Coated and uncoated electrode arrays were implanted into rat visual cortex and subjected to daily cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) for 11 days. Coated electrodes experienced a significant decrease in 1 kHz impedance within the first two days of implantation followed by an increase between days 4 and 7. Equivalent circuit analysis showed that the impedance increase is the result of surface capacitance reduction, likely due to protein and cellular processes encapsulating the porous coating. Coating’s charge storage capacity remained consistently higher than uncoated electrodes, demonstrating its in vivo electrochemical stability. To decouple the PEDOT/MWCNT material property changes from the tissue response, in vitro characterization was conducted by soaking the coated electrodes in PBS for 11 days. Some coated electrodes exhibited steady impedance while others exhibiting large increases associated with large decreases in charge storage capacity suggesting delamination in PBS. This was not observed in vivo, as scanning electron microscopy of explants verified the integrity of the coating with no sign of delamination or cracking. Despite the impedance increase, coated electrodes successfully recorded neural activity throughout the implantation period.Neural electrodes hold tremendous potential for improving understanding of brain function and restoring lost neurological functions. Multi-walled carbon nanotube (MWCNT) and dexamethasone (Dex)-doped poly (PEDOT) coatings have shown promise to improve chronic neural electrode performance. Here, we employ electrochemical techniques to characterize the coating  Neural prostheses have seen effective use in a variety of applications, including auditory prostheses, visual prostheses, brain-computer interface, and even opto-electrical applications ,2,3,4,5.In light of these observations, novel intracortical electrode design has largely focused on improving electrical characteristics , reducinin vivo on demand drug delivery. PEDOT has exhibited excellent electrochemical stability [in vivo application, and studies of chronically implanted PEDOT-coated electrodes show that the coated electrodes exhibited lower impedance and improved recording characteristics compared to uncoated controls [Poly, or PEDOT, is an example conducting polymer applicable to neural prosthesis employed for tability ,56,57 antability , and hastability ,63,64,65tability ,54,55,62tability ,60,66,67tability ,56,67 antability . The bencontrols ,59,61,62controls .in vivo chronically in rat brain over 6 weeks, where they demonstrated excellent coating stability, reduced astrocyte activation, and increased local neuronal density compared to bare platinum implants, though recording ability was not evaluated [in vivo stimulation , along with reduced inflammation and improved neuronal survival around electrodes compared to those left uncoated [Multi-walled or single-walled carbon nanotubes (MWCNTs or SWCNTs) may be incorporated into PEDOT coatings as a sole dopant or co-dopant to mechanically reinforce the polymer and prevent the spallation and cracking previously observed during chronic stimulation ,70. MWCNvaluated . In a movaluated . Additiovaluated . We haveuncoated . Dexametuncoated  and by wuncoated .in vivo within the sub-chronic period, using impedance spectroscopy and equivalent circuit modeling. Several earlier studies noted that while the 1 kHz impedance of PEDOT-coated electrodes was significantly lower than uncoated electrodes in vitro and at early time points in vivo, within a week of implantation into cortex coated electrode impedances raise rapidly, sometimes to the point of being statistically indistinguishable from impedances of uncoated controls [The purpose of the work presented here is to build upon our previous study by performing a more in-depth analysis of the biological and electrochemical material changes that occur at the MWCNT-doped PEDOT-coated tissue/electrode interface controls ,59,66. Wcontrols ,18, the in vivo and immersed within a PBS bath in vitro were studied with CV and EIS. The neural recording performance before and after CV stimulations and the coating integrity after 11 days of implantation were examined.While 1 kHz impedance is commonly used to characterize electrodes, EIS allows for the measurement of changes to impedance magnitude and phase across an interface over a wide range of frequencies ,9,18, an3 and H2SO4 , carboxylating the nanotube surfaces. The solution was then stirred at ambient temperature for 12 h. Treated nanotubes were collected by decantation following ultracentrifugation  and sonicated for 10 min in DI water . Centrifugation was repeated until the pH of the supernatant solution was 6.0. Nanotubes were then collected and the remaining solvent was evaporated off in an oven at 60 °C.Multi-walled carbon nanotubes were purchased  and functionalized using a previously published method . In summet al. [vs. Ag/AgCl) coating potential for 30 s. The array sites to be coated were selected using an alternating arrangement to prevent positional bias. After coating, the array was rinsed using DI water and impedance measurement was repeated in sterile PBS. Arrays were allowed to continue soaking in PBS for 30 min to remove adsorbed dexamethasone, given a final rinse using DI water, and were then stored dry in a sterile enclosure.Floating Microelectrode Arrays  were sterilized using an ethylene oxide gas sterilizer  after which they were transferred to a sterile environment. Initial quality-control impedance testing of all array sites was performed in a sterile PBS bath using a potentiostat  with a platinum counter and Ag/AgCl reference . If measured impedances differed substantially from manufacturer-reported values, the array was electrochemically cleaned (constant −2 V for 20 s). After cleaning, impedance was re-measured and cleaning repeated if necessary. Following testing, each array was immersed in a sterile polymerization solution prepared identically as that used in Kolarcik et al. , prepare2 area, Microprobes for Life Science) identical to those within FMAs implanted in vivo were used to characterize the coating morphology using scanning electron microscopy (SEM). SEM was conducted at The University of Pittsburgh’s Center for Biological Imaging on a field emission SEM . Coating adhesion was evaluated by inserting and removing coated microelectrodes from Long Evans rat cortex in vivo or from an agarose gel using a micromanipulator . Coating integrity was evaluated using impedance spectroscopy and SEM. Agarose gel was prepared by heating a stirred 5 mg/mL agarose  solution to 85 °C until clear, at which point it was allowed to cool and set.In addition, single microelectrodes  and in accordance with regulations specified by the division of laboratory animal resources.Prepared FMAs were implanted unilaterally into the right primary visual cortex, monocular area (V1M) of three male Long Evans rats. Each animal was anesthetized under 3% isoflurane and mounted onto a stereotaxic frame . The skull was exposed and a 3 × 3 mm craniotomy centered at 6.5 mm post Bregma and 3.5 mm lateral to midline was made over V1 using a high speed drill and fine rongeurs. Saline was applied continuously onto the skull to suppress heat. The dura was resected using fine Vannas scissors, and the brain surface was moistened using gelfoam while stereotaxic hardware was put into place. Insertion of the FMA array was performed using a vacuum tip mounted to a hand-driven micromanipulator . The craniotomy was sealed using a low-viscosity silicone   and recording. All coated and uncoated array sites were subjected to an identical CV program each session. Before and after CV, both spontaneous and evoked neural activity were recorded and impedance was measured across the entire array. This protocol allowed all metrics to be measured immediately before and immediately after each CV session, and tracked daily for the duration of the experiment. Each component of the session is described in detail below.vs. Pt. counter electrode) at a 1 V/s scan rate, anode-first. Redox behavior of each site was qualitatively observed in terms of reduction and oxidation peak height and potential shift. Charge storage capacity and charge balance were computed by integrating the area under cathodic and anodic curves, and cathodic and anodic charge capacities were summed to yield the total charge storage capacity of each electrode.CV was performed using a PGSTAT128N potentiostat. Sequentially on each channel, CV was performed using 20 cycles from −0.9 V to 0.6 V  and NOVA  were used for measurement and analyses.Coating integrity of the explanted probes was evaluated using scanning electron microscopy. Following array extraction, electrodes were soaked in a 5% trypsin solution for twenty minutes at ambient temperature to remove tissue residue and reveal the underlying coating surface. Arrays were then rinsed with DI water and dried for high resolution SEM.in vivo electrochemical behavior from the material property changes of the coating, PEDOT/MWCNT/Dex coatings were evaluated in vitro in a controlled environment. A custom array was ordered from Microprobes for Life Science featuring ten 4 cm long platinum/iridium microelectrodes with composition and tip geometry identical to those implanted in vivo. Each electrode shank featured an additional polyimide sleeve to minimize capacitive shunting, in addition to the parylene-C insulation. Electrode tips were coated with MWCNT and dexamethasone-doped PEDOT in a manner identical to that described in In order to decouple the underlying mechanisms behind the sub-acute changes to t-test. Comparison between multiple groups was performed using one-way standard ANOVA with a Tamhane T2 post-hoc test. Tamhane T2 was selected in place of Tukey due to the large difference in variances within in vivo impedance data. For all tests, α < 0.01 indicated a significant result.Comparison between two groups was performed using a two-tailed Welch’s p = 0.0003) the 1 kHz impedance modulus of the coated microwire tips (276 kΩ ± 147 kΩ) compared to those left uncoated (446 kΩ ± 153 kΩ) in PBS. Coating adhesion testing demonstrated no apparent changes to electrode impedance or surface morphology following insertion and removal of a coated microwire from in vivo rat cortex. Insertion and removal of a coated microwire from agarose gel resulted in a clinging residue of agarose to the surface visible by SEM, but no change in electrode impedance.Dexamethasone (Dex) and MWCNT-doped PEDOT coatings were characterized with regard to morphology and impedance . Represein vivo performance of PEDOT/MWCNT/Dex-coated probes against conventional non-coated microwires, Long Evans rats were implanted with 16-channel floating microwire arrays unilaterally into V1 monocular cortex. The layout of the implanted arrays is illustrated . Subsequently, the impedances of coated probes increased rapidly to the point that they on average became indistinguishable from uncoated values for the remainder of the experiment. Dynamic impedance behavior over the first three days of the experiment is shown in detail , while uncoated probes on average exhibited steadily increasing impedance values. Daily values for the average change in probe 1 kHz impedance measured immediately before CV were compared to those measured immediately after CV .Average daily pre-CV 1 kHz impedance modulus values for coated and uncoated probes are shown b. Impedan detail c, which after CV d. Averagin vivo. Typical in vivo CV curves are displayed  are shown for both coated and uncoated probes . The average anodic-to-cathodic charge balance ratio of all coated electrodes across all days was 1.32 ± 0.22 (n = 176), indicating a degree of anodic charge buildup. The average percent change in TCSC of coated electrodes between day 1 and day 11 was found to be 4.6% ± 21.0% (n = 16), indicating only a small change of charge storage capacity over the course of the experiment, with no definite trend upwards or downwards.Average daily values of total charge storage capacity , and β, a parameter defined by the phase angle of the CPE. β has a value between 0 and 1, where β = 1 represents an ideal capacitor with phase angle 90° and β = 0.5 represents a CPE with phase angle 45° . The physical correlate of β is not well understood, and is thought to be related to surface roughness, charge uniformity, coating bulk properties, or varying reaction rates along the electrode surface [SERR represents the solution resistance of the bulk saline/tissue environment. For typical microelectrodes composed of a blocking material such as platinum, CTR is expected to be extremely high, leading to the electrode behavior being dominated by the probe capacitance [in vitro or at very early time points in vivo, in plots where multiple time-constant behavior was not observed.Curve fitting and equivalent circuit analysis was applied to the measured data using a method developed by Bisquert ,83 that sulation  as well in vitro ,85. Usinin vitro ,87. In tDZ, a double-channel transmission line distributed element representing a superposition between a solid and a liquid continuum, extending from the electrode surface with thickness L. A schematic representation of DZ is provided (r0 is the resistance per unit length (Ω·cm−1) of the electrolyte fluid, r1 is the charge transfer resistance length (Ω·cm) of the electrolyte/solid interface, and q1 is the coefficient of the interface CPE per unit length (F·sα−1·cm−1). An extensive mathematical treatment of this element can be found in the literature [DZ is generally expressed as i = −1). If L is equated to 1 for fitting purposes, R0 becomes the total resistance of the electrolyte phase and represents restriction to ionic motion within the pores of the coating, while R1 and Q1 represent the total charge transfer resistance and CPE capacitance of the electrolyte/conducting polymer boundary with α representing the CPE exponential parameter, analogous to β above. An important stipulation of this model is that the resistivity of the solid phase must remain negligible, requiring the assumption that the PEDOT coating remains oxidized and highly conductive for the duration of the experiment [TCSC, which suggest intact conduction between the coating and the metal electrode.Model B b is simiprovided c, where terature ,84. In speriment . This asRepresentative Nyquist plots of recorded impedance data are shown d–i, withModel parameters were fitted to experimental data using a complex linear least squares fitting program. Before fitting, the thirteen lowest frequency impedance measurements from each impedance spectrum were removed to eliminate scatter due to low-frequency noise, which was found to be a consequence of the multisine measurement method. Also, impedance spectra that were found to contain enough broadband noise or measurement artifact to interfere with consistent fitting were removed.CTR, is an example, as it is most relevant to frequencies much lower than those measured here (f < 1 Hz). Parameters demonstrating the most dynamic and consistent behavior in coated electrodes were found to be CPEC and Q1, representing the metal substrate and the coating or tissue encapsulation surface capacitance coefficients respectively. Their behavior compared to the same parameters modeled from uncoated electrode data is shown in CPEC (β) also demonstrated dynamic change in the coated electrodes but remained at consistent elevated values in uncoated electrodes, as shown in Q1 (α) maintained a high value of between 0.85 and 1 for the duration of the experiment for both coated and uncoated electrodes. Pore resistance R0 and conducting polymer charge transfer resistance R1 of coated electrodes demonstrated a small degree of variation over time that did not correlate with impedance. The solution resistance SERR was found to be inconsequentially small compared to other elements and did not contribute substantially to quality of fits when varied manually.Multiple trends were observed in the fitted model parameters, as shown in p > 0.01 for all). Performance was also not observed to be correlated with impedance during the initial week, as uncoated and coated probes exhibited the same noise amplitude and SNR despite having significantly different 1 kHz impedance. In general, the coated channels performed similarly in comparison with uncoated channels.Neurophysiological recording capability of coated electrodes compared to uncoated electrodes was evaluated through the visual stimulation of the array-implanted animals. Visual stimulation evoked robust firing rate change during the entire period of experimentation. A representative filtered (0.3–5 kHz) spike data stream from a coated channel on the last day of implantation is shown in Scanning electron microscope images of representative explanted electrodes are shown in in vivo, this disparity can be attributed to variations in exposed tip surface area and coating thickness. Among the electrodes that were not subjected to CV, impedances were seen to remain consistent throughout the experiment, changing at most 35% between day 1 and 11 (TCSC) information. Initial TCSC values for these electrodes ranged between 5.8 and 38.0 mC·cm−2. The three electrodes that exhibited the greatest increases in 1 kHz impedance, electrodes 1, 2, and 3, also exhibited large decreases in TCSC, at 54%, 52%, and 48%, respectively. The electrodes that exhibited smaller impedance increases, electrodes 4 and 5, showed less charge storage capacity loss, at 16% and 37%, respectively. It should be noted that while electrode 5 exhibited the smallest impedance increase, it also exhibited the greater degree of TCSC reduction at 37% and also had the highest initial TCSC. Nyquist plots of a non-CV stimulated electrode  was employed to coat Pt/Ir microelectrodes as lower potentials resulted in inconsistent impedances. Deposition on Pt/Ir was carried out to a charge density of approximately 0.29 C/cm2. Careful MWCNT functionalization and suspension preparation were critical to achieving consistent and robust coatings. Insufficient MWCNT carboxylation was found to result in clumping of the nanotubes in solution, poor dispersion, and poor, non-uniform integration into the coating.We found that electrodeposition using constant potential produced the most consistent PEDOT/MWCNT/Dex coatings with the greatest degree of impedance reduction on both gold macroelectrodes and Pt/Ir microelectrodes. This is in contrast with observations by Zhou et al.  who founet al. ,65, whilet al. [et al. [SEM imaging of coatings revealed an open and porous morphology a,b compaet al. , who pre [et al. , who fouet al. [Coated microelectrodes exhibited a significant decrease in 1 kHz impedance and an increase in charge storage capacity, which were observed by other studies using similar compositions ,70,85. Tin vivo due to the increased anesthesia time required as well as the likelihood of increased charge buildup and faradaic damage to the coating and tissue [vs. Pt/Ir) PEDOT reduction potential could lead to a violation of the water window [in vivo environment and the potential-controlled nature of stimulation. A fast CV scan rate minimizes the charge accumulation that may result in permanent damage of the electrode tissue interface. The continued presence of the PEDOT coating was verified by the reduction peak during CV stimulation in all coated channels throughout the experimental period. Material stability following CV was verified using SEM imaging of explants. SEMs showed that electrode dimensions and morphology remained visually unchanged after over 200 cycles. Interface integrity was monitored through post-CV impedance measurement. Neither the post-stimulus impedance nor gross anatomical histology revealed any obvious evidence of stimulation-induced lesion or gas evolution. Most importantly, the CV stimulus did not have any quantifiable impact on recording SNR, though a small but significant reduction in noise amplitude was observed from coated electrodes on the final day of implantation. CV scans also demonstrated consistent redox behavior and charge storage characteristics throughout the experiment. This evidence suggests that the majority of charge was transduced by way of safe, reversible mechanisms, and that the application of CV did not generate an observable degree of hydrolysis.In order to generate enough current to drive dexamethasone release without damaging the coating, a conservative electrochemical analysis paradigm was developed. The stimulus employed a cyclic voltammetry (CV) waveform which approximated charge balance while providing electrochemical feedback on both redox behavior and charge storage capacity. CV has been commonly employed to study conductive polymer properties, often as a stimulation for active drug release ,90,91 thd tissue . As mentr window  and the et al. [in vivo performance of PEDOT-coated electrodes [in vivo.The coated probes demonstrated dynamic multimodal changes in broad-spectrum impedance over the eleven day period of implantation, suggesting a progression of changes to physiological or material factors at the tissue/coating interface. This is contrasted with the behavior of uncoated probes, which exhibited a gradual increase in 1 kHz impedance typical of chronically implanted uncoated microelectrodes during the first week post-implantation in rat cortex ,59,92. Tectrodes ,66. We oCPEC, CTR, and β, and high frequency behavior characterized by an encapsulation element modeled using DZ. This is contrasted against Nyquist plots of uncoated electrodes in PBS, which in the measured frequency range (10 Hz to 32 kHz) demonstrate nearly linear constant-phase behavior characterized by CPEC, CTR, and β. The reduction in 1 kHz impedance between coated and uncoated probes in vitro most strongly correlates with CPEC, suggesting that the principal benefit of the coating is that of increasing the effective surface area of the double-layer capacitance. However, the coating also seems to contribute a diffusion barrier to the interface which is most apparent at high frequencies. It should be noted that within the frequency range measured, the ability of fitting techniques to distinguish between CTR and β is limited, particularly in vivo where substantial low frequency noise is often encountered during impedance measurement.Nyquist plots collected from coated electrodes in PBS before implantation reveal the characteristic bimodal frequency response typically observed in conducting-polymer-coated electrodes, with low frequency behavior dominated by metal interface parameters in vivo 1 kHz impedance of uncoated electrodes is typically characterized by subtle changes in CTR and β which dominate low-frequency behavior, as well as with a gradually emerging high-frequency diffusion barrier and encapsulation element modeled using DZ. These changes coincide with known physiological events thought to play a role in evolving electrode in vivo impedance, with CTR and β representing changes to electrode surface properties due to the protein adsorption that takes place immediately upon implantation, and DZ representing the growing boundary effect of inflammation, microglial encapsulation, and edema [in vivo studies using uncoated microelectrodes have shown that 1 kHz impedance tends to peak at 9–15 days and then reduces to an intermediate magnitude where it typically remains at a fluctuating plateau. This is thought to correspond with the reduction of initial acute inflammation and tissue swelling, and the transformation of the interface to a stable chronic inflammatory state [The gradual increase of average nd edema ,26. Chrory state ,59.CPEC and Q1 correlate with this impedance low-point when averaged, suggesting that the coating required a one day “maturation” period following implantation to achieve its full benefit. The drop of impedance in the two days following implantation is possibly due to the time required for electrolyte to fully penetrate the pores of the coating, or for the fluid and tissue around the probes to stabilize post-implantation. Between day 3 and day 5 post-implantation, the average 1 kHz impedance of coated probes increased substantially to the point of equivalence with that of uncoated probes. Nyquist plots reveal that this increase is distinct from the increase observed in uncoated probes, and is principally due to large decreases in parameters CPEC and Q1, which allowed the encapsulation element DZ to dominate increasingly greater portions of the measured frequency range. These modeling results suggest that beginning at day 3–5, the surface area enhancement of the coating was sharply reduced and that a barrier composed of coating and inflammatory tissue elements began to dominate impedance behavior by reducing the exposed surface area, thereby reducing the capacitance of the conducting polymer. This hypothesis is supported by explant SEM imaging which revealed the presence of a dense membranous substance enveloping and interpenetrating the coating pores of all coated electrodes. Due to explant preparation for imaging, this substance was compromised before identification could be performed, but it is speculated to be a combination of cellular processes from fibroblasts, macrophage and microglia as well as dense extracellular matrix. Despite the impedance changes, the recording performance of the coated electrodes did not appear to be detrimentally affected, suggesting that the encapsulation element is limited to the area immediately surrounding and within the coating.In contrast to the behavior of uncoated electrodes, the coated electrodes demonstrated an initial low-impedance period followed several days later by a rapid increase consistent between all coated electrodes. The initial low-impedance period persisted for between 3 to 4 days, with the nadir occurring between days 1 and 2. Fitted model parameters DZ to model both the encapsulation component of the conducting polymer coating as well as the ionic diffusion barrier of tissue encapsulation. DZ has been used to model each of these elements individually in different previously-published studies [in vitro chronic PBS soak test was performed using comparably-coated electrodes  will the PEDOT/MWCNT coating interfere with the recording capability; (2) will the CV stimulation cause any reversible or irreversible to the activity and health of the neurons nearby. Both spontaneous and evoked neural activity was recorded before and after daily CV and impedance measurement. A variety of different drifting solid grating visual stimulation programs were employed to drive neural activity, though for the purposes of this study all measures were averaged together across spontaneous and evoked blocks. In order to provide an assessment of raw recording performance, metrics of signal-to-noise ratio and noise amplitude were quantified. In general, only sparse unit activity was observed across both coated and uncoated probes over the initial week of implantation. However, both coated and uncoated probes exhibited well-defined units. Activity in both coated and uncoated probes increased substantially within recordings taken during the final days of implantation, with recording quality being essentially equivalent. This suggests that the multicomponent organic coating did not compromise the recording capability of the electrodes. Furthermore, we observed no different in SNR and yield immediately before and after the CV stimulation, indicating the stimuli applied here are mild enough to not cause any activity change. The inconsistent probe performance from day to day during the initial week post-implantation is likely due to the progression of acute inflammation and edema local to the implanted electrodes and the eventual stabilization of the interface tissue as it enters the chronic inflammatory stage, and is a rationale behind the common practice of delaying neural recording for the initial week post-implantation.in vivo are due to the development of an encapsulation element, observed through analysis of the complex impedance behavior. We further demonstrate that this encapsulation element is not due to intrinsic changes within the coating itself, as comparable behavior was not observed in coated electrodes subjected to the same time period of electrolyte exposure and daily cyclic voltammetry in vitro. Despite the development of the encapsulating element, the coating was not observed to hinder neural recording and performed comparably with uncoated electrodes. This is in agreement with our longitudinal recording experiment with MWCNT-doped PEDOT sites [We demonstrate that the rapid sub-acute increases in the 1 kHz impedance of MWCNT-doped PEDOT coatings OT sites . Additio


GDM rats showed possible insulin resistance as evidenced by high expression of proinflammatory cytokines, PCK-1 and CRP. Furthermore, low levels of IRS-2 and AKT-1 genes and downregulation of GLUT-4 from the initial to final phases indicate possible defect of insulin signaling. GDM rats also showed an impairment of antioxidant status and a hyperlipidemic state. Additionally, GDM rats exhibited significantly higher body weight and blood glucose and lower plasma insulin level and C-peptide than control. Based on the findings outlined, the current GDM animal model closely replicates the disease state in human and can serve as a reference for future investigations.This study attempts to develop an experimental gestational diabetes mellitus (GDM) animal model in female Sprague-Dawley rats. Rats were fed with high fat sucrose diet, impregnated, and induced with Streptozotocin and Nicotinamide on gestational day 0 (D0). Sleeping patterns of the rats were also manipulated to induce stress, a lifestyle factor that contributes to GDM. Rats were tested for glycemic parameters , lipid profiles , genes affecting insulin signaling , glucose transporters (GLUT-2 and GLUT-4), proinflammatory cytokines (IL-6, TNF- Gestational diabetes mellitus (GDM), a common pregnancy complication, is defined by the American Diabetes Association as diabetes that is not clearly apparent diabetes, diagnosed in the second or third trimester of pregnancy . During Observations of tissues and organs from pregnant women with and without GDM would expand our understanding of the disease. Yet, the scarcity of the samples and the lack of modalities make understanding the molecular mechanism and finding possible therapeutics for GDM difficult. For these reasons, animal models deliver an attractive alternative in studying the molecular mechanisms and treatment options for GDM. Initially, GDM animal models were induced solely by injecting diabetogenic agents such as Streptozotocin (STZ) and alloxan in low and high dosages –12. HoweIn addition to the combination of STZ, NA, and HFSD in our previous GDM animal model, we now include stress in the current model. GDM pregnancies are linked with a heightened level of oxidative stress, where sleeplessness or disturbance in sleep increases stressand consequently impairs antioxidant defense system . MothersHence, the current study investigates whether the combination of nutritional manipulation, pharmacological treatment, and stress induction can create appropriate immunometabolic changes in pregnant rat, which can be developed as a model for understanding the consequences of GDM as well as providing insights into potential treatments and preventative measures. To the authors' knowledge, GDM animal model induced with the previously mentioned factors has not been published in the scientific literature and hence recorded as a pilot study. Changes on glycemic and lipid parameters along with proinflammatory cytokines level and oxidative stress in the rats were observed in this study to evaluate the appropriateness of our animal model.Female Sprague-Dawley rats aged 8-9 weeks were procured from the Laboratory Animal Facility and Management (LAFAM), UiTM Puncak Alam Campus, Malaysia. A total of 45 female rats were randomly taken for the study, where 10 rats were assigned as control and the rest were assigned as GDM group. Animals were maintained in an experimental room under the following conditions: (a) temperature of 22 ± 2°C, (b) humidity of 50 ± 10%, and (c) illumination of a 12-hour light/dark cycle for control and 18-hour light/4-hour dark cycle for GDM group. Changes in sleeping cycle and environmental status have been shown to induce abnormally high levels of oxidant stress . All expStandard diet  was given to control rats. Meanwhile, GDM group rats were fed with HFSD  from Week 10 onwards. HFSD consist of 25% sucrose, 40% beef tallow, and 20% of casein protein.After one week of acclimatization, animals were examined for estrous cycles for 2 consecutive weeks. Rats follow a 4-day pattern of estrous cycle, namely, estrous (E), metaestrous (M), diestrous (D), and proestrous (P). Rats in estrous stage  were allIn this technique, vaginal secretion from each rat was collected using a plastic pipette containing ~1-2 mL of normal saline (NaCl 0.9%) every morning. Vaginal secretion was collected by inserting the pipette tip into the rat's vagina and flushing the cells from the vaginal lining. One or two drops of vaginal secretion were placed onto a clean glass slide. Separate glass slides were used for each cage of animals. Unstained vaginal secretions were directly viewed under a light microscope at 40x magnification. Three types of cells were observed, which were round and nucleated epithelial cells, nonnucleated irregular cornified cells, and small round cells . These cStreptozotocin (STZ)  was induced on D0 by a single intraperitoneal (ip) injection at a dose of 35 mg/kg bw in 0.1 mol/L citrate buffer (pH 4.5). Nicotinamide (NA)  120 mg/kg bw was induced ip 15 minutes prior to STZ. Control rats received an equal volume of citrate buffer only. The rats were returned to their respective cages and blood glucose levels were analysed 72 hours after STZ administration. Rats with stable hyperglycemia were selected for further study where on D6 half of the animals were euthanized after initial results were collected and the rest were sacrificed after the final results were collected.Blood samples were collected through cardiac puncture for biochemical and hematological analysis on D6 and D21. Serum was collected into plain tubes while plasma was collected into heparin tubes. Collected blood in plain tube was left to clot for half an hour. Subsequently, heparin tubes were kept cold in an ice box to prevent clotting. Both tubes were centrifuged at 2500 rpm for 12 min at −4°C. Serum and plasma were collected in microcentrifuge tubes and kept at −80°C for further analysis.Food intake was calculated daily by calculating the difference between the amount of food given and the amount of residual food at the end of each day.Six animals were sacrificed on D6 to acquire the initial results, and the rest was sacrificed on D21. Rats were anesthetized with diethyl ether and thoracic, abdominal, and pelvic regions were dissected. Abnormalities in the internal organs were examined. Uterus and ovaries were excised and exposed. The number of corpora lutea on each ovary was identified and total gravid weight was noted prior to opening the uterus. To find out the implantation loss, uterus was kept in ammonium sulphide (10%) solution for 5 minutes . The weiTail incision method was used to measure weekly fasting glucose level using hemoglucometer  and glycemic levels were monitored throughout the experiment. Serum glucose was measured upon sacrifice using commercially available reagent kits .Insulin level was assayed using Mercodia Rat Insulin ELISA kit (Sweden). The optical densities of the samples were read at 450 nm. The concentration of insulin level was obtained by computerized data reduction of the absorbance for the calibrators, except for calibrator 0, versus the concentration using cubic spline regression.Plasma C-peptide level was assayed by Mercodia Rat C-peptide ELISA kit (Sweden). The optical densities of the samples were read at 450 nm. The concentration of C-peptide was obtained by using cubic spline regression the same way as the Insulin ELISA kit mentioned above.μL plasma was transferred to each Selectra tube, and the results were obtained using a Vita lab Selectra machine.Blood plasma was used for the determination of C-reactive protein, using full range CRP (frCRP) commercial kit purchased from Randox (All Eight (M) Sdn. Bhd). Liquid assayed specific protein control levels 1 and 2 were used as control. After preparation of blood samples, 500 Serum levels of triglycerides, total cholesterol, LDL-cholesterol, and HDL-cholesterol were measured using ILab Chemistry Analyzer 300 PLUS .α) were isolated from spleens RNase Mini Kit  according to the manufacturer's instructions. Purity of the extracted RNA was determined by measuring the ratio of the optical density at 260 and 280 nm using a spectrophotometer , which ranged between 1.8 and 2.0. mRNA expressions of the genes were determined by qPCR as described in our previous study, John et al. [Rattus norvegicus) adapted from NCBI  GenBank Database [α-_1 (NM_012675), IL-6 (NM_012589), GPX (NC_005107.4), SOD (NM_017050.1), CAT (NM_012520.1), and ACTB (NM_031144.2) as housekeeping gene. The expression for each sample was measured according to the quantity of β-actin expressed, while the number of fold expressions was calculated using 2−ΔΔCt.Organs for gene expression studies were harvested upon sacrifice and were immediately stored in liquid nitrogen for further investigations. Total RNA of tissues of glucose related genes  and antioxidant genes  was isolated from the liver while tissues of inflammatory genes  than control, confirming the hyperglycemic state at both initial (D6) and final (D22) phases of pregnancy. Additionally, insulin and C-peptide levels of GDM group showed a significant increase from the initial phase to the final phase of pregnancy, indicating increase of insulin production in the final phase of pregnancy. A slight decrease in body weight was observed from Week 1 to Week 2 and from Week 2 to Week 3 of GDM group as shown in Weekly fasting serum glucose levels are shown in P < 0.001) from the initial to the final phases of pregnancy in the GDM group compared to the control group. Additionally, HDL-cholesterol was significantly decreased in the GDM group compared to the control group. From both hierarchical clustering and PCA  at the initial and final phases of pregnancy in GDM and control groups. The level of plasma CRP is higher in GDM group compared to control group at both phases of pregnancy which stimulates the acute phase inflammatory response (see α levels increase (P < 0.001) from the initial and final phases of pregnancy in GDM group, but no significant changes were observed in the control group. In addition, in both phases, the level of IL-6 and TNF-α in GDM groups was higher than control.onse see . As showGenes involved in glucose transporter (GLUT-2 and GLUT-4) and insulin signaling pathway  at the initial and final phases of pregnancy in the control and GDM group are shown in Figures P < 0.001) in the GDM group compared to control, in both initial and final phases.The expressions of antioxidant genes for SOD, CAT, and GPX were shown in Histology section of pancreas was shown in Current animal model combines multiple factors that cause insulin resistance in gestation. This study provides data associated with glycemic parameters, lipid parameters, glucose transporters parameters, genes affecting insulin signaling, proinflammatory cytokines parameters, and oxidative parameters on the initial and final phases of pregnancy in both control and GDM groups. In our study, body weight of rats on first week in the GDM group was higher compared to the control group, which can be attributed to consumption of high fat sucrose diet . A decreA state of hyperlipidemia is commonly observed in GDM . This waα level and IL-6 levels are higher in the GDM group compared to control  was also higher in GDM than control (see α) in stimulating the acute phase inflammatory response [In the evaluation of proinflammatory cytokines, our results show that TNF-trol see . Obesityl , PCK-1, and serum CRP level were higher than control. Furthermore, low concentration of IRS-2 genes and AKT-1 and reduced level of GLUT-4 from the initial to final phases indicate the possible defect of insulin signaling in our GDM animal model. The impaired antioxidant status in our GDM model showed that inducing stress through changing sleeping cycle could induce oxidative stress, which is associated with diabetes. Our GDM animal model showed a higher body weight than control during Week 1, which was due to HFSD feeding. The GDM animal then showed a decrease in body weight from Week 1 to Week 3, due to the destruction of beta cells by STZ. Blood glucose level was also higher in GDM group than control, indicating the hyperglycemic state of the GDM rats. Higher level of lipid parameters  in GDM group then confirms the state of hyperlipidemia in GDM rats. Based on these results, it can be concluded that suitable GDM animal model can be created through nutritional, pharmacological, and lifestyle manipulations. This model can then be used to further understand the pathophysiology of GDM and consequently finding novel therapies for GDM.In this study, we administered STZ and NA to pregnant rats, along with feeding them with HFSD and altering sleep patterns to induce GDM. The GDM animal model showed signs of insulin resistance where expressions of both proinflammatory cytokines (IL-6 and TNF-


Drosophila melanogaster shows a pattern of aging similar to human beings and recapitulates the arrhythmogenic conditions found in the human heart. Moreover, the kinase CaMKII has been characterized as an important regulator of heart function and an arrhythmogenic molecule that participate in Ca2+ handling. Using a genetically engineered expressed Ca2+ indicator, we report changes in cardiac Ca2+ handling at two different ages. Aging prolonged relaxation, reduced spontaneous heart rate (HR) and increased the occurrence of arrhythmias, ectopic beats and asystoles. Alignment between Drosophila melanogaster and human CaMKII showed a high degree of conservation and indicates that relevant phosphorylation sites in humans are also present in the fruit fly. Inhibition of CaMKII by KN-93 (CaMKII-specific inhibitor), reduced HR without significant changes in other parameters. By contrast, overexpression of CaMKII increased HR and reduced arrhythmias. Moreover, it increased fluorescence amplitude, maximal rate of rise of fluorescence and reduced time to peak fluorescence. These results suggest that CaMKII in Drosophila melanogaster acts directly on heart function and that increasing CaMKII expression levels could be beneficial to improve contractility.Aging is associated to disrupted contractility and rhythmicity, among other cardiovascular alterations.  Drosophila melanogaster has emerged as a model for studies related to cardiac function and dysfunction. Moreover, it is known that Drosophila melanogaster has similar proteins responsible for Ca2+ handling in the cardiomyocyte, 2+ regulation observed in human myocardium Due to the similarity of genes between flies and humans and the rapid genetic manipulation that offers the fruit fly, 2+-calmodulin-dependent protein kinase II (CaMKII) is an ubiquitous Ser/Thr-directed protein kinase that is expressed from a family of four closely related genes, α, β, γ and δ whose official symbols are: CAMK2A, CAMK2B, CAMK2G and CAMK2D. All CaMKII isoforms in humans and other mammals have numerous splice variants 2+i) increases, CaMKII is activated through the binding to the Ca2+-calmodulin complex. Once activated, autophosphorylation or oxidation of CaMKII can sustain its activity regardless of the levels of Ca2+i2+ channel (LTCC) or voltage-gated sodium channels Na(V)1.5 CaDrosophila melanogaster CaMKII is codified by a single gene, with several splicing products. This protein also becomes independent of Ca2+/calmodulin upon autophosphorylation 2+ handling, excitability, contractility and cardiac pathology in humans, and being that Drosophila melanogaster constitutes a good model organism, it is of great importance to assess the role of this kinase in the fruit fly heart. However, a possible participation of CaMKII in Drosophila melanogaster heart regulation has never been considered.Drosophila melanogaster semi-intact heart model that express the Ca2+ reporter system GCaMP3 , with two main goals: 1. To characterize the intracellular Ca2+ behavior of Drosophila melanogaster heart in young (7 days) and old (60 days) flies; 2. To investigate a possible role of CaMKII in the regulation of cardiac function in young adult flies.In the present study we used a 2+ dynamics in young and senescent Drosophila melanogaster hearts. Moreover, CaMKII appears to play a significant role in Drosophila melanogaster heart function regulation and deregulation.It will be shown that the fly model expressing GCaMP3 is a convenient experimental tool to assess intracellular Ca2+/calmodulin binding domain and a peptide from myosin light chain kinase, M13. According to what has been described, the genetic indicator GCaMP3 is brighter, has greater protein stability and a larger dynamic range and higher affinity for Ca2+ compared to GCaMP2 We used transgenic flies harboring UAS-associated GCaMP3 construct and the GAL4 transcriptional activator protein, under the control of the cardiac-specific tinC driver. GCaMP3 is a green fluorescent protein that, like GCAMP2, includes a Ca2+-sensing protein, GCaMP3. Typical recordings of Ca2+ transients are also shown in panel B of Using fluorescence microscopy we observed semi-intact heart preparations from 7 and 60 days old flies maintained in oxygenated supplemented artificial hemolymph. Drosophila melanogaster2+ dynamics. Using the genetic approach developed by Lin et al. 2+ dynamics with aging. 2+ transients obtained in heart flies at 7 and 60 days. As can be appreciated, senescence did not modify Ca2+ transient amplitude and maximal rate of rise of fluorescence (+dF/dtmax), meanwhile, maximal rate of fluorescence decay (–dF/dtmax) was significantly diminished. This finding is consistent with a prolongation of full duration at half maximum (FDHM) of Ca2+ fluorescence produced by a significant increase in half relaxation time. Taken together, these findings indicated that senescence produces a significant decline in the relaxation process. Moreover, senescence decreased spontaneous frequency and increased what has been called arrhythmicity index 2+ handling in the fruit fly heart and indicate that major changes produced by senescence are related to a decrease in spontaneous frequency, the increased incidence of arrhythmogenic events and the slowing of intracellular Ca2+ decay. Interestingly, these results recapitulate typical characteristics of mammalian senescent myocardium Different cardiac functional changes during aging have already been described in Drosophila melanogaster CaMKII, a BLAST search alignment using the HomoloGene tool was performed. Interestingly, CAMK2D variant, which is present in heart tissue, has the highest homology with the Drosophila melanogaster CaMKII. Furthermore, in comparison with other mammalian species, human CaMKII has one of the highest scores of similarity to fruit fly ortholog [Table S1]. We then determined the degree of similarity between the different isoforms of CaMKII from Drosophila melanogaster and the isoforms of the human CaMKIIδ. To this purpose, all splice variants of CaMKIIδ from human and CAMKII from fruit fly were obtained and aligned. Scores ranged between 74 and 81%. All Drosophila melanogaster isoforms showed the highest percentage of similarity with the isoforms 1 and 2 of the human CaMK2D, with a slightly predominance of the isoform 2  is relevant to regulate the auto-activated form of the enzyme Drosophila melanogaster has similar phosphorylation sites to that of the neural CaMKIIα from rat 281/Met282, capable to be oxidized by reactive oxygen species (ROS). Activation of CaMKII by ROS evokes, similar to autophosphorylation, Ca2+ autonomous activity Drosophila melanogaster CaMKII. Instead, methionines are replaced by valine residues in the fruit fly.The regulatory domain of human CaMKIIδ includes features of calmodulin binding and autophosphorylation through the threonineDrosophila melanogaster. Moreover, the fly fruit conserves fundamental residues for CaMKII function. This prompted us to continue with functional analysis of this protein in the fruit fly heart.Taken together the above results indicate a high degree of similarity between human and Drosophila melanogaster heart may provide a useful tool to gain insights into the mechanisms of cardiac failure and arrhythmias in human myocardium. We therefore explored the effects of CaMKII inhibition in Drosophila melanogaster heart. As already stated, CaMKII regulates many of the key proteins that are involved in the excitation-contraction coupling (ECC) of human heart. There is an increasing awareness that disorders of ECC are at the basis of different type of arrhythmias and may conduct to cardiac hypertrophy and heart failure. Thus, knowledge of the effect of CaMKII in a model like The incidence of flies that showed ectopic beats with CaMKII inhibition was slightly lower than that observed without KN-93, while ectopic beats, expressed as percentage of total beats, was similar in both groups  and individuals that overexpress CaMKII . Cardiac CaMKII overexpression increased the amplitude of intracellular Ca2+ transients and spontaneous heart rate. The increase of amplitude in CaMKII-OE flies was associated with a significant enhancement in +dF/dtmax and a shortening of TTP of Ca2+ transient  and shortening of heart walls by recording of high speed videos, provided information about functional parameters 2+ handling with aging and the possible role of the multifunctional CaMKII on cardiac function.In the present report, we used the transgenic flies that harbor genetic codified calcium indicator 2+ dynamics, prolonging the decay of Ca2+ transient in Drosophila melanogaster. 2. Aging increases the incidence of ectopic beats and asystoles. 3. CaMKII gene from Drosophila melanogaster is homolog to mammalian CaMK2D gene and CaMKII from the fruit fly conserves important fundamental phosphorylation residues for CaMKII function. 4. CaMKII from Drosophila regulates spontaneous frequency, frequency variability and intracellular Ca2+ dynamics.Our results showed for the first time that 1. Aging alters intracellular cardiac CaDrosophila melanogaster cardiac preparation as a useful tool to gain important insights into human cardiac health and disease.These findings emphasize the importance of 2+ dynamics by slowing cardiac relaxation. Similar findings have been described in humans, whose myocardial contraction is also prolonged and relaxation is incomplete in aged individuals compared with younger adults An important finding of this study is that aging modified intracellular Caet al., using OCT and high speed movies of movement of heart wall The present results also showed that old flies exhibited a reduction in spontaneous cardiac frequency than younger flies. Moreover frequency variability was increased in the older flies, in agreement with previous reports by Ocorr et al. in this model Drosophila homolog of human KCNQ1- encoded K+ channel α subunits. Whether the predominance of ectopic beats described here can also be ascribed to these channels is not apparent from the present findings.Importantly, our results also demonstrated an increase in ectopic beats and asystoles with aging. The study carried out by Ocorr CaMKII can phosphorylate different key proteins of the ECC, like RyR, PLN, the Na(V)1.5 channels and LTCC. Phosphorylation of these proteins has been involved in the pathogenesis of different type of arrhythmias in mammals Drosophila CaMKII and the human product of gene CaMK2D, the most predominant isoform in heart tissue. Previous reports about similarities of CaMKII in Drosophila and mammals have referred to its presence and functionality in brain Drosophila heart through mass spectrometry Drosophila heart was not identified.In the present study, we showed a high degree of similarity between 2+ dynamics. These results are in agreement with studies in mice hearts in which either the absence of CaMKIIδ, a major myocardial CaMKII isoform, or the cardiac expression of a CaMKII inhibitory peptide, does not alter ECC under basal conditions Drosophila melanogaster did not affect heart function 2+ transient amplitude and reduced time to peak of Ca2+ transient amplitude, a result that demonstrated a direct role of CaMKII on heart function in Drosophila melanogaster adult heart. These results are at odds with the results obtained with chronic overexpression of CaMKII in mouse hearts. In these mammals, overexpression of CaMKII produced changes in ECC, like a decrease in Ca2+ transient amplitude and SR Ca2+ content, which predispose these animals to the progression to heart failure stage Drosophila vs. mammalian heart function is not apparent from the present results. Whether the different circulatory systems: open (Drosophila) vs. closed  can influence the dissimilar impact of CaMKII in Drosophila and mammalian heart is difficult to ascertain at present. Possible other reasons for this difference may be searched on putative different phosphorylations targets for CaMKII in both species, different regulation of CaMKII activity , or different long term effects of the overexpression of the kinase through the activation of different transcription factors. Future work need to be performed along these lines to definitively understand this phenomenon.In the present report, we found that inhibition of CaMKII in young adults did not significantly affect CaMoreover, the present results showed that CaMKII inhibition reduced spontaneous frequency and increased the fraction of asystoles. Moreover, CaMKII overexpression increased spontaneous frequency and reduced frequency variability  with little effect on ectopic events.2+ current (ICa) The underlying mechanism of CaMKII role on spontaneous frequency and arrhythmias remains to be elucidated. It has been reported in rabbit heart, that spontaneous rhythmic excitation of sinoatrial (SA) node cells is critically dependent on CaMKII, which is highly localized beneath the surface membrane in the vicinity of L-type channels and modulates L-type CaDrosophila heart, i.e., inotropism and lusitropism, because it increases the amplitude of intracellular Ca2+ transients and the speed relaxation and chronotropism, because it regulates spontaneous frequency and heart rate variability. Ongoing experiments in our laboratory are studying the possible influence of CaMKII activation in aging, with the goal of providing further insights in this possible and still unknown relationship.Taken together, the results indicate that CaMKII modifies different properties of Drosophila heart. Future exhaustive analysis will permit to explore possible compensatory mechanisms of chronic overexpression of CaMKII in Drosophila melanogaster.In summary, the results indicate that CaMKII exerts a beneficial effect by improving cardiac performance and reducing heart rate variability. Although CaMKII is described in mammalian heart as an arrhythmogenic molecule, we did not find evidence that this was also the case for http://www.ncbi.nlm.nih.gov/pubmed/Gene), uploaded to UGENE program , and the multiple sequence alignment was performed using integrated CLUSTAL and Kalign algorithms.Sequences were obtained from the NCBI Gene database (http://www.ncbi.nlm.nih.gov/homologen).Pairwise alignment scores of the different species were obtained using the HomoloGene tool of the NCBI . These transgenic flies express the reporter system GCaMP3 downstream the UAS sequence, and the Gal4 protein under control of TinC promoter, specifically expressed in the heart. 7 and 60 days old individuals were selected for experiments.w[*]; P{w[+mC] = UAS-CaMKII.R3}2 that contains the CaMKII gene downstream UAS sequence. This strain was obtained from Bloomington Stock Center . We collected virgin females to cross with the stock that contains the reporter system. Offspring selectively expresses CaMKII in the heart, under the control of TinCGal4. Individuals heterozygous of 7 days were collected to obtain semi-intact preparations.We also amplified flies with the genotype 2+ fluorescence measurements. Hearts were prepared according to previously described methods 2 and placed on a soft gel plate dorsal side down. Thorax and legs were removed from the abdomen using iridectomy scissors. In this way, we studied the myogenic contraction without influences from neuronal input, as described in previous reports 2, 8; NaH2PO4, 1; NaHCO3, 4; HEPES, 5 (pH 7.1); sucrose, 10; Trehalose, 5; and CaCl2, 2, at room temperature. The ventral abdominal cuticle was removed and abdominal organs were gently dissected away from the heart. Surrounding fat was removed by liposuction using glass capillaries. The heart remained attached to the dorsal cuticle and was readily identified as a beating structure along the midline from abdominal segments A1 to A4. We registered the signal on the conical chamber, positioned on the first abdominal segment, where the fluorescence intensity was higher than in other regions of the preparation.Flies collected at 7 and 60 days after eclosion were used for cardiac Ca2+ that precedes contraction. Recordings of Ca2+ transients were carried out for as long as 26 seconds. Images were analyzed with ImageJ  and LabChart software .Dissected hearts were observed using a confocal microscope (Carls Zeiss 410). We scanned changes in fluorescence of the conical chamber, localized in the first abdominal segment. Each fluorescence increase followed by a fluorescence decrease represents the transient elevation of cytosolic CaExperiments were performed on individuals carrying the reporter system only, which we called control or wild type (WT) and individual heterozygous carrying one additional copy of CaMKII (CaMKII-OE). In a group of experiments, we used 5 µMol/L of KN-93  to inhibit CaMKII in WT flies.2+ transients: peak Ca2+ transient amplitude, expressed in arbitrary units of fluorescence intensity, maximal rate of rise of fluorescence (+dF/dtmax), maximal rate of fluorescence decay (−dF/dtmax), full duration at half maximum (FDHM), time to peak (TTP) and half-time of peak fluorescence decline (t1/2). We measured heart period, as the interval between two consecutive peaks of maximal fluorescence, to analyze irregularities in heart rhythm, i.e. frequency variability, through the arrhythmicity index according Ocorr et al.The following parameters were analyzed from CaA summary of experimental procedures and parameters described is shown by Hearts of WT and CaMKII-OE flies were removed, collected and maintained in RNA later (Quiagen Inc). RNA extraction was carried out with RNAeasy kit, according to the manufacturer’s instructions (Quiagen Inc). RT-PCR was performed using SuperScript reverse transcriptase (Quiagen Inc). PCR reaction conditions were as follows: step 1: 95°C for 30 seconds, Step 2: 40 cycles of 95°C for 30 sec followed by 55°C for 1 min and 72°C for 30 sec.Primers to identify a 101 pairs of bases fragment of CaMKII cDNA were designed with the following sequences:ACAACCATTTTAAATCCTCATGTGCForward primer: TGTGCATGTCCTTGTTATCAReverse primer: Tubulin α-1 was used as control and primers to identify a 182 pairs of bases fragment of the cDNA were designed with the following sequences:ATCAACTACCAGCCTCCCACForward primer: TCCTCCATCCCCTCCCCAACReverse primer: Samples containing 40 ng of RNA were separated by electrophoresis on 2% agarose gel and visualized with SYBR Safe DNA Gel Stain .t test. Values of P≤0.05 were considered statistically significant. Due to the exploratory nature of the present study, multiple, unrelated hypothesis were tested using a single control group.All results are expressed as mean ± SEM. Comparisons were made using Student’s unpaired Figure S1Summary of experimental procedures to obtain semi-intact preparation and recording of fluorescent signal. A: Intact fly anesthetized, positioned with dorsal side down, on the Petri dish. B: Head, thorax and abdominal organs were removed. C: abdominal region with beating heart attached to cuticle through alary muscles was exposed. Preparation was bathed with artificial oxygenated hemolymph. Heart was visualized by bright field microscopy and then, fluorescent image was obtained by excitation of preparation with laser 488. Emission was recording and transformed in digital signal to be analyzed. D. Parameters measured are indicated.(TIF)Click here for additional data file.Figure S2Detection of transcripts for CaMKII in heart tissue from control and CaMKII-OE flies. Total RNA was extracted from 120 isolated hearts from control and CaMKII-OE flies. CaMKII was amplified and visualized by electrophoresis on gel of agarose 2%. The observed amplified product of 101 base pairs (bp) correspond to CaMKII detected in brain and hear tissue form control and heterozygous with one extra copy of gene. Fragment of alpha-tubulin of 182 bp was utilized as control.(TIF)Click here for additional data file.Table S1Pairwise alignment scores between CaMKII from Drosophila melanogaster against different mammalian species. Official gene symbol for each species is indicated. Drosophila melanogaster and Homo sapiens have a greater percentage of identity compare with other common mammalian species used as model for cardiac studies.(DOC)Click here for additional data file.


Secale cereale L. accessions using 22 previously published simple sequence repeat (SSR) markers. The plant material included 367 rye accessions comprising historical and contemporary cultivars, cultivated materials, landraces, and breeding strains from the Polish breeding company Danko. The studied accessions represented a wide geographical diversity. Several methods were employed to analyze genetic diversity among the Secale cereale L. accessions and to determine population structure: principal coordinate analysis (PCoA), neighbor-joining (NJ), and Bayesian clustering. We also defined a core collection of 25 rye accessions representing over 93 % of SSR alleles. The results of these analyses showed that accessions from the rye gene bank are clearly divergent in comparison with materials received directly from European breeding companies. Our findings suggest also that the genetic pool of current rye cultivars is becoming narrower during breeding processes. The selected panel of SSR markers performed well in detection of genetic diversity patterns and can be recommended for future germplasm characterization studies in rye.The primary aim of this study was to estimate genetic diversity among The online version of this article (doi:10.1007/s11105-015-0896-4) contains supplementary material, which is available to authorized users. Secale comprises typical representatives of the Mediterranean flora, and its members are widely distributed from Central Europe and the Western Mediterranean through the Balkans, Anatolia, Israel, and the Caucasus to Central Asia. An isolated population also occurs in South Africa. The genus includes annuals, short-lived and long-lived perennials, wild, weedy, and cultivated taxa  is one of the most important cereal crops cultivated in Eastern and Northern Europe. It is characterized by the ability to produce high yields even when grown under environmental stress conditions, i.e., low temperatures, drought, and low soil fertility. The presence of disease resistance genes reduces the need for intensive chemical protection of this crop ; 27 population cultivars, 12 hybrid cultivars, and 26 breeding strains from European breeding companies; and 11 rye accessions from the collection of A. Lukaszewski from the University of California, Riverside (USA). Out of 367 accessions used in this study, 362 were examined previously using DArT markers . Equal amounts of DNA from the 96 plants representing each accession were pooled into one sample. DNA purity and concentration were evaluated using a NanoDrop 2000 spectrophotometer .Forty-two public SSR markers  under conditions specified by the high-stringency protocol of Pillen et al. , 1.5x DreamTaq buffer, 0.2 mM dNTP’s, 0.5 mM MgCln et al. . The ampn et al. . The conFor ease of analysis, we labeled rye accessions according to their geographical origin, source of the seeds, and assignation to one of the heterotic pools. We also conducted separate analyses only for accessions from breeding companies and from PAS BG.Based on the PCR results, a binary matrix was constructed, where the presence of an amplified product was scored as 1 and the absence of a product as 0. Because the templates used in SSR analysis were pooled DNA from 96 plants and more than two alleles per sample were observed, separate bands were treated as individual alleles. The bands were scored as present or absent, and allele frequencies within individual accessions were not established. The number of alleles, number of polymorphic alleles, proportion of polymorphic products , and percentage of frequent (appearing in >50 % of rye accessions) and rare (appeared in <5 % of accessions) alleles were counted. Polymorphic information content (PIC) was calculated according to the formula of Roy et al. .The total number of alleles, number of alleles with a frequency of <5 %, number of private alleles, number of alleles found in more than 25 and 50 % of the accessions from subgroups created based on source of seeds and improvement status, mean diversity, and unbiased diversity were calculated using GenAlEx 6.501  was calculated using Jaccard and Dice coefficients with NTSYSpc 2.1 software Rohlf . CorrelaK) capturing the major structure in the data, a burn-in period of 100,000 Markov Chain Monte Carlo (MCMC) iterations was used, with 100,000 run length and admixture model following the Hardy-Weinberg equilibrium. Five independent runs were performed for each simulated value of K, ranging from 1 to 20. The most likely number of K was determined using the DeltaK method in Structure Harvester  in GenAlEx 6.501.M) algorithm implemented through a modified heuristic algorithm in PowerCore 1.0 was used  (Table h = 0.113), while this value was nearly two fold higher for the collection of A. Lukaszewski (h = 0.218).Analysis of the total allele pattern within groups of accessions sorted according to the source of the seeds and improvement status Table  showed tr = 0.991 and P = 0.001  and cultivars from PAS BG (34 %), especially from Western and Central Europe. Only cultivar Gonello (KWS Lochow) belonged to the P2 population. Breeding strains from the company Danko were the most homogeneous group and were characterized by an inferred ancestry level of >80 %. The P1 population also contained landraces, cultivated materials from European countries, and accession from the A. Lukaszewski collection.Population P2 was comprised mainly of landraces from PAS BG (79 %), while the representation of cultivars and cultivated materials from PAS BG was relatively lower: 8 and 10 %, respectively. The geographical origins of the majority of accessions classified as P2 were Asia, the Balkans, and Southern Europe. The remaining P2 accessions were cultivars and cultivated materials originating from Eastern and Central Europe, and South and North America. Two accessions from the A. Lukaszewski collection originating from North America were also assigned to P2.Accessions characterized by mixed ancestry were mainly from PAS BG (90 %). The geographical origin of accessions with mixed ancestry was very diverse.Several clusters were visible in the NJ dendorgram Figs.  and 3. TTwo groups could be distinguished in the PCoA plot Figs.  and 5. APCoA comparing cultivars and breeding materials showed clear dissimilarity between accessions from the gene bank and from breeding companies. Two separate clusters were visible both in the dendrogram Fig.  and PCoA1) cultivars were dispersed throughout the PCoA plot.In the PCo analysis with labeling according to the assignation to one of the heterotic pools (Petkus or Carsten), the majority of accessions with confirmed Petkus pedigree were closely grouped (Online Resource P < 0.001). The pairwise PhiPT value  was 0.137 and indicated relatively high degree of differentiation between populations P1 and P2. Much greater proportion of variation within than among populations was found for groups of accessions from different sources and with different improvement status: 87 and 13 %, respectively (P < 0.001). When accessions were grouped based on geographical origin, these values were 92 and 8 % (P < 0.001). Pairwise PhiPT values estimated between groups of accessions with different source and improvement status indicated a wide range of genetic differentiation between them ranging from low (0.073) between cultivated materials and landraces from PAS BG to very high (0.275) between breeding strains from Danko and accessions from the A. Lukaszewski collection. In the case of plants grouped according to geographical origin, very low 0.003 PhiPT, indicating little genetic differentiation, was detected between accessions from South America and Northern Europe. The highest genetic differentiation (PhiPT = 0.217) was found between accessions from Western Asia and Central Europe  were selected as core entries. Core collection entries represented all sources and geographical origins .In general, the presented results were largely consistent with those of Bolibok-Brągoszewska et al. . In the In our study, we did not analyze the genetic diversity of rye accessions on a single plant basis. Instead a bulking strategy with 96 plants for each accession pooled into one sample was applied. In consequence of DNA pooling some information is lost, for example, it is not possible to estimate allele frequencies within individual accessions. While this limitation is true for both SSR markers used in the present study and for DArT markers applied earlier  which indicates their high informativeness. The average percentage of polymorphic alleles for the set of 22 markers was also high (93 %). This was predictable because the public markers were not selected randomly, but based on the results of analyses using six diverse rye accessions and 42 SSR markers. The high values of the marker informativeness parameters and the congruency of the results reported here with the results obtained based on over 1000 genome-wide DArT markers  and also from population and hybrid varieties from other breeding companies. Similar results for Gonello were obtained earlier using DArT markers . While this result could be attributed to a mistake in a labeling of seed samples, an alternative explanation may be found in the work of Chebotar et al. , where iAs discussed above, factors such as reproduction or the manner of storage of plant material can greatly influence genetic variation. This may reflect differences in the procedures of plant reproduction employed by gene banks and breeding companies. In the case of open-pollinated plants, it is important to consider regeneration management in ex situ collections, particularly the optimal population size and distance between regeneration plots  and unbiased diversity (uh = 0.118) were the lowest in this germplasm group. Evidence indicating low genetic diversity in Polish rye was previously obtained by Rafalski et al. . Genetic similarity within cultivars from breeding companies and within breeding strains from Danko was relatively high. It is also noteworthy that no unique alleles were observed in accessions obtained from Danko and the values for mean diversity  is one of the major determinants of the population structure in cereals 367 Online Resource 2SSR markers used in preliminary analyses. (PDF 83 kb)Online Resource 3The result of Mantel test showing a strong positive correlation between Jaccard and Dice coefficient. (PDF 541 kb)Online Resource 4GS values (according to the Jaccard coefficient) for pairs of accessions. (PDF 6710 kb)Online Resource 5Delta K values (number of populations assumed) ranging from 1 to 20. (PDF 1347 kb)Online Resource 6Results of Analysis of Molecular Variance. (PDF 34 kb)Online Resource 7PCoA of 367 rye accessions based on 22 SSR markers. Accessions labeled according to their pedigree. (PDF 1369 kb)


Ochre is the common archaeological term for prehistoric pigments. It is applied to a range of uses, from ritual burials to cave art to medications. While a substantial number of Palaeolithic paint mining pits have been identified across Europe, the link between ochre use and provenance, and their antiquity, has never yet been identified. Here we characterise the mineralogical signature of core-shell processed ochre from the Palaeolithic paint mining pits near Lovas in Hungary, using a novel integration of petrographic and mineralogical techniques. We present the first evidence for core-shell processed, natural pigment that was prepared by prehistoric people from hematitic red ochre. This involved combining the darker red outer shell with the less intensely coloured core to efficiently produce an economical, yet still strongly coloured, paint. We demonstrate the antiquity of the site as having operated between 14–13 kcal BP, during the Epigravettian period. This is based on new radiocarbon dating of bone artefacts associated with the quarry site. The dating results indicate the site to be the oldest known evidence for core-shell pigment processing. We show that the ochre mined at Lovas was exported from the site based on its characteristic signature at other archaeological sites in the region. Our discovery not only provides a methodological framework for future characterisation of ochre pigments, but also provides the earliest known evidence for “value-adding” of products for trade. Red ochres are relatively common in natural geological and soil formations.Ochre is the colloquial term used by archaeologists to describe an earth or rock containing red or yellow oxides, most commonly hydroxides of iron , 2. Red Ochres have long been used for a range of applications by prehistoric people. The best known examples of Palaeolithic use of ochre are from cave paintings  and rituPotential symbolic purposes of ochres are demonstrated by present-day hunter-gatherers, yet remain hypothetical or circumstantial in the case of Palaeolithic use. Ochres are used by modern hunter-gatherers for both internal and external medications, food and wood preservatives, insect repellents, and for tanning of hides , 3, 8–12Regardless of whether ochres have long held a symbolic purpose, early evidence of intentional processing of raw ochres into higher value products has been demonstrated by identification of ochre powder as an ingredient in the ancient manufacture of compound adhesives . In EuroThe study of prehistoric ochre has mainly been focused on the analysis of raw materials and their uses , 18–23. The natural sources of prehistoric ochres are not easily identifiable due to the ubiquity of raw material sources, combined with high mineralogical variability. In this study we address this challenge to provenancing raw ochre sources by investigating the crystallographic and microstructural features of archaeological pigments, in order to identify the actual origin of red pigments based on hematite. Previous hypotheses regarding the provenance of archaeological ochres have typically been based on geographical proximity rather than considering their mineralogical and microscopic-scale features, which require a mineralogical or materials science approach. The fundamental characteristics of natural and artificial hematite must, however, be taken into account in order to fingerprint an ochre source. For example, hematite can be obtained artificially by heating the mineral goethite to moderate temperatures 300–350°C) , 23, 29,0–350°C ,Here we put forward an alternative hypothesis for pigment production: the “core-shell” technique. We suggest that pigments were not only derived from pure natural ochre or heat-treated goethite, but also that prehistoric people ground the highly pigmented red material to produce finer particles, and then exploited natural oxidation processes whereby the fine hematite nano-particles—the “shell”–coated non-pigmented sand or silt-sized grains—the “core”. This process would have resulted in substantially higher volumes of pigmented material than was available by using the pure ochre. Most modern-day core-shell nanostructures are developed with the idea to combine two materials—and thus two properties—within one structure. In this way, the nanostructure influences the properties of both core and shell, offering a variety of new possibilities and combinations, and expanding the availability of ochre for a wider range of activities. A typical example of modern core-shell pigments are those which contain a core of cheap oxides covered with a layer of phosphates, which are produced as a more economically feasible solution for anticorrosive pigments than pure pigment . The corThe core-shell hypothesis is best investigated by investigating at the microscopic scale the mineralogical and internal structure characteristics of ochre from a known raw material source, in order to establish its formation, provenance, intentional post-extraction processing. Of further interest is its antiquity, to determine precisely how early the method was adopted as a means of “value-adding” a raw material product and therefore better understand how long people have been employing enterprising and innovative means of improving products for trade.We use the Hungarian ochre quarry site of Lovas as a case study for our approach. This is the second known instance of Palaeolithic ochre exploitation in eastern central Europe, following Rydno in Central Poland . BecauseIn this study we clarify the age of mining activity at Lovas with a new set of radiocarbon dates, characterize the petrographic and mineralogical attributes of the Lovas core—shell type red ochre, and establish a database for further investigations of the distribution of processed material throughout the region coeval with its extraction. X-ray diffraction (XRD) is used to identify and characterise the pigments , 36.The Lovas quarry is located 3 km north of Lake Balaton in central western Hungary, in a shallow basin immediately behind the bedrock-dominated Balaton Highland. The small quarry was operated for dolomite grit until the 1960s Figs , and lieOchre has formed within the dolomite as rare thin, yellow, yellowish red or red marly dolomite with thin calcite veins . The och3 of ochre was mined [The outcropping of iron ore—ochre—veins within the dolomite at the Lovas locality drew the attention of Palaeolithic people who exploited these veins for ochre production. The main prehistoric mine site follows one of these veins, forming a pit approximately 6 meters long, 2.5 m deep, and 2.5 m wide. Approximately 25 mas mined . Rubble as mined .Archaeological traces found within the rubble infill from the prehistoric ochre mining pit provides evidence both for ochre mining and processing methods, as well as for the antiquity of mining activities. Archaeological traces include antler picks and shovels, stones for grinding the ore, and tubes made from hollow bones which are interpreted to have acted as carrying tubes to transport the powder produced from mining activities . Of the Alces (Eurasian elk), comprising 80% of the tools when excluding six items indistinguishable from Cervus elaphus (red deer). Elk skeletal parts in the toolkit are derived from ribs, ulna, metapodial, metacarpal, antler, tibia, humerus and femur [A large proportion of the archaeological assemblage comprises bone remnants and bone tools. A number of worked elk bones , 45 and nd femur , the shand femur , 45. Boand femur . The tapThe lithic assemblage is typologically inconsistent, which led to the conclusion that the mine was operated by different lithic “cultures” over a long period of time. The lithic assemblage is dominated by blank flakes of relatively fresh radiolarite. One patinated and relatively weathered bifacial tool, attributed to the Late Middle Palaeolithic Jankovichian period , is form14C date, and stone tool typology based on a single bifacial leaf-shaped tool characteristic of the Late Middle Palaeolithic Jankovichian culture [Until now the antiquity of mining activity at Lovas was interpreted based on the faunal assemblage, a single  culture . These i culture , 44–47.Alces sp. (Eurasian Elk)–the most common raw material—were derived from the same stratigraphic unit as all other bone fragments, and bore clear impacts of human activity. Since the different samples were weathered to varying degrees, it was assumed that the resulting chronology would reflect the full duration of mining activities at Lovas, and that this may have been of significant duration. No dry bone breakage was reported by the taphonomy study [In this study, radiocarbon dating was undertaken on 5 bone artefacts collected from the rubble overburden pit 2, layer 5, in order to establish the antiquity, and if possible the duration, of ochre mining activities at Lovas. The bone fragments taken as samples were all from my study  and ther14C years BP (1σ error). These results are in full agreement with the previous radiocarbon date on elk bone  produced by the Eidgenössische Technische Hochschule (ETH) Zurich, which yielded a date of 11,740±100 14C BP [The uncalibrated radiocarbon dates range from 11,941±44 to 11,469±40 0 14C BP  Fig 5)..14C yearThe calibrated radiocarbon dates yield a modelled range for the duration of mining activities of between 13.2–13.8 kcal BP . These rThe Lovas ochre was characterised based on sampling from the Palaeolithic ochre grit found on the quarry floor, the in situ red dolomite veins, and from ochre traces next to a bone tool found at the site. The three types of samples were investigated in detail by means of X-ray diffraction (XRD) and microscopic analyses.Our quantitative evaluation of XRD spectra shows that the ochre mineralogy is dominated by dolomite (about 80 w/w %), with smaller proportions of quartz (~10%), clay (~5% kaolinite) and mica (~1% muscovite). The mineralogical characterisation of the ochre by XRD is summarised in The natural pigments do not dominate, but instead are intermixed with clays and quartz . The intIn order to determine how such a small proportion of hematite can dominate the ochre material, the internal structure of the pigment was investigated at microscopic level. Polarization microscopy shows that the majority of ochre mineral grains are finer than 150 μm in diameter, mostly ranging from 50 to 150 μm , and comAt the sub-micron scale, SEM images of the dolomite surfaces show hematite as bright particles and aggregates sitting on the darker gray dolomite crystal surface . Hematitcore of dolomite, and occasionally quartz grains, with an external shell of pigment. This core-shell structure of the ochre grains is unlikely to have been produced naturally, since it contrasts with the more consolidated nature of the in situ pigment. The coarser core grains are angular, suggesting post-depositional fragmentation, most likely due to post-extraction processing of the raw ochre material. The distribution of the pigmented shell hematite, which gives the grains there colour, also suggests post-depositional redistribution of the naturally occurring fine hematite. This raises the question as to how this process was undertaken, and why.The ochre therefore can be characterised as having a Mining activities at Lovas appear to have taken place over a relatively short period of time, from ca. 13.8–13.2 kcal BP. This period corresponds to the Allerød interstadial 1b-d. Comparison of the timing of this site with palaeoenvironmental and archaeological records enables hypotheses to be formed with respect to the implications for human behaviour of the mining activities at this particular site.Alces bones, as was the case for Lovas, although there is otherwise no cultural similarity between the sites. In Poland, Magdalenian radiocarbon dates are mostly no later than 14.0 kcal BP except for a few instances which are contemporaneous with Lovas [The archaeological record at the time of the Allerød interstadial 1b-d is as yet poorly understood in the Carpathian basin. An Epigravettian site at Nadap, northeast of Lovas between Lake Balaton and the Danube, yielded a date of 15.6 kcal BP from a horse phalange . At Szekth Lovas , 57. Froth Lovas . DespiteThe environmental conditions prevailing in the region during the Allerød interstadial 1b-d are best known from the surrounding loess records , 42, andThe absence of archaeological sites contemporaneous with Lovas in the Carpathian basin, combined with the change in climatic conditions described above, may indicate that the area was abandoned during the Allerød climatic amelioration in favour of a wider territory beyond the Pannonian basin. Based on this hypothesis, the hunter-gatherer population would have visited the area solely in order to exploit mineral dyes without including the territory in their usual foraging range. Mining activity at Lovas was of short duration, but was nevertheless intensive.total, whereas red ochres may exceed 90% FeOtotal [This study uses petrographic techniques to provide the first evidence for post-depositional treatment of ochres during the late Palaeolithic. Previous investigations interpreted the red material from Lovas as being not hematite, but limonite , a yelloFeOtotal , 49. Our2O3). In general, the goethite and hematite responsible for red coloration of rock particles form by combined hydrolysis and oxidation of Fe(II) bearing minerals , which afterwards dehydroxylate into hematite [There is in fact no evidence to suggest that heating played a role in the processing of the raw ochre. The red coloration is, in our case, due to hematite coating of quartz and dolomite grains. The two most common low-temperature iron(III) oxides on Earth are goethite (α-FeOOH) and hematite , with a proportional detector containing a graphite monochromator in the reflected beam. The following analysis parameters were used: 40 kV, beam current of 35 mA, scan range of 4°–70° 2θ in continuous scan of 0.02° 2θ steps, with 1 s per step. The diffraction spectra were compared with the International Centre for Diffraction Data database and evaluated for quantitative phase composition using full pattern fit software.Individual ochre particles were investigated using a field-emission scanning electron microscope (JEOL JSM-5800), fitted with an energy-dispersive X-ray analysis (EDS) to distinguish different mineral particles and their shape, size and elemental composition.Vacuum impregnation was used for the soft ochre samples. The dried samples were put into mounting cup holders and then placed in a vacuum impregnation unit (Struers CitoVac). The mounting cups in the vacuum chamber were filled with impregnation material (EpoFix resin) under vacuum. Specimens impregnated with resin were then bonded onto a glass slide. Transmitted light microscopy and imaging was performed on polished thin sections using a Nikon Eclipse E600 POL microscope fitted with Nikon D90 digital camera.Alces alces which comprise ~70% of the total bone assemblage, including the bone tools [The five bone samples (Pb.53/96.3 (100); Pb.53/98.2; Pb.53/97.3 157); Pb.53/99 (2); Pb.53/99 (1); see 7; Pb.53/ne tools . The sam2 effervescence was observed. 0.1M NaOH was added for 30 minutes to remove humic material. This was followed by a final 0.5M HCl digestion for 15 minutes. The resulting solid was gelatinized following [The samples were pretreated at the Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology (MPI-EVA), Leipzig, Germany, using published methods . The outollowing  at pH3 iollowing . Prior tollowing . The samStable isotopic analyses were undertaken at MPI-EVA, Leipzig (Lab Code S-EVA), using a Thermo Finnigan Flash EA coupled to a Delta V isotope ratio mass spectrometer. For good quality collagen, the C:N ratio should be between 2.9 and 3.6 and the collagen yield no less than 1% of the sample weight , 84. At After all the quality criteria were evaluated, between 3 mg to 5 mg of the collagen extracted were sent to the Klaus-Tschira-AMS facility of the Curt-Engelhorn Centre in Mannheim, Germany (Lab Code MAMS), where they were graphitized and dated .14C free bone samples, provided by the MAMS and pretreated in the same way as the archaeological samples.The resulting radiocarbon dates are listed in S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableThe results of AMS radiocarbon dating of 5 samples from Lovas. δ13C values are reported relative to the vPDB standard and δ15N values are reported relative to the AIR standard. The human modified bones are indicate by an asterisk (*) on the MPI Lab Code.(DOCX)Click here for additional data file.