Huntington's disease (HD) is a progressive neurodegenerative disorder for which there is no effective disease modifying treatment. Following-on from studies in HD animal models, histone deacetylase (HDAC) inhibition has emerged as an attractive therapeutic option. In parallel, several reports have demonstrated a role for histone deacetylase 6 (HDAC6) in the modulation of the toxicity caused by the accumulation of misfolded proteins, including that of expanded polyglutamine in an N-terminal huntingtin fragment. An important role for HDAC6 in kinesin-1 dependent transport of brain-derived neurotrophic factor (BDNF) from the cortex to the striatum has also been demonstrated. To elucidate the role that HDAC6 plays in HD progression, we evaluated the effects of the genetic depletion of HDAC6 in the R6/2 mouse model of HD. Loss of HDAC6 resulted in a marked increase in tubulin acetylation throughout the brain. Despite this, there was no effect on the onset and progression of a wide range of behavioural, physiological, molecular and pathological HD-related phenotypes. We observed no change in the aggregate load or in the levels of soluble mutant exon 1 transprotein. HDAC6 genetic depletion did not affect the efficiency of BDNF transport from the cortex to the striatum. Therefore, we conclude that HDAC6 inhibition does not modify disease progression in R6/2 mice and HDAC6 should not be prioritized as a therapeutic target for HD. HTT gene, resulting in an expanded polyglutamine (polyQ) tract in the N-terminus of the huntingtin protein Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder with a mean age of onset of 40 years 2+ dependent catalytic domains and are homologous to yeast Rpd3 and Hda1, respectively. HDAC11 is also Zn2+ dependent but has been placed in a separate category (class IV) due to a lack of sufficient sequence similarity to any other group + as a co-factor Histone deacetylases (HDACs) have been proposed as possible therapeutic targets for HD Drosophila melanogaster model of spinal and bulbar muscular atrophy (SBMA) HDAC6 expression rescued neurodegeneration in an autophagy-dependent manner HDAC6 is a target of some of the broad range HDAC inhibitors including suberoyl anilide hydroxamic acid (SAHA) and trichostatin A (TSA) Hdac6 knock-out background. As Hdac6 is located on the X-chromosome, hemizygous male mice are nullizygous for Hdac6HdhQ150 knock-in model of HD Based on the previous studies in cell culture models of HD, we might expect that loss of HDAC6 would increase aggregate load and exacerbate the phenotype in a mouse model of HD. On the other hand, an increase in cortico-striatal BDNF transport might be expected to improve some HD-related phenotypes. To determine whether HDAC6 regulates aggregate formation and turnover and/or plays a role in BDNF transport in a mouse model of HD, we crossed the R6/2 transgene onto an Hdac6 knock-out mice (Hdac6KO) are viable and fertile, and do not show any overt phenotypes Hdac6KO mice and analysed by real-time quantitative RT-PCR (qPCR). Hdac6 was not expressed in tissue from Hdac6KO mice  mice to WT, Hdac6KO and R6/2 littermates. As Hdac6 is an X-linked gene Hdac6  = 14.28, p<0.001) and gained weight at a significantly slower rate  = 55.99, p<0.001). HDAC6 depletion by itself had no impact on the weight  = 0.567, p = 0.454) and weight gain  = 0.247, p = 0.741) when compared to WT mice. Absence of HDAC6 did not affect the loss of weight in R6/2  = 0.007, p = 0.931) or the decreased rate in weight gain  = 0.512, p = 0.567). These data indicate that depletion of HDAC6 does not affect weight loss in the R6/2 mice  = 39.74, p<0.001) and that performance deteriorated over the course of the study  = 18.23, p<0.001). The performance of Hdac6KO mice was similar to WT  = 0.32, p = 0.573) but the variability in their performance over time was significantly different to that of WT mice  = 3.53, p = 0.020). Nevertheless, the Hdac6 knock-out genotype did not modify the performance of R6/2 mice overall  = 0.40, p = 0.529) or with age  = 1.09, p = 0.352).RotaRod measures motor coordination and balance, both of which become significantly impaired in R6/2 mice. Here the animals were subjected to RotaRod analysis at 4, 8, 10, 12 and 14 weeks of age . As expe = 142.41, p<0.001) and deteriorated over time  = 50.69, p<0.001). The grip strength of Hdac6KO mice was no different from WT mice either overall  = 0.621, p = 0.434) or with time  = 1.97, p = 0.121). Nullizygosity for Hdac6 did not have an effect on the overall grip strength of R6/2 mice  = 3.17, p = 0.081) nor did it influence the deterioration in grip strength over the course of the experiment  = 0.856, p = 0.463). Therefore, genetic depletion of HDAC6 does not modify loss of forelimb muscle strength in R6/2 mice.Forelimb grip strength was measured at 4 weeks of age and weekly from 11 to 14 weeks of age . As expep-values are presented in Hdac6KO Genotype), but a significant difference in the pattern of activity over time was already evident for activity (p = 0.011) and rearing (p = 0.016) for mice carrying the R6/2 transgene. At 7 weeks of age the R6/2 genotype significantly affected the activity and mobility (p<0.001) of the mice. A clear hypoactivity was evident in both the R6/2 and Dble mice at 9, 11 and 13 weeks of age  and that of the Dble mutant mice was similar to R6/2 (p = 0.784). Thus, absence of HDAC6 does not modify the loss in brain weight exhibited by the R6/2 mice at end-stage disease.Mice were sacrificed at 15 weeks of age and brains weighed to the nearest 0.001 g . As expeAggregation of mutant huntingtin is a prominent feature of HD neuropathology and occurs in all of the HD rodent models that have thus far been developed. Aggregates in the R6/2 mouse appear prior to the onset of behavioural symptoms and continue to accumulate throughout the course of the disease. At the same time, a steady decrease in the levels of soluble transprotein can be observed. In cells, HDAC6 acts as a linker between ubiquitinated protein and/or aggregates and the dynein/dynactin motor complex enabling their transport to the aggresome for autophagic degradation We measured the amount of SDS insoluble aggregates by the Seprion ligand ELISA Our data show that HDAC6 ablation does not influence aggregate load or levels of soluble mutant huntingtin in the cortex, hippocampus or brain stem at 4, 9 or 15 weeks of age  or in quHdac6KO and Dble mutant mice  do not express HDAC6 at the mRNA or protein levels and have hyperacetylated tubulin in the cortex, striatum and cerebellum from 4 to 15 weeks of age. We show that in the presence of the polyQ expanded R6/2 transprotein, levels of acetylated tubulin do not differ from that in wild type animals and similarly, the HD mutation does not interfere with the effect that HDAC6 ablation has on tubulin hyperacetylation. We found that depletion of HDAC6 had no effect on the aggregate load or BDNF levels in the R6/2 brain and did not modify physiological or behavioural HD-related phenotypes in R6/2 mice.Previous studies in cell culture models of HD have shown that modulation of HDAC6 levels alters HD-related phenotypes Hdac6 gene has been disrupted Hdac6 knock-out mice used here are a suitable model. There are numerous mouse models of HD, which include mice transgenic for an N-terminal fragment of HTT, YAC and BAC mice that are transgenic for full-length HTT gene and knock-in mice in which an expanded CAG repeat has been introduced into the mouse Hdh gene Hdac6 levels on HD phenotypes in these more genetically precise models might be considered important. However, before embarking on such a project, it would be wise to determine whether a reduction in HDAC6 levels has an effect in cell culture models expressing a mutant version of full-length huntingtin.In this study, we have used mice in which the endogenous Hdac6KO mouse embryonic fibroblasts as compared to wild type cells indicating that HDAC6 is important for autophagy Drosophila melanogaster model of SBMA exacerbated retinal degeneration and overexpression of HDAC6 decreased aggregate load and ameliorated retinal degeneration, effects that required HDAC6 catalytic activity Hdac6KO mice by 6 months of age HDAC6 has been extensively characterised as being required for the formation and subsequent autophagic degradation of the aggresome BDNF mRNA expression Hdac6KO and double mutant mice. We did not observe any change in BDNF levels. Strategies that increase BDNF levels have been shown to be beneficial in R6/2 mice and other N-terminal fragment models In cultured cells, an increase in tubulin acetylation has been shown to enhance kinesin-1 binding and microtubule-based transport Drosophila melanogaster models of Parkinson's disease Hdacs3, 5, 7 and 9 have not resulted in a phenotypic improvement . Based on our data, we can conclude that HDAC6 inhibition would not be a valid therapeutic strategy for HD.There has been an increased focus on HDAC6 in neurodegenerative disease. In addition to the polyglutamine diseases, over-expression of HDAC6 was protective in All experimental procedures performed on mice were approved by the King's College London Ethical Review Process Committee and carried out under the UK Home Office License 70/6545.Hdac6 knock-out (Hdac6KO) mice Hdac6KO genetic cross, R6/2 males were bred to Hdac6 heterozygous females. At 4 weeks of age, mice were weaned into cages of 5, each containing at least one representative of each genotype. Animals were housed under 12 h light/12 h dark cycle, with unlimited access to water and chow . Cages were environmentally enriched as described Hdac6KO cross were given mash food consisting of powdered chow mixed with water during 4–6 and 12–15 weeks of age and sacrificed at 15 weeks.Hemizygous R6/2 mice were maintained by backcrossing R6/2 males to CBAxC57BL/6 F1 (CBF) females  Hdac6 genotyping, the primers were forward: 5′-GTACAATGTGGCTCACAGAA, reverse wt: 5′-CAGGCACAGGAATATGAGTT and reverse KO: 5′-CAACTCTGCCTCTCCTGG each used 1 µL at 10 µM in one multiplex 10 µL reaction, containing also 1 µL of 100 ng/µL DNA, 0.8 µL 25 mM MgCl2, 1 µL 2 mM dNTP, 1 µL Sigma 10×PCR buffer and 0.1 µL Sigma Taq Polymerase . Cycling conditions were as follows: 94°C for 5 min, ×40 followed by 10 min at 72°C.Mice were genotyped by PCR of tail-tip DNA. R6/2 mice were genotyped and their repeat sizes determined as described Hdac6KO cross were assessed blind to genotype. Mice were weighed weekly to the nearest 0.1 g. RotaRod performance was measured at 4 weeks of age for 4 consecutive days, 3 runs a day and after that at 8, 10, 12 and 14 weeks of age for 3 consecutive days, 3 runs a day, using an accelerating (4–44 rpm in 5 min) Ugo Basile 7650 Rotarod,  modified as described The phenotypes of the mice from the R6/2xThe HDAC6 antibody was a kind gift from Dr. Tso-Pang Yao For tubulin acetylation analysis, tissues were homogenised using 1% Triton X-100 buffer with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 5 µM TSA  and 10 mM nicotinamide (Sigma), supplemented with protease inhibitor cocktail (Roche). For HDAC6 immuno-detection, tissues were homogenised in RIPA buffer  supplemented with protease inhibitor cocktail (Roche). Samples were sonicated on ice for 10 s at 80 Hz (Vibracell Sonicator). Lysates were cleared by centrifugation at 16 200 rcf for 15 min at 4°C. Protein concentration was measured with Pierce BCA assay kit (Thermo Scientific). Samples were diluted with 2× protein Laemmli buffer  and denatured for 5 min at 95°C.Equal amounts of protein were loaded onto SDS polyacrylamide gel with a size reference . Proteins were transferred onto Protran nitrocellulose membrane (Whatman) at 120 V for 90 min by submerged transfer apparatus (Bio-Rad) in transfer buffer . Membranes were blocked in 5% non-fat dried milk in PBS for at least 1 hour. Primary antibodies were applied in 0.02% PBS-Tween 20 (PBST) for 20 min  or 1 hour (1∶5000 S830) at room temperature or overnight (1∶250 HDAC6) at 4°C. Blots were washed thrice for 5 min in 0.2% PBST and incubated with appropriate HRP coupled secondary antibody . For signal detection, GE Healthcare Enhanced Chemiluminescence detection system and Amersham Hyperfilms  were used according to the manufacturer's instructions. Signals were quantified using a GS-800 densitometer (Bio-Rad).For aggregate detection 2.5% lysates (w/v) were prepared by homogenising tissue in RIPA. Aggregate capture and detection were performed in Seprion ligand coated plates (Microsens) as described Time resolved - Förster resonance energy transfer experiments (TR-FRET) were performed as described BDNF protein content was measured by the commercially available ELISA kit (Promega) according to manufacturer's instructions modified as described Hdac6 expression analysis were forward: 5′ - GGAGACAACCCAGTACATGAATGAA; reverse: 5′ - CGGAGGACAGAGCCTGTAG and the probe was 5′-FAM-TATCTGCATCCGAACTCATATTCCTGTGCCTG-TAMRA. Primers for Bdnf coding region were forward: 5′ - GGGTCACAGCGGCAGATAAA; reverse: 5′ - GCCTTTGGATACCGGGACTT; and the probe was: 5′ - FAM - TCTGGCGGGACGGTCACAGTCC - TAMRA.RNA extraction, cDNA synthesis, Taqman RT-qPCR and ΔCt analysis were performed as described previously Whole brains were snap frozen in isopentane at −50°C. Immunohistochemistry was performed as described Hdac6KO cross were analysed with SPSS using one way ANOVA or General Linear Model ANOVA with Greenhouse-Geisser correction for non-sphericity. Data from qPCR and tubulin acetylation assay were analysed with Microsoft Excel using Student's t-test (two tailed).Data from the R6/2xFigure S1Hdac6 knock-out has no effect on spontaneous motor activity in WT or R6/2 mice. Five minute moving averages for Activity, Mobility, Rearing and Centre rearing at 5, 7, 9, 11 and 13 weeks of age for WT, Hdac6KO, R6/2 and Dble mice. n≥16/genotype (as shown in (TIF)Click here for additional data file.Figure S2Hdac6 knock-out does not change aggregate load in the brain or muscle. (A) Aggregate load was measured by Seprion ligand ELISA with the MW8 antibody in the cortex, hippocampus and brain stem of R6/2 and Dble mice at 4 weeks. n≥5/genotype. (B) Representative western blot with S830 anti-huntingtin antibody showing aggregated and soluble transprotein (Htt) in the cortex at 4 weeks. α-tubulin (Tub) was used as a loading control. Specificity of S830 staining is confirmed by lack of signal in WT and Hdac6KO cortices. (C) Densitometric quantification of soluble Htt from western blots shown in (A) and D) Soluble  and aggregated  transprotein levels were measured by TR-FRET in the cortex at 9 and 15 weeks. Data normalised to R6/2 at 9 weeks. * p<0.05; n≥8/genotype. (E) Aggregate load was measured by Seprion ligand ELISA with the MW8 antibody and soluble transprotein levels were measured by TR-FRET (2B7-MW1 antibodies) in the quadriceps muscle at 9 and 15 weeks. Data normalised to R6/2 at each time point. n≥7/genotype. Error bars represent SEM.(TIF)Click here for additional data file.Figure S3The increase in tubulin acetylation in brain is comparable between Hdac6KO and Dble mice. (A) Representative western blots showing acetylated tubulin (Ac-tub) in Dble and Hdac6KO mice at 4, 9 and 15 weeks in cortex, striatum and cerebellum with α-tubulin (Tub) as a loading control. There was insufficient tissue to perform the analysis on striatum at 9 and 15 weeks. (B) Densitometric quantification of western blots presented in (A). Acetylated tubulin was normalised to α-tubulin and the relative signal for Dble mice expressed as fold change to Hdac6KO. Error bars represent SEM. KO - Hdac6KO, Dble - Hdac6KOxR6/2; n≥3/genotype.(TIF)Click here for additional data file.Figure S4Similar levels of BDNF protein in striatum, cortex and hippocampus despite very low Bdnf mRNA striatal expression. (A) Bdnf mRNA coding region expression in WT mice at 9 weeks between cortex, striatum and hippocampus. Data normalised to Ubc and expressed as fold change of cortex. n≥3/genotype. ** p<0.01 (to cortex) *** p<0.001 (to cortex and to hippocampus) (B) BDNF protein content measured by ELISA in cortex, striatum, hippocampus and liver in WT mice at 9 weeks of age. Data normalised to cortex. * p<0.05; n = 3/genotype. Error bars represent SEM.(TIF)Click here for additional data file.Table S1p - values calculated via ANOVA General Linear Model with Greenhouse-Geisser correction.Statistical analysis of the influence of time (30 min duration) and genotype(s) on the spontaneous motor activity parameters: activity, mobility, rearing and centre rearing presented as (DOC)Click here for additional data file.


It is well accepted that insulin-induced hypoglycemia can result in seizures. However, the effects of the seizures, as well as possible treatment strategies, have yet to be elucidated, particularly in juvenile or insulin-dependent diabetes mellitus (IDDM). Here we establish a model of diabetes in young rats, to examine the consequences of severe hypoglycemia in this age group; particularly seizures and mortality. Diabetes was induced in post-weaned 22-day-old Sprague-Dawley rats by streptozotocin (STZ) administered intraperitoneally (IP). Insulin IP (15 U/kg), in rats fasted (14–16 hours), induced hypoglycemia, defined as <3.5 mM blood glucose (BG), in 68% of diabetic (STZ) and 86% of control rats (CON). Seizures occurred in 86% of STZ and all CON rats that reached hypoglycemic levels with mortality only occurring post-seizure. The fasting BG levels were significantly higher in STZ (12.4±1.3 mM) than in CON rodents (6.3±0.3 mM), resulting in earlier onset of hypoglycemia and seizures in the CON group. However, the BG at seizure onset was statistically similar between STZ (1.8±0.2 mM) and CON animals (1.6±0.1 mM) as well as between those that survived (S+S) and those that died (S+M) post-seizure. Despite this, the S+M group underwent a significantly greater number of seizure events than the S+S group. 25% glucose administered at seizure onset and repeated with recurrent seizures was not sufficient to mitigate these continued convulsions. Combining glucose with diazepam and phenytoin significantly decreased post-treatment seizures, but not mortality. Intracranial electroencephalograms (EEGs) were recorded in 10 CON and 9 STZ animals. Predictive EEG changes were not observed in these animals that underwent seizures. Fluorojade staining revealed damaged cells in non-seizing STZ animals and in STZ and CON animals post-seizure. In summary, this model of hypoglycemia and seizures in juvenile diabetic rats provides a paradigm for further study of underlying mechanisms. Our data demonstrate that severe hypoglycemia (<2.0 mM) is a necessary precondition for seizures, and the increased frequency of these seizures is associated with mortality. The brain stores minimal glucose, mainly in the form of astrocytic glycogen Studies evaluating the relationship between hypoglycemia and cognitive dysfunction have yielded conflicting results. Some studies have suggested that hypoglycemia results in cognitive decline ATP channel expression decreases in overnight fasted rodents, therefore contributing to neuronal excitability and possibly to hypoglycemic seizures Critically, both hyperglycemia and hypoglycemia can upset the balance between inhibition and excitation of neuronal networks and have been demonstrated to increase seizure susceptibility. A high-glucose medium is associated with reduced seizure threshold in vitro In the developing brain, excitation is predominant EEG abnormalities are more frequent in patients with IDDM than in the general population ad libitum access to water, a standard rat chow diet and under a 12-hour light/dark cycle. The rats were ear-punched for identification.All studies were done in accordance with and approved by the Animal Research Council at the University Health Network . Male Sprague Dawley rats from Charles River Laboratories , weighing 40–60 g, were housed in pairs in a temperature-controlled environment with Streptozotocin (STZ) was dissolved in a citric acid buffer (0.1 mM sodium citrate (Fisher Scientific) buffered with 1 M citric acid (Fisher Scientific) to a pH of 4.5) making a 10 mg/ml solution just prior to injection. The 22-day-old rats were fasted overnight (14–16 hrs) and received IP injections with one of the following doses of STZ: 60 mg/kg, 75 mg/kg or 80 mg/kg, to induce diabetes. Controls (CON) were randomly selected and injected with the 0.1 mM sodium citrate buffer vehicle. Following STZ or citric buffer vehicle injections, body weights and tail vein blood glucose levels were measured (BG) using a Hemocue Glucose 201 glucometer . Measurements were made 2 days after STZ IP and every 4 days subsequently to confirm stable diabetes.Animals were fasted overnight (14–16 hours), administered insulin IP  and video-monitored for 5 hours to detect motor seizures or convulsive seizure-like events, these are referred to as “seizures” for the rest of this paper. Fasting BG was measured (see methods above) prior to insulin IP and hourly, after receiving insulin, leading up to seizures. The BG measures were conducted hourly, as the glucose meter (Hemocue) used was more accurate but required substantially more blood volume than over-the-counter glucose meters. “Lowest BG” was considered as the minimum blood sugar measured in the time period leading up to seizures or up to 3 hours in animals that did not seize. In some cases this measure was taken 15–30 mins prior, as blood samples were difficult to acquire when rats were lethargic. In a subset of animals (22 diabetic and 16 control rats), BG was measured at seizure onset before administering treatment to determine the BG threshold for seizures. After seizures, rats that appeared healthy as defined by resumption of normal grooming habits as well as eating and drinking, survived. Rats that continued seizing despite treatment, were not responsive and displayed agonal breathing  were eutTable 1. CON rats were used in the glu treatment group and STZ rats were used in all three of the treatment groups.The strategies that were employed to treat seizures are described in A seizure score was developed to characterize and quantify the SLEs observed in this model Table 2.The seizure score that was obtained in 5-minute epochs was used for further analysis. Animals were subsequently grouped according to the types of seizures that they underwent. Two types of categorization were performed. Firstly, rats were segregated by whether or not the brainstem was recruited (defined by the loss of righting reflex) during the seizure (seizure score ≥ 4.0). Secondly, rats were also placed in three categories (1) partial seizures: only involving one limb (seizure score  =  2.5); (2) partial to secondary generalization: the seizure spreads from one limb to other areas (3) generalization without prior evident partial seizures (seizure score ≥ 3.0).th version; Systat Software Inc.). Comparison of data between two groups was carried out via Students’ T-test. To compare proportions of the various treatment groups, a Chi-squared (n ≥ 5) or Fisher’s exact test (n < 5) was used. Significance was set at P<0.05. All error bars indicate ± SEM.All statistical tests were performed with Sigma Stat software  and 40 mL 4% paraformaldehyde. The brain was removed at either 48 hours post-hypoglycemic insult or in age-matched STZ and CON animals. Whole brains were cut along the coronal plane into 3 mm sections and embedded in paraffin. Subsequently, paraffin blocks were cut into coronal slices, 10 microns in thickness every 50 microns with a total of 3 slices taken from each block. Fluorojade C  staining was performed to mark degenerating neurons. Staining methods were in accordance with the manufacturer’s protocol (millipore.com) and cells that displayed fluorescence were counted as having undergone neurodegeneration.While the STZ model of diabetes induction has been widely demonstrated in adult rodents A week after STZ administration, the diabetic (STZ) animals had a significantly decreased weight gain (p<0.005) body weight; 78.7±1.2 g (n = 40) than the age-matched control (CON) animals; 100.5±2.5 g, (n = 17).To assess the prevalence of seizures and other concomitant effects of hypoglycemia, insulin IP (15 U/kg), was administered to overnight-fasted rats 1 week after STZ  or CON  induction. Overnight fasting, prior to receiving insulin, has been demonstrated to increase seizure incidence ), which is a non-significant difference. In addition, there was no significant difference between the proportions of hypoglycemic CON (100%: n = 19/19) and STZ (86%: n = 37/43) rats that displayed convulsions .Both CON and STZ rats, attained hypoglycemia (BG < 3.5 mM). Incidence of hypoglycemia was 68% (n = 43/63) in STZ and 86% (n = 19/23) in CON rats  or STZ rats , for predicting the incidence of seizures. Concurrently, prevalence of mortality was not significantly affected by fasting pre-insulin BG levels in CON .Blood glucose (BG) levels were measured prior to insulin administration and every subsequent hour until seizure onset. Fasting BG levels, prior to insulin administration, were significantly higher in STZ than in CON animals (p<0.001). Consequently, the latency to seizures was significantly increased in STZ (2.4±0.2 hours) compared to CON rats . However, fasting BG levels were not a significant factor in CON .After insulin administration, both STZ and CON rats that eventually developed seizures had a significant drop in BG levels within the first hour compared to the NS groups , reached significantly lower (p<0.002) BG levels than the 6 NS hypoglycemic animals (lowest BG  =  2.8±0.3 mM). There was no significant difference in the lowest BG measured between seizing rats that survived (S+S  =  2.0±0.1 mM) or died . Despite this, at the onset of seizures, the BG levels were similar in STZ (1.8±0.2 mM) and CON rats . No significant difference was measured in the seizure onset BG levels of STZ surviving  and non-surviving rats . The BG of CON rats did not differ significantly between those that survived  and those that did not  or subsequent seizures (despite treatment). In CON rats, the BG levels were 1.7±0.2 mM (n = 11) and 1.6±0.1 mM (n = 5) in groups with single and multiple seizures, respectively. Likewise, in STZ rats, BG levels at seizure onset of rats with a single seizure and multiple seizures were 1.9±0.2 mM (n = 10) and 1.9±0.3 mM (n = 12), respectively . Prior to reaching seizure-inducing hypoglycemic levels, all rats that reached moderate hypoglycemia (<3.5 mM) became lethargic. In the absence of EEG recording, it was difficult to discern less severe seizure-like activity (seizure score: 0.5–2) from lethargic behavior. Therefore, only rats that reached a seizure score of ≥2.5 (single limb clonus) were treated and classified as having seized (see methods). Seizures in the STZ and CON animals exhibited variable progression, whether or not the animals survived . Notably, mortality occurred in all rats that reached a seizure score of 7.In order to establish a seizure model, the observed seizure behaviours must first be described. Reliable video recordings were obtained in 22 STZ and 16 CON rats that displayed seizures. All rats were treated with glucose at seizure onset. Seizure scoring was performed to quantify the severity of the observed seizures Table 2Figure S4). The S+M rats in both the STZ and CON groups trended toward a higher initial and maximum seizure score compared to the respective S+S rats. The lack of significance is potentially due to the low sample size of the S+M animals.While it is evident that hypoglycemia and the resulting seizures are associated with mortality, it is not clear whether seizures were the sole cause .A stronger predictor of mortality was the number of seizures that an animal underwent. The number of seizures observed was significantly higher in the CON rats: S+M  =  7.8±2.7 (n = 5) compared with S+S  =  1.6±0.3 . This difference was also observed between STZ rats: S+S  =  1.6±0.2 (n = 17) and S+M  =  4.4±1.2 (n = 5); p<0.001  were administered to STZ rats. The trend in BG decrease after insulin was similar in the treatment groups, indicating that the BG decline was not a confounding factor of treatment outcome .To evaluate the efficacy of glucose, the current treatment strategy, and whether outcomes differed with anticonvulsant treatment, the treatments described in glu treated animals exhibited seizures after treatment compared with 24% of animals in the ac+1xglu treatment group (n = 5/21)  undergoing subsequent seizures (p<0.02) (ac+1xglu (n = 5/21) and the ac+multiple glu (n = 2/14).Treatment with glucose at seizure onset was not always successful in mitigating seizures. 55% (n = 12/22) of ac+multiple glu: 93% (n = 13/14), compared with glu: 77% (n = 17/22) was not significant . As the anticonvulsants appeared to be mitigating the motor component of the seizures , despite continued hypoglycemia , improved survival rate was still dependent on continued glucose administration.Despite the success of anticonvulsant treatment in mitigating seizures with multiple glucose administrations, the impact on survival, ac+1xglu-treated group the number of motor seizures were similar whether or not mortality resulted  and significantly lower (p<0.05) than the glu-treated rats where mortality resulted  . In ac+1xglu group, the 8 animals in the S+M group died despite having a reduced number of seizures, as glucose was necessary even though the anticonvulsants prevented the behavioural seizure manifestations. However, multiple glucose administrations (glu) alone were not sufficient to prevent continued seizures that eventually led to mortality . These rats may have had a temporary increase in BG levels and returned to hypoglycemia. In the ac+multiple glu group only one animal died and could not be statistically compared to the other 2 groups .As demonstrated in ac+1xglu-treated group was significantly higher in S+M rats: 6.1±0.6 (n = 8) compared to S+S rats: 4.0±0.6 (n = 8) (p<0.01). While anticonvulsants were able to transiently stop seizing, since the rats were not treated with continuous glucose, 5 rats eventually had more severe seizures. In contrast, the maximum score observed in the glu-treated rats that survived  was statistically similar to those that died  . This was also the case in ac+multiple glu-treated rats: S+S: 4.0±0.4 (n = 13) and S+M: 5.5 (n = 1), though the sample size of the latter was not adequate for statistical analysis.Interestingly, the maximum seizure score in the ) of EEG, characteristic of severe hypoglycemia (Auer 2004). However, behavioural seizures occurred independently of EEG suppression as only 2 out of 9 STZ and none of the CON rodents displayed such pronounced suppression prior to exhibiting convulsions. Burst suppression was characteristic of EEGs in all animals that reached hypoglycemic levels. Animals that seized showed intermittent, non-rhythmic spikes, often with a positive polarity. Two animals’ recordings also contained low-amplitude (50 uV) polyspike bursts. Brief electrographic seizures were recorded in the CA1  region in one animal and brainstem  in another. Although, rats at this stage of hypoglycemia were lethargic and immobile, no overt convulsive behaviour was observed. The rat exhibiting the hippocampal electrographic seizure had previously received a “rescue” administration of glucose, diazepam and phenytoin after the prior incidence of a behavioural seizure. The other had not been treated as a behavioural seizure had not yet been observed.EEG recordings were obtained in 17 STZ and 12 CON rats. 11/12 CON and 13/17 STZ animals reached hypoglycemic levels following insulin administration of which 10 CON and 9 STZ rats had behavioral seizures. An example of baseline recording prior to insulin administration is shown in Consistent with previous studies Fluorojade staining was performed to visualize degenerating cells and assess the effects of hypoglycemic seizures on the neurons of STZ and CON animals. The number of fluorescent cells was minimal in the four groups: CON and STZ rats dissected 48 hours after insulin-induced hypoglycemic seizures and age-matched non-seizing CON and STZ animals. STZ rats without seizures  exhibited a significantly higher number of damaged cells than CON rats . There was no significant difference between CON  and STZ rats  particularly due to the high variability in the number of damaged cells in the STZ animals. Two STZ rats displayed numerous (>10) fluorojade (+) cells while this others exhibited more minimal damage Figure 8This study has established a model of hypoglycemic seizures in young diabetic animals to examine seizure manifestations and consequences in this age group. As Type 1 diabetes is diagnosed primarily during childhood, these hypoglycemic episodes begin early in life and the associated neurological complications are particularly harmful /B). In STZ animals, the higher baseline fasting BG levels resulted in lower incidence of hypoglycemia and seizures (/B). These higher initial BG levels in STZ rats combined with the similar rates of BG decline following insulin administration resulted in an increased latency to hypoglycemia and subsequent seizures in these animals.In our model, the drop in blood glucose (BG) within the first hour post-insulin administration was predictive of those animals that would attain severe hypoglycemia and progress to seizures . This was further evidence for the association between seizures and mortality. Velisek et al, Mortality was only observed in animals post-seizure. Additionally, the animals that survived, despite having similar seizure scores, underwent a significantly lower number of seizures . This temporary effect suggests that other seizure-causing mechanisms, such as enhanced NMDA activity, may trigger these seizures in the presence of continued hypoglycemia Diazepam, a GABA agonist, and phenytoin, which act on sodium channels, were only able to transiently stop seizures without glucose replenishment and the seizures eventually become more severe with mortality as an end result  were brief (10–30s), failed to spread to the cortex, and occurred in the context of EEG suppression that is associated with neuronal damage ATP channels that impair potassium buffering has been postulated to enhance excitability +/K+-ATPase and thus the cell’s ability to restore intracellular potassium and extracellular sodium levels, leading to an enhanced glutamate receptor function There are several factors that potentially explain the mechanisms for seizures during hypoglycemia. The downregulation of KIn summary, hypoglycemic seizures due to exogenous insulin administration can occur in juvenile diabetic and non-diabetic animals. A blood glucose threshold of approximately 2mM was associated with the occurrence of seizures in both control and diabetic animals after an acute hypoglycemic event. Most importantly, mortality only occurred following seizures, and it was the frequency of seizures rather than blood glucose levels that were predictive of mortality. Treatment of seizures with glucose, helped prevent seizure-related mortality with no added protection from anticonvulsants. While electrographic seizures were recorded in two animals, it was not correlated with behavioural manifestations. Although there have been improvements in treatments such as better insulin, insulin pumps, and improved glucose monitoring techniques, hypoglycemic seizures remain a major problem in insulin-treated diabetic children and adolescents, and a better understanding of these is crucial for improved patient care. The description of the seizures and outcomes performed in our STZ-model is the first step toward accomplishing this goal.Figure S1Blood Glucose (BG) measured post seizure of glu-treated CON and STZ animals. A: BG post-seizure in CON rats at 1.0 hour (n = 8): 4.4±0.2 mM, 2.0 hours (n = 8): 5.5±0.5 mM B: BG post-seizure in STZ rats 1.0 hour (n = 11): 7.8±1.9 mM, 2.0 hours (n = 12): 13.7±2.9 mM, 3.5 hours (n = 5): 15.9.3±4.1 mM.(TIF)Click here for additional data file.Figure S2Evolution of seizures over the course of hypoglycemia . A: CON; S+S; n = 11 B: CON; S+M; n = 5 C: STZ; S+S; n = 17 D: STZ; S+M; n = 5.(TIF)Click here for additional data file.Figure S3Blood Glucose (BG) post seizure of glu and ac+multiple glu-treated STZ animals. A: BG post-seizure in glu rats 1.0 hour (n = 11): 7.8±1.9 mM, 2.0 hours (n = 12): 13.7±2.9 mM, 3.5 hours (n = 5): 15.9.3±4.1 mM B: BG post-seizure in ac+multiple glu rats at 0.5 hours (n = 12): 3.1±0.5 mM, 1.5 hours (n = 10): 3.2±0.4 mM, 2.5 hours (n = 6): 3.2±0.6 mM, 3.5 hours (n = 4): 6.3±3.3 mM.(TIF)Click here for additional data file.Figure S4Total duration in which the rodents demonstrated SLEs as an indication of hypoglycemia. No significant difference between glucose-treated control rats that seized and died; CON_GLU_S+M (n = 5): 124.3±31.5 and survived; CON_GLU_S+S (n = 11): 42.1±20.1 No significant difference between glucose-treated diabetic rats that seized and died; STZ_GLU_S+M (n = 5): 113.6±38.0 and survived; STZ_GLU_S+S (n = 17): 55.8±16.2 No significant difference between anticonvulsants+1 dose of glucose-treated diabetic rats that seized and died; STZ_ACGLU1_S+M (n = 8): 30.7±11.4, and survived; STZ_ACGLU1_S+S (n = 8): 36.1±12.0 Anticonvulsants+multiple glucose-treated diabetic rats that seized and survived; STZ_ACMULTGLU_S+S (n = 13): 97.04±26.2 could not be statistically compared with ones that seized and died; STZ_ACMULTGLU_S+M (n = 1).(TIF)Click here for additional data file.Table S1Reasons for sacrificing 2 of 5 glu Treated CON rats, 2 of 5 glu treated STZ rats and 5 of 8 ac+1xglu treated STZ rats.(DOC)Click here for additional data file.


Acinetobacter baumannii are very common in hospital environments. It usually causes soft tissue infections including urinary tract infections and pneumonia. It is difficult to treat due to acquired resistance to available antibiotics is well known. In order to design specific inhibitors against one of the important enzymes, peptidyl-tRNA hydrolase from Acinetobacter baumannii, we have determined its three-dimensional structure. Peptidyl-tRNA hydrolase (AbPth) is involved in recycling of peptidyl-tRNAs which are produced in the cell as a result of premature termination of translation process. We have also determined the structures of two complexes of AbPth with cytidine and uridine. AbPth was cloned, expressed and crystallized in unbound and in two bound states with cytidine and uridine. The binding studies carried out using fluorescence spectroscopic and surface plasmon resonance techniques revealed that both cytidine and uridine bound to AbPth at nanomolar concentrations. The structure determinations of the complexes revealed that both ligands were located in the active site cleft of AbPth. The introduction of ligands to AbPth caused a significant widening of the entrance gate to the active site region and in the process of binding, it expelled several water molecules from the active site. As a result of interactions with protein atoms, the ligands caused conformational changes in several residues to attain the induced tight fittings. Such a binding capability of this protein makes it a versatile molecule for hydrolysis of peptidyl-tRNAs having variable peptide sequences. These are the first studies that revealed the mode of inhibitor binding in Peptidyl-tRNA hydrolases which will facilitate the structure based ligand design.The incidences of infections caused by an aerobic Gram-negative bacterium,  Acinetobacter baumannii is an aerobic Gram-negative bacterium commonly found in hospital environments Acinetobacter baumanniiAcinetobacter baumannii is responsible for a number of diseases such as pneumonia Acinetobacter baumannii is increasingly becoming resistant to antibiotics Acinetobacter baumannii (AbPth) plays an essential role in the survival of bacteria, the inhibition of its function will lead to the death of Acinetobacter baumannii. Therefore, the design of tight inhibitors of AbPth will help in developing useful antibacterial drugs against infections caused by Acinetobacter baumannii. In this regard, it is fortunate that the structures of eukaryotic and prokaryotic peptidyl-tRNA hydrolases are not similar Peptidyl-tRNA hydrolase (Pth) is involved in the processing of prematurely dissociated peptidyl-tRNAs from ribosome during protein biosynthesis into peptide and tRNA AbPth as the target protein, it is essential to obtain the three-dimensional structure of AbPth. So far, structures of three other prokaryotic peptidyl-tRNA hydrolases from Escherichia coli (EcPth) Mycobacterium tuberculosis (MtPth) Mycobacterium smegmatis (MsPth) Pseudomonas aeruginosa (PaPth) (PDB: 4FNO (4th July 2012), PDB: 4FYJ (12th December 2012), EcPth with tRNA CCA-acceptor-TΨC domain AbPth with these enzymes vary in the moderate range of 58% to 39%  for further enhancing the understanding of Pth structures as well as for the purpose of carrying out the structure based design of antibacterial compounds using peptidyl-tRNA hydrolase from Acinetobactor baumannii as the target protein. We report here the three-dimensional structures of unbound AbPth and two of its complexes with cytidine and uridine including binding studies using fluorescence spectroscopic and surface plasmon resonance studies. These structures revealed that cytidine and uridine bound to AbPth in the active site region and caused several conformational changes in the side chains of residues at the active site region in the protein resulting in the induced fits. The observed modes of binding of cytidine and uridine clearly provided a very useful information for the design of ligands as tight inhibitors of AbPth.In order to carry out structure based drug design using Acinetobacter baumannii was isolated and Pth gene was amplified using forward and reverse primers (Pthf: 5- GGAATTCCATATGTCAAATATTTCGCTAATTG-3′) and (Pthr: 5- CCGCTCGAGTTAAGCTGGTTTATACGCATT-3′) respectively. The forward primer contained the site of recognition of Nde1 (CATATG) and reverse primer contained the site of recognition of xho1 (CTCGAG). The amplification of Pth gene was done with Taq DNA polymerase. A reaction was made using 5U of Taq DNA polymerase  using PCR buffer containing 75 mM Tris-HCl, pH 8.8, 20 mM (NH4)2SO4, 0.01% Triton X-100, 1.5 mM MgCl2, 0.2 mM dNTPs and the genomic DNA of Acinetobacter baumannii. The amplified AbPth gene was used for ligation with linearized pGEM-T Easy vector. The ligation reaction was set up using the pGEM-T easy vector kit . The reaction mix was incubated at 4°C overnight. The amount of amplified AbPth DNA required for ligation was calculated. The molar ratio of amplified AbPth gene and vector DNA was taken at a ratio of 5∶1. The pET vector DNA was completely digested with appropriate restriction enzymes (Nde1 and Xho1). The digested plasmid was resolved on 0.8% agarose gel to remove the stuffer fragment. The plasmid band was purified by QIAEX-II gel extraction kit  and stored at −20°C. The AbPth gene released from the recombinant pGEM-T Easy plasmid carrying complete open reading frame by digesting with Nde1 and Xho1 was then purified and ligated in the right reading frame  into already digested and purified pET-28a vector and transformed in E. coli DH5α cells. The recombinant clone was checked for the insert by colony PCR and also by restriction enzyme digestion with Nde1 and Xho1 in a 20 µl reaction containing 1.5 µl each of Nde1 and Xho1 and 2 µl NE Buffer (10X) and 15 µl of isolated plasmid (approximately 1 µg) which was incubated at 37°C for 4 h. In the pET prokaryotic system, the protein was expressed as an N-terminal fusion to 6 Histidine residues. The construct obtained with pET-28a was transformed in E. coli (Bl21 DE3) cells for expression of Pth. A single freshly transformed colony was inoculated in 10 ml LB containing 100 µg/ml kanamycin and was kept in water bath overnight at 37°C in shaking condition.The genomic DNA from Overnight culture from primary inoculation (1%) was added in 1000 ml LB medium containing 100 µg/ml kanamycin and kept in water bath at 37°C in shaking condition till the optical density of the culture at 600 nm reached 0.4–0.6. 5 ml of this secondary culture was removed and kept at 4°C as an uninduced culture. The uninduced cells were used as the control. The remaining secondary culture of recombinant cells containing the inserts was induced with 1 mM IPTG at 37°C for 4 h. The induced and uninduced cells were harvested by centrifugation at 5000 rpm for 15 min, the supernatants were discarded and the pellet was stored at −70°C. The induction was checked on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The His-tagged protein containing 6 Histidine residues at the N-terminus was purified using Ni-NTA affinity resin chromatography.AbPth as indicated by its molecular mass were pooled. The imidazole was removed by dialysis in lysis buffer and the sample was concentrated using centricon with cut-off 3 kDa molecular mass . The concentrated protein was further purified using Fast protein liquid chromatography  using Superdex G50 column in buffer containing 20 mM Tris-HCl, 50 mM NaCl, 1.0 mM ethylenediaminetetraacetic acid (EDTA) and 5 mM of 2-mercaptoethanol (MPD), pH 8. The protein was concentrated using a centricon ultrafiltration of 3 kDa molecular mass cut off . The purity of protein was established using SDS-PAGE which showed a single band  was dissolved in 5 ml of Tris-HCl, pH 8.0 containing protease inhibitor cocktail . The sonication was done at 28 amplitude, 4 s pulse and 2 s rest was given. The cleared lysate was applied to a Ni-NTA Super-flow column pre-equilibrated in lysis buffer and purified using stepwise washing with 30 mM imidazole in lysis buffer followed by 300 mM imidazole in lysis buffer. The protein contents of each fraction were examined using 10% SDS-PAGE. The fractions corresponding to AbPth with cytidine and uridine were carried out using fluorescence spectroscopic technique with spectrofluorometer, FP-6200 . The fluorescence experiments were conducted by keeping the entrance and exit slit widths at 5 nm each and scanning it at the speed of 240 nm/min. AbPth was excited using an excitation wavelength of 280 nm. The fluorescence emission spectra of the protein were measured in the range of 300–550 nm at 298 K. The concentrations of ligands were varied as 5 µl, 10 µl, 15 µl, and 20 µl which were prepared from stock solutions of cytidine and uridine already prepared at a concentration of 1×10−8 mol/l. The protein concentration was kept constant at 1×10−9 mol/l. The spectral changes of AbPth with various concentrations were recorded and plotted. The least-squares fit of the fluorescence intensity changes for AbPth-cytidine and AbPth-uridine binding curves were obtained by Sigma Plot The binding studies of AbPth was covalently immobilized by a standard amine coupling procedure using the amine coupling kit supplied by the manufacturer. Throughout the immobilization experiment the flow rate of the running buffer was fixed at 10 µl/min with HBS-EP . The surface was activated using 70 µl of freshly mixed 100 mM NHS and 391 mM EDC for 7 min at 1∶1 (v/v) ratio. Upon activation, a 500 µg/ml solution of AbPth in 50 mM HEPES (pH 7.5) was injected for 8 min. The remaining active esters on the surface were quenched using 70 µl of 1.0 M ethanolamine (pH 8.5) for 7 min. Four different concentrations  of each of the analytes, cytidine  and uridine  were passed over the immobilized AbPth at a flow rate of 10 µl/min. The regeneration of bound analytes was carried out by injecting 10 mM NaOH, pH 11.4 for 60 seconds, at a flow rate of 30 µl/min. These experiments were repeated six times and standard errors were calculated. The association rate constants, kon (M−1s−1) and dissociation rate constants koff (s−1) for the bindings and dissociations of cytidine and uridine to AbPth were obtained and the values of the equilibrium binding constants, KD (M) were determined by the mass action relation, KD = koff/kon M using BIA evaluation 3.0 software as provided by the manufacturer.Surface plasmon resonance (SPR) measurements were carried out for analyzing the bio-specific interactions using BIAcore T200 biosensor system . The CM-5 research grade sensor chip, immobilization reagents  carbodiimide (EDC), N-hydroxy succinimide (NHS), HBS-EP buffer and ethanolamine were procured from BIAcore Inc. All solutions were filtered using 0.22-µm membrane syringe filter and degassed before using. −1 in 0.1 M HEPES buffer at pH 6.0. The diffractable crystals were obtained by equilibrating 10 µl protein drops (containing 7 µl protein solution and 3 µl reservoir solution) against a reservoir solution containing 25% (v/v) PEG-10000, 0.3 M MgCl2 and 0.1 M HEPES buffer at pH 6.0. Crystals grew to approximate dimensions of 0.4×0.4×0.2 mm3 in about 8 to 10 days. Fresh crystals were also obtained using a 1∶4 ratio of PEG-400 and PEG-1500 respectively in place of PEG-10000. The rest of the conditions were same. The crystals were soaked in the reservoir solutions containing 20 mg/ml cytidine and uridine in separate experiments. The crystals grown using 25% PEG-10000 were not stable in soaking conditions while those grown with PEG-400 and PEG-1500 were stable and were used for data collections on the two complexes. The soaking experiments were carried out for a minimum of 72 hours before mounting the crystals in the loops for data collections.The purified samples of the unbound protein were crystallized using hanging drop vapor-diffusion method at 298 K by employing 24 well linbro crystallization plates. The initial crystallization screening experiments were carried out using Hampton research crystallization kit (HR2110 - HR2112). The protein was concentrated to 10 mgml3/Da, 2.07 Å3/Da and 2.02 Å3/Da respectively. These corresponded to solvent contents of approximately 53.1%, 40.6% and 39.1% respectively.Three X-ray intensity data sets were collected using 345 mm diameter MAR Research imaging plate scanner  mounted on a rotating anode X-ray generator  operating at 50 kv and 100 mA. The crystals of unbound protein diffracted to 1.9 Å resolution while those of its complexes with cytidine and uridine diffracted to 1.87 Å and 1.40 Å respectively. The data were processed using program package HKL - 2000 EcPth cryst and Rfree factors. The C-terminal segment consisting of residues 181–193 which was reported to be flexible in the structures of peptidyl-tRNA hydrolases from other species was found to be well defined in the electron density of AbPth  as the search model. Since the space group of the crystals of the unbound protein was different from those of the complexes with cytidine and uridine, the use of molecular replacement method was essential for obtaining the solution of the structures of the complexes. Several cycles of refinement calculations carried out with REFMAC cryst factors of approximate value of 0.25, the Fourier maps with (Fo–Fc) and (2Fo–Fc) coefficients were calculated for both structures. The extra non-protein electron densities were observed in the active site region into which molecules of cytidine /Fo, where F is the measured fluorescence and Fo is the fluorescence in the absence of ligands. The values of Q were plotted in percentage against the different concentrations of ligands. The R2 values, which provide an index of the goodness of fit of the curves were obtained using Sigma Plot 8.0 −9 M and 2.11±0.32 × 10−9 M respectively indicating that both compounds bound to AbPth with high affinities in the range of nanomolars.The binding studies of AbPth with uridine and cytidine were also carried out using real time SPR spectroscopy. The SPR sensograms illustrating the nature of association and dissociation curves for cytidine and uridine with immobilized AbPth are given in −9 M and 1.4±0.2 × 10−9 M for the equilibrium binding constants (KDs) for cytidine and uridine respectively.The binding studies of AbPth consists of 1476 protein atoms from 193 amino acid residues, 200 water oxygen atoms, 18 atoms from three glycerol molecules, two molecules of 1, 2-ethanediol, two molecules of diethyleneglycol and one acetate ion. The polypeptide chain of AbPth adopted an α/β fold with seven β-strands, β1 (Ser5 - Leu10), β2 (Lys41 - Asp43), β3 (Gly48 - Arg52), β4 (His58 - Leu64), β5 (Ala89 - Glu96), β6  and β7 (His130 - Ile136) and six α-helices, α1 (Ala24 - Tyr36), α2 (Ser72 - Phe82), α3 (Leu116 - Ile125), α4 (Ser146 - Gly151), α5 (Asn156 - Gln179) and α6 (Pro181 - Asn188) (The refined model of  Asn188) . A twist Asn188) . W1 is lAbPth is similar to that of PaPth (PDB: 4FNO), EcPth MtPth MsPth α traces being 1.0 Å, 1.0 Å, 1.5 Å and 1.7 Å respectively. As seen from AbPth (green), PaPth (pink), EcPth (cyan), MtPth (yellow) and MsPth (grey) differ greatly in several regions including segments with residues: Met1 - Leu6, Pro65 - Ser80, Gly111 - Leu118, Asp120 - Pro127, His138 - Val149 and Pro181 - Ala193. These structural variations in Pth enzymes from different species  were found to be 6.1 Å, 6.8 Å, 6.6 Å, 3.3 Å and 4.5 Å in AbPth, PaPth, EcPth, MtPth and MsPth respectively. Another set of distances measured between Gly113 Cα and Ser146 Cα, also from opposite sides were found to be 15.3 Å, 15.0 Å, 14.9 Å, 11.6 Å and 11.3 Å in AbPth, PaPth, EcPth, MtPth and MsPth respectively. The distance between Asn70 Oδ1 and Asp98 Oδ2 is found to be 15.7 Å in the unbound AbPth. The corresponding distances in PaPth, EcPth, MtPth and MsPth were 20.9 Å, 16.9 Å, 16.2 Å and 17.2 Å respectively. The lengths of Pth molecules as measured in one of the directions in terms of distances between Tyr83 Cα and Gly140 Cα were found to be 41.0 Å, 41.1 Å, 40.0 Å, 42.1 Å and 43.1 Å for AbPth, PaPth, EcPth, MtPth and MsPth respectively. These values indicated that AbPth, EcPth and PaPth are slightly more compact molecules but possessed wider opening of the gate to the active site cleft than those of MtPth and MsPth. The minimum distance between the side chains of two tyrosine residues, Tyr17 and Tyr68 which are believed to be involved in the stacking with RNA bases is 4.1 Å in AbPth. The corresponding distances in PaPth, EcPth, MtPth and MsPth are 3.7 Å, 3.8 Å, 6.8 Å and 5.7 Å respectively. In AbPth, Tyr68 adopted a conformation with φ, ψ values of 76.6°, 148.3° which falls in the disallowed region of Ramachandran map EcPth with φ, ψ values of 92.5°, 147.5° was also observed in the disallowed region EcPth formed a complex with CCA-acceptor-TΨC domain of tRNA moiety adopted a conformation with φ, ψ values of −60.1°, 157.3° which belonged to the fully allowed region. In this regard, it may be mentioned that the structure of the so-called unbound state in EcPth contained the C-terminal tripeptide of a symmetry related neighboring molecule at the active site cleft EcPth adopted a conformation in the disallowed region with external chemical moiety in the active site region and it adopted the conformation in the allowed region when empty. The φ, ψ values for the corresponding residues in PaPth [4FNO], MtPth MsPth The overall structure of AbPth indicated a considerable deviation from the normal values for α-helical conformations. This may be due to the presence of Pro123 in the middle of the segment. The corresponding residues in Pth from other species are either Ala or Ser  in AbPth which also includes α-helix, α6  was found to be very well defined in the electron density . The values of φ, ψ  for the residues in this segment of AbPth showed the presence of six water molecules in the active site region of which W1 is hydrogen bonded to both Asn70 and His22 . The pyrimidine ring of cytidine molecule is seen well stacked with the side chain of Tyr68 which was found to adopt a conformation in the disallowed region with φ, ψ values of 74.9°, 148.3°. This conformation is similar to that observed in the unbound state. It appears that the observed disallowed conformation of Tyr68 is favorable to generate good the stacking interactions with RNA bases.The structure of the unbound nd His22 . Howevernd His22  as a resAbPth with uridine is shown in α and Asp98 Oδ2 and between Asn70 Oδ1 and Asp98 Oδ2 increased from 6.1 Å to 7.8 Å and 15.7 Å to 22.7 Å respectively as the segments containing residues Gly113 and Asp98 as well as the side chains of Asn70 and Asp98 move away to accommodate the ligands. The binding of uridine was stabilized with the help of seven hydrogen bonds and more than 100 van der Waals contacts (distances upto 4.2 Å). As observed in the structure of the complex of AbPth with cytidine, the pyrimidine ring of uridine is also well stacked with the aromatic side chain of Tyr68. The conformation of Tyr68 with φ, ψ values of 75.4°, 148.3° is similar to that observed in the structure of unbound AbPth and its complexes with cytidine.The structure of the complex of Ds) for the binding of cytidine and uridine to AbPth using fluorescence spectroscopic technique were found to be 4.79×10−9 M and 3.11×10−9 M respectively. Similar values of 5.3×10−9 M and 2.4×10−9 M for the equilibrium constants (KDs) for the binding of cytidine and uridine respectively were obtained using SPR techniques. The high values of binding constants showed that the two compounds bound to AbPth firmly. The buried surface areas as estimated from the structures of the complexes of AbPth with cytidine and uridine were estimated to be 202 Å2 and 198 Å2 respectively AbPth.The equilibrium constants (KAbPth the minimum distance between the atoms on the opposite sides of the entry point (Asp98 Oδ2– Gly113 O) was 4.9 Å while the shortest separation between the opposite walls at the active site (His22 Nε2– Asn70 Oδ1) was 4.8 Å  were (i) to determine structural features of Pth including the stereochemical details of the active site, (ii) to explain the mechanism of catalytic action and (iii) to understand the mode of ligand binding for the design of inhibitors. The goals (i) and (ii) had been partially understood from previous studies while the information on goal (iii) was completely missing. The present studies have provided a clear understanding of the mode of inhibitor binding in peptidyl-tRNA hydrolases. The structure determinations of the complexes of occurred . Furthercytidine  and uridcytidine . TherefoFile S1Acinetobacter baumannii (AbPth) with Pseudomonas aeruginosa (PaPth) Escherichia coli (EcPth), Mycobacterium tuberculosis (MtPth) and Mycobacterium smegmatis (MsPth) whose crystal structures are known. The overall sequence identities vary from 58% to 39%. The flexible regions consisting of residues, Met1 - Leu6, Pro65 - Ser80, Gly111 - Leu118, Ile121 - Pro127, Pro139 - His148 and Pro181 - Ala193 are highlighted in grey. The probable residues involved in the catalysis are highlighted in yellow. The secondary structure elements α-helices (cylinders) and β-strands (arrows) are also indicated above the sequences. Figure S2. Showing results of polyacrylamide gel electrophoresis for AbPth. Lane A: molecular weight markers, 116.0 kDa - β-galactosidase, 66.2 kDa - bovine serum albumin, 45.0 kDa - ovalbumin, 35.0 kDa - lactate dehydrogenase, 25.0 kDa - ribonuclease, 18.4 kDa - β-lactoglobulin and 14.4 kDa - lysozyme; Lane B: purified protein, AbPth. Figure S3. Showing electron densities for the C-terminal segment, Pro181– Ala193 (A) |Fo–Fc| map at 2.5 σ cut off and (B) |2Fo–Fc| map at 1 σ cut off. Figure S4. Bindings of cytidine (A) and uridine (B) showing large conformational changes with side chains of Asn70, Asp98 and Asn116. The electron densities from the initial (Fo–Fc) map at 2.5 σ cut off.Figure S1. Sequence alignments of peptidyl tRNA hydrolases (Pths) from (DOC)Click here for additional data file.


P < .001), while SPAr had longer arterial ischemia 103 ± 42 min (P = .0004). One-year patient's and graft survival were, respectively, 89% and 95% versus 94% and 100% (P = .29). At median followup of 13 ± 6 versus 14 ± 7 months biliary complications were anastomotic stenosis in 15% versus 19% (P = .78), and intrahepatic nonanastomotic biliary strictures in 26% versus none (P = .01), respectively, in SPAr and CPAr. CPAr reduces the incidence of intrahepatic biliary strictures by decreasing the duration of arterial ischemia.We prospectively compared sequential portal-arterial revascularization  versus contemporaneous portal-hepatic artery revascularization  in 40 consecutive liver transplantation (LT). There were no differences in the demographics characteristics, MELD score, indication to LT, and donor's parameters between the two groups. CPAr had longer warm ischemia 66 ± 8 versus 37 ± 7 min ( Sequential portal-arterial revascularization (SPAr) of the graft is the most common technique in liver transplantation (LT), based on the experience that portal blood flow is sufficient for adequate liver function , 2. The P = .83). All grafts were whole liver except one extended right liver split including segment one in group 1. Portal vein and arterial anastomosis between the graft and the recipient have been always performed with terminal-terminal interrupted suture. In the SPAr group the graft was revascularized through the portal vein, and the hepatic artery was subsequently performed; the time interval between portal and arterial revascularization was recorded and defined as arterial ischemic time. In the CPAr the graft was revascularized simultaneously through the portal vein, and hepatic artery. Cold and warm ischemia time were determined, respectively, as the period between cross-clamp and graft inside the abdomen and between the graft in the abdomen and reperfusion. Reperfusion syndrome was defined as a drop in mean arterial pressure greater than 30% within 1 minute from reperfusion of the graft and lasting more than 5 minutes. Primary nonfunction (PNF) was defined as the need for retransplantation or death due to graft failure within 7 days in absence of technical problems. Delayed graft function (DGF) was defined as AST or ALT peak greater than 2000 U/ml within 72 hours from LT. Biliary complications were analyzed when clinically evident or through magnetic resonance cholangiopancreatography (MRCP) every 6 months after transplantation in asymptomatic patients. From June 2008 to December 2009, 40 consecutive LT from heart-beating donors were randomized 1 : 1 to SPAr  or CPAr . No differences between the groups for indications to OLT have been evidenced during statistical analysis. Cases requiring an aortohepatic bypass for arterial revascularization, and reOLT were excluded from the analysis. The LT was always carried out by piggy-back technique in all cases. The bile duct was reconstructed in all cases by termino-terminal duct to duct anastomosis; the T-tube was used, respectively, in 32% versus 29% of cases  years old and 74% versus 75% (P = .93) in group 1 and 2. No differences in the indication for LT were encountered with HCC as the main indication in 32% versus 42%, HCV in 26% versus 24%, and alcohol in 16% versus 19% (P = ns). The MELD score was 17 ± 7 versus 14 ± 5 (0.12). Donor's age was, respectively, 52 ± 18 versus 51 ± 15 years old in groups 1 and 2 (P = .85). Twenty-one percent of the grafts in groups 1 versus 19% in group 2 had macrovacuolar steatosis greater than 15% (P = .92). Duration of LT was not different between groups 1 and 2  as well as cold ischemia time , while warm ischemia time was statistically significant longer in the CPAr group . Arterial ischemic time in the SPAr group was statistically significant longer than warm ischemia time in the CPAr group . Units of blood and amount of plasma transfusion were, respectively, 6 ± 4 versus 7 ± 4 (P = .60) and 1,300 ± 1,175 ml versus 2,382 ± 1,853 ml (P = .045) in groups 1 and 2. There were no PNF in both groups; DGF was diagnosed in 10% versus 9% in groups 1 and 2 (P = .91). ICU and total hospital stay were, respectively, 7 ± 4 versus 5 ± 2 and 17 ± 5 versus 18 ± 6 days, respectively, in groups 1 and 2 (P = .12 and P = .68) (P = .29 and P = .53). At a median follow-up of 13 ± 6 versus 14 ± 7 months (P = .71) biliary complications diagnosed were anastomotic stenosis in 15% versus 19% (P = .78), and intrahepatic nonanastomotic biliary strictures in 26% versus none (P = .01), respectively, in SPAr and CPAr.Recipient's age and male gender were, respectively, 52 ± 9 versus 56 ± 9 (P = .68) . AST andP = .68) , total b5% P = .9 in groupIn all cases biliary stenosis occurred within 1 year after OLT, and these were successfully treated through ERCP when anastomotic  and through PTC (5 cases out of group 1) when intrahepatic nonanastomotic strictures have been diagnosed.The most commonly used procedure for revascularization of the liver graft is initial reperfusion via the portal vein and subsequent reconstruction of the hepatic artery –7. The d


Genotyping of human papillomarvirus (HPV) is crucial for patient management in a clinical setting. This study accesses the combined use of broad-range real-time PCR and high-resolution melting (HRM) analysis for rapid identification of HPV genotypes.Genomic DNA sequences of 8 high-risk genotypes (HPV16/18/39/45/52/56/58/68) were subject to bioinformatic analysis to select for appropriate PCR amplicon. Asymmetric broad-range real-time PCR in the presence of HRM dye and two unlabeled probes specific to HPV16 and 18 was employed to generate HRM molecular signatures for HPV genotyping. The method was validated via assessment of 119 clinical HPV isolates.A DNA fragment within the L1 region was selected as the PCR amplicon ranging from 215–221 bp for different HPV genotypes. Each genotype displayed a distinct HRM molecular signature with minimal inter-assay variability. According to the HRM molecular signatures, HPV genotypes can be determined with one PCR within 3 h from the time of viral DNA isolation. In the validation assay, a 91% accuracy rate was achieved when the genotypes were in the database. Concomitantly, the HRM molecular signatures for additional 6 low-risk genotypes were established.This assay provides a novel approach for HPV genotyping in a rapid and cost-effective manner. Human papillomavirus (HPV), a small and nonenveloped double-stranded DNA virus, is established as the key etiological factor in cervical neoplasms HPV infection can be monitored by detection of thirteen high-risk oncogenic HPV types  using a commercially available HPV testing method such as the Hybrid Capture 2 assay , the only HPV assay approved by the US Food and Drug Administration Recently, a high-resolution melting (HRM) analysis method which incorporates double-stranded DNA saturating dye and specifically designed data collection and analyzing software has been developed. HRM analysis was first developed as a closed-tube technology for genotyping DNA variants and mutation screening with advantages over the other techniques such as high-throughput, rapid and non-destructive nature In this study, we reported a novel method based on the use of HRM analysis and unlabeled probes to rapidly identify and differentiate HPV genotypes in clinical specimens. Without multiplexing, HPV genotypes can be completed with one PCR within 3 h from the time of viral DNA isolation.All cervical samples were collected from the Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital with the approval by the Institutional Review Board (IRB 99-0112B) and with informed consent from the patients. The genomic DNAs of 140 consecutive HPV clinical specimens were obtained from the sample bank for this study. The individuals who performed the experiments do not know the genotype of these clinical specimens until completion of HRM analysis.The QIAamp DNA mini kit was purchased from Qiagen . The LightCycler 480 and the LightCycler 480 High Resolution Melting Master were purchased from Roche Applied Science . The T&A cloning vector was purchased from RBC Bioscience . The McTaq DNA polymerase was purchased from One-Star Biotechnology . The UniPOL-Long Range PCR enzyme mix containing Taq DNA polymerase and AccuPOL with 3′→5′ exonuclease activity was purchased from Ampliqon ApS . The EasyChip HPV genotyping system was purchased from KingCar .2), 2 µl of 10 mM dNTP, 0.5 µl of McTaq DNA polymerase (5 U/µl), 2 µl of 5 µM forward primer GAPDH-F, 2 µl of 5 µM reverse primer GAPDH-R  in a 50 µl of reaction containing 5 µl of 10× PCR buffer , 1 µl of 25 mM MgCl2, 2 µl of 10 mM dNTP, 0.5 µl of the UniPOL-Long Range PCR Enzyme mix (5 U/µl), 2 µl of forward primer FRG5 (5 µM), 2 µl of reverse primer FRG2 (5 µM) and 2 µl of template DNA. The PCR condition was 95°C for 5 min followed by 50 cycles of 95°C for 30 sec, 46°C for 40 sec, and 72°C for 30 sec. An additional 3 min of extension at 72°C was performed after the last PCR cycle to replenish PCR product followed by cooling at 4°C. The PCR product was then cloned into the T&A cloning vector as described by the manufacturer's instruction (RBC Bioscience) and was confirmed by DNA sequencing.Partial HPV L1 region was amplified by PCR using the clinical DNA samples of HPV16, 18, 39, 45, 52, 56, 58 and 68 and the primer pair FRG5/FRG2 . Briefly2, 0.8 µl of 5 µM forward primer FRG5, 0.8 µl of 5 µM reverse primer FRG2, and 2 µl of template DNA. The amplification condition was optimized for the use of FastStart Taq DNA polymerase by incubating the reaction mixtures at 95°C for 15 min, followed by 50 cycles of 95°C for 15 sec, 46°C for 20 sec, and 72°C for 30 sec with the ramp to 95°C at 4.8°C/s, to 46°C at 2.5°C/s, and to 72°C at 4.8°C/s. An additional 1 min at 72°C was added to replenish the PCR product.For symmetric amplification of the HPV genomic DNA, broad-range real-time PCR was performed in a 384-well format using LightCycler 480. Briefly, the PCR reaction (20 µl) was composed of 10 µl of 2× HRM master mix, 2 µl of 25 mM MgCl2, 0.4 µl of 2.5 µM forward primer FRG5, 2 µl of 5 µM reverse primer FRG2, 2 µl of 5 µM unlabeled probe for HPV16 and HPV18 when indicated, and 2 µl of template DNA. The unlabeled probe was modified by C6-amine or inverted dT at the 3′-end to prevent the probe from self-extension. The amplification condition was optimized for the use of FastStart Taq DNA polymerase by incubating the reaction mixtures at 95°C for 15 min, followed by 65 cycles of 95°C for 15 sec, 46°C for 20 sec, and 72°C for 30 sec with the ramp to 95°C at 4.8°C/s, to 46°C at 2.5°C/s, and to 72°C at 4.8°C/s. An additional 1 min at 72°C was added to replenish PCR product.For asymmetric amplification of HPV genomic DNA, the PCR reaction (20 µl) was composed of 10 µl of 2× HRM master mix, 2 µl of 25 mM MgClFor HRM analysis, the PCR product was denatured by rising temperature to 95°C at 4.8°C/s and was then cool down to 55°C at 2.5°C/s for hybridization. The melting curve was acquired by increasing the temperature from 55°C to 95°C at a ramp rate of 4.8°C/s with 25 acquisitions per degree of temperature.To assess the combined use of real-time PCR and HRM analysis for rapid detection and differentiation of HPV genotypes, genomic DNA sequences for 8 high-risk HPV genotypes  were subject to bioinformatic analysis. These genotypes represent 98% and 75% of the clinical isolates in Southeast Asia and Europe/US, respectively We determined whether symmetric PCR of the L1 fragment followed by HRM analysis provides distinguishable melting profiles for HPV genotyping. To facilitate the assay, the PCR products corresponding to L1 fragment of various HPV genotypes were subcloned into the T&A cloning vector. These plasmids were used as the template for PCR amplification using the primers FRG5 and FRG2. Despite that most of the HPV genotypes displayed unique HRM profiles, the melting profiles for some HPV genotypes were not distinguishable. For example, HPV18 and HPV45 exhibited almost identical derivative plots and the genotypes were not likely to be determined accordingly .To overcome this problem, we determined whether appropriate use of type-specific unlabeled probe(s) generates HRM profiles sufficient for differentiating HPV genotypes. To facilitate the binding of unlabeled probe to the PCR product, asymmetric PCR was performed to selectively amplify the DNA strain complementary to the unlabeled probe. An unlabeled probe specific to HPV18 was designed  and was The detection limit of this method was evaluated by asymmetric PCR amplification of 10-fold serially diluted plasmid DNA (from 1 pg to 10 ag) that contained 221-bp of HPV18 L1 fragment. The amplification reaction was performed in the presence of 40 ng of human genomic DNA to mimic the co-presence of HPV and human genomic DNA in clinical specimens. As revealed by amplification plots, as little as 10 ag plasmid DNA equivalent to 3 copies of HPV DNA was detectable in this assay condition . The staIt is known that the last 6–7 nucleotides of PCR primers are critical for amplification specificity and efficiency The HRM profiles for 8 high-risk HPV genotypes were then generated by asymmetric broad-range real-time PCR in the presence of the unlabeled probes specific for HPV16 and HPV18. The melting temperatures and melting profiles provide molecular signatures for the 8 high-risk genotypes that can be unambiguously identified through high-resolution derivative plots . When clWe further assessed our method in HPV genotyping of clinical specimens retrospectively. A total of 140 clinical samples from patients who were suspected of having precancerous lesion were blind tested. Due to the nature of sample storage, only 119 of the 140 DNA samples were informative and generated PCR products that were suited for HRM analysis . As reveDuring our analysis of clinical specimens, several low-risk HPV genotypes including HPV42, 62, 70, CP8304, CP8061 and MM8 were found to display their unique high-resolution derivative plots . Of the HPV is well recognized as a major cause of cervical cancer. The development of high grade pre-cancer and invasive cervical cancer could be due to persistent high-risk HPV infection. A rapid, accurate and sensitive method to detect and differentiate HPV genotypes is essential to identify high risk patients who are otherwise found to have normal cytological results and women with cervical cancer potential from the screened population. HPV genotyping provides a reference point for HPV vaccination and HPV prevalence in natural history studies during clinical practice. Unlike hepatitis B vaccine, there is no acceptable antibody test for HPV vaccines. If it is affordable, HPV genotyping assay should be the first choice for HPV testing. In this study, a novel approach for HPV genotyping is developed. This method is based on the combined use of asymmetric broad-range real-time PCR and HRM analysis. Accordingly, 8 high-risk and 6 low-risk HPV subtypes can be identified that offers a novel approach for HPV genotyping.A number of HPV DNA genotyping methods including real-time multiplex PCR, Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR, Roche Amplicor MWP HPV test and Digene HC2 assay have been reported in the literature Multi-infection of HPV strains with various genotypes accounts for approximate 8–22% of the HPV-infected patients In conclusion, the molecular signatures from HRM analysis of the broad-range real-time PCR products are useful for detection and genotyping of HPV infection. This approach can be extended further to cover all the 13 high-risk HPV genotypes. With an appropriate HRM molecular signatures database, this method should allow rapid and cost-effective differentiation of HPV genotypes.Figure S1Effects of human genomic DNA on the amplification of HPV DNA. A–C. Serial dilution of the plasmid harboring the L1 fragment of HPV18 (panel A and C) or HPV68 (panel B and C) corresponding to the indicated copy number of template DNA were subject to amplification by asymmetric broad-range real-time PCR in the presence or absence of 20 ng of human genomic DNA. The copy numbers of the template DNA were plotted against Ct values.(DOC)Click here for additional data file.Table S1The genotypes for the samples with multiple infection.(DOC)Click here for additional data file.


Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα. Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage  compared with controls (30.5 ± 2.4%) or embryos developed in TNFα plus indomethacin (25.8 ± 2.8%). There was no difference between control embryos and embryos developed in TNFα plus indomethacin. These results indicate that TNFα is inhibitory to the in vitro development of bovine embryos and that this inhibition may be mediated by prostaglandins because it can be blocked by indomethacin.Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α (TNFα) are released in response to infection and may have negative effects on embryo development. In the current study the effect of exposure to TNFα on the development of  Cytokst stage . This efst stage . Exposurin vitro  and progin vitro . Bovine in vitro . The objTissue culture medium (TCM-199) was purchased from Gibco-BRL . Fatty-acid free BSA was purchased from Boehringer-Mannheim . Recombinant bovine TNFα was purchased from Pierce Endogen  and reconstituted in sterile saline to a final concentration of 4 ng/mL. All other reagents and media supplements were of tissue culture grade and were obtained from Sigma Chemical Co. . Indomethacin was reconstituted to a 1000X stock solution of 1 μg/mL in sterile absolute ethanol. With the exception of the fertilization medium, all culture media contained 50 μg/mL of gentamicin.In vitro maturation (IVM), fertilization (IVF) and development (IVD) were performed as the method described by [2. After maturation, COC were washed and held in fertilization medium containing heparin-supplemented Tyrodes albumin lactate pyruvate (TALP) with 6 mg/mL BSA [ribed by . Ovariesribed by . The COCg/mL BSA .6 cell per ml. Gametes were co-incubated for 18 hs 39°C in 5% CO2. At 18 h post insemination (hpi) presumptive zygotes were washed six times in TL-Hepes and approximately 25-30 were randomly distributed to one well of a four well culture plate containing the development medium with one of the treatments. All treatments were contained within development medium containing M199, 10% FBS and 50 μg/mL of gentamicin 39°C in 5% CO2 and 100% humidity. Development medium was changed at 48 h intervals throughout culture.Frozen/thawed sperm from the same bull were used for fertilization. Swim-up sperm were collected and used at a concentration of 0.1 × 10Treatments included: Control development medium alone, 25 ng/mL TNFα, 1 μg/mL indomethacin, and 25 ng/mL TNFα plus 1 μg/mL indomethacin. All treatments were replicated four times except indomethacin alone which was replicated twice. Total number of oocytes used in the experiment were control (225), TNFα (232), indomethacin (114) and TNFα plus indomethacin (225).Development of the embryos was checked at 168 and 216 hpi and evaluated for embryo quality grade and stage of development. Developmental stages were classified as compact morula, early blastocyst, mid-blastocyst, expanded blastocyst and hatched blastocyst. The quality of the embryo was graded as 1 for excellent, 2 for moderate and 3 for poor quality .Data for the percent embryos developing to the blastocyst stage were arc-sin transformed and analyzed by one way ANOVA using PROC GLM of SAS 9.1  with Duncan's Multiple Range Test used for mean separation.In preliminary experiments, bovine presumptive zygotes were placed into medium containing 0, 10, 25, 50 or 100 ng/mL TNFα and evaluated for development to the blastocyst stage at 168 hpi . Fewer blastocysts developed in the 25 ng/mL (19.6 ± 2.2%) 50 (18.5 ± 2.7%) and 100 (14.1 ± 1.9%) ng/mL treatments than the 0 ng/mL dose group (27.5 ± 2.6%) so the 25 ng/mL dose was chosen for subsequent experiments as the lowest effective dose. The 1 μg/mL dose of indomethacin was tested in two replicates in the system and had no effect by itself on the percentage of embryos reaching the blastocyst stage (control 25.2 ± 2.1% vs. indomethacin 25.6 ± 2.4%). As ethanol was used for the vehicle for indomethacin, absolute ethanol was tested in two replicates and had no effect on development to the blastocyst stage at 168 hpi at the concentration used (29.7 ± 2.3% control vs. 30.4 ± 2.2% ethanol).Treatment with 25 ng/mL TNFα reduced the percentage of bovine embryos reaching the blastocyst stage at 168 hpi (17.2 ± 2.4%) compared to controls (30.2 ± 2.7%) and embryos treated with 25 ng/mL TNFα plus 1 μg/mL indomethacin (25.8 ± 2.3%) (Figure Soto et al.  reportedIn vivo, bovine endometrium and cumulus cells can produce prostaglandins [The research presented herein indicates that TNFα works through the prostaglandins since the inhibitory effect of TNFα was blocked by co-treatment with indomethacin. In previous work , PGF addglandins ,15. Prodglandins  but embrCows with mastitis had higher concentrations of PGF2α in their milk than cows that did not have mastitis  IncreaseThe authors declare that they have no competing interests.LJ conducted the oocyte maturation and embryo culture. CF participated in the design of the experiment and provided technical expertise for oocyte maturation and embryo culture. CW conceived the experiment and conducted the statistical analysis. All authors participated in writing the manuscript. All authors have read and approved the final manuscript.


Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia. Low levels of MAC increase cell mitogenesis and VEGF release c7 cells . PPADS mectively . At 208 d by NHS . We concp = 0.0002), 84.42±16.9% (p = 0.0001) and 93.17±11.67% (p<0.0001), respectively  ectively . 20.8 µMp<0.0001, The most commonly used animal model of wet AMD is laser induced CNV p = 0.0091) reduction in levels of MAC relative to vehicle control  were maintained in 200PRF (phenol red free) medium supplemented with 2% Low Serum Growth Supplement . Mouse Hepatoma cells (Hepa1c1c7) were purchased from ATCC  and maintained in Minimal Essential Medium  supplemented with 10% fetal bovine serum (FBS). Cells were propagated at 37In Vitro Angiogenesis) protocol . Briefly, 9×104 HUVECs were seeded per well on Geltrex™  coated wells of a 24-well plate. HUVECs cultured in 200PRF/2% LSGS served as negative controls and vehicle controls contained a total of 0.0045% NaCl. Treatments were performed by adding PPADS to final concentrations of 0.417 µM, 1.043 µM, 2.085 µM and 4.17 µM to 200PRF/2% LSGS medium. After 16 hours the cells were analyzed and images captured with an inverted microscope . Number of master junctions, master segments or meshes in each image were quantified using the “HUVEC angiogenesis analyzer plugin” of ImageJ software.Tube formation assays were carried out according to the Endothelial Tube Formation Assay  containing PPADS at concentrations of 20.8 µM, 52 µM, 104 µM, 208 µM. Lysis was induced with 1% normal human serum (NHS) for 1 hour at 37°C with continuous gentle rotatory motion. GVB containing a total of 0.225% NaCl, which equals the NaCl content in PPADS containing samples, served as control. Cell lysis was detected as uptake of propidium iodide in 2.5×104 cells by flow cytometry . Analysis was done with the FlowJo software .FACS lysis assays were performed as previously described 4 Hepa1c1c7 cells were seeded per 8 well chamber slides and grown in MEMα containing 2% FBS for 2 days. Medium was exchanged to GVB/10% NHS containing PPADS at concentrations of 20.8 µM, 52 µM, 104 µM, 208 µM. After incubation for 5 minutes at 37°C, the reaction was terminated with ice-cold PBS and cells fixed in 4% formaldehyde. For MAC detection, cells were blocked in 6% normal goat serum, 0.5% Triton X-100, 1x PBS (blocking solution). MAC was labeled with a mouse anti-C5b9 antibody  and detected with a Cy3-conjugated goat anti-mouse antibody . Nuclei were counterstained with DAPI. Images were captured using an inverted microscope  attached to a fluorescence unit. Integrated Cy3 densities and DAPI signals were quantified using ImageJ software. The amount of MAC deposition was defined as integrated density of C5b9 labeling (Cy3) per cell (DAPI).MAC deposition assays were carried out as previously described 2 inhalation, followed by cervical dislocation.This study was carried out in strict accordance with the Statement for the Use of Animals in Ophthalmic and Vision Research, set out by the Association of Research in Vision and Ophthalmology (ARVO) and was approved by Tufts University Institutional Animal Care and Use Committee (IACUC) protocol B2011-150 and Tufts University Institutional Biosafety Committee registration 2011-BRIA68. For experiments 6 weeks old C57Bl/6J mice  were utilized. Animals were anesthetized by intraperitoneal injections of a mixture containing 0.1 mg/g body weight Ketamine  and of 0.01 mg/g body weight Xylazine . Mice were kept constantly warm during anesthesia. At indicated time points mice were sacrificed by COLaser induced CNV was performed as previously described A total of 20 µl of a 4.17 mM PPADS solution was topically applied to each eye of anaesthetized mice. Control mice received 20 µl of 0.9% NaCl (vehicle control). Treatment was performed after laser burn for 3 consecutive days in intervals of 24 hours. Mice were sacrificed 7 days after laser, eyes enucleated and passed to histological processing.Cornea, iris, lens and the neuronal retina were removed from enucleated eyes. Posterior eye cups were fixed by immersion in 4% PFA and cut four times from the edge to the center to obtain flat mounts. Specimens were blocked in PBS/2.5% BSA and stained with FITC-conjugated Griffonia Simplicifolia Lectin I  10 mg/ml in PBS. Flat mounts were washed with PBS, re-blocked with blocking solution , incubated with a rabbit anti-human C5b9 antibody  and detected with a Cy3-conjugated goat anti-rabbit antibody . Flat mounts were imaged using an inverted microscope  attached to a fluorescence unit and images were captured using a Qimaging RETIGA200R camera . Areas of GSL-I labeling (CNV areas) and signal intensities of C5b9 labeled areas (MAC areas) were measured using ImageJ software. Data points for MAC deposition are depicted as C5b9 intensities per C5b9 area ratios. C5b9 intensities were corrected for background intensities before calculations.in vivo experiment, 5 mice per group were utilized. In vitro experiments were performed in duplicate. All experiments were repeated at least three times. All data are presented as mean ± SEM.Statistical analysis was performed with the GraphPad5 software . Statistical significance between PPADS treatment groups and control groups were determined by the unpaired student’s t-test. For each


We applied T2 mapping in 72 volunteers compared to 15 patients with myocardial edema. T2 of myocardial edema was 70 ms, whereas normal myocardium had a T2 of 55ms. However, female volunteers showed a wider range of myocardial T2 with overlap into values considered abnormal.T2-mapping has been proposed to overcome limitations of T2-weighted imaging of myocardial edema. We applied T2-mapping in normal volunteers to reveal the inter- and intra-individual homogeneity of myocardial T2 and to compare it to patients with myocardial edema.We scanned 72 healthy volunteers  in a 1.5 T scanner. All had no cardiac disease and no signs of inflammation. We applied a FLASH and a SSFP-based T2-mapping sequence resulting in 3 single-shot images with echo times of 0, 24 and 54 ms and a pixel size of 2.7 x 2.1 mm (slice thickness 8 mm). Images were obtained in midventricular short axis and 4-chamber-view (4CV). We measured global and segmental T2 according to a 16-segment model. Contours were manually drawn on images by two independent observers and then copied to the map. 26 volunteers underwent additional mapping with reduced pixel size of 2.2 x 1.8 mm and automatic motion correction. 6 volunteers were scanned twice on separate days. Results were compared to 15 patients with myocardial edema .We excluded 4 volunteers due to obvious pathologies. In patients myocardial edema had a T2 of 71±4 ms (p<0.001 compared to volunteers). In volunteers mapping based on FLASH resulted in lower T2-values than SSFP . T2 values were independent from heart rate, age or body weight. Apical septal segments had higher values than basal lateral segments in 4CV . Women below 36 years had a 95% confidence interval of 43-86 ms based on SSFP mapping on 4CV images.Mean intraobserver variability was 0.8±0.9 ms (correlation coefficient r=0.99), mean interobserver variability was 1.6±1.8 ms (correlation coefficient r=0.98). Variability for repeated scans was 2.3±1.8 ms. Improved spatial resolution and the applied motion correction did not significantly change the T2-measurements (p=0.3).T2 mapping is feasible with low intra- and interobserver variability. T2 of myocardial edema was 70 ms, whereas normal myocardium had a T2 of 55ms. However, female volunteers showed a wider range of myocardial T2 with overlap into values considered abnormal. This is most likely due to myocardial contamination with blood (T2 of about 180 ms). Motion correction and spatial resolution needs to be improved before T2-mapping can widely be applied for exclusion of edema.The scanner manufacturer provided a preliminary version of the mapping sequence based on the local research agreement. Funding was provided by the university.