Arabidopsis thaliana. Here we show that plant architecture generates fine scale spatial variation in the systemic induction of invertase and phenolic compounds. We examined whether the arrangement of leaves along the stem (phyllotaxy) produces predictable spatial patterns of cell-wall bound and soluble invertase activities, and downstream phenolic accumulation following feeding by the dietary specialist herbivore, Pieris rapae and the generalist, Spodoptera exigua. Responses were measured in leaves within and outside of the damaged orthostichy (leaves sharing direct vascular connections), and compared to those from plants where source-sink transport was disrupted by source leaf removal and by an insertional mutation in a sucrose transporter gene (suc2-1). Following herbivore damage to a single, middle-aged leaf, induction of cell-wall and soluble invertase was most pronounced in young and old leaves within the damaged orthostichy. The pattern of accumulation of phenolics was also predicted by these vascular connections and was, in part, dependent on the presence of source leaves and intact sucrose transporter function. Induction also occurred in leaves outside of the damaged orthostichy, suggesting that mechanisms may exist to overcome vascular constraints in this system. Our results demonstrate that systemic responses vary widely according to orthostichy, are often herbivore-specific, and partially rely on transport between source and sink leaves. We also provide evidence that patterns of induction are more integrated in A. thaliana than previously described. This work highlights the importance of plant vascular architecture in determining patterns of systemic induction, which is likely to be ecologically important to insect herbivores and plant pathogens.The induction of systemic responses in plants is associated with the connectivity between damaged and undamaged leaves, as determined by vascular architecture. Despite the widespread appreciation for studying variation in induced plant defense, few studies have characterized spatial variability of induction in the model species,  Attack by insect herbivores elicits chemical defenses as a critical step in the development of resistance in plants. This induction can occur locally, within a damaged leaf, or in undamaged, system leaves. Many studies have found that systemic defense responses are often highly variable in space and time. Such variation has been attributed to developmental plasticity and plant modularity, to variation in transport of systemic signals, to within-plant genetic variation, and to constraints arising from vascular architecture , 2. BecaIf vascular constraint determines the pattern of plant defense heterogeneity, plant responses to their insect herbivores should often be related to the arrangement of leaves along the stem (phyllotaxy), and the vascular connectivity between these leaves (orthostichy), which are dynamically linked to plant ontogeny . The impNicotiana spp., where salicylic acid transport fluoro-2-deoxy-D-glucose, and 11C-photosynthate administered to plants as 11CO2, to link the movement of potential defense-related substrates to plant architecture in response to wounding and herbivore-related elicitation fluoro-2-deoxy-D-glucose, is capable of traveling to leaves that are non-orthostichous to the tracer administration site, despite that plant’s highly sectorial vascular architecture, which consists of the leaf petiole that received the radiolabel, and the vertical column of younger and older leaves above and below it [inducible than older leaves) or reflect greater connectivity among orthostichies in young leaves whose vascular connections are not fully matured [A. thaliana rosette leaves, it is also likely that within-plant volatile signaling may act as a mechanism to bypass vascular constraints in systemic induction [Populus [A. thaliana uses such mechanisms to facilitate signaling across orthostichies.While vascular architecture was the most consistent predictor of systemic patterns of induction, we found that both a specialist and generalist herbivore frequently induced changes in invertase and phenolic compounds in undamaged leaves located outside of the attacked orthostichy. While plant phyllotaxy links source and sink tissues by a common transport stream, minor connections among other leaves on the stem still exist that may facilitate such responses . Indeed,below it . Inducti matured . Given tnduction . While t[Populus  and lima[Populus , furtherA. thaliana rosettes were best predicted by orthostichy, were often herbivore-specific, and relied, in part, on intact vasculature between source and sink leaves. While induction was most apparent within leaves of the herbivore-damaged orthostichy, our results also provide evidence that induction is sometimes less constrained and more integrated in A. thaliana than previously described. Nevertheless, future studies which aim to describe herbivore-induced plant responses will benefit from taking into account the potential constraints imposed by plant vascular architecture.In summary, we found that systemic responses in S1 FileA. thaliana plants. Summary of 3-way ANOVA comparing total phenolics within two herbivore-damaged leaves and young, systemic leaves of intact A. thaliana rosettes or those with source leaves removed. Table B, Source leaf removal reduces anthocyanin accumulation in A. thaliana plants. Summary of 3-way ANOVA comparing anthocyanin accumulation within two herbivore-damaged leaves and young, systemic leaves of intact A. thaliana rosettes or those with source leaves removed. Table C, Source leaf removal does not influence flavonoid accumulation in A. thaliana plants. Summary of 3-way ANOVA comparing flavonoid accumulation within two herbivore-damaged leaves and young, systemic leaves of intact A. thaliana rosettes or those with source leaves removed. Table D, Middle-aged leaves of suc2-1 plants contain lower levels of total phenolic compounds relative to WT plants. Summary of 3-way ANOVA comparing total phenolics within two herbivore-damaged leaves and young, systemic leaves of wild-type (Ws-2) and mutant (suc2-1) lines. Table E, Middle-aged leaves of suc2-1 plants contain lower levels of anthocyanin relative to WT plants. Summary of 3-way ANOVA comparing anthocyanin accumulation within two herbivore-damaged leaves and young, systemic leaves of wild-type (Ws-2) and mutant (suc2-1) lines. Table F, Middle-aged leaves of suc2-1 plants contain lower levels of flavonoids relative to WT plants. Summary of 3-way ANOVA comparing flavonoid accumulation within two herbivore-damaged leaves and young, systemic leaves of wild-type (Ws-2) and mutant (suc2-1) lines.White arrows point to petioles of young leaves where color differences were evident. Table A, Source leaf removal increases total phenolics in (PDF)Click here for additional data file.


Whether hepatocytes can convert into biliary epithelial cells (BECs) during biliary injury is much debated. To test this concept, we traced the fate of genetically labeled [dipeptidyl peptidase IV (DPPIV)-positive] hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by 4,4’-methylene-dianiline (DAPM) and D-galactosamine  and in DPPIV-chimeric liver model subjected to acute  or chronic biliary injury caused by DAPM and bile duct ligation (DAPM+BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and proliferation of DPPIV-deficient hepatocytes is restricted by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs after biliary injury. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated, and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their hepatocytic identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. In DPPIV-chimeric liver model, periportal hepatocytes expressing hepatocyte nuclear factor-1β (HNF-1β) were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. We conclude that mature hepatocytes contribute to biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation. The liver has an enormous capacity to regenerate after injury –3. In moSeveral major assays have been used to document the transdifferentiation potential of hepatocytes: in vitro clonogenic growth, cell transplantation, forced activation of Notch signaling, and in vivo lineage tracing –19. In vCell proliferation is thought to facilitate cell type conversions . TransplDPPIV-deficient F344 rats were kindly provided by Professor Sanjeev Gupta from the Albert Einstein College of Medicine. Male DPPIV-deficient rats were used as recipient animals. Normal male DPPIV-positive F344 rats  were purchased from National Laboratory Animal Center, Taiwan and used as donor animals. These animals were in-house bred and maintained on standard laboratory chow and daily 12 hour light/dark cycles. All of the animals received humane care in compliance with the guidelines of the National Science Council of Taiwan . All animal experiments were approved by the Institutional Laboratory Animal Care and Use Committee of the National Taiwan University, College of Medicine and College of Public Health and Institutional Animal Care and Use Committee (IACUC), Taipei Tzuchi Hospital, The Buddhist Tzu Chi Medical Foundation .The retrorsine , 4,4’-methylene-dianiline (DAPM) , and D-galactosamine   working solutions were prepared as described previously and used immediately after preparation ,22,26,277/ml) through the portal cannula 1 day or 4 days after D-galactosamine treatment. The rats were left to recover and were sacrificed at 1, 2, and 4 weeks after cell transplantation  two weeks apart, at six and eight weeks of age. D-galactosamine  or DAPM /D-galactosamine  were used to induce acute hepatic injury  or acute hepato-biliary injury  two weeks after the second retrorsine treatment. A long-term cannulation of the main portal vein was implanted one week before induction of acute hepato-biliary injury . The ratantation .7/ml) intraportally 24 hours after D-galactosamine treatment. The rats were left to recover for the next one month. In the DPPIV-chimeric livers, hepatocytes derived from the donor DPPIV-positive cells appear positive for DPPIV, whereas host hepatocytes and bile ductules are uniformly negative  DAPM /D-galactosamine  treatment , (2) DAPM /bile duct ligation  treatment (Chimeric liver+DAPM+BDL), (3) two intraperitoneal injection of retrorsine (30 mg/kg) two week apart, followed by DAPM /D-galactosamine  treatment , or (4) two intraperitoneal injection of retrorsine (30 mg/kg) followed by DAPM /BDL treatment (Chimeric liver+R+DAPM+BDL). The rats were allowed to recover and were sacrificed at the indicated time points.All histochemical and immunohistochemical stainings were performed according to previously described protocols. Primary antibodies are listed in The combination of retrorsine, DAPM, and D-galactosamine was used to induce acute hepato-biliary injury in DPPIV-deficient rats. In our previous study, retrorsine+D-galactosamine treatment  causes acute hepatic injury and induced oval cell response ,30. The Liver tissues removed at 1, 2, and 4 weeks after hepatocyte transplantation were stained for DPPIV and GGT (a marker of BECs)  in seriaTo determine the characteristics of the DPPIV-positive cells arranged in lines or in ducts and their lineage relationship with transplanted DPPIV-positive hepatocytes, we used double immunofluorescence stainings for DPPIV with laminin, hepatocyte marker  , BEC marTransplanted hepatocytes have been thought to be more amenable to lineage conversion than resident hepatocytes . To corrAlthough the transplanted hepatocytes we used were highly pure population, it could not be ruled out that contaminating BECs/hepatic oval cells were the sources of DPPIV-positive BECs in the liver after hepatocyte transplantation and injured DPPIV chimeric liver. To test this possibility, we first performed transplantation experiments using enriched BECs/hepatic oval cells populations containing 40–50% of GGT(+)/CK-19(+) cells, that were isolated from wild-type F-344 rats five days after D-galactosamine treatment. We expected to find easily DPPIV-positive BECs. Unexpectedly, however, DPPIV-positive BECs were found only in low frequency, with a variation from none to five per liver lobe, at both two and four weeks after cell transplantation (n = 4–5 rats at each time point) . At two We next treated rats with DPPIV-chimeric livers with additional two doses of retrorsine to inhibit the proliferation capacity of DPPIV-positive hepatocytes , and then subjected the rats to the two experimental protocols: DAPM+D-gal and DAPM+BDL (n = 4–5 rats at each time point). We expected that the regenerating BECs in these rats with DPPIV-chimeric livers would be uniformly DPPIV-deficient. This was the case. None of the numerous regenerating BECs was stained positive for DPPIV in all analyzed samples from the rats with DPPIV-chimeric livers. Notably, most DPPIV-positive hepatocyte clusters were located distantly from portal areas. Few DPPIV-positive hepatocyte clusters located in the periportal areas, and none of them expressed CK-19 and HNF-1β .These results together support our findings that mature hepatocytes, but not contaminating BECs/hepatic oval cells, are the sources of periportal DPPIV-positive BECs.In this study, we clearly demonstrate that mature hepatocytes can undergo a stepwise conversion into BECs in the liver during repair from acute hepato-biliary injury and chronic biliary injury. Our findings in DPPIV chimeric livers subjected to chronic biliary injury are consistent with previous studies ,17,18. MIn the hepatocyte transplantation experiment in acute hepato-biliary injury model, we observed that the conversion of transplanted DPPIV-positive hepatocytes into BECs is marked by cell proliferation and formation of two-layered plates in the periportal areas. This finding is reminiscent of the ductal plate development observed during embryology . HoweverHowever, it is unknown whether the hepatocyte conversion process in chronic biliary injury requires cellular proliferation. Previous studies and our present study have shown that only hepatocytes residing in the periportal areas are able to convert into BECs ,18,38. IWe observed that in transplantation experiment or DPPIV-chimeric liver model the conversion of DPPIV-positive hepatocytes into BECs occurred in the periportal regions under acute and chronic biliary injury. This finding is in line with the studies in mice that only those hepatocytes residing around the periportal areas are able to convert into BECs ,18,38, eThe findings that only a portion of the DPPIV-positive two-layered plate cells participated in the formation of bile ducts and the two-layered plates connected with DPPIV-positive hepatocyte clusters on the parenchymal side reinforce the phenotypic plasticity of hepatocytes. Indeed, hepatocyte dedifferentiation has been shown to be reversible with changes in the composition of the extracellular matrix . SimilarDespite established transdifferentiation of hepatocytes to BEC in rats with biliary injury in previous studies and ours, the studies in mice using DDC model have generated considerable controversy. In addition, the contribution of hepatocytes to BECs varies largely among studies. The proliferation of host bile ductules is robust in DDC-induced biliary injury in mice and in DAPM-induced biliary injury in rats. Therefore, hepatocytes transdifferentiation may be an injury evasion strategy . We specIt is difficult to estimate the net contribution of mature hepatocytes to biliary regeneration in this study. We can count only DPPIV-positive BECs. However, biliary injury induced by single-dose DAPM was not universal or homogeneous. DPPIV- positive hepatocytes in the transplantation experiment were not exactly entrapped in the injured portal areas. Moreover, DPPIV-positive hepatocytes did not completely occupy the DPPIV chimeric liver mass and uniformly abut on the injured portal region. We may underestimate the numbers of hepatocyte-derived BECs. Our study is more a proof of concept.DPPIV-positive BECs were not observed in DPPIV chimeric liver treated with additional retrorsine and then subjected to the identical acute and chronic biliary injuries. Both DPPIV-positive and DPPIV-deficient hepatocytes are inhibited to proliferate by retrorsine and are unable to undergo transdifferentiation . NotablyA basic concern must be addressed is whether contaminating BECs/hepatic oval cells that composed less than 1% in the transplanted hepatocyte population could be the source of DPPIV-positive BECs in the acute injured livers receiving hepatocyte transplantation. Several evidences argue against this possibility. First, transplantation with enriched DPPIV-positive BECs/hepatic oval cells  produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency (0–5 strings or ducts per liver lobe). Second, these DPPIV-positive BECs or bile ducts were located in the mid-lobular zones. Third, they expressed strong GGT and exclusively BEC-specific markers throughout the study time. These characteristics were remarkably distinct from those of DPPIV-positive hepatocyte- derived BECs. The low efficiency with enriched DPPIV-positive BEC/hepatic oval cell transplantation is consistent with previous studies –25. A poWe did not observe DPPIV-positive hepatocytes acquiring mesenchymal morphology during their conversion into BECs based on histopathological analysis. This could be a limitation of this study, since recent work showed that conversion of hepatocytes into BECs was marked by induction of mesenchymal markers in vitro . HoweverGGT expression was weak in DPPIV-positive plates and BECs derived from transplanted DPPIV-positive hepatocytes throughout the study time. This GGT expression pattern further supports their hepatocyte origin instead of hepatic oval cells/BECs origin. GGT expression is driven by several promoters during liver development. Hepatoblasts and hepatic precursor cells lose the promoters that drive GGT expression when they differentiate into hepatocytes . Recent A recent study by Isse et al. showed that hybrid transitional hepatocytes existed in the periportal area of normal human liver . We cannBased on this study, we conclude that mature hepatocytes contribute to the biliary regeneration in the environment of acute and chronic biliary injury through a ductal plate configuration without the need of exogenously genetic or epigenetic manipulation. Our finding should be valuable in developing hepatocyte transplantation therapy for hepato-biliary diseases.All histochemical and immunohistochemical stainings were performed according to previously described protocols. Primary antibodies are listed in S1 Fig(A) Hepatocytes and bile duct epithelial cells are stained red (positive) for DPPIV in the normal Fisher rat liver, the former in a bile canalicular pattern and the latter in a diffuse cytoplasmic expression pattern. (B) Hepatocytes and bile duct epithelial cells are negative for DPPIV staining in the DPPIV-deficient rat liver. (C) Bile canaliculi of donor hepatocytes are stained red for DPPIV, and bile ductules are uniformly negative for DPPIV staining in the DPPIV chimeric liver of DPPIV-deficient rats. .(TIF)Click here for additional data file.S2 FigLiver sections are analyzed using double immunofluorescence staining for CK-19 (green)/C/EBP-α (red) and OV6 (green)/Sox9 (red) in retrorsine+D-galactosamine treated rats , and retrorsine+DAPM+D-galactosamine treated rats . .(TIF)Click here for additional data file.S3 FigShown are original single color and merged images of (TIF)Click here for additional data file.S4 FigShown are representative figures of double immunofluorescence staining for DPPIV (green)/Sox9 (red) in serial sections in R+DAPM+D-gal treated liver at 2 weeks after hepatocyte transplantation. Transplanted DPPIV-positive cells express Sox9 (arrow) at 2 weeks after hepatocyte transplantation in R+DAPM+D-gal treated liver. .(TIF)Click here for additional data file.S5 FigShown are representative figures of (A) Lgr5(+) (arrow) staining in normal colon (positive control), (B) dual immunofluorescence staining for CK-19(+)/Lgr5(+) in bile duct ligation liver (BDL) at 3 weeks (positive control), and (C) dual immunofluorescence stainings for CK-19(+)/DPPIV(+) and DPPIV(+)/Lgr5(-) in R+DAPM+D-gal treated liver sections at 2 weeks after hepatocyte transplantation. Transplanted DPPIV-positive cells did not express Lgr5 during their conversion into BECs in R+DAPM+D-gal-treated livers. .(TIF)Click here for additional data file.S6 FigShown are representative figures of vimentin staining in bile duct ligation liver (BDL) at 4 weeks (positive control) and of dual immunofluorescence stainings for DPPIV (green)/vimentin (red) in R+DAPM+D-gal treated liver sections at 1 and 2 weeks after hepatocyte transplantation. .(TIF)Click here for additional data file.


The levels of circulating miRNAs are affected by metabolic changes in obesity, as well as in diet-induced weight loss. Circulating miRNAs are transported by high-density lipoproteins (HDL) but the regulation of HDL-associated miRNAs after diet-induced weight loss has not been studied. We aim to determine if HDL-associated miR-16, miR-17, miR-126, miR-222 and miR-223 levels are altered by diet-induced weight loss in overweight and obese males.HDL were isolated from 47 subjects following 12 weeks weight loss comparing a high protein diet  with a normal protein diet . HDL-associated miRNAs  at baseline and after 12 weeks of weight loss were quantified by TaqMan miRNA assays. HDL particle sizes were determined by non-denaturing polyacrylamide gradient gel electrophoresis. Serum concentrations of human HDL constituents were measured immunoturbidometrically or enzymatically.miR-16, miR-17, miR-126, miR-222 and miR-223 were present on HDL from overweight and obese subjects at baseline and after 12 weeks of the HP and NP weight loss diets. The HP diet induced a significant decrease in HDL-associated miR-223 levels (p = 0.015), which positively correlated with changes in body weight . Changes in miR-223 levels were not associated to changes in HDL composition or size.HDL-associated miR-223 levels are significantly decreased after HP diet-induced weight loss in overweight and obese males. This is the first study reporting changes in HDL-associated miRNA levels with diet-induced weight loss. A recent study from our laboratory showed that both a long-term low-fat high protein (HP) diet and a low-fat high carbohydrate (HC) diet reduce body weight in overweight and obese males. HoweverMicroRNAs (miRNAs) are small non-coding RNAs that suppress gene expression through post-transcriptional regulation, 3. PlasMultiple miRNAs have been associated with obesity and metabolic disorders in humans. These include miR-16, miR-17, miR-126, miR-222 and miR-223–11. In fet al. showed that baseline expression of several miRNAs including miR-223, in peripheral blood mononuclear cells of obese women can predict the response to a hypocaloric diet[It has been shown that circulating miRNAs can predict weight loss in obese subjects and therefore could be used as prognostic biomarkers in response to diet. In fact, Milagro oric diet. Interesoric diet.As there are no reports of changes in HDL-associated miRNA levels with diet-induced weight loss in the literature, we have examined the effects of weight loss on HDL levels of miR-16, miR-17, miR-126, miR-222 and miR-223, all of which have a known association with obesity and are HDL-associated[47 men were selected from a cohort of 120 men who participated in a weight loss study contrasting a high protein (HP) diet (30% of energy) with a normal protein (NP) diet (20% of the energy). The samples were selected randomly based on sample availability (n = 27 for NP group and n = 20 for HP group) for both week 0 and week 12. The outcomes of the weight loss study are fully reported[Body weight was measured using calibrated electronic digital scales  and body composition  was measured by dual-energy X-ray absorptiometry [Serum lipids , triglycerides, and plasma glucose were measured using commercial enzymatic kits  on a Hitachi 902 autoanalyzer . Low-denHDL were isolated from 600 μl of serum by immunoprecipitation using goat anti-apoA-I beads as previously described. Briefly, human serum was applied to a column containing goat anti-human apoA-I antibody covalently coupled to Cyanogen bromide (CNBr)-activated Sepharose 4B (Amersham Pharmacia Biotech). To remove proteins non-specifically bound to the beads, the column was washed 10 times with 1X Tris Buffered Saline (TBS). HDL that bound to the column were eluted with stripping buffer (0.1 M acetic acid) and neutralized immediately with 1 M Tris, pH 11 . Samples were further concentrated using Amicon Ultra-15 centrifuge filter unit  and Amicon Ultra-0.5, ultracel-10 membrane .HDL were isolated from serum using the Polyethylene Glycol 6000 (PEG-6000) method. BrieflyHDL particle sizes were determined using 3/40% non-denaturing polyacrylamide gradient gel electrophoresis (GGE) and Coomassie staining, with reference to high molecular weight standards of known diameter.Caenorhabditis elegans (Cel) miR-39 (which was spiked into the samples after the Qiazol step) and HDL total protein concentration determined by the BCA assay . Results are expressed as 2-(Ct(microRNA)-Ct(Cel-miR-39)).HDL-miRNA levels at baseline and after 12 weeks of weight loss were assessed by real-time PCR TaqMan miRNA assays. Briefly, total RNA was isolated from HDL using Qiazol miRNAeasy kits (Qiagen) as previously described. Total Rp<0.05.Data analysis was performed using SPSS 22 . Data were examined for normality based on skewness and kurtosis before analysis. Non-normally distributed variables (fold changes of miR-222 and miR-223) were normalised prior to analysis using natural logarithmic (ln) transformation. Comparison of clinical characteristics between two independent groups at baseline was performed using student t-test. To evaluate the effect of diet treatment on clinical characteristics, two-tailed paired t-test was used to assess significant differences between baseline and after 12 weeks of treatment. As miRNA levels at baseline were highly skewed, analysis focused on the fold changes over 12 weeks, which were calculated as the ratios of the miRNA levels at 12 weeks to those at baseline, and were assessed by one-sample t-test. Multiple testing correction was performed by Bonferroni correction for the two diet treatment groups, in which the threshold of p value for significance was <0.025. Correlations between fold changes of miRNA levels and changes in clinical characteristics over 12 weeks were performed using bivariate Pearson correlation coefficients. Results for the serum concentration of human HDL constituents were analyzed by 2-way ANOVA with Tukey’s multiple comparison test. Statistical significance was set at p = 0.013). Over the 12 weeks of weight loss, body weight, total cholesterol, LDL cholesterol, triglyceride levels, fasting glucose, total fat mass, total lean mass, total body fat percentage, calculated fat mass, calculated lean mass, calculated fat free mass, total abdominal fat mass and abdominal fat percentage significantly decreased, whereas total body fat free mass percentage increased in both HP and NP groups . When compared to the NP diet, the HP diet group showed a greater decrease in total body fat percentage and calculated fat mass, and a greater increase in total body fat free mass percentage .The 47 individuals included in this study were older than the other 73 individuals who commenced the weight loss study  miR-223 levels predict the response to diet-induced weight loss with basal levels of miR-223 being lower in people that did not respond to the weight loss intervention[et al. was performed in women, who received a hypocaloric diet  for 8-weeks and the results were reported for miRNAs in PBMCs[Circulating miR-223 levels have previously been associated with diet-induced weight loss. Indeed,ervention. Contrar in PBMCs. This su in PBMCs and imme in PBMCs.Although circulating miRNAs are stable in plasma, and their levels can predict human diseases , 21, we The present study also examined other HDL-associated miRNAs  that are associated with obesity and/or weight loss but they were not significantly altered with diet. The explanation for this lack of change could be related to the type of weight loss intervention. Indeed, the effects of weight loss interventions on plasma (as opposed to HDL-associated) miRNA levels suggest that different weight loss interventions  may differentially affect circulating miRNAs levels, 23. ForFurther studies in other clinical settings with larger sample size are needed to confirm the findings in the present study. Further studies targeting the role of HDL-associated miR-223 will also provide insight into the effect of dysregulated HDL-miRNAs in obesity-associated diseases and the metabolic changes associated with weight loss.


Among the gene ontology (GO) terms significantly enriched in Macro-Dissected-DEGs (FDR < 10%), 18 immune-related terms were enriched in Inconsistent-DEGs. DEGs associated with proliferation could reflect common changes of malignant epithelial and stromal cells; DEGs associated with immune functions are sensitive to the percentage of malignant epithelial cells in macro-dissected tissues. A prognostic signature which was insensitive to the cellular composition of macro-dissected tissues was developed and validated for ER+ breast patients.Cancer tissue sampling affects the identification of cancer characteristics. We aimed to clarify the source of differentially expressed genes (DEGs) in macro-dissected cancer tissue and develop a robust prognostic signature against the effects of tissue sampling. For estrogen receptor (ER)+ breast cancer patients, we identified DEGs in macro-dissected cancer tissues, malignant epithelial cells and stromal cells, defined as Macro-Dissected-DEGs, Epithelial-DEGs and Stromal-DEGs, respectively. Comparing Epithelial-DEGs to Stromal-DEGs  < 10%), 86% of the overlapping genes exhibited consistent dysregulation (defined as Consistent-DEGs), and the other 14% of genes were dysregulated inconsistently (defined as Inconsistent-DEGs). The consistency score of dysregulation directions between Macro-Dissected-DEGs and Consistent-DEGs was 91% (P-value < 2.2 × 10 Macro-dissected cancer tissues contain both carcinoma cells and stromal cells with distinct gene expression patternsTo avoid this uncertainty, several deconvolution algorithms have been proposed to decompose gene expression profiles of macro-dissected samples into cell type-specific subprofiles4810111214In this study of estrogen receptor (ER)+ breast cancer patients, we identified DEGs for macro-dissected cancer tissues, malignant epithelial cells and stromal cells, defined as Macro-Dissected-DEGs, Epithelial-DEGs and Stromal-DEGs, respectively, and compared them to reveal the cellular source of Macro-Dissected-DEGs. Then, we evaluated the correlation between expression measurements of DEGs identified in macro-dissected cancer tissues and the proportions of tumor cells in the tissues. Finally, we developed a prognostic signature based on the relative order of gene expression values that commonly occur in malignant epithelial cells and stromal cells compared with normal controls.−12, hypergeometric test, see Methods the equation −16, Using the Rankprod algorithm (see Methods), with 10% FDR control, we extracted DEGs in macro-dissected ER+ breast cancer tissues compared with normal controls from three datasets , among which 97% were dysregulated in the same direction in the two lists. This result indicates that the DEGs of epithelial cells in two independent datasets were significantly reproducible , among which 92% were dysregulated in the same direction  among the 790 overlapping genes between Macro-Dissected-DEGs and Consistent-DEGs, which suggested that Consistent-DEGs for both epithelial and stromal cancer cells can be largely reflected in macro-dissected breast cancer tissue. In contrast, among the 91 overlapping genes between Macro-Dissected-DEGs and Inconsistent-DEGs, the consistency score was only 51.7% , which suggested that the differential expression signals of such Inconsistent-DEGs, when detected in macro-dissected tissues, were sensitive to the tissue compositions of epithelial and stromal cells. Obviously, the differential expression signals detected in macro-dissected tissues would be consistent with the epithelial DEGs only when the proportion of stromal cell is sufficiently small; otherwise, they would be affected by the stromal cells. Thus, when detected in macro-dissected tissues, the differential expression signals of these Inconsistent-DEGs would be different on datasets of macro-dissected tissues with different composition of epithelial and stromal cells and lack biological interpretation.Among the 1251 overlapping genes between Epithelial-DEGs and Stromal-DEGs, 86.2% exhibited consistent dysregulation directions (defined as Consistent-DEGs), and the remaining 13.8% were dysregulated in opposite directions (defined as Inconsistent-DEGs). Then, we compared the Consistent-DEGs and Inconsistent-DEGs with Macro-Dissected-DEGs. The consistency score was 90.6%  of Gene Ontology (GO), using the GO-function algorithmFor the 376 gene expression profiles extracted from TCGA for ER+ breast cancer tissues which contained 60–100% tumor cell, we evaluated the correlation between the expression measurements of DEGs and the proportions of tumor cell by Pearson correlation analysis (see Methods). The results indicate that, when detected in macro-dissected tissues, the expression levels of 39.8% Consistent-DEGs and 47.8% Inconsistent-DEGs were significantly correlated with the proportions of tumor cell in the macro-dissected cancer tissues . Thus, the measurement values of both Consistent-DEGs and Inconsistent-DEGs expression were sensitive to the tissue composition of epithelial and stromal cells.Nagalla et al.Reyal et al.We extracted the immune signatures developed by −16, binomial test). We defined these gene pairs as Consistent-Gene-Pairs. When the Consistent-Gene-Pairs were compared with those extracted from Lcm-Data2, M-Data1, M-Data2 and M-Data1, the consistency scores were all greater than 99.70% . The similar process was performed for stromal cells. These two lists contained 56,268 overlapping gene pairs, among which 99.9% exhibited the same reversal patterns in malignant epithelial and stromal cells compared with normal controls, which was significantly more than expected by chance http://cancergenome.nih.gov/)28298323334The ten gene expression datasets used in this study were downloaded from the Gene Expression Omnibus  from each dataset was processed using the Robust Multi-array Average (RMA) algorithm for background adjustment with quantile normalizationThe Bioconductor package RankProdL1 genes and DEG list 2 with L2 genes have k overlapping genes, the probability (P1) of observing at least k overlapping genes by chance can be calculated according to the following cumulative hypergeometric distribution model:If DEG list 1 with L represents the number of the background genes commonly detected in the datasets from which the DEGs are extracted. The two DEG lists were considered to be significantly overlapping if P1 < 0.05.where P2) of observing at least s DEGs with the same dysregulation direction across the two datasets from k randomly selected genes was calculated according to the following cumulative binomial distribution modelIf a DEG exhibited the same dysregulated direction (up- or down-regulated) in the two DEG lists, it was considered consistent across the datasets. We defined a dysregulation consistency score as the percentage of consistent DEGs in the overlapping DEGs between the two DEG lists. The probability (p represents the random possibility (here 0.5) of one DEG having the same dysregulated direction across two DEGs lists. A dysregulation consistency score was considered significant if P2 < 0.05.where To derive biologically relevant, non-redundant terms from statistically significant terms for a disease, GO-functionExtracted from TCGA, the 376 gene expression profiles for ER+ breast cancer tissues included the data of tumor cell proportions. Using these samples, for the Consistent-DEGs and Inconsistent-DEGs respectively, we applied Pearson’s correlation analysis to detect genes whose expression levels were significantly correlated with tumor cell proportions. Then, the percentages of DEGs that were significantly correlated with tumor cell proportions were calculated for Consistent-DEGs and Inconsistent-DEGs, respectively.For a pair of genes, gene A and gene B, we used Fisher’s exact test to evaluate whether the frequency of samples with a higher (or lower) expression level of gene A than gene B in disease samples was significantly different from that in the corresponding normal controls. The P-values were adjusted using the the Benjamini-Hochberg procedureThen, based on the expression profiles of ER+ breast cancer with RFS information, a univariate Cox regression model was used to select gene pairs among the Consistent-Gene-Pairs with a relative order of expression that was significantly correlated with the RFS; these pairs were defined as prognostic gene pairs. The prognostic classifier was constructed according to the following rule: a patient was classified into the low risk group if there were significantly more prognostic gene pairs classifying her as low risk ; otherwise, the patient was classified into the high risk group. The multivariate Cox proportional hazards regression model was performed to determine whether prognostic gene pairs are an independent prognostic factor in predicting RFS after adjusting for clinical factors, such as age, grade and tumor size.http://www.r-project.org/).All statistical analyses were performed using the R 2.15.3 .


Tumors employ multiple mechanisms to escape immune surveillance and thus hamper cancer immunotherapies. Even when immune responses are generated with tumor vaccines the anti-tumor therapeutic outcome is often not feasible due to tumor-mediated immune suppression. These inhibitory mechanisms involve co-inhibitory receptor-ligand interactions, such as PD-1/PD-L1, secretion of inhibitory molecules, such as TGFβ, IL-10, IDO, and recruitment of suppressive cells, such as regulatory T cells (Treg), myeloid derived suppressor cells (MDSC), etc. Thus, successful cancer immunotherapy requires not only induction and enhancement of effector immune response but also simultaneous targeting of suppressor arm of immune system.Therapeutic and immune efficacy of mono- and combinational immunotherapies were tested in E7 antigen expressing TC-1 mouse tumor model. Tumor growth, survival, as well as peripheral and tumor-infiltrating immune cell profiles after immunotherapy were assessed.We developed multiple immune corrective strategies to target various tumor-mediated immune inhibitory mechanisms that enhance anti-tumor immunity and restructure tumor microenvironment to allow effector cells to function potently. We evaluated the immune and therapeutic efficacy of multiple combinational therapies, including blocking and agonist antibodies to co-inhibitory/co-stimulatory molecules, such as PD-1, PD-L1, OX40, CTLA-4, GITR, inhibitors and neutralizing antibodies to inhibitory cytokines/molecules, such as IL-10, TGFβ, IDO, and small molecules for selective inhibition of Tregs. In addition to evaluation of anti-tumor efficacy we also investigated cellular and molecular mechanisms of action for these agents when combined with different vaccine formulations and explored the interactions between compounds within combinational immunotherapies in animal tumor models.We are demonstrating the importance of treatment sequence and scheduling when multiple agents are combined which requires full understanding of mechanisms of action for each component and can lead to the successful translation of developed treatment into the clinic.


Treatment options for patients with Epstein-Barr Virus-driven lymphoproliferative diseases (EBV-LPD) are limited. Chemo-immunotherapeutic approaches often lead to immune suppression, risk of lethal infection and EBV reactivation, thus it is essential to identify agents that can deliver direct anti-tumor activity while preserving innate and adaptive host immune surveillance. Silvestrol possesses direct anti-tumor activity in multiple hematologic malignancies while causing minimal toxicity to normal mononuclear cells. However, the effects of silvestrol on immune function have not been described. We utilized in vitro and in vivo models of EBV-LPD to simultaneously examine the impact of silvestrol on both tumor and normal immune function. We show that silvestrol induces direct anti-tumor activity against EBV-transformed lymphoblastoid cell lines (LCL), with growth inhibition, decreased expression of the EBV oncogene latent membrane protein-1, and inhibition of the downstream AKT, STAT1 and STAT3 signaling pathways. Silvestrol promoted potent indirect anti-tumor effects by preserving expansion of innate and EBV antigen-specific adaptive immune effector subsets capable of effective clearance of LCL tumor targets in autologous co-cultures. In an animal model of spontaneous EBV-LPD, silvestrol demonstrated significant therapeutic activity dependent on the presence of CD8-positive T-cells. These findings establish a novel immune-sparing activity of silvestrol, justifying further exploration in patients with EBV-positive malignancies. Epstein-Barr Virus (EBV) is an oncogenic B-lymphotropic virus associated with Burkitt's lymphoma, non-Hodgkin's and Hodgkin's lymphomas, nasopharyngeal and gastric carcinomas, and post-transplant lymphoproliferative disease (LPD) . Followiin vivo activity in the B-cell malignancies chronic lymphocytic leukemia, acute lymphoblastic lymphoma [Silvestrol  is a unilymphoma  and mantlymphoma , and alslymphoma . To datein vitro and in vivo models of EBV-LPD. This highly unusual activity suggests that silvestrol may provide an entirely new immune-potentiating therapeutic strategy for this histologic subset of aggressive lymphomas.Here we show that silvestrol promotes direct anti-tumor activity against EBV-LPD by blocking oncogenic pathways driven by the EBV gene product, latent membrane protein-1 (LMP-1). Furthermore, we demonstrate that silvestrol preserves the anti-tumor function of innate immune effectors as well as antigen-specific adaptive immune effectors in both 50) of approximately 40 nM at 72 hr. Recent pharmacokinetic work in mice indicates that a 10 nM plasma concentration of silvestrol is attainable in vivo [We first evaluated silvestrol's direct anti-tumor activity in LCL derived from malignant EBV-LPD tumors that spontaneously developed in SCID mice engrafted with PBMC from EBV-seropositive donors , 16, 21. in vivo . TherefoThe virally-encoded transmembrane oncoprotein LMP-1 acts as a constitutively active receptor of the TNF-R family , promoteLMP-1 is known to constitutively activate multiple pro-survival signaling pathways including NF-κB, PI3K/AKT, STAT1 and STAT3 through its cytoplasmic C-terminal-activating regions (CTAR1 and 2), biologically mimicking the TNF family receptor CD40 , 34-37. We next examined the effects of silvestrol on known short half-life proteins, as we and others have reported in other cell types , 14. Cyc+ cytotoxic T-cells (CTL) and CD4+ helper T-cells with silvestrol treatment  were created by lethally irradiating LCL and plating in the presence of autologous PBMC (1:1 ratio). Under these conditions, memory adaptive components of PBMC become activated and expand in response to the antigenic stimuli from the LCL , 16, 21.pulation . These r−/CD19+) appear in the bottom right quadrant of each plot; PBMC-derived effector populations (CD19-) appear in the left quadrants. In untreated (vehicle control) conditions, transformed LCL cells proliferated and matched the expansion of effector cell subsets, approximately maintaining the 1:1 ratio . Figure o Figure , althougs Figure  and CD56s Figure  expandeds Figure . Althougs Figure .Similar cultures were set up and treated with a single dose of fludarabine (active metabolite 2-fluoro-ara-A), as this agent has been used clinically to treat LPD. However, unlike silvestrol, 2-fluoro-ara-A treatment resulted in outgrowth of LCL . It alsoWe next worked to identify which effector subsets mediate the anti-LCL effect in the presence of silvestrol. Cells expressing CD8 (CTL), CD14 (monocytes) or CD56 (NK cells) were depleted from PBMC using immunomagnetic beads; mock depletion with biotin-only beads was included as a control. Effector cell numbers equivalent to the total number in the mock-depleted condition were added 1:1 with autologous LCL and incubated with 0 or 10 nM silvestrol for 10 days. Depleted cultures in the absence of silvestrol showed varying outgrowth of LCL; however, silvestrol-treated CoCx all produced a similar loss of LCL. These results indicate that each of these immune cell subsets participates in the anti-tumor activity of the autologous effector population .To further explore the differential effect of silvestrol on the proliferative capacity of tumor targets and effector populations, LCL or PBMC were stained with the membrane dye CFSE and equal numbers were plated with their unstained autologous counterparts . Cultures were treated with 0 or 10 nM silvestrol for 3 or 5 days Figure . ConversWe next analyzed immune cell function as part of the indirect anti-tumor activity of silvestrol. Viable LCL cells were stained with CFSE, and then incubated at an effector-to-target ratio of 20:1 with autologous PBMC that had been expanded in the presence of irradiated LCL, with or without silvestrol, for 14 days. After a 4 hr incubation, cells were stained with the viability dye 7-AAD and washed. Direct cytotoxic activity of effectors against the CFSE-labeled LCL targets was measured by gating on CFSE-positive events and measuring 7-AAD-positive cells. As shown in Figure + NK cells) and fresh, viable LCL were then incubated separately with silvestrol or vehicle control for 18 hr and washed. Cells were then combined, and after a 4 hr incubation, cytotoxicity assays were performed as described above. Silvestrol-treated effectors showed no decrease in their ability to kill LCL targets compared to untreated effectors  or the negative control antibody herceptin (anti-HER2) in the presence of NK cells. After 4 hr, ADCC was evaluated by gating on CFSE events (stained targets) and analyzing the percentage of 7-AAD positive cells. Rituximab-mediated ADCC of NK cells was similar between effectors expanded with versus without silvestrol , CD27 (co-stimulatory molecule that binds CD70), CD28 (CD80/86 activating binding partner), or LFA-1 (integrin that is bound by ICAM-1 to stabilize the synapse). Although silvestrol treatment decreased expression of the inhibitory molecule CTLA-4 on some CTL, these effects were more variable between samples and thus overall did not reach significance  was performed on spleen cells from 2 mice in each group. Mice in the vehicle control group showed substantial tumor infiltration of the spleen without obvious nodal involvement, whereas no tumor cells could be detected in the spleens of mice from the silvestrol-treated group (not shown). By 8 weeks post-engraftment, spleens from mice in the control group showed profound splenomegaly characterized by human B-cell infiltration, while spleens from silvestrol-treated animals exhibited normal spleen mass , CD14 (monocytes), or CD56 (NK cells). Mock depletion was performed using biotin-conjugated beads as a negative control. Efficiency of depletion was verified by flow cytometric analyses to be greater than 90% , nor did it reduce tumor burden as measured by spleen mass (not shown). Mice receiving CD14+ (monocyte) or CD56+ (NK cell) depleted engraftments exhibited an intermediate response to silvestrol treatment  that did not reach statistical significance with this number of animals. These data indicate that silvestrol is indirectly mediating protection through immune surveillance mechanisms, and that at least CD8+ T-lymphocytes are essential in the silvestrol-mediated clearance of tumor.To further investigate the importance of immune effector cells in silvestrol's dramatic protective effect in EBV-LPD, the previous than 90% . A totaleviously , were eneviously . Two weein vitro and in vivo models of EBV-LPD.EBV is associated with a broad spectrum of benign and malignant diseases that, collectively, represent a growing number of cases in immunocompromised individuals and in our aging population -48. Currin vivo EBV-LPD model revealed a remarkable survival advantage with silvestrol treatment, far greater than we and others have previously shown with silvestrol using xenografts in immune deficient mice [in vivo studies with silvestrol to date have used tumor xenografts in immune deficient mice, this crucial aspect was previously unknown.Our principle findings show that direct anti-proliferative activity of silvestrol toward LCL at physiologically achievable concentrations is moderate. However, these low concentrations of silvestrol, when added to co-cultures of LCL and autologous PBMC, produce complete ablation of the malignant LCL cells. Importantly, the ent mice , 14. TogThe direct growth inhibitory activity of silvestrol as well its ability to sensitize LCL to immune-mediated killing appears to be associated with a loss of the LMP-1 oncoprotein. Drugs and cytotoxic T-cell preparations directly targeting LMP-1 have been shown to prevent metastasis, promote apoptosis and enhance radiosensitivity, identifying LMP-1 as a potential therapeutic target for EBV-driven malignancies -30. The in vitro model in which EBV-driven lymphoma cells (LCL) are incubated with autologous PBMC. Left untreated, the LCL ultimately outpace the expanding effector populations, and overtake the culture. With the addition of as little as 10 nM silvestrol, we observed complete ablation of viable LCL in these autologous co-cultures while effector cell populations (i.e. CD8+ CTL and CD56+ NK cells) were maintained; importantly, this effect of silvestrol on LCL is greatly reduced in the absence of effector cells. Furthermore, proliferation assays in silvestrol-treated co-cultures show a substantial decrease in proliferation of LCL targets without a corresponding change in proliferation of effector cells. We are currently investigating the differential activity of silvestrol on cell cycle checkpoints in tumor versus immune effector populations.It has been previously reported that silvestrol sensitizes tumor cells to classical chemotherapeutic agents including doxorubicin , 11. HowBesides affecting absolute cell numbers, classical chemotherapy is known to have a deleterious effect on cancer-relevant immune cell function including NK cell-mediated ADCC , 52. Ourin vivo studies using the hu-PBL SCID model provide significant insight into the efficacy of silvestrol in treating EBV-LPD. Importantly, our in vivo experiment using grafts depleted of effector cell subsets establishes that CD8+ CTL are necessary for full silvestrol efficacy in this model. The moderate loss of protection when other lymphocyte subsets are depleted suggests that silvestrol is sensitizing tumor cells for clearance through other immune effectors as well, although a larger study is required to address this. Furthermore, in vivo experiments using animals with intact immune systems did not show significant alteration in total lymphocytes or modulation in lymphocyte populations with silvestrol treatment, consistent with previously reported data [Our ted data . TogetheIn summary, we present new information regarding the efficacy of the translation inhibitor silvestrol in EBV-LPD and demonstrate that this efficacy is largely due to the maintenance of number and function of both innate and adaptive anti-tumor immune components. Additionally, silvestrol promotes modest direct anti-tumor activity against EBV-transformed lymphoma cells, likely by depleting the oncogenic viral protein LMP-1 and interfering with downstream survival pathways. Other chemotherapeutic regimens currently in use for the treatment of EBV-LPD display a non-specific cytotoxicity both to the tumor target as well as immune effectors, leading to elimination of potent memory anti-tumor responses. This effect leaves patients vulnerable to disease relapse and at increased risk of serious and potentially lethal opportunistic infections. The ability of silvestrol to simultaneously exert direct anti-proliferative activity against EBV-transformed tumor cells while sparing adaptive and innate immune effector function is highly unusual if not unique, and shows promise for future clinical translation.Investigations were conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines, and were approved by the OSU institutional review board.Silvestrol was isolated as described . The actin vivo by engrafting severe combined immune-deficient (SCID) mice  with human peripheral blood mononuclear cells (PBMC) from healthy EBV-positive donors [EBV-transformed lymphoblastoid cell lines (LCL) were derived e donors , 16. Co-LCL were subjected to immunoblotting as described . AntibodTM assay .MTS assays to measure mitochondrial function were performed using the CellTiter96Cells were co-stained with LIVE/DEAD reagent  and hu-CD3-APC, as well as hu-CD4-PE, hu-CD8-PE, hu-CD19-PE , or hu-CD56-PE . Events were gathered by gating on cells negative for the LIVE/DEAD stain on an FC500 cytometer (Beckman-Coulter). Cell viability also was measured by flow cytometry using annexin-V-FITC and propidium iodide (PI) (BD Biosciences). Human HLA-B8 tetramers complexed with immunodominant peptide from BZLF-1 (RAKFKQLL) and conjugated with allophycocyanin (APC)  were proTM, Invitrogen) prior to being mixed with effectors from the irradiated CoCx at an effector:target ratio of 20:1. Cells were stained with the viability dye 7-aminoactinomycin D (7-AAD) (BD Biosciences) and washed. Cytotoxicity was measured by gating on CFSE-positive events and measuring 7-AAD-positive cells. For antibody-dependent cell-mediated cytotoxicity (ADCC) assays, co-cultured effectors were natural killer (NK) cell-enriched and were incubated in the presence or absence of 5 μg/ml rituximab or the irrelevant control antibody herceptin .Non-radioactive flow cytometry-based cytotoxicity assays were performed as described , 20. CoCCFSE-stained PBMCs were incubated with unstained autologous LCLs, and CFSE-stained LCLs were incubated with autologous unstained PBMCs. At days 3 and 5, cells were stained with anti-CD19-APC for CFSE-LCL, or anti-CD56-APC or anti-CD8-APC for CFSE-PBMC, and analyzed by flow cytometry.The Hu-PBL-SCID model has been described , 16, 21.ICAM-1 (CD54) mRNA levels were determined using the Viia7 Real-Time PCR System , using TaqMan Gene Expression Assays for ICAM-1 and TBP (control) (Applied Biosystems).To account for correlations among observations from the same donor, linear mixed effects models were used to estimate the effect of silvestrol on apoptosis and cell proliferation. Similarly, differences in rituximab-mediated NK cell ADCC activity in effectors expanded in the presence versus absence of silvestrol were examined using mixed effects models. Differences in cell numbers of each of the four subsets  were compared between cells incubated with versus without silvestrol using paired t-tests. Differences in surface expression of immunological synapse proteins in activated effectors incubated with versus without silvestrol were also examined using paired t-tests, with an adjusted significance level of 0.01 to control overall Type I error. The proportions of mice alive at end of study were compared between the vehicle control and silvestrol-treated groups using Fisher's exact test. Finally, spleen size differences between silvestrol-treated and control mice were assessed using a two-sample t-test. All analyses were performed using SAS/STAT software version 9.2 .


Bortezomib inhibits the ubiquitin/proteasome pathway to achieve its anti-cancer effect and its well characterized activity is the NF-κB inhibition through which the anti-apoptotic bcl-2 expression is down-regulated and apoptosis is subsequently induced. However, the downstream molecular targets of bortezomib are still incompletely defined. Because telomere stabilization via activation of telomerase, induction of telomerase reverse transcriptase (hTERT) and appropriate expression of shelterin proteins is essential to cancer development and progression, we investigated the effect of bortezomib on telomere homeostasis/function in malignant cells. The bortezomib treatment of leukemic (HEL) and gastric cancer cells (BGC-823) led to significant inhibition of hTERT and telomerase expression, widespread dysregulation of shelterin protein expression, and telomere shortening, thereby triggering telomere dysfunction and DNA damage. hTERT over-expression attenuated bortezomib-induced telomere shortening, abnormal shelterin expression and telomere dysfunction. Importantly, bortezomib-mediated apoptosis of malignant cells was partially prevented by hTERT over-expression. Mechanistically, hTERT first robustly enhances bcl2 expression and maintains significantly high residual levels of bcl2 even in bortezomib-treated HEL cells. Second, hTERT protects against bortezomib-induced DNA damage. Our findings collectively reveal a profound impact of bortezomib on telomere homeostasis/function. Down-regulation of hTERT expression and telomere dysfunction induced by bortezomib both contribute to its cancer cell killing actions. It is evident from the present study that hTERT can confer resistance of malignant cells to bortezomib-based target cancer therapy, which may have important clinical implications. The ubiquitin/proteasome system is the major proteolytic site in mammalian cells and plays essential parts in cellular homeostasis and various physiological processes. Evidence has accumulated that the aberrant proteasome-dependent proteolysis contributes to oncogenesis, and targeting the proteasome pathway is thus an attractive anti-cancer strategy. Indeed, bortezomib, as the first proteasome inhibitor for cancer therapy, has proved successful in the treatment of hematological and other malignancies –6. It ishTERT gene, which encodes the key telomerase catalytic component [Human telomeres are nucleoprotein complexes consisting of up to 15 - 20 kb tandemly repetitive TTAGGGs and associated proteins –10. Six omponent –9. In shomponent –9. In adomponent –17.Because hTERT/telomerase-mediated telomere stabilization plays a key role in cancer development and progression, we are interested in potential effects of bortezomib on telomere homeostasis and function. A previous study showed that bortezomib down-regulated hTERT expression and telomerase activity in subsets of multiple myeloma (MM) cells , howeverhTERT and hTER are the core of the telomerase complex and essential to telomerase activity. hTERT expression was previously shown to be down-regulated by bortezomib in subsets of myeloma cell lines . To see In addition to hTERT/telomerase, shelterin proteins binding to telomere are also essential to telomere length maintenance and function . We thusP = 0.046) and BGC-823 cells  in the presence of bortezomib , although the difference was close to but did not reach a statistically significant levels .Having observed widespread effects of bortezomib on the expression of hTERT and shelterin proteins, we then wanted to determine whether telomere length and function was affected in bortezomib-treated cells. Flow-FISH analyses revealed that a significant telomere shortening did occur in HEL cells .Given the above results, we wanted to determine whether the ectopic expression of hTERT/telomerase is capable of preventing telomere dysfunction induced by bortezomib. HEL and BGC-823 cells were infected with hTERT lenti-viral and retro-viral vectors, respectively, to make hTERT-over-expressed variants, and they were then treated with bortezomib. Cells expressing ectopic hTERT contain approximately the double amount of telomerase activity compared with their empty vector-infected counterparts Figure . Upon thWe further sought to determine whether hTERT over-expression was capable of antagonizing bortezomib-induced dysregulation of shelterin expression, too. In HEL-hTERT and BGC-823-hTERT cells treated with bortezomib, POT1 was the only shelterin protein that was still significantly down-regulated, while TRF1 and TRF2 expression was stabilized Figure , differiIt is well known that bortezomib treatment induces apoptotic death of cancer cells . To deteP = 0.005 and 0.01, respectively)  Figure . Moreove) Figure .Caspase 3 activation/cleavage and bcl-2 down-regulation via the NK-kB signalling inhibition is observed in bortezomib-treated MM and other malignant cells [Bortezomib exerts its anti-cancer effect by inhibiting the ubiquitin/proteasome pathway, however, the exact mechanisms remain incompletely understood . In the Weise et al recently reported that bortezomib down-regulated telomerase activity accompanied by a decline in hTERT expression in subsets of MM cell lines . The inhThe maintenance of telomere length and function depends on not only hTERT/telomerase, but also appropriate levels of shelterin protein expression , 11. We Recent studies unveil multiple biological activities of telomerase or hTERT in addition to its telomere lengthening function. For instance, hTERT is capable of facilitating the recruitment of DNA repair factors to sites of double-stranded breaks , while dIt has been well characterized that bortezomib inhibits the anti-apoptotic NF-κB signaling pathway by repressing its target gene expression (such as bcl-2 and others) , 29. We In summary, we demonstrate that bortezomib induces down-regulation of hTERT/telomerase and broad dysregulation of shelterin protein expression, thereby disrupting telomere stabilization and leading to telomere dysfunction in malignant cells. Moreover, hTERT over-expression protects from apoptotic cell death mediated by bortezomib. Collectively, the telomerase/telomere system is an important target of bortezomib. We thus suggest that future clinical trials designed to evaluate the efficacy of bortezomib include telomerase/telomere function inhibition as one indicator of response.2. Bortezomib was bought from Selleck Chemicals  and exponentially growing cells were incubated with bortezomib at different concentrations for various time periods. Cells were counted for numbers and viability using trypan blue exclusion test.The erythroid leukemia cell line HEL and the gastric cancer cell line BGC-823 were used in the present study. Cells were grown in 10% foetal calf serum-containing RPMI-1640 with addition of 2 mM L-glutamine and antibiotics  in a humid atmosphere at 37°C/5% COA lenti-III-HA-GFP-hTERT vector was constructed and a Lenti-BMN-GFP vector  was used as control . The lenTotal cellular RNA was extracted using Trizol kits according to the manufacture's instruction . cDNA was synthesized using RevertAid First Strand cDNA Synthesis kits  and M-MLV reverse transcriptase. qPCR was carried out in an ABI7700 sequence detector  using SYBR Green kit  and the primer pairs specific to hTERT, hTER, TRF1, TRF2, TPP1, POT1, RAP1 and TIN2 as described  , TFR2 , POT1 , Caspase3 , or Bcl-2  followed by anti-mouse or rabbit horseradish peroxidase-conjugated IgG and developed with the enhanced chemiluminescent method . GAPDH Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, #sc-47724) immunoblotting was performed in parallel as a loading control.Telomerase activity was assessed using a commercial Telomerase PCR ELISA kit  as recommended by the manufacturer. For each assay, one μg of protein was used, and 23 PCR cycles were performed after the telomerase-primer elongation reaction. The PCR products were detected using ELISA color reaction and the level of telomerase activity was expressed as absorbance in arbitrary units.6 cells were measured. The control gate was set based on the negative control.Cells were treated with bortezomib as decribed above, and then harvested for apoptosis assay. The cells were stained with a kit from BD pharmingen using a FlowCytometer (BD). For each sample 1 × 10Immuno-FISH was carried out as described . Briefly−ΔCt, where ΔCt = average Cttelomere − average Ctβ-globin and relative telomere length was expressed as the percentage of that in BGC-823 cells without bortezomib.Flow FISH of cells was performed according to a previous protocol by Baerlocher et al –35 with t-test or Mann-Whitney U test was used to compare cell numbers, apoptotic cells, hTERT, hTERC and shelterin protein mRNA levels and telomere length between control and bortezomib-treated cells. All the tests were two-tailed and computed using SigmaStat3.1® software . P values of < 0.05 were regarded as statistically significant.Student's


Further functional study demonstrated that miR-506 could augment the response to cisplatin and olaparib through targeting RAD51 and suppressing homologous recombination in a panel of ovarian cancer cell lines. Systemic delivery of miR-506 in an orthotopic ovarian cancer mouse model significantly augmented the cisplatin response, thus recapitulating the clinical observation. Therefore, miR-506 plays a functionally important role in homologous recombination and has important therapeutic value for sensitizing cancer cells to chemotherapy, especially in chemo-resistant patients with attenuated expression of miR-506.Ovarian carcinoma is the most lethal gynecologic malignancy. Resistance to platinum is considered the major problem affecting prognosis. Our recent study established that microRNA-506 ( Epithelial ovarian cancer remains the most lethal gynecologic malignancy , 2. The Recently, it has been reported that microRNAs (miRNAs), which are single-stranded, non-coding endogenous RNAs, play a crucial role in regulating drug resistance. Possible mechanisms in regulating drug resistance involve the regulation of cell cycle distribution, drug transport, DNA repair, epithelial-mesenchymal transition (EMT), and apoptosis pathways. Platinum-based drugs can form intra- and inter-strand adducts with DNA, which cause DNA double-strand breaks and trigger DNA damage and repair pathways. Thus, enhancement of the DNA repair system plays a major role in platinum resistance. Homologous recombination (HR) is a critical pathway for DNA double-strand break repair; cells with compromised HR machinery are highly sensitive to DNA-damaging drugs .miR-506) expression was associated with an increased response to therapy and prolonged progression-free survival (PFS) and overall survival (OS). This result was confirmed in another clinically annotated genomics dataset (Bagnoli) [miR-506 can inhibit EMT and proliferation by targeting the snail family zinc finger 2 (SNAL2) and cyclin-dependent kinase 4/6-Forkhead box protein M1 (CDK4/6-FOXM1) axes, respectively [miR-506 in a panel of ovarian cancer cell lines, we analyzed the down-regulated genes via a microarray and observed a decrease in RAD51 levels. When we used the miRNA target prediction algorithm TargetScan 6.0 , we found that RAD51 had one predicted binding site for miR-506. We next performed a set of functional validation experiments using reporter gene assays. We found that miR-506 suppressed the reporter activity linked to the 3′- untranslated region (UTR) of RAD51 that contains the predicted binding site. The use of control miRNA or removal of the binding site did not produce the same regulation exhibited by miR-506. These results showed that RAD51 is a direct target of miR-506. Furthermore, the inverse association between miR-506 and RAD51 expression was confirmed in three different cohorts of clinical samples .Our recent investigation of The Cancer Genome Atlas (TCGA) database network for high-grade serous ovarian carcinoma showed that microRNA-506  against RAD51 significantly reduced the HR efficiency. In addition, ectopic overexpression of miR-506 led to higher residual DNA damage (as shown by single-cell gel electrophoresis) and less RAD51 foci formation (as shown by immunofluorescence microscopy imaging) as compared with controls after cisplatin treatment, confirming the defect in HR.We next sought to understand how important the regulation of equences . Becauseequences –13. By pBRCA2) mutations, and to a lesser extent breast cancer early onset 1 (BRCA1) mutations/methylation, are associated with improved survival and responses to therapy in serous ovarian cancer patients [miR-506 on the sensitivity to cisplatin and olaparib (a PARP inhibitor) in ovarian cancer in vitro and in vivo and found that miR-506-transfected cells were more sensitive to cisplatin or olaparib than control cells in both 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT) and clonogenic survival assays. Conversely, anti-miR-506 locked nucleic acid (LNA) transfection enhanced RAD51 expression and induced resistance to cisplatin and olaparib in cells. Moreover, the effect of miR-506 on cisplatin and olaparib sensitivity was rescued by overexpressing RAD51 without its 3′-UTR, suggesting that miR-506-mediated sensitivity to cisplatin and olaparib is the result of the suppression of RAD51 expression. Consistent with the results of in vitro experiments, delivery of miR-506 incorporated in 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) nanoliposomes effectively enhanced the effect of cisplatin and olaparib in an orthotopic ovarian cancer model.HR-deficient cells are sensitive to DNA-damaging drugs and poly(ADP) ribose polymerase (PARP) inhibitors, and this effect has been termed synthetic lethality , 15. Recpatients –18. We nmiR-506 can enhance the sensitivity to cisplatin and PARP inhibitors through suppression of the RAD51-HR axis. Furthermore, nanoparticle delivery of miR-506 enhanced the effects of cisplatin and olaparib in orthotopic ovarian cancer models. Thus, miR-506 not only is a robust clinical marker for the chemotherapy response and survival of serous ovarian cancer patients but also has important therapeutic value in sensitizing cancer cells to chemotherapy. This new discovery shows that miR-506 combined with DNA-damaging agent treatment may benefit patients with high-grade serous ovarian carcinoma. Taken together with the findings of our previous study on this small molecule, we have shown that miR-506 has multiple therapeutic effects on regulating the biological behavior of cancer cells (Fig. In summary, our recent investigations have revealed that


The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as “methionine dependence” or “methionine stress sensitivity” in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential.This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions. Reprogrammed metabolism in cancer has gained greater attention in recent years, beyond the well-known Warburg effect . While m5-methyltetrahydrofolic acid or toward cysteine and glutathione synthesis in the transsulfuration pathway . To. To53]. In conclusion, the present study reveals dynamic changes in the metabolic phenotype associated with methionine dependency of breast cancer and further highlights the importance of SAM as a major regulator of tumorigenesis. Importantly, using a labeled precursor, we discovered that in both MB468 and MB468res-R8 cell types, homocysteine does not significantly contribute to formation of methionine and ultimately S-adenosylmethionine. Our study indicates that SAM metabolism, which is required for all methylation reactions, depends on availability of methionine in the cancer cell lines investigated and cannot be substituted by homocysteine-forming pathways. Moreover, these findings shed light on the metabolic reprogramming, which accompanies methionine stress beyond methylation potential. Such knowledge is instrumental for elucidating novel targets of therapeutic intervention, which can be exploited in future studies. Given the importance of SAM in lipid biogenesis and our observed changes in LLS, assessing the impact of methionine stress on lipid composition and synthesis is of considerable interest and warrants further investigation.


Pinus radiata D. Don) branches and genes differentially transcribed in CW and OW were investigated using cDNA microarrays.Formation of compression (CW) and opposite wood (OW) in branches and bent trunks is an adaptive feature of conifer trees in response to various displacement forces, such as gravity, wind, snow and artificial bending. Several previous studies have characterized tracheids, wood and gene transcription in artificially or naturally bent conifer trunks. These studies have provided molecular basis of reaction wood formation in response to bending forces and gravity stimulus. However, little is known about reaction wood formation and gene transcription in conifer branches under gravity stress. In this study SilviScan® technology was used to characterize tracheid and wood traits in radiate pine , higher density and lower stiffness. However, CW and OW tracheids had similar diameters in either radial or tangential direction. Thus, gravity stress largely influenced wood growth, secondary wall deposition, cellulose microfibril orientation and wood properties, but had little impact on primary wall expansion. Microarray gene transcription revealed about 29% of the xylem transcriptomes were significantly altered in CW and OW sampled in both spring and autumn, providing molecular evidence for the drastic variation in tracheid and wood traits. Genes involved in cell division, cellulose biosynthesis, lignin deposition, and microtubules were mostly up-regulated in CW, conferring its greater growth, thicker tracheid walls, higher density, larger MFA and lower stiffness. However, genes with roles in cell expansion and primary wall formation were differentially transcribed in CW and OW, respectively, implicating their similar diameters of tracheid walls and different tracheid lengths. Interestingly, many genes related to hormone and calcium signalling as well as various environmental stresses were exclusively up-regulated in CW, providing important clues for earlier molecular signatures of reaction wood formation under gravity stimulus.The first comprehensive investigation of tracheid characteristics, wood properties and gene transcription in branches of a conifer species revealed more accurate and new insights into reaction wood formation in response to gravity stress. The identified differentially transcribed genes with diverse functions conferred or implicated drastic CW and OW variation observed in radiata pine branches. These genes are excellent candidates for further researches on the molecular mechanisms of reaction wood formation with a view to plant gravitropism.The online version of this article (doi:10.1186/1471-2164-14-768) contains supplementary material, which is available to authorized users. Trees can change their growth orientation in response to various environmental stresses  , and durMolecular basis of CW formation has been previously studied in the bent trunks of several conifer species. Using the semi-quantitative RT-PCR technique a few cell wall structural protein genes and several lignin-related genes were identified with differential transcription in the bent trunks of loblolly pine  and JapaTo our knowledge all published researches on the properties and gene transcription of reaction wood in conifer species (CW) and angiosperms  were based on the bent trunks under artificial bending or natural inclining forces. It should be noted that these trees had experienced both external forces and gravity stimulus. Particularly, the bending force in young trees and seedlings could be much larger than gravity stress. Thus, wood properties and gene transcription observed in bent trunks may not accurately reflect gravity stress. In contrast, naturally grown branches are mostly under gravity stress, providing excellent materials for the study of wood formation in response to gravity stimulus. Although one previous study with eucalyptus branches identified a few cell wall genes in response to gravity stress in angiosperms , wood prPinus radiata Don.) is the most important conifer species for commercial forestry in Australia, New Zealand and Chile. CW properties of radiata pine have been previously characterized using inclined trees [Radiata pine  was 4.6 cm, significantly longer than that of total OW formed on the upper side of branches . The six branch discs had 10 growth rings in the cross-section. Within a ring formed in a growing circle CW was significant wider than OW  except for the first four rings from pith. These results indicated that gravity stress significantly increased wood formation on the lower side of branches. The larger wood growth in CW could generate compression force to maintain branches at certain orientation.SilviScan measurement of the six branch discs showed significant differences between CW and OW in wood growth, tracheid characteristics and wood properties in terms of average ring values Figure . SignifiThe xylem transcriptomes of CW and OW sampled in spring  and autumn  were compared respectively using radiata pine cDNA microarrays. In the first comparison between CW and OW sampled in spring, 944 out of 3,320 xylem unigenes (28.4%) on the microarrays had differential transcription, including 781 and 163 unigenes preferentially transcribed in CW and OW, respectively  Figure . This reactin depolymerizing factor (ADF) and ADF-like genes were exclusively up-regulated in CW of branches. In the microtubule development, different members of the same gene families encoding tubulin folding cofactors, microtubule-associated proteins (MAPs) and MAP kinases were differentially transcribed in CW and OW, respectively. Interestingly, seven tubulins  were exclusively up-regulated in CW sampled in the two seasons (three tubulins) or a single season (four).From a total of 1,728 genes with differential transcription identified in this study, 28 genes were involved in actin filaments and microtubules  were up-regulated in CW; while only one gene (cyclin-like F-box domain) was preferentially transcribed in OW. In cell expansion, three expansin and extensin genes were up-regulated in CW ; while only one up-regulated in OW . In pectin biosynthesis, three genes  were up-regulated in OW; while only a pectin lyase-like had higher transcription in CW. In primary wall modification, two xyloglucan endotransglucosylase/hydrolases (XET8 and 32) were up-regulated in CW of branches, and a gene encoding ovule/fiber cell elongation proteins was more highly transcribed in OW.Genes related to cell wall formation at various developmental stages were identified with differential transcription in CW and OW of radiata pine branches. Interestingly, these genes were more frequently up-regulated in CW than in OW Table . In cellcellulose synthases , PrCesA-like and sucrose synthases (SuSy and SuSy1) were preferentially transcribed in CW. Four of these genes  were up-regulated in CW sampled in both spring and autumn. In the lignin-related genes, 4-coumarate:CoA ligase (4CL), cinnamic acid 4-hydroxylase (C4H), cinnamoyl CoA reductase-like (CCR-like), caffeic acid ortho-methyltransferase (COMT), phenylalanine ammonia-lyase 2 (PAL2), methionine synthases, S-adenosylmethionine synthetase (SAMS), S-adenosylmethionine decarboxylase (SAMDC), laccases (LAC2 and 4) and dirigent-like were up-regulated in CW. A wide range of cell wall structural protein genes were up-regulated in CW, such as arabinogalactan proteins , fasciclin-like arabinogalactan proteins (FLA1 and 8), glycine-rich protein, proline-rich protein and so on  were preferentially transcribed in OW of branches.Secondary cell wall genes were mostly up-regulated in CW of radiata pine branches Table . Of geneauxin-induced protein, auxin-regulated protein (ARP) and ARP-like. Other hormone-related genes up-regulated in CW included genes encoding gibberellins-induced receptor-like kinase, cytokinin-binding protein, ethylene response factor-like, ethylene responsive element binding factor and ethylene-forming enzyme. In addition, six calcium-related genes  were up-regulated in CW of branches in both spring and autumn  and disease resistance  protein and TIR/P-loop/LRR) were exclusively up-regulated in CW formation. Surprisingly, light-inducible protein ATLS1, light-induced protein-like and phytochrome were exclusively up-regulated in CW, suggesting CW formation may be affected by light signals.Fifteen genes involved in various environmental stresses  were up-regulated in CW of branches; while only two genes related to environmental stresses were preferentially transcribed in OW Table . Of geneDrastic variation between CW and OW of radiata pine branches indicated that gravity stimulus affects cell division, secondary wall deposition, cellulose microfibril orientation and overall wood properties. The larger MFA in CW greatly contributes to its lower wood stiffness despite of its higher density. This is because MFA rather than density has a predominant and adverse effect on wood stiffness . The greDifferential gene transcription could provide molecular evidence for the drastic variation between CW and OW. The xylem transcriptome changes in CW and OW of radiata pine branches 28-29%) are among the highest in a number of microarray comparisons with regard to radiata pine wood development, including earlywood vs. latewood (11-30%) , juvenil8-29% arealpha- and beta-tubulins in CW (with larger MFA) of radiata pine branches is in agreement with previous study using bent trunks of maritime pine [alpha-tubulin with MFA was observed in secondary xylem of loblolly pine [alpha- and beta-tubulins were highly transcribed in TW (with reduced MFA) formed in bent poplar trunks [beta-tubulin gene in transgenic xylem directly influenced MFA [tubulin genes involved in cellulose microfibril orientation of secondary xylem have been conserved in both gymnosperms and angiosperms.The cytoskeleton is made up of microtubules, actin filaments, and intermediate filaments . There iime pine . Associally pine . In angir trunks  and eucar trunks . Over-trnced MFA . Taken tADFs (ADF and ADF-like) that were exclusively up-regulated in CW of branches. ADF plays an important role in regulating the optimum balance between unpolymerised actin molecules and assembled actin filaments [actin polymerizing factors up-regulated in CW of bent trunks in spring were not identified in CW of branches in either spring or autumn, suggesting their responses exclusive to bending forces rather than gravity stimulus. In contrast, genes  with differential transcription in branches were not identified in bent trunks, highlighting their possible roles in response to gravity stress.Actin filaments are much less rigid compared to microtubules . Interacilaments . Genes iilaments  in conifPrCesA genes  up-regulated in CW were previously clustered as secondary wall genes and PrCesA10 preferentially transcribed in OW is a primary wall gene [CesAs were also up-regulated in CW of bent maritime pine [CesA-like and SuSy genes were up-regulated in CW of both branches (this study) and bent trunks of conifers [SuSy gene increased cellulose content, secondary wall thickness and wood density in poplars [This study identified many secondary cell wall genes with preferential transcription in CW of radiata pine branches Table  and 4. Tall gene . Severalime pine , TW of bime pine , 37 and ime pine , 39. Besconifers . In Scotconifers . Over-tr poplars .shikimate kinase 2, chorismate synthase, chorismate mutase, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) and 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS)  were up-regulated in CW of radiata pine branches. Preferential transcription of genes related to shikimate pathway and SAM metabolism in CW may result in more production of monolignol precursors. Finally, monolignol synthesis could be also enhanced in CW due to up-regulation of several key genes involved in monolignol pathway . Most of these lignin-related genes were also up-regulated in CW of bent trunks in maritime pine [laccases, COMT and methionine synthase) [Lignin biosynthesis consists of three major steps: shikimate pathway, monolignol pathway and monolignol polymerization . Phenylaime pine  and TW oime pine . Besidesime pine  and in Jynthase) . In summAGPs, AGP-like, glycine-rich proteins and proline-rich proteins) were exclusively up-regulated in CW of radiata pine branches. In loblolly pine six PtaAGPs were predominantly transcribed in secondary xylem development [FLAs were differentially transcribed in either CW (FLA1 and 8) or OW  of radiata pine branches. In angiosperms FLAs were up-regulated in TW of bent poplar trees [FLAs affect MFA and tensile stiffness in transgenic eucalypts and Arabidopsis by altering cellulose deposition and the integrity of the cell wall matrix [A number of cell wall structural protein genes  and bent trunks of maritime pine [XETs can cut and rejoin xyloglucan (XG) chains, and are believed to be important regulators of primary wall expansion [pectin lyase-like) or OW  of radiata pine branches. Differential transcription of these genes in CW and OW could provide molecular evidence for their similar tracheid diameters . On the other hand, a gene encoding ovule/fiber cell elongation protein with up-regulation in OW could suggest its possible function in the longitudinal growth of tracheids.Tracheids are structures of three dimensions  which are determined in cell expansion during primary wall formation. Several ime pine . XETs caxpansion . DifferePlant gravitropism is a complex process including three major stages: gravity perception, signal transduction, and growth response. In this study many genes related to hormone and calcium signalling as well as environmental stresses were up-regulated in CW of branches; while only a few genes in these categories were preferentially transcribed in OW Table  and 6. TAcacia mangium seedlings [Some hormone signalling genes with up-regulation in CW of radiata pine branches Table  had prefeedlings ). Cytokieedlings . The ide2+) signalling has a strong relationship with plant gravitropism [calcium/calmodulin-dependent protein kinase from maize showed light-regulated gravitropism [Calcium  in CW of radiata pine branches could be a result of reduced light radiation on the lower side of branches. Phytochromes are red and far-red light photoreceptors, and they regulate a large number of genes involved in hormone signalling or enzymes involved in cell wall modification [The majority of differentially transcribed genes involved in various environmental stresses  were up-regulated in CW of branches Table . This isfication . In hypofication . Thus, pfication , 61.Compression wood formed in radiata pine branches showed greater radial growth, thicker tracheid walls, larger microfibril angle (MFA), higher density and lower stiffness, but similar tracheid diameters compared to its opposite wood. Extensive remodelling of the xylem transcriptomes (29%) observed in compression and opposite wood could provide molecular evidence for their drastic variation in tracheid and wood traits. Many genes involved in cell division, cellulose biosynthesis, lignin pathway and microtubules were exclusively up-regulated in compression wood, conferring its greater radial growth, thicker tracheid walls, higher density, larger MFA and lower stiffness. In contrast, genes related to cell expansion and primary wall modification were differentially transcribed in either compression or opposite wood, implicating their similar tracheid diameters but different tracheid lengths. Of particular interest, a broad range of genes related to hormone and calcium signalling and various environmental stresses were exclusively up-regulated in compression wood, suggesting possible earlier molecular signatures of plant gravitropism during reaction wood formation in conifers. The first transcriptome profiling of radiate pine branches provides more accurate insights into the molecular basis of reaction wood formation in response to gravity stimulus without external bending forces.Six trees with well-developed branches were selected from a radiata pine commercial plantation located at Bondo, NSW, Australia . These trees were originated from seedlings with different genotypes and they were 13 years old at the time of sampling. The largest branch from each tree was further selected for study, including three branches sampled in autumn and three sampled in spring. Bark was removed from the base part (about 10 cm in length) of each branch. Developing xylem tissues were scraped from the exposed upper and lower side surface respectively with a sharp chisel. Samples were immediately placed into 50 ml BD Falcon™ tubes filled with liquid nitrogen. One branch disc (approximately 5 cm in length) was then cut off from the larger end of each branch adjacent to the base part used for developing xylem sampling. Location of upper and lower side zones was immediately marked on all discs collected from branches.After removing the bark from each branch disc a block of wood  was cut from the top of upper side to the bottom of lower side through pith. A twin-blade saw was used to trim the wood blocks to produce strips (containing pith) of 2 mm in the tangential direction and 7 mm in the longitudinal direction. The wood strips were characterized using the SilviScan® instrument , 19. A tTotal RNA was extracted from developing xylem tissues using a modified CTAB method . TranscrConstruction of cDNA microarrays, synthesis of probes and microarray hybridization were performed in methods described previously –24. Hybrcellulose synthase 3 (PrCesA3), PrCesA11, cinnamic acid 4-hydroxylase (C4H) and plastocyanin-like (PCL); and three genes for OW: peroxidase (PER), E3 ubiquitin protein ligase (UPL1) and retinoblastoma-like protein (RBL)  method . A totalApproximately 400 ng of purified total RNA was reverse transcribed into first strand cDNA using the ImProm-II Reverse Transcription System . The cDNA was hybridized at 60°C overnight with bulked RPO (right probe oligo) and LPO (left probe oligo) probes designed for the selected genes  and opposite wood (OW) of radiata pine branches sampled in spring and autumn. In total, 846 genes were identified with differential transcription in CW (693) and OW (153) of radiata pine branches sampled in spring; while 872 genes were preferentially transcribed in CW (511) and OW (361) sampled in autumn. (XLS 506 KB)Additional file 1: Transcription factors differentially transcribed in compression (CW) and opposite wood (OW) of radiata pine branches. (XLS 25 KB)Additional file 2: LPO (left probe oligo) and RPO (right probe oligo) of selected differentially transcribed genes involved in the validation. A total of seven differentially transcribed genes identified in the microarray experiments were selected in the validation by reverse transcriptase-multiplex ligation dependent probe amplification (RT-MLPA). Their LPO and RPO sequences were listed in the table. (XLS 24 KB)Additional file 3: