In this review, we delineate the mechanisms relevant for pemphigus pathogenesis based on the histology and the ultrastructure of patients’ lesions. We then dissect the mechanisms which can explain the ultrastructural hallmarks detectable in pemphigus patient skin. Finally, we reevaluate the concept that the spectrum of mechanisms, which induce desmosome dysfunction upon binding of pemphigus autoantibodies, finally defines the clinical phenotype.Pemphigus is a severe autoimmune-blistering disease of the skin and mucous membranes caused by autoantibodies reducing desmosomal adhesion between epithelial cells. Autoantibodies against the desmosomal cadherins desmogleins (Dsgs) 1 and 3 as well as desmocollin 3 were shown to be pathogenic, whereas the role of other antibodies is unclear. Dsg3 interactions can be directly reduced by specific autoantibodies. Autoantibodies also alter the activity of signaling pathways, some of which regulate cell cohesion under baseline conditions and alter the turnover of desmosomal components. These pathways include Ca Pemphigus is a severe autoimmune-blistering skin disease caused by autoantibodies primarily targeting the desmosomal adhesion molecules desmogleins (Dsgs) 1 and 3 –5. The pCultured keratinocytes or mouse models can only partially reproduce the situation in patients. We believe that for the identification of relevant mechanisms in pemphigus pathogenesis, the careful evaluation of patients’ lesions is the gold standard. On the histological level, skin blistering in PV occurs by suprabasal splitting Figure , whereasvia Stat3-induced Dsg3 transcription increase  in the absence of ultrastructural alterations of desmosomes , 10. By increase .On the ultrastructural level, smaller desmosomes were found only in conditions when patients presented with antibodies against Dsg1 such as in mcPV and PF but not in mPV , 13, 14,Split desmosomes both with and without attached keratin filaments were detected by electron microscopy and SIM on the keratinocyte surface facing blisters in PF and mcPV , 14. DesApoptosis is not a major mechanism because cells displaying signs of apoptotic cell death are absent or sparse in PV and PF skin lesions and therefore cannot explain acantholysis of a significant epidermal area , 18, 19.As outlined earlier, split desmosomes, reduced desmosome numbers and size, and keratin retraction are ultrastructural hallmarks in pemphigus skin. Reduced desmosome size or numbers cannot be explained by the direct interference of pemphigus autoantibodies with Dsg binding but rather are a consequence of the altered turnover of desmosomal proteins. These changes are likely steered by intracellular-signaling pathways, which are modulated in response to autoantibody binding and represent potential pharmacologic targets. In principal, reduced desmosome size and numbers can result either from interference with desmosome assembly or from the enhanced disassembly of desmosomes. Available data suggest that in pemphigus, both mechanisms contribute to impaired desmosome turnover, shifting the balance toward an overall reduction of desmosomal components .Desmosome assembly is tightly interwoven with adherens junction formation and appears to proceed in distinct steps  Figure , which, Vice versa, keratin filaments were shown to influence both desmosome stability and Dsg3-binding characteristics in a signaling-dependent manner. Under physiologic conditions, the keratin-dependent suppression of p38MAPK signaling increases Dsg3-binding strength, whereas the suppression of PKC signaling through the adapter protein Rack-1 prevents DP phosphorylation, stabilizes Dsg3 in the desmosome (Pemphigus autoantibodies interfere with desmosome turnover by enhancing the internalization of desmosomal components Figure , 36. Conesmosome , 45, andesmosome , promoteesmosome , which mesmosome . TogetheThe occurrence of desmosome disassembly can be concluded from observations that Dsg3 together with PV-IgG is excluded from desmosomes and internalized . This isSplit desmosomes are rarely detectable in keratinocyte cultures incubated with pemphigus antibodies in the absence of mechanical stress  but are All mechanisms described earlier finally lead to desmosome dysfunction, which impair keratinocyte cohesion. However, the functional interplay of the different mechanisms is not well understood at present. It is also unclear why epidermal splitting affects the deep epidermis in PV, whereas PF blisters are restricted to superficial epidermal layers Figure . BesidesIn the mucosa, Dsg3 is the predominant desmosomal cadherin, and in the superficial epidermis Dsg1 is strongly expressed, whereas other isoforms of desmosomal cadherins are largely absent except of Dsc1 Figure . The cad2+ as well as Erk activation was induced only by PV-IgG and PF-IgG containing Dsg1 autoantibodies, which was paralleled by p38MAPK activation (2+, which has been shown to be associated with PKC activation in keratinocytes treated with pemphigus antibodies (The direct inhibition of Dsg3 binding by PV-IgG has been described under cell-free conditions as well as on the surface of living keratinocytes; however, it was ineffective to induce the complete loss of cell cohesion –64. By ctivation . In thistibodies , 69, as via incubation with AK23 is sufficient to cause skin blisters in mice (ex vivo indicate that the significance of p38MAPK for blister formation may be different in PV and PF. When using PV-IgG, the inhibition of p38MAPK was sufficient to abrogate blistering (2+, PKC, or Erk may be necessary to induce superficial epidermal blistering in PF (Figure For most of the pathways described earlier, it was shown that modulation is sufficient to largely reduce skin blistering in mice . However in mice , 70. Thiistering , 56 but istering . On the istering . HoweverF Figure . HoweverVS and JW conceived the perspective and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glucocorticoids promote fat accumulation in visceral compared to subcutaneous depots, but the molecular mechanisms involved remain poorly understood. To identify long-term changes in gene expression that are differentially sensitive or responsive to glucocorticoids in these depots, paired samples of human omental (Om) and abdominal subcutaneous (Abdsc) adipose tissues obtained from obese women during elective surgery were cultured with the glucocorticoid receptor agonist dexamethasone  for 7 days. Dex regulated 32% of the 19,741 genes on the array, while 53% differed by Depot and 2.5% exhibited a Depot*Dex concentration interaction. Gene set enrichment analysis showed Dex regulation of the expected metabolic and inflammatory pathways in both depots. Cluster analysis of the 460 transcripts that exhibited an interaction of Depot and Dex concentration revealed sets of mRNAs for which the responses to Dex differed in magnitude, sensitivity or direction between the two depots as well as mRNAs that responded to Dex only in one depot. These transcripts were also clearly depot different in fresh adipose tissue and are implicated in processes that could affect adipose tissue distribution or functions . Elucidation of the mechanisms underlying the depot differences in the effect of Dex on the expression of specific genes and pathways that regulate adipose function may offer novel insights into understanding the biology of visceral adipose tissues and their links to metabolic health. The mass of visceral fat, defined as those depots located within the abdominal cavity and associated with digestive organs , is associated with risk for type 2 diabetes and cardiovascular disease in both men and women . GlucocoGCs integrate a wide variety of regulatory signals that control cell proliferation, metabolism, and inflammation . The actADIPOQ, LEP, GLUT4, LPL) is synergistically upregulated by GCs and insulin, and maintained at initial levels for at least 7 days ) was included in the model. [Dex] was modeled as a categorical, unordered variable so as not to exclude genes with a nonlinear dose response. The interaction effect measures whether dose effect on the expression of a given gene differs between the depots. For example, the concentration of Dex that consistently affects a given gene or the magnitude of the effect is dependent on depot. After calculation of p values for each term for each gene, the Benjamini-Hochberg False Discovery Rate (FDR) correction was used to obtain FDR-corrected p values .A cluster analysis was conducted on a subset of 460 genes with a FDRq less than 0.05 for the interaction of Depot and [Dex] that also showed an effect size over 1.2 fold up or down in at least one concentration of Dex in one depot, and expression values greater than a cutoff of 20 (out of a maximum ~6000) arbitrary units. The mean values for expression of these at each Dex concentration were scaled across both depots (-ΔCT) and presented as the mean and SEM of relative abundance. All values were log-transformed prior to statistical analysis. The effect of Dex within a depot was tested with a one way repeated measures ANOVA (on dose), with post-hoc paired t-tests as indicated, within each depot. Paired t-tests were used to compare values in Abdsc and Om depots within subjects.Expression levels of selected genes of interest were verified by qPCR using 7 paired Om and Abdsc samples. Data are normalized by PPIA gene expression  were significantly regulated by Dex, as indicated by a significant main effect. 10,424 transcripts (53%) showed a Depot effect and 513 (2.5%) showed a Depot*[Dex] interaction. Gene lists, average expression values and FDRq values for the LME analysis are provided in Dex upregulated pathways: PCK1 which is of importance in adipocytes because it functions as a glyceroneogenic enzyme that regulates esterification of FA ) indicated that overall, values for both Om control and Om Dex 1 nM clustered with Abdsc control (0 Dex). This finding indicates that Om 1 nM values were more similar to Om control (0 Dex), but that Abdsc cultured with 1 nM Dex was different from Abdsc control and clustered with Abdsc 10 and 1000 nM and is consistent with the conclusion that overall Om is less sensitive to a low concentration of Dex. The two-way clustered dendrogram is shown in INHBA and GREM1, PKHD1L1 and ITLN1, ITGB8  depot differences in the Dex regulation of two genes that were much more highly expressed at baseline in Om and remained higher despite suppression by Dex , and those for which a clear depot-difference was induced by culture with Dex  would also exhibit depot-difference in fresh adipose tissue. INHBA, GREM1, PKHDL1, ITLN1, and ITGB8, were also higher in snap frozen Om, while NRN1, which was increased by Dex only in Abdsc, was substantially higher in fresh Abdsc tissue.To determine if our results in tissues cultured with Dex for 7 days ex vivo were relevant to the in vivo, we examined expression levels in tissues that were snap frozen immediately after excision. We reasoned that genes that were more highly expressed in one depot at baseline  also showed substantial depot differences in fresh tissue, supporting the physiological relevance of the organ culture approach and the important role of GCs in driving depot differences in adipose tissue function. These results were easily detected with a small sample size of almost exclusively female obese subjects, indicating the differences are robust and consistent.The current dataset and pathway (GSEA) analyses confirm and expand our prior observations that many metabolic  and immune-related pathways are regulated similarly by Dex in both depots. More importantly, with the mixed model statistical approach and cluster analysis of dose-dependent changes in the adipose tissue transcriptome, we were able to identify depot-dependent differences in genes and pathways that vary in sensitivity to low Dex concentrations and others that exhibit depot differences in the magnitude or direction of the Dex effects. We verified with qPCR that selected transcripts showing depot-dependent responses to Dex in vitro , which inhibits adipogenesis via increased production of lysophosphatidic acid  interaction (p < 0.05) were used for this cluster analysis. Cluster and sub-cluster numbers as well as LME results and expression values are given for each gene [Data are the mean of the expression values  for the 3 paired samples of Om and Abdsc from the microarray analysis].(XLSX)Click here for additional data file.


PA tissue content seems to be controlled around a well-defined concentration, and changes in its intake do not influence significantly its tissue concentration because the exogenous source is counterbalanced by PA endogenous biosynthesis. Particular physiopathological conditions and nutritional factors may strongly induce DNL, resulting in increased tissue content of PA and disrupted homeostatic control of its tissue concentration. The tight homeostatic control of PA tissue concentration is likely related to its fundamental physiological role to guarantee membrane physical properties but also to consent protein palmitoylation, palmitoylethanolamide (PEA) biosynthesis, and in the lung an efficient surfactant activity. In order to maintain membrane phospholipids (PL) balance may be crucial an optimal intake of PA in a certain ratio with unsaturated fatty acids, especially PUFAs of both n-6 and n-3 families. However, in presence of other factors such as positive energy balance, excessive intake of carbohydrates (in particular mono and disaccharides), and a sedentary lifestyle, the mechanisms to maintain a steady state of PA concentration may be disrupted leading to an over accumulation of tissue PA resulting in dyslipidemia, hyperglycemia, increased ectopic fat accumulation and increased inflammatory tone via toll-like receptor 4. It is therefore likely that the controversial data on the association of dietary PA with detrimental health effects, may be related to an excessive imbalance of dietary PA/PUFA ratio which, in certain physiopathological conditions, and in presence of an enhanced DNL, may further accelerate these deleterious effects.Palmitic acid (PA) has been for long time negatively depicted for its putative detrimental health effects, shadowing its multiple crucial physiological activities. PA is the most common saturated fatty acid accounting for 20–30% of total fatty acids in the human body and can be provided in the diet or synthesized endogenously via  Particular physiopathological conditions and nutritional factors may strongly induce DNL, resulting in increased tissue content of PA and disrupted homeostatic control of its tissue concentration  is the most common saturated fatty acid found in the human body and can be provided in the diet or synthesized endogenously from other fatty acids, carbohydrates and amino acids. PA represents 20–30% of total fatty acids (FA) in membrane phospholipids (PL), and adipose triacylglycerols (TAG) , which catalyzes the DNL limiting step reaction, and the FA synthase (FAS). The main sources of citrate for DNL are glucose and glutamine-derived α-ketoglutarate (α-KG), especially under hypoxia or disruption of the mitochondrial oxidative machinery , is regulated in parallel with the DNL pathway  is a dominant regulator of metabolism, and key energy sensor of intracellular AMP/ATP ratios. When there is an increase of this ratio (low energy conditions), two molecules of AMP bind, and consequently AMPK is activated  in high affinity tissues and contributes, under excessive fat supply, to lipid accumulation and metabolic dysfunction signaling  and phosphatidylethanolamine (PE), that are 16:0/18:2, 16:0/18:1, 16:0/22:6, and 18:1/18:2  and the studied diet is either lacking, or overloaded with the particular FA. Relatively few studies attempted to examine the normal situation . Marginal EFAD is also known to exist in patients with cystic fibrosis (CF) that revealed an elevated level of PE and PG, whereas the degree of saturation of PC was reduced . Indeed, it derives directly by PA esterified in sn-1 position of a donor membrane PL like PC Hansen, . UsuallyDifferent studies have evaluated whether PA tissue concentrations, particularly in membrane PL, are able to influence PEA biosynthesis and whether tissue levels could be influenced by variation in intake of dietary FA. In a previous study, we found that incubation of cultured adipocytes with PA was able to induce a 3-fold increase of PEA  and 2-arachidonoylglycerol (2-AG), even PEA tissue concentrations are changed in various pathologies   pathway (Iannotti et al., As a whole, these findings suggest that PEA may exert its physiological activities through many mechanisms of action, and PA either of dietary origin or formed from DNL, may influence its biosynthesis.PA exerts multiple fundamental biological functions at cellular and tissue levels and its steady concentration is guaranteed by its endogenous biosynthesis by DNL, a metabolic pathway present from the lower steps of the evolutionary scale. However, in the mammals the endogenous biosynthesis may not be sufficient in the long term. Contribution by dietary means is physiologically relevant and may be crucial an optimal intake of PA in a certain ratio with unsaturated fatty acids, especially PUFAs of both n-6 and n-3 families Figure . In factAll the Authors contributed to the conception or design of the work, drafting the work and revising it critically for important intellectual content, and approved the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There has been substantial progress in the management of patients with osteoporosis and the prevention of osteoporotic fractures. Currently available strong anti-resorptive agents are bisphosphonates and an anti-receptor activator of nuclear factor-kappa B ligand (RANKL) antibody, denosumab. Although bisphosphonates and denosumab both inhibit bone resorption and prevent vertebral and non-vertebral fractures, their mechanisms of action are different. Whereas bisphosphonates’ effects on bone mineral density and fracture peak around 3 to 5 years and become plateaued, those of denosumab are maintained for up to 10 years. There are differences in the modes of action of these two drugs. Bisphosphonates accumulate on the mineralized bone surface and are released by the acid environment under osteoclastic bone resorption, whereas denosumab is not accumulated on bone but directly binds RANKL and inhibits its binding to the receptor RANK. Thus, the reduction in denosumab concentration 4 to 6 months after injection may enable RANK to bind to RANKL, where it is highly expressed, such as in damaged bone regions. As anabolic agents, only teriparatide has been available for a long time, but abaloparatide, a synthetic analog of PTHrP(1–34), is currently under development. Because of the difference in the preferential binding conformations of PTH1 receptor between teriparatide and abaloparatide, the latter shows anabolic effects with fewer bone resorptive effects. Romosozumab, an anti-sclerostin antibody, inhibits the action of sclerostin, a canonical Wnt signal inhibitor secreted from osteocytes, and enhances canonical Wnt signaling. Romosozumab robustly increases vertebral and proximal femoral bone mineral density within 12 months and inhibits vertebral and clinical fractures in patients with osteoporosis by enhancing bone formation and inhibiting bone resorption. In this review, we summarize the recent advances in therapeutic agents for the treatment of osteoporosis and discuss future prospects with their use. Osteoporosis is a disease characterized by reduced bone mass and strength. Adult bone is continuously remodeled by osteoclastic bone resorption and osteoblastic bone formation. Advances in the treatment of osteoporosis have made substantial improvement in the management of patients with osteoporosis and the prevention of osteoporotic fractures. Many of these drugs increase bone mass and strength by inhibiting bone resorption, but there are drugs that increase bone formation. In this review, we summarize recent advances in the development of anti-osteoporotic drugs by focusing on bisphosphonates, denosumab, teriparatide, abaloparatide, and romosozumab. There are other drugs being used for the treatment of osteoporosis in some areas of the world but not discussed in this review, such as selective estrogen receptor modulators, active vitamin D compounds, vitamin K compound, strontium ranelate, calcitonin, and parathyroid hormone (PTH) (1–84).1. In contrast, the increase of BMD in proximal femur or total body reached a plateau after 3 to 5 years1. Similarly, the increase of proximal femoral BMD by once-yearly intravenous administration of zoledronic acid was evident for up to 4 years2. However, femoral BMD remained stable after that period, even with continuing administration of zoledronic acid. In addition, phase 3 clinical trials have shown that several nitrogen-containing bisphosphonates prevent vertebral, non-vertebral, and proximal femoral fractures during treatment for 3 years. However, the effects of long-term administration of nitrogen-containing bisphosphonates on fracture prevention, especially of non-vertebral bones, are not clear. In a study comparing the effects of continuous administration of alendronate for 10 years and alendronate for 5 years followed by placebo for 5 years, clinical vertebral fractures were reduced in patients who received alendronate for 10 years3. However, there was no difference in the frequency of morphometric vertebral fractures, non-vertebral fractures, proximal femoral fractures, and wrist fractures between patients who received alendronate for 10 years and 5 years3. Similarly, there was no difference in the frequency of morphometric vertebral fractures and clinical fractures in patients who received intravenous zoledronic acid for 9 years and 6 years followed by placebo for 3 years2, although fracture risk remained lower in the patients receiving zoledronic acid compared with those without zoledronic acid treatment. These results suggest that the efficacy of bisphosphonate on fracture prevention reaches a plateau after several years.The first bisphosphonate developed for the treatment of osteoporosis was etidronate, which had weak anti-resorptive activity compared with its mineralization inhibitory activity. Thereafter, nitrogen-containing bisphosphonates with much stronger anti-resorptive activity have become the main drugs for patients with osteoporosis. Bisphosphonates bind to hydroxyapatite during mineralization or demineralization and are released under acidic condition during osteoclastic bone resorption. Nitrogen-containing bisphosphonates suppress bone resorption by inhibiting the activities of osteoclasts and inducing their apoptosis. These drugs mainly increase the bone mineral density (BMD) of trabecular bones. For example, it has been shown that alendronate increased lumbar BMD for up to 10 years4. Large doses of intravenous bisphosphonates for the treatment of cancer-associated bone diseases are associated with an increased risk of osteonecrosis of the jaw (ONJ). The incidence of ONJ in subjects whose osteoporosis was treated with low doses of oral or intravenous bisphosphonates is very low, and there is a report suggesting that the incidence of ONJ is not increased among patients whose osteoporosis was treated with bisphosphonate5. Because of these adverse events, a drug holiday is proposed for some patients receiving bisphosphonates6. From those results, it is clear that there are several limitations for nitrogen-containing bisphosphonates, although they certainly increase BMD and prevent fractures, especially during the first several years.In addition to bisphosphonates’ plateauing efficacy, their long-term use has been shown to be associated with atypical femoral fractures (AFFs)7. FPPS is a rate-limiting enzyme for the formation of geranylgeranyl pyrophosphate, which prenylates and activates small GTP-binding proteins such as Rab, Rho, and Rac, and inhibition of FPPS leads to the suppression of osteoclastic bone resorption. Thus, if the affinity of a bisphosphonate for the mineralized surface of bone under the circulating blood pH is low, only an insufficient amount of bisphosphonate could be accumulated on the bone surface. Alternatively, even though a low amount of bisphosphonate is accumulated on the bone surface, if the affinity of bisphosphonate is low at the Howship’s lacunae under an acidic environment, enough bisphosphonate could be released and taken up by osteoclasts. If a similar amount of bisphosphonate is taken up by osteoclasts, the anti-resorptive activity of bisphosphonate would be determined by the affinity and inhibitory activity of that bisphosphonate for FPPS.The anti-resorptive activity of bisphosphonates is determined by the balance among several processes, and the behavior of each bisphosphonate is quite different. These include (a) binding of bisphosphonates in the circulation to the mineralized surface of bone matrix, (b) release from the bone surface under an acidic environment by osteoclastic bone resorption, and (c) inhibition of farnesyl pyrophosphate synthase (FPPS) after being taken up by osteoclasts8. Given the long-term accumulation of bisphosphonate in bone, the ideal property of bisphosphonate would be low affinity at neutral pH with less accumulation on the bone surface but low affinity at acidic pH with enough bisphosphonate to be released from bone and strong inhibitory activity to FPPS with inhibition of osteoclastic bone resorption9. Most long-term adverse events such as AFF were reported using alendronate or zoledronic acid, which have high affinity to mineralized bone at neutral and acidic pH, but there are bisphosphonates such as minodronate and risedronate that have physico-chemical characteristics that are substantially different from those of alendronate or zoledronic acid. Therefore, we have to take into consideration that the long-term efficacy on vertebral and non-vertebral fractures, as well as safety issues such as ONJ and AFF, may be different among bisphosphonates with different pharmacological and physico-chemical characteristics.On the mineralized bone surface, hydroxyapatite crystal is bound to collagen matrix, and the affinity of bisphosphonate for the bone surface is determined by its affinity for hydroxyapatite. The affinity for hydroxyapatite at neutral pH is highest for zoledronic acid, followed by alendronate and minodronate, and is lowest for risedronate followed by ibandronate. Under acidic pH, the affinity for hydroxyapatite is highest for alendronate, followed by zoledronic acid and risedronate, and is lowest for minodronate10. There was a close relationship between femoral neck BMD change and fracture risk reduction11. Denosumab showed long-term continuous effects on lumbar and proximal femoral BMD and on vertebral and non-vertebral fracture prevention for up to 10 years13. Denosumab also increased BMD of 1/3 radius consisting of mainly cortical bone almost in a linear fashion. This continuous increase of radial BMD was not reported in patients receiving oral bisphosphonates14. These results demonstrate that denosumab has potent effects on not only trabecular bone but also cortical bone, effects that were not observed using bisphosphonates. Long-term use of denosumab also has anti-fracture effects different from those of bisphosphonates. The yearly incidence of non-vertebral fractures was lower in patients who continued denosumab in the FREEDOM extension study. Whereas the annual incidence of non-vertebral fractures was more than 2% in the first 3 years of denosumab treatment in the FREEDOM study, it became less than 2% during 4 to 7 years of denosumab treatment and 0.7% in year 813. These results are consistent with the assumption that the prevention of non-vertebral fractures by denosumab becomes more prominent with long-term use of denosumab. However, it should be noted that a randomized controlled trial was performed for 3 years in the FREEDOM study, and there were no placebo controls in the extension study.Receptor activator of nuclear factor-kappa B ligand (RANKL) expressed in osteoblastic cells binds to its receptor, RANK, on the surface of osteoclasts and their precursor cells. The binding of RANKL to RANK is essential for osteoclast formation, activity, and survival. Denosumab is a human monoclonal antibody against RANKL and has been used for patients with osteoporosis, bone metastases, multiple myeloma, and giant cell tumor of bone. The pivotal phase 3 clinical trial Fracture REduction Evaluation of Denosumab in Osteoporosis every 6 Months (FREEDOM) in postmenopausal women with osteoporosis indicated that denosumab prevented vertebral, non-vertebral, and hip fractures15. RANKL expression by osteoblasts and osteocytes is expected to increase at sites of bone with microcracks or deteriorated strength. Thus, there is a possibility that after 4 to 6 months of denosumab injection, bone remodeling can take place, especially where RANKL expression is high. Such a possibility needs to be tested in the future.The clearance of denosumab from the blood circulation is very slow because it is not excreted from the kidney or metabolized in the liver but is cleared by the reticuloendothelial system. Consistent with the gradual decrease in the circulating denosumab concentration, bone resorption markers start to increase 4 months after denosumab injection and keep increasing toward the next injection at 6 months16. Thus, treatment with other anti-resorptive drugs is required after discontinuation of denosumab. It should also be noted that switching to teriparatide after denosumab treatment further enhances bone remodeling and causes a temporal reduction in vertebral and proximal femoral BMD and a more prolonged reduction in radial BMD17.Because denosumab rapidly inhibits RANKL-induced bone resorption, if patients do not receive enough vitamin D or calcium or both, there is a risk of hypocalcemia, especially during the first week after administration. Denosumab is also reported to be associated with an increased risk of AFF and ONJ. The non-accumulating, reversible nature of the action of denosumab also causes a rapid increase in bone turnover after discontinuation, resulting in a rapid loss of bone and a possible increase in fractures18. Teriparatide treatment markedly increases trabecular bone mass but is associated with bone loss at sites composed of mainly cortical bone, such as distal radius. After several months of teriparatide treatment, teriparatide stimulates bone resorption by increasing bone remodeling18. This enhanced bone resorption causes reduced bone mass and increased cortical porosity in peripheral bones19. The use of teriparatide is limited to 2 years in any life span, and this is because of the development of osteosarcoma in pre-clinical animal studies and the decrease of effects to increase BMD. Because long-term teriparatide use is currently not possible, it is necessary to gain maximal benefit of teriparatide during the 2 years. Previous studies indicated that a combination of alendronate with teriparatide does not have an overall superior effect on BMD21. The combination of zoledronic acid and teriparatide induced faster gain in lumbar and hip BMD than the respective drug alone until 26 weeks. After 52 weeks, lumbar BMD in the teriparatide group and hip BMD in the zoledronic acid group were similar to those in the combination group22. In contrast, the recent Denosumab and Teriparatide Administration (DATA) study indicated that the combination of denosumab and teriparatide produced a more prominent effect on BMD than each drug did alone24. The combination of teriparatide and denosumab may have the strongest anti-fracture efficacy among currently available regimens. As mentioned earlier, the DATA-Switch study indicated that teriparatide treatment after denosumab was associated with transient bone loss in lumbar spine and proximal femur and with prolonged BMD decrease in distal radius17. In contrast, teriparatide followed by denosumab continuously increased the BMD of lumbar spine and proximal femur. Thus, it is necessary to consider the timing of teriparatide use, as well as the order of sequential use of teriparatide, in the long-term management of patients with osteoporosis.Teriparatide is a peptide corresponding to the 34 N-terminal amino acids of PTH. Daily subcutaneous injections of teriparatide were shown to increase lumbar BMD and prevent vertebral and non-vertebral fractures34 to Ala-NH2. PTH and PTHrP bind to the same PTH1 receptor through their N-terminal portions. PTHR1 can take two conformations, R(0) and RG; R(0) binding results in prolonged signaling, whereas RG binding causes more transient responses. Abaloparatide binds more preferably to the RG conformation and is shown to cause a more transient response than does teriparatide25. The more transient action of abaloparatide via binding to the RG conformation of PTHR1 appears to favor the anabolic effect of abaloparatide with fewer bone resorptive effects. Because abaloparatide is currently under development, most of its clinical data come from a phase 3 clinical study . In that study, abaloparatide was shown to increase BMD and reduce vertebral and non-vertebral fractures in postmenopausal women with osteoporosis over 18 months26. The reduction of non-vertebral fractures by abaloparatide was significant when compared with placebo, but there was no difference in non-vertebral fractures between the teriparatide and placebo groups. The effects of abaloparatide on the increase in BMD of lumbar spine and proximal femur were shown to be greater than those of teriparatide. Hypercalcemia was lower in the abaloparatide than the teriparatide group26. The superior anti-fracture effects, especially on non-vertebral fractures with less hypercalcemic effect, may render abaloparatide a safe and effective anabolic agent when approved for the treatment of osteoporosis.Abaloparatide is a synthetic analog of the 34 N-terminal amino acid region of PTH-related protein (PTHrP) with substitution of eight amino acids from amino acid 22 to 31 and Ala27. Although Wnt signaling ubiquitously affects almost all types of cells, Wnt–β-catenin signaling in bone is controlled by sclerostin, a glycoprotein selectively secreted from osteocytes. Sclerostin is an inhibitor of Wnt signaling and potently inhibits bone formation. Romosozumab is a humanized monoclonal antibody against sclerostin. Romosozumab is also under clinical development, and a phase 3 study demonstrated that romosozumab was shown to markedly increase the BMD of lumbar spine by 13.3% and proximal femur by 6.8% in just 12 months and prevent vertebral and clinical fractures in postmenopausal women with osteoporosis28. Although non-vertebral fractures were not significantly reduced by 12-month romosozumab treatment, the low rate of non-vertebral fracture in the placebo group among the Latin American participants might have affected these results. In apost hoc analysis including only subjects outside Latin America, there was a significant 42% reduction in non-vertebral fractures in 1 year because of the higher incidence of non-vertebral fractures in the placebo group (2.7%)28. Among bone turnover markers, a bone formation marker, PINP, increased rapidly 14 days after the first injection and gradually decreased to baseline by 9 months, and there was a transient increase after repeated romosozumab injections28. A bone resorption marker, β-CTX, decreased rapidly on day 14 after the first injection and remained suppressed for 12 months28. Thus, romosozumab increased bone formation and suppressed bone resorption. It must be emphasized that this therapeutic profile is unique to romosozumab. Given the robust anti-fracture effect after only 12 months of treatment, romosozumab can become a strong therapeutic option for the treatment of osteoporosis.Canonical Wnt signaling plays a pivotal role in maintaining bone homeostasis. Mouse and human genetic data indicate that canonical Wnt–β-catenin signaling enhances osteoblastic bone formation and inhibits bone resorption via direct and indirect mechanisms. Enhanced Wnt–β-catenin signaling in osteoblastic lineages increases bone mass, whereas reduced signaling decreases bone mass29. The development of new bone-forming agents such as abaloparatide and romosozumab will increase treatment choice in patients with various severities of osteoporosis and will facilitate the understanding of the necessity and rationale for the combined or sequential treatment of osteoporosis.There are differences in the duration of anti-fracture effect among the currently available anti-resorptive agents. It is important to analyze the difference in the mode of action, efficacy in fracture prevention, and safety profiles of these agents in order to develop safe and effective management of patients with osteoporosis. After the report on ONJ and AFFs among bisphosphonate and denosumab users, there has been increasing concern about the use of drugs for osteoporosis treatment. The current crisis of osteoporosis treatment, owing to the reduction in the number of patients under treatment and the increase in hip fractures, can be overcome by these effortsAFF, atypical femoral fracture; BMD, bone mineral density; DATA, Denosumab and Teriparatide Administration; FPPS, farnesyl pyrophosphate synthase; FREEDOM, Fracture REduction Evaluation of Denosumab in Osteoporosis every 6 Months; ONJ, osteonecrosis of the jaw; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; RANK, receptor activator of nuclear factor-kappa B; RANKL, receptor activator of nuclear factor-kappa B ligand.


The limitations and side effects associated with current antidiabetic therapies have necessitated the search for novel therapeutic agents. Due to the multipathogenicity of diabetes mellitus, plant-derived compounds with proven multiple pharmacological actions have been postulated to “hold the key” in the search for an affordable, efficacious, and safer therapeutic agent in the treatment of the disease and associated complications. Resveratrol, a phytoalexin present in few plant species, has demonstrated beneficial antidiabetic effects in animals and humans through diverse mechanisms and multiple molecular targets. However, despite the enthusiasm and widespread successes achieved with the use of resveratrol in animal models of diabetes mellitus, there are extremely limited clinical data to confirm the antidiabetic qualities of resveratrol. This review presents an update on the mechanisms of action and protection of resveratrol in diabetes mellitus, highlights challenges in its clinical utility, and suggests the way forward in translating the promising preclinical data to a possible antidiabetic drug in the near future.Despite recent advances in the understanding and management of It is the most relevant among stilbenes, due to its well-known bioactivity. It was first identified in 1940 by Michio Takaoka from hellebore roots (Veratrum grandiflorum O. Loes) and it was from this source that the name “resveratrol” was derived  related factor 2), a transcription factor responsible for the constitutive and inducible expression of antioxidant response element- (ARE-) regulated genes . The actand HO-1 . Under nAs part of a coordinated response to the onslaught of ROS and other cellular stress signals, inflammatory cells  are recruited. These cells propagate their activities through the activation of inflammatory mediators such as cytokines/chemokines, cell adhesion molecules, growth factors, and prostaglandins . Altoget via the inhibition of the nuclear factor NF-κB pathway. NF-κB is a proinflammatory master switch, which activates proinflammatory cytokines gene expression and apoptosis cascade [κB can mediate the transcription of inflammatory genes. Production and release of proinflammatory cytokines and chemokines induce ROS and RNS production and further increase tissue oxidative stress resulting in a vicious cycle of inflammation and oxidative stress. Thus, modulation of NF-κB may be an attractive treatment strategy in the treatment and management of DM. RSV administration has been reported to significantly decrease NF-κB activity in the retinas of diabetic rats [κB activity and ameliorated the elevated levels of inflammatory proteins, TNF-α (tumour necrosis factor-α), interleukin- (IL-) 6, and COX-2 (cyclooxygenase 2), thus contributing to reduction in neuroinflammation and protection against functional and behavioural deficits in diabetic neuropathy [α, IL-1β, IL-6, IL-4, IL-17, COX-2 intercellular adhesion molecule 1 (ICAM), and vascular cell adhesion protein 1 (VCAM1) [κB. In addition, RSV significantly decreased the activation and recruitment of macrophages thereby retarding the inflammation process in diabetic rats [ via the downregulation of mRNA and protein expression of TNF-α, a known inflammatory trigger resulting in the inhibition of TNF-α-induced NAD(P)H oxidase activation, a key cellular source of O2∙−.RSV has been shown to elicit its anti-inflammatory properties in DM mainly cascade . Upon actic rats . RSV alsuropathy . The ant (VCAM1) , 108 whitic rats , 109. Zhtic rats  reported in vitro, limited studies have specifically investigated the antidiabetic effects of RSV in humans [Although numerous data exist on the beneficial outcomes of RSV in diabetic animals andn humans , 111–113β-cell function (HOMA-β) and therefore proposed that the mechanism of antidiabetic effects might be due to reduction in oxidative stress and a more efficient insulin signaling. Recently, Movahed and others [β at the administered dose, data from the study of Movahed et al. revealed a decrease in HOMA-β as well as homeostasis model of assessment of insulin resistance (HOMA-IR). Another important observation from the two studies is that RSV not only complemented but also provided additional protection over standard antidiabetic medication.Bhatt and colleagues demonstrated that RSV (250 mg/day for 3 months) administered along with glibenclamide and/or metformin demonstrated improvement in glycaemic parameters in diabetic patients . The stud others  reportedd others  who obseα protein content in the muscle [ VCAM, ICAM, IL-8, and IFN-γ was observed following incubation of human coronary artery endothelial cells with plasma from participants [Some beneficial outcomes have also been reported with RSV treatment in nondiabetic humans. In obese subjects, Timmers and colleagues  reportede muscle . In a doicipants .Although several studies have reported the beneficial effects of RSV in clinical trials, some other studies have also shown no benefit. The daily administration of RSV at a dose of 150 mg for 30 days improved metabolic parameters and modulated the putative molecular targets of RSV in obese men with no endocrine disorder . However in vitro and in animal models but there is so little human evidence. Unfortunately, studies conducted so far on the clinical utility of RSV in DM have been confronted with several limitations. The inherent cellular mechanisms underlying the antidiabetic effects of RSV deduced from preclinical data were not investigated properly in most of the studies. Single doses of RSV were employed in the studies, resulting in the lack of a proper understanding of dose-response relationships.Numerous studies on the biological activity of RSV have been carried outAnother limitation in the RSV clinical trials is sample size, involving only small cohorts of diabetic patients and the short duration of the trials. Long-term intervention studies are required to prove the efficacy of RSV and these are usually very expensive. RSV is a natural substance that is commercially available to everyone at a relatively cheap cost. This may partly explain the low interest of the pharmaceutical industry in investing funds on clinical studies to perform large-scale trials that would clarify the efficacy of RSV in DM.μM was reported to activate AMPK in a SIRT1-dependent manner, whereas a higher dose of 50 μM activated AMPK in a SIRT1-independent manner [In addition to the aforementioned limitations, the low bioavailability of RSV in humans poses another challenge in its use in DM . The lowt manner .There is a strong correlation between RSV metabolism and its effectiveness. New approaches to increase the bioavailability of RSV can help to actualise its potentials as a therapeutic agent in DM and related complications. One of such approaches is the coadministration of RSV with compounds that act as substrate for enzymes involved in RSV metabolism. UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) are key enzymes of RSV metabolism which catalyse glucuronidation and sulfation of RSV, respectively, enhancing its clearance from the body . The comPiperine is one of such compounds and has been shown to improve the effects and bioavailability of RSV in cell cultures and animal models. However, the outcome of the clinical trial on coadministration of RSV and piperine so far failed to improve bioavailability . The lowIn addition, the combination of RSV with genistein was reported to synergistically enhance antioxidant action in high-glucose treated Madin-Darby canine kidney (MDCK) epithelial cells when compared with RSV only and this combination may also have a therapeutic potential in diabetic nephropathy . However via delivery systems such as liposome-mediated delivery or nanotechnological approaches may result in improved therapeutic outcome. Also, intravenous injection as an alternative to traditional oral route of administration of RSV may bypass gastrointestinal absorption, conjugation, and hepatic metabolism, thus resulting in increased bioavailability and improved outcomes in diabetic patients.Long-term studies should be designed to determine appropriate dosage regimen and conclude on the antidiabetic effects of RSV in humans. Also, different new strategies such as the use of RSV nanoformulations, prodrugs, liposome-mediated delivery, and implantable devices could be used to improve the antidiabetic efficacy of RSV. Some of these technologies have been investigated in animal studies , 143 witRSV analogues with improved bioavailability, which can be administered either alone or in combination with other antidiabetic drugs, should be developed. The RSV molecule could be the primer for the development of other synthetic compounds that can be more bioavailable and of interest to pharmaceutical companies. In the bioavailability studies of RSV, red blood cells and tissue samples (such as muscle and adipose biopsy) should be included when it is ethical and clinically possible for a better assessment of RSV pool and tissue distribution. in vitro and in animals coupled with the reported health benefits in type 2 diabetic humans should be a driving force for conducting clinical research. The time is ripe to embark on large, well-controlled clinical studies to confirm the efficacy of resveratrol in the management of diabetes mellitus and gain a better insight into its biological effects in humans. In addition, the effective doses of resveratrol and treatment duration may differ between preventative and therapeutic trials. Therefore, it is important to investigate the dose dependent effects at varying treatment periods. A combinational approach as well as improved formulations of resveratrol may help to overcome the challenge of maintaining an effective concentration at the site of action for an appropriate period. By considering the aforementioned factors limiting clinical utility of resveratrol in subsequent studies, we may be able to use resveratrol or its analogue to treat or prevent diabetes mellitus in humans in the nearest future.The divergent mechanism of actions of resveratrol


The rapid and accurate determination of specific circulating biomarkers at different molecular levels with non- or minimally invasive methods constitutes a major challenge to improve the breast cancer outcomes and life quality of patients. In this field, electrochemical biosensors have demonstrated to be promising alternatives against more complex conventional strategies to perform fast, accurate and on-site determination of circulating biomarkers at low concentrations in minimally treated body fluids. In this article, after discussing briefly the relevance and current challenges associated with the determination of breast cancer circulating biomarkers, an updated overview of the electrochemical affinity biosensing strategies emerged in the last 5 years for this purpose is provided highlighting the great potentiality of these methodologies. After critically discussing the most interesting features of the electrochemical strategies reported so far for the single or multiplexed determination of such biomarkers with demonstrated applicability in liquid biopsy analysis, existing challenges still to be addressed and future directions in this field will be pointed out. Breast cancer is one of the three most common invasive cancers in females and one of the leading causes of cancer mortality among women worldwide. Since distant metastases are regarded as the major reason of death, early diagnosis becomes vital for improving this cancer type survival rate ,2.Traditional breast cancer diagnostic tools include clinical and physical examinations, histopathology, imaging mammography, and ultrasound magnetic resonance imaging. Insufficient clinical and physical examinations drive patients into mammography or ultrasound studies. Ultrasound is a non-invasive and safe tool, but since it is unable to screen many of the cancers it cannot replace mammograms, especially in women above 40. Mammography has limited sensitivity, a high rate of false positive results and may also lead to an accumulated exposure to radiation, which is considered an additional risk factor. Histopathology is an invasive approach to examining cancerous tissues once the disease is installed. All these limitations demand urgently the development of non-invasive, simple and low risk methods suitable for point-of-care (POC) screening/diagnosis of breast cancer. Undoubtedly, the international oncology community is in active pursuit of non-invasive methods adequate to be implemented in clinical routines for the diagnosis of breast cancer patients. Currently, strong efforts are being developed to monitor specific bodily fluid biomarkers for early and minimally invasive detection of this type of cancer .In this sense, electrochemical biosensors, combining selective biochemical recognition with the high sensitivity assumed for electrochemical detection, show particularly attractive merits such as disposability, portability, simplicity, low cost, small size, rapidity of response, ease of use, possibility of reading small sample volumes directly over a wide range of concentrations and compatibility with POC testing ,4,5,6. The need for ultrasensitive determination of cancer biomarkers of different natures in minimally invasive samples has led to the coupling of the benefits of electrochemical biosensing with those of nanotechnology to generate a new class of low cost, robust, reliable, easy-to-use, and ultra-sensitive diagnostics tools. Nanomaterials with unique features, such as noble metal and metal oxide nanoparticles (NPs), nanochains (NCs) and nanospheres, and integrated nanostructures including graphene or reduced graphene oxide (rGO), or composed of metallic NPs or oxides and multiwalled carbon nanotubes (MWCNTs), do provide biosensors for the determination of circulating breast cancer biomarkers with high sensitivity and precision and low detection limits (LODs) .A biomarker is defined as “a biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process or of a condition or disease” . Breast BCRA1), cell-free DNAs, messenger and microRNAs (mRNAs and miRNAs), proteins (soluble or membrane-associated proteins and glycoproteins), exosomes and tumor cells. Sensitive and rapid analytical methods for the reliable determination of these biomarkers in liquid biopsy samples are crucial since their level is useful for understanding the pathogenesis of disease prognosis, for early and reliable diagnosis and in the development of new therapeutic treatment regimens . I. I2O2 an3-Gr) coupling with catalyzed hairpin assembly target recycling and enzyme signal amplification, was developed by Shuai et al. ) between 15 and 100 ng mL−1 and a LOD of 4.4 ng mL−1  were achieved. Marques et al.  proposed3O4 NPs) to form stable highly loaded bioconjugates which were subsequently laid over a modified gold electrode surface 3+ bonded electrostatically to the probe immobilized on a gold surface and was used to determine the target cells. The charge density increased with the concentration of the target cells because the DNA probe bound to cells was protected from ExoI-catalyzed digestion and higher amounts of [Ru(NH3)6]3+ were bound to the intact DNA probe. The proposed method showed a linear detection of MCF-7 cells in a wide range from 102 to 106 cells mL−1 with a low LOD of 67 cells mL−1. An electrochemical cytosensor for highly selective and sensitive detection of cancer cells by using folate-linked DNA probes was reported by Zhao et al. . The met6]3−/4−. The microfabricated glass chip device provided rapid, label-free electrochemical detection of prostate cancer cells with high sensitivity and selectivity. Moscovici et al.  develope6]3−/4− DPV peak current in the presence of the sandwich system was employed to quantify the breast cancer cells. The aptamer used in this work was against the MUC1 protein, which is overexpressed in MCF-7 cells. The polydA modified aptamer was immobilized on both bare gold electrode and GO modified with AuNPs due to the intrinsic affinity between multiple consecutive adenines of polydA sequences and this metal. The aptasensor demonstrated a high selectivity in discriminating MCF-7 cells against non-tumorigenic cells and other cancer cells, and provided a linear range of 10–105 cells mL−1, a LOD of 8 MCF-7 cells mL−1, and successful applicability to the detection of 10-times diluted human serum samples spiked with the target cells.A sandwich electrochemical aptasensor was developed for the label-free and selective detection of breast cancer cells (MCF-7) using a polyadenine (polydA)-aptamer modified gold electrode and a polydA-aptamer functionalized AuNPs/GO hybrid  47]. Th. Th47]. Due to population variations in the expression of a single biomarker, the lack of totally specific biomarkers, and the multiple complex molecular events involved in breast cancer development and prognosis, the detection of a single biomarker does not support reliable diagnosis and prognosis of this neoplastic disease . Accordi2O2/3, 3, 5, 5-tetramethylbenzidine system allowed obtaining LODs of 3.9 fM and 7.4 pg mL−1 for IL-8 mRNA and IL-8 protein, respectively, in saliva samples. The biosensor was applied to the determination of both target biomarkers in 28 cancer and 28 matched control saliva samples.Wei et al.  develope−1 (IL-8 protein) in undiluted saliva samples. The dual amperometric biosensor was successfully applied to the direct determination of these two biomarkers in raw saliva samples spiked with known concentrations of synthetic IL-8 mRNA. An MBs-based dual amperometric disposable platform for the direct determination of IL-8 protein and IL-8 mRNA was also developed by Torrente-Rodríguez et al.  involvin2O2 addition using HQ in solution as redox mediator was employed to monitor each receptor concentration providing LODs at the level of low pg mL−1 for both receptors. The usefulness of the dual immunosensing platform was evaluated by analyzing spiked human serum and cancer cell lysates. Results obtained demonstrated the reliability of the approach to accurately determine the endogenous content of such biomarkers in these minimally pretreated complex samples without any matrix effect and the absence of significant cross-talking between the adjacent working electrodes. Eletxigerra et al.  developeThe previous sections in this review article provide useful insights into the latest advances and current tendencies in the electrochemical biosensing strategies reported so far for circulating breast cancer biomarkers with applicability in liquid biopsies analysis. The highlighted contributions describe the development and preliminary applicability of versatile electrochemical bioscaffolds for determining circulating breast cancer biomarkers at different molecular levels: gene mutations, miRNAs, mRNAs, proteins and even intact cells using single or multiplexed methods although still mainly in spiked serum and saliva samples. The wide variability of the selected approaches demonstrates the possibility of using different bioreceptors, bioassay formats and amplification strategies in connection with different electrode substrates and electrochemical techniques. It is worth mentioning that the variable complexity of the reported methodologies, ranging from a wide number of quite simple label-free approaches, such as signaling mechanism linked to target-induced conformational change of specific aptameric probes, to more complicated but extremely attractive strategies. Although most amplification methods make use of nanomaterials and/or enzymes, other less explored strategies based on the use of DNA concatamers and ECC redox cycling, nuclease assisted target recycling and target catalyzed hairpin assembly have also been proposed. 2/rGO, PPDNSs, organosilica@chitosan nanospheres, graphene, WO3), as signaling carriers , or even as artificial enzymes (Pt NCs and Pd–Ir and PB NPs). Most of the reported methods have been developed using integrated formats, both at conventional and disposable electrodes. Considering the high sensitivity required for the determination of circulating breast cancer biomarkers, and the remarkable current progress made in bioconjugation and nanotechnology techniques, most of the reported biosensing strategies have been developed in connection with the use of different nanomaterials taking advantage of the unique properties they offer when employed as electrode modifiers (AuNPs, CNTs, Pt NCs, SnOTaking into account the number of strategies described so far in this field, the determination of circulating breast cancer protein and miRs biomarkers have shown to be of special relevance. Other less explored but extremely interesting biosensing methods have been described recently for the determination of intact circulating breast cancer cells. All the compiled approaches have demonstrated compatibility with the clinical relevance range of the target biomarker and to be suitable for practical purposes through the analysis of real samples.When electrochemical techniques are compared with optical techniques, which are also commonly used in biosensing of circulating breast cancer biomarkers, it can be said that the former have been more and more used in recent years because they are able to overcome some limitations of optical techniques in terms of portability, cost and compatibility with POC devices. These features make electrochemical biosensors especially promising candidates for non-invasive clinical diagnosis of breast cancer with easy adaptability to a broad range of settings. Due to the inherent miniaturization ability of electrochemical biosensors and their compatibility with standard microfabrication and semiconductor technologies, simple, accurate, portable, and inexpensive biosensing platforms have been developed for circulating breast cancer biomarkers at different molecular levels. However, a major limitation is the need for multiple steps and labeled reagents in the preparation of the biosensor before electrochemical detection takes place. While electrochemical detection often achieves the highest sensitivities due to the use of surface-bound probes and the preconcentration of the analyte on the sensor surface, single-step optical readout strategies—generally performed in homogeneous solution—are often less sensitive. In addition, electrochemical detection is extremely compatible with high multiplexing capability and both low- and high-density electrochemical sensor arrays can be conveniently fabricated by adopting the standard microfabrication technology. These breakthroughs and the superseding capabilities already demonstrated allow envisioning faster progress and greater applications of electrochemical compared to optical biosensing for non-invasive breast cancer diagnosis.However, despite the encouraging capabilities demonstrated, all the electrochemical biosensors reported so far for the determination of circulating breast cancer biomarkers remain in the proof of concept or prototype stages of development, and none of them has been evaluated with a large enough number of real patient samples to assess their reliable clinical validation. Therefore, an exhaustive validation through the analysis of large numbers of real patient samples and the comparison against other available competitive methodologies remains to be addressed. The future translation into commercial products demands also the improvement of reproducibility in the preparation of different batches, the robustness against environmental variables by optimizing the best storage stability and transportation conditions to ensure appropriate functionality, and their simplicity of preparation and use. Additional efforts should be focused also on the biosensors’ integration into automated and miniaturized systems. It is worth mentioning also that, despite the significant progress made regarding the non-specific adsorption and multiplexed requirement issues, further efforts will be needed to detect a larger number of analytes in scarcely diluted liquid biopsies. Therefore, although the replacement of techniques currently used for the determination of circulating breast cancer biomarkers by electrochemical biosensing devices is clearly not imminent, they promise a paradigmatic shift in the way to conduct disease diagnosis and health monitoring in the near future, allowing for more rapid clinical decision-making, with the corresponding healthcare costs and patient stress reduction it represents. This shift will be facilitated by developments in molecular biology, nanofabrication methods and labeling, and likely will cause electrochemical biosensors to find an important niche for determining circulating breast cancer biomarkers in the hospital routine. In summary, although extremely sensitive and accurate clinical measurements of circulating biomarkers for early detection of breast cancer pose a formidable challenge, the wide range of possibilities and interesting capabilities described so far make feasible the future availability of inexpensive devices for reliable on-the-spot cancer diagnosis using electrochemical biosensors. These methodologies are envisaged to provide, apart from a reliable earlier diagnosis which will lead to improved therapeutic outcomes, also a better fundamental understanding of this disease progression.


The growing incidence of non-melanoma skin cancer (NMSC) necessitates a thorough understanding of its primary risk factors, which include exposure to ultraviolet (UV) wavelengths of sunlight and age. Whereas UV radiation (UVR) has long been known to generate photoproducts in genomic DNA that promote genetic mutations that drive skin carcinogenesis, the mechanism by which age contributes to disease pathogenesis is less understood and has not been sufficiently studied. In this review, we highlight studies that have considered age as a variable in examining DNA damage responses in UV-irradiated skin and then discuss emerging evidence that the reduced production of insulin-like growth factor-1 (IGF-1) by senescent fibroblasts in the dermis of geriatric skin creates an environment that negatively impacts how epidermal keratinocytes respond to UVR-induced DNA damage. In particular, recent data suggest that two principle components of the cellular response to DNA damage, including nucleotide excision repair and DNA damage checkpoint signaling, are both partially defective in keratinocytes with inactive IGF-1 receptors. Overcoming these tumor-promoting conditions in aged skin may therefore provide a way to lower aging-associated skin cancer risk, and thus we will consider how dermal wounding and related clinical interventions may work to rejuvenate the skin, re-activate IGF-1 signaling, and prevent the initiation of NMSC. Non-melanoma skin cancers (NMSCs) comprise the most common types of cancers in humans worldwide and originate from keratinocytes within the epidermal layer of the skin. In the United States alone, more than 2 million people are diagnosed with a NMSC each year ,2. The mThe single greatest risk factor for NMSC development is exposure to ultraviolet (UV) wavelengths of sunlight, which induce the formation of UV photoproducts in DNA. When not properly dealt with, these photoproducts may lead to mutations in genomic DNA that provide a growth advantage to epidermal keratinocytes and initiate a NMSC. The observed correlation between skin cancer and aging has traditionally been attributed to a lifetime of exposure to UVR that begins during childhood, which results in an accumulation of mutations that eventually drive tumorigenesis later in life. However, even in adults, sun avoidance and the application of sunscreens have been shown to reduce the incidence of actinic keratoses ,5,6. ThuIn this review, we will therefore summarize how DNA photoproducts induced by UV wavelengths of light generate mutations in DNA and highlight the primary mechanisms by which cells respond to this DNA damage. This discussion will include an overview of nucleotide excision repair and DNA damage checkpoint signaling, which together allow cells to cope with a genome damaged by UV. Where appropriate, we will focus on published work that has addressed these issues in the context of aging and specifically within epidermal keratinocytes, which have the potential to become transformed and give rise to skin cancers. We will then review a growing body of literature that supports a role for the insulin-like growth factor (IGF-1) in keratinocyte responses to DNA damage and evidence that this system is de-regulated in geriatric skin. Lastly, we will discuss clinical interventions that can be employed to counteract this IGF-1-deficiency and the tumor-promoting environment of geriatric skin , which mPhotons of light with wavelengths in the range of 100–400 nm fall within the UV spectrum, which can be further subdivided as UVA (320–400 nm), UVB (280–320 nm), or UVC (100–280 nm) radiation. Though the sun emits UVR within all of these wavelengths, most UVC is absorbed by the Earth’s ozone layer. Thus, the UV wavelengths of sunlight that humans are typically exposed to on a daily basis are primarily composed of UVA (90%–95%) and UVB (5%–10%). A variety of cellular biomolecules absorb these wavelengths of light, including genomic DNA.The direct absorption of UV photons by DNA induces the formation of adducts between adjacent pyrimidine nucleotides ,11, withThese UV photoproducts are problematic to irradiated cells because the DNA lesions are potentially mutagenic and/or lethal. For example, cytosines and 5-methylcytosines within CPDs spontaneously deaminate to uracils and thymines, respectively, at a million-fold increased rate relative to the undamaged nucleotides ,17,18,19Humans and other placental mammals possess a single system for removing UV photoproducts from genomic DNA known as nucleotide excision repair ,32,33. TMutations in NER gene products give rise to both the disease xeroderma pigmentosum, which is characterized by a several thousand-fold increased risk of skin carcinogenesis, and to a neurodegenerative and premature aging disorder known as Cockayne syndrome ,36. VariIn addition to NER, cells have additional systems for detecting the presence of UV photoproducts throughout the genome that are thought to reduce the likelihood of introducing incorrect nucleotide opposite CPDs and (6-4)PPs during DNA replication and to provide additional time for damage removal by NER. These systems are termed DNA damage checkpoints and are comprised of protein kinases that regulate the activities of effector proteins that control DNA replication and cell cycle progression ,38. The The two most well-recognized signals for activation of the ATR-CHK1 signaling network are unfilled gaps generated by NER that subsequently get enlarged by exonucleolytic action ,51,52 anOnce activated, ATR and CHK1 phosphorylate numerous protein targets that control DNA synthesis on UV-damaged templates ,67,68,69The process of aging can cause a number of changes to the morphology and physiology of skin ,85,86,8732P-postlabeling method to quantify the induction and subsequent removal of a number of UV photoproducts from the epidermis of 30 human subjects of diverse age and skin type [2 of solar simulating radiation, excising a small punch biopsy of the area, and then purifying the genomic DNA from the epidermis. This genomic DNA was then treated with a panel of nucleases to produce nucleoside-3′-phosphates that were subsequently 5′-labeled with T4 polynucleotide kinase and detected as trinucleotides (containing an unmodified 5′-thymidine) by high performance liquid chromatography. The use of photoproduct standards provided the investigators the means to quantify the abundance of TT<>T and TT<>C trinucleotides containing CPDs and (6-4)PPs within the subjects’ epidermal genomic DNA.Nonetheless, one relevant study used the kin type . The met7 nucleotides. As will be discussed in greater detail below, several other studies that have addressed UV photoproduct levels in the epidermis as a function of subject age have typically only done so in the context of DNA repair and have not provided sufficient information regarding the levels of initial DNA damage caused by UV exposure. Consideration of this issue in future work may therefore provide a more complete picture regarding how skin aging affects both the generation of UV photoproducts and the subsequent cellular responses. The use of deep sequencing technologies such as Damage-seq [Interestingly, when the authors classified the subjects by age, the levels of all four of these adduct-containing damages were found to be higher in individuals over the age of 50 than in individuals under the age of 50 , though mage-seq  and relamage-seq ,93,94,95Though there has long been interest in understanding the association between aging and the repair of UV photoproducts , many pr32P-postlabeling and HPLC method described earlier [In addition to quantifying DNA adduct levels immediately following UV exposure, the study employing the  earlier  also exaNonetheless, the notion that nucleotide excision repair of UV photoproducts occurs at a slower rate in the skin of older individuals was supported by a more recent study that compared CPD removal rates between subjects in their 20 s and 70 s, all of whom had a type III or IV skin type . These a2 of UVB , geriatric skin from subjects >65 years of age displayed many such cells [The three studies described above examined the loss of UV photoproducts from whole epidermis. Though keratinocytes throughout the different layers of the epidermis will contain DNA damage following UV exposure, the effect of UV on the proliferating cells within the basal layer is potentially most relevant to skin carcinogenesis. Using anti-CPD and anti-Ki67 antibodies to stain replicating keratinocytes containing UV photoproducts, a more recent study showed that whereas skin from subjects between the ages of 20 and 28 years displayed very few replicating keratinocytes containing DNA damage 24 h after exposure to 350 J/mTogether, these several studies indicate that the efficiency by which epidermal keratinocytes in human skin are able to remove CPDs may decrease as people age. However, it should be noted that the different approaches for measuring CPD repair that were used in the studies above have a number of limitations that are relevant to UV-induced mutagenesis and skin carcinogenesis. The first issue is that these studies have generally focused on CPDs and ignored (6-4)PPs, which are also capable of introducing mutations into genomic DNA. Although (6-4)PPs are generally repaired at a much faster rate than CPDs, this repair largely takes place within the same time frame as the bulk of ATR-CHK1 signaling (during the first 4 h post-UV). Thus, defects in (6-4)PP removal, ATR-CHK1 signaling, and in the suppression of chromosomal DNA synthesis after UV could in principle be associated with mutagenesis that gives rise to skin cancers. Furthermore, the accuracy of assays for measuring CPD removal, which in many studies takes place on the time scale of days, may be complicated by effects of cell proliferation, epidermal cell turnover, and apoptosis. These processes may dilute CPD content within genomic DNA in a DNA repair-independent manner, and this complication therefore affects the accuracy of CPD quantitation as a reliable measure of nucleotide excision repair capacity in the skin. Thus, the application of novel technologies, including assays that directly detect the sedDNA products of nucleotide excision repair ,104,105,Whereas a number of studies have explored UV photoproduct formation and repair in human skin in vivo, much less is known regarding the effects of UVR on epidermal keratinocyte DNA synthesis and DNA damage checkpoint signaling in human epidermis. Nonetheless, an early study employed tritiated thymidine injection into the skin of human subjects following UVR exposure to monitor how UVR affects both normal, chromosomal DNA replication and DNA synthesis associated with DNA repair . These tAs the most abundant cell type in the epidermis, keratinocytes are the major target of UVR and are also the cell type of origin for the development of NMSCs. Though keratinocytes are capable of responding to UV in a cell autonomous manner, interactions of keratinocytes with other cell types within the skin are also expected to influence keratinocyte responses to UVR. An important goal of skin carcinogenesis research is therefore to understand how the physiological environment of the skin contributes to NMSC development. Given that many aspects of skin biology change as human age ,85,86, aThrough the regulation of various intracellular signaling pathways that control cell proliferation and other cellular phenotypes, growth factors play fundamental roles in general cell biology, including within the epidermis of the skin. Local paracrine signaling in particular may affect keratinocyte growth and response to exogenous stress. One such factor of relevance to the epidermis and its response to UV-induced DNA damage is insulin-like growth factor-1 (IGF-1). In human skin, keratinocytes express the IGF-1 receptor (IGF-1R) but do not produce IGF-1 ,112,113.Extensive DNA damage caused by UVR may lead cells to undergo either apoptosis or senescence ,118. BotWhen UV-induced DNA damage is less extensive, cells may undergo a permanent growth arrest known as senescence ,118. TheTo better understand the link between IGF-1 and UV photoproduct removal in human keratinocytes, additional studies were recently carried out using primary and telomerase-immortalized human adult or neonatal foreskin-derived keratinocytes that were cultured in vitro ,124. In Studies with cultured keratinocytes in vitro demonstrated that disruption of IGF-1R signaling was associated with a reduction in the expression of the NER factors XPC and XPF/ERCC4 at both the level of mRNA and protein . These fThough nucleotide excision repair is a major system that protects keratinocytes from DNA damage and associated mutagenesis associated with UV exposures, it is not the only protective barrier to cancer initiation. Cells also possess various DNA damage signaling pathways that sense DNA damage and transiently arrest cell cycle progression and DNA synthesis to provide additional time for DNA repair. Together, DNA repair and DNA damage checkpoint signaling therefore limit mutagenesis and carcinogenesis. As described above, the ATR and CHK1 kinases play a major role in regulating these various DNA damage responses following UVB exposure, and partial disruption of their activities is linked to tumorigenesis ,81,82,83Two recent studies have examined how the activation status of the IGF-1R affects ATR-CHK1 signaling following UVB exposure in cultured keratinocytes ,122. BotThe observation that both nucleotide excision repair and ATR-CHK1 signaling are disrupted in keratinocytes with inactive IGF-1Rs indicates that both phenotypes may be caused by a single, common defect in DNA metabolism. Indeed, both systems utilize the ubiquitous DNA metabolic protein RPA, which plays critical roles in DNA replication, repair, and recombination ,56,57,58Though RPA is generally thought to be an abundant nuclear protein, the fact that it functions in so many diverse DNA metabolic processes may become problematic under conditions in which its availability becomes limiting. Consistent with this notion, several recent studies have suggested that “RPA exhaustion” is a frequent problem in cells undergoing extensive replicative stress ,150,151,There has long been an interest in the use of cosmetic dermal rejuvenation approaches to create more youthful-appearing skin ,153. TheGiven that an increase in dermal fibroblast senescence in geriatric skin relative to young skin is correlated with a decrease in the expression of IGF-1 , the effNonetheless, these novel findings suggest that wounding therapies have the potential to be useful in preventing the initiation of NMSCs in geriatric patients ,156. It The fact that the majority of NMSCs occur in patients over the age of 60 indicates that the physiology of aged skin may contribute to the risk of UVB-induced skin carcinogenesis. As summarized here, the skin of geriatric individuals is characterized by an increase in dermal fibroblast senescence and a corresponding decrease in IGF-1 production. This phenotype is associated with the decreased activation of the IGF-1R in epidermal keratinocytes and in altered cellular responses to UVB-induced DNA damage, including defects in UV photoproduct removal rate by NER, ATR-CHK1 kinase signaling, and in the suppression of DNA synthesis following UVB exposure. Together these altered responses to DNA damage increase the likelihood of mutagenesis and NMSC development. Fortunately, these negative outcomes may be counteracted by dermal wounding methods that rejuvenate the skin and restore the IGF-1/IGF-1R system to that found in young skin. Thus, dermal wounding has the potential to become a cost effective method for preventing NMSC initiation in aging populations.


Gliomas are devastating brain cancers that have poor prognostic outcomes for their patients. Short overall patient survival is due to a lack of durable, efficacious treatment options. Such therapeutic difficulties exist, in part, due to several glioma survival adaptations and mechanisms, which allow glioma cells to repurpose paracrine signalling pathways and ion channels within discreet microenvironments. These Darwinian adaptations facilitate invasion into brain parenchyma and perivascular space or promote evasion from anti-cancer defence mechanisms. Ultimately, this culminates in glioma repopulation and migration at distances beyond the original tumour site, which is a considerable obstacle for effective treatment. After an era of failed phase II trials targeting individual signalling pathways, coupled to our increasing knowledge of glioma sub-clonal divergence, combinatorial therapeutic approaches which target multiple molecular pathways and mechanisms will be necessary for better treatment outcomes in treating malignant gliomas. Furthermore, next-generation therapy which focuses on infiltrative tumour phenotypes and disruption of the vascular and perivascular microenvironments harbouring residual disease cells offers optimism for the localised control of malignant gliomas. High-grade gliomas remain one of the most aggressive and difficult to treat cancers in adults, with a very poor prognosis of 14 months . HoweverGlioma aggressiveness and therapeutic resistance has led to many transcriptome- or genome-wide studies aiming to unravel the complex molecular pathways of gliomas to better understand disease progression and to design novel targeted therapeutic agents. For instance, efforts have been made to research and analyse thousands of genetic profiles of tumour samples uploaded in a shared online space called The Cancer Genome Atlas Research Network in order to identify appropriate tumour targets ,11,12. AThe invasive ability of gliomas has been the focus of pathologists since 1938, when German neuropathologist Hans Joachim Scherer suggested that the tumour cells of glioma malignancies infiltrate the normal brain parenchyma with distinct morphological patterns . Of thesAmong all spaces provided by the Scherer model of invasion, the perivascular space has the major share of glioma invasion, and more than 85% of the invading malignant cells migrate around blood vessels to form the satellite tumour shape ,27. Furt2+ concentrations through inositol-1,4,5-triphosphate receptor 3 [2+ oscillations [2+ resulted in the activation of ion channels necessary for volume and morphology changes of glioma cells during migration through narrow spaces. Calcium oscillations results in reprogramming of the Cl− and K+ channels which are normally set to regulate neural membrane potential. This enables the glioma cell to reduce its volume down to 33% of its original size, and thus facilitating the infiltration through small compartments by removing free cytoplasmic water outside the cell via Cl− efflux [2+ which, in turn, induces the contraction of cellular cytoskeleton, cytosolic flow and ultimately the formation of bleb protrusions at glioma cell membranes. Both BK receptor 2 inhibitor, Hoe-140, and bleb retraction blocker, blebbistatin, were both effective in inhibiting glioma cell invasion [2+ levels, but it can also enhance human and rat glioma cell migration in vitro via binding to BK receptor 1. This leads to the activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT cascade signalling pathway [Despite a predilection in gliomas to vascularity, it is not clear yet whether glioma cells infiltrate this perivascular space preferentially as a response to appropriate nutrients and environmental cues which promote tumour survival and growth, or whether this space represents the least physical barrier that resists tumour cell propagation to surroundings. According to our current knowledge, this question is yet to be answered and therefore needs to be comprehensively addressed, as it affects the trends and the prioritisation of tumour treatment modalities. It could be argued that the availability of certain chemo-attractive molecules in the perivascular and endothelial cell niches could explain the preferential habitation of glioma cells around the vascular tree . For exaceptor 3 ,36. Calcceptor 3 ; i.e., lceptor 3 ,37, wherllations ,38. Thes− efflux ,39. Seif pathway , involve pathway , or the  pathway ,45,46,47 pathway .As a consequence of the significant role of BK in glioma perivascular satellitosis, a BK receptor inhibitor, Icatibant , has been tested in preclinical studies using glioma rat models and shown to be effective in impairing the migration of glioma cells through cerebral parenchyma and ultimately resulting in a smaller tumour mass . SimilarIntegrins likely also promote perivascular invasion through the activation of the chemokine (C-X-C motif) ligand 12 (CXCL12)/CXCR4) signalling pathway . CXCL12,+/CD133+ GSCs are located around CD31+ endothelial cells [The GSC theory postulates an explanation for tumour aetiology, albeit the dynamic nature of GSC subpopulations cautions against reductionist descriptions of static populations. It suggests that the bulk of genotypically diverse tumour could be generated by a small population of self-renewing cancer stem-like cells that in turn can differentiate into multiple tumour cell lineages . Howeveral cells ; likewisal cells . Recentlal cells . In addiDespite the anti-tumour activity of endothelial cells advocated by studies conducted in other tumours such as colorectal and prostate cancers ,59, seveCurrent consensus in the glioma field suggests that GSC are located within two main compartments; firstly, GSC could be located where hypoxia is evident, and pseudopalisading glioma cells  are gathered with this characteristic form around the necrotic regions in the glioma core ; the sec2+-dependant release of K+. Moreover, this also leads to several dysfunctional consequences on the delicate neurovascular unit such as the decrement of shuttling energetic metabolites such as lactate from astrocytes to neurons [Current studies indicate that the infiltration of glioma cells through the perivascular space of the adjacent surrounding parenchyma has several dismal effects on the locally invaded niche of brain tissue. In normal physiological conditions, astrocytic end-feet circumferentially covers almost the entire surface of blood vessels  to form  neurons , and the neurons . The rep neurons . The dis neurons .Although the definitive mechanism of glioma invasion through the perivascular space is largely unclear, we advocate that gliomas and GSC seek to invade the perivascular compartment to reach the abluminal surface of the blood vessels via tracking different chemotactic ligands, activating signalling pathways, expressing extracellular adhesive molecules, and even reprogramming the normal protective mechanisms towards tumour benefit. Such mechanisms hint at the adaptive nature of glioma cells as the perivascular compartment provides several advantages for the tumour, in addition to nutrients provided in the serum leaked trough fragile basement membranes of the invaded blood vessels. For instance, the generation of new vascular stem microenvironments by the migrating GSC could lead to considerable repopulation of tumour, with a completely distinct genetic and epigenetic profile and facilitate further tumour growth and invasion at the new site . This ni


Inflorescence development in cereals, including such important crops as rice, maize, and wheat, directly affects grain number and size and is a key determinant of yield. Cytokinin regulates meristem size and activity and, as a result, has profound effects on inflorescence development and architecture. To clarify the role of cytokinin action in inflorescence development, we used the NanoString nCounter system to analyze gene expression in the early stages of rice panicle development, focusing on 67 genes involved in cytokinin biosynthesis, degradation, and signaling. Results point toward key members of these gene families involved in panicle development and indicate that the expression of many genes involved in cytokinin action differs between the panicle and vegetative tissues. Dynamic patterns of gene expression suggest that subnetworks mediate cytokinin action during different stages of panicle development. The variation of expression during panicle development is greater among genes encoding proteins involved in cytokinin metabolism and negative regulators of the pathway than for the genes in the primary response pathway. These results provide insight into the expression patterns of genes involved in cytokinin action during inflorescence development in a crop of agricultural importance, with relevance to similar processes in other monocots. The identification of subnetworks of genes expressed at different stages of early panicle development suggests that manipulation of their expression could have substantial effects on inflorescence architecture. Inflorescence development in cereals, including such important crops as rice, maize, and wheat, directly affects the number and size of seeds and is a key determinant of yield –3. The fN6 position of ADP/ATP, hydroxylate the isoprenoid side chain, and then activate the cytokinin by converting it to the free base form . Al. AlIPT g qRT-PCR . Group ILOG gene as well as for CKX11, the most abundant of the CKX family member in the early panicles, both patterns being confirmed by RNA-Seq  relative to the NanoString data  affecting grain yield [CKX2 encodes a cytokinin oxidase and its reduced expression results in increased cytokinin levels [CKX2 expression and grain yield has been observed based on natural variation between different rice strains or when CKX2 expression is specifically down-regulated by RNAi or CRISPR-based approaches [CKX2 is of medium abundance in early panicles within the CKX family. This may allow for greater variation in its expression level without severely perturbing panicle development and, in this respect, it will be of interest to determine whether mutations in the more abundant CKX family members negatively affect panicle development. We find that CKX2 is a member of group Ic based on cluster analysis, with maximal expression during ST2 a major stage for primary branch formation, and thus the effects of CKX2 mutations on panicle size and yield are likely related in part to panicle branching.A second gene involved in cytokinin metabolism, in yield . CKX2 enn levels , which cproaches , 43, 44.AHPs and type-A RRs. The two rice AHPs are found in group Ia (AHP2) and II (AHP1) based on cluster analysis, but there is only limited variation of expression during the panicle stages and thus they can be considered as expressed throughout panicle development. AHP2 is the most highly expressed of the two family members. The role of the AHPs was examined by using RNAi with the AHP2 sequence, which substantially reduced expression of AHP2, the closely related AHP1, as well as the PHPs to some extent [Effects on panicle development have also been found by taking targeted approaches to modify the expression of cytokinin signaling elements, in particular e extent . The mutRR6, like many of the type-A RRs is a member of group II, and is most highly expressed during the stages when the branch meristems are formed. The type-A RRs are negative regulators and so ectopic overexpression of RR6, as examined by Hirose et al. [e et al. , would be et al. , consistThe results obtained from the NanoString expression analysis provide insight into how genes involved in cytokinin action regulate inflorescence development in a crop of agricultural importance, with relevance to similar processes in other monocots. The expression levels for many genes involved in cytokinin action differ between the panicle and vegetative tissues, suggestive of differing functional roles tailored to the specific needs of these tissues. Substantial differences in expression were identified among gene-family members, and these may point the way toward which genes play predominant roles in early panicle development. The identification of subnetworks of genes expressed at different stages of early panicle development points to how manipulation of their expression could have differential effects on inflorescence architecture.S1 FigThe average gene expression value during early panicle development was compared to that found in rice roots and shoots following treatment for 2 h with 5 μM BA or a vehicle control.(PDF)Click here for additional data file.S2 Fig# Significant differences based on a T-Test between the two stages with maximum and minimum expression (P < 0.05). *Significant differences when comparing expression across all stages based on ANOVA with Holm post-test (P < 0.05).A cluster analysis was performed based on Euclidian distance between gene expression at stages 0–5 of early panicle development. This is plotted as a heat map with a dendrogram for each family of genes. (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.