthetic miRNAs of negative control, miR‐24‐1* and let‐7a*, and siRNAs of ARP2, ARP3, RAC1 and RAC2  were used in cell transfection coupled with Lipofectamine 2000 Reagent  following the manufacturer's instructions. RNA oligos mentioned in this study were listed in Table Human 293T and SH‐SY5Y cell lines were purchased from American Type Culture Collection , and were cultured in a complete growth medium DMEM  supplemented with 10% foetal bovine serum (10% FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C, 5% CO6 cells/ml), 100 μl of which was added to the upper compartment of the chambers . A conditioned medium with 10% (v/v) FBS was used as a chemoattractant and placed in the bottom compartment of the chamber. After 24 hrs of incubation, the invaded cells were stained with the crystal violet staining solution (Beyotime) for 15 min., and the cells were quantified from five different fields under 40× magnification (five views per well). All these experiments were replicated three times.After transfection, the harvested cells were suspended in serum free medium .Cell proliferation was determined by the Cell Counting Kit 8  after transfection for 24 hrs. 10BD Biosciences FACS Calibur Flow Cytometry  and Annexin V‐FITC/Propidium Iodide Kit  were used for detection of apoptosis and cell cycle, respectively, and all the tests were repeated three times.The 3′‐UTR sequences of ARP2 and ARP3, containing the putative target sites for miR‐24‐1* and let‐7a*, respectively, were inserted into the KpnI and SacI sites of pGL3 promoter vector . The resulting constructs were named pGL3‐ARP2, pGL3‐ARP2‐mut, pGL3‐ARP3 and pGL3‐ARP3‐mut. Cells were plated in 24‐well plates and cotransfected with 100 ng of pGL3‐ARP2, pGL3‐ARP2‐mut and 50 nM miR‐24‐1* mimics and negative control using Lipofectamine 2000 (Invitrogen). For pGL3‐ARP3, pGL3‐ARP3‐mut and let‐7a* mimics, we performed the same procedure towards pGL3‐ARP2, pGL3‐ARP2‐mut and miR‐24‐1*. A renilla luciferase vector pRL‐SV40 (5 ng) was also cotransfected to normalize the difference in transfection efficiency. Luciferase activity levels after 48 hrs incubation were measured using a Dual‐Luciferase Reporter Assay System  according to manufacturer's instructions. Transfection was repeated in triplicate.Cells were transfected for 24 hrs with siRNA‐ARP2 or siRNA‐ARP3 for co‐immunoprecipitation (co‐IP). Cells were harvested and lysed in cold RIPA Lysis Buffer (Beyotime) in the presence of 1 mM PMSF. The cell extracts were centrifuged at 10,000 × g at 4°C for 15 min., and supernatants were incubated with protein A/G agarose beads (Beyotime) or a control IgG (Beyotime). Cell lysates containing 1 mg of protein were incubated with 2 μg of anti‐ARP2 or anti‐ARP3 antibody at 4°C for 1 hr, followed by incubation overnight with protein A/G agarose beads. The immunoprecipitates were collected and washed twice with the lysis buffer. 5× SDS loading buffer was added and incubated at 95°C for 5 min. for SDS‐PAGE and Western blotting was subsequently performed with indicated antibodies.U‐test, and the data obtained from the cell lines are presented as mean ± S.E.M. from three separate experiments, which are analysed by double‐sided Student's t‐test. P < 0.05 was considered statistically significant.The data from the tissue samples in this study are presented as box plot of the median and range of log‐transformed relative expression level using Mann–Whitney Colon tissues taken from 70 patients pathologically diagnosed with HSCR were used for this study. Seventy‐four colon tissues from normal individuals were used as controls. The ages of patients with HSCR and of control groups were 3.12 ± 0.23 and 2.82 ± 0.20 months old respectively. The gender ratio was 54/16 and 61/13 , and the bws were 4.86 ± 0.10 and 4.75 ± 0.14 kg respectively. The 70 patients included in this study consisted of 33 short‐segment and 37 long‐segment. All the information demonstrated no statistical differences in the specimens obtained between HSCR patients and controls . The data showed that down‐regulation of ARP2 and ARP3 resulted in reduced cell proliferation and migration  and MICRORNA.ORG (http://www.microrna.org/) to backward predict the upstream regulators of ARP2 and ARP3. The results indicated that miR‐24‐1* and let‐7a* have an ilka combination with the 3′‐UTR of ARP2 and ARP3 mRNAs.We used DIANA‐MICROT .The ENS originates from the neural crest cells, and the migration of ENCCs from the foregut to the rectum takes 7 weeks to occur in human embryonic development. During this process, the alteration of internal environment and aberrant expression of genes or molecules may contribute to aganglionosis etc. We found that no significant difference was presented between the case and control group. Besides the classic disease‐causing genes, such as RET and SOX10, many other genes have been found to be associated to ENS development include Meis3, FOXA1, NTF‐3 The pathogenesis of HSCR contains different aspects, but one main reason underlying its development is the aberrant migration of ENCCs during embryogenesis. Current research on the disease has a variety of directions, including epigenetics, environmental chemical exposure, enteral environment and so on. An unpublished study in our group aims to investigate the single nucleotide polymorphisms in the patients with HSCR. The patients used in the detection covered the patients in this study. The Exome‐wide scan was performed by using the Illumina Human Exome Bead Chips. We analysed the genes which were reported to be associated with HSCR including RET, EDNRB, NRG1, SOX10, via RAC in glial cells Based on the reported function of the ARP2/3 complex in vitro experiments. We transfected ARP2 and ARP3 siRNAs into both cell lines, and the results showed that RAC1 and RAC2 were down‐regulated, accompanied by the inhibition of migration and proliferation. With such findings, we speculated that the RAC isoforms are potential downstream targets of ARP2 and ARP3 and might play a significant role in the etiologic mechanism of HSCR. The cotransfected group was set to investigate a possible synergistic action of ARP2 and ARP3 siRNAs. Our data showed that RAC1 and RAC2 were not significantly down‐regulated compared with each single siRNA‐transfected groups. Meanwhile, as ARP2 and ARP3 were two subunits of a complex, we performed co‐IP experiments to ensure their association after inhibition by the corresponding siRNAs. The observations from this study indicates that a decrease in the levels of ARP2 or ARP3 could lead to a reduced association, which further support an involvement of the ARP2/3 complex.To further support the possible presence of this pathway, we determine the levels of the RAC isoforms in cases and control samples. The results demonstrated the both RAC1 and RAC2 were down‐regulated in HSCR, whereas RAC3 showed no difference, which suggested that RAC1 and RAC2 may be involved in the process of HSCR. In our study, three members of RAC isoforms presented different expression level in HSCR. According to previous studies In vitro experiments showed that increased levels of miR‐24‐1* and let‐7a* suppressed migration and proliferation, which also decreased ARP2, ARP3 and RAC isoforms at mRNA and protein levels without a noticeable synergistic action. We finally ascertained our assumption that the miR‐24‐1*/let‐7a*‐ARP2/3 complex‐RAC isoform pathway plays a role in HSCR.As we transfected both siRNAs of RAC1 and RAC2 into both cell lines, the proliferation and migration were inhibited, without the synergistic action observed. To examine the initial status of miR‐24‐1* and let‐7a*, we first confirmed their expression levels in tissues samples, both of which were shown to be decreased in HSCR patients. Dual‐luciferase reporter assay was then performed to illustrate the potential regulatory relationships of miR‐24‐1*/ARP2 and let‐7a*/ARP3. In conclusion, our study identified a novel pathway that is involved in the pathogenesis of HSCR. Some small molecular inhibitors of ARP2/3, miR‐24‐1* and let‐7a* regulate the heptamer. Meanwhile, the ARP2/3 complex suppresses migration and proliferation by down‐regulating RAC isoforms in aganglionosis Fig. . MoreoveThere are no conflicts of interest.Figure S1 RNA oligos showed no influence on apoptosis and cell cycle.Click here for additional data file.Figure S2 Transfection efficiency of siRNA‐ARP2 and siRNA‐ARP3.Click here for additional data file. Click here for additional data file.


However only a few reports showed the tumor forming ability of SaOS2 cells on CAM.Osteosarcoma is the most common primary tumor that affects usually children. Due to its cellular complex and osteoid formation it is very difficult to understand the mechanism behind the progressiveness of osteosarcoma. Various animal models are available to study the issue but they are time consuming and costly. We aimed to understand the progressiveness and invasiveness of osteosarcoma induced by SaOS2 cells using chicken chorioallantoic membrane. CAM is a well-established model which allows Angioquant software under different cellular volume. The invasiveness was analyzed by histological staining. Involvement of angiogenic factors at differential stage of progressiveness was confirmed by the molecular and protein level expression analysis.Angiogenic ability of SaOS2 cells on CAM was validated by various methods. Angiogenic ability was scored by direct visualization and scanning microscopic analysis. The sprouting ability and growth of the vessel was measured by SaOS2 cells induces sprouting angiogenesis on CAM and shows its aggressiveness by rupturing the ectodermal layer of the CAM. Growth and development of osteosarcoma depends mainly on the activation of VEGF165, MMP2 and MMP9. CAM able to reproduce angiogenic response against the stimulation of SaOS2 cells exactly as in other animal models without inflammatory reactions.in vivo model for studying the aggressiveness and tumor progression of osteosarcoma using various angiogenic techniques in an easily, faster and affordable way. We further provided insight about the involvement of various angiogenic growth factors on the development of osteosarcoma which will enable to find the suitable therapeutic molecule for the treatment of osteosarcoma. CAM model could provide a wide space using modern techniques like micro array or in situ hybridization to have a better understanding about the progression and invasiveness of osteosarcoma cells to develop suitable therapeutic molecules.CAM is an excellent alternative  Osteosarcoma is the most common malignant bone tumor that affects mostly children. So far tremendous advanced therapies are available for the management of osteosarcoma, but the survival rate is comparatively poor . VariousCAM is a well-established model in the field of angiogenesis and is widely used for the monitoring of tumor angiogenesis . CAM hasin vivo animal models this assay can be useful for having wide range of experimental analysis to have a better understanding about osteosarcoma for therapeutic approach.In this concern, we aimed to validate the angiogenic efficacy of human osteosarcoma derived SaOS2 cell line using chicken late CAM assay as a model system. The model has been used to understand the interactive and invasive character of SaOS2 cells with its surrounding stroma as an indication of its angiogenic ability which in turn favor tumor growth and metastasis. The angiogenic and tumor progressive effect of SaOS2 cells was analyzed under three different cellular volumes on CAM to have a better understanding about the progressiveness of the tumor in stepwise manner. We demonstrated that SaOS2 cells are able to induce rich vascularization at the implanted area which can be directly visualized and quantified and also provided idea about the invasiveness and progressiveness with reproducible similar key features of human osteosarcoma growth at its different stages of progression with large scale analysis. Since, CAM assay is reproducing the characterization of osteosarcoma exactly as in various 5, 6 × 105 and 12 × 105 cells/volume) to induce angiogenesis on CAM vascular bed was analyzed and is compared with control incubated with DMEM alone. Figure 1e, f, h, i, k and l). CAM incubated with 6 × 105 and 12 × 105 cells/volume shows numerous scattered and spoke wheel pattern of allantoic vessel growth around the sponge  suggestive of the sprouting angiogenic ability of SaOS2 cells within shorter time period of incubation. Reddishness around the sponges directly indicates the richness in the vascularization which correlates with the ability of SaOS2 cells to form solid tumors on CAM within 96 h of incubation. The newly formed vessels grow perpendicular to the plane of the CAM inside the sponge. The average number of allantoic vessels between the sponge and the surrounding mesenchyme is significantly higher (p = < 0.001 and p = 0.005) for 6 × 105 and 12 × 105 cells/volume respectively   and graph (C) shows that the number of vessels junctions was significantly increased (p = < 0.001) for all three volumes of cell line when compared with control at 96th hour of incubation. Al together our data suggest that the ability of SaOS2 cells to accelerate the growth and sprouting of blood vessels is irrespective of the number of cells but it depends on the incubation period. In this study we found that SaOS2 cells are able to induce and accelerate significant level of angiogenic responds within 96 h of incubation. This pattern of experiment is useful to analyze the differential stage of progression of tumor development and also helpful for understanding the anti angiogenic potential of agents with beneficial therapeutic approach against osteosarcoma within shorter incubation period with a direct approach on the tumor growth. The value for length, size and junctions at 0 h was taken as one.Next we quantified the angiogenic ability of SaOS2 cell line from the images taken at 0 and 96 h of incubation using Image J and es) Fig.  suggestiaph Fig.  indicateb, c and d). The key finding is that the ectodermal layer is ruptured due to the invasiveness of the sarcoma cells and it also shows the growth of blood vessels at the ruptured area. Presence of nucleated erythrocytes (slightly pink colored) at the blood vessel of the ruptured area indicates that these vessels were originated from the CAM vascular bed and is penetrated towards the progressive front of the tumor to support the growth and invasiveness of the tumor. The ruptured area also shows large number of monocytes  without inflammatory signs. Control CAM shows uniform thickness at the ectodermal layer along with active proliferation of fibroblast cells under culture media  and also shows the presence of nucleated erythrocytes (pink in colour) in between of the ectodermal and mesodermal layer. Individually, CAM incubated with 6×105 and 12 × 105 cells/volume shows more prominent evidences of invasiveness of osteosarcoma  and CAM incubated with 3×105 cells/volume shows clumsiness at the invading area because of early penetration and invasiveness of the tumor cells. This kind of experimental approach provided a step by step analysis of the progression of tumor growth that will enables to study the mechanism of early progression and invasion of the osteosarcoma. Followed by the morphological changes we also calculated the average tissue thickness of the CAM from haematoxylin and eosin stained cross vertical sections. The distance between the chorionic and allantoic epithelial layers was measured in micrometer excluding the tumor growth part. Figure p = < 0.001) both for 6 × 105 and 12 × 105 cells/volumes of cells due to capillary migration from stroma to the inner shell membrane (ISM). This data give direct evident about the ability of SaOS2 cells to penetrate deep into the ectoderm of the CAM towards the progression and invasiveness of tumor growth which is majorly supported by the angiogenic response from CAM. Since CAM doesn’t have immunoreaction at this developmental stage study using this model could provide a pour analytical data without immune compromise for the aid of anticancer therapy for osteosarcoma.The penetrating and invading ability of SaOS2 cells into the ectodermal layer of the CAM and also its potential to attract CAM blood vessels at the invading area for the progression of tumor growth and survival was analyzed. Morphological changes were observed from haematoxylin and eosin stained cross sections of CAM after 96 h of incubation Fig. . The com35, 6 × 105 and 12 × 105 cells/volume) and control with DMEM alone. Result shows that, CAM incubated with 6 × 105 and 12 × 105 cells/volume induces a significant increase in the mRNA level expression of VEGF165 (p = < 0.001), MMP2 (p = < 0.001) and MMP9 (p = < 0.001) after 96 h of incubation. The expression of FGF2 and NOS were found to increase significantly with 12 × 105 cells/volume (p = < 0.001and p = 0.001) after 96 h when compared with control. The RT-PCR analyzes indicates that the direct contact of SaOS2 cells with CAM is sufficient to induce significant level of angiogenic response after 96 h of incubation. It was also noticed that the development of tumor growth by SaOS2 cells on CAM is majorly depends on the activation of VEGF165, MMP2 and MMP9 and is in concordance with other animal model studies. The result also indicates that CAM vasculature is highly responsive towards the angiogenic signals produced by the tumor cells implanted on it and this positive response is important to have a better understanding about the development of specific angiogenic groth factor targeted anti angiogenic molecules for the treatment of osteosarcoma at it early stage of development.Potential of SaOS2 cells to upregulate the transcription level of main angiogenic growth factors like VEGF165, FGF2, MMP2, MMP9 and NOS on CAM vasculature was analyzed using reverse Transcriptase PCR method. Figure 5 cells/volume shows increased capillary growth in the form of vascular mesh like structures with extensive inter connecting capillaries together with multiple junction points  supplemented with 10 % fetal bovine serum (FBS) and 1 % penicilline/streptomycine,  at 37 °C in a humidified 5 % CO2 incubator until it get full confluent .Prior implantation on CAM, cell suspensions were prepared by detaching cells with trypsin/EDTA . Cells were centrifuged at 1200 rpm for 5 min, washed twice in culture medium without FBS and suspended in DMEM alone medium at a final concentration of each 30, 60 and 120 lakhs cells per 20 μl volume .2 was opened in the egg shell at the opposite to blunt edge and sealed with paraffin film to prevent dehydration. The eggs were returned for further incubation. On embryonic day 9, gelatin sponges of size (Jhonson & Jhonson Pvt Lmtd) cut into a size of 1 mm3 were soaked with 30, 60 and 120 lakhs cells per 20 μl volume of medium were placed on the top of growing CAM under sterile condition. Control CAM was incubated with 20 μl of DMEM alone. The window was closed with a transparent adhesive tape and the eggs were returned for incubation till day 13 (for 96 h). The experimental groups were divided into 4 of each containing 40 numbers of eggs. Group1 treated with DMEM alone as control, group 2, 3 and 4 were treated with 20 μl of 30, 60 and 120 lakhs of SaOS2 cells per volume respectively. In ovo CAM was photographed at 0, 24, 48, 72 and 96 h using Cannon digital camera of 12 × 5.0 Mega Pixel (Power Shot A95) and the images were subsequently analyzed with Image J and Angioquant Toolbox, MATLAB 6.5 Software’s to measure the growth of the vessels by means of its length, size and number of vessels junctions [Fertilized White Leghorn chicken eggs weighing 50 ± 2 g  were incubated at 37 °C in a humidified atmosphere (at >60 % relative humidity) as per the protocol for the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) method. On day 3 of post incubation, 2 to 3 ml of albumin was withdrawn, using a 21 gauge needle, through a small opening at the large blunt edge of the egg to minimize adhesion of the shell membrane with CAM. A square window of 1 cmunctions , 19. TheCAM). In paraffin-embedded tissue, material shrinkage is estimated to be ~25 % relative to the fresh material. As all tissues were prepared similarly, tissue shrinkage is same for all CAM zones. Thus, shrinkage corrections are unnecessary for the comparisons of tissue thickness [CAM incubated with SaOS2 cell line was flooded with Bouin’s fixative solution after 96 h of incubation and the treated area was removed carefully, dehydrated and embedded in paraffin wax. Vertical cross sections of 7 μm in thickness were taken using Rotary Microtome . After staining with haematoxylin and eosin, sections were mounted with DPX and observed using light microscope at 40× magnification for qualitative assessment. The images were recorded at 10× magnification using Nikon D70 DSLR (6.1 megapixel) camera attached with light microscope . Thicknehickness .zol reagent according to the manufacture’s protocol . The quantity and the purity of the isolated RNA was checked with UV-Visible spectrophotometer and after running on 1 % agarose gel electrophoresis, respectively. cDNA was synthesized for each group from 5 μg of total RNA using ImProm-11™ Reverse Transcriptase kit with Oligo (dt)  based on manufacture’s protocol . PCR amplification was performed using GoTaq Green Master Mix kit with 1.5 μl of cDNA from each group . PCR reaction was set up based on manufacture’s protocol. Variation in the mRNA expression of pro-angiogenic molecules namely VEGF165 [Scion Image release α 4.0 3.2 software. Specific primer sequences and PCR reaction set up was given in Tables Total RNA was isolated from CAM (10 numbers each) after incubation with SaOS2 cells using TRI VEGF165 , FGF2 [2 VEGF165 , MMP2 [2 VEGF165 , MMP9 [2 VEGF165 , NOS [22 VEGF165  and GAPD VEGF165  were evaAfter 4th day of post incubation with 120 lakhs of cells/volume of SaOS2 cell line, CAM at the area of incubation was dissected out and washed with 1× PBS. The CAM was dried at room temperature without disturbances. The unfolded air dried membranes were glued onto stubs with carbon, spattered with gold  and observed under a Hitachi S-3400 N Variable Pressure Scanning Electron Microscope at an accelerating voltage of 15–30 kV. The images were recorded at a magnification of 200 μM .The deparaffinised and dehydrated CAM incubated with 120 lakhs of cells/volume was allowed to undergo antigen retrieval process using Sodium Citrate (10 mM-pH 6.0) in a microwave oven for 20 min and then washed with DDH2O for 3 × 5 min in 1× PBS (pH 7.3). Normal Goat Serum Blocking Solution  of 50 to 75 μl was added immediately on the sections and incubated for 1 h in a humidified chamber. After washing with 1× PBS, primary antibody of VEGF A , MMP2 and MMP9 (1:200 dilution) was applied on the sections and after overnight incubation rinsed with 1× PBS with 0.05 % of Tween-20. Diluted FITC and HRP conjugated secondary antibodies of 1:40 dilution was applied for 1 h according to manufacturer’s instruction . Counterstaining with haematoxylin and eosin was performed for those sections incubated with HRP conjugated secondary antibody. Images were recorded at 40× magnification using B×51 Olympus Fluorescence Microscope at a wavelength of 515 nm with ASI FISH View 5.5 software for FITC and Light Microscope was used for HRP .n = 3) unless otherwise specified. Data are presented as mean ± SEM and were analysed by Descriptive analysis for ± SEM and One-Way ANOVA analysis of Holm-Sidak Test for appropriate using SigmaPlot 12. P values of p = < 0.001, p = 0.001, p = 0.010 and p = 0.005 were considered for statistical significance.All the experiments were performed in triplicate (


Open beam (free field) and optical-fiber-based measurements at wavelengths of 1302 nm and 1546 nm are reported. Both laboratories’ reference standards were compared by means of a temperature-controlled optical trap detector. Measurements showed a largest difference of less than 3.4 parts in 10 In our previous work –5, we reFor optical fiber power meter measurements, the primary standard for each laboratory is a cryogenic radiometer described in , 8. MostFor this comparison we used a transfer standard designed and built by NIST . The traThe NIST and CENAM measurement systems are very similar; therefore in this section we describe the common measurement system used by both laboratories. The measurement system is depicted in −3 at both wavelengths of 1302 nm and 1546 nm. At CENAM, nine measurement runs were performed with a relative standard deviation of 4 × 10−4 at 1302 nm and a relative standard deviation of 1 × 10−3 at 1546 nm. The results of the comparison are presented in Both participating laboratories used the same optical fiber cable with FC/PC connectors. At NIST, six measurement runs were performed with relative standard deviations of 1.2 × 103, and at 1546 nm the difference was 3 parts in 104 (minus sign for the difference indicates that the Ge trap detector responsivity measured by CENAM is lower than that measured by NIST). The NIST combined standard uncertainty was 2 parts in 103 at 1302 nm and 2.5 parts in 103 at 1546 nm, while that of CENAM’s was 1.4 parts in 103 at both wavelengths of 1302 nm and 1546 nm. k = 1) uncertainties for the laboratories’ reference standards.The standard uncertainties for the optical power measurements were evaluated in accordance with ISO document standards . At 1302−3 at a wavelength of 1302 nm and a relative standard deviation of 1.5 × 10−3 at a wavelength of 1546 nm. At CENAM, nine measurement runs were performed with a relative standard deviation of 6 × 10−4 at 1302 nm and a relative standard deviation of 5 × 10−4 at 1546 nm. The beam size at both wavelengths was 1.7 mm ± 0.1 mm in diameter at the 1/e2 intensity points. The results of the comparison are presented in Each participating laboratory used the same beam size at both wavelengths. At NIST, six measurement runs were performed with a relative standard deviation of 0.7 × 103, and at 1546 nm the difference was 3.4 parts in 103 (minus sign for the difference indicates that the Ge trap detector responsivity measured by CENAM is lower than that measured by NIST). The NIST combined standard uncertainty (k = 1) was 1.7 parts in 103 at 1302 nm and 2.3 parts in 103 at 1546 nm, while that of CENAM’s was 9 parts in 104 at 1302 nm and 1 part in 103 at 1546 nm. k = 2) uncertainties for the laboratories’ reference standards.At 1302 nm, the difference between the NIST and CENAM results was 1.4 parts in 10This optical power meter comparison shows reasonably good agreement within the combined expanded uncertainty between NIST and CENAM. The agreement between these two laboratories is in harmony with the previous international comparisons described in –5. Such


Dr. Chaudhury puts forward an interesting hypothesis that reductions in sarcolemmal nNOSμ might parallel reductions in enteric neuron nNOSα expression, localization, and activity . In otheKey assumptions of this hypothesis include (1) the molecular similarity of the nNOSα and nNOSμ splice variants; 2) the mechanisms regulating nNOSα localization in neurons are similar to those regulating nNOSμ in skeletal muscle cells. The molecular similarity assumption is reasonably based on high protein homology between nNOSα and nNOSμ. Muscle nNOSμ contains an internal 34 amino acid insert in the autoregulatory domain not found in nNOSα  protein is the key scaffold in different neuronal cells of the central nervous system  and the Department of Defense (MD140021). The content is solely the responsibility of the author and does not necessarily reflect the official views of NIAMS or Department of Defense.


To achieve tumor control for radiotherapy, a dose distribution is planned which has a good chance of sterilizing all cancer cells without causing unacceptable normal tissue complications. The aim of the present study was to achieve an accurate calculation of dose for small field dimensions and perform this by evaluating the accuracy of planning system calculation. This will be compared with real measurement of dose for the same small field dimensions using different detectors. Practical work was performed in two steps: (i) determination of the physical factors required for dose estimation measured by three ionization chambers and calculated by treatment planning system (TPS) based on the latest technical report series (IAEATRS-398) and (ii) comparison of the calculated and measured data. Our data analysis for small field is irradiated by photon energy matched with the data obtained from the ionization chambers and the treatment planning system. Radiographic films were used as an additional detector for the obtained data and showed matching with TPS calculation. It can be concluded that studied small field dimensions were averaged 6% and 4% for 6 MV and 15 MV, respectively. Radiographic film measurements showed a variation in results within ±2% than TPS calculation. Human tissue is considered similar to water in terms of its physical properties as it represents 90% of its contents and how it interacts with radiation. For this reason, radiation doses (energy imparted per mass) prescribed to cancer patients typically refer to doses in water. Fundamental to small field dosimetry is the difficulty of linking the measurement in the clinic to that at the international standards laboratory so that the measurement signal is converted to absorbed dose in water . The pre2 is considered outside the conventional treatment field size that needs special attention both in dose measurements and in dose calculations. A more scientific approach is needed to set the criteria which define a small field condition based on the beam energy and the density of the medium [The definition of a small field in radiation dosimetry is currently very subjective and ad hoc. There is no clear consensus definition as to what constitutes a small field. Commonly, a field size of less than 3 × 3 cmthe dimensions of beam the same as projected upon the detectors,suitable dimension of detector used in measurements,small scattered radiation due to small field dimensions.Small field dosimetry plays an important role in modem radiotherapy for many reasons. Treatment planning system commissioning requires the input of beam data, specific to the treatment units. This requires the acquisition of depth dose profiles, beam profiles, and RDFs, as introduced in the previous sections [There are essentially three “equilibrium factors” that determine the scale of the field as small field it is consideration as small field or not:sections .The difficulty in achieving accurate small field dose measurements is similar to the factors which affect specific measurements in the dosimetry of larger fields  but they are accentuated. The loss of lateral charged particle equilibrium generally results in a decreased dose at central axis, rather than just at the beam edges and in the build-up regions. In addition, the narrowing of the beam results in a more peaked lateral dose profile, which escalates the requirement for higher spatial resolution not only in the penumbral regions but also at the central axis.The Linear accelerator used was Elekta model—dual energies 6 and 15 MV photon beam and four electron energies .The treatment planning system used in this work is Precise Plan. The ionization chambers used were Farmer ionization chamber, vented cylindrical ionization chamber for measuring high-energy photon and electron radiation in water or in solid-state material, Semiflex chamber (0.125 cm³), and Pinpoint chamber (0.015 cm³). The type of waterproof chambers that has been specially designed for relative beam profile measurements was done by motorized water. The Pinpoint chamber is ideally suited for measurements of small fields of inner diameter 2.9 mm. Farmer dosimeter model (2570/1B (# 1164)) andradiographic Filmmodel Kodak X-OmatV.The absorbed dose was determined after the mechanical check to ensure the suitability of the machine to perform the dosimetric measurements to ensure the suitability of the machine to perform the dosimetric measurements. The laser lines that compromise the cross wires in the light field area should be checked. The isocentre point for gantry, collimator, and couch rotation should be checked to ensure. Then, adjust the water phantom at 100 cm SSD and locate the ionization chamber at 10 cm depth for 6 and 15 MV photon beam with 90-degree gantry angle, zero-degree collimator angle, and zero-degree couch angle according to IAEA protocol (TRS 398).KT,p (correction factor temperature and pressure) which estimate the effect of pressure and temperature on measurement.Measure the pressure and temperature to calculate the factor When different small fields by field were irradiated to measure absorbed dose for each field. The data carried out by TPS were compared with practical data of ICs.Estimating the standard film to be the reference dose gradient by irradiating different films to gradual from 20 to 100 monitor units. Where the irradiated film placed in the percpix sheets placed at the depth of maximum dose for each energy (1.6 cm for 6 MV and 2.7 cm for 15 MV), process the film and draw an isodose curve through which we can determine the absorbed dose for each irradiated film optical density value can be determined.When irradiating different fields, determining the absorbed dose for each field, and comparing the results with the TPS data.The calculated data of absorbed dose taken by the precise treatment planning system versus the measured data for the three ionization chambers were represented in 2 dimensions at 100 cm SSD, at reference depth of 10 cm in a standard phantom.2) were averaged to 6% for all Ionization chambers used in the current study. The second region shows the difference averaged to 41% between the calculated data by TPS and measured data by ICs for 2 × 2 cm2 field dimensions. The third region illustrates discrepancy exceeding the average of 80% between the calculated data by TPS and measured data by ICs for 1 × 1 cm2 field dimensions.The comparison of the TPS calculated data and measured parameters by different ionization chambers described the difference between calculated and measured data which were observed in three regions. The first one represents the difference between the calculated data by TPS and measured data by ionization chambers data for two small field dimensions  averaged to 4% for the three types of ICs used in present work. These data are in agreement with data published by [2). The measured doses in standard phantom were less than the TPS values for small field dimensions, less than 3 × 3 cm2; this underestimation is due to electronic equilibrium which is not sufficient for fully scattered photons from linear accelerator of photon beam energies 6 and 15 MV [The discrepancies between calculated data and measured values which were observed in two regions are illustrated as follows. The first one represents difference between the calculated data by TPS and measured data by ICs for small field dimensions, (3 × 3 and 4 × 4 cmished by  with res2) at Dmax (depth for measured max dose) 1.6 cm at 50 Mu.The calculated data of absorbed dose taken by the precise treatment planning system versus the measured data by radiographic film were represented in Dmax 1.6 cm, 100 cm SSD, and 50 Mu in solid phantom.2; this difference within ±2% is due to good accuracy of measurement planning system. These data are in agreement with results obtained by [ained by , the cha2, the measured data was decreased within 7% and, for field 4 × 4 cm2, the measured data increased within 6%. This is due to the quantity of scattered radiation which is directly proportional to field area.For field area 1 × 1 cm2) at Dmax 2.7 cm and 50 Mu.The calculated data of absorbed dose taken by the precise treatment planning system versus the measured data by radiographer film were tabulated in Dmax 2.7 cm, 100 cm SSD, and 50 Mu in solid phantom.2. Measured data was increased as field area enlarged from 2 × 2 to 4 × 4 cm2 by constant value of 5% [ue of 5% , 8. Filmue of 5% .2, this underestimation was due to electronic equilibrium which is not sufficient for fully scattered photons from linear accelerator of photon beam energies 6 and 15 MV. On the other hand, field dimensions more than 3 × 3 cm2 have an average of 6% for three types of ionization chambers  used in the current study to 6 MV while 15 MV shows an average of 4% for three types of ionization chambers.The measured doses in standard phantom were less than the TPS values for small field dimensions; for fields less than 3 × 3 cm2; this difference within ±2% is due to good accuracy of measurement planning system. For field area 1 × 1 cm2, the measured data was decreased within 7% and for field 4 × 4 cm2 the measured data increased within 6%. This is due to the quantity of scattered radiation which is directly proportional to field area.The measured data by radiographic film for 6 MV shows mild differences between the TPS and the experimental data for fields 2 × 2 and 3 × 3 cm2. Measured data was increased as field area enlarged from 2 × 2 to 4 × 4 cm2 by constant value of 5%.Small difference existed between the TPS and the experimental data using radiographic film for 15 MV; the results were the same for TPS and measured data, at only field dimension of 1 × 1 cm


Many anticancer treatments, including radiotherapy, act by damaging DNA and hindering cell function and proliferation. H2AX is a histone protein directly associated with DNA that is phosphorylated to produce γH2AX that accumulates in foci in an early response to DNA double‐strand breaks, the most deleterious lesion caused by anticancer therapy. This study reports a γH2AX detection assay that has the potential to be used as a biomarker of response to guide cancer treatment. γH2AX immunostaining was applied to tumour cell specimens obtained using fine needle aspiration (FNA). Liquid‐based cytology and direct smear cytology methods were evaluated and immunostaining protocols established using FNA samples from five cancer patients. The assay was then applied to three patients before and after radiotherapy. Results demonstrate induction of γH2AX foci following treatment, persisting for as long as one week after therapy. Immunostaining for γH2AX has been successfully applied to FNA samples, providing an opportunity to evaluate γH2AX as a treatment response marker in cancer. Diagn. Cytopathol. 2016;44:141–146. © 2015 The Authors Diagnostic Cytopathology Published by Wiley Periodicals, Inc. It is estimated that between 2% and 25% of human cell H2A protein consists of the H2AX variant, depending on cell type.ex vivo (data not shown). There was no evidence of cross‐reactivity between anti‐rabbit secondary and the mouse anti‐γH2AX primary or between anti‐mouse secondary and rabbit anti‐53BP1 primary. There was no evidence of autofluorescence in the absence of secondary antibodies. Nuclear counterstaining was achieved using Vectorshield mounting medium  containing 4',6‐diamidino‐2‐phenylindole (DAPI).Nine patients were recruited to the study. Fine needle aspirate (FNA) samples from the first three participants were used for liquid‐based cytology (LBC) and immunostaining method development. For the next two study subjects the methodology was modified, and direct smear cytology was used. This provided a greater number of cells per sample and more specific immunostaining. A mouse monoclonal anti‐γH2AX antibody  was diluted 1:800 in blocking buffer  and paired with an Alexa Fluor 488 conjugated goat anti‐mouse secondary antibody , diluted 1:250. A rabbit polyclonal anti‐53BP1 antibody  was paired with an Alexa Fluor 594 conjugated goat anti‐rabbit secondary antibody, diluted 1:250. Both antibodies demonstrated a focal intranuclear staining pattern in human peripheral blood mononuclear cells irradiated The remainder of the study was restricted to participants commencing anticancer therapy to enable FNA samples to be taken before and after the first treatment. One patient withdrew from the study prior to sample acquisition leaving three patients for evaluation . To ensure reproducibility and minimise intra‐observer variability, samples were obtained using three separate passes into different parts of the tumour for each time point before and after radiotherapy treatment. The first patient, DSB07, underwent a course of radical radiotherapy to a cervical lymph node recurrence of a squamous cell carcinoma of the parapharyngeal space. FNA samples were taken immediately before and 20 minutes after the first 2 Gy fraction of radiotherapy to the area of recurrence. The samples taken after radiotherapy showed a marked increase in the number of intranuclear foci, when compared with those taken before treatment Fig. C‐. Given tP < 0.0001).Confocal microscopy images from participant DSB07 were used to optimise image recognition softwareParticipant DSB08 had a long history of non‐Hodgkins lymphoma and received palliative radiotherapy to a malignant lesion in the posterior triangle of the neck. Three FNA samples were collected immediately before and three immediately after a 3 Gy fraction of radiotherapy. Synchronous staining for 53BP1 was introduced as it has been demonstrated to co‐localise with γH2AX.There was marked co‐localization of 53BP1 with γH2AX foci, before and after irradiation, however, background staining was subjectively greater than for γH2AX, making foci identification difficult. Consistent results for automated 53BP1 foci recognition could not be achieved. Baseline γH2AX foci numbers were lower for participant DSB08 compared to participant DSB07, and post‐treatment foci more numerous Fig. C‐.Participant DSB09 had skin metastasis from non‐small cell lung cancer on the right upper arm. This was to be irradiated with 17 Gy divided in two fractions delivered one week apart. Samples were taken immediately before the first fraction 8.5 Gy), 20 minutes after this, and one week later, before the second fraction was delivered. The results  for the transformed number of foci for each patient were calculated to show how much of the variability was because of actual differences before and after treatment rather than measurement error. They were all greater than 0.7, suggesting that in this study most of the variability was between time points. However, ICC values may be limited in their interpretation, given the small sample size of this current study.This study was designed to assess the feasibility of γH2AX quantification in tumours before and after radiation treatment using FNA cytology. We also sought to evaluate the reproducibility of the assay under a variety of conditions.The ability to track DNA damage in a tumour following genotoxic treatment may provide valuable insight into a patient's response to therapy. Data from the literature have demonstrated that tracking DNA damage using radioimmunoconjugates to target γH2AX is in fact possible.Samples from two participants were used to develop a LBC method for the γH2AX immunocytochemical assay. LBC is useful for the standardisation and preservation of cell quality during sample processing. Commercial LBC sample processing platforms developed for cervical cytology are routinely applied to different cytological specimen types including FNA,Samples from participant DSB07 were obtained before and after a 2 Gy fraction of radiotherapy. They provided the first indication of the success of the assay in identifying an increase in γH2AX signal in a focal staining pattern. The magnitude of this increase was quantified using automated image recognition. Setting the parameters of the image recognition algorithm does depend on the subjective judgement of the operator, in the same way that manual foci counting is subjective. However, the automated method then provides for rapid, standardised foci counting. This approach has been adopted by other researchers seeking to translate a γH2AX foci assay for possible clinical applications.The persistence of γH2AX foci observed in samples from the study subject with skin metastasis DSB09) was a particularly interesting finding since there is contrary data to suggest that the majority of ionizing radiation (IR) induced γH2AX foci resolve rapidly following repair. was a paThere are few published data from tumour samples in cancer patients with which to compare these results for γH2AX expression. One study reported γH2AX immunohistochemistry in 18 sets of formalin‐fixed paraffin‐embedded cervical biopsies before and 24 hours after 1.8–2.0 Gy fractions of radiotherapy.There are reports of cytological γH2AX assay, using circulating tumour cells (CTCs).The current study considers the role of γH2AX assays as a general marker of treatment‐induced DNA damage. Further data on assay variability using a larger number of samples would be needed in order to judge the assay's potential for application to clinical questions, including informing sample size calculations. γH2AX immunostaining of FNA samples could then be considered as a response biomarker in studies of new radiotherapy or cytotoxic treatment protocols.


Ionic current carried by hERG channels (IhERG) is known to exhibit a paradoxical dependence on external potassium concentration ([K+]e), but effects of acute [K+]e changes on the response of IhERG to premature stimulation have not been characterized. Whole‐cell patch‐clamp measurements of hERG current were made at 37°C from hERG channels expressed in HEK293 cells. Under conventional voltage‐clamp, both wild‐type (WT) and S624A pore‐mutant IhERG during depolarization to +20 mV and subsequent repolarization to −40 mV were decreased when superfusate [K+]e was decreased from 4 to 1 mmol/L. When [K+]e was increased from 4 to 10 mmol/L, pulse current was increased and tail IhERG was decreased. Increasing [K+]e produced a +10 mV shift in voltage‐dependent inactivation of WT IhERG and slowed inactivation time course, while lowering [K+]e from 4 to 1 mmol/L produced little change in inactivation voltage dependence, but accelerated inactivation time course. Under action potential (AP) voltage‐clamp, lowering [K+]e reduced the amplitude of IhERG during the AP and suppressed the maximal IhERG response to premature stimuli. Raising [K+]e increased IhERG early during the AP and augmented the IhERG response to premature stimuli. Our results are suggestive that during hypokalemia not only is the contribution of IKr to ventricular repolarization reduced but its ability to protect against unwanted premature stimuli also becomes impaired.Potassium channels encoded by  hERG potassium channels are important for ventricular repolarization and for protecting the ventricles of the heart from unwanted premature stimuli. This study shows that, in addition to reducing the contribution of hERG channel current to ventricular repolarization, hypokalemia impairs the protective response of hERG to premature stimulation. For experiments on coexpressed hERG1a/1b, 0.25 μg of the hERG 1a construct were cotransfected with the same amount of hERG 1b, together with 0.5 μg of CD8 as a transfection marker. Successfully transfected cells were detected using Dynabeads® (Invitrogen). After transfection cells were incubated at 37°C (5% CO2) for 6 h before plating them on small dry‐heat sterilized glass coverslips. Electrophysiological experiments were conducted after at least 24 h of further incubation at 37°C (5% CO2). Throughout the Results section, hERG refers to hERG1a, except for data in HEK‐293 cells stably expressing WT hERG or transiently expressing S624A‐mutant constructs were maintained and passaged as described previously    and a CV‐4/100 or CV203BU head‐stage. Voltage‐clamp commands were generated with Clampex 8 or Clampex 9.2 . Pipette series resistance was compensated between 70% and 80%. Data were acquired through a Digidata 1200B or a Digidata 1320A . Data digitization rates were 10–25 kHz during all protocols and an appropriate bandwidth of 2–10 kHz was set on the amplifier.Coverslips with plated cells were placed in a recording chamber mounted on an inverted microscope . The chamber temperature was kept at 37°C and cells were continuously superfused with a standard Tyrode's solution containing (in mmol/L): 140 NaCl, 4 KCl, 2.5 CaCl+]e solution was made by lowering the KCl in the Tyrode's solution from 4 to 1 mmol/L, while the raised [K+]e solution contained 10 mmol/L KCl. In both cases, the NaCl concentration was adjusted accordingly to maintain the same total external [K+] + [Na+]: when [K+]e was reduced to 1 mmol/L, [Na+]e was increased by 3 mmol/L and when [K+]e was increased to 10 mmol/L, [Na+]e was reduced by 6 mmol/L. All the solutions were warmed at 37°C and superfused over the cells using a homemade, multibarreled perfusion system that allowed rapid exchange of extracellular solutions , Prism v4.03 and Excel 2003 and 2007. All data are presented as the mean ± SEM.hERG “pulse” and “tail” currents was determined using the equation: IhERG‐Altered[K+] and IhERG‐Control represent “pulse” or “tail” currents in altered  and normal external potassium concentration. In both altered potassium conditions, a steady‐state was reached within ≈2 min and therefore no run‐down correction was needed.The effect of different external potassium concentrations on II is amplitude of the peak current elicited by the third depolarizing step of the protocol after a brief 2 msec conditioning step (Vm) that relieves the inactivation caused by the first depolarizing step. IMAX is the maximal current amplitude during the third pulse observed during the protocol, and V0.5 and k are the half‐maximal inactivation voltage and the slope factor for the fit to the plotted relation.The voltage dependence of inactivation was assessed using a three‐step protocol Fig. A, inset y is the current amplitude at time x, τ is the time constant for the decay of the transient current, A represent the total fitted current, and C is the residual unfitted current component after the decline of the transient current.To calculate the time constant of inactivation the transient current elicited by the third step of the three‐step protocol after a 2 msec step to −120 mV was fitted with a mono‐exponential equation: IhERG protocol  with Bonferroni post‐test, as appropriate. P values less than 0.05 were considered to be statistically significant.Statistical analysis was performed using a paired, unpaired +]e from 4 to 1 mmol/L and elevating it from 4 to 10 mmol/L were assessed using a conventional voltage protocol, employed in a number of prior studies of IhERG from our laboratory . Reducing [K+]e from 4 to 1 mmol/L did not significantly alter τfast of deactivation and produced only a small (~10%) decrease in τslow . Raising [K+]e from 4 to 10 mmol/L did not significantly alter τslow of deactivation and produced only a small (~4%) increase in τfast .In initial experiments, the effects of reducing e and a ~13.0 mV positive shift in inactivation V0.5 on switching from 4 to 10 mmol/L [K+]e. However, time‐matched control measurements  showed a modest ~3.1 mV (n = 5) positive shift in inactivation at time points correlating to those used to evaluate effects of 1 and 10 mmol/L [K+] and therefore derived V0.5 and k values were corrected accordingly. In paired experiments, V0.5 and k values in 4 mmol/L [K+]e were −60.3 ± 1.3 mV and 21.7 ± 0.4 mV, while in 1 mmol/L [K+]e, V0.5 and k values were −61.1 ± 1.6 mV and 21.5 ± 0.6 . For cells used to evaluate the effect of 10 mmol/L [K+]e, control V0.5 and k values in 4 mmol/L [K+]e were −53.4 ± 2.0 mV and 25.0 ± 1.2 mV, while in 10 mmol/L [K+]e, V0.5 and k values were −43.6 ± 4.6 mV and 27.4 ± 0.9 . Thus, under our conditions, reducing [K+]e from 4 to 1 mmol/L did not significantly alter the voltage dependence of IhERG inactivation, while increasing [K+]e from 4 to 10 mmol/L produced a ~+10 mV positive shift in inactivation V0.5. τinact) for the development of inactivation at +40 mV, following relief of inactivation at −120 mV .The voltage dependence of  a brief  msec rep a brief  msec rep a brief  msec rep a brief  msec rep+]e has been proposed to decrease surface membrane IKr/hERG through induction of a novel nonconducting state and promotion of channel internalization/degradation  was suggested to be able to induce the nonconducting state for wild‐type (WT) hERG within minutes, but not to be able to do so for channels comprising the S624A hERG pore mutant . The similar responses of WT and S624A IhERG to acute exposure to 1 mmol/L [K+]e under our conditions indicate that WT current amplitude reductions with lowered [K+]e in our experiments are unlikely to be attributable to induction of the (S624A‐hERG sensitive) nonconducting state reported by Massaeli et al.  and a decrease of 23.1 ± 3.4% of the IhERG tail . The lack of significant difference between the response of WT and S624A IhERG during the +20 mV test command is suggestive that any difference in tail current response likely resulted from differences in gating of the two channels rather than in surface expression (though any such differences were beyond the intended scope of this study and so were not pursued).Chronic exposure to low [Ki et al. . Mean da+]e on the IhERG response to premature stimulation, a pulse protocol was used that comprised paired AP waveforms, in which an initial and second AP command were separated by varying intervals following the application of the first AP, with the second AP applied both before and following completion of initial AP repolarization . The overall pattern of rapid IhERG transients was similar between 4 and 1 mmol/L [K+]e .In order to ascertain the effect of e was raised from 4 to 10 mmol/L. The response of IhERG during the initial AP was mixed: current immediately following the phase 1 repolarization was increased (by 26.8±8.3% n = 7 cells), while the maximal current during repolarization was insignificantly reduced . The differential effects of raised [K+]e on IhERG at positive voltages early in the AP and the peak current later in repolarization (which occurred at ~−30 to −40 mV) are analogous to those seen with conventional voltage‐clamp in Figures +]e due to the positively shifted equilibrium potential for K+ compared to the −80 mV holding potential. The response to premature stimuli was augmented, however  and statistically significant increases were seen between APD90 and 60 msec following APD90 of the first AP.Kr channels comprised hERG1a coassembled with the shorter hERG1b isoform , while maximal IhERG during AP repolarization was reduced by 23.7 ± 4.8% . As shown by the representative traces in IhERG transients induced by premature stimulation were reduced by exposure to 1 mmol/L [K+]e. +]e the relationship descended more steeply following the maximal response that seen for hERG1a; this is attributable to the known more rapid deactivation kinetics for hERG1a/1b than hERG1a alone  and IhERG transient amplitude was significantly smaller in 1 mmol/L than 4 mmol/L [K+]e between 70 ms preceding APD90 and 40 ms following APD90 of the initial AP command. Consequently, lowering [K+]e reduced the IhERG response to premature stimulation both for hERG1a and for hERG1a/1b.Although most studies of recombinant hERG focus on the hERG1a isoform, there is some evidence that native Il/L Fig. Ai and ii+]e on IhERG amplitude was observed in early studies of IKr and hERG. Thus, acutely lowering [K+]e from 4 to 0 mmol/L was reported to increase the amplitude of slow delayed rectifier current, IKs, in guinea‐pig ventricular myocytes , while it reduced IKr from the same preparation  was reported to drive WT hERG channels into a nonconducting state, followed by subsequent internalization and degradation  [K+]e both during imposed AP clamp commands and in response to premature AP stimuli  . If pathological ischemia/K+ accumulation is localized, then the localized effect of raised [K+]e on hERG/IKr could contribute to heterogeneity in repolarization and in tissue sensitivity to premature excitation. On the other hand, global hypokalemia is strongly associated with risk of arrhythmia and is known to exacerbate the risk of acquired (drug‐induced) LQTS and associated Torsades de Pointes (TdP)  in patients with hERG mutation‐linked congenital LQTS  Viskin . In prof) Viskin . Thus, wNone declared.