Cdkl5 knockout (Cdkl5 +/−) mouse, has been little characterized. The lack of detailed behavioral profiling of this model remains a crucial gap that must be addressed in order to advance preclinical studies. Here, we provide a behavioral and molecular characterization of heterozygous Cdkl5 +/− mice. We found that Cdkl5 +/− mice reliably recapitulate several aspects of CDKL5 disorder, including autistic-like behaviors, defects in motor coordination and memory performance, and breathing abnormalities. These defects are associated with neuroanatomical alterations, such as reduced dendritic arborization and spine density of hippocampal neurons. Interestingly, Cdkl5 +/− mice show age-related alterations in protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling, two crucial signaling pathways involved in many neurodevelopmental processes. In conclusion, our study provides a comprehensive overview of neurobehavioral phenotypes of heterozygous female Cdkl5 +/− mice and demonstrates that the heterozygous female might be a valuable animal model in preclinical studies on CDKL5 disorder.CDKL5 disorder is a severe neurodevelopmental disorder caused by mutations in the X-linked CDKL5 (cyclin-dependent kinase-like five) gene. CDKL5 disorder primarily affects girls and is characterized by early-onset epileptic seizures, gross motor impairment, intellectual disability, and autistic features. Although all CDKL5 female patients are heterozygous, the most valid disease-related model, the heterozygous female  CDKL5 gene. Primary clinical features include early-onset intractable epileptic seizures, gross motor impairment, severe intellectual disability, and autistic-like features   females . Cdkl5 +/− females achieved an intermediate body weight between that of Cdkl5 +/+ and Cdkl5 −/− female mice.We first evaluated whether mutant reported , no signCdkl5 +/− and Cdkl5 −/− female mice to smell volatile odors. We found that 3 month old Cdkl5 +/− and Cdkl5 −/− female mice showed a reduced olfactory performance compared with wild-type (Cdkl5 +/+) females   phenotypes, we first analyzed home-cage social behaviors (nest building ability and marble burying). Cdkl5 +/− and Cdkl5 −/− displayed impaired nesting compared to control mice. After 3 h, Cdkl5 +/+ female mice showed initial structured nests with a median score of 1, while Cdkl5 +/− and Cdkl5 −/− mice showed a significantly lower median nest quality with most of the mice leaving the material untouched   = 3.591, p = 0.0339; Fisher's LSD: p = 0.0013 and p = 0.0274, resp.] , rotations in which the mouse does not perform any coordinated movement and is passively transported from the rotating apparatus. Rotarod performance notably differed among groups, with Cdkl5 +/− and Cdkl5 −/− mice showing significantly more passive rotations during the four trials   = 11.90, p < 0.0001; Fisher's LSD: p = 0.0116 and p < 0.0001, resp.]  = 11.89, p < 0.0001; Fisher's LSD: p = 0.0116 and p < 0.0001, resp.] (Cdkl5 +/+), indicating elevated locomotor activity in Cdkl5 mutants. To determine whether this hyperactivity was caused by increased anxiety, we compared the time spent in the border area, near the walls, and in the center of the arena  = 1.135, p = 0.3289], near the walls , and in the center  , suggesting that the stereotypic behavior was not caused by the stressful situation (the new environment).In addition to motor dysfunction, stereotypic movements frequently cooccur in CDKL5 patients . Therefo, resp.] . An incrCdkl5 +/− and Cdkl5 −/− female mice show cognitive impairment, hippocampus-dependent learning and memory were evaluated using the Morris water maze (MWM). Mice were tested for their ability to find a hidden platform for 5 days (learning phase) and were subjected to the probe test on day 6. A repeated ANOVA on escape latency during the learning phase revealed a significant genotype effect . In the learning phase, both Cdkl5 +/− and Cdkl5 −/− mice showed a reduced ability to learn over time compared to Cdkl5 +/+ mice   = 0.4431, p = 0.6445]  = 28.21, p < 0.0001; Fisher's LSD: p = 0.0003 and p < 0.0001, resp.]  was not observed among Cdkl5 female genotypes.Performance in the MWM is influenced by sensorimotor function and motivation, and these parameters can be assessed using swimming patterns such as swim speed and floating (rest time). No difference in maximum swim speed was observed among  0.6445] , indicat, resp.] . Even re, resp.] . An anal 0.0001] , while C 0.1716] . These rCdkl5 +/− and Cdkl5 −/− mice. Both showed an increase in the latency to enter the former platform zone   = 22.72, p < 0.0001; Fisher's LSD: p = 0.0003 and p < 0.0001, resp.]  = 11.33, p < 0.0001; Fisher's LSD: p = 0.0195 and p < 0.0001, resp.]  = 10.25, p = 0.0002; Fisher's LSD: p = 0.0176 and p < 0.0001, resp.]  latency to enter the former platform zone , (ii) fr, resp.] , a reduc, resp.] , a reduc, resp.] , and a r, resp.] .p = 0.8028]  and 24 h later are tested for recollection of that experience. Figures  0.8028] , Cdkl5 +, resp.] , suggestCdkl5 +/− and Cdkl5 −/− females, hypnic and respiratory phenotypes were assessed using noninvasive whole-body plethysmography (WBP). Cdkl5 +/− and Cdkl5 −/− females had more frequent apneas during NREM sleep  and during total sleep time  compared to Cdkl5 +/+ female mice  = 1.384, p = 0.2691]  from 3–4 month-old females. Total dendritic length  and number of dendritic branches  of granule neurons were reduced in Cdkl5 +/− and Cdkl5 −/− females compared to their wild-type counterparts  = 9.355, p = 0.008; Fisher's LSD: p = 0.0445 and p = 0.0026, resp.] in comparison with wild-type (+/+) mice  compared to +/+ mice  = 30.66, p = 0.0002; Fisher's LSD: p < 0.0001] compared to Cdkl5 +/+ female mice   Cdkl5 KO females revealed no change in P-AKT-Ser473 levels   = 0.1222, p = 0.8863]  = 7.897, p = 0.0057; Fisher's LSD: p = 0.0071 and p = 0.0027, resp.] and cerebellum  from both Cdkl5 +/− and Cdkl5 −/− female mice, when compared to their wild-type counterparts  = 4.685, p = 0.0294; Fisher's LSD: p = 0.0385 and p = 0.0119, resp.]  and young adult (8 week old) Cdkl5 KO female mice. In line with previous results  and β-catenin levels   = 1.241, p = 0.3299] and P-MSK1-Thr581  levels were observed in juvenile Cdkl5 KO female mice  = 5.076, p = 0.0274; Fisher's LSD: p = 0.0566 and p = 0.0093, resp.] and P-ERK1/2  levels  = 6.907, p = 0.0090; Fisher's LSD: p = 0.0560 and p = 0.0026, resp.]  = 8.202, p = 0.0094; Fisher's LSD: p = 0.0110 and p = 0.0036, resp.]  female mice is of utmost importance in order to advance preclinical and translational studies. In the present study, we found that heterozygous Cdkl5 female mice recapitulate several behavioral aspects of CDKL5 disorder including autistic-like behaviors, motor impaired behavior, learning and memory disability, and abnormal breathing pattern. These phenotypes have been described in CDKL5 patients [Cdkl5 KO female mice, similarly to Cdkl5 male mice [Cdkl5 KO female mice impaired, in an age-related manner, phosphorylation levels of the protein kinase B (Akt) and the extracellular signal-regulated kinase (ERK1/2), important signaling mediators in brain function. Therefore, we believe that heterozygous Cdkl5 KO female mice are a valuable tool for further studies aimed at improving the neurological phenotype due to mutations in CDKL5.Animal models of neurological disorders serve as one of the most valuable tools for the development and validation of potential therapies. They enable scientists to test the safety and efficacy of new therapeutic agents patients , 4, 7 anale mice , 33–35, Cdkl5 female mice , observed in heterozygous and homozygous Cdkl5 female mice, could influence body weight by increasing energy expenditure. With regard to reduced olfaction, it remains to be established whether alterations in the neuronal circuitry of the olfactory bulb and/or a reduced neurogenesis underlie this defect in Cdkl5 KO mice.Although no significant difference was observed in body weight among young adult wild-type, heterozygous, and homozygous le mice , decreased interest toward the environment (reduced marble burying), and increased stereotypic behaviors  suggests that the heterozygous Cdkl5 female mouse model mirrors the ASD-like features of CDKL5 female patients.Autistic-like (ASD-like) features, including deficits in social interaction, disinterest for the surrounding environment, and stereotypic/repetitive behaviors, have been commonly described in CDKL5 patients , 45 and e models , 32. OurCdkl5 KO mice [Cdkl5 KO female mice. Since female Cdkl5 KO mice had a tendency to ride the rotarod beam around rather than walk forward, in addition to the latency to fall from the rotating rod, we scored the frequency of these passive rotations. While hanging onto the rotating rod is not commonly seen in wild-type mice, we found that both homozygous and heterozygous Cdkl5 KO female mice showed an increased frequency of uncoordinated rotations, indicating a serious difficulty in motor coordination. Cdkl5 KO female mice showed higher levels of activity in the open field similar to what has been described in male Cdkl5 KO mice [Motor dysfunction is a prominent feature of CDKL5 deficiency, with the majority of patients having some degree of impairment in gross and fine motor skills, including gait dysfunction and defects in sitting, standing, walking, and grasping . Consist KO mice , 31, 32, KO mice , 32, 35.Cdkl5 KO mice [Cdkl5 females was attained in the spatial learning and memory paradigm. The acquisition of allocentric learning was markedly impaired in Cdkl5 KO female mice, thus reinforcing the concept that loss of Cdkl5 is affecting hippocampal learning and memory function. Since learning/memory defects evaluated in the Morris water maze task might be influenced by the differences in swimming speed or in the motivational/motor (floating behavior) aspects observed between wild-type and Cdkl5 KO female mice, we also tested memory in a passive avoidance test [Cdkl5 KO female mice showed a substantial memory impairment (−40%), reinforcing the concept that loss of Cdkl5 leads to alterations in the acquisition process.Severe intellectual disability is a central feature of human CDKL5 deficiency . Impairm KO mice , 32, 35.nce test . Again, Cdkl5 KO mice [Cdkl5 KO female mice, strengthening the statement that the sleep apnea occurrence rate can be a significant biomarker for validation of future therapies in CDKL5 disorder.To complete our neurobehavioral studies, we monitored breathing pattern, a physiological parameter. Respiratory problems, including the presence and frequency of breathing irregularities , are a reported feature in CDKL5 patients , 47. Mor KO mice , 48. We Cdkl5 KO male mice, in addition to various behavioral abnormalities, exhibit profound changes in neuronal morphology [Cdkl5 KO females show altered neuronal maturation and synapse development confirm that cellular phenotypes, in addition to behavioral phenotypes, are useful outcome measures in preclinical studies.Several studies have shown that rphology , 33–35. rphology , 33, 35.Cdkl5 KO female mice show an alteration in the AKT pathway but no differences in the phosphorylation levels of ERK1/2 in the hippocampal region. Our present findings that young adult (8 week old) Cdkl5 KO female mice show defects in both AKT and ERK signaling pathways, while adult (3–4 months of age) Cdkl5 KO females show a strong reduction in ERK1/2 phosphorylation, but no alterations in AKT phosphorylation levels might be explained by age-related changes in the activation and/or modulation of these pathways [Cdkl5 KO mice, indicate that the changes in signaling pathways occurring during the stage of early brain development due to loss of Cdkl5 differ from those that present later on in adulthood. The direct functional effects of these changes on CDKL5 pathogenesis are not yet known. However, the age dependence of these alterations suggests that the efficacy of signaling pathway-targeted therapeutics may be limited to different developmental periods.In line with previous evidence , 25, we pathways , 50. Intpathways . On the pathways . Consistpathways , mental pathways , and sevpathways . Our datCdkl5 +/− mice show average decreased levels of Cdkl5 to ∼42% of those contained in wild-type counterparts. Our results, which show little variability in Cdkl5 protein expression among the Cdkl5 +/− brain samples, provide a first indication of balanced XCI patterns in heterozygous Cdkl5 KO mouse brains. This might explain the relatively small degree of phenotypic variability among heterozygous female mice. Moreover, the putative skewing toward the mutated X chromosome found in the hippocampus of heterozygous females (decreased levels of Cdkl5 ~ 36%) could explain the severe phenotypic outcome in hippocampus-dependent behaviors that is more similar to homozygous Cdkl5 KO female and hemizygous Cdkl5 KO male mice  female and hemizygous Cdkl5 KO (Cdkl5 −/Y) male mice. The assessment of specificity and sensitivity of the behavioral tests to discriminate between control and Cdkl5 KO female mice  in female mice. Our study provides a comprehensive overview of neurobehavioral and physiological phenotypes of the heterozygous female Cdkl5 KO mouse and demonstrates that this mouse model recapitulates multiple features observed in CDKL5 patients; therefore, it might be considered a valuable animal model for future therapeutic studies.Recent studies in  in mice , 24, 33.


Lawsonia intracellularis is one of the most economically important pathogens in swine production. This study tested the hypothesis that the composition of diets for pigs has an impact on the excretion of L. intracellularis in a natural infection model.L. intracellularis infection up to the beginning of the final fattening period were transported, regrouped and randomly allotted to groups of five animals each at the research facility. After a 1-week acclimatisation period groups were fed one of five diets 4 weeks before slaughter. These were either a finely ground pelleted diet (FP) or a coarsely ground meal diet (CM), both consisting of wheat (40.0%), barley (39.3%), soybean meal (16.0%), soybean oil (2.0%) and minor components. In the other meal diets parts of wheat, barley and soybean meal were substituted either with 22% cracked corn (CORN), 16.9% dried whey (WHEY) or 30% raw potato starch (RPS). The animals had a comparable serological status in a blocking-ELISA immediately before the start and at the end of the feeding experiment. Values increased significantly during the trial. In all subgroups (FP/CM/CORN/WHEY/RPS), shedding was detected in week 0 . The average number of L. intracellularis microbes in faeces during the trial period did not differ between the groups (log10 GE L. intracellularis/g faeces: 3.40 ± 1.53/3.01 ± 1.41/3.80 ± 1.71/3.98 ± 2.20/4.08 ± 2.13). In animals fed the WHEY-diet, significantly lower counts of L. intracellularis were found in the caecal content. The acetate content in the caecum was negatively correlated with the serological results at the end of the trial . Butyrate concentrations in the caecal content were negatively correlated with the number of L. intracellularis in the caecum .Fifty boars (~ 90 kg BW) from a SPF-farm with a strict hygiene and management regime for reducing the spread of an L. intracellularis infection.Therefore, this study provides preliminary evidence that there might be specific dietary effects on the course of a  Lawsonia intracellularis is a disease present in all pig-producing countries regardless of husbandry systems [L. intracellularis. For Germany, an even higher prevalence was achieved. About 94% of all fattening farms and 99% of the breeding units investigated had been in contact with the pathogen [The infection with  systems –7. In a pathogen .L. intracellularis in faeces [L. intracellularis does not seem to have any effects on daily weight gains [L. intracellularis in faeces means the probability of that pig having a lower growth rate by a factor of 1.97 [6 pathogens per gramme of faeces were detected, this was also a significant risk factor for a lower body weight gain [The degree of severity of the clinical symptoms is reflected by the level of excretion of n faeces . Increasn faeces . Excretiht gains . Howeverht gains . An incr of 1.97 . EspeciaL. intracellularis infection: on the one hand, the effect of packaging , on the other hand the possible effects of lactic acid-rich diets. On the basis of a survey conducted on 79 Danish pig farms, a reduced in-herd prevalence of L. intracellularis was associated with home-mixed and (or) non-pelleted diets [L. intracellularis colonisation would appear possible. This has already been reported in pigs [L. intracellularis in the total ileal microbiota of pigs fed a non-pelleted diet could be shown [L. intracellularis infection was found [L. intracellularis failed [L. intracellularis delayed the excretion of the organism [So far, only two main dietary factors have been investigated more systematically depending on the course of an ed diets . On farmed diets . Therefo in pigs , 12 as w in pigs . A relatbe shown . OverallL. intracellularis infections in pigs. A natural infection model was used because it simulates the conditions in practice more than an experimental infection approach. Transport and regrouping of subclinically infected individuals from a high health herd was the starting point for the model. This was intended to simulate the phenomenon observed under natural conditions [L. intracellularis infection model in fattening boars in the final finishing phase.The present study was based on the hypothesis that both, the packaging and the potential of a diet to foster the concentrations of specific fermentation products  specifically minimise the effects of nditions ; namely,The study consisted of three stages of sampling, which require an explanation regarding classification in accordance with the German Animal Protection Act or the Council Regulation (EC) No 1099/2009 on the protection of animals at the time of slaughter.L. intracellularis serostatus of boars) previously carried out on the supplier farm for boars were provided by the corresponding field veterinarian prior to the trial. The sampling was thus not subject to an approval in accordance with the Animal Protection Act.First, serological results from routine examinations of organ samples of boars . The slaughtering process took place at the abattoir in Hannover. The pigs were subsequently killed by bleeding after finishing the trial.Fifty boars from a specific pathogen-free (SPF) pig fattening unit were chosen for the experiments. The animals were selected at a specific date. At ~ 90 kg body weight (BW) at the farm, already 50% of the animals had been serologically positive for the pathogen in the blocking ELISA  in the past. This was also the case in the experiment. At this time, it can be assumed that an intensive pathogen exchange can be expected within the following few weeks. In particular, if transport and regrouping additionally take place. Under these circumstances, it can be assumed that the pathogen excretion is high, especially if the herd of origin has a high health status and the infection occurs late .The animals were transported to the Institute for Animal Nutrition, University of Veterinary Medicine Hannover.The 50 boars from the German Federal Breeding Programme were genetically defined animals of five lines with regard to inheritance factors for androstenone and skatole. Groups of five boars each were newly formed, with one representative of each genetic line per group. The boars were kept in groups of five animals each Table  in 2 × 3The experiments were carried out for a period of about 5 weeks, this being divided into a 1-week acclimatisation phase and a 4-week experimental phase. During the 1-week acclimatisation phase (Time point 1 = TP1), the boars were given a pelleted diet . This diet had previously been fed on the farm. The five complete diets used during the 4-week trial period TP2–5) had been produced at the research institute itself. These differed from one another either in their composition or in their packaging . Body weight gain and feed intake on group basis were used to calculate the feed conversion ratio (FCR) per kg gain.L. intracellularis qPCR).Once a week, samples from all individual animals were collected during defaecation or directly from the rectum. One part of the sample was used for analysis (DM), another part being frozen for further analyses  at the abattoir in Hanover. The slaughtering process was done in small groups. In each group, genetic lineage and feeding variants were equally represented. On the morning before the dissection all the boars were given their respective experimental diets. In order to avoid unnecessary stress for the animals, only those boars were loaded and transported to the abattoir, which were also going to be killed directly afterwards in the chamber.At the end of the 4-week experimental period (TP6), the animals were killed in a mobile chamber by means of a CODuring the bleeding, blood samples were taken. After the evisceration by the abattoir personnel, the caecum was ligated before at the base of the caecum with a double ligature. Subsequently, the organ was separated with a pair of scissors and removed. The tip of the caecum was opened. The entire contents were collected and cooled until storage for further processing. For the histological examination, a piece tissue from the intestinal wall (about 3 × 2 cm in size) was taken of each boar in the vicinity of the appendix of the caecum. Care was taken to save the taenia-area. The samples obtained from the caecum wall were clamped on cork plates with needles. Afterwards, tissue was transferred to a cup filled with 10% formalin. After a fixation time of about 24 h, the tissue samples were further processed.®, Elementar, Hanau, Germany). The crude fat content was determined after acid digestion in the soxhlet apparatus. The content of crude fibre was determined after washing in dilute acids and alkalis. Starch determination was carried out polarimetrically . The sugar content was analysed using the Luff-Schoorl method by titration with sodium thiosulphate. The mineral content was determined by atomic absorption spectrometry . Amino acids were determined by ion-exchange chromatography . The content of volatile fatty acids in the homogenised caecal chyme was measured by means of a gas chromatograph . After the sample had been mixed with an internal standard , the mixture was centrifuged and then subjected to gas chromatography with a column temperature of 155 °C .Diets were analysed by standard procedures in accordance with the official methods of the VDLUFA . The dryThe serological tests were carried out using a sandwich blocking ELISA . This ELL. intracellularis content was determined from the aliquot of the homogenised faeces via quantitative PCR using established methods [The mean  methods . Results® plus, Menzel GmbH & Co. KG, Brunswick, Germany) per block.For histopathological analysis, the fixed tissue specimens were processed by routine methods, embedded into paraffin, and sectioned at 2 μm; three series (with three cuts per series) with a minimum distance of 25 μm were prepared and fixed on microscope slides . For each slide, ten completely cut crypts were measured from their base to the opening to the intestinal lumen.To measure the crypt depth of the caecal epithelium, a hematoxylin–eosin (HE) staining was performed in accordance with a standard protocol . For each boar, these two HE stained sections were examined with regard to the crypt depth of the caecal epithelium. The measurement was carried out with a photomicroscope  and the program analysisSIS®, version 9.3, . The group comparison for performance parameters , results of serological analyses , quantitative detection of L. intracellularis in faeces and caecal content, DM content in faeces, short chain fatty acids in caecal content and crypt depth of caecal wall were performed by a one-way analysis of variance (ANOVA) for independent samples. The Ryan-Einot-Gabriel-Welsch multiple range test was used for the multiple pairwise means comparisons. The distributions of the residuals from the linear models belonging to the analyses of variance were close to the normal one.The statistical analyses were performed using the Statistical Analysis System for Windows SASIn the case of non-normalised data, overall differences in parameters between groups were assessed by the Kruskal–Wallis equality of populations rank test followed by comparisons with a Wilcoxon signed-rank test. The latter was carried out in pairs to investigate differences in the mean values. The transition from the comparative to the experimental-related error probability was achieved by the so-called “α-adjustment” according to Bonferroni.L. intracellularis counts at different time points depending on group as well as differences between DM content in faeces between groups were examined by means of one-way analyses of variance for repeated measures.Differences in concentration between For the correlation-analysis of data with normal distribution, the correlation coefficient of Pearson was used. In non-normally distributed residuals the Spearman`s rank correlation coefficient was calculated.All statements of statistical significance were based upon P values smaller than 0.05. This approach controls the comparison-wise error rate.L. intracellularis, were not treated with antibiotics due to the short duration of the clinical manifestation.The experiment ran as planned. There were no animal losses. Changes in faecal consistency, which were associated with the detection of The experimental diets could be regarded as almost isoenergetic (15.6 ± 0.33 MJ ME/kg DM) and isonitrogenous 187 ± 4.02 g/kg DM). The lysine contents were comparable (13.1 ± 0.55 g/kg DM) as intended when formulating the diets .Overall, there were no significant differences in the final body weight (Ø at group level: 134 ± 3.42 kg), or in the weight development between the groups  and at the time of slaughter  did not differ between groups at each time point. There was a significant increase in each single group between PI values from the start of the experiment to slaughter Table . The comL. intracellularis with the faeces did not differ between the groups at the different time points, nor did the mean excretion in the experimental period. The difference between the mean excretion in the experimental period (TP2–5) and the initial value (TP1) was positive with the exception of group CM. Group CM, however, showed the highest numbers of L. intracellularis in faeces at time TP1 in numerical terms. Within some of the groups , significant differences were seen as a function of time. In the FP group, the most significant excretion was found for TP3 (4.32 ± 3.16 log10 GE/g) and TP5 (4.55 ± 3.33 log10 GE/g). In group CM, on the other hand, the excretion at TP3 was the lowest (0.59 ± 1.87 log10 GE/g), and the highest at TP4 (5.83 ± 2.36 log10 GE/g). In group RPS, a significantly higher excretion with faeces was observed at the end of the trial phase .The excretion of The DM content in the faeces of the boars did not differ at the beginning of the experiment (Ø DM content at group level: 236 ± 16.8 g/kg). At TP2 in groups WHEY and RPS, at TP3 , TP4 and TP5, the DM content in group RPS was the lowest, whereas at TP2, TP4 and TP5, the DM content in the control group (FP) was highest. When comparing the average DM content during the experimental period (TP2–5), group FP showed the highest DM content in faeces, group RPS the lowest (P < 0.001). Depending on the time, there were no significant differences within each group.L. intracellularis genome equivalents were significantly highest  occurred between the PI TP1 and the PI Δ TP1 to TP6  as well as between the logL. intracellularis status in faeces, there were significances between DM at TP1 and crypt depth in the caecum , as well as between the log GE in the caecal content and the butyrate concentration at this location . These correlations were moderate (DM) and weak (butyrate).In terms of L. intracellularis (log10 GE) at TP1 correlated negatively with the acetate content in the caecum for the WHEY group . The average log10 numbers of L. intracellularis during the experimental period correlated positively with the DM at TP1  and negatively with the average DM content  for the FP group during the experimental phase. For the CORN group, the mean log GE content in ZP2–5 correlated positively with the butyrate content . For the CORN group, the difference in the log10 GE L. intracellularis between TP2–5 and TP1 correlated negatively with the average DM content during the experimental period . The L. intracellularis counts in the caecal content correlated negatively with the DM at TP1 for the WHEY group , there also being a negative correlation for the RPS group to all volatile fatty acids acetate , propionate  and butyrate .At a feeding group level, significant correlations were found between PI TP6 and the mean faecal DM TP2–5  for group CM as well as positive correlations between butyrate and PI TP6 for the CORN group  was shown [L. intracellularis was minimal. In pigs with faecal DM above 25%, no effect on ADWG was reported [In the present study, no effect of diet on the performance parameters could be seen. If, however, independent of the diet, the mean e faeces . Howeveras shown . The autL. intracellularis counts in faeces were significantly negatively correlated with ADWG (r = − 0.78). The present investigations thus confirm that the known relationship applies to RPS. The numerically lowest ADWG in the comparison between the feeding groups was associated with the numerically highest excretion in this group.In the experimental period of the present study, the group RPS showed the lowest average DM content in faeces (225 ± 35.4 g/kg). The correlation analysis showed only for the group RPS that the average L. intracellularis between TP2 and TP5 . At group average level a higher L. intracellularis excretion could be the reason for a lower performance. Nonetheless, also factors such as the palatability of the feed can provoke a different feed intake. Thus, this relation could also be a reason for the numerically different weight development between the different feeding groups.In the present study, a negative correlation was observed between the mean values of ADWG of the different feeding groups and excretion levels of L. intracellularis spread within the animal group is to be assumed. In each feeding group, a significant increase in the serological response provided evidence of this confrontation with the pathogen. It is known from the literature that the antibodies can fall off already 2 weeks after the excretion peak [The results of the serological tests are clear in the present study. During the acclimatisation and experimental period, in each group an intense ion peak , 18. ThiL. intracellularis infection is termed proliferative haemorrhagic enteropathy [L. intracellularis infection occurred during the experiment . These animals showed numerically significant serological characteristics. The initial PI values were lower (20.2 ± 8.74). The final values tended to be higher (59.1 ± 6.69). The ΔPI values were more pronounced during the experiment (∆PI: 38.9 ± 7.39). The average excretion was higher in the experimental phase (TP2–5: 5.86 ± 3.45 log10 GE/g) and the ADWG was lower (0.946 ± 0.477). This also indicates the intensive infection in the groups.The haemorrhagic form of an eropathy . This foeropathy . In the L. intracellularis in faeces as a function of the diet. Feeding of whey powder showed, however, significantly lower L. intracellularis levels in the caecum.Overall, however, during the experimental phase in the present study there was no difference in the quantitative excretion of L. intracellularis [In literature, it could be proven that a fermented liquid diet (uncontrolled fermentation for 3–4 days at 24 °C) delayed the excretion of llularis . Furtherllularis . High lallularis . For a rllularis .L. intracellularis in the intestine. This could be very promising for future dietetic approaches in swine.Against this background, for the WHEY group higher concentrations of lactate in the gastrointestinal tract can be presumed. Therefore, it can be assumed that the lactic acid has a certain influence on the level of L. intracellularis in the caecum. This is a first link between parameters describing the extent of an infection and the substrate properties at the site of the main infection (caecum). In both cases, however, the correlation was weak.Regardless of the diet`s nature, two significant correlations were identified. First, the acetate content in the caecum was negatively correlated with the serological results at the end of the trial. Second, the butyrate contents in the caecum were negatively correlated with the number of L. intracellularis infections. In a US study, a reduction in feed intake  in animals aged 28 to 49 days could be observed for different inoculation doses  [10L. intracellularis organisms in the inoculum) was demonstrated. As a control animals were treated with 50 ppm tylvalosin for 14 further days after experimental infection [A direct effect is not yet known . However2 × 106) . In pigsnfection .L. intracellularis excretion in the experimental phase. At the same time, however, there was a negative correlation between DM content and L. intracellularis excretion during the experimental phase. Animals show more severe clinical symptoms during initial L. intracellularis infection [L. intracellularis excretion. During the experimental phase, a negative correlation between the DM content in faeces and the amount of excretion is to be expected. A previous study described that higher excretion is associated with softer faeces [In the present trial, for group FP a positive correlation was found between the DM content in faeces in the acclimatisation phase and the average nfection . The DM nfection . Conversr faeces , 7. In tL. intracellularis was reflected relatively clearly in the faecal consistency. Both, the final PI value and the change in the PI value were negatively correlated with the DM content in the faeces during the experimental phase. An effect of L. intracellularis is only relevant to performance when it is also reflected in the DM content [For the CM group, the serological response to the infection with  content . This re content .L. intracellularis could be observed. Also, in this group there are indications that a clearer response to the pathogen (ΔPI ↑) leads to changes in fecal consistency (softer).For the CORN group, there were positive correlations of serological results (PI TP6 and Δ PI) with the butyrate concentration in the caecal content. However, this group was characterised by the numerically lowest butyrate concentrations. At low butyrate concentrations, no positive effect on the excretion of L. intracellularis in faeces at TP1 was negatively associated with the acetate content in the caecum. On the other hand, there was a negative correlation between the DM content in faeces in the acclimatisation phase and L. intracellularis counts in the caecum. The period between acclimatisation and slaughter is relatively long (more than 30 days). A possible effect can certainly only be achieved if the corresponding feeding regime creates constant conditions in the animal. The measured parameters should also be causally related to the living conditions and characteristics of the pathogen. This is not proven for acetate. For the WHEY group, no correlation between the excretion and DM content in faeces could be determined for TP1 . It is also known from the literature that high amounts of pathogens in the caecum or adjacent areas need not be found in the colon [When feeding whey (WHEY), few correlations were detectable. On the one hand, the number of he colon  and therL. intracellularis counts in the caecum as well as the deepest crypt in the caecum. For this group a negative correlation of all volatile fatty acids with the L. intracellularis content in the caecum could be shown. The PI at TP6 as well as the average L. intracellularis content in the faeces were negatively correlated with the ADWG (r = − 0.66 and r = − 0.78), respectively. Thus, the question is whether the disease leads to less substrate or if less substrate favours the disease. The fact that less L. intracellularis was detected at absolutely lower butyrate concentrations in the caecal content of the CORN group rather suggests that the butyrate content within this group is more likely to be an indicator function than it being causally related to L. intracellularis counts.The RPS group had the highest concentrations of butyrate and the highest L. intracellularis infections. Dietetics can be a starting point for limiting the extent of infection at an early stage. Nevertheless, further tests over longer periods have to be performed first. In this context, feeding concepts with higher original lactic acid contents or provoking lactic acid production, such as from controlled fermentation, could prove promising to limit the effects of an L. intracellularis infection on performance and health.The present investigations indicate that the model of a natural infection in the finishing boars fundamentally works. Therefore, it is a good tool to analyse possible feeding influences on


Tg(flk1:RFP)is18/+ adults develop nonmalignant retinal tumors. Key pathways driving injury-induced glial reprogramming and regeneration contributed to tumor formation. In this study, we examine a time course of proliferation and present new analyses of the Tg(flk1:RFP)is18/+ dysplastic retina and tumor transcriptomes. Retinal dysplasia was first detected in 3-month-old adults, but was not limited to a specific stem cell or progenitor niche. Pathway analyses suggested a decrease in cellular respiration and increased expression of components of Hif1-α, VEGF, mTOR, NFκβ, and multiple interleukin pathways are associated with early retinal dysplasia. Hif-α targets VEGFA (vegfab) and Leptin (lepb) were both highly upregulated in dysplastic retina; however, each showed distinct expression patterns in neurons and glia, respectively. Phospho-S6 immunolabeling indicated that mTOR signaling is activated in multiple cell populations in wild-type retina and in the dysplastic retina and advanced tumor. Our results suggest that multiple pathways may contribute to the continuous proliferation of retinal progenitors and tumor growth in this optic pathway tumor model. Further investigation of these signaling pathways may yield insight into potential mechanisms to control the proliferative response during regeneration in the nervous system.In the central nervous system injury induces cellular reprogramming and progenitor proliferation, but the molecular mechanisms that limit regeneration and prevent tumorigenesis are not completely understood. We previously described a zebrafish optic pathway tumor model in which transgenic  Proper regulation of regenerative programs is necessary to limit proliferation and prevent cellular transformation and neoplasia. In injured tissue dying cells and damaged vasculature lead to inflammation and a hypoxic environment.2 The pro-inflammatory cytokine and hypoxia inducible factor Hif-α pathways are critical for mediating the cellular response to cell death and hypoxia.4 How activation of hypoxia and inflammatory pathways contribute to proliferation and regeneration in the nervous system is still not completely understood.E5 VEGF receptor tyrosine kinase signaling is essential to embryonic blood vessel development, adult angiogenesis, tumor vascularization, and disease-related vascular retinopathies.6 In addition, multiple nonvascular functions have been described for VEGF in the developing and adult central nervous system (CNS).8 VEGF is expressed by glial progenitors and astrocytes in response to hypoxia in the neonatal subventricular zone and stimulates proliferation of glial progenitors.9 In the subgranular zone/neurogenic niche of the adult mouse hippocampus, secretion of VEGFA and VEGFB by neural stem and progenitor cells is thought to influence the microenvironment and maintain the stem cell pool.10 Treatment of embryonic cortical neural precursors in vitro with VEGF induces expression of E2F family members and cell cycle regulators cyclin D1, cyclin E, and cdc25, supporting the above observations that VEGF may have a direct role in promoting progenitor proliferation in vivo.11 In the retina, VEGF has been shown to act directly on chick retinal progenitors to promote proliferation;12 however, whether VEGF mediates the proliferative response to hypoxia in the retina has not previously been shown.Hypoxia has been shown to activate regeneration gene programs in injured peripheral sensory neurons by Hif1-α induced expression of its direct target, the vascular endothelial growth factor (VEGF).13 and has been shown to stimulate proliferation of neural progenitors14 and regulate angiogenesis15–17 and wound healing.18 The presence of Leptin in the eye vitreous is associated with inflammation and vision loss in patients with diabetic retinopathy.19 Experimentally, Leptin expression is increased as part of the inflammatory response in a guinea pig model of uveitis20 and induces accumulation of reactive oxygen species in human endothelial cells.21 This suggests that Leptin and VEGF together might integrate hypoxia and inflammatory signaling pathways, which are both implicated in solid tumor initiation and cancer progression.22–24 Most recently it has been shown that extended exposure of normal bronchial epithelial cells to hypoxia and pro-inflammatory cytokines TNF-α and IL1-β induces cancer-like phenotypic changes, providing a link among hypoxia, inflammation, and cellular transformation in vitro.25 How sustained signaling due to a combination of hypoxia and chronic inflammation contributes to transformation in solid tumor initiation in vivo remains an open question. Moreover, the connection between hypoxic induction of Hif-α targets, such as VEGF and Leptin, to neoplastic transformation and tumor induction is less clear than the well-documented role of these factors in vascularization supporting tumor growth.26A second transcriptional target of Hif-α, the cytokine Leptin, is expressed in response to hypoxia28 Two populations of progenitor cells contribute to the growth of the zebrafish retina; latent progenitors derived from Müller glia in the inner nuclear layer and neural progenitors from neuroepithelial stem cells present in the ciliary marginal zone at the retina periphery.28 Chemical, mechanical, genetic, and light-induced injury of the zebrafish retina has revealed multiple growth factor, and cytokine signaling pathways stimulate latent progenitor proliferation and Müller glia dedifferentiation and reprogramming, both of which contribute to regeneration.29–31 Together these studies reveal the importance of coordinated activation of multiple pathways, including heparin-binding EGF-like growth factor, Wnt, Leptin, Interleukin-6, and Jak/Stat signaling, in progenitor proliferation and subsequent fate specification and differentiation.32Zebrafish has been used extensively as a model system for studying activation of latent progenitor populations and the molecular pathways controlling injury induced regeneration in the vertebrate nervous system.33 The limited regenerative response indicates that control mechanisms are enacted to prevent unregulated growth and dysplasia. Hif-α and mTOR signaling have been reported to work together in regulating ciliary marginal zone progenitor proliferation in Xenopus retina after nutrient starvation,35 and mTOR has been shown to be required for Müller glia-derived progenitor proliferation in injured chick retina.36 A role for Hif-α or mTOR signaling in zebrafish retinal development and regeneration has not previously been reported.The initial response to injury in the zebrafish retina involves transient expression of the pro-inflammatory cytokine TNF-α, which prevents gliosis and stimulates induction of inner nuclear layer progenitor proliferation and regeneration.Tg(flk1:RFP)is18/+ adults develop nonmalignant retinal tumors at ∼80% penetrance.37 The molecular basis for tumor induction is not known; however, our analyses showed that the retinal tumors may originate, in part, from Müller glia-derived progenitors, and activated signaling pathways in the tumor transcriptome are similar to injury induced regeneration pathways driving Müller glia reprogramming and progenitor proliferation. In contrast to injury induced regeneration models, our retinal tumor model is distinct in that TNF-α expression is not upregulated and proliferation is not transient. Once initiated, proliferation is sustained and contributes continuously to the growth of dysplastic tissue and the development of nonmalignant glial-like tumors. These observations indicate that other molecular pathways can stimulate retinal progenitor proliferation and override the normal controls that limit regeneration.We previously reported the characterization of a zebrafish optic pathway tumor model in which transgenic Tg(flk1:RFP)is18/+ retina. Early proliferation and dysplasia were not restricted to the ciliary marginal zone or to Müller glia-derived progenitors in the inner nuclear layer, the normal stem cell/progenitor niches of the teleost retina.38 Ingenuity pathway analysis (IPA) of the early dysplastic retina identified Hif-α signaling targets VEGF and Leptin, components of inflammation pathways NFκβ, IL-1, IL-6, and IL-8, and pathways required for Müller glia-derived progenitor proliferation in injured retina, including HBEGF39 and mTOR.36 Novel pathways that act in tissue repair and regeneration,40 but not previously implicated in retinal regeneration, were represented, such as GADD45 growth arrest and DNA damage response, endothelin-1 signaling, and caveolin-1-mediated endocytosis. Our results suggest that multiple signaling pathways might trigger progenitor proliferation and contribute to tumor growth. The Tg(flk1:RFP)is18 tumor model may provide insight into the molecular mechanisms that prevent neoplastic transformation during regeneration in the nervous system.To identify pathways that might be associated with proliferation and the responsive retinal cell populations, we performed additional immunocytochemical and differential gene expression analyses on adult Tg(flk1:RFP)is18 transgenic zebrafish line predisposed to optic pathway tumors was previously described.37 Zebrafish were housed in an Aquatic Habitat system  and maintained on a 14-h light/14-h dark cycle at 27°C. Transgenic fish predisposed to tumor formation were raised side by side with nontransgenic siblings. Heterozygous and homozygous transgenic fish and sibling fish were monitored daily during routine feeding for viability and morbidity, and monitored bi-weekly for gross presentation of ocular tumors. Juvenile and adult fish were anesthetized and euthanized in MS-222 Tricaine Methanesulfonate according to experimental protocols approved by the Iowa State University Institutional Animal Care and Use Committee (Log # 11-06-6252-I) in compliance with the American Veterinary Medical Association and the National Institutes of Health guidelines for the humane use of laboratory animals in research. Adult fish were anesthetized and euthanized in MS-222 Tricaine Methanesulfonate before sacrifice and tissue dissection for RNA isolation, histopathology, and immunolabeling.The One month old intact juvenile fish or whole heads dissected from adult zebrafish were fixed in Davidson's fixative  for 16 h at 4°C, decalcified in Cal-Ex (Fisher) for 2 days at 4°C, and processed and embedded in paraffin blocks at the Clinical Histopathology Laboratory at Iowa State University. Paraffin blocks were serial sectioned at 6 μm on a Shandon Finesse 325 microtome. Slides were stained with Hematoxylin 7211 Richard-Allan Scientific (Fisher) and 3% Eosin Y (Argos Organics) and mounted in Permount (Fisher). To aid antigen retrieval for PCNA labeling, slides with sectioned tissue were pretreated with 10 mM Sodium Citrate. Mouse monoclonal anti-PCNA (Sigma P8825) was used at 1:1000; rabbit anti-Sox2 polyclonal (Sigma-Aldrich/Millipore AB5603) was used at 1:200; rabbit anti-phospho-S6 ribosomal protein  was used at 1:300; and Alexa-488 and Alexa-594 conjugated secondary antibodies (Invitrogen) were used at a dilution of 1:500. Antibody-labeled slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Fluorogel (EMS). Hematoxylin and Eosin (H&E) stained slides were imaged on a Zeiss Axioskop II using a Nikon Rebel camera. Immunofluorescence was imaged on a Zeiss LSM700 laser scanning confocal microscope. Images of histological staining and antibody labeling from the same fish were captured from tissue sections separated by ∼100 μm.Tg(flk1:RFP)is18/+ age-matched siblings . The total number of positively labeled cells was counted separately in two regions of the retina: at the ciliary marginal zone and in a 300 μm length of the inner retina. To quantify phospho-S6 labeling, three individual wild-type and three individual Tg(flk1:RFP)is18/+ retinal tumors were examined. From each individual, three serial sections were imaged at 63 × for a total of 18 images. The total number of DAPI positive cells and phospho-S6 positive cells were counted in a 100 μm × 100 μm region of wild-type retina and Tg(flk1:RFP)is18/+ tumor tissue in each section and used to calculate the percentage of phospho-S6 positive cells. Statistical comparison of quantification in wild type and Tg(flk1:RFP)is18/+ was performed using student's t-test in GraphPad Prism software.For analysis of proliferation in early dysplastic regions of the retina, sections of retinal tissue adjacent to a section stained with H&E  were labapoe forward 5′ TAGCGGCCGCGAATTCGCCC 3′, apoe reverse 5′ TAGTCCTGCAGGTTTAAACGA 3′; lepb forward 5′ TGCTTGTTAATATCATCCCTGGT 3′, lepb reverse 5′ GAGAATGAATGTCTCAGCCACA 3′; lepr forward 5′ CGCTGTAAAGACGTGAACGA 3′, lepr reverse 5′ TCTGCCTGAAGTCCATTCCT 3′; flk1 forward 5′ AAGTGGCTAAAGGCATGGAGTTC 3′, flk1 reverse 5′ GACACTCCATCTCCGAGTCAAGG 3′; vegfab forward 5′ CGCGTGCTCCAGTTATTTATTGTG 3′, vegfab reverse 5′ CACCTCCTTGGTTTGTCACATCTG 3′; vegfaa forward 5′ TGATACAGTTATTTCTCGCGGCTC 3′, vegfaa reverse 5′ TTTGCAGGAGCATTTACAGGTGAG 3′. Digoxigenin-labeled probes for in situ hybridization were synthesized using a DIG RNA labeling mix (Roche #11277073910) and T3 RNA polymerase (Roche #11031163001) and stored in 50% formamide at −20°C. Adult zebrafish tissues were dissected, fixed in 4% paraformaldehyde, and embedded in optimal cutting temperature medium. in situ hybridization on 12–16 μm cryosections of head and eye tissue was performed as described.41 Tissues were photographed on a Zeiss Axioskop II microscope using a Nikon Rebel camera.cDNA was amplified by reverse transcription-polymerase chain reaction out of total RNA isolated from wild-type 5 days post-fertilization embryos and was cloned into the pCR4-TOPO vector (Invitrogen). Primers for amplification were as follows: Tg(flk1:RFP)is18/+ 6-month-old adults was reported previously37 and the data deposited at the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2886. Briefly, total RNA from dissected retinas or tumor tissue from age-matched 6-month-old sibling fish was isolated using an RNeasy RNA Isolation Kit (Qiagen). Three dissected wild-type sibling retina, heterozygous Tg(flk1:RFP)is18/+ dysplastic retina, and heterozygous Tg(flk1:RFP)is18/+ retinal tumor tissue were pooled and used for total RNA isolation. Tg(flk1:RFP)is18/+ retinas were classified as dysplastic or tumor based on the gross appearance of the tissue. Dissected Tg(flk1:RFP)is18/+ retinas that were thicker than wild type but did not have obvious lesions were used for the dysplastic retina sample. Dissected Tg(flk1:RFP)is18/+ eyes that contained large masses filling the vitreous were used for the retinal tumor tissue sample. A single RNA-Seq library for 100 bp paired end sequencing was prepared for each sample and the three libraries sequenced at the Genome Sequencing and Analysis Core Resource, Duke Institute for Genome Sciences and Policy, Duke University. Wild-type, dysplastic retina, and retinal tumor RNA-Seq libraries contained 436,511,378, 444,838,528, and 422,324,454 reads, respectively.The transcriptome of retinal and tumor tissue from age-matched wild-type sibling and 37 sequences were mapped to the zebrafish reference genome v9 using GSNAP, counted with HTSeq-count, upper quartile normalization applied, and the Fisher's exact test used to determine differential gene expression. q-value estimation of false discovery rate was performed in R using the open source software qvalue (http://bioconductor.org/biocLite.R). In the present study genes with at least 10 read counts were used for downstream analyses. Gene ontology GO Term42 analyses were done using the Princeton GO Term Finder website (http://go.princeton.edu/cgi-bin/GOTermFinder). Cluster frequency was calculated as the percentage of genes from the dataset significantly associated with a particular term, while genome frequency (or background frequency) was calculated as the percentage of all genes in the Danio rerio genome associated with a particular term. p-value cutoffs were set at 0.01. p-values were Bonferroni corrected with an estimated false discovery rate of <0.01.As previously described,www.qiagen.com/ingenuity) using genes having a read count of at least 10 in all samples and a three-fold change in expression between wild-type retina and Tg(flk1:RFP)is18/+ dysplastic retina or retinal tumor samples. Ensembl gene IDs for human homologs were extracted using Biomart43 at (http://useast.ensembl.org/biomart/martview/da30878652120cac4e352ddac342a16e).Transcriptome data were analyzed with QIAGEN's IPA . An excel file containing normalized read counts for all genes was previously published in Solin et al.37Raw transcriptome dataset supporting the conclusions of this article is available in the ArrayExpress repository (E-MTAB-2886 Tg(flk1:RFP)is18 retina, we performed histopathology on serial sectioned head tissue from cohorts of age-matched wild-type sibling and Tg(flk1:RFP)is18/+ zebrafish. Wild-type and transgenic siblings were sacrificed at 4 weeks , 3 months/11 weeks , and 4 months  of age. In all individuals 6 μm sections extending from the anterior to posterior limits of the eyes were examined for the frequency and location of ectopic cell proliferation and abnormal retinal architecture.To examine morphological changes and dysplasia in the Tg(flk1:RFP)is18/+ with 10 age-matched wild-type siblings did not reveal any obvious morphological abnormalities or ectopic proliferation in the retinas, indicating that during juvenile stages in Tg(flk1:RFP)is18/+ heterozygotes, the growth of the retina and the rate of progenitor proliferation and production of neurons and Müller glia proceed normally. At 3 months of age, in comparison to wild-type retina (Tg(flk1:RFP)is18/+ fish had regions of ectopic proliferation in the retina in at least one eye that contained clusters of proliferating cells in the inner nuclear layer, inner plexiform layer, or retinal ganglion cell layer is18/+ fish, with 10 out of 15 individuals presenting with ectopic proliferation in one or both retina. However, phenotypes ranged from retina with a normal appearance to retina containing multiple regions of proliferation and disorganization with gaps in the inner nuclear layer or large lesions distending the nuclear layers (Tg(flk1:RFP)is18/+ fish.37A similar frequency of retinal abnormalities was observed in 4-month-old r layers  and retir layers , which wr layers . Mitoticr layers , arrows.r layers , arrows Tg(flk1:RFP)is18/+ 4-month-old adults were histologically stained with H&E and adjacent sections labeled with PCNA and Sox2 antibodies is18/+ adults had regions of ectopic proliferation in multiple serial sections (Tg(flk1:RFP)is18/+ retina compared to wild type. The diameter of the ventral ciliary circumferential arteries was increased (Tg(flk1:RFP)is18/+ compared to age-matched wild-type siblings was significant is18/+ retina, neurogenesis proceeds normally through juvenile stages of development. Abnormal proliferation begins in early adulthood and occurs in the inner nuclear layer and ganglion cell layers of the retina (Tg(flk1:RFP)is18/+ retina are stimulated to proliferate and may include cells in the pigmented epithelium or ganglion cell layer, in addition to the known progenitor populations in the inner nuclear layer and at the ciliary marginal zone.38In summary, the histological analysis of tumor onset indicates that in the e retina . The none retina . In conte retina  and 2g. Tg(flk1:RFP)is18/+ dysplastic retina (formerly designated Pretumor) and Tg(flk1:RFP)is18/+ retinal tumor tissue were consistent with activation of signal transduction pathways known to be required for Müller glia reprogramming during injury induced regeneration.37 GO Term analysis of the RNASeq dataset using genes with an fragments per kilobase of transcript per million mapped reads (FPKM) of ≥1 and a significant change in expression level between samples (p ≤ 0.01) revealed translation; vascular development and blood vessel development processes were upregulated in Tg(flk1:RFP)is18/+ dysplastic retina (www.liebertpub.com/zeb). In retinal tumor the analysis revealed a significant decrease in phototransduction and ion transport and an increase in cellular processes necessary for proliferation, including DNA replication, cell division, and ribonucleoprotein complex biogenesis  were identified using BLAST, leaving 2570 genes for IPA analysis , Phototransduction Pathway (p = 4.65E-15), mTOR signaling (p = 2.28E-08), and Cell Cycle Control of Chromosomal Replication (1.46E-07). General categories of disease, disorders, and molecular and cellular function were consistent with injury and tumorigenesis and included cancer, organismal injury and abnormalities, and neurological disease and predicted an increase in cell movement, growth, and proliferation and a decrease in cell death and apoptosis. Specific canonical signaling pathways represented in the dysplastic retina included caveolin-mediated endocytosis, EIF2 translation, Hif1-α, endothelin-1, VEGF, mTOR, and GADD45B (Tg(flk1:RFP)is18/+ retina.Pathways with the most significant overlap in the dysplastic tissue were EIF2 Signaling ( GADD45B . NFκβ an GADD45B . A gliom GADD45B  as expecTg(flk1:RFP)is18/+ dysplastic retina are direct targets of hypoxia-inducible factor Hif1-α45–48 or components of the Hif1-α , VEGF , or JAK/Stat signaling pathways (socs3a) . The level of hif1aa and hi1ab gene expression was not significantly changed between wild type, Tg(flk1:RFP)is18/+ dysplastic retina, and tumor tissue. Together these observations suggest that a hypoxic environment is one possible trigger associated with ectopic proliferation in the Tg(flk1:RFP)is18/+ retina. Because both VEGF and Leptin are direct targets of Hif1-α and have been shown to promote neural progenitor proliferation,50 we examined these two signaling molecules in more detail.A number of genes with the greatest change in expression between wild-type and (socs3a) . Other hTg(flk1:RFP)is18/+ retina tissue, the level of vegfaa increased 2.9-fold and vegfab increased 11-fold. IPA analysis showed increased expression or activation of many components in the VEGF regulatory pathway that promotes tumor cell invasion , cytokines , matrix metalloproteases, and peptidases , and enzymes involved in prostaglandin synthesis . In the retina, VEGF promotes proliferation of retinal neural progenitors through activation of E2F using the MEK-ERK signal pathway.51 The E2F family of transcription factors has been shown to activate expression of gene networks regulating cell cycle progression, DNA damage and mitotic checkpoints, chromosome maintenance, and chromatin assembly.52 Many direct targets of E2F were significantly increased in the dysplastic retina transcriptome, including cyclin dependent kinase cdk1 and cdk2, minichromosome maintenance deficit family members mcm3, mcm5, mcm6, and mitotic kinases aurora kinase aurkb and polo-like kinase plk1  and remained elevated in tumor tissue is18/+ retina, either contributing to dysplasia and tumorigenesis or as a result of increased proliferation.A second target of Hif1-α signaling, the cytokine Leptin,r tissue . IPA ana socs3a; . Leptin vegfaa, vegfab, and lepb, in situ hybridization was performed on retinal sections from wild type and Tg(flk1:RFP)is18/+ adults . The panel of in situ hybridization results shown in Tg(flk1:RFP)is18/+ retina, to illustrate expression for multiple genes in a retina with dysplasia, as well as an advanced tumor. As shown in To examine which cell types in the retina express vegfaa signal was weak in all three nuclear layers is18/+ retina with dysplasia, a high level of vegfab labeling was present and appeared to be expressed in the inner and outer nuclear layers, but was absent from the ganglion cell layer is18 dysplastic retina. Together these results indicate that in the Tg(flk1:RFP)is18 retina, neurons and glia respond by expression of distinct growth factors and cytokines. vegfab expression is highly induced in neurons, and possibly Müller glia, in the inner nuclear layer. In contrast, lebp expression overlapped with apoeb and appeared restricted to the Müller glia and astrocytes in the nerve fiber layer.Apolipoprotein ApoE is expressed by activated Müller glia and astrocytes in the injured retina and promotes neurite outgrowth of various neuronal cell types.al cells , furtherTg(flk1:RFP)is18/+ transcriptome was mTOR signaling is18/+ retina tissues were labeled with an antibody specific for phosphorylated ribosomal protein S6 (phospho-S6) [n = 7 wild type and n = 8 Tg(flk1:RFP)is18/+ biological replicates]. In wild-type adult zebrafish retina phospo-S6 labeling was detected in numerous cell types. In the inner nuclear layer of the mature retina phospho-S6 labeled putative horizontal cells (56 Regions of the Tg(flk1:RFP)is18/+ retina with dysplasia (Tg(flk1:RFP)is18/+ tumor tissue was not significantly different than in wild-type retina . In the Tg(flk1:RFP)is18/+ retina phospho-S6 was also detected in the nerve fiber layer on the vitreal side of the retina (Tg(flk1:RFP)is18/+ retina.To examine mTOR signaling in the al cells , arrows.ysplasia  or advanysplasia  showed pe retina , arrows.Tg(flk1:RFP)is18/+ zebrafish predisposed to nonmalignant retinal tumors and analyzed activation of signaling pathways that correlate with retinal dysplasia and tumor growth. A major finding from our time course analysis of proliferation is that multiple cell populations proliferate in the early dysplastic Tg(flk1:RFP)is18/+ retina, including cells in the pigmented epithelium, ganglion cell layer, and inner nuclear layer, as well as at the ciliary marginal zone. We also observed aberrant cell migration across the inner nuclear layer in the very early dysplastic retina, which could contribute to dysplasia and tumor growth. These cells may originate from angiogenic sprouting of blood vessels in the nerve fiber layer in response to release of VEGF by inner nuclear layer neurons. Together, our data indicate that cells outside of the zebrafish retina ciliary marginal zone, in addition to Müller glia-derived progenitors, have the potential to proliferate.In this study we examined the onset of ectopic proliferation in the retina of adult vegfab expression upregulated in neurons and lepb expression overlapping with the glial marker apoeb. How this contributes to dysplasia in the retina, and separating and evaluating the distinct roles VEGF plays in neurogenesis directly versus indirectly through enhanced angiogenesis, will require additional investigation. Leptin has previously been reported to function in Müller glia reprogramming,32 and our results showing increased expression of lepb in Tg(flk1:RFP)is18/+ retinal glia support a role for leptin signaling in glial-derived progenitor proliferation. Recent studies have identified a tissue regeneration enhancer element in the zebrafish lepb promoter that mediates high levels of leptin expression during fin and heart regeneration,57 suggesting that the elevated expression of lepb in Tg(flk1:RFP)is18/+ retina may simply be due to enhancer activation. It is well documented that Hif1-α activates the VEGF and Leptin promoters,53 consistent with elevation of the Hif1-α pathway in the dysplastic retina transcriptome. However, it is equally likely that additional upstream triggers may activate VEGF and Leptin expression in the Tg(flk1:RFP)is18/+ retina, leading to proliferation and dysplasia.A second major finding of our study is that expression of VEGFA and Leptin is significantly increased in early dysplastic retina, before formation of large lesions or tumors. Within the dysplastic retina neural and glial cell populations appear to respond differently, with Tg(flk1:RFP)is18/+ retina phospho-S6 positive cells were present in dysplastic regions with high cellularity and large tumor lesions. Compared to VEGF and Leptin signaling, mTOR activation may not be a factor in initiating inappropriate proliferation in the early dysplastic retina, but could contribute to the sustained growth of retinal tumors. mTOR has been shown to be required for proliferation of Müller glia progenitors in response to injury in chick retina.36 While multiple signaling pathways most likely drive proliferation and tumor growth in the Tg(flk1:RFP)is18/+ model, our data are consistent with our previous analyses which suggest that mTOR signaling could influence activation of Müller glia regeneration pathways in the Tg(flk1:RFP)is18/+ retina.Our study also provides evidence that in wild-type adult retina mTOR signaling is active in numerous cells in the inner nuclear layer and retinal ganglion cell layer, which has not previously been reported in zebrafish. mTOR activation was detected in putative horizontal cells, amacrine cells, and retinal ganglion cells, but was absent from the ciliary marginal zone. In Tg(flk1:RFP)is18/+ zebrafish predisposed to optic pathway tumors reveal that ectopic proliferation is associated with elevated expression of markers in the VEGF, Leptin, and mTOR pathways. The causative molecular mechanism inducing retinal dysplasia and tumor formation in the Tg(flk1:RFP)is18 line is not known. Our previous molecular characterization of the Tg(flk1:RFP)is18 line revealed a 500-copy number <flk1:RFP> transgene inserted into a lncRNA. Isolation of a 147 kb deletion allele that removed the majority of the lncRNA gene did not cause tumor formation in heterozygous or homozygous adults.37 It is possible that the nature of this particular high copy number transgene is linked to deregulation of multiple signaling pathways, and this underlies proliferation and dysplasia. One possibility is that hundreds of copies of the flk1 promoter alter global gene expression or a subset of vascular genes, including VEGF. Elevated levels of VEGF can cause vascular permeability and leakage,7 which would disrupt exchange of nutrients and metabolic waste. Alternatively, high levels of membrane targeted RFP expressed from the transgene in vascular endothelial cells may compromise vascular integrity, leading to defective retina function and metabolism. One of the most highly overexpressed genes in the Tg(flk1:RFP)is18 dysplastic retina transcriptome is homologous to Plasmalemma Vesicle Associated Protein (plvap), a marker of vascular permeability. This supports the idea that defective vasculature and metabolic recycling could contribute to persistent stimulation of neural progenitor proliferation, leading to retinal dysplasia and nonmalignant tumor formation. Determining whether similar mechanisms regulate stem and progenitor cells throughout the nervous system will be important for a better understanding of CNS injury and repair.In summary, differential gene expression and cytological analyses of retinal dysplasia in adult


The well mixed molybdenum oxides at the microscale and the involvement of both mechanisms are considered as the key to the better electrochemical properties. The strategy can be applied to other transition metal oxides to enhance their performance as electrode materials.A simple and effective carbon-free strategy is carried out to prepare mixed molybdenum oxides as an advanced anode material for lithium-ion batteries. The new material shows a high specific capacity up to 930.6 mAh·g Bulk molybdenum oxides, however, show much lower capacities compared with their theoretical ones, which can be attributed to the significant volume change during the charge and discharge processes and the intrinsic slow kinetics for the conversion reactionsAs one of the solutions for our new energy-based economy, lithium ion batteries are widely used in mobile devices, such as cellular phones and laptops, because of their relatively high volumetric and gravimetric energy density1−1 after 50 cycles at 50 mA·g−1 −1 after 30 cycles even at 3 C rate2 hollow structures coated with Mo2N show a capacity of 815 mAh·g−1 after 100 cycles at 100 mA·g−1 Recently, metal oxide nanostructures, especially nanostructured oxides have been prepared as anode materials for lithium ion batteries due to their short diffusion length, larger reaction surface area between the electrode and electrolyte, as well as extra space for better accommodation of the strain induced by large volume changes6789103O8 generate LixV2O5/LiV3O8 composite with significantly improved cycling performancs and rate capabilityx, 2 < x < 3) by moderately oxidizing molybdenum dioxide nanoparticles under mild oxidizing atmosphere. The obtained MMO exhibits a discharge capacity as high as 930.6 mAh·g−1 at a current density of 200 mA·g−1 after 200 cycles as an advanced material, indicating this material has potential for applications in lithium-ion batteries.Partial oxidation or reduction strategy has been employed to improve the electrochemical properties of cathode materials, for example, controlled reduction of LiV2 nanoparticles were prepared by using a hydrothermal method reported by Liu et al.MoO2 nanostructured powders was heated in a tube furnace at a rate of 5 °C·min−1 to 500 °C and kept at the temperature for 100 min. A mixed gas containing a small fraction of oxygen in nitrogen was flowed into the system during heating at a rate of 20 mL/min. The fractions of oxygen were controlled as 0.2% and 1%, in order to achieve the final product MMO and MoO3, respectively.To prepare MMO, the as prepared MoO5/2 peak to the Mo 3d3/2 peak is fixed to 1.5 while the binding energy separation is set to 3.0 eV. A Bruker Advance III 400 MHz spectrometer with a 3.2 mm Magic Angle Spinning (MAS) probe was used for acquiring the 7Li solid-state NMR spectra at a resonant frequency of 155.46 MHz. A spin-echo and a 2D exchanged pulse sequence were used in the data acquisition. In order to select reduce the intensity of the resonance due to the SEI, a T2 filter was used in the spin-echo NMR data acquisition. After discharging to 5 × 10−3 V, the self-supported electrodeThe XRD patterns were acquired with an X-ray diffractometer  at a 2θ range of 10–70°. The diffractometer was equipped with a Cu Kα radiation (λ = 1.5418 Å) and operated at 40 kV and 40 mA. The air sensitive samples were protected by Kapton films in the XRD measurement. The morphology of the samples were investigated using a field-emission scanning electron microscope (Hitachi FE-SEM S4800). X-ray photoelectron spectroscopy (XPS) was used to survey the surface compositions and the chemical states of the samples . The hydrocarbon C 1 s peak at 284.5 eV was chosen as the reference for the binding energies. The XPS spectra were analyzed with the software XPSPeak 4.1. The ratio of the peak area of the Mo 3d6 in a mixture of ethyl methyl carbonate (EMC), ethylene carbonate (EC) and dimethyl carbonate (DMC) (1:1:1 in weight) solution while a Celgard 2325 film was used as the separator. An electrochemical station (CHI660D) was used to obtain cyclic voltammetry (CV) at ambient temperature. CV measurements were performed over a potential range of 5 × 10−3–3.0 V (vs. Li+/Li) at a scan rate of 2 × 10−4 V s−1. Galvanostatic discharge–charge measurements were performed at different rate on a battery testing system  with a voltage window range from 5 × 10−3 to 3.0 V (vs. Li+/Li).Electrochemical measurements were performed on coin cells (model 2032) with lithium metal foils as the reference and counter electrodes at ambient temperature. 70 wt% of as prepared samples, 20 wt% acetylene black (conducting additive), and 10 wt% polyvinylidene fluoride (binder) were blended with N-Methyl-2-pyrrolidone (NMP) to make a slurry and then spread onto a copper foil current collector. After drying at 80 °C for 6 hours, the electrode was punched and then dried under vacuum at 120 °C for 12 hours. The electrodes were then transferred to an argon-filled glove box where both the moisture and oxygen concentrations were kept below 1 ppm. The electrolyte was prepared by dissolving 1 M LiPF2 nanoparticles annealed with pure N2 can be indexed to MoO2 (2 (1% O2 in N2) in the flow is completely oxidized to MoO3  show mixed phases which can be attributed to MoO2, MoO3, as well as a small fraction of Mo4O11 . The SEM image of MMO shows this sample is composed of small particles with different sizes, resembling a mixture of the above two materials. The results indicate that MMO are not uniformly oxidized, in agreement with the XRD data.6+, and can be attributed to Mo6+ (3d5/2) and Mo6+ (3d3/2), respectively4+ (3d5/2), suggesting that the Mo ions on the surface of MMO are in mixed valence states6+ (3d5/2)/Mo6+ (3d3/2) and Mo4+ (3d5/2)/Mo4+(3d3/2)  measurement was performed in order to investigate the surface valence states of Mo in MMO, and the results are shown in +(3d3/2) . The Mo6x) to MoO3 in the air and a wide exothermic peak is associated with this process. The total weight increase is 4.30%  was used to study the Li insertion/extraction behaviors in different samples and the results are presented in articles  and prevarticles  and lite−1. The initial discharge capacity is 1068.2 mAh·g−1, slightly higher than the theoretical capacity of MMO (1021 mAh·g−1), which can be assigned to some irreversible reactions, e.g. the generation of an SEI layer23133 nanostructuresxMoO2, the decrease in voltage can be ascribed to the transformation of crystalline phase to amorphous-like structure13To further investigate the electrochemical performance of the samples, galvanostatic experiments were performed on the coin cells. of MMO 10 mAh·g−1,of MMO 10 mAh·g−1,2 or MoO3. For MoO2, the first discharge capacity is only 670.5 mAh·g−1 while the capacity gradually increases upon cycling and attains 781.7 mAh·g−1 after 15 cycles. This activation process has been reported by other groups and may be attributed to the partial loss of crystallinity of the material during the cycling1328xMoO2 phase−1 remains after 50 cycles. The initial capacity of MoO3 is 1017.6 mAh·g−1, however, it drops to 593.4 mAh·g−1 after only 30 cycles due to the big volume changes during cycling. Such decrease can also be observed in MMO, which has a large fraction of MoO3. However, the drop in capacity is much less. The capacity decreases from the first discharge capacity of 1068.2 mAh·g−1 to 842.3 mAh·g−1 after 20 cycles, corresponding to a capacity loss of 12% . After that, the capacity increases slowly and reaches 976.3 mAh·g−1 after 90 cycles. The discharge capacity of the MMO only decreases slightly to 930.6 mAh·g−1 after 200 cycles, which is equivalent to a capacity retention of 95% of the maximum value of 977 mAh·g−1, demonstrating high capacity and excellent stability. Such high reversible capacity of more than 930.6 mAh·g−1 at 200 mA·g−1 is almost three-fold that of commercial graphite (~330 mAh·g−1). Although the overall profile of MMO’s cycling curve is similar to that of MoO3, the key difference is the capacity decay is much smaller for MMO than MoO3. This decay, which can be seen in many reports of MoO3, is generally assigned to the huge volume and/or structure change during the conversion reaction23xMoO2 in MMO is expected to help stabilize the whole electrode and avoid the capacity loss. To further investigate the hypothesis above, the electrochemical performance of the mixed MoO2 and MoO3 sample was prepared by ball milling (MMM)  . Althougoportion , the eleraphite ~0 mAh·g−1−3–3.0 V at 0.1 A·g−1, 0.2 A·g−1, 0.5 A·g−1, 1 A·g−1, 2 A·g−1, 5 A·g−1 and 0.1 A·g−1 for 10 cycles each. The specific capacity reaches 994 mAh·g−1 at 0.1 A·g−1, 883 mAh·g−1 at 0.2 A·g−1, 735 mAh·g−1 at 0.5 A·g−1, 644 mAh·g−1 at 1 A·g−1, 538 mAh·g−1 at 2 A·g−1, 397 mAh·g−1 at 5 A·g−1, respectively. A specific capacity of 397 mAh·g−1 at 5 A·g−1 for this material is as high as 40% of its capacity at 0.1 A·g−1 (994 mAh·g−1). The results show that MMO has a better rate capability compared to previous reports26The rate capability of MMO was further investigated and the results are shown in −1, 0.2 A·g−1) and cycling performance of MMO in this work excels most of the molybdenum oxide based materials previously reported in the literature, and even those with carbon coating3536373839407Li NMR spectroscopy proves to be a powerful method to investigate the relationship between local structures and electrochemical performances of materials for lithium ion batteries and to reveal the electrochemical reaction mechanisms7Li MAS NMR spectra of the MMO and MoO3 lithiated to 5 × 10−3 V are given in 3, which can be attributed to Li2O which is the product of the conversion reaction. The resonance of the lithiated oxide is broader compared to the spectrum of commercial Li2O standard sample, indicating more complex local structures of the lithium ions after the lithiation process, and/or the magnetic fields generated by the Mo particles2O formed in the conversion reaction. A sharp peak at −7.0 ppm can also be observed for MMO, indicating that there is another local environment of lithium ions.To further understand lithium storage mechanisms of MMO, Solid-State NMR and XRD experiments on lithiated samples were performed. 3, suggesting the poor crystallinity of the product after lithiation. However, pronounced peaks due to Li0.98MoO2, which was previously reported as the product of the insertion reaction of MoO27Li MAS NMR spectra of MMO after lithiation, it is clear that two mechanisms including both conversion and insertion reactions are involved in the lithiation of MMO (MoOx) while only conversion reaction is associated with MoO3. Assuming MoO3 in MMO only undergoes a conversion reaction while MoO2 is associated with both insertion/extraction and conversion reaction, the fraction of MoO2 (x) which underwent insertion/extraction reaction in MMO can be estimated in the following equation :The presence of the other lithium compound is further supported by XRD data of lithiated MMO . The Kapx can be determined as 0.058, implying 16% of the MoO2 material underwent the insertion/extraction reaction in the MMO material. The capacity of MMO increased after 50 cycles, indicating that more MoO2 underwent conversion reaction20Thus, the value of 7Li spectra provides more detailed information on the structure of MMO and additional evidences for its better electrochemical properties432O and Li0.98MoO2 can be observed in the 2D EXSY spectrum  during cycling272 nanoparticles282 and MoO3 species are not well mixed at the microscale for MMM, inhomogeneous distribution causes phase isolation during cycling, which will lead to pulverization and rapid capacity decay. It has been demonstrated that superior stability can be achieved for the sample with a closer combination between mixed valence lithium vanadates formed in an in-situ transformation than other common coating methods14344748492 to Li0.98MoO2 in insertion reaction only involves small volume/structure change in the very initial cycles, the well dispersed MoO2 helps to keep the integrity of the electrode and prevent the loss of active materials.Two-dimensional exchange (EXSY) spectrum . Howeverspectrum  while nospectrum . It sugg2, nanostructured MoO2 often undergoes conversion reaction in further lithiation, presumably due to faster kinetics2 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.