Maternal stress programs offspring disease in a sexually dimorphic manner with males often more adversely affected. Previous studies of maternal glucocorticoid exposure suggest male vulnerability may derive from placental alterations. The hexosamine signalling pathway and O-linked glycosylation (O-GlcNAcylation) are part of an essential adaptive survival response in healthy cells. The key enzyme involved is O-linked-N-acetylglucosamine transferase (OGT), a gene recently identified as a sex-specific placental biomarker of maternal stress. Using a mouse model of maternal corticosterone (Cort) exposure, we examined components of hexosamine biosynthesis/signalling and O-GlcNAcylation in whole placentae at E14.5. Our results demonstrate sex-specific differences in OGT levels and O-GlcNAcylation during Cort exposure which impacts on key mediators of cell survival, in particular AKT as well as the stress responsive OGT/GR transrepression complex. In male placentae only, Cort exposure increased Akt O-GlcNacylation which correlated with decreased phosphorylation. Female placentae had higher basal OGT and OGT/GR complex compared with male placentae. Cort exposure did not alter these levels in female placentae but increased global O-GlcNacylation. In male placentae Cort increased OGT and OGT/GR complex with no change in global O-GlcNacylation. These findings suggest that sex-specific differences in placental OGT play a key role in the sexually dimorphic responses to stress. Male fetuses are often reported as being more adversely affected than female fetuses and often demonstrate more severe disease outcomes than their female counterparts. We have previously shown that excessive maternal glucocorticoid administration results in more significant and profound effects on renal and cardiovascular health in male offspring2 that appear to derive from sex-specific placental adaptations in response to the same maternal challenge4. These sex-specific placental stress responses include differences in placental architecture as well as molecular changes such as altered expression of the enzyme 11β-hydroxysteroid dehydrogenase type 2, which acts as a barrier ameliorating the impact of glucocorticoids. Additionally, altered expression of vasculogenic and other growth factors such as VEGF and IGF-II also occurs in a sex-specific manner. Differences in how male and female placentae respond to stress suggest that sex-specific adaptations of the placenta may be a critical determinant of sexually dimorphic differences in programming outcomes6.A sub-optimal or stressful environment 7 and also implicated as a driver of sex disparity in disease8. This key nucleoplasmic enzyme catalyses the terminal transfer step in the hexosamine signalling pathway which results in the post-translational modification of numerous cellular regulatory proteins in the nucleus and cytoplasm with a single moiety of N-acetyl glucosamine (N-GlcNAc) through O-linked glycosylation (O-GlcNAcylation) of key serine and threonine residues9. This is a dynamic modification exhibiting properties that are commonly functionally reciprocal to phosphorylation at these same sites to regulate cellular function in response to nutrient and environmental cues10. O-GlcNAcylation appears to promote cell/tissue survival by regulating a multitude of biological processes including: the phosphoinositide 3-kinase/Akt pathway, heat shock protein expression, levels of reactive oxygen species, ER stress, protein stability, mitochondrial dynamics, and inflammation11. Inhibition of hexosamine biosynthesis, which results in the formation of the donor substrate UDP-GlcNAc for O-GlcNAcylation, eliminates cellular stress responsiveness leading to reduced cell survival in numerous systems12 including the early mouse embryo14. Whilst deletion of the key enzymes involved in hexosamine signalling is found to be lethal in mammals18, targeted placental deletion of OGT results in profound disruption of early hypothalamic gene expression and elevated hypothalamic-pituitary-adrenal axis responsivity that closely mirrors the phenotype of a mouse model of prenatal stress19.This leads us to question which stress responses are differentially regulated in placentae of female fetuses compared to male fetuses that may infer these differential outcomes. Recent studies suggest that the X-linked OGT may be a placental biomarker of maternal stress20, we sought to examine whether key elements of this signalling system or some of its known targets were implicated in placental adaptations in our model of maternal corticosterone (Cort) exposure in the mouse. We have previously demonstrated that this Cort exposure increases maternal plasma Cort concentrations by approximately threefold compared to Untr levels4. We have recently demonstrated a similar magnitude of change in Cort concentrations in rats that were subjected to acute restraint stress21 and previous studies have demonstrated a similar relative increase in women with a stressful labour22. Using this physiologically relevant model of Cort exposure, our results lead us to conclude that changes in OGT and hexosamine signalling in the placenta in response to maternal stress is likely to be a key mechanism through which sex-specific fetal programming occurs.Given the essential role that hexosamine signalling and O-GlcNAc cycling play in regulating the cellular stress and survival responseSlc2a1 , and Slc2a2  were not altered significantly by Cort exposure. Gene expression of the rate-limiting enzyme in hexosamine biosynthesis Gfpt1  was investigated in the fetal liver and kidney. We have previously reported that maternal Cort exposure induced changes in the gene expression in the fetal kidney and heart23 but had not investigated stress responsive genes in these tissues. In the current study, Hsp90a1 was increased in livers of both male and female fetuses , Hsf1  or Ogt  in the fetal kidney compared to Untr controls.To demonstrate that maternal glucocorticoid exposure induced cellular stress in fetal tissues, mRNA expression of a number of stress related genes  and female (P < 0.001) fetuses of dams exposed to Cort  and heat shock factor 1 (Hsf1) were unaffected at E14.5 during exposure to corticosterone . We have previously reported that maternal Cort exposure increases mRNA and protein levels of Hsd11b2 in placentas of female fetuses4. In the current study, Hsd11b1 mRNA levels tended to be increased by maternal Cort exposure but this did not reach statistical significance .Placental exposure to stress was also confirmed as indicated by increased expression of one Fig. . MoreoveGfpt1 . There was also a significant interaction between sex and treatment and post-hoc analysis demonstrated a significant increase in Gfpt1 mRNA levels only in female placentae (P < 0.0001). Despite these changes in mRNA levels, GFPT1 protein levels were unaffected by maternal exposure to corticosterone in both male (P = 0.81) and female (P = 0.74) placentae  isoform of the les Fig.  nor betwles Fig. . As wellsue Fig. . Moreove4. These adaptations were characterised by upregulation of the glucocorticoid barrier in females but dysregulated expression of growth and vasculogenic factors such as Igf2 and Vegfa together with structural alterations of the placenta at E17.5 in males only4. This was associated with impaired renal28, adrenal29 and cardiovascular function2 only in male offspring. While this suggests that sex differences in the level of fetal glucocorticoid exposure may be contributing to the sex specific disease outcomes, it is clear that placental adaptations mediate these sex differences. In the current study, we have identified sex specific placental adaptations in OGT and associated cell signalling pathways which may provide the female fetus with a greater level of protective capacity to maternal stress and long term disease outcomes30.It is well established that excessive maternal stress as well as synthetic glucocorticoid administration during pregnancy are associated with altered growth and organ development and adverse postnatal outcomes in offspring  within these two fetal tissues to regulate cellular stress responses. Despite this tissue specific stress response to maternal corticosterone exposure, the fetal response was not affected by fetal sex. Similarly, Ogt levels in both the fetal liver and kidney were not affected by maternal treatment or fetal sex highlighting that sex differences in OGT expression may be specific to the placenta and that sex specific programmed disease outcomes in offspring related to these organs28 may occur indirectly as a response to altered placental function.The Cort exposure in this model increased maternal plasma Cort levels by two-three fold and caused significant negative feedback on maternal adrenal steroidogenesis32. The current study demonstrated that maternal Cort exposure did not alter placental Hsf1 or Hsp70 expression but caused uniform increases in HSP90α both at the gene and protein level in placentae of both sexes. HSP90 is a constitutively expressed protective molecular chaperone involved in suppressing protein aggregation of mis-folded proteins that occurs in response to a range of cellular stresses. In addition, HSP90 can regulate cellular responses to glucocorticoids themselves by facilitating glucocorticoid receptor nuclear translocation and function34. This suggests that the sex specific placental adaptations and fetal outcomes are regulated not by the level of cellular stress that the placenta is exposed to but by how the placenta responds to this stress. Indeed, we have previously demonstrated that in response to maternal Cort exposure, mRNA and protein concentrations of the glucocorticoid metabolising enzyme Hsd11b2 are increased to a greater extent in females compared to males4. Hsd11b1 on the other hand, is known to convert inactive glucocorticoids to active glucocorticoids. The current study demonstrates that unlike Hsd11b2, placental Hsd11b1 mRNA levels were not affected by maternal Cort exposure.We then investigated if placentas from male and female fetuses experienced similar levels of cellar stress by examining expression of members of the heat shock protein (HSP) family. Activation of the cellular stress response is usually accompanied by a robust upregulation of heat shock proteins, including HSF-1 and HSP70 (Hsp1a1), which drives increased expression and activity of HSP90Ogt in female embryonic stem cells35 in accordance with higher resting levels of the gene and protein found in female human placentae7, suggests that the Ogt gene may escape X-inactivation in the placenta. Consistent with this suggestion we find basal OGT protein levels in the female placenta are twice that of the male. Indeed, the Cort exposure elevated OGT protein levels in the male placenta to similar levels to those observed in the females under basal conditions. The higher basal levels observed in female placentae may therefore be explained by this capacity of the X-linked Ogt gene to escape X-inactivation17 and importantly, may provide the female fetus with a greater protective capacity, given the rapid ability to respond to stress via this pathway is associated with adaptive cellular survival20. It is therefore likely that differential OGT expression and activity may contribute to different disease susceptibility in male versus female offspring in response to stress. In support of this proposition in both humans and animal models where sex chromosome gene dosage is altered as a result of chromosomal abnormalities associated with Turner’s (45X) or Klinefelter’s (47XXY) syndromes, the data suggest that dysregulation of OGT may be implicated in disease disparity8. Interestingly, despite no change in OGT in females, there was an overall increase in O-GlcNAcylation in female placenta in response to the Cort exposure. This suggests additional factors other than OGT may contribute to the sex specific alterations in O-GlcNacylation. As discussed further below, we hypothesize a role for the association of OGT with the glucocorticoid receptor. Levels of cellular O-GlcNAcylation increase in response to multiple forms of stress36 but must be tightly regulated for cellular homeostasis to be maintained as both inappropriately high and low levels of O-GlcNAcylation are linked to multiple chronic diseases37.A major finding of the current study is that maternal Cort exposure induces sexually dimorphic changes in placental OGT with levels increasing only in the placenta of males. This may be due in part to differences in basal levels of OGT. Bi-allelic expression of 38. The three isoforms (Akt1/2/3) have two major activation sites: T308, T309 and T305 and S473, S474 and S472 respectively. Activity of AKT, which depends upon the degree of phosphorylation at these sites, impacts on cell survival but also processes such as angiogenesis, as seen in endothelial cells39 and other stress models40. Disruption of AKT signalling results in structural abnormalities of the placenta and intrauterine growth restriction41. Previous work in our Cort exposure model resulted in changes in placental morphology consistent with decreased placental efficiency in male but not female fetuses4. This was associated with a significant decrease in protein expression of VEGF in male but not female placentae at E14.5, which is likely to have a significant impact on angiogenesis and therefore placental efficiency4. In the rat, significant decreases in VEGF were observed in response to synthetic glucocorticoid exposure and this was linked to decreased AKTS473 phosphorylation42. Those studies however did not examine differences between male and female placentae or provide mechanistic insight as to how this may be mediated. In the current study, although maternal Cort exposure had no impact on total levels of placental AKT1 and AKT2, there was a significant decrease in phosphorylation of AKT1 (AKT1S473) and AKT 2 (AKT2S474) in the placentas from male fetuses only. This is important as the O-GlcNAc modification is functionally reciprocal to phosphorylation43 and as such O-GlcNAc modification of a protein decreases its activity but increases its stability46. Studies using rat adipocytes have demonstrated this specifically with AKT2, demonstrating that O-GlcNAcylation of AKT2 reduces its activity38. Indeed our co-immunoprecipitation experiments in the current study demonstrated that Cort exposure increased O-GlcNAc modification of total AKT in male but not female placentae, confirming the reciprocal nature of this modification and suggesting that the sex specific Cort induced increase in OGT likely mediates this outcome. Collectively this suggests that the decreased phosphorylation of AKT1 at S473 and AKT2 at S474 in males may be occurring by sex-specific alterations of O-glycosylation and may be contributing to the differential alterations in placental growth morphology47.Akt is central to development and is a key target of O-GlcNAcylation45. Overexpression of OGT in HEK cells has similarly been shown to reduce phosphorylation levels of AKT at S473 but not at T30848. Furthermore, specific blunting of AKT1 activation at T308 through preventing glycosylation of Keratin 18 resulted in apoptosis49. It is therefore likely that the Cort induced changes to placental morphology result from OGT induced glycosylation of AKT1 which in turn prevented AKT1 Phosphorylation at S473 to dysregulate placental growth rather than at T308 which may have more strongly affected apoptosis. In contrast, while placentas of female fetuses have higher OGT levels under basal conditions, OGT levels are not affected by Cort exposure and so the phosphorylation status of AKT1 and 2 are similar between both treatment groups.Interestingly, we saw no effect of corticosterone treatment on phosphorylation of AKT1 at T308 in male placentae in response to stress. Maximal activation of AKT1 is regulated by phosphorylation at both T308 and S473 however studies have suggested distinct functions depending on which residue is phosphorylated. While AKT1 is phosphorylated at S473 by TORC2, DNA-PK and TORC1, phosphorylation at T308 is mediated by PDK1 and PIP3Nr3c1 mRNA expression4, it was somewhat surprising that GR protein levels were unaffected by prenatal Cort exposure. Previous studies similarly demonstrated that glucocorticoids can increase Nr3c1 mRNA but not GR protein in the fetal sheep kidney50. Given the complex nature of the Nr3c1 gene , this discrepancy warrants further investigation but likely indicates that Cort dysregulates placental GR in an isoform specific manner51. While classical signalling through the multiple isoforms of the GR can induce a variety of effects on both positive and negative GREs, additional regulation of glucocorticoid mediated cell signalling can be regulated by GR interactions with other protein. Indeed, many nuclear receptors are known to interact with other proteins and transcription factors such as NF-κB to inhibit their function in a process known as transrepression51. Recent in vitro studies have demonstrated that ligand bound GR interacts with OGT to potentiate GR transrepression pathways in a dose dependant manner26. To the best of our knowledge the current study demonstrates for the first time in an in vivo model, that there is a direct physical interaction between OGT and the GR. In contrast to the previously discussed in vitro studies, our results in placental tissue indicate that the degree of association between OGT and GR is governed by the availability of OGT. This is evident as female placentae have higher basal OGT levels than males and also demonstrate a 30% higher level of OGT/GR complex than male placentae. In addition, in female placentae exposed to Cort, there is no increase in OGT/GR complex formation compared with control placentae. An increase in GR ligand therefore, does not appear to increase complex formation in the placenta. In male placenta on the other hand, Cort exposure does increase OGT protein levels by approximately 40% which in turn increases the OGT/GR complex by approximately 70%.Given that we have previously demonstrated that Cort exposure leads to the immediate and prolonged upregulation of 52. Indeed, GR regulation of the transcriptional regulator NFκB has been shown to be at least partially mediated by the OGT/GR complex in vitro as evidenced by increased OGT induced O-GlcNAcylation and decreased phosphorylation of RNA polymerase II26. This may also occur in vivo whereby glucocorticoids that bind to the OGT/GR complex induce nuclear translocation and direct OGT activity to specific promoter regions, increasing O-GlcNacylation of transcriptional regulators and thereby suppressing transcription of target genes. The findings from the current study suggest that this O-GlcNacylation induced inhibition of gene transcription may be increased in response to Cort only in placenate of females. In contrast, Cort exposure increases the level of OGT/GR interaction but does not give rise to any increase in global protein O-GlcNacylation, perhaps due to insufficient OGT/GR complex upon initial Cort exposure and the requirement to synthesise additional OGT. As there are no basal differences in placental GR protein levels between sexes or between control and Cort exposed male placentae, there is likely to be GR protein which is not complexed with OGT in male placentae. Thus, Cort may bind to “free” GR (Hsp90 bound) to induce nuclear localisation and transcriptional regulation of genes by binding to glucocorticoid responsive elements or interacting with additional coactivator complexes53. This proposition is supported by our previous findings of increased expression of a number of genes, such as Igf2 and Vegfa in male but not female placentae upon exposure to Cort. In addition, the increased synthesis of OGT in male placentae in response to Cort, may account for the increased O-GlcNacylation of AKT which would also likely impact gene expression48. Although further studies would be useful to confirm subcellular localisation of the OGT/GR complex and O-GlcNac modification, it is clear that there are sex specific differences in OGT levels both basally and in response to Cort.Interestingly, despite similar levels of OGT/GR complex in control and Cort exposed placentae of females, there was a significant increase in global protein O-GlcNacylation in response to Cort in placentas of female but not male fetuses. Studies have demonstrated that proteins involved in mRNA processing can be O-GlcNacylated which may regulate transcriptional activityIn conclusion, the current study provides new insight into the mechanisms which underlie the sexually dimorphic placental responses to maternal stress. Our results confirm that placentae of female fetuses have two-fold OGT protein level compared with male placentae. This gives rise to differential complex formation with GR, global O-GlcNacylayion, AKT activity and gene expression, which negatively impacts male placental growth and morphology. This suggests that the higher basal levels of OGT potentiates the OGT/GR complex in female placentae which in turn affords protection by giving greater capacity to rapidly respond to elevated levels of maternal stress.28. Pumps were filled to capacity with Cort and primed for a 60-h infusion (E12.5–E15) at a rate of 33 μg/kg·h. The control group for this study was an untreated group not exposed to the stress resulting from anesthesia and surgery. Food and water intake were measured daily throughout pregnancy and extra care was taken to minimise all stress in both the Untr and Cort groups. On the 14th day of gestation (E14.5) dams were killed at 10am by cervical dislocation  and blood glucose concentrations immediately collected. Maternal livers were collected and Placentae, livers and kidneys collected from fetuses for analysis. We have previously reported the Cort concentrations of maternal plasma in these pregnant female mice4.All experiments were approved by The University of Queensland Anatomical Biosciences ethics committee. All experiments were conducted in accordance with the NHMRC guidelines and the Australian Code for the Care and use of Animals for Scientific Purposes. Briefly, Virgin C57BL/6 mice were time mated over a 3-h period with pregnancy being confirmed by the presence of seminal plugs and recorded as embryonic day 0.5 (E0.5). At E12.5, dams were anaesthetized for implantation of an osmotic pump as previously describedSry  as previously described4.Fetal tails were removed and incubated in lysis buffer with proteinase K overnight at 55 °C prior to heat inactivation. After DNA purification, this extract was analysed using Real-time PCR with primers designed for 4. Maternal liver mRNA levels were measured using Assays-on-Demand primer/probe sets  for Slc2a1 (Solute carrier family 2 (facilitated glucose transporter), member 1 (encodes for Glucose Transporter 1 (GLUT1)), Mm 00441473_m1), Pck1 , Slc2a2 (Solute carrier family 2 (facilitated glucose transporter), member 2 (encodes for Glucose Transporter 2 (GLUT2)), Mm 00446229_m1), Gfpt1  and Ogt (O-linked N-acetylglucosamine (GlcNAc) transferase, Mm00507317_m1).RNA was extracted from maternal livers (n = 6–7 per group), fetal livers (n = 9–11 per sex from 6–7 litters), fetal kidneys (n = 6–7 per sex) and placental quarters containing both junctional and labyrinth zone (n = 9–11 per sex from 6–7 litters) using the RNeasy mini kit . When more than one male or female was used per litter, all results were averaged such that only one value per sex was used per litter. RNA was treated with deoxyribonuclease 1, and reverse transcribed for qPCR analysis using 25 ng of cDNA per reactionHsp90aa1 , class A member 1, Mm00658568_gH) and Ogt were obtained. The gene expression of Hsp90aa1, Ogt and Hsf1  were measured in the fetal kidney. Placental mRNA levels were measured for Hsp90aa1, Hspa1a , Hsf1, Sp1 , Hsd11b1 , Ogt and Gfpt1. Real-time PCR results were analyzed using the delta delta cycle of threshold fluorescence method using the most appropriate endogenous controls for each tissue from either Rn18s (18s) or Actb. All groups were compared to the average of the male untreated animal group. All qPCR experiments were performed in triplicate.mRNA expression of key stress responsive genes were measured in both the fetal liver and kidney. Within the fetal liver, mRNA levels of Protein was extracted from maternal and fetal livers as well as placentae (n = 6 of each sex and treatment group) using commercial Cell Lysis buffer  supplemented with protease inhibitor cocktail (Sigma) but also additionally supplemented with sodium fluoride, sodium vanadate and sodium pyrophosphate and O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate , a nonselective inhibitor of O-GlcNAcase to maximize recovery of O-GlcNAc modified proteins. 20 μg of protein was loaded per well in 12% polyacrylamide gels and subjected to SDS-PAGE. Protein was transferred to Immobilon FL polyvinylidene difluoride membranes for Western blotting. After blocking, membranes were incubated overnight at 4 °C with primary antibodies seen in Table Ptrt) and sex (Psex) as factors and either Bonferroni or Tukey’s post hoc multiple comparison tests were carried out as appropriate. For levels of gene expression in maternal liver two-tailed student’s t tests were used between untreated control and corticosterone treated animals. Western blots were analysed using Student’s t tests between Control and Cort groups of the same sex or between male and female Control groups. For gene expression data, if more than one animal per litter were used in a group, data was analysed using litter averages such that n = 7 for both the Untr and Cort group per sex.Results are represented as the mean ± sem. Statistical analyses were performed using GraphPad Prism version 6 for Mac. Gene expression results were analysed by two-way ANOVA with treatment (Supplementary Information


Rubus plants of different geographic origins, cultivars, and cultivation systems, as well as in putative insect vectors like leafhoppers.Rubus stunt is an economically important disease in the production of raspberries, blackberries, and loganberries. A fast, sensitive, and reliable diagnosis of phytoplasmas, the causal agent of the disease, is of prime importance to stop its spread by vegetative propagation and by insect vectors. Therefore, multiplex qPCR assays using TaqMan probes with different kinds of fluorophores in one reaction were developed, allowing the detection of phytoplasmas in general as well as a more specific detection of phytoplasmas belonging to group 16SrV and host DNA (either plant or insect). This assay now provides a practical tool for the screening of motherplants and monitoring the presence and distribution of phytoplasmas in  Rubus species like raspberry (Rubus ideaeus L.), blackberry (Rubus fruticosus L.), loganberry (Rubus x loganobaccus), and European dewberry (Rubus caesius L.) they cause a disease referred to as Rubus stunt. Symptoms include stunted growth, shoot proliferation, small leaves, short internodes, enlarged sepals, phyllody, flower proliferation, as well as fruit malformations . In this case, the purified cloned pGEM-T Easy Vector (3015 + 149 bp) had a concentration of 1.24 × 10−7 g/μl. Hence, the number of copies was 3.59 × 1010 molecules/μl. Ten-fold serial dilutions from 1 × 109 to 1 × 105 molecules/μl of the purified plasmids were used to generate the standard curve. As the secY gene is a single copy gene in phytoplasma genomes [To generate a qPCR standard curve for the elm yellows phytoplasma specific primers and probe a PCR with primer pairs RuS-F02 and RuS-R02 was carried out with  genomes  the copyIn addition, the same serial dilution of plasmid DNA was run with 1 μl of a DNA extract (at 100 ng/μl nucleic acid) from an uninfected raspberry plant on the same qPCR plate as the standard curve, to evaluate the degree of interference of plant DNA extracts in the quantification of phytoplasma copy numbers. All samples were run in triplicate.Oligonucleotides were combined in a qPCR assay to detect DNA of elm yellows phytoplasmas , phytoplasma DNA in general , and either plant  or insect  host DNA as an internal control. Assays were run in 25 μl reactions using the KAPA PROBE FAST Master Mix (2X) Universal  on an iQ5 real-time thermal cycler , with an initial denaturation step of 20 sec at 95°C followed by 40 cycles with 3 sec denaturation at 95°C and 30 sec annealing and elongation at 60°C. Optimal final concentrations of primer and probe pairs were initially determined empirically and are shown in q) values of samples that were run both in singleplex and multiplex assays on the same 96-well plate. One validation assay was run for plant samples and one for insects. For the plant assay DNA from a Rubus stunt symptomatic infected raspberry plant was adjusted to 250 ng/μl of total nucleic acid and then used in six 10-fold (from 1 to 10−5) serial dilutions. Since there was no insect sample with DNA from Ca. Phytoplasma rubi available, an artificial reference sample was created by mixing DNA from a phytoplasma-free leafhopper (same sample as the one used as a negative control in the assays) with Ca. Phytoplasma rubi DNA from an infected periwinkle (Catharanthus roseus) plant in equal parts. This sample was adjusted to 200 ng/μl of total nucleic acid and was used in the same serial dilutions as the plant DNA sample. All samples were run in triplicate in qPCR assays with 2 μl of DNA solution.The multiplex assays were validated by comparing quantification cycle  were able to detect their respective targets along a 10-fold serial dilution gradient from undiluted DNA  up to a dilution of 10−3. The internal control for plant host DNA yielded positive results for all six 10-fold serial dilutions. There were no obvious differences in Cq-values between multiplex and singleplex assays  and U5/U3 (nested PCR), an amplification product of the expected size could be obtained up to a dilution of 10−4 . HoweverFor routine purposes in diagnostics of phytoplasmas, material of several asymptomatic plants (whether infected or not) is often mixed, resulting in different amounts of phytoplasma infected material. When the qPCR assay for plant samples was run with different proportions of infected and healthy leaf material , it was evident that it is possible to reliably detect phytoplasma DNA in samples containing only 1% of infected leaf material .When the assay was run with phytoplasma DNA from aster yellows (16SrI-B), ash yellows (16SrVII-A), Western X (16SrIII-A), elm yellows (16SrV-A), palatinate grapevine yellows (16SrV), flavescence dorée (16SrV), Rubus stunt (16SrV-E), and apple proliferation (16SrX-A), all were positive for phytoplasmas in general. A positive signal for the 16SrV specific primer-probe combination was only obtained for elm yellows, palatinate grapevine yellows, flavescence dorée, and Rubus stunt DNA .−4 .When the multiplex TaqMan assay was applied to insect DNA artificially mixed with phytoplasma DNA, all respective targets  could be detected up to a serial DNA dilution of 10−4 . Cq-valu−4, the same dilution as the multiplex TaqMan qPCR assay  obtained from 9 different commercial plantings throughout Germany were tested for the presence of phytoplasmas using both the newly developed multiplex TaqMan qPCR assay and the standard nested PCR assay. Some of the plants were showing distinctive symptoms pointing to a phytoplasma infection, however, for the majority of plants, these symptoms were not clearly visible . Of thesEuscelidius and six from leafhopper species identified as Macrosteles spp.In the course of monitoring putative Rubus stunt insect vectors in five different commercial raspberry and blackberry fields throughout Germany in 2014, 1293 individual phloem sucking insects were caught on raspberry and blackberry plants. After sorting and classification, 379 DNA samples of putative Rubus stunt insect vectors representing the full range of sampled locations, dates, and phloem sucking insects were extracted and analysed with the multiplex TaqMan qPCR assay as described above. Nine DNA samples were tested positive for the presence of phytoplasmas in general, but negative for Elm yellows phytoplasmas . Three oMalus domestica [Candidatus Phlomobacter fragariae, and plant DNA [Phytoplasmas are known as serious pathogens of a variety of commercial crop plants, with their diagnosis often being challenging due to unspecific symptoms, a long period of latency and low concentrations in sample tissues because they are limited to the phloem. Therefore, a fast, sensitive, and reliable diagnostic method is of prime importance to minimize their spread by insect vectors and by vegetative propagation. Nested PCR has been the most commonly used tool for the detection of phytoplasmas since the early 1990s ,32. Howeomestica ; aster yomestica ; flavescomestica ; stolburlant DNA ; phytopllant DNA ; phytopllant DNA ; Europealant DNA . Here, wlant DNA ,30,38, blant DNA . Accordilant DNA ,37. MoreRubus motherplants for the presence of both elm yellows phytoplasmas (to which Rubus stunt Ca. Phytoplasma rubi belongs) and phytoplasmas in general in nurseries or during plant propagation.The possibility of multiplexing by equipping TaqMan probes with different fluorogenic dyes allows detecting multiple targets in a single reaction. In the assays presented here, multiplexing was used not only to detect specifically elm yellows phytoplasmas and phytoplasmas in general at the same time, but also to include internal controls detecting either insect or plant host DNA. This enables the confirmation of a successful DNA extraction, and excludes false negative results resulting from a potential inhibition of the PCR. This, together with the lower risk of contamination due to detection of amplification products in a closed-tube system, makes the assays much more reliable than nested PCR. Furthermore, multiplex TaqMan assays are more time-saving than nested PCR because there is no need for post-PCR processing, and only one round of PCR has to be carried out. Using the KAPA PROBE FAST Master Mix as described here, once PCR reactions are fully set up on a 96-well plate, about 1 hour is needed until results can be obtained. In comparison, with nested PCR approximately about 6–8 hours are needed to get results from the same number of samples. In addition, the results obtained here using different proportions of phytoplasma infected and healthy leaf material showed that it can be a valid option for plant nurseries to pool multiple leaf samples of different plants while still being able to reliably detect phytoplasmas and thus saving even more time and resources. Accordingly, the assays developed in this study provide a rapid and practical tool for screening of Euscelidius and Macrosteles who apparently had previously sucked on phytoplasma infected plants were present in commercial Rubus plantings. Both species are known as vectors of phytoplasmas [Rubus species. In any case, the multiplex qPCR TaqMan assay developed in the present study will now allow a quick and reliable identification of phytoplasma insect vectors in orchards. Since measurements targeting insect vectors of phytoplasmas are the only options for managing phytoplasmas and particularly for preventing their spread within and among orchards, a detailed knowledge of respective vectors is a prerequisite for improvement of plant protection strategies against phytoplasma diseases.Screening of putative insect vectors showed that leafhoppers of the genus oplasmas ,40, howeS1 Fig(AM397299) Rubus stunt 16SrV-E, (AY197686) flavescence dorée 16SrV, (AY197690) elm yellows 16SrV-A, (GU004329) ash yellows 16SrVII-A, (GU004354) Western X 16SrIII-A, (GU004335) apple proliferation 16SrX-A, and (AY803177) aster yellows 16SrI-B.(TIF)Click here for additional data file.S1 TableLocations used for sampling as well as tissue types are presented. The same DNA extract was used for qPCR and nested PCR analysis.(XLSX)Click here for additional data file.S2 TableIndividual insect samples with positive qPCR results for the presence of phytoplasmas are shaded in grey.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 TableEach leaf sample was extracted in 3 independent replicates (designated as 1–3) and each DNA extract was analysed in triplicate in the qPCR.(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold’s regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cellremoving detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology.molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.Preservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules  during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques  are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the  The CHP is designed to have a strong propensity to fold into a triple helix, a unique super-secondary protein structure that is nearly exclusively found in collagen. A CHP strand can readily bind to unfolded collagen chains by forming a hybridized triple helix through inter-strand hydrogen bonds, in a fashion analogous to primers binding to DNA strands during PCR  and the sequence-scrambled control peptide  were synthesized using standard solid-phase Fmoc and HBTU chemistry as described previously  was synthesized, purified and heated at 80 °C for 5 min followed by addition to a solution of citrate-stabilized gold nanoparticle (NP), as described previously [The CHP with an N-terminal acetylated cysteine [C-GGG-(GPO)eviously . The mixvia gamma irradiation at a dose of 2 × 106 RADS.Porcine urinary bladder matrix was prepared according to previous reported methods ,43. BrieHealthy porcine medial cruciate ligaments were a kind gift from the Jeffrey Weiss group (University of Utah). Ligaments were frozen in optimal cutting temperature (OCT) compound and cut to 5 μm thick sections which were mounted on glass slides. Before treating with detergents, the OCT compound was removed by two rounds of 5 min rinsing in 1 × PBS. All detergents were dissolved in deionized water to the predetermined weight/volume concentrations. Tissue slides were completely immersed in detergent solutions and incubated at room temperature overnight. Slides were rinsed with deionized water twice and with 1 × PBS three times to remove leftover detergents. Because CHP can self-assemble into homotrimers in solution during storage at 4 °C leaving no driving force to hybridize with unfolded collagen chains, in order to stain denatured collagen in tissue sections, CHP needs to be dissociated to monomers by heating immediately prior to use. A solution containing 15 μM of CF-CHP in 1 × PBS were heated at 80 °C for 5 min, and immediately cooled down to room temperature in an ice/water bath. One hundred microliter of the quenched CF-CHP solution was quickly added onto each tissue section. In this way, most CHP peptides were expected to remain as active monomers during the staining process, because CHP trimerization follows a third order reaction rate and takes several hours to complete at the concentration of 15 μM , based oThe UBM samples were flash frozen in OCT compound and sectioned. The sections were blocked with 10% goat serum in 1 × PBS for 30 min at room temperature, and treated with 15 μM of CF-CHP as described above. An anti-collagen I antibody  was diluted (1:500) into the quenched CF-CHP solution before the mixture was added to the sections. The slides were incubated in a humidity chamber overnight at 4 °C, and rinsed in 1 × PBS for 5 min three times. Afterwards, the sections were stained by Alexa Fluor555 labeled donkey anti-rabbit IgG H&L  for 1 h at room temperature, rinsed and mounted.Ligament tissue sections treated with predetermined concentrations of SDS were scanned using an automated Nikon wide-field fluorescence microscope . The sections of UBM stained with CF-CHP and anti-collagen I antibody were imaged by an EVOS FL auto cell imaging system (Thermo Fisher Scientific) with a 20 × objective lens (GFP light cube for CF-CHP and RFP light cube for anti-collagen I antibody). Images within each experiment were obtained under identical microscope settings  to allow direct comparison.The detergent-treated ligament sections were imaged by a custom Prairie View Ultima multiphoton microscope (Brucker). A single excitation wavelength at 800 nm was used to simultaneously visualize collagen fibers and CF-CHP binding in the tissue. The resulting SHG and CF-fluorescence signals were detected at 435–485 nm and 500–550 nm respectively. Images were scanned with Z-stack for the entire 5 μm section depth. Images were acquired from three different locations within one section from each treatment category. Images for both SHG and CF-fluorescence were analyzed in ImageJ software using measurements of the mean intensity and area of all remaining pixels after a background subtraction. The signal quantification was expressed as integrated intensity (the mean intensity times area). The image of the heat denatured ligament section was not included in the CF-fluorescence quantification because of over saturated intensity.The collagen-detergent mixture solutions were freshly prepared from acid solubilized collagen to minimize potential collagen fibril self-assembly and to allow direct monitoring of the effect of a detergent on individual collagen molecules. Rat tail, type I collagen  in blank and four different detergent solutions were freshly prepared immediately prior to the CD measurement . CD spectra (200–260 nm) were recorded on a JASCO J-1500 CD spectrometer at 22 °C with a scanning speed of 20 nm/min, a data integration time of 16 s, and a bandwidth of 5 nm. After heating all solutions up to 65 °C, the spectra of the denatured samples were measured again at 22 °C.Proteins in detergent solutions can aggregate and interfere with CD measurements. In this experiment, the combinations of detergent and buffer shown above were carefully chosen for minimal background CD signals. Unfortunately, we were unable to find a condition that provides a noise-free CD spectrum for collagen solutions containing 3% Triton X-100.A neutralized solution containing 0.6 mg/mL of rat tail, type I collagen was prepared by diluting the acidic stock solution  in 1 × PBS. The neutralized collagen solution was incubated at room temperature under magnetic stirring for 2 h to allow collagen fibril self-assembly. Subsequently, the neutralized collagen solution was aliquoted into four vials, and 1 × PBS buffer solutions containing four different detergents at 2 × concentrations were added into individual vials to make four final solutions . The collagen fibrils were incubated with detergents under magnetic stirring at room temperature for 3 h to allow detergent-fibril interaction. The collagen fibrils assembled in the detergent-free PBS solution were denatured in 80 °C water bath for 1 min as a control sample. TEM samples were prepared by adding a drop of a collagen-detergent mixture solution onto a copper grid covered with a thin carbon film (EMS). The excess solution was wicked off with a piece of blotting paper. The samples were air dried at room temperature. To visualize unfolded collagen molecules under TEM, a drop of CHP-NP (10 nM) solution containing 2% bovine serum albumin was applied to the grid. After 10 min of incubation at room temperature, the grid was washed with deionized water three times. Finally, the samples were stained with a drop of 2% uranyl acetate (w/v) and air-dried at room temperature. TEM images were collected on a FEI Tecnai T12 microscope operated at 120 kV, and processed by a Gatan digital micrograph software. The collagen-bound nan-oparticles in the TEM images were carefully identified and manually marked as red dots using the overlay brush tool in ImageJ.t-tests. When there were more than one pair of comparison in an experiment, the t-tests were performed with the Benjamini-Hochberg correction for multiple comparisons with a significance  of 0.05 [Unless indicated, all experiments were repeated three times. All quantitative results are shown as means + standard error. All statistical analyses were performed using Kaleidagraph 4.0 (Synergy Software). Sample groups were compared using  of 0.05 .SDS is a well-known protein denaturant commonly used in electrophoresis. It is also one of the most widely utilized detergents for tissue decellularization, often in the concentration range of 0.1% to 1% (w/v), and its disrupting effect on the ECM has been recognized . We enviWe selected four detergents  with representative concentrations (w/v%) because of their frequent use in the decellularization process ,25,48,49fibrillar organization of collagen in the tissue while leaving the collagen molecules intact. This result was unexpected, since previous studies using SEM and SHG indicated that CHAPS and SD clearly disrupt collagen structure in the decellularized tissues [To corroborate our findings, we conducted second harmonic generation imaging, a classic technique that can interrogate the collagen fiber integrity, in conjunction with the fluorescence microscopy. The ligament samples were imaged using multiphoton confocal microscopy under a single excitation wavelength, acquiring the collagen fiber’s SHG signal on one channel, and the CF-CHP’s two-photon excitation fluorescence (TPEF) signal on another channel simultaneously. Comparing the stacked SHG micrographs and their quantified signals acquired from the same location within each tissue section , we were tissues . To confTo verify the intact structure of collagen molecules in 8 mM CHAPS and 4% SD, we acquired the CD spectra of collagen molecules in the detergent solutions , since CTo verify the disruption to the fibrillar structure, we analyzed the morphology of the collagen fibrils (reconstituted and assembled from acid-soluble type I collagen) after they were mixed with each of the four detergents . The repTo verify whether these detergent-modified collagen structures contain denatured collagen molecules, we probed the TEM samples with gold nanoparticles that have CHPs on their surface (designed as CHP-NP). Previously using TEM, we demonstrated that CHP-NPs bind to both thermally denatured  and mechFinally, we conducted a field test of staining detergent treated UBM samples with CF-CHP in order to detect denatured collagen. Four pieces of tissues from the same porcine urinary bladder were decellularized by each of the four detergents (see Methods), cryosectioned, and stained by the CF-CHP. The levels of cell removal by the four detergents have been evaluated by dsDNA quantification and mass spectroscopy in the previous studies ,43. HereIn this study, CHP was used to investigate the denaturation of triple helical collagen molecules in ECM-based tissue scaffolds after they were decellularized by common detergent treatment protocols. The reduced SHG signals  and non-The protein-denaturing power of detergents depends on a combination of structural factors such as charge density of hydrophilic groups, size and rigidity of hydrophobic groups, as well as specific interactions between detergents and proteins –53. The in vivo digestion of the decellularized ECM have potent bioactivities and contribute to the tissue repair processes [Cells can readily recognize denaturation of the collagen triple helix. Many cell receptors, such as DDR1, DDR2, and integrins α2β1, α1β1, α10β1, α11β1, only bind to sites on triple helical collagen and not the denatured collagen chains . These rrocesses . The denOur study provides an opportunity to reconcile the conflicting literature reports about the denaturing effect of common decellularization agents. For example, a study by Gratzer and coworkers showed that 1% SDS disrupted collagen in porcine anterior cruciate ligament and altered its mechanical strength , while aCHP staining is a reliable and highly sensitive method to examine the unfolding of collagen molecules in decellularized ECM, and it has many advantages over conventional methods. The mechanical or trypsin digestion method involves too many error-prone steps that undermine the detection sensitivity and accuracy as can be seen in the above-mentioned example. Microscopic methods such as SEM and TEM are not quantitative. Moreover, as demonstrated in our previous SEM work on gelatin, type I collagen, and their mixtures, highly denatured collagen (gelatin) are invisible on SEM . In contWe characterized the molecular denaturation of collagen in tissues as a result of common detergent treatment . The study was made possible by a novel collagen hybridizing peptide that specifically anneals to the unfolded collagen chains through triple helix formation. By staining tissue samples with fluorescently labeled CHP and subsequent quantitative image analysis, we showed that while all four detergents alter the fibrillar structure of collagen, only SDS and Triton X-100 have the propensity to denature the collagen molecules. The results were consistent in all test samples: self-assembled type I collagen fibrils, decellularized porcine ligament, and urinary bladder matrix. The results were also in agreement with other characterization methods, including SHG, CD and TEM. The triple helix is the most fundamental structural motif of collagen, and its integrity, which is known to affect cellular responses, is directly assessed in the decellularized ECM in our study. There are growing clinical interests in the use of decellularized biologic scaffolds for healing and regeneration of damaged tissues. The CHP technique introduced herein can help identify one of the key variables in the decellularization process — the molecular level destruction of collagen, and is expected to bring us one step closer to the understanding of how the decellularization process results in structural changes in the ECM and how that change influence the functional outcome of the ECM scaffolds. These new insights will ultimately lead to rationally optimized tissue decellularization protocols for specific regenerative applications.


The leucine- and valine-raising allele was not associated with AD (p = 0.46). These data suggest that a genetic predisposition to raised plasma isoleucine levels is positively associated with AD.We conducted a two-sample Mendelian randomization study to test the hypothesis that raised plasma levels of the branched-chain amino acids isoleucine, leucine, and valine are associated with Alzheimer’s disease (AD). From a genome-wide association study of 16,596 individuals of European ancestry, we obtained summary statistics for four independent single nucleotide polymorphisms (SNPs) associated with isoleucine levels and one SNP associated with both leucine and valine levels at genome-wide significance. Summary statistics of the associations of the five SNPs with AD were obtained from the International Genomics of Alzheimer’s Project . Based on four SNPs, the odds ratio of AD per genetically predicted one standard deviation higher isoleucine levels was 1.35 (95% CI, 1.08–1.69;  The neuronal damage that leads to AD may in part be caused by toxic effect of Aβ peptides or alterations in Aβ processing1. Thus, modifiable environmental factors or medical therapies that modify the accumulation of amyloid plaques or hippocampal neurogenesis may influence AD development.Alzheimer’s disease (AD) is a neurodegenerative disease characterized by synaptic loss and neuronal cell death, primarily in hippocampus, and manifests as progressive memory loss and cognitive decline. The pathological hallmarks of AD are amyloid plaques containing deposits of amyloid-β (Aβ) peptides, and neurofibrillary tangles containing hyperphosphorylated tau protein5 and for enhancing neurogenesis8. It has been observed that AD patients have decreased serotonin receptor levels12 and that activation of serotonin receptors decreases Aβ production13 and brain Aβ levels5 and increases neuronal survival2. There are also data showing that selective serotonin reuptake inhibitors, a class of antidepressants that increase serotonin levels, reduce brain Aβ levels4 and stimulate neurogenesis in hippocampus8.The neurotransmitter serotonin is a candidate for reducing amyloid plaque load16. Long-term raised circulating BCAA levels could conceivably lead to decreased brain tryptophan levels as well as reduced neuronal serotonin synthesis and serotonergic signaling17  isoleucine, leucine, and valine are essential amino acids that are obtained from the diet through protein-containing foods. Uptake of BCAAs into the brain occurs at the blood-brain barrier through a competitive transport carrier that they share with other large neutral amino acids, including tryptophan, the precursor of serotonin18. The aim of the present study was to use MR to test the hypothesis that plasma BCAA levels are positively associated with AD.Mendelian randomization (MR) is a genetic epidemiologic approach that utilizes genetic variants associated with the modifiable exposure  as proxies for the exposure to evaluate whether the exposure is causally associated with the outcome . This method reduces some of the crucial limitations of observational studies, including confounding and reverse causation bias. A recent genome-wide association study (GWAS) identified several genetic variants that are associated with plasma BCAA levelsp < 5 × 10−8)18. We selected the lead SNP from each of these genomic regions. Five independent SNPs (R2 < 0.03) were associated with isoleucine levels. These SNPs were located near PPM1K (rs7678928), DDX19A (rs75950518), TRMT61A (rs58101275), CBLN1 (rs1420601), and GCKR (rs1260326). Only one SNP (rs1440581), near the PPM1K gene, was associated with leucine and valine levels at genome-wide significance. To investigate whether the BCAA-associated SNPs had pleiotropic associations with other phenotypes, we searched PhenoScanner, which is a publicly available GWAS database19.A GWAS of 16,596 individuals of European ancestry identified five independent genomic regions with single-nucleotide polymorphisms (SNPs) associated with plasma BCAA levels at genome wide significance level , the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium (CHARGE), the European Alzheimer’s disease Initiative (EADI), and the Genetic and Environmental Risk in AD consortium (GERAD)], including a total of 17,008 cases of AD and 37,154 controls. All studies included in IGAP had been approved by a relevant Institutional Review Board. Informed consent was obtained from participants themselves or, for those with considerable cognitive impairment, from a caregiver, legal guardian, or other proxy.Summary statistics (beta coefficients and standard errors) for the associations of the BCAA-related SNPs with AD were obtained from the International Genomics of Alzheimer’s Project (IGAP)21. To explore and adjust for pleiotropy, sensitivity analyses were performed using the weighted median and MR-Egger regression methods21. Results are reported as odds ratios (OR) with their 95% confidence intervals (CI) of AD per genetically predicted 1 standard deviation (SD) increase in plasma isoleucine, leucine, and valine levels. All statistical tests were two-sided. We applied a Bonferroni-corrected significance threshold for analyses of five BCAA-raising SNPs (p = 0.05/5 = 0.01). Tests were otherwise considered statistically significant at p < 0.05. The statistical analyses were conducted using Stata, version 14.2  and R (R Foundation), version 3.3.3.An instrumental variable was constructed for each BCAA-associated SNP by dividing the SNP’s effect (beta coefficient) on AD by the corresponding effect on BCAA levels. The instrumental variable estimates for isoleucine were combined using a fixed-effect inverse-variance weighted meta-analysisAll data generated or analyzed during this study are included in this published article and its supplementary information file.GCKR gene) had strong pleiotropic associations with numerous phenotypes (Table p = 0.007), without heterogeneity among estimates from different SNPs  . The MR-Egger analysis showed no evidence of pleiotropy  but the causal estimate from this analysis was very imprecise and the CI included the null . The leucine- and valine-raising allele was not significantly associated with AD (p = 0.46)  Table . The ORsThis MR analysis showed that a genetic predisposition to higher plasma isoleucine levels was positively associated with AD. This finding suggests that lifelong raised isoleucine levels may increase the risk of AD. Genetically predicted higher levels of leucine and valine, based on a single genetic variant, were not associated with AD.22. This effect reflects the competitive nature of the blood-brain barrier large neutral amino acid transporter, which is almost fully saturated at normal plasma levels of these amino acids22. Hence, the observed association of genetically raised isoleucine levels with AD may be mediated by decreased brain serotonin levels and diminished serotonin signaling, potentially leading to higher amyloid plaque load5 and decreased neuronal survival2 and adult hippocampal neurogenesis8. Experimental data have shown that dietary BCAA supplementation leads to lowered hippocampal levels of nerve growth factor, which is involved in growth and survival of cholinergic neurons23. High BCAA levels also result in reduced brain uptake of tyrosine and its conversion to the catecholamine neurotransmitters dopamine and norepinephrine22, but their role in AD is uncertain.It is well known that an increase in circulating BCAA levels leads to decreased brain levels of tryptophan and its conversion to serotonin Fig. 15,16,22.GCKR gene. The genetic risk scores for isoleucine, leucine and valine were specifically associated with the three BCAAs and not with any of 172 other metabolites measured18, suggesting that pleiotropic effects with other metabolites unlikely explain our findings. We also found no evidence of pleiotropy in sensitivity analyses using the weighted median and MR-Egger methods. However, the MR-Egger method had low statistical power and could not reliably detect pleiotropy. The genetic variants were weakly associated, at nominal statistical significance, with some diseases and disorders, most notably psychiatric and mood disorders that are related to impaired serotonin signaling, such as schizophrenia, bipolar disorders, and major depressive disorders. This observation reinforces the hypothesis that genetically raised BCAA levels might increase AD risk by modifying the serotonergic system.A strength of the MR approach is that potential confounding from other modifiable risk factors were reduced by the use of genetic variants as proxies for BCAA exposure. However, we cannot rule out the possibility that the BCAA-associated genetic variants may affect the risk of AD through pathways other than through BCAA levels. For this reason, we excluded the pleiotropic variant near the A limitation of this study is that our genetic instrument for leucine and valine comprised only one genetic variant, leading to low statistical power to detect an association. Although the odds ratio estimates for the leucine- and valine-raising allele was in the same direction as for isoleucine, the precision was low and the associations were not significant. Likewise, none of the individual SNPs associated with isoleucine levels was significantly associated with AD at the Bonferroni-corrected significance level.In conclusion, using data from a large genetic consortium on AD, we found that a genetic predisposition to raised plasma isoleucine levels was associated with AD. The role of BCAAs in the development of AD merits further investigation.Supplementary Material


Thalassia testudinum (turtle grass) is the dominant and climax-successional seagrass species in the subtropical/tropical Atlantic and Caribbean region. Two die-offs of T. testudinum in Florida Bay, United States have raised concerns regarding the resilience of this species to environmental disturbances. Seedlings are important in recovery of T. testudinum, following disturbance events. Leaf spectral reflectance [R(λ)] was measured in T. testudinum seedlings exposed for 2 weeks to three salinities  and two light levels (full sun and 50–70% light reduction) in experimental mesocosms. Multivariate analyses indicated that hypersalinity had a greater effect on spectral reflectance than hyposalinity or light reduction. There was an increase in variability and flattening of reflectance spectra at the highest salinity. All three salinity treatments had distinct reflectance spectra across green wavelengths (530–580 nm), with additional discrimination between 20 versus 50 and 35 versus 50 treatments across red wavelengths (630–690 nm). Red:Green reflectance ratios were highest and photochemical reflective index values were lowest for the salinity 50 treatment, but were not significantly different between the salinity 20 and 35 treatments. The changes in the R(λ) spectra for the salinity 50 seedlings were consistent with previously observed reductions in leaf pigments and maximum photochemical efficiency of photosystem II. These observations indicate that leaf spectral reflectance is a sensitive indicator of plant stress in T. testudinum seedlings and that seedlings are more sensitive to short-term exposures to hypersalinity than hyposalinity. Thalassia testudinum Banks ex König (turtle grass) is the dominant seagrass species in the subtropical/tropical Atlantic and Caribbean region  was initiated to increase freshwater inflow to Florida Bay back to historical patterns . DecreasT. testudinum, following disturbance events  under hypersaline , but not hyposaline  conditions, suggesting a greater sensitivity to high-salinity stress  compared to the salinity 20 and 35 treatment seedlings, while shading resulted in increases in chlorophyll b resulting in significant reductions in chlorophyll a:b ratios ] is a non-destructive optical technique that has long been used as an indicator of plant stress (R(λ) is an apparent bio-optical property that varies largely in response to changes in leaf pigments (R(λ) may correspond with changes in relative photosynthetic efficiency and capacity under various environmental conditions (a to b ratio (R(λ) is rapid and can be applied across multiple spatial scales  and two light treatments (full sun and 50–70% reduction from ambient light). Based on our previous observations on photochemical efficiency and pigment contents (R(λ) would increase in response to hypersalinity, but decrease under shaded conditions.The measurement of spectral reflectance  is the fraction (%) of downwelling plane irradiance that reflects upward as a function of wavelength (λ) [R(λ) = Eu(λ)/Ed(λ)]. Reflectance measurements employed an Ocean Optics USB2000 fiber optic spectrometer coupled to a 400 μm diameter UV-VIS reflection probe (Ocean Optics R400-7-UV-VIS). To measure upwelling irradiance [Eu(λ)], the probe was positioned 0.5 cm above the surface of each rank 2 leaf at a 45o zenith angle using a clear lexan leaf clip. Immediately following the upwelling irradiance measurement, downwelling irradiance [Ed(λ)] was measured using a Spectralon diffuse reflectance target (Ocean Optics WS-1) placed adjacent to, and in the same plane as, the leaf. It was assumed that the Spectralon target is Lambertian . Ten spectra per sample were averaged and then smoothed with a 5 nm boxcar filter window to ensure an optimal signal-to-noise ratio and preserve the spectral features , which provides an effective estimate of the ratio of anthocyanins to chlorophyll (R531-R570)/(R531+R570)], which is an indicator of epoxidation of xanthophylls cycle pigments and photochemical efficiency ficiency . Among-ta and b . Reflectance was relatively low and constant from 400 to 500 nm then increased toward a peak around 550 nm followed by a decrease to another low around 675 nm and then rapidly increasing to 700 nm. There was an increase in variability among spectra as salinity increased  and a flattening of spectral reflectance at the highest salinity . Shade-treatment seedlings generally exhibited higher reflectance between 525 to 575 nm and lower reflectance around 675 nm compared to the unshaded (Sun) treatment.Normalized reflectance spectra for the 20 and 35 salinity treatment leaves were typical of green vascular plant leaves containing chlorophylls Figure 4). There was clear separation of the hypersalinity treatment seedlings along the primary ordination axis while most of the lower two salinity treatment samples were clustered. All of the Sun 50 replicates and 3 of the 4 Shade 50 replicates are separated from the other treatments on the right side of the ordination plot. Two-way crossed ANOSIM indicated that reflectance spectra for Sun and Shaded treatments were not significantly different . In contrast, salinity had a significant effect on spectral reflectance , with both the salinity 20  and 35  treatments being significantly different from the salinity 50 treatment, but not different from each other .Multivariate (nMDS) ordination indicated that hypersalinity had a greater effect on spectral reflectance than hyposalinity or shading . Consistent differences among all three salinity treatments occurred across the green wavelengths (530–580 nm), with additional discrimination between 20 versus 50 and 35 versus 50 treatments in red wavelengths (630–690 nm). The Red:Green reflectance ratio was significantly affected by salinity , being highest in the salinity 50 treatment and not significantly different between the salinity 20 and 35 treatments . The photochemical reflectance index (PRI) was also significantly affected by salinity  being lowest in the salinity 50 treatment and similar between the salinity 20 and 35 treatments .Similarity percentage analysis of the salinity data with light treatments pooled indicated that no single wavelength contributed more than 1% to the dissimilarity among salinity treatment reflectance spectra (R(λ)] for T. testudinum seedlings exhibited a greater response to increased salinity than to low salinity or light reduction, after 2 weeks exposure. Despite a 50–70% reduction in ambient irradiance, daily mean PAR quantum flux for the Shade treatments was approximately 8 mol quanta m-2 day-1, which exceeds the daily light requirements estimated for tropical seagrasses (R(λ) in the Shade treatments exhibited only slight increases in the green wavelengths and small decreases in the red wavelengths. These changes are consistent with the small, but non-significant increases in chlorophyll a and b contents in the shaded seedlings relative to the full sun treatments (R(λ) spectra, with reduced reflectance in the green and increased reflectance in the red. This resulted in a significant increase in the Red:Green ratios in the hypersalinity treatment relative to the control and hyposalinity treatments. Increased Red:Green ratios generally correlate with elevated anthocyanin contents (T. testudinum is associated with high-light environments (T. testudinum shoots have shorter and narrower leaves than green-leafed shoots and higher effective quantum yields (R(λ) spectra and the two reflectance indices for the salinity 50 seedlings were consistent with the reduced leaf chlorophyll and carotenoid contents in these leaves  spectra, Red:Green ratios and PRI values for the salinity 20 and 35 treatment seedlings were not significantly different, reflecting similar pigments contents and photochemical efficiencies of the leaves in these two treatments (R(λ) spectra for T. testudinum after 24 h exposure to hyposaline  conditions  reported high mortality in both hyper- and hypo-salinity treatments (After 14 days at target salinities, eatments ,b. Our rnditions . These deatments . In conteatments . Thus, tT. testudinum seedlings. The results of multivariate analyses of reflectance spectra from 400 to 700 nm and statistical comparisons of two reflectance indices all suggested greater sensitivity of seedlings to hypersalinity than to hyposalinity. These results are highly consistent with previous measurements of chlorophyll fluorescence (T. testudinum in Florida Bay (Our observations indicate that leaf spectral reflectance is a sensitive indicator of plant stress in rescence  and pigmrescence . The occrida Bay . Our datMD and JH together designed and executed the research project. MD and JH conducted the spectral reflectance measurements. MD lead the reflectance data analysis and drafted the manuscript with the assistance of JH. All co-authors commented on and approved the final manuscript draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glucocorticoid replacement therapy is the mainstay of treatment for congenital adrenal hyperplasia (CAH) but has a narrow therapeutic index and dose optimisation is challenging. Metabolomic profiling was carried out on plasma samples from 117 adults with 21-hydroxylase deficiency receiving their usual glucocorticoid replacement therapy who were part of the CaHASE study. Samples were profiled by using hydrophilic interaction chromatography with high resolution mass spectrometry. The patients were also profiled using nine routine clinical measures. The data were modelled by using both multivariate and univariate statistics by using the clinical metadata to inform the choice of patient groupings. Comparison of 382 metabolites amongst groups receiving different glucocorticoid doses revealed a clear distinction between patients receiving ≤5 mg (n = 64) and >5 mg (n = 53) daily prednisolone-equivalent doses. The 24 metabolites which were statistically significantly different between groups included free fatty acids, bile acids, and amino acid metabolites. Using 7 metabolites improved the receiver operating characteristic with area under the curve for predicting glucocorticoid dose of >0.9 with FDR adjusted P values in the range 3.3 E-04 -1.9 E-10. A combination of seven plasma metabolite biomarkers readily discriminates supraphysiological glucocorticoid replacement doses in patients with CAH. Glucocorticoids are also employed commonly in a variety of inflammatory diseases such as rheumatoid arthritis, obstructive lung diseases, and asthma3. Although highly efficacious, treatment with glucocorticoids is generally associated with adverse effects such as obesity, hyperglycaemia, hypertension, cardiovascular disease4 and osteoporosis5, and in children, retarded linear growth. These dose-related adverse effects are observed even amongst CAH patients when the goal is physiological replacement rather than pharmacological anti-inflammatory therapy8. The efficacy of glucocorticoid therapy can be assessed with disease-related endpoints, including adrenal androgen levels in CAH. However, given the narrow therapeutic index, objective monitoring of glucocorticoid toxicity would also be valuable to assist with dose optimisation; unfortunately, the pharmacokinetics of oral glucocorticoids preclude maintenance of blood steroid concentrations within physiological reference ranges, and no sensitive pharmacodynamic biomarkers exist with which to assess glucocorticoid toxicity.Glucocorticoid replacement therapy is the mainstay of treatment for congenital adrenal hyperplasia (CAH)9. The aim of this study was to employ metabolomics in plasma samples which were available from patients with CAH10 firstly to establish whether the metabolomics profile varies across the range of glucocorticoid replacement regimes employed in these patients, and secondly to identify metabolites which might be useful for monitoring glucocorticoid toxicity.Metabolomic screening has previously been applied to glucocorticoid therapy only for inflammation using urine biomarkersIn order to examine relationships between glucocorticoid dose and metabolomic profiles, patients were grouped by their daily dose; 1) 1–2.5 mg, (2) >2.5–5 mg, (3) >5–7.5 mg and (4) >7.5–15 mg prednisolone equivalents 15 and might be expected to reduce levels of tryptophan and its metabolite N-methylnicotinamide but this is not observed in the current case. TDO has haem at its active centre and enzyme activity is regenerated by coupling with the superoxide anion16, since a major source of superoxide is from xanthine oxidase, which converts hypoxanthine via xanthine to uric acid16, the elevated hypoxanthine and inosine in the high GC group could indicate inhibition of xanthine oxidase and thus possibly reduced TDO activity. Palmitoleic acid has been used as a plasma marker of stearoyl CoA desaturase (SCD) activity which is required for the secretion of triglycerides by the liver17, lower levels, and desaturation of C16:0 to C16:1, in the high GC group are consistent with glucocorticoid inhibition of SCD and induction of fatty liver disease18.The glucocorticoid dose-related biomarkers were plausibly associated with glucocorticoid action. Chenodeoxycholic acid is representative of bile acid biosynthesis, which is both regulated by glucocorticoids and may influence glucocorticoid metabolism19, tyrosine and phenylalanine were increased in patients receiving high GC dose. The bacterial-derived metabolite 4-Hydroxy-2-oxopentanoate was also higher with insulin resistance19. In the current study this metabolite also increases with glucocorticoid dose. In our study C15:0, C16:0, C20:3 and C22:6 fatty acids were all elevated in patients receiving high GC dose while C13:0 and C16:1 fatty acids were reduced  has a strong positive association with type 2 diabetes, although the odd chain pentadecanoic acid (C15:0) has an inverse association with type 2 diabetes21. Urinary excretion of N-methylnicotinamide (NMN), a metabolite of tryptophan which is increased with high GC dose in the current study, has been found to be elevated in type 2 diabetes along with its metabolites the N-methyl pyridine carboxamides, and knock down of nicotinamide N-methyl transferase protects against obesity22. Patients with impaired glucose tolerance (IGT) have reduced levels of phenylacetyl-glutamine and increased levels of acylcarnitines and α-ketoglutarate, a pattern indicative of TCA cycle intermediate depletion which interferes with insulin action23, as well as reduced tryptophan, xanthine, methionine and nucleotides; patients with diabetes also have a higher plasma level of octanoylcarnitine compared to non-diabetic individuals24. We found these diabetes-related metabolites to be altered with glucocorticoid dose  and hypoxanthine as observed in the current study. The two studies are not entirely comparable since in the previous study a high and a low dose of corticosteroid was used rather than a gradation of doses as in the current case. What is absent in the current case is a clear effect on branched chain amino acids which in the previous study were elevated by increased HC dose. These metabolites are also established markers of a pre-diabetic state25 but are not highlighted as important markers in the current study. The value of a multivariate statistical approach is confirmed in the current study, particularly since the metabolite markers are not normally distributed, and the final OPLSDA model is very strong considering that the seven biomarkers can be used to largely distinguish between the two groups in this large co-hort.This observational study cannot distinguish metabolites which are directly affected by glucocorticoids from those which are indirectly affected, for example by the documented differences in body composition with variation in glucocorticoid dose, or by differences in efficacy of suppression of adrenal androgensExperimental details for sample preparation and analysis are given in supplementary material along with details for data extraction and metabolite identification.10. All participants gave written informed consent. All methods were performed in accordance with the relevant guidelines determined by the protocols approved by the ethics committee. This study was not a clinical trial but was an observational clinical study and therefore is not categorised as a clinical trial and is not registrable as one.The UK Congenital adrenal Hyperplasia Adult Study Executive (CaHASE) cohort is a cross-sectional study of adult CAH patients (aged ≥18 years) recruited from 17 specialized endocrine centres across the UK. The study protocol was approved by West Midlands research ethics committee (MREC/03/7/086) and registered with ClinicalTrials.gov (NCT00749593) and has been previously published in detail26.Participants attended the research unit of their respective centre after an overnight fast having taken their regular medication, followed by medical history, physical examination  and blood sampling (including for 17-hydroxyprogesterone (17OHP), androstenedione). All laboratories participate in the UK NEQAS scheme for quality control of steroid assays. Inclusion criteria for the metabolomics analysis were as follows: known 21-hydroxylase deficiency; additional serum sample collected at time of recruitment; full anthropometric and biochemical data available for each participant. Samples from 117 patients were used for metabolomics analysis; subjects were treated with hydrocortisone, prednisolone and dexamethasone or combination therapy. Glucocorticoid therapies were converted to daily prednisolone equivalents based on the relative potencies of the steroids reported in the British National Formulary (PredEqBNF)20.The methods used for statistical analysis are described in supplementary material and also in our previous publicationThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary information


However, zingerone and geraniol ameliorated the fibrosis in diabetic prostate through suppressing the elevated prostate transforming growth factor beta 1 (TGFβ1) and collagen IV. Therefore, ginger ingredients could be beneficial in alleviating diabetic prostatic complications through suppressing oxidative stress and tissue fibrosis.Prostatic complications are common in patients with diabetes. This study investigated the effect of different ginger ingredients: zingerone, geraniol, and 6-gingerol on the prostate in diabetic rats. Diabetes was induced in Wistar rats by streptozotocin intraperitoneal injection (50 mg/kg), and the rats were left for 10 weeks to develop prostatic complications. In diabetic treated groups, rats received daily oral zingerone, geraniol, and 6-gingerol in doses of 20, 200, and 75 mg/kg, respectively, in the last 8 weeks. Treatment with the compounds caused changes in the ventral prostate of diabetic animals as indicated by the columnar ductal epithelium and dense secretions. There was an amelioration of oxidative stress as evidenced by the lowering of prostate malondialdehyde and elevating prostate oxidized to reduced glutathione (GSH/GSSG) ratios by geraniol and 6-gingerol. None of the three ginger ingredients affected the hyperglycemia, reduction in body weight gain, and testosterone deficiency seen in diabetic animals. Interleukin-1 Prostatic complications are commonly reported in patients with diabetes mellitus . The patOxidative stress is the excessive release of reactive oxygen species (ROS). When associated with hyperglycemia, oxidative stress has been postulated to be one of the hallmarks of the development of complications associated with diabetes, mainly via the polyol pathway , 7. Oxid  is a plant which is popular in homeopathy and alternative medicine and is used to treat a number of diseases [Gingerdiseases . Gingerodiseases . In partdiseases –15. Gingdiseases . Since 6diseases . Geraniodiseases –20.These studies suggest that microvascular changes may occur in the prostate gland due to oxidative stress as a result of hyperglycemia. They also suggest that the components of ginger, namely, 6-gingerol, zingerone, and geraniol, could provide a protective effect on the prostate gland by acting as antioxidants and improving the overall male reproductive state. The aims of the current study were to (a) evaluate the protective effect of 6-gingerol, zingerone, and geraniol on the histological changes that occur in the prostate gland; (b) to evaluate the protective effect of 6-gingerol, zingerone, and geraniol on serum levels of various markers of oxidative stress as well as some inflammatory cytokines; and (c) to evaluate the protective effect of 6-gingerol, zingerone, and geraniol on key markers of fibrosis using immunofluorescence.The research methodologies are in accordance with the Regulations of Research Bioethics on the Living Creatures of the National Committee of Bio. & Med. Ethics, Kingdom of Saudi Arabia.Male Wistar rats  were randomly divided into five groups ; the control (C) group received the vehicle for a total duration of 10 weeks. Streptozotocin (50 mg/kg) was injected IP to induce diabetes. The diabetic (D) group received streptozotocin and the vehicle. The diabetic-6-gingerol (DG) group received 6-gingerol (75 mg/kg/day) orally by gavage two weeks after streptozotocin injection for 8 weeks. The diabetic-zingerone (DZ) group received zingerone (20 mg/kg/day) orally by gavage two weeks after streptozotocin injection for 8 weeks. The diabetic-geraniol (DR) group received geraniol (200 mg/kg/day) orally by gavage two weeks after streptozotocin injection for 8 weeks.−1 two weeks after STZ injection confirmed diabetes and diabetic complications developed after an additional 8 weeks [μm thick sections from each sample were prepared and mounted on poly-L-lysine coated glass slides.A glucose meter was used to check tail blood glucose levels . A blood sugar level of 250–350 mg dL 8 weeks . AnimalsAt the end of study, the effects of 6-gingerol, zingerone, and geraniol administration on prostatic changes due to diabetes mellitus were determined through the following.μm thick sections were obtained by rotatory microtome; then sections were stained with Harris' hematoxylin & eosin and Masson's trichrome stains, according to standard procedures. Stained sections were examined by a light microscope to detect histopathological changes.Samples of the ventral prostate were collected from all animal groups (6 rats/group) and fixed in a 10% buffered formalin solution overnight. The samples were then processed and embedded in paraffin. 5 Epithelial heights were obtained by using image analyzer software (Leica Qwin 500). Twenty readings from random fields of each stained section (5 rats/group) were recorded at high magnification (×400).3VO4; 1 mM NaF; 20 mM Na4P2O7 and 0.02% NaN3  was used to homogenize the snap frozen tissues under cooling. Centrifugation at 10,000 rpm was carried out for 5 minutes with cooling. An analysis of the supernatant was carried out to determine the level of malondialdehyde and glutathione-glutathione reductase ratio in the prostate homogenate using commercially available kits . Prostatic IL-6 and IL-1β and serum testosterone were measured by ELISA kit  using primary antibodies raised against rat IL-6 and IL-1β and testosterone, respectively.Tissue extraction reagent I of the following composition: 50 mM Tris, pH 7.4; 250 mM NaCl; 5 mM EDTA; 2 mM Naμm sections  of the ventral lobe of the prostate were stained using TGFβ1 and collagen IV antibodies for immunofluorescence studies. The slides were heated using a hot plate and subsequently deparaffinized using xylene and placed in ethanol and distilled water. The slides were then placed in hydrogen peroxide kept at −20°C for half an hour. Antigen retrieval was carried out using a citrate buffer at 95°C for half an hour and then washed using PBS. The slides were then kept in a humidified chamber. Nonspecific binding sites were blocked using bovine serum albumin in PBS with 5% normal goat serum, 1% BSA, and 0.1% Triton for half an hour at ambient temperature. Subsequently the sections were kept in PBS for 35 min for washing. Incubation overnight with the primary antibody diluted in the blocking buffer at 4°C was next performed. The sections were then washed three times (5 min) using PBS and then incubated with a fluorescent conjugated secondary antibody that was diluted 1 : 200 in blocking buffer for 1 hour in the dark. The sections were then washed three times (5 min) using PBS, dried, and mounted with “Prolong” mounting media . The slides were kept in the dark until the next day before they were studied using Zeiss LSM 780 immunofluorescence microscope  at wavelengths (488 and 561 nm excitation and 497–542 emission filters). Minimal excitation and gain using the same parameters were used to obtain the images. For quantification, one image was used for each animal. Image fluorescence intensity was measured by Zen software  on the captured unmanipulated images. Representative of the captured whole unmanipulated fluorescence images were presented in results figures. No fluorescence was detected in the sections treated only with the secondary antibody. The primary chicken polyclonal antitransforming growth factor beta 1 (TGFβ1) and the mouse monoclonal anti-collagen I antibody  and the secondary Alexa Fluor  conjugated goat anti-chicken and goat anti-mouse  were used.Five p < 0.05 indicated statistical significance. Statistical analyses were conducted using Prism 5 .Data were analyzed by ANOVA and Newman-Keuls' post hoc test. They are shown as mean ± SE of the mean. Significant changes in glandular epithelial heights were recorded . In diabEpithelial folding in diabetic and diabetic treated groups was not a prominent feature. Although epithelial folds were always present in the superficial layers of prostatic ductules in all examined groups Figures , it was Changes in the ductal epithelium of the rat ventral prostate in control rats, diabetic rats (D), zingerone treated rats (DZ), geraniol treated rats (DR), and 6-gingerol treated rats (DG) were presented in H&E stained sections Figures , and 4. In diabetic rats (D), walls of prostatic ducts were thin and undulated Figures ; relativIn the diabetic group (D), wider interductal spaces (IS) appeared between prostatic ducts Figures  when comPostprandial glucose levels were found to be threefold higher in diabetic rats than in control rats . These lp < 0.05) (p < 0.05). Zingerone treatment, however, had no significant effect on the prostate GSH/GSSG ratio in comparison to the diabetic animals (p < 0.05), and these levels were not affected by treatment  . Treatin animals . The levreatment . The levreatment .β1 (p < 0.05) when compared to the control group   whereas 6-gingerol treatment had no effect.Sections of the prostate displayed a clear increase in the immunofluorescence for TGF Figures . Zingero Figures . TreatmeThe aim of the current study was to investigate the prostatic changes that occur in a streptozotocin-induced diabetic rat model, as well as the protective effect of 6-gingerol, zingerone, and geraniol on these changes. This was carried out by isolating the ventral lobe of the prostate gland from each group of rats and examining the histological changes that occur, in addition to immunofluorescence studies.Upon histopathological examination of stained sections of the ventral prostate, signs of necrosis and leukocytic infiltration were absent. The gland of diabetic rats looked less active in comparison with the normal prostate in control rats. Ductules were enlarged with irregular outlines and thin walls. The epithelial lining was low in height ranging from being cuboidal to being flat. Epithelial folds were absent and lumina were filled with scanty lightly stained acidophilic secretions. Wide interductal spaces between ductules were a prominent feature; these spaces were filled with connective tissue.Diabetic rats treated with the tested substances showed improvement of the profile picture of the ventral prostate. This improvement was apparent in the ductules, where the lining epithelium ranged from cuboidal to columnar cells. The tallest columnar epithelial cells were present in prostate of zingerone treated rats. Secretions were dense darkly stained acidophilic fluids (pink with H&E and blue with MT). The interductal spaces were narrow and connective tissue elements between ductules were sparse if compared with that of diabetic rats. These changes reflect an amelioration of the histopathological changes that occurred in diabetic rats.It is known that the rat ventral prostate is a compound ductal gland and its ductules are lined mainly by columnar epithelium . InductiPrevious studies stated that diabetes caused adverse effects in different body systems including urogenital system  and impaA constant finding in streptozotocin-induced diabetic rats was the decreased weight of ventral prostate –32; thisIn the current study, connective tissue elements specially collagen increased in the wide interductal spaces in diabetic rats; it was detected by histological and immunofluorescence techniques; similar results were recorded in the same species , 34; it  Z. officinale reduced malondialdehyde levels and showed a protective antioxidant effect by a reduction in the level of malondialdehyde in male rats [Malondialdehyde levels serve as a measure of ROS, which can have detrimental effects on male reproductive organs , 38. Theale rats  and imprThe prostate GSH/GSSG ratio was significantly lower in diabetic rats in comparison to control rats. Administration of geraniol and 6-gingerol improved the prostate GSH/GSSG ratio to levels comparable with controls. These results are similar to previously published work in which ginger was found to have powerful antioxidant properties due to the action of its active ingredients including zingerone, gingerols, and shogaols , 40, 41.α, and IGF-1 and clusterin levels were all reported by other studies to be elevated in prostatic inflammation [IL-6 has been reported as a major mediator of acute inflammatory reactions . Previouammation .β1 is a major cytokine in inflammation that is as a key player in the regulation of stromal differentiation and proliferation in benign prostatic hyperplasia. The expression of TGFβ receptor II protein expression in patients suffering from BPH was studied and a correlation between TGFBRII stromal staining and the volume of the prostate was reported [β1 which was reduced by zingerone. In addition, we observed a significant increase in the fluorescence of prostate collagen IV in diabetic rats in comparison to controls. This level was lowered by zingerone and geraniol treatment indicating reduced fibrosis.TGFreported . In our In men who suffer from diabetes mellitus and prostate complications, the polyuria caused by hyperglycemia in combination with lower urinary tract symptoms could be problematic and this combination may potentially interfere with activities of daily living leading to social isolation and dependency. This may feasibly lead to further deterioration of the patient's health as well as psychological state. While treatments for the underlying problems do exist, many patients prefer to minimize the number of medications they are taking while leaning towards therapies derived from nature. Our study demonstrated that the products studied while overall safe had the added benefit of exhibiting a protective effect on prostate tissue exposed to toxic environment in the form of hyperglycemia-induced inflammation. We demonstrated that the tested products ameliorated the tissue damage caused by the inflammatory milieu in addition to effectively lowering ROS products. Further human research is needed to confirm that these findings translate into clinical outcome.This study has provided direct evidence of a link between diabetes mellitus and prostatic complications and demonstrated that the three active ginger compounds, namely, zingerone, geraniol, and 6-gingerol, provide a protective effect on the prostate as evidenced by an improvement of the histopathological profile as well as a reduction in inflammatory processes.