Major depressive disorder (MDD) is associated with a significantly elevated risk of developing serious medical illnesses such as cardiovascular disease, immune impairments, infection, dementia and premature death. Previous work has demonstrated immune dysregulation in subjects with MDD. Using genome-wide transcriptional profiling and promoter-based bioinformatic strategies, we assessed leukocyte transcription factor (TF) activity in leukocytes from 20 unmedicated MDD subjects versus 20 age-, sex- and ethnicity-matched healthy controls, before initiation of antidepressant therapy, and in 17 of the MDD subjects after 8 weeks of sertraline treatment. In leukocytes from unmedicated MDD subjects, bioinformatic analysis of transcription control pathway activity indicated an increased transcriptional activity of cAMP response element-binding/activating TF (CREB/ATF) and increased activity of TFs associated with cellular responses to oxidative stress . Eight weeks of antidepressant therapy was associated with significant reductions in Hamilton Depression Rating Scale scores and reduced activity of NRF2, but not in CREB/ATF activity. Several other transcriptional regulation pathways, including the glucocorticoid receptor (GR), nuclear factor kappa-B cells (NF-κB), early growth response proteins 1–4 (EGR1–4) and interferon-responsive TFs, showed either no significant differences as a function of disease or treatment, or activities that were opposite to those previously hypothesized to be involved in the etiology of MDD or effective treatment. Our results suggest that CREB/ATF and NRF2 signaling may contribute to MDD by activating immune cell transcriptome dynamics that ultimately influence central nervous system (CNS) motivational and affective processes via circulating mediators. These pathways include the cAMP response element-binding/activating transcription factor (CREB/ATF) family of transcription factors (TFs),22 the glucocorticoid receptor (GR)30 and early growth response (EGR) family TFs.34 Other studies have linked MDD to altered activity of circulating mediators that regulate TF activity, including glucocorticoids,38 pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and tumor necrosis factor-α,40 Type I interferons43 and oxidative stress.46Major depressive disorder (MDD) is a pressing public health problem, with up to 16% of the US population experiencing at least one episode in their lives.For both biological and statistical reasons, variations in the activity of signal transduction pathways, rather than small variations in expression of individual downstream gene targets, might show more reliable associations with MDD. We hypothesize that even if correlations between depression and transcriptionally activated pathways exist, it may be difficult to identify significant changes in the expression of individual genes activated through those pathways because of the additional effects of individual differences in genetic background, early developmental influences  and current physiologic conditions . Statistically, reliability in measurements will increase when levels of expression of individual genes are aggregated into a single measure of common signaling pathway activity. The activity of transcriptional control pathways may also help clarify the biological mechanisms by which gene expression in immune cells correlates with MDD. Regulatory pathway analysis, rather than individual gene analysis, may thus be a more reliable biomarker of MDD and its sequelae.49 The conceptual logic of the analysis is as follows: if a TF is activated, it will only modulate the expression of those genes that bear the specific TFBM to which that TF binds. Thus, the subset of upregulated genes will show a relative enrichment of promoter TFBMs for the activated TF  relative to that observed in a control set of genes . In the present study, we conducted such promoter TFBM enrichment analyses to test a series of a priori theories regarding the transcription control pathways that may be activated in the context of MDD, including the CREB/ATF family,22 the GR,52 EGR family TFs,34 the nuclear factor kappa-B cell (NF-κB)/Rel family of TFs that mediate signaling by many pro-inflammatory cytokines,40 interferon regulatory factor (IRF) family TFs that mediate signaling by Type I interferons53 and the nuclear factor erythroid-derived 2-like 2 (NRF2) TF that mediates transcriptional responses to oxidative stress.46In the present study, we applied a promoter-based bioinformatic strategy to assess transcriptional control pathway activity via its functional impact on genome-wide transcriptional profiles, to test several emerging theories regarding immune system and oxidative stress involvement in MDD pathophysiology and its remediation by antidepressant treatment. In this bioinformatics approach, differentially expressed genes are first identified, and the promoter DNA sequence of each differentially expressed gene is then scanned to identify the prevalence of TF-binding motifs (TFBMs) that are hypothesized to be activated in association with the condition under study . The identification of such 'promoter TFBM enrichment' within a set of differentially expressed genes has been shown in several previous validation studies to provide a generally accurate indication of whether a given TF contributes to the observed difference in gene expression profiles.54 and 20 healthy controls matched by sex, ethnicity and age , newspaper ads and through clinical referrals. Subjects were seen in an outpatient setting, and were paid for their participation. SCID diagnostic interviews were conducted by an experienced clinical psychologist and were clinically verified by a separate psychiatric interview with a board-certified psychiatrist. Depressed subjects with psychosis, bipolar histories and post-traumatic stress disorder were excluded from participation in the study; other comorbid anxiety disorders were allowed when the depressive diagnosis was considered to be the primary diagnosis. MDD subjects were required to have a rating of 17 or greater on the 17-item Hamilton Depression Rating Scale (HDRS).57 Healthy control subjects were also screened with the SCID, and were required to have no present or past history of any DSM-IV Axis I diagnosis. Potential subjects were also excluded if they met SCID criteria for alcohol or substance abuse within 6 months of entering the study. Subjects in both groups were medically healthy , had no acute illnesses or infections and had not had any vaccinations within 6 weeks of entering the study. Depressed and control subjects were free of any psychotropic medications, including antidepressants, antipsychotics and mood stabilizers, hormone supplements, steroid-containing birth control or other medications  or vitamin supplements above the US Recommended Daily Allowances  for a minimum of 6 weeks before entry into the study; use of short-acting sedative hypnotics was allowed as needed, up to a maximum of three times per week, but none within 1 week before testing.We recruited 20 subjects with MDD, diagnosed with the Structured Clinical Interview for DSM-IV-TR (SCID),Subjects were admitted as outpatients to the University of California, San Francisco Clinical and Translational Science Institute’s Clinical Research Center at 0800 hours, having fasted (except water) since 2200 hours the night before. Subjects were required to test negative on a urine toxicology screen (measuring the presence of abused drugs) and, in women of child-bearing capacity, a urine pregnancy test. While subjects sat or reclined in a resting position, blood samples were obtained for leukocyte RNA preparation.N-desmethylsertraline at week 4 was 46±23 ng ml−1; range: 10–97 ng ml−1 and at week 8 was 67±37 ng ml−1; range: 10–146 ng ml−1. All individuals had plasma concentrations within the range of published steady-state concentrations for sertraline at therapeutic doses,59 indicating good compliance with medication treatment. One depressed subject prematurely dropped out of the study because of clinical worsening while on sertraline, and two were excluded because of developing exclusionary criteria unrelated to sertraline  during the 8-week follow-up period, leaving 17 treated MDD subjects.Following the baseline evaluation and blood draw, depressed subjects were treated for 8 weeks, in an open-label manner, with sertraline, dosed according to the clinicians’ judgment, based on efficacy and tolerability, beginning with 50 mg per day, increasing to a maximum of 200 mg per day. In two cases, the beginning dose was lowered to 25 mg per day because of transient side effects. Medication compliance was monitored by pill counts and by plasma levels at weeks 4 and 8 of treatment. The mean plasma concentration of sertraline+m ammonium chloride, 10 mm potassium bicarbonate and 1 mm EDTA) and was incubated on ice for 15 min. Samples were centrifuged at 500 g for 10 min at 4 oC, the supernatant was discarded, 1 ml of lysis buffer was added to the pellet and the leukocytes were resuspended by vortexing. The leukocyte suspension was centrifuged, the supernatant was removed, the leukocyte pellet was resuspended in TRIzol Reagent , extracted with chloroform and RNA was precipitated with isopropanol at −20 oC, followed by centrifugation. The RNA pellet was resuspended in 10 mm Tris-Cl pH 7.5/1 mm EDTA; total RNA was tested for quantity , integrity and purity . All samples met quality criteria: RIN⩾7.0 ; 28 S:18 S rRNA ratio⩾1.0. Subsequently, 5 μg of total RNA per sample were used for genome-wide transcriptional profiling using Affymetrix U133 Plus 2 high-density oligonucleotide arrays, following the manufacturer’s protocol . Low-level gene expression data were normalized by robust multi-array averaging61 and were log2-transformed for analysis. Differentially expressed genes were identified by >15% difference in average expression level across groups . Functional characteristics of differentially expressed genes were identified by enrichment analysis of Gene Ontology annotations and Entrez-Gene/RefSeq annotations.68 To test the hypothesis that differential gene expression was mediated in part by altered activity of specific TFs, TELiS bioinformatics analysis62 analyzed the promoters of differentially expressed genes for over-representation of TFBMs targeted by CREB/ATF factors (TRANSFAC V$CREB_01 position-specific weight matrix)69 the GR (V$GR_Q6), NF-κB/Rel factors (V$CREL_01), EGR1–4 , interferon-responsive TFs (V$ISRE_01) and the oxidative-stress-responsive TF NRF2 (V$NRF2_01). TFBM prevalence was ascertained within the promoters of genes selectively upregulated in association with MDD, and separately within the promoters of genes selectively downregulated in association with MDD , and the magnitude of differential TF activity was estimated by the ratio of TFBM prevalence in promoters from upregulated genes versus downregulated genes. This approach ensures that all genes analyzed are potentially expressible within the PBMC pool , and thereby eliminates potential biases that may stem from using the genome-wide prevalence of promoter TFBMs as a reference point . TFBM ratios were calculated in analyses using nine alternative TFBM scan parameters involving varying signal detection stringency 70 and promoter length ,71 and log-transformed ratios were averaged to estimate TFBM prevalence ratios robust to methodological variations. Average relative prevalence ratios significantly exceeding the null hypothesis of 1.0 indicate greater TFBM prevalence among genes upregulated in association with MDD , and average TFBM ratios significantly less than the null hypothesis value of 1.0 indicate lower TFBM prevalence among genes upregulated in association with MDD . These bioinformatics indications have been found to provide accurate inferences of TF activity as assessed by direct assays of TF binding  and experimental manipulation of TF activity.72 To assess the generality of the present findings, we also repeated TF bioinformatics analyses on data from two archival studies that compared peripheral blood cell RNA samples from MDD patients versus controls (GSE38206) and cancer patients with low versus high levels of depressive symptoms (GSE36957).Genome-wide transcriptional profiling was carried out on peripheral blood mononuclear cells (PBMCs) obtained from 20 individuals meeting diagnostic criteria for MDD (HDRS>17) and 20 age- sex- and ethnicity-matched non-depressed controls. All participants were unmedicated at baseline, and 17 MDD subjects subsequently initiated antidepressant treatment and were assessed again for changes in PBMC gene expression profile after 8 weeks of treatment. Blood was collected into vacutainer tubes containing EDTA. RNA was extracted from 10 ml whole blood, after red cell lysis in a hypotonic buffer  following the recommended protocol supplied with the kit. Lysis buffer was added to solubilize the sample and added to the nucleic acid-binding columns supplied with the kit. RNA bound to the column was washed and eluted, and the concentration of the RNA was measured with a Nanodrop Spectrophotometer .oC and combined with dNTPs, reaction buffer and Superscript III to synthesize cDNA at 42 oC.Invitrogen Superscript III  was used to generate complementary DNA (cDNA), using the manufacturer’s protocols. Samples with yields close to 4–5 μg of total RNA were used to synthesize cDNA. cDNA reactions containing RNA and random primers were annealed at 50 GAPDH) was monitored as an internal control. Gene expression differences were estimated using the 'Comparative Ct method' of relative quantification, normalizing the Ct values relative to the reference gene. This was performed by calculating ΔCtsample=Cttargetgene−Ctreferencegene. The relative fold change was represented as 2-ΔΔCt, where ΔΔCt=mean ΔCtsamples−mean ΔCtcontrolsamples. The amplification efficiencies (close to 100%) of the genes of interest and the housekeeping genes were similar (within 5–10%).Primers used for quantitative PCR qPCR;  gave sin58 and the mean household income were significantly lower in the depressed subjects . Average activity/exercise levels per month, as measured by the Yale Physical Activity Survey,73 were not different between groups. The mean 17-item HDRS57 rating in the depressed subjects at baseline was 18.71±3.22 and after 8 weeks of antidepressant treatment was 10.24±6.32, P<0.001, and the mean chronicity of depression  was 14.02±11.75 years (range 0.77–35 years), corresponding to a mean ratio of lifetime depression to chronological age of 0.36±0.27 (range 0.02–0.88).There were no significant differences in the mean age of the depressed versus control subjects, their sex distribution, ethnicity or body mass index . The groIFI44, IFI44L, IFIT1, IFI6, MX1, OAS3, OASL and RNASE2), antimicrobial responses/tissue remodeling , cytokine and chemokine signaling , immunoglobulin production (IGJ and IGH@), cellular activation and proliferation , skewing of macrophage responses toward M2 profiles (ARG1) and cellular responses to oxidative stress (GSTM4). Prominent among the 189 downregulated transcripts in subjects with MDD were genes involved in cAMP/PKA signaling , genes encoding antibody immunoglobulin heavy chains (IGHD and IGHM) and antibody receptors , the T-cell receptor alpha gene (TRA@), chemokine receptors (CCR7 and CXCR6), multiple leukocyte cell surface antigens  and EGR1. The prominent differential expression of leukocyte cell type-specific transcripts suggested potential alterations in subset distribution within the leukocyte pool in MDD versus control subjects. However, we found no significant difference in the leukocyte pool level of mRNA for any canonical leukocyte subset marker .In comparisons of depressed individuals with controls, microarray gene expression analysis identified 346 distinct transcripts showing >15% difference in the average expression level across groups . The 157Promoter-based bioinformatics analyses  showed sP<0.001). Transcriptional profiling of leukocytes collected after 8-week treatment identified 183 transcripts showing >15% upregulation relative to baseline and 101 showing >15% downregulation . Downregulated transcripts also included the oxidative stress response gene SOD2.After 8 weeks of antidepressant therapy, average HDRS depression scores were reduced significantly from baseline levels in MDD subjects  analysis (primers shown in P=0.059) over-representation of NRF2 TFBMs. Results from these two archival data sets were also consistent among themselves in indicating elevated prevalence of NF-κB and interferon-responsive TFBMs . Analysis of the archival MDD versus control comparison also indicated highly significant signals for all of the other pathways analyzed; however, these indications were not corroborated in the cancer/depressive symptoms' sample.To assess the generality of the TF bioinformatics findings from this sample, we compared results from contrasting MDD patients versus controls with results from parallel analyses of peripheral blood cell RNA in an archival data set comparing MDD versus controls in another population (GSE38206) and cancer patients with high versus low levels of depressive symptoms (GSE36957). Results  showed gThe present analyses utilize a transcriptome-driven bioinformatic strategy to evaluate the activity of several transcriptional control pathways that have been hypothesized to be activated in association with MDD. Results comparing unmedicated MDD subjects versus control subjects support previously proposed roles for CREB and the oxidative stress response factor NRF2. However, only NRF2 showed a significant reduction in activity following 8 weeks of antidepressant therapy. Indices of CREB-mediated gene transcription were not reversed by antidepressant therapy, and continued to trend upward after 8 weeks of sertraline treatment. Thus, the NRF2 transcriptional pathway is distinct from the other candidate transcriptional control pathways evaluated here, as it tracked with both disease (MDD) and effective antidepressant treatment.74 However, our failure to see positive signals for NFκB and GR activation, when other transcription control pathways such as CREB/ATF and NRF2 show detectable differences in activity, suggests that NF-κB and GR do not constitute the most sensitive or clinically useful TF pathway biomarkers of MDD.These analyses do not support a role for activation of the master pro-inflammatory TF NF-κB, interferon regulatory factors, the GR or the EGR family of TFs in the basic pathogenesis of MDD. Significant associations did emerge for EGR family factors in analyses adjusting for age, sex and leukocyte subset distributions; however, these effects trended in the opposite direction of previous hypotheses . Because our sample size is small, and thus has limited statistical power to detect small effects, we cannot conclude that our results definitively refute roles for NF-κB, interferons or the GR in the pathophysiology of depression. Indeed, altered hypothalamic pituitary axis activity may be restricted mainly to the subset of MDD cases associated with trauma.79 that MDD is associated with reductions in tonic CREB activity, possibly secondary to reduced activity of neurotrophic factors  and/or neurotransmitters . Alterations in CREB activity in the central nervous system (CNS) in animal models of depression85 and findings from clinical studies linking increased leukocyte CREB activity to successful behavioral or antidepressant therapy89 support this view. However, our results find no tonic inhibition of CREB/ATF-mediated gene expression in leukocytes from untreated MDD patients versus control subjects, but rather indicate upregulation of CREB/ATF activity at baseline before treatment. Moreover, comparisons of CREB/ATF-associated gene expression before and after antidepressant treatment are consistent with previous observations that successful MDD treatment is associated with increased CREB phosphorylation in leukocytes.91 Thus, although the pathophysiologic role of leukocyte CREB activity remains uncertain, our results are consistent with previous studies, suggesting that increasing leukocyte CREB activity levels may potentially serve as a biomarker of treatment response. Our data are also consistent with studies, suggesting that the mRNAs for ATF3 TFs are increased in MDD compared with healthy controls.31Our results have significant implications for five published hypotheses regarding peripheral immune system function in the genetic basis of MDD. First, it has been proposed43 Administration of Type I interferons induces symptoms of major depression, suggesting that some cases of depression may stem from brain responses to subclinical viral infections or other physiologic dynamics that elicit systemic Type I interferon signaling . Our results are partially consistent with this hypothesis, as they show increased expression of several individual interferon-responsive genes in untreated MDD subjects, including genes involved in the interferon-mediated 'antiviral state'. However, TFBM analyses found no evidence of an aggregate-level elevation in interferon-responsive TF activity in unmedicated MDD patients relative to healthy controls. Moreover, genes that were downregulated in association with antidepressant treatment showed a significant under-representation of interferon-related TFBMs, and individual interferon target transcripts continued to show an upward trend during treatment. This treatment-associated upregulation of interferon signaling activity is consistent with previous observations of increased activation of the interferon response factor 7 TF following citalopram treatment.93 These data suggest that if subclinical infectious dynamics or other immunoregulatory disturbances underlie the aberrant interferon signaling associated with some cases of MDD, antidepressants may potentially block the downstream neurobiological consequences of interferon activity, but they are not likely to have an impact on the underlying infectious/physiologic elicitors of interferon activity. This interpretation would be consistent with studies showing that antidepressant prophylaxis can block the emergence of depressive symptoms and sickness behavior following pharmacologic interferon treatment.101A second hypothesis suggests that dysregulated activity of innate immune response signaling pathways may activate CNS 'sickness behavior' systems that trigger motivational deficits and affective alterations characteristic of MDD.104 This hypothesis would predict that MDD should be linked to TFs involved in monocyte/macrophage activation such as NF-κB and EGR family factors. Although our study replicated previous observations of increased EGR3 mRNA in leukocytes from MDD patients, analyses of NF-κB and EGR TFBM distributions provide no evidence that EGR3, other EGR family members or NF-κB/Rel family members show increased transcriptional activity in MDD. By contrast, we found modest indications of baseline reductions in NF-κB- and EGR3-mediated gene expression in leukocytes from MDD patients. These results are not likely to differ from previous findings because of differences in the cell types examined (leukocytes versus isolated monocytes in previous studies) because our adjusted analyses controlled for the relative prevalence of monocytes in the assayed leukocyte pool and because our present study replicated previous indications of increased EGR3 mRNA. Nevertheless, our results suggest reduced functional activity of that pathway. The differing conclusions likely stem from our study’s focus on TF activity, whereas previous studies only focused on EGR3 mRNA abundances. This distinction is consistent with data in other systems showing that TF mRNA abundances do not correlate perfectly with functional TF activity, which can also be substantially regulated by post-translational modifications of TFs.105Third, the 'macrophage theory of depression' suggests that upregulated monocyte/macrophage production of pro-inflammatory cytokines such as IL-1β, tumor necrosis factor-α and IL-6 activates CNS sickness behavior functions in a manner analogous to that hypothesized for Type I interferons.122 Our results are highly consistent with that hypothesis. We found baseline indications of increased activity for the oxidative stress response TF NRF2 in MDD, and reduced NRF2 transcriptional activity following antidepressant treatment. The mRNA abundance of SOD2, a redox-sensitive enzyme, also declined following antidepressant treatment. Those effects could not be attributed to differences in body mass index, age or other demographic variables. The implication of NRF2 and SOD2 in altered leukocyte transcription in MDD subjects suggests a potential leukocyte gene transcriptional biomarker in MDD, a potential target for therapeutic intervention, and a potential biomarker of effective treatment. Given that NRF2 emerged as the only TF in our analysis that directly tracked both depression under basal conditions and the effects of antidepressant therapy, identification of the etiology of peripheral reactive oxygen species in MDD and its relationship to other immune-related transcriptional dynamics  may provide deeper insights into the biological basis for depression.A fourth hypothesis is that peripheral oxidative stress signals to the CNS to evoke symptoms of depression.130 We found no significant alterations in GR-mediated gene expression in control versus untreated MDD subjects, either before or after 8 weeks of antidepressant treatment. Because the GR hypothesis primarily concerns glucocorticoid activity in the CNS, our data from circulating leukocytes cannot be taken as a direct contradiction of this hypothesis. However, our results suggest that leukocyte GR activity may not be a useful proxy biomarker of alterations in glucocorticoid response in the CNS.A fifth hypothesis that is not supported by our study suggests that chronically high cortisol levels, and consequent alterations in glucocorticoid-responsive transcription contribute to the pathogenesis of MDD.72 Our bioinformatic findings can suggest targets for such analyses, but do not provide direct indications of TF functional activity . Our study is also not powered to discover statistically significant associations between individual gene transcripts and MDD versus control status or sertraline treatment. The results in 43 as well as in studies of socioenvironmental conditions that can precipitate depression.134Our data provide new insights into the potential immunobiological and oxidative processes that might contribute to, or serve as biomarkers of, the neurobiological substrates of MDD. However, our study has several limitations, especially small sample size. Replication with larger samples will be needed to resolve small effects that might remain undetected in the present study. Indeed, such power limitations may explain why some significant TFBM patterns that emerged in analyses of the archival data sets were not confirmed in the present sample . However, this study’s primary findings regarding CREB/ATF and NRF TFs did prove replicable in the two independent archival data sets. Our target gene mRNA read-out provides a genome-wide functional framework for assessing TF activity. However, we inferred TF activity from promoter sequence analyses; future studies may use more direct assays of TF activity, such as chromatin immunoprecipitation or nuclear protein assays.Our results provide valuable new insights into the pathobiology of MDD by confirming that cells of the immune system show coherent and highly focal transcriptional alterations in association with MDD and effective antidepressant treatment. These results add to a burgeoning literature implicating immune system dynamics in MDD, and they provide information that helps to discriminate among several of the most prominent current theories of MDD. Future studies should address the roles of oxidative stress and CREB/ATF signaling in crosstalk between the peripheral immune system and CNS biology in the context of MDD.


The use of rapamycin to extend lifespan and delay age-related disease in mice is well-established despite its potential to impair glucose metabolism which is driven partially due to increased hepatic gluconeogenesis. We tested whether a combination therapeutic approach using rapamycin and metformin could diminish some of the known metabolic defects caused by rapamycin treatment in mice. In genetically heterogeneous HET3 mice, we found that chronic administration of encapsulated rapamycin by diet caused a measurable defect in glucose metabolism in both male and female mice as early as 1 month after treatment. In female mice, this defect was alleviated over time by simultaneous treatment with metformin, also by diet, such that females treated with both drugs where indistinguishable from control mice during glucose tolerance tests. While rapamycin-mediated glucose intolerance was unaffected by metformin in males, we found metformin prevented rapamycin-mediated reduction in insulin and leptin concentrations following 9 months of co-treatment. Recently, the Interventions Testing Program showed that mice treated with metformin and rapamycin live at least as long as those treated with rapamycin alone. Together, our data provide compelling evidence that the pro-longevity effects of rapamycin can be uncoupled from its detrimental effects on metabolism through combined therapeutic approaches. Pharmaceutical inhibition of the mechanistic target of rapamycin (mTOR) is now well established as a valuable tool to better understand ways by which the aging process can be delayed in mammals. In multiple studies, chronic treatment with the mTOR inhibitor rapamycin significantly extends mouse lifespan in both males and females even when begun late in life -5. MoreoAnother alternative approach may be to utilize a combination of therapeutics in which the side-effects of chronic rapamycin are treated simultaneously with an additional pharmaceutical. In regards to metabolism, co-treatment with rosiglitazone, a PPARγ ligand and insulin sensitizer, has been shown to partially rescue glucose intolerance and insulin resistance in rapamycin-treated rats . While rAs part of the Interventions Testing Program (ITP), the 2011 longevity cohort tested the effect of combined metformin and rapamycin treatment on the lifespan of HET3 mice . In thesFour month old male and female HET3 mice were randomly assigned to one of four groups fed the following diets based on LabDiet 5LG6: diet only (Control), 14 ppm encapsulated rapamycin (eRapa), 0.1% metformin (Met), or both 14 ppm encapsulated rapamycin and 0.1% metformin (eRapa+Met). For the duration of the experiment, mice were given access to diet and water ad libitum.In male mice, the four groups did not differ in weight through 3 months of treatment. After 9 months of treatment, eRapa males weighed significantly less than all other groups including eRapa+Met mice suggesting that metformin abolishes this effect of rapamycin. Females treated with rapamycin, either eRapa or eRapa+Met, weighed significantly less than control and Met-treated females starting at 3 months of treatment and continuing through 9 months . The masThe primary goal of this study was to determine whether metformin could improve the reported defects in glucose metabolism that are caused by short-term and chronic treatment with rapamycin. For both males and females, metformin alone had no effect on glucose tolerance compared to controls at any point during this study. Martin-Montalvo et al. showed a similar outcome in male C57BL/6 mice using the same 0.1% metformin dose . In contIn females, rapamycin had a significant effect on glucose tolerance at all points tested. Metformin had no significant effect through 3 months of treatment, but did have a significant effect at 9 months of treatment. Ad hoc multiple comparison (Holm-Sidak) testing of female treatment groups found a significant effect of metformin among those mice treated with rapamycin (p=0.001) but no effect of metformin within mice not treated with rapamycin (p=0.52). Thus, metformin at this dose reduces the glucose metabolic dysfunction caused by rapamycin in female, but not male, HET3 mice.In males, rapamycin increased fasting blood glucose concentrations as early as 1 month on the diet and continued throughout the duration of the experiment . Metformi.e., each treatment group as an independent variable) the eRapa treated mice had significantly reduced insulin concentration compared to control (Holm-Sidak multiple comparison p = 0.024). Neither metformin nor rapamycin had any effect on insulin concentrations in female mice. Interestingly, insulin concentrations in females were ~1/5 of that of males corresponding to the relative insulin sensitivity of female HET3 mice compared to males. Plasma triglycerides in both male and female mice were unaffected by treatment . Rapamycin also significantly reduced leptin concentrations in males which appeared to be restored by concurrent metformin treatment  . In fema). G6P was unaffected by either treatment in females, but rapamycin increased expression of PEPCK in females with no effect of metformin .Previous studies suggest rapamycin-induced impairments in glucose metabolism are largely due to hepatic insulin resistance and failure of suppression of hepatic gluconeogenesis ,34. GeneFigures 4D and 4EIn males, rapamycin generally reduced mTORC1 signaling (as measured by p-S6) in liver, skeletal muscle and adipose tissue . MetformWe found that rapamycin had much less effect on mTORC2 signaling (as measured by Ser473 phosphorylation of Akt) in both sexes with the only significant effect on a small increase in female adipose. Metformin also had little effect on mTORC2 with the only significant effect a small increase in male liver .While the mechanism action of metformin is incompletely understood, it has been reasonably well-accepted that metformin activates the AMP-activated protein kinase (AMPK) signaling pathway particularly in the liver. In male mice, phosphorylation of the AMPKα subunit was unchanged by either rapamycin or metformin  . We founOne potential reason for the differences between males and females in the outcomes we report above might be sex-dependent differences in metabolism of rapamycin . Unlike Our main finding here is that a multi-drug approach utilizing metformin can alleviate common metabolic deficits associated with chronic rapamycin treatment as a pro-longevity therapeutic. Moreover our data are in line with recent studies suggesting the metabolic dysfunction of rapamycin can be dissociated from its molecular effects on inhibition of mTOR either pharmaceutically or through alternative pharmaceutical regimens ,16. Thisin vivo [The effects of rapamycin on glucose intolerance have been largely attributed to the inhibition of mTORC2, rather than mTORC1, that occurs with chronic administration of rapamycin . Geneticin vivo . MoreoveInterestingly, we show several disparities between male and female mice in terms of both rapamycin as well as combined rapamycin and metformin treatment. It has been well-established that rapamycin extends lifespan to a greater degree in female compared to male mice ,2,4,37. Perhaps most interesting in this study is the finding that combined treatment with metformin and rapamycin reduces the effective concentration of rapamycin  and metformin (in both sexes) relative to treatment with either drug alone. While mathematically the diet which contains both drugs would have less of each drug available on a per gram basis, this difference is relatively tiny and unlikely to produce such a large difference. More likely, there is likely drug interaction between metformin and rapamycin that in some way impairs absorption or metabolism of these compounds. While these drugs have not been reported to interact directly, there are several drug interactions known to alter metabolism of metformin . ImportaIn contrast to previous studies, we found no evidence that metformin inhibited mTOR signaling when given as a single therapeutic . InteresDetails of breeding and husbandry have been described elsewhere ,2,37. Ma-1) or insulin (0.75U kg-1) intraperitoneally as previously described [Glucose and insulin tolerance tests were performed by fasting the mice for 6 hours (8:00-14:00) and then injecting either glucose , followed by centrifuged at 14,000g at 4°C for 15 minutes. Protein content of the resultant supernatant was measured by BCA assay and equal amounts of protein samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore) and subjected to immunoblot using the following antibodies and catalog numbers : phosphorylated  and total ribosomal protein S6 (#CS2217), phosphorylated  and total Akt (#CS9272), phosphorylated AMPKα  and total AMPKα (#CS5831), and phosphorylated Acetyl-CoA carboxylase  and total ACC (#CS3676). Protein bands were visualized using alkaline phosphatase-conjugated secondary antibodies  and ECL reagent. All immunoblots were quantified using ImageJ (NIH).Total RNA was prepared from liver using TriReagent (Sigma) which was then treated with DNase I . cDNA was generated using Retroscript for RT-PCR . Primers for PEPCK: (F) 5’-3’:TCTCTGATCCAGACCTTCCAA; (R) 5’-3’: GAAGTCCAGACCGTTATGCAG. Primers for G6P: (F) 5’-3’: GAAGGCCAAGAGATGGTGTGA; (R) 5’-3’:TGCAGCTCTTGCGGTACATG. Expression of actin as a housekeeping gene was used for normalization. Real-time PCR was performed in Applied Biosystems 7900 Real-Time PCR system with default PCR program. Triplicate samples were run in in 384-well real time PCR plate using a final volume of 10 µl with 16.26 µl SYBR Green PCR Master Mix , 160 nM forward primer, 160 nM reverse primer, and 8 ng template cDNA in each well. Ct values generated from these PCR were analyzed by relative quantification to values generated for control mice of each sex.Whole blood was collected at sacrifice and analyzed for rapamycin and metformin concentrations by the San Antonio Nathan Shock Center Bioanalytical Pharmacology Core ,48.Male and female data were analyzed separately by 2 way ANOVA using rapamycin and metformin treatment as the two subject factors. Male and female data were analyzed separately due to well-known sex difference in both the basic physiology of this strain of mice (HET3) as well as the difference in effect of rapamycin published elsewhere. Post-hoc analyses were performed using the method of Holm-Sidak. Where applicable, Student t-tests were used to compare 2 groups of interest. Statistical significance was attributed to all data for which a p value of less than or equal to 0.05.Supplementary File


VS38c is a monoclonal antibody that recognises a rough endoplasmic reticulum (rER) intracellular antigen termed cytoskeleton-linking membrane protein 63. rER is typically found in viable tumour cells and is abundant in osteosarcoma cells. The aim of this study was to determine the diagnostic and prognostic utility of VS38c in the histological assessment of osteosarcoma and other bone tumours/tumour-like leisons.Immunohistochemical staining with VS38c was carried out on formalin-fixed specimens of osteosarcoma (pre/post-chemotherapy) and a wide range of benign and malignant bone lesions. In addition, VS38c staining of cultures of MG63 and Sa0S2 osteosarcoma cell cultures. (±cisplatin and actinomycin D-treatment) was analysed.VS38c strongly stained tumour cells in all low-grade and high-grade osteosarcomas and in undifferentiated sarcomas and high-grade chondrosarcomas. There was little or no VS38c staining of low-grade chondrosarcomas or chordomas and variable staining of Ewing sarcomas. Osteoblasts in benign bone-forming tumours and mononuclear stromal cells in chondroblastomas, giant cell tumours and non-ossifying fibromas strongly stained for VS38c. VS38c staining was absent in cisplatin and actinomycin D treated Sa0S2 and MG63 cells. In specimens of osteosarcoma post-neoadjuvant therapy, VS38c staining was absent in most morphologically necrotic areas of tumor although some cells with pyknotic nuclei stained for VS38c in these areas. Most tumour cells exhibiting atypical nuclear forms were not stained by VS38c.Our findings show that VS38c is a sensitive but not specific diagnostic marker of osteosarcoma. Staining with VS38c identifies viable osteosarcoma cells, a feature which may be useful in the assessment of percentage tumour necrosis post-neoadjuvant chemotherapy. VS38c is a monoclonal antibody that recognises an intracellular antigen identified as the rough endoplasmic reticulum (rER) cytoskeleton-linking membrane protein P63 (CLIMP 63) which has also been termed P63 and cytoskeletal-associated protein 4 . CLIMP-6Regular protein production is a functional characteristic of viable cells, and loss or inactivation of ribosomes and rER has been noted in necrotic tumour cells , 9. SeveIn this study, we have sought to determine the diagnostic and prognostic utility of VS38c in the assessment of osteosarcoma and other primary bone tumours. We have examined VS38c staining in a wide range of benign and malignant bone tumours, including osteosarcoma subtypes, in order to assess the sensitivity and specificity of this marker for the diagnosis of osteosarcoma. In addition, as ER changes have been noted in necrotic tumour cells, and active protein production is a feature of cell viability, we have examined VS38c staining in osteosarcomas that have received neoadjuvant chemotherapy in order to determine if expression of this marker can be used to identify viable tumour cells and to facilitate the evaluation of percentage tumour necrosis in osteosarcoma specimens.Paraffin-embedded tissue from 178 specimens of bone tumours as well as specimens of normal compact and cancellous bone, femoral articular surface and growth plate, were retrieved from the files of the Nuffield Orthopedic Centre Histopathology Department, Oxford . After blocking endogenous peroxidase, sections were incubated with the monoclonal antibody VS38C  with antigen unmasking pretreatment. Antigens were detected by incubation with labelled polymer and diaminobenzidine. Sections of myeloma were employed as a positive control. Negative controls consisted of sections which were incubated with normal mouse IgG1  substituted for the monoclonal antibody. The VS38c staining was scored as strong or weak relative to the positive control. Where the majority of lesional cells were not stained with VS38c, an assessment of the percentage of VS38-positive cells relative to the total number of  cells was noted.5 cells) or high (106 cells) density in a 6 well plate and allowed to adhere overnight. Cells were then treated for 24 h in the presence of either cisplatin (1 µg/mL), actinomycin D (10 µg/mL) or vehicle control. Cultures were fixed in formalin and stained as above by immunohistochemistry with the antibody VS38c.MG63 and SAOS2 human osteosarcoma cells were cultured in Iscove’s Modified Dulbecco’s Medium  supplemented with 10% fetal calf serum . To deteStrong cytoplasmic staining of osteoblasts with VS38c was noted in compact and cancellous woven and lamellar bone in areas of bone remodelling Fig. . A few oTumour cells in all high-grade osteoblastic osteosarcomas Fig. a, as welNo staining or weak staining of a few (<10%) cartilage cells was noted with VS38c in benign cartilage tumours including enchondroma, osteochondroma and chondromyxoid fibroma; some cellular enchondromas of the hand, however, showed more prominent staining of cartilage cells. In contrast, VS38c strongly stained chondroblasts in chondroblastoma. Atypical cartilaginous tumour/chondrosarcoma grade 1 showed a similar pattern of VS38c staining to that seen in most enchondromas, but malignant cells in high-grade chondrosarcomas strongly and diffusely stained with VS38c  influence disease survival in osteosarcoma, the extent of chemotherapy-induced necrosis has been shown to correlate most strongly with prognosis. Multi-drug neoadjuvant chemotherapy for high-grade osteosarcoma is now routinely employed and systematic pathological evaluation of the resection specimen of a treated osteosarcoma has been shown to yield important prognostic information about the chemotherapeutic response and to provide a guide as to whether adjuvant chemotherapy is required –19. In cQualitative assessment of the percentage area of tumour necrosis in pathological specimens of osteosarcoma is believed to correlate well with quantitative morphometric analysis –19. HoweIn conclusion, our findings indicate that VS38c is a highly sensitive but not specific diagnostic marker for osteosarcoma. VS38c staining was seen in other primary benign and malignant bone tumours but it was not prominent in some low-grade tumours such as atypical cartilaginous tumour. VS38c staining outlined areas of residual viable tumour in post-chemotherapy osteosarcoma specimens and aided assessment of percentage tumor necrosis. However, although there was little or no VS38c staining in most areas of tumour necrosis in osteosarcoma specimens, there was variable staining of atypical cells with clumped chromatin and vacuolated cytoplasm and cells with pyknotic nuclei. The status of these VS38c positive cells in these necrotic areas is uncertain and more studies are needed to determine whether the morphological assessment of percentage tumour necrosis truly reflects the extent of viable and necrotic tumour in osteosarcoma specimens.


This study aims to investigate the genetic control of hepatic PNPLA3 transcription and to explore its impact on NAFLD risk in humans. Through a locus-wide expression quantitative trait loci (eQTL) mapping in two human liver sample sets, a PNPLA3 intronic SNP, rs139051 A>G was identified as a significant eQTL (p = 6.6×10−8) influencing PNPLA3 transcription, with the A allele significantly associated with increased PNPLA3 mRNA. An electrophoresis mobility shift assay further demonstrated that the A allele has enhanced affinity to nuclear proteins than the G allele. The impact of this eQTL on NAFLD risk was further tested in three independent populations. We found that rs139051 did not independently affect the NAFLD risk, whilst rs738409 did not significantly modulate PNPLA3 transcription but was associated with NAFLD risk. The A-G haplotype associated with higher transcription of the disease-risk rs738409 G allele conferred similar risk for NAFLD compared to the G-G haplotype that possesses a lower transcription level. Our study suggests that the pathogenic role of 148MPNPLA3 in NAFLD is independent of the gene transcription in humans, which may be attributed to the high endogenous transcription level of PNPLA3 gene in human livers.The increased expression of  Two SNPth] Fig. .2r ≤ 0.50 for all tests), rs2294918 was in high LD with the remaining eQTLs (2r ≥ 0.80), suggesting an independent role of rs139051. The plotted data indicated that the A allele of rs139051 and G allele of rs2294918 were associated with increased PNPLA3 gene expression   analysis of variants across the locus revealed that whilst rs139051 was in low LD with all other significant eQTLs  Fig. .PNPLA3 gene expression. Analysis of the HapMap data  demonstrated that, although there is low LD among these three SNPs as measured by the 2r values , the D’ values (coefficient of linkage disequilibrium)  suggested alleles of these SNPs may share the same haplotype(s). Similar data  were observed in both Asian  and African  populations (data not shown). The phased HapMap haplotypic data (CEU) suggest that the rs139051A and rs2294918G alleles (both of which are associated with increased PNPLA3 expression) are almost always co-segregated in the same haplotype (data not shown). Further multivariate haplotypic association with gene expression in liver tissue demonstrated that the association between the rs2294918 G allele and PNPLA3 expression was actually driven by the rs139051 A allele. In rs139051 G/G homozygous individuals, rs2294918 genotypes did not significantly contribute additional variability in gene expression, which was in contrast with the association between rs139051 genotypes and gene expression among the rs2294918 A/A homozygous individuals , while after conditioning all other SNPs, rs139051 remained significant (p = 8.18×10−6). Taken together, these data suggest a potential causal role of rs139051 in regulating hepatic PNPLA3 transcription.We further analyzed the inter-dependence of rs139051, rs2294918 and rs738409 in altering als Fig. . Similarion Fig. . The indPNPLA3 mRNA expression, we used another set of liver samples among which genome-wide transcriptome data has been previously collected in a subset of samples (N = 54) [PNPLA3 transcription . After controlling for age, gender and BMI, this association at rs139051 remained significant (p = 0.029).To confirm the association between rs139051 and (N = 54) . These s(N = 54) . We founp = 0.017). This allelic binding specificity to nuclear protein was supported by a much weaker signal in the reaction with proteins extracted from the cytoplasm as compared to the nuclear extracts  affinity to nuclear factor(s) compared to the G allele , NAFLD Activity Score  (p = 1.5×10−8) and stage of fibrosis (p = 7.7×10−5) after adjusting for relevant covariates . A less strong but statistically significant association between rs139051 and steatosis (p = 1.8×10−4), steatohepatitis (p = 2.1×10−4) but not fibrosis (p = 0.435) was also observed. Similarly, rs2294918 was also significantly associated with steatosis (p = 2.1×10−5), steatohepatitis (p = 2.8×10−4) and fibrosis (p = 0.01). However, in the multivariate analysis conditioning on the rs738409 genotype, neither rs139051 nor rs2294918 was significantly associated with these phenotypes , suggesting that neither rs139051 nor rs2294918 is independent of rs738409 as a risk factor for NAFLD.To test whether rs139051 or rs2294918 contributes to NAFLD risk, we performed association analyses between rs139051, rs2294918 as well as rs738409 and NAFLD phenotypes in a large cohort of European Caucasian NAFLD patients with histologically characterized disease collected by the FLIP Consortium (N = 949). Consistent with previous studies , rs73840p = 0.51)  allele, conferred similar risk for NAFLD phenotypes as compared to the G-G haplotype which contains the rs738409 risk allele, but possesses a relatively lower expression level , and no significant independent association between rs139051 and NAFLD phenotypes was observed after conditioning on rs738409 and adjusting for age, gender and BMI. Also, no significant difference between A-G and G-G haplotypes in conferring risk for NAFLD or influencing NAFLD histological features was observed  (data not shown). The PNPLA3 mRNA level did not significantly modify the risk of NAFLD conferred by rs738409 .To further verify these observations in an independent sample, we performed similar analysis in the aforementioned bariatric patient cohort (N = 212) in which quantitative pathological evaluation of NAFLD phenotypes has previously been described , 24, 25.p = 2.0×10−4 and 1.3×10−3, respectively). Once again, after controlling for rs738409, rs139051 was no longer associated with NAFLD (p = 0.22). In testing the association between rs139051-rs738409 haplotypes and NAFLD, we again found that the A-G and G-G haplotypes confer similar risk of NAFLD (p = 0.27)  and healthy control (N = 384) population with matched age and gender that are reported in our previous study . After a84 and hep > 0.2)  variant is solely attributed to the amino acid change or whether transcriptional regulation is also as relevant in humans as it appears to be in mice. Our analyses suggest that PNPLA3 gene expression does not significantly modify the risk for NAFLD in humans.Our data clarifies a number of critical points raised by previous translational studies using animal models but not yet clarified in humans. Studies in animal models have demonstrated that: 1) The pathogenic role of the 148M allele is not via altering the PNPLA3 transcription. Knockin (KI) of the 148M allele into the mouse Pnpla3 gene did not cause a significant change to the gene expression . 2) VariOur study in humans confirmed some of these observations in mice. First, we show that carriage of the rs738409 G allele does not alter PNPLA3 transcription in humans, suggesting that, as in mice, the effect of the rs738409 variant in humans is more likely related to the functional impact of the amino acid substitution rather than any quantitative effect on gene expression. This assertion is further supported by reports that 148M directly alters the enzymatic activity of PNPLA3 , 18. SecOn the other hand, we also observed significant differences in the pathogenic effect of PNPLA3 between human and mouse. First, our study indicates that the pathogenic function of the PNPLA3148M isoform in humans does not rely on the transcriptional variability. The apparent changes in hepatic PNPLA3 expression due to rs139051 carriage do not modify the effects of rs738409 G-allele carriage in promoting NAFLD and related phenotypes. This is supported by the analysis of associations between rs139051-rs738409 haplotypes and histological features of NAFLD. While the A-G haplotype represents a higher expression level of the disease risk allele than the G-G haplotype, there is no significant difference between these two haplotypes in conferring risk to NAFLD or disease severity Table . Taken tThe divergence between our findings in humans and what have observed in animal models may reflect a fundamental difference in disease pathogenesis between species, and highlights the limitations of using animal models in recapitulating human pathophysiology. Given the differences in diet and other environmental factors between human and mouse, it is possible that the two species are under different selection pressure during evolution, which further leads to differential transcriptional regulation of hepatic genes. Indeed, the PNPLA3 gene undergoes a higher baseline hepatic expression in humans than in mice. This has been demonstrated in recent studies that the PNPLA3 mRNA expression is highly expressed in human livers , while iWe note that rs2294918 has been associated with PNPLA3 gene expression in human liver and was reported to confer additional risk for NAFLD and NASH independent of rs738409 in a recent study by Donati et al . HoweverIn conclusion, our study revealed a genetic basis underlying variation in human hepatic PNPLA3 transcription. Using this information, our study further clarified the role of this variation, as well as its impact on the established role of the 148M variant in NAFLD susceptibility. The apparent divergence between our results and what has previously been reported in animal models highlights the complexity underlying the role of the PNPLA3148M variant in the development of NAFLD and potentially offers important new insights into the consequent pathogenic mechanisms both in mice and humans.This study consists of samples obtained from several sources and parent studies as described below. Samples were collected with written informed consent obtained from participants with approval of institutional review boards (IRBs) at each participating institution. The Purdue University IRB has approved the use of the liver samples for the purpose of this study . The study was carried out in compliance with the Helsinki Declaration.FLIP samples: A cohort of 949 unrelated patients of European-Caucasian descent with histologically characterized NAFLD representing the full spectrum of disease from simple steatosis through steatohepatitis to advanced fibrosis and cirrhosis was assembled from centers across Europe as part of a GWAS study by members of the FLIP consortium. The study had the necessary ethical approvals in all recruiting centers and all participants gave informed consent. Alternative diagnoses were excluded, including excess alcohol intake, chronic viral hepatitis (hepatitis B and hepatitis C), autoimmune liver diseases, hereditary hemochroma-tosis, α1-antitrypsin deficiency, Wilson's disease and drug induced liver injury.2 or > 35 kg/m2 with significant comorbidities) and prior unsuccessful attempts to lose and maintain weight, who underwent bariatric surgery. A protocol intraoperative liver biopsy was performed on all patients for histological phenotyping. Patients with alcohol intake >20 g/d and those with other liver diseases based on positive disease-specific serological tests and suggestive liver histology were excluded. Patients using drugs associated with NAFLD prior to liver biopsy were excluded [Bariatric NAFLD samples: The samples (N = 212) were collected under a study protocol that had been previously reviewed and approved by the Medical College of Wisconsin's Institutional Review Board. Each participating subjected gave a written Informed consent for participation. Subjects were of northern European descent, morbidly obese  and healthy controls (N = 384) were collected in the health examination center of Shanghai Jiaotong University Affiliated Hospital  based on a previously study approved by the Shanghai Jiaotong University Affiliated Hospital . The NAFThe study populations were summarized in Table p < 0.001), identity-by-decent analysis, sex imputation for each sample, etc., as previously described [PNPLA3 gene eQTL mapping, we focused only on the cis-acting eQTLs within the ±1Mb region of PNPLA3 gene. The eQTL mapping was performed using the --assoc function for quantitative traits in PLINK [The liver sample set (N = 206) used to perform eQTL mapping has been described in our previous study  Table 55. Brieflescribed , 38. Ageescribed  to quantin PLINK , which cin PLINK . Plottinin PLINK .Genotyping of rs738409 and rs139051 in DNA samples were performed using Taqman assays according to the instructions provided by the manufacturer .ATAACTTC TCTCTCCT and Biotin/GGGTGCTCTCGCCTGTAAC TTCTCTCTCCT, respectively. Non-biotin labeled probes in a higher concentration (200×) than the labeled probes were used as competitors. EMSA bands were visualized by exposed to the CL-XPosure film  for 3 min. Films were then scanned and the intensity of the band interested was quantified using the ImageJ software [EMSA assay was performed according to our protocol published previously . Brieflysoftware . EMSA asHaplotype frequency between rs139051 and rs738409 among the eQTL mapping population as well as the diplotype for each individual were estimated and assigned by using PHASE  under dePNPLA3 mRNA expression and interaction between polymorphisms and diabetes was examined using linear (for quantitative phenotypes) or logistic (for binary phenotypes) regression analysis within the R package. The genetic association between SNPs or haplotype and NAFLD or NAFLD-related phenotypes  was analysed using the software programs R, PLINK [Linkage disequilibrium and haplotype analyses between rs139051, rs738409 and rs2294918 in HapMap samples  were performed using Haploview 4.2 . CompariR, PLINK , 48. The


Dry Eye disease causes discomfort and pain in millions of patients. Using a mouse acute desiccating stress (DS) model we show that DS induces a reduction in intraepithelial corneal nerve (ICN) density, corneal sensitivity, and apical extension of the intraepithelial nerve terminals (INTs) that branch from the subbasal nerves (SBNs). Topical application of 0.02% Mitomycin C (MMC) or vehicle alone has no impact on the overall loss of axon density due to acute DS. Chronic dry eye, which develops progressively as C57BL/6 mice age, is accompanied by significant loss of the ICNs and corneal sensitivity between 2 and 24 months of age. QPCR studies show that mRNAs for several proteins that regulate axon growth and extension are reduced in corneal epithelial cells by 24 months of age but those that regulate phagocytosis and autophagy are not altered. Taken together, these data demonstrate that dry eye disease is accompanied by alterations in intraepithelial sensory nerve morphology and function and by reduced expression in corneal epithelial cells of mRNAs encoding genes mediating axon extension. The cornea is innervated by a dense collection of sensory axons termed intraepithelial corneal nerves (ICNs) that consist of two components: subbasal nerves (SBNs) ensheathed by corneal epithelial basal cells and intraepithelial nerve terminals (INTs) which branch from the SBNs and extend perpendicularly towards the apical squames. While sympathetic nerves have been reported by some groups in the normal mouse cornea , others Advances in imaging allow the acquisition of high-resolution images of the ICNs on mouse corneas using confocal microscopy ; similarThe ICNs have been implicated in the discomfort associated with dry eye. Pain can be caused by increased friction between the eyelid and cornea due to reduced tear volume or quality and is exacerbated by inflammation . The disAdvances in our understanding of dry eye disease have been made using both chronic and acute mouse models to study the disease and it's progression . GeneticIn the current study, we assess the impact of acute DS and aging on ICN density, thickness, and sensitivity as well as the ability of ICN axons to extend apically towards the epithelial surface. In addition, we assess the impact of the topical application of 0.02% Mitomycin C or vehicle alone (PBS) on axon density in C57BL/6 mice subjected to acute DS. Topical application of MMC (0.02%) is used clinically in ophthalmology during glaucoma and refractive surgical procedures to reduce scarring . Using aAll studies performed were approved by The Institutional Animal Care and Use Committees at Baylor College of Medicine and comply with the ARVO Statement for the Use of Animals in Vision Research. Female C57BL/6 mice aged 6–8 weeks old were purchased from Jackson Laboratories . DS was induced by subcutaneous injection of scopolamine hydrobromide , QID , for 3, 5 or 10 consecutive days in 6–8 week old female C57BL/6 mice and euthanized at the end of each variable , as previously published . Mice weMice (DS5 and DS10) received one topical application of Mitomycin-C (0.02%), 2µl/eye, Green Park Pharmacy, Houston, TX) or vehicle  at d1 and d3 after initiation of DS.Corneal sensitivity was measured under a surgical loupe using a modified Cochet-Bonnet filament in the right eye only of each mouse. The standard Cochet-Bonnet nylon filament has a 0.12 mm diameter; we used a 9–0 nylon that has 0.03 mm diameter to compensate for smaller and thinner corneas in mice as compared to humans . The nylNaturally aged C57BL/6 mice of both sexes were maintained in specific pathogen free vivarium and used at 24 months of age. Only mice free of visible cornea pathology were used. Male and female mice were used unless otherwise indicated.Corneal surface irregularity was assessed in young and aged C57BL/6 mice  as previously published . BrieflyFixing and staining of mouse corneas for identification of the intraepithelial nerve terminals has been described previously . CorneasConfocal microscopy was performed at the GW Nanofabrication and Imaging Center at the George Washington University Medical Center. For Sholl (25× magnification) and Thickness (40× magnification) analysis, images were acquired using the Zeiss Cell Observer Z1 spinning disk confocal microscope , equipped with ASI MS-2000  scanning stage with z-galvo motor, and Yokogawa CSU-X1 spinning disk. A multi-immersion 25×/0.8 and 40×/1.4 objective lens, LCI Plan-Neofluor, was used for imaging, with oil immersion. Evolve Delta  512 × 512 EM-CCD camera was used as detector (80-msec exposure time). A diode laser emitting at 568 nm was used for excitation (54% power). Zen Blue software  was used to acquire the images, fuse the adjacent tiles, and produce maximum intensity projections. The adjacent image tiles were captured with overlap to ensure proper tiling. All images were acquired using the same intensity settings. Sholl analysis was performed using ImageJ as described previously .z = 0.5 µm or 1 µm) were acquired sequentially with a 63× objective lens. For quantification of intraepithelial nerve terminals, 3D images were rotated to generate cross section views using Volocity software  and images were presented as cross sections projected through the length of the acquired image (135 µm). Pixel intensity data for apical and basally projecting nerve fibers were obtained as described previously  equipped with a krypton-argon laser was used to image the localization Alexa Fluor 594 . Optical sections  and stored at −80 °C until used. QPCR was performed using a Bio-Rad CFX384 Real-Time PCR detection system. The primers used were ordered from Bio Rad, unless otherwise specified: Bec1(qMmuCID0005981), LC3 (qMmuCED0045817), LAMP1 (qMmuCID0027030), LAMP2 (qMmuCID0011408), CXCL1(qMmuCED0047655), BDNF(qMmuCED0050333), NTN1(Qiagen #QT00128478), DCC(Qiagen #QT00135100), Unc5b(Qiagen #QT00167846), Efna4(Qiagen #QT00100681), Efna5(Qiagen #QT00116494), Rgma (Qiagen #QT00310583) and GAPDH (qMmuCED0027497). QPCR data was normalized against GAPDH.For quantitative polymerase chain reaction (QPCR) studies, epithelium was scraped using a dulled blade and frozen immediately. Four corneas per sample and at least 3 samples per time point were used for the QPCR studies. RNA was extracted using a QIAGEN RNeasy Plus Micro RNA isolation kit (Qiagen) following the manufacturer's protocol. After isolation, the concentration of RNA was quantified using a NanoDropp value < 0.05 was considered statistically significant.Quantitative data are presented as mean ± standard error of the mean. All data were analyzed using one-way ANOVA; when standard deviations were significantly different from one another as determined by Barlett's test, the Kruskal-Wallis Multiple Comparisons test was performed as specified by Graphpad Prism, Version 6 . All Statistical calculations were performed using Prism, . A DS was induced acutely in 8-week-old C57BL/6 mice as described previously using cholinergic receptor blockade (scopolamine) and a low humidity drafty environment . MMC  and MMC treatment. When axon density data for the three d5 and d10 subgroups were compared to one another by the Kruskal Wallis Multiple Comparisons test, no significant differences were seen between subgroups at either time point. Axon density data for the subgroups were combined into a single group that included 38 (d5) and 30 (d10) mice. Relative to controls, the combined data shows that axon density is significantly reduced by d5 and remains decreased at d10.Next, we assessed mean axon thickness. Representative images from these assessments are presented in The method used to quantify axon density uses flattened projection confocal image stacks and does not permit assessment of the intraepithelial nerve terminals (INTs). To look at apical INT extension, we obtained high-resolution confocal image stacks from 3 to 5 corneas for each variable, generated 3D images, and rotated them to generate cross sectional views projecting through 135 µm of cornea tissue  corneal staining, reduced conjunctival goblet cell density and corneal surface irregularity noted in reflected rings . To confWe next assessed corneal sensitivity in control and 24-month-old mice The reduced numbers of axons present in the 24-month-old mouse cornea could be secondary to a number of factors. It could be caused by increased turnover and phagocytosis by the corneal epithelial cells of older, shed axon fragments, reduced expression of mRNAs for proteins that regulate axon extension and targeting, or increased expression of proteins that repel axons. To study the mechanism underlying the loss of axons in older mice, corneal epithelium of 6–8 wk and 24-month-old mice was harvested by debridement and used to isolate RNA. QPCR studies were performed using primers for mRNAs whose expression was associated with increased phagocytosis and autophagy as well as primers for mRNAs associated with axon targeting and extension. Data are presented in Mitomycin C (MMC) is an antibiotic that induces DNA strand breaks and crosslinks in bacteria ; in mammThe corneal epithelial basal cells maintain the intraepithelial corneal nerves (ICNs) and act as surrogate glial cells to stabilize both the subbasal nerves (SBNs) and the intraepithelial nerve terminals (INTs) that extend towards the apical squames . Here weBetween 2 and 24 months of age, axon thickness and nerve terminal apical extension remain the same despite significant loss of axon density. While nerve terminals are present near the apical aspect of the corneal epithelium, corneal sensitivity at 24 months of age is either undetectable or low.The disappearance of the majority of the sensory axons in the cornea by 24 months of age reported in our study is consistent with an earlier study ; but extQPCR was performed to quantify RNAs implicated in the growth, inflammation, and/or phagocytosis of axons. We find that at 24 months, Ntn1, DCC, Unc5b, and Efna5 are all significantly reduced relative to 2-month-old mice and Rgma is increased. By contrast, levels of several RNAs involved in phagocytosis and autophagy are not altered.Netrins (Ntns) are mammalian homologs of UNC6, an ECM protein containing laminin motifs first identified in C. elegans; netrin receptors, referred to as “dependence receptors”, include several integrins, Dcc, Unc5B, and Neo1 . Ntn1 anRgma induces retraction of retinal axons  and it'sNeuroinflammation impacts development of small fiber neuropathy and alters sensory axon morphology. Taken together with the data obtained on mRNAs for axon targeting, these results suggest that older corneas have fewer axons because corneal epithelial cells do not produce the proteins that enhance axon targeting  at high enough concentrations and/or produce proteins that inhibit axon targeting (Rgma) at higher levels leading to reduced axon density. It is not clear whether reduced corneal epithelial cell proliferation in older corneas is caused by the loss of sensory nerves, increase in immune cells seen in these corneas , or to oHere we show that axon density is reduced in corneas of mice subjected to acute DS as well as in older mice previously shown to have chronic dry eye. Changes in sensory axon morphology and numbers after initiation of acute DS and with aging are accompanied by loss of corneal touch threshold.supplement


Transient hypertension may be an adverse event of intravenous methylprednisolone. Hypotension, a known adverse effect of interferon (INF), is a known risk factor of ischemic colitis and ischemic colitis is one of the serious adverse events of treatment with IFNs type I. Ischemic colitis should be considered in INF and acetylcholine inhibitors (AChI) and calcium channel blockers (CCB) co-administration.10Treatment with IFN type I could predispose the patient to develop an autoimmune disease.11 Similarly, new-onset hypertension could be a sign of INF-induced systemic lupus erythematosus (SLE).12Some reports define INF-induced de novo Raynaud’s phenomenon, sometimes with progression to systemic sclerosis. A new-onset accelerated arterial hypertension could be a part of systemic sclerosis triad.13Thrombotic microangiopathy is a known rare adverse event of INF-therapy and new-onset hypertension is one of its important presentations advised to be evaluated carefully and controlled regularly in patients with MS receiving IFN-β.14Hypertension is reported in approximately 10% of patients with MS exposed to glatiramer acetate in premarketing studies. During post marketing period, there are reports of hypertensive crisis with glatiramer acetate complicated with acute pulmonary edema and myocardial ischemic injury.15-18 As a result, in some patients experiencing a slight transient hypotension after the initiation of fingolimod therapy, it is not strange. Sometimes, this is followed by a small hypertension (~3 mmHg systolic and ~1 mm Hg diastolic blood pressure); but after 6 months of treatment, hypertension is placed in a stable plateau level.19Fingolimod could cause vasodilation and associated hypotension via activation of the endothelial nitric oxide synthase/nitric oxide (eNOS/NO) pathway.20After the infusion of natalizumab and typically following two days, there are some reports of hypertension but much less frequent; this side effect is defined as probable and very likely.21 In a phase-II teriflunomide clinical trial, high blood pressure was a cause of withdraw.22 European medical agency recommends careful hypertension history taking and appropriate management during the treatment with teriflunomide.23 Hypertension could be a common side effect of alemtuzumab.24In teriflunomide trials, hypertension is reported in 3.1 and 4.3% of the patients treated with 7 or 14 mg of teriflunomide compared with 1.8% for the placebo.Up to now, there is not any information on arterial hypertension induced by dimethyl fumarate.Essential hypertension is common in patients with MS similar to general population and probably could affect mortality, morbidity and final disability. New-onset hypertension could be a presenting sign of a treatment adverse event. MS healthcare professionals should measure and observe patients’ blood pressure in follow-up visits and manage it appropriately.


Actinidia chinensis var. deliciosa ‘Hayward’ fruit are very responsive to CPPU treatments, but the mechanism underlying the significant fruit weight increase and associated decrease in DM is unclear. In this study, we hypothesised that CPPU-enhanced growth increases fruit carbohydrate demand, but limited carbohydrate supply resulted in decreased fruit DM. During fruit development, CPPU effects on physical parameters, metabolites, osmotic pressure and transcriptional changes were assessed under conditions of both standard and a high carbohydrate supply. We showed that CPPU increased fruit fresh weight but the dramatic DM decrease was not carbohydrate limited. Enhanced glucose and fructose concentrations contributed to an increase in soluble carbohydrate osmotic pressure, which was correlated with increased water accumulation in CPPU-treated fruit and up-regulation of water channel aquaporin gene PIP2.4 at 49 days after anthesis. Transcipt analysis suggested that the molecular mechanism contributing to increased glucose and fructose concentrations was altered by carbohydrate supply. At standard carbohydrate supply, the early glucose increase in CPPU fruit was associated with reduced starch synthesis and increased starch degradation. When carbohydrate supply was high, the early glucose increase in CPPU fruit was associated with a general decrease in starch synthesis but up-regulation of vacuolar invertase and fructokinase genes. We conclude that CPPU affected fruit expansion by increasing the osmotically-driven water uptake and its effect was not carbohydrate supply-limited.Exogenous application of a cytokinin-like compound forchlorfenuron (CPPU) can promote fruit growth, although often at the expense of dry matter (DM), an important indicator of fruit quality.  However, a number of studies have shown how the use of growth regulators can negatively affect fruit quality.3–5 The growth regulator forchlorfenuron (N1-(2-chloro-4-pyridyl)-N3-phenylurea, CPPU) is an example of a synthetic cytokinin that influences fruit set and/or development of several fruiting crop species, such as apples,6 grapes,7 watermelon,8Lagenaria leucantha,9 macadamia10 and kiwi fruit.12Fruit size (fresh weight) and dry matter  are important quality traits for horticultural crops affecting their value, determining their economical returns and driving consumer purchases.11–19 using CPPU, there has been no clear explanation of its molecular mechanism in any species. These studies agree that CPPU application to kiwifruit dramatically increased fruit fresh weight but this desired effect was often accompanied by an undesirable decrease in final fruit DM.5 Antognozzi et al.11 also found CPPU-treated fruit to be lower in DM, despite having higher carbohydrate concentration  and enhanced starch metabolism. Patterson et al.5 showed CPPU promoted fruit fresh weight through cell expansion and Ainalidou et al.20 confirmed it affected small cell size, rather than by increasing cell division as later observed by Cruz-Castillo et al.,16 despite having a similar timing of application. Together, these findings suggest that CPPU has a role in the modulation of both carbohydrate metabolism and water accumulation.While a large number of studies have been conducted in kiwifruitAPL4) and beta-amylase 9 (BAM9), important during kiwifruit development and DM accumulation, were previously identified and their transcripts investigated in genotypes with contrasting carbohydrate metabolism.21 In high starch and high DM genotypes, the higher and extended transcription of APL4 was associated with enhanced starch concentration in fruit, whilst an involvement of BAM9 in starch turnover was proposed during early fruit development.Key carbohydrate metabolic genes, such as ADP-glucose pyrophosphorylase large subunit 4  have been attributed to having a large role in controlling water movement across membranes.27 These proteins may also contribute to cell osmotic adjustments following continuous sugar accumulation and cell expansion during fruit growth.29 The molecular mechanisms of water accumulation have not been investigated in kiwifruit to date.The role of aquaporins, transmembrane water channel proteins that facilitate water transport across membranes,30) promoting carbohydrate accumulation in the fruit but that the dramatic fruit fresh weight increase is not well supported by carbohydrate supply from leaves.19 In conditions of high carbohydrate supply, kiwifruit vines were able to redistribute carbohydrates to fruit, including to fruit growing on leafless shoots.31 Experiments have shown that on average between two to three leaves were required to satisfy the normal carbohydrate demand of a fruit,32 and on a whole plant basis fruit fresh weight increased up to a leaf-to-fruit ratio of five.33Despite the low DM observed in CPPU-treated fruit, current literature suggests that CPPU increases fruit sink strength (the capacity to attract carbohydratesThe aim of this study was to find what limits kiwifruit DM accumulation after CPPU application. We proposed that carbohydrate supply limitation could be the cause of low DM for CPPU-treated fruit. The leaf-to-fruit ratio of girdled 1-year-old fruiting canes was adjusted to test the effect of two different controlled carbohydrate supplies (standard and high) on CPPU treatment. We verified that CPPU-treated fruit had higher fruit weights and lower DM. We hypothesised that the lower DM was the result of a reduction in starch concentration. We then investigated how the changes in the fresh weight growth and carbohydrate metabolism in the CPPU-treated fruit were modulated by examining transcriptional variation of genes involved in metabolism and/or movement of carbohydrates and water transport.Actinidia chinensis var. deliciosa (A. Chev.) A. Chev. ‘Hayward’ 15-year-old vines planted in an orchard block at Plant & Food Research (PFR), Te Puke Research Centre . All vines were grown on clonal Actinidia hemsleyana Dunn ‘Kaimai’ rootstocks. The vines were managed for normal commercial production  on a pergola-trained system with 6 m spacing within the row and 5 m spacing between rows.34 The date of anthesis (defined as 50% open flowers) was recorded as the 23rd of November. Treatment and sampling dates were referred to as days after anthesis (DAA).The experiment was carried out on The experiment was a two by two factorial design with equal numbers of canes allocated to two carbohydrate supplies (standard and high) by two CPPU treatments (treated and untreated). A total of 120 uniform 1-year-old canes (8 canes per vine) were selected. Five canes per treatment were randomly allocated to five sampling times. All canes were girdled at 28 DAA and all the leaves and fruit distal from the girdle counted. CPPU was applied to fruitlets 28 DAA as a 10 ppm dip. On the same day the leaf-to-fruit ratio was adjusted on girdled canes to either three leaves per fruit (standard carbohydrate supply) or six leaves per fruit (high carbohydrate supply). The girdles were kept open throughout the experiment and leaf-to-fruit ratios maintained by immediately removing any new growth on canes.et al.35 DM, the ratio of fruit dry weight to fruit fresh weight, was determined by drying 2-mm fruit slices at 65 °C for 24 h36 and expressed as g kg−1. Mean absolute growth rate (AGR) was calculated for fruit fresh and dry weights as described by Opara.37Fruit samples were collected 35, 49, 70, 126 and 154 DAA. At each sampling date six randomly selected fruit were harvested from each of five selected canes per treatment. Fruit fresh weight, fruit dry weight and DM were measured on all fruit as described in Nardozza Fruit samples, consisting of longitudinal slices representative of all fruit tissue types, were collected from four biological replicates, snap-frozen in liquid nitrogen, and stored at −80 °C for carbohydrate and transcript analysis.et al.38 Soluble carbohydrates  were analysed as per Klages et al.39 and organic acids as per Cheng et al.40 by gas chromatography (Carlo Erba GC 6000) with a DB1701 column and FID detection. The individual starch, sugar and acid contents were expressed as milligrams per gram fresh fruit weight (mg gFW−1). Osmotic pressures were calculated as described in Nardozza et al.35 Briefly, the contribution of soluble carbohydrates and organic acids to total fruit osmotic pressure were calculated from the solute concentrations applying the van‘t Hoff relation41 with the same conditions described by Bertin et al.42Starch was colourimetrically determined following enzymatic digestion as reported by Smith 43 DNase treatment  and cDNA synthesis , transcript analysis was performed by real-time PCR on a LightCycler 480 detection system (Roche) using LightCycler 480 SYBR Green master mix (Roche) as described by Nardozza et al.21 Gene-specific primers, designed to span exon junctions, were used as reported by Nardozza et al.21 or as listed in EF1α, and protein phosphatase 2A, PPRSA.Following RNA isolation from 2 g of fruit tissues,The effect of CPPU treatment, carbohydrate supply and fruit age on fruit fresh weight, fruit dry weight, DM, starch, glucose, fructose and sucrose concentrations, total starch content per fruit, osmotic pressure and DM components were analysed by a general three-way factorial ANOVA using GenStat software version 17.1.0.14731 . For variables characterised by large developmental changes, such as fruit fresh weight, fruit dry weight, dry matter and starch, the data analysis considering fruit age as a factor produced standard errors that were larger than the measured value at 35 DAA (7 days after the treatment), therefore hiding significant 2-fold variations between treatments. For this reason, the effect of CPPU treatment and carbohydrate supply on fruit fresh weight, fruit dry weight, DM and starch concentration at 35 DAA were also tested using a two-way ANOVA.CPPU significantly affected kiwifruit fruit weight, at both standard and high carbohydrate supply. The CPPU effect on fruit fresh weight was evident from 49 DAA (~20 days after the treatment) through to harvest for fruit from both carbohydrate supplies. CPPU-treated fruit had a final fruit fresh weight 28 percent greater than untreated fruit with a standard carbohydrate supply, and 37 percent greater than untreated fruit with a high carbohydrate supply .Fruit dry weight was also affected by CPPU treatment, being higher in CPPU-treated fruit, with both carbohydrate supplies. Dry weight differences were detectable from 70 DAA (~40 days after the treatment) until final harvest. The relative change in dry weight accumulation with CPPU treatment was less than the effect on fresh weight  and 54.3 g H2O/fruit (41 percent) at the standard and high carbohydrate supply, respectively.By final harvest, CPPU treatment led to an increase in 38.6 g H−1 by CPPU treatment with both carbohydrate supplies .Differences in DM were first detectable at 70 DAA (~40 days after the treatment) and continued through until harvest. At the final harvest, fruit DM was significantly lowered by approximately 25 g kgP=0.03, fruit age x treatment interaction). Sucrose  until final harvest, irrespective of carbohydrate supply . Treated Sucrose , galactotrations  were unaA two-way ANOVA analysis carried out on small young fruit at 35 DAA (7 days after the treatment) also showed significant treatment affects. CPPU-treated fruit were significantly larger, had higher dry weights and lower starch concentrations than untreated fruit , whilst P<0.001; P=0.026) whilst it had no overall and consistent effect on starch and other components of DM. However, it is worth noting that between 126 and 154 DAA the starch component decreased in CPPU-treated fruit with high carbohydrate supply  metabolism, as well as sugar and water movement , showed that application of CPPU and/or high carbohydrate supply contributed to a variation in transcript abundance of some genes in comparison to the untreated control with a standard carbohydrate supply . In contAPL2, APL4, large subunit 4 and APS1, small subunit 1; FK4  started at 126 DAA, earlier than in fruit from other treatments (data not shown due to low transcription at early stages), suggesting an early onset of maturation in these fruit. This is further supported by the significantly higher total soluble carbohydrate concentration of CPPU-treated fruit from 126 DAA onwards  (PIP1.3 peaked at 49 DAA and then decreased throughout the rest of fruit development (PIP1.3 transcription was delayed to 70 DAA and the transcription decreased more slowly than the untreated fruit at standard supply (At 49 DAA the water channel protein ctively) . Transcrelopment . With thd supply . These td supply .−1 reduction in DM caused by CPPU application to fruit. The reduction in DM was the result of a higher water accumulation in CPPU-treated fruit osmotically driven by increased glucose and fructose concentrations, through a molecular mechanism influenced by carbohydrate supply.Exogenous cytokinin treatment affected ‘Hayward’ fruit development as expected by increasing fresh weight. An unexpected result was the inability of doubling of carbohydrate supply to compensate for the 25 g kg44–46 The observed 28 and 37% increases in fresh weight at standard and high carbohydrate supply, respectively, are in agreement with previous observations. However, the controlled system created by application of girdles enhanced the average fruit weight from all treatments, with the untreated fruit at standard carbohydrate supply having a fresh weight of about 150 g, which is higher than commercially available kiwifruit47 The combination of both CPPU and high carbohydrate supply resulted in a 54 percent higher fresh weight, which equated to an average fruit weight of 225 g, a figure that is nearly double that of a commercially available kiwifruit47 and probably close to the maximum potential size for these fruit.The most obvious and widely reported CPPU effect is the dramatic increase in fruit fresh weight, for kiwifruit as well for other fruit crops.11 The higher carbohydrate supply was not sufficient to supply the fruit with adequate photosynthate to balance the dramatic fruit fresh weight increase promoted by CPPU treatment, and DM was decreased by 25 g kg−1 DM with either carbohydrate supply. This undesirable reduced DM ‘side-effect’ of CPPU treatment was consistent with previous studies.11 In a commercial orchard in New Zealand, a 25 g kg−1 DM decrease by a CPPU treatment would translate in to large financial loss per hectare  through reduced DM quality payments.48Isolating the cane by girdling (and thus preventing photosynthate redistribution from other parts of the vine) also amplified the effect of CPPU on lowering fruit DM at both carbohydrate supplies, with a much greater decrease than reported in previous studies.et al.49 showed that a 50% increase in carbohydrate supply led to an enhancement in both fruit fresh weight and DM, although DM was increased by only 4–5 g kg−1. However, in our study, where girdling prevented carbohydrate redistribution from canes to the rest of the vine,31 doubling carbohydrate supply increased fruit DM by 12 g kg−1 in both CPPU-treated and untreated fruit.Using whole vine manipulations, Richardson et al.5 Starch is one of the major components of DM in Actinidia chinensis var. deliciosa fruit, representing about 40% of the total DM at harvest time.35 Our data  in CPPU-treated fruit. In CPPU-treated fruit with a standard carbohydrate supply, apart from 35 DAA starch concentration was unaffected until later in development (126 DAA onwards), when the lower DM reflected the decrease in starch concentration. In fruit from high carbohydrate supply, starch concentration did not differ between treated and untreated fruit from 35 DAA until harvest, when the lower starch in CPPU-treated fruit was probably due to an early onset of fruit maturation, as previously observed by Patterson Our data  and 2 cl50 For this reason, we will discuss the two models separately.Combining the transcript accumulation data and the carbohydrate analysis, we propose two models for carbohydrate metabolism following CPPU treatment under standard or high carbohydrate supply. In our study, carbohydrate supply treatments contributed to the variation of transcript accumulation, as previously observed in citrus.−1) and glucose concentration increased by about 1.5  mg gFW−1. Although from a metabolite point of view the outcome was similar between the two different fruit carbohydrate supplies, transcript analysis suggests that control of carbohydrate metabolism in fruit may have been affected differently than carbohydrate supply to fruit. At a standard carbohydrate supply, transcription of a gene coding for an enzyme critical for starch synthesis (APL2) was decreased and transcription of a beta-amylase gene involved in starch degradation (BAM9) increased in CPPU-treated fruit. Therefore starch synthesis was reduced and its degradation increased in CPPU-treated fruit, consistent with the overall decrease in starch in these fruit early in fruit development. This is consistent with previous results with kiwifruit, where BAM9, coding for a cytosolic enzyme, was associated with starch turnover during early fruit development.21 When carbohydrate supply to fruit was high, CPPU-treated fruit showed a general decrease in starch concentration (decreased accumulation of ADP-glucose pyrophosphorylase subunit transcripts) throughout fruit development. These changes in carbohydrate metabolic genes are consistent with observed low starch and high glucose concentration in CPPU-treated fruit.At 35 DAA (7 days after the treatment), during the stage of rapid fruit growth, CPPU-treated fruit starch concentration had halved  gene expression in early fruit development. An effect of CPPU stimulating both invertase expression and activity has been observed previously in cucurbitaceae.51 While invertase produces equimolar amounts of glucose and fructose by an irreversible reaction,52 the increased concentrations of fructokinase (another irreversible reaction) could be responsible for the phosphorylation of the excess fructose produced, channelling it towards further metabolism. Sucrose synthase, another sucrose cleaving enzyme and fructokinase54 have high activity early in fruit development.21 These two enzymes are both inhibited by fructose so it is important that excess free fructose produced by an increase in invertase cleavage of sucrose is quickly removed via fructokinase. In tomato, transgenic fruit lacking LeFRK2 transcripts (homologous to kiwifruit FK4) had an increased fructose concentration,55 and the authors suggested a role for fructokinases in maintaining sugar import into sink tissues.In CPPU-treated fruit with high carbohydrate supply increased vacuolar invertase INV3 transcription. At this time, as a consequence of the general physiological decrease in FK4 transcription,21 fructose also accumulated in fruit and by 126 DAA fructose and glucose had similar concentrations. At high carbohydrate supply, INV3 transcripts did not differ between CPPU-treated and untreated fruit, although transcripts in fruit from both treatments were elevated by an increase in carbohydrate supply compared with untreated fruit at standard carbohydrate supply. It is possible that the increase in glucose and fructose concentrations in fruit with high carbohydrate supply is determined by the lower SPSA1 transcription at 70 DAA and SUS1 down-regulation from 70 DAA onwards, which might have affected sucrose cycling,56 and the early onset of fruit maturation suggested by soluble carbohydrate concentrations and earlier BAM3 transcription.Later in development , enhancement in glucose and fructose concentrations in CPPU-treated fruit at standard carbohydrate supply was associated by the higher 21 is mainly caused by cell enlargement, due to both increased turgor pressure generated by accumulating osmotically active molecules attracting water and cell wall relaxation.57 Vacuolar invertase is a key enzyme for driving cell expansion through osmotic regulation, as shown for cotton fibres’ elongation.58 Enhanced glucose (and fructose) in CPPU-treated fruit led to an increase in the soluble carbohydrate component of the fruit osmotic pressure, despite the total osmotic pressure being unchanged in CPPU-treated fruit. This apparent discrepancy could be explained by cellular compartmentalisation: while organic acids are mainly stored in the vacuole,59 small soluble carbohydrates can move, actively or passively, between the symplast, apoplast and vacuole, to control cell osmotic potential and turgor pressure.60 The movement of soluble carbohydrates between compartments would create an osmotic imbalance likely to attract more water, hence promoting fruit growth through cell expansion. This hypothesis is consistent with the higher water content observed in CPPU-treated fruit.The fruit expansion phase, which occurs in kiwifruit from about 50 DAA,PIP2.4 at 49 DAA in CPPU-treated fruit is also consistent with the higher water importation inferred in these fruit and driven by the higher glucose and fructose concentrations. Aquaporins control trans-cellular water conductance and have been previously associated with cell expansion.61 PIP are divided into two groups: PIP2, with high water transport activity 62 and PIP1, the function of which has been debated as being either non-functional63 or requiring a physical interaction and co-expression with PIP2 to reach the plasma membrane and function as a water channel.64 Yanef et al.65 recently reviewed the complexity of aquaporins and concluded that the PIP1-PIP2 pair should be considered as a functional unit when investigating their role. Our data show that PIP1.3 transcript accumulation in kiwifruit was delayed and prolonged by CPPU or high carbohydrate availability, and we could speculate that it possibly played a controlling role on the PIP2.4 water channels, keeping them open for longer and contributing to the higher water uptake of these fruit.The higher transcription of transmembrane aquaporin PIP2.4 transcription by sugar signalling, which may discriminate between osmotic pressure generated by soluble carbohydrates or organic acids. This sensing mechanism could be triggered by higher concentrations of glucose, via hexokinase, for example, as hexokinase is a well-known sugar sensor in plants.66 A similar interaction between aquaporins and hexokinase has been described in photosynthetic tissues of transgenic tomato plants.67 This aspect is worth of further investigation, given the positive and significant correlation between PIP2.4 and HK3 transcripts .An alternative mechanism for the disproportional increase in water accumulation in CPPU-treated fruit could involve the regulation of We have investigated the effects of CPPU on ‘Hayward’ fruit development under controlled standard and high carbohydrate supply to determine whether DM reduction is caused by limited carbohydrate supply to fruit. Despite the presence of high carbohydrate supply, CPPU-treated fruit still showed a dramatic reduction in DM as a consequence of extreme fruit growth osmotically driven by an increase in glucose and fructose concentrations during the early stage of rapid fruit growth. The examination of transcript accumulation of a limited set of candidate genes suggests how changes in carbohydrate metabolism and water uptake in CPPU-treated fruit may occur.