The gastrointestinal (GI) tract is separated from the body’s internal environment by a single layer of epithelial cells, through which nutrients must pass for their absorption into the bloodstream. Besides food and drink, the GI lumen is also exposed to bioactive chemicals and bacterial products including short-chain fatty acids (SCFAs). Therefore, the GI tract has to monitor the composition of its contents continuously to discriminate between necessary and unnecessary compounds. Recent molecular identification of epithelial membrane receptor proteins has revealed the sensory roles of intestinal epithelial cells in the gut chemosensory system. Malfunctioning of these receptors may be responsible for a variety of metabolic dysfunctions associated with obesity and related disorders. Recent studies suggest that SCFAs produced by microbiota fermentation act as signaling molecules and influence the host’s metabolism; uncovering the sensory mechanisms of such bacterial metabolites would help us understand the interactions between the host and microbiota in host energy homeostasis. In this review, the contribution of colonic SCFA receptors in energy metabolism and our recent findings concerning the possible link between SCFA receptors and host energy homeostasis are discussed. Part of the gastrointestinal (GI) tract, the intestinal lumen is one-way tube where food materials from the external environment are progressively converted into molecular products. Approximately 100 trillion bacteria, which are termed the gut microbiota, are present in the intestinal lumen, especially in the colon. A single layer of epithelial cells separates this diverse bacterial community from the internal environment. Thus, host–microbiota interactions occur at the mucosal surface of the intestine. The genome of the gut microbiota contains an estimated 150 times as many genes as in the host genome, and continuously produces large amount of various chemicals, including short-chain fatty acids (SCFAs), which can be beneficial or harmful to the host  cells  Figure . Lamina i/o) and toxin-insensitive (Gq) G proteins, whereas FFA3 couples only to Gi/o protein. We hypothesized that FFA2 and FFA3 function as molecular interfaces between the gut microbiota and host colon. Indeed, SCFAs produced by bacterial fermentation in the colon affects local colonic functions; luminal application of propionate and butyrate induces Cl− secretion and muscle contraction in rat distal colons  and FFA3 (GPR41) were deorphanized as SCFA receptors –10. Thesl colons , 12. Thel colons .Using RT-PCR and western blotting analysis, mRNA and protein for FFA2 were found in the distal ileum and colon in extracts from separated rat mucosa as well as in human colon extracts , 14. HowImmunohistochemistry was used to identify the cellular distribution of FFA2 and FFA3 in the colon. FFA2-immunoreactive epithelial cells were found in rat, guinea pig, and human colonic epithelia, with particularly strong expression in PYY and GLP-1-producing enteroendocrine L-cells but not 5-HT containing enterochromaffin (EC) cells –16. Immuin vitro. The results from single cell cultures, however, are difficult to extrapolate to understand receptor physiological function because intestinal tissue contains different epithelial cell types as well as the enteric nervous system and mucosal immune system, which influence the secretion of gut hormones. In addition, even in vivo experiments have inconsistent results; intravenous acetate infusion in innervated and denervated loops in conscious pig did not change the concentration of GLP-1 but did for PYY  Most cultured cells in in vitro cell culture system experiments cannot maintain cell polarity. (3) Many studies cannot directly differentiate whether specific gut hormone-containing enteroendocrine cells are activated to secrete hormones through direct or indirect chemical sensing, particularly if non-enteroendocrine cells also express chemosensory receptors. Indeed, our morphological data suggest that enterocytes also express FFA2 and FFA3.The morphological data suggest that gut microbiota-derived SCFAs in the colonic lumen function as stimuli for GLP-1 and PYY release. SCFAs are known to trigger the release of gut hormones, but results have been inconsistent due to differences in the systems used. Enteroendocrine cell culture systems, such as murine STC-1 , GLUTag  for PYY . On the  for PYY . There ain vitro and in vivo conditions (We used the Ussing chamber system to investigate whether SCFA stimulation induces GLP-1 secretion and to define precise stimulation and secretion sites of FFAs. This preparation maintains the polarity of epithelial cells and contains other cellular elements like intact intestine. In addition, an advantage of this system is that it allows simultaneous measurement of physiological phenomena and hormone release. In muscle-stripped mucosa–submucosal preparations, luminal application of 5 mM propionate induced GLP-1 release into the basolateral side of the rat distal colon . Simultanditions . UnfortuBesides the direct effects of SCFAs on gut hormone release, some studies have shown a relationship between dietary fiber intake – the substrate for SCFA production by microbiota – and GI hormone release. Indeed, non-digestible and fermentable dietary fibers, as well as SCFAs themselves, have been shown to induce GLP-1 secretion in humans  and rodeThe CNS plays an important role in the maintenance of body weight and energy balance within a narrow range by regulating energy intake and expenditure. A reduction in body weight requires long-term negative energy balance by reducing appetite and food intake or by increasing energy expenditure, or both. GLP-1 and PYY are postulated to be hormonal signals from the gut to the brain to inhibit food intake and appetite control because reduced food intake in part reflects increases in hormone release in conjunction with decreased secretion of ghrelin, which increases food intake through effects on hypothalamic, brainstem, and reward-related circuitry . ReleaseGLP-1 is generally believed to be expressed mainly in a limited population of enteroendocrine cells in the ileum and the colon . HoweverSince obesity and metabolic disorders are associated with changes in gut microbiota, the integral role of the gut microbiota in the regulation of host energy homeostasis is an important issue. SCFAs are primary metabolites of gut bacteria and are present in the colon at high concentrations. The SCFA receptors, FFA2 and FFA3 are located in rodent and human colonic L-cells containing GLP-1 and PYY, whose release are involved in the regulation of host energy homeostasis. Furthermore, luminal application of SCFA induces GLP-1 secretion and long-term ingestion of FOS influences the density of FFA2-expressing L-cells. Therefore, it seems likely that the colonic epithelia communicate with the gut microbiota through products and receptors to maintain long-term host–bacterial interactions. The studies that aimed at elucidating physiological phenomena and underlying mechanisms in the colon discussed in this review are important contributions in the race to develop new therapeutic strategies for obesity and its related disorders in future.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Synechococcus is among the most important contributors to global primary productivity. The genomes of several strains of this taxon have been previously sequenced in an effort to understand the physiology and ecology of these highly diverse microorganisms. Here we present a comparative study of Synechococcus genomes. For that end, we developed GenTaxo, a program written in Perl to perform genomic taxonomy based on average nucleotide identity, average amino acid identity and dinucleotide signatures, which revealed that the analyzed strains are drastically distinct regarding their genomic content. Phylogenomic reconstruction indicated a division of Synechococcus in two clades (i.e. Synechococcus and the new genus Parasynechococcus), corroborating evidences that this is in fact a polyphyletic group. By clustering protein encoding genes into homologue groups we were able to trace the Pangenome and core genome of both marine and freshwater Synechococcus and determine the genotypic traits that differentiate these lineages. Cyanobacteria are unique among prokaryotes due to their ability to perform oxygenic photosynthesis. Members of this phylum are important contributors of global primary production, since they are responsible for a significant fraction of carbon fixation at aquatic habitats (Synechococcus and Prochlorococcus are often the most abundant members of the picophytoplankton (reaching concentrations up to 105 and 106 cells ml−1 respectively), being considered the most important contributors to CO2 fixation taking place in several oceanic regions . It has been demonstrated that this process plays a significant role into the evolution of Cyanobacterial genomes , G+C content (48.20%–65.40%) and amount of protein encoding genes   , suggestin silico DNA-DNA hybridization (rrsA gene sequences (16S rRNA gene) and VI) Multi-locus sequence analysis (MLSA) of genes rrsA, gyrB, pyrH, recA and rpoB. The Cgview comparison tool  Average Nucleotide Identity , II) Avedization , V) Idenson tool  was usediveMauve .sourceforge.net/projects/gentaxo/. This tool receives as input genome sequences in FASTA format to calculate three metrics for genome comparison: Average Nucleotide Identity (ANI), Average Amino acid Identity (AAI) and distances between genomes based on dinucleotide signature.To facilitate analyzes for genomic taxonomy we developed a program written in Perl named GenTaxo, freely available at Synechococcus and 13 Prochlorococcus genomes  were aligned through MUSCLE . The results obtained from DDH and dinucleotide signatures corroborated these patterns as all the strains had an estimated level of DNA-DNA hybridization below 70% (expect for the aforementioned pair) and the majority of genome pairs yielded distances based on dinucleotide content above the 0.01 cutoff. The same trend was observed through MLSA and 16S rRNA gene comparisons.The results from the different methods of comparative genomic analysis all indicated the same pattern: the analyzed strains were extremely distinct regarding genomic content. With the exception of Synechococcus was reconstructed based on the sequences of 607 orthologous genes, with no paralogs, shared between Synechococcus and Prochlorococcus genomes , as demonstrated by the dominance of black and dark red colors of the outermost circle, several segments appeared to be exclusive of PCC7942 and not present in PCC6301, which are examples of variation of genomic content between organisms that have very short divergence times.A whole-genome identity map was created by comparing all the genomes against that of h blastx . This anh blastx  revealedSynechococcus revealed little to no synteny between the genomes of these lineages and a significant amount of genome rearrangement events occurring between strains . Carbonic anhydrase converts 2 around RuBisCO that in turn utilizes this CO2 molecule to synthesize phosphoglycerate. The compartmentalization brought by the carboxysome, enhances the efficiency of carbon fixation by elevating the levels of intracellular CO2 around RuBisCO , which are characteristic of β-Carboxysomes were found exclusively and in all of the members freshwater Synechococcus clade. These findings indicate that marine and freshwater Synechococcus use distinguished apparatus for carbon concentration, meaning that these groups could have differential contributions regarding their roles as primary producers and to the global biogeochemical carbon cycle. Although, despite the different protein composition of α and β carboxysomes, functional differences between them are still poorly characterized , an inorganic pyrophosphatase (phosphorus metabolism), a carboxypeptidase (protein degradation and maturation), an aspartocyalase (aspartic acid biosynthesis), GMC oxidoreductase (carbohydrate metabolism), a Ribonuclease (tRNA maturation), and an RpoD like sigma factor . A total of 71 orthologue groups are found exclusively and in all genomes of freshwater Synechococcus, the ones with well characterized functions include: a Lycopene cyclase (carotenoid biosynthesis), a RecJ exonuclease (DNA repair), an oligopeptide permease (peptide transport), a citramalate synthase (aminoacid biosynthesis), a folate transporter and several ABC transporters.A total of 46 orthologue groups are found exclusively on genomes of Synechococcus by: 16S rRNA gene homology to marine strains of Synechococcus, absence of Fructose-1,6-bisphosphatase and 6-phosphofructokinase and presence of DNA polymerase II (not detected in any of the other Synechococcus genomes) and also by the presence of 101 identified homologs groups that are specific to either one of these two lineages. Many of these genes are involved in essential metabolic processes, i.e. carbohydrate metabolism, nutrient uptake, transcriptional regulation and DNA replication. Nevertheless, molecular assays would be required to describe how the physiology of these organisms is affected by the presence/absence of these proteins, which unfortunately is outside of our scope. However it is reasonable to assume that these organisms make use of alternative proteins to fulfill those roles, which could belong to the many poorly characterized and hypothetical proteins that are present in these genomes . These orthologue groups are indicated by stars on column 4 of Two-component response regulators are among the mechanisms used by bacteria to sense environmental stimulus and adapt accordingly by modulating protein activity through alteration of transcriptional patterns . FreshwaSynechococcus strains based on phenotypical and ecological traits. Most of this work has been focused on marine and coastal strains, and unfortunately freshwater strains remain poorly characterized. Nevertheless, those phenotypic analyzes corroborate our results, as they all point to significant distinction between the Synechococcus lineages. A time-series study of the abundance patterns of the clades that include lineages CC9311, CC9605, WH8102 and CC9902 at a coastal Pacific ocean environment revealed that these organisms have distinctive temporal distribution patterns throughout the year, with the clades that include strains CC9311 and CC902 emerging as dominant members of the community while the clades of WH8102 and CC9605 appear as low-abundance members throughout most of the time series  across oceanic provinces provided strong evidence for distinctive distribution signatures across the oceans for each one of the analyzed clades, which could be driven by differences in the capacities of these organisms to adapt to nutrient availability and temperature  Freshwater Synechococcus (C) Prochlorococcus.(A) Marine Click here for additional data file.


Homeobox genes are essential for both the development of the blood and lymphatic vascular systems, as well as for their maintenance in the adult. Homeobox genes comprise an important family of transcription factors, which are characterized by a well conserved DNA binding motif; the homeodomain. The specificity of the homeodomain allows the transcription factor to bind to the promoter regions of batteries of target genes and thereby regulates their expression. Target genes identified for homeodomain proteins have been shown to control fundamental cell processes such as proliferation, differentiation, and apoptosis. We and others have reported that homeobox genes are expressed in the placental vasculature, but our knowledge of their downstream target genes is limited. This review highlights the importance of studying the cellular and molecular mechanisms by which homeobox genes and their downstream targets may regulate important vascular cellular processes such as proliferation, migration, and endothelial tube formation, which are essential for placental vasculogenesis and angiogenesis. A better understanding of the molecular targets of homeobox genes may lead to new therapies for aberrant angiogenesis associated with clinically important pregnancy pathologies, including fetal growth restriction and preeclampsia. Placental angiogenesis has become a focus for the development of diagnostic tools and potential therapeutics for pregnancy complications. Strategies for pro-angiogenic therapies are grounded on our knowledge of normal placental angiogenesis and our understanding of the angiogenic pathways that are disrupted in pregnancy pathologies. However, it is clear that our comprehension of normal angiogenesis in the placenta is lacking in comparison with other tissues and organs, such as the cardiovascular system. Furthermore, unique aspects of placental angiogenesis offer the potential for identifying novel angiogenic pathways from which new pro-angiogenic factors could be identified as potential therapeutics for various obstetric complications associated with aberrant angiogenesis. This review summarizes the genetic and molecular aspects of normal placental angiogenesis with a focus on placental endothelial cells. Our laboratory has major interest in understanding the transcriptional control of placental angiogenesis, with a specific focus on a family of transcription factors called “homeobox genes” and their expression in placental endothelial cells.Homeobox genes play an essential role in regulating the function of vascular systems . They coFigure 1; An efficient and high capacity materno-fetal exchange system is crucial for the growth and development of the fetus and the outcome of a healthy baby . The plade novo process of vasculogenesis , which is a common and clinically significant disorder of pregnancy. FGR is defined as failure of the fetus to achieve genetically determined potential size to an extent where its health is adversely affected . FGR affDespite extensive research, it is unknown whether vascular changes cause FGR or whether these changes are a consequence of aberrant biological mechanisms in the FGR-affected placenta . ClearlyFigure 2 shows the two distinct processes of vasculogenesis and angiogenesis involved in fetoplacental vascular development in human pregnancy.Angiogenesis involves distinct changes in the phenotype of endothelial cells, the central cellular organizational units of vascular structures. In a multi-step event, quiescent endothelial cells are first activated to re-enter the cell cycle . As a coin vitro and in vivo  in placental angiogenesis  signaling pathway  to extraembryonic angiogenesis, particularly in the human placenta, remains largely unexplored.Studies by Esx1  and gestation-matched control (n = 25) pregnancies, we reported an overall decrease in homeobox gene HLX and ESX1L expression in all cell types, including endothelial cells, in FGR-affected placentae compared with GMC , we used a modified methodology based on the perfusion-based technique described by DLX3, DLX4, MSX2, GAX, and HLX (formerly known as HLX1 or HB24) in both PLEC and HUVEC. Our study was the first to show DLX3, DLX4, and MSX2 are expressed in macrovascular HUVEC. We also reported that the mRNA levels of HLX mRNA in HUVEC were significantly lower compared with PLEC  was used to detect mRNA levels of homeobox genes ith PLEC . These dHLX is important in the proliferation and lineage commitment of haematopoietic cells  and Dlx families in the mouse embryo. HLX is expressed with MSX2, GAX  and DLX4 in the trophoblast cell layers of the human placenta (The reported the co-expression of  in PLEC  and thisplacenta . Quinn eplacenta  further To further expand our knowledge of the repertoire of homeobox genes expressed in placental endothelial cells, in a subsequent study by HEX, PHOX1, LIM6, HOXB7 and TGIF, have not been previously detected in the placenta and were selected because they exhibited the greatest relative expression in the microarray data (TLX1 and TLX2 homeobox gene expression data was obtained from the GNF Microarray Analysis Data for the Human U95A microarray, Version 2 dataset (http://expression.gnf.org; LIM6, HOXB7, TGIF, PHOX1, and HEX were expressed in the endothelial cells of the placenta.Microarray expression data as reported in ray data . Novel hHEX (PHOX1/Prx1 (HOXB7 (TLX1, TLX2, PHOX1, MEIS2, and TGIF are expressed in PLEC. In addition, in the same study, we have also reported a differential expression of TLX1, TLX2, PHOX1, MEIS2, and TGIF mRNA levels in macrovascular and microvascular endothelial cells.Expression of these homeobox genes in the placenta or in placental endothelial cells has not been previously reported. HEX , PHOX1/POX1/Prx1 , and HOX1 (HOXB7  homeobox1 (HOXB7 , the micThus, we have identified novel homeobox genes in microvascular endothelial cells, and consistent with our previous studies reported in Figure 3 summarizes the association between the detection of homeobox gene expression and the regions of angiogenic potential in the human placenta. In the microvasculature, where angiogenesis is predominant, we have identified increased expression of homeobox genes TLX1, TLX2, and PHOX1. In the macrovasculature, where there is limited angiogenesis, the level of TGIF and MEIS2 are significantly increased suggesting that the heterogeneity in homeobox gene expression between PLEC and HUVEC that could reflect differences in the angiogenic potential in the two different endothelial environments. Functional studies in cultured endothelial cells are underway in our laboratory to determine the role of novel homeobox genes.In summary, our studies have reported homeobox genes that are novel not only in placental microvascular endothelial cells but also in the macrovascular endothelial cells of the placenta . Figure Table 1 provides examples of downstream target genes of homeobox genes that are required for the regulation of endothelial function in general.Homeobox genes control transcription by binding to regulatory elements in the promoter regions of target genes. Studies using umbilical or uterine artery Doppler for identifying FGR, in the absence of maternal hypertensive disease, show that maternal serum sFLT-1 is increased in these pregnancies compared with pregnancies of normotensive women delivering average for gestational age infants , 2013. MDLX3 as regulators of trophoblast differentiation; GATA2 and PPARγ (GATA2 and PPARγ were further assessed in placental tissues and showed increased expression in FGR-affected tissues compared with gestation-matched controls. Our studies showed that DLX3 orchestrates the expression of multiple regulators of trophoblast differentiation and that expression of these regulatory genes is abnormal in FGR.Studies from our laboratory, using a real-time PCR-based gene profiling, recently identified candidate target genes of homeobox gene nd PPARγ . The expPPARγ ligands suppress VEGFR1 and VEGFR2 expression in HUVECs. Anti-angiogenic actions of 15-lipoxygenase on angiogenesis is regulated by PPARγ and VEGF by inhibiting the expression of VEGFR2 in endothelial cells (PPARγ ligands thiazolidinediones have been used widely for the treatment of type 2 diabetic patients, many of whom experience vascular diseases, clarifying the precise role of PPARγ in defective placental angiogenesis may be of clinical significance.al cells . BecauseTGIF, MEIS, HOXB7, and HHEX in human placental endothelial cells, which may reveal molecular pathways responsible for fundamental cellular functions such as endothelial cell migration, invasion, proliferation, and tube formation that are important for placental angiogenesis. As depicted in Figure 4, in response to angiogenic stimuli or insult, as in the case of FGR or PE, altered expression of homeobox genes in the placental microvascular endothelial may directly or indirectly alter the expression of angiogenic molecules. These angiogenic molecules in turn may regulate genes responsible for cell adhesion, migration, proliferation, growth arrest, and the expression of cytokines needed for extracellular matrix interactions all of which are functions characteristic of the angiogenic phenotype.Current studies in our laboratory are also focused on identifying target genes of homeobox genes Clearly, identifying target genes regulated by homeobox genes in placental microvascular endothelial cells will reveal the biological pathways regulated by homeobox genes. These pathways will provide important information on the function of homeobox genes in placental angiogenesis. Although homeobox gene nuclear transcription factors are unlikely to be ideal disease biomarkers or therapeutic targets, their target genes, if secreted, may provide viable biomarkers or diagnostic markers. A better understanding of cellular and molecular mechanisms that regulate homeobox genes in placental endothelial cells may lead to new approaches for correcting aberrant angiogenesis observed in pregnancy pathologies, including FGR and preeclampsia.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.


Ageing is associated with functional, structural and mechanical changes in arteries that closely resemble the vascular alterations in hypertension. Characteristic features of large and small arteries that occur with ageing and during the development of hypertension include endothelial dysfunction, vascular remodelling, inflammation, calcification and increased stiffness. Arterial changes in young hypertensive patients mimic those in old normotensive individuals. Hypertension accelerates and augments age-related vascular remodelling and dysfunction, and ageing may impact on the severity of vascular damage in hypertension, indicating close interactions between biological ageing and blood pressure elevation. Molecular and cellular mechanisms underlying vascular alterations in ageing and hypertension are common and include aberrant signal transduction, oxidative stress and activation of pro-inflammatory and pro-fibrotic transcription factors. Strategies to suppress age-associated vascular changes could ameliorate vascular damage associated with hypertension. An overview on the vascular biology of ageing and hypertension is presented and novel molecular mechanisms contributing to these processes are discussed. The complex interaction between biological ageing and blood pressure elevation on the vasculature is highlighted. This article is part of a Special Issue entitled: CV Ageing. Diagram demonstrating how vascular function and structure change with ageing and how this process impacts on development of hypertension. With increasing ageing and in the presence of compounding factors, such as environmental stressors, activation of the renin–angiotensin–aldsoterone system (RAAS) and/or activation of the sympathetic nervous system (SNS), molecular and cellular processes in the vasculature are impaired leading to endothelial dysfunction, vascular remodelling and inflammation. These events contribute to vascular injury, which leads to increased total peripheral resistance (TPR) in small arteries and increased stiffness in large arteries, contributing to increased blood pressure, which itself promotes vascular injury. •Ageing and hypertension are characterised by vascular dysfunction and remodelling.•Molecular processes involve activation of MAP kinases and oxidative stress.•Novel signalling molecules implicated in vascular ageing include FoxO and p66Shc.•Biological ageing per se may not be fundamental in vascular changes of hypertension. Calcification of the vascular media is a hallmark of vascular ageing 4With ageing there is a shift towards a proinflammatory vascular phenotype with upregulation of inflammatory cytokines, chemokines and adhesion molecules in the vascular wall 5Functionally, vascular contraction is altered during ageing and is determined in large part by changes in vascular smooth muscle cell cytoskeletal organisation and impaired contractile signalling. Mesenteric arteries from aged rats demonstrate hypercontractility in response to phenylephrine compared to young controls 2 + concentration ([Ca2 +]i), which occurs following activation of phopholipase C (PLC) leading to the generation of second messengers insitol trisphosphate (IP3) and diacylglycerol (DAG) 2 + binds to calmodulin facilitating an interaction with myosin light chain kinase (MLCK) leading to its activation. Activated MLCK triggers phosphorylation of the regulatory light chains of myosin (MLC20) promoting cycling of myosin cross-bridges with actin and consequent contraction. Dephosphorylation of MLC20 by myosin light chain phosphatase (MLCP) results in VSMC relaxation. As such, the relative activities of MLCK and MLCP determine vascular smooth muscle tone by influencing the degree of MLC20 phosphorylation. Arteries from aged animals display altered responses to various contractile agents including norepinephrine, serotonin and KCL At the cellular level, with ageing, vascular smooth muscle cells, which are normally contractile, undergo phenotypic changes to become stiff and pro-migratory. Subsets of apoptotic, senescent and proliferative cells as well as hyper-contractile cells may co-exist in the vascular media. A major trigger for these functional changes is an increase in intracellular free Ca6The vascular endothelium is a monolayer of cells that lines blood vessels and plays a key role in arterial function through synthesis and release of biologically active molecules that can influence endothelial function in an autocrine or paracrine fashion. The healthy endothelium is characterised by a vasodilatory, anti-inflammatory and anti-thrombotic phenotype. Endothelial dysfunction is characterised by reduced vasodilatory responses to flow or agonists and is pro-inflammatory. Independent of the occurrence of other pathologies, ageing results in altered endothelium-dependent relaxation of both the aorta and resistance arteries in rodents 7Molecular mechanisms and cell signalling events underlying the structural and functional alterations observed during ageing are similar to those that occur in hypertension . Many ag7.1Sirtuins (SIRTs) are a family of NAD-dependent protein deacetylases and ribosyl transferases consisting of 7 members which are localised in the cytoplasm (SIRT1 and SIRT2), nucleus  or mitochondria . SIRTs have been implicated in various cellular processes associated with ageing, including, apoptosis, inflammation and mitochondrial biogenesis. SIRTs are able to modulate the ageing process in a number of species 7.2Another emerging candidate implicated in age-related signalling in the vasculature, is peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), which plays an important role in regulating mitochondrial biogenesis and turnover 7.3The FoxO family of Forkhead transcription factors are involved in tumour suppression, energy metabolism, and longevity. Mammals express four FoxO isoforms, FoxO1, FoxO3, FoxO4 and FoxO6. FoxO1, FoxO3 and FoxO4 are phosphorylated in an Akt-dependent manner that promotes FoxO export from the nucleus to the cytoplasm, thereby repressing FoxO transcriptional function. FoxO targets include genes that have pivotal roles in cell cycle progression  and ROS detoxification (MnSOD) and thus may be important in regulation of the ageing phenotype in the vasculature 7.4β and VEGF, resulting in recognition by the prolyl isomerase Pin1, allowing translocation and entrance into mitochondria where it interacts with cytochrome C resulting in production of H2O2. Levels of p66shc in heart, kidney and vascular smooth muscle increase with ageing Mitochondrial dysfunction and increased mitochondrial-derived ROS have been implicated in vascular changes in ageing 7.5In culture, vascular cells respond to prolonged series of replication and stresses by eventually entering an irreversible growth arrest or senescent state Considering the remarkable plasticity of vascular smooth muscle cells, there is a requirement for tight control of transcriptional, metabolic and ultrastructural processes, events that are coordinated through autophagy. Autophagy is the basic cellular mechanism that involves cell degradation of unnecessary or dysfunctional molecules through lysosomes 7.6Protein kinases are major regulators of signal transduction that catalyse the phosphorylation of other proteins, thus regulating their activity. Primary targets of protein kinases include transcription factors which modulate intracellular signalling via specific alteration of downstream gene expression/activity 7.7Common to many of the molecular and cellular processes described above that underlie changes in the vasculature with ageing is oxidative stress Changes in cellular anti-oxidant systems are also important. The expression and activity of antioxidant enzymes, including SOD, decline as tissues age. Decreased anti-oxidant capacity is further promoted by downregulation of Nrf2, the master transcription factor regulating anti-oxidant genes Multiple oxidases generate ROS in the vascular wall and endothelium, including NADPH oxidases (Nox), xanthine oxidase, uncoupled NOS and mitochondrial oxidases. Of these, mitochondria seem to play a major role in processes related to ageing. Noxs, of which there are 7 isoforms , have also been shown to contribute to oxidative stress in vascular ageing With biological ageing, mitochondria become dysfunctional leading to reduced energy production and increased ROS formation. Mechanisms related to mitochondrial dysfunction during ageing include decreased ATP synthesis, increased apoptosis and mutations of mitochondrial DNA by oxidation 7.82 + storage and cholesterol/lipid biosynthesis. Due to the large amount of unfolded protein in the ER, a control system that avoids protein aggregation and accumulation of unfolded proteins is necessary. In experimental models of ageing, the expression and activity of ER chaperones or folding enzymes decay, whilst oxidative damage, such as carbonylation, is exacerbated, leading to accumulation of misfolded/unfolded proteins and ER stress. This activates signalling mechanisms that are part of the UPR. Induction of ER stress leads to endothelial cell apoptosis, but not senescence, implicated in endothelial dysfunction in ageing Prolonged perturbation of the endoplasmic reticulum (ER) leads to ER stress and unfolded protein response (UPR) and contributes to pathogenic processes associated with vascular damage and endothelial dysfunction 7.91R is increased and sensitivity of the mineralocorticoid receptor to aldosterone is enhanced, phenomena that are also observed in hypertension Many of the signalling pathways associated with vascular changes during ageing are also activated in hypertension leading to endothelial dysfunction, vascular inflammation, remodelling and increased arterial stiffness. With normal physiological ageing the process is gradual and regulated but in itself represents a strong and independent risk factor for hypertension and future cardiovascular events 7.101 receptor. Expression of various components of the RAS, including angiotensinogen, chymase, angiotensin converting enzyme (ACE) and the AT1 receptor is increased in arteries of aged rodents and humans 1 receptor blockers decrease ageing-associated vascular damage. Mice treated with enalapril or losartan demonstrated vasoprotection and an increase in life span 1 receptor blockers doubles the lifespan by improving endothelial function and alleviating complications of hypertension 1 receptor in oxidative stress, vascular injury and ageing, studies in mice with targeted disruption of the Agtr1a gene, which encodes the AT1A receptor, resulted in prolonged life span The RAAS plays an important role in functional, structural and mechanical changes of the vasculature that occur with ageing and hypertension Clinical and experimental studies demonstrate that many pro-hypertensive systems influence processes of vascular ageing, including aldosterone, ET-1 and growth factors 7.11S-nitroso-penicillamine significantly reduces endothelial cell senescence and age-dependent inhibition of telomerase activity NO is a potent vasodilator produced by endothelial cells that mediates vascular relaxation and thus plays a critical role in the regulation of blood pressure. Abnormalities in endothelial production of NO occur in hypertension and are due, in large part, to decreased eNOS activity 2S) produced by cystathionine g-lyase (CSE) or cystathionine b-synthase (CBS) has recently emerged as a novel antihypertensive factor based on the observations that exogenous H2S is vasoprotective in pulmonary hypertension The gaseous messenger hydrogen sulphide  can inhibit leukocyte adherence in mesenteric venules whilst inhibition of endogenous H2S synthesis promotes leukocyte adhesion and vascular inflammation In endothelial cells stimulated with TNF-α, NaHS (H2S donor) suppressed pro-inflammatory responses by reducing the TNF-induced increase in expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin. Furthermore, TNF-α-induced NF-κB was decreased in the presence of NaHS 7.12Although there are many signalling pathways and functional and structural characteristics that are common in vessels during ageing and hypertension, these processes are dynamic and change throughout life and as such may not necessarily be superimposable. For example, with advancing age arterial stiffness and blood pressure start to diverge rather than parallel There is also some evidence to show that structural alterations in the vascular wall occur before the development of hypertension. For example rates of pulse wave velocity (PWV) increase are accelerated with both advancing age and elevated blood pressure. However, the effect of blood pressure on PWV increase occurs primarily during the prehypertensive phase and suggests that these vascular alterations precede the phase of established hypertension 7.13Vascular changes that occur with ageing may be independent of biological ageing in hypertension. This is highlighted in studies that have examined vascular function and arterial structure in children with hypertension. Endothelial dysfunction, arterial stiffening and structural alterations of the arterial wall may precede evidence of high blood pressure, as quantified by systolic and diastolic blood pressure, and may be independent of the ageing process Taken together, emerging experimental and clinical evidence indicates that although the molecular and cellular processes that characterise the vascular phenotype in hypertension resemble those that occur with normal healthy ageing, age per se may not be a critical factor. However, ageing may be a compounding factor that amplifies vascular injury that occurs with blood pressure elevation. In the presence of other co-morbidities, such as diabetes, dyslipidemia, smoking and obesity, these processes may be further exaggerated.8Ageing is associated with a progressive deterioration in endothelial function, vascular remodelling, inflammation and increased arterial stiffness. Processes underlying these processes include activation of pro-inflammatory transcription factors, oxidative stress, cell senescence and apoptosis, aberrant signalling cascades and a shift from a vasoconstrictor to a proliferative vascular cell phenotype. Many of these phenomena are also relevant in the pathophysiology of hypertension, which is characterised by a vascular phenotype of impaired endothelium-dependent vasorelaxation, arterial remodelling, increased stiffness and vascular inflammation. Through such vascular changes, ageing and hypertension are closely interlinked: ageing promotes hypertension and pro-hypertensive factors promote vascular ageing. Whilst many of the molecular processes and signalling pathways contributing to vascular dysfunction are common in ageing and in hypertension, biological age per se, may not be a fundamental factor, since vascular damage is already present in children and young adults with hypertension. A better understanding of the vascular biology of ageing will facilitate development of strategies to promote healthy vessels and suppress age-associated changes, especially in pathological conditions. Such approaches could prevent or ameliorate vascular damage in hypertension and hence reduce cardiovascular diseases, commonly linked to ageing.There are no disclosures to declare.


In vivo and in vitro studies have provided insights on the molecular mechanisms underlying the neurogenic and osteogenic processes in adulthood. This knowledge appears fundamental for the development of targeted strategies to manipulate stem cells. Here we review recent literature dealing with the effects of electromagnetic fields on stem cell biology that lends support to their use as a promising tool to positively influence the different steps of neurogenic and osteogenic processes. We will focus on recent studies revealing that extremely-low frequency electromagnetic fields enhance adult hippocampal neurogenesis by inducing epigenetic modifications on the regulatory sequences of genes responsible for neural stem cell proliferation and neuronal differentiation. In light of the emerging critical role played by chromatin modifications in maintaining the stemness as well as in regulating stem cell differentiation, we will also attempt to exploit epigenetic changes that can represent common targets for electromagnetic field effects on neurogenic and osteogenic processes.In the recent years adult neural and mesenchymal stem cells have been intensively investigated as effective resources for repair therapies.  Any adult tissue with repair/regenerative capabilities contains tissue-specific stem cells (SCs) defined as clonogenic, self-renewing cells that retain proliferative and differentiation potential allowing to preserve tissue homeostasis and to repair injury  stimulation that is emerging as an effective tool to positively modulate neurogenic and osteogenic processes.Ascl1 , Neurogenin1 and NeuroD1.In the adult mammalian brain, NSCs reside mainly in two discrete regions: the subgranular zone of the hippocampal dentate gyrus and the subventricular zone of the lateral ventricles Gage, . Through2+ signaling triggers phosphorylation of CREB, that, once activated, promotes NSC differentiation  gene, during differentiation of osteoprogenitor cells to mature osteoblasts. In particular, signaling molecules such as bone morphogenetic proteins (BMPs) and Wnt pathway favor osteoblastogenesis, while Notch1 and its downstream target Hes1 inhibit osteoblast differentiation. Recently, it has been shown that the transcriptional factor Runx2, a major target of BMP pathway, induces osteoblast differentiation by repressing Increasing body of evidence supports the view that epigenetic mechanisms including DNA methylation and histone modifications orchestrate SC self-renewal, lineage commitment, cell fate specification and terminal differentiation. These regulatory mechanisms promote the formation of relatively “open” and “poised” epigenetic states that, by regulating transcriptional activity, mediate the execution of lineage-specific gene expression programs.Ascl1, Neurogenin1 and NeuroD1 . DNA methylation is catalyzed by DNA-methyl-transferase (DNMT) and usually results in gene repression. DNMT3a and DNMT3b establish OC locus has been associated with its repression. Accordingly, during osteoblast differentiation this CpG methylation significantly decreased, resulting in enhanced OC expression  Polycomb (PcG), that promotes H3 lysine 27 tri-methylation (H3K27me3); and (ii) Trithorax (TrxG), that promotes H3 lysine 4 tri-methylation (H3K4me3).In NSCs, depletion of PcG components, such as Ezh2, largely removed H3K27me3 markers, de-repressed a wide panel of genes, and delicately altered the balance between self-renewal and differentiation as well as the timing of neurogenesis  , and double-labeled for BrdU and the immature neuronal marker doublecortin. Interestingly, 30 days after the end of the ELFEF stimulation protocol ~50% of the newborn neurons became mature granule cells that were functionally integrated in the dentate gyrus network, as demonstrated by neurophysiological indexes. In particular, in hippocampal brain slices from ELFEF exposed mice, long-term potentiation at medial perforant path-dentate granule cell synapses in the presence and in the absence of GABAA receptor blockade was significantly greater than that observed in unexposed control mice  enhanced differentiation of adult cortical NSCs  that are known to critically regulate SC self-renewal and differentiation  demonstrated that exposure to PEMF stimulates cell proliferation as well as osteogenesis by increasing early osteogenic markers including Runx2/Cbfa1 and alkaline phosphatase  have been more recently investigated. In particular, PEMF treatment enhanced the expression of bone matrix genes (OC and collagen type I in ASC) as well as bone mineralization (Ceccarelli et al., 2+ flux and expression/activation of Ca2+-binding proteins such as calmodulin in human osteoblast-like cells and MSCs (Fitzsimmons et al., 2+ concentration is the starting point for signaling pathways targeting specific bone matrix genes and, in keeping with this, the application of the electromagnetic waves was shown to increase the level of transcripts of osteogenesis-related genes including those encoding for decorin, osteopontin, collagen type-I and Runx2 (Figure At present the mechanism by which PEMFs/ELFEFs promote the formation of bone remains elusive and future studies are highly demanded. Some evidences indicate that, as documented for NSCs, the electromagnetic stimulation raises the net Ca2 Figure .The recent findings in stem cell biology have opened a new window in the expanding area of regenerative medicine based on tissue engineering and cell therapy derived from a variety of SCs, including NSCs and MSCs.With regard to neurogenesis and ostegenesis it is becoming increasingly clear that these processes rely on the activation of specific and complex transcriptional programs whose regulation may provide a cellular candidate for therapeutic intervention. In this context epigenetic mechanisms play a critical regulatory role translating a wide array of endogenous and exogenous signals into persistent changes in gene expression in both NSCs and MSCs. ELFEF stimulation has been recognized as effective tool in promoting both neurogenesis and osteogenesis and studies performed so far on NSCs point to chromatin remodeling as a critical determinant in ELFEF’s induced pro-neuronal gene expression. The literature here reviewed suggests that epigenetic regulation of bone-related gene may seemingly mediate the effects exerted by EFs on osteogenesis.It is our opinion that future research on different types of SCs may benefit from higher degree of communication between the different fields that would contribute to uncover more than expected common molecular pathways and stimulation paradigms of potential relevance for therapeutic interventions.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.


Proteolytical processing of membrane bound molecules is a fundamental mechanism for the degradation of these proteins as well as for controlling cell-to-cell communication, which is at the basis of tissue development and homeostasis. Members of families of metalloproteinases and intra-membrane proteases are major effectors of these events. A recent workshop in Baeza, Spain, was devoted to discuss how this mechanism coordinates brain development and how its dysfunction leads to brain pathologies. Herein we summarize the findings presented during this workshop, which illuminate the role of metalloproteinases, including matrix metalloproteinase, A Disintegrin and Metalloproteinase-proteases and intra-membrane proteases, in the regulation of neurogenesis, axon guidance, and synaptogenesis as well as in neurodegeneration. Indeed, there is increasing evidence that proteolysis at the membrane is directly linked to neuropathologies such as Alzheimer Disease and autism spectrum or prion disorders. These proteolytic events are tightly regulated and we are just at the beginning of understanding how these processes could be exploited to design therapeutic treatments aimed at alleviating psychiatric and neurodegenerative pathologies. The highest functions of the nervous system, including the cognitive capabilities of the human brain, are rooted on stereotyped and yet plastic interactions among a huge variety of neurons and glial cells, which compose the vertebrate brain. The specification of neural cells occurs with an apparently invariable precision during a protracted period of the embryonic and early postnatal life . In turnProteolytical processing of membrane bound molecules acquires particular importance in the context of the brain since its function strongly depends on well-orchestrated interactions among many different cell types, including neurons and glial cells. Members of families of metalloproteinases are major effectors of these events. The meeting “Proteases at work: cues for understanding neural development and degeneration” which took place in Baeza, Spain from October 20th until October 23rd, 2014 brought together internationally recognized leaders in the field to summarize the most recent evidence demonstrating that proteases are essential for the development of the CNS, underscoring their relevance in neurodegeneration and discussing whether endogenous or pharmacological modulation of their activity represents a therapeutic tool.Carl Blobel  initiated the conference by providing an overview about proteinases belonging to the A Disintegrin and Metalloproteinase Family (ADAMs). ADAMs regulate the fate of cell surface expressed membrane proteins . He focused on a specific member of this family, ADAM17, and its well-established role in liberating Epidermal Growth Factor Receptor ligands from their membrane-anchor. As for the other proteolytically active members of the ADAM family, their regulation still remains a field with many open questions. In this regard, the inactive Rhomboid1 and 2 (iRhom) proteins turned out to be instrumental upstream regulators for ADAM17/EGFR signaling. iRhom2 controls the substrate selectivity of stimulated ADAM17-dependent ectodomain shedding . Disruption of both iRhoms genes in mice led to a phenotype comparable to the ADAM17 and EGFR-knockout mice providing clear evidence that ADAM17 is tightly controlled by both iRhoms.shedding . Its intPaul Saftig  reported about a number of additional neuronal substrates of ADAM10. Conditional knockout mice with disruption of ADAM10 in the developing or adult brain revealed different and stage-dependent functions of this protease. ADAM10 controls cell fate decision events with a process that depends on the cleavage of the Notch receptor and that discriminates between neurogenesis and gliogenesis . Spine development, synapse plasticity, synaptic network function, memory, and learning all rely on proper cell-autonomous activity of ADAM10  family, including TSPAN15, associate with ADAM10 and are co-transported at the cell surface .Closely related to ADAM17, ADAM10 exerts a number of essential functions related to the development and function of the brain. ADAM10 sheds the amyloid precursor protein (APP), the Notch receptor, and the prion protein . Paul Saogenesis . In adulf ADAM10 . ADAM10  surface , where tElena Marcello from Monica Di Lucaˊs  laboratory also supported the idea that trafficking of ADAM10 is central to its regulation. The Synapse-Associated Protein-97 (SAP97) interacts with the cytoplasmic domain of ADAM10  presentation. The postsynaptic adhesion molecule neuroligin (NLG) binds to its presynaptic counterpart neurexin. Interestingly, NLG mutations have been linked to psychiatric disorders and mental retardation . Therefore, ADAM proteases may be interesting targets to treat autism spectrum disorders, because, as mentioned above, NLGs have been strongly linked to these neuropsychiatric diseases . This powerful approach not only validated a number of already described substrates but extended this list to previously unrecognized surface proteins involved in synapse function, axon guidance and intercellular adhesion. A similar approach previously and successfully identified new neuronal substrates for BACE-1, the β-secretase involved in APP processing  described the pivotal importance of matrix metalloproteinase-9 (MMP-9) in the regulation of synaptic plasticity , learning, and memory. MMP-9 belongs to the family of metzincins, which are enzymes secreted by neurons and glia . MMP-9 expression is tightly regulated and it is rapidly released near excitatory synapses in response to synaptic stimulation  illustrated this idea presenting a novel enzymatic mechanism, which controls Notch signaling, a classical example of a pathway activated by the generation of a transcriptionally active intracellular fragment (trans-membrane protein with an extracellular glycerophosphodiester phosphordiesterase (GDPD) domain  domain . GDE2 ex) domain .Avraham Yaron  instead discussed the relevance of proteolytical inactivation of axon guidance signaling mediated by the Neuropilin-1 (Nrp1) receptor and its Sema3A ligand. Sema3A acts as an axonal repellent for sensory axons projecting to the spinal cord. Genetic inactivation of ADAM10/ADAM17 causes guidance errors of proprioceptive axons, because the otherwise developmentally regulated shedding of Nrp1 at the sensory growth cones does not occur. This failed shedding abnormally prolongs the growth cone repulsive response to Sema3A. Accordingly, this repulsive response is no longer observed when growth cones express a form of Nrp1 highly susceptible to cleavage, further underscoring the importance of ectodomain shedding in switching growth cone responsiveness to guidance cues  presented data supporting that Secreted Frizzled Related Proteins (Sfrps), highly diffusible secreted modulators of cell–cell communication , nication , particinication . These cth cones  and, on in or L1 , which aPatricia Maness . Alterations in the function of GABAergic basket interneurons inhibiting pyramidal cells of the prefrontal cortex are thought to be responsible for working memory deficits in schizophrenia patients. On the other hand, ADAM-mediated proteolysis of NCAM is known to participate in synaptic plasticity during learning and memory  showed that N-cadherin-mediated adhesion to ependymocytes contributes to the quiescence of neural stem cells in the sub-ependymal niche. More interestingly, MT5-MMP, a membrane-type metalloproteinase, sheds N-cadherin ectodomain in this niche, ensuring the proper activation of the stem cell population in both physiological and regenerative conditions  demonstrated that two members of this family, MMP-2 and MMP-3, are required for the on-time migration of granule cells and GABAergic interneurons in the cerebellum as well as for a proper dendritic arborisation of the Purkinje cells and pyramidal neurons of the neocortex  discussed the possible causes of a failed phase III clinical trial using semagacestat, a γ-secretase inhibitor. Most likely owing to the inhibition of the Notch-1 signaling pathway, patients treated with this small compound presented serious side effects, including skin cancers. Treated AD patients were also subjected to infections and paradoxically experienced further cognitive decline  showed that reduction of Sfrp1 expression shifts APP processing toward a non-amyloidogenic pathway  in the adult mouse brain  instead showed that ADAM10 sheds PrPC , capable of binding, and mediating the toxicity of Aβ oligomers  raised several important considerations that need to be solved before pursuing ADAM10 as a therapeutic target. She underscored that it is still unclear to what extent a deficient ADAM10 catalytic activity or a reduced expression of its gene participates in AD pathogenesis. We also do not know the full network of factors regulating ADAM10 expression in humans and whether pharmacological interference with ADAM10 enzymatic activity can be achieved in patients. More importantly, is it safe? Kristina reported encouraging results on both fronts with the identification of transcription factors that influence ADAM10 expression .pression . She alsElena Cattaneo  reported the power of combining evolutionary and developmental approaches to study disease-genes such as that encoding the ancestral protein Huntingtin. Expansion over 36 residues of a polymorphic tri-nucleotide CAG repeat, translated into polyglutamine, causes Hungtington Disease (HD), a genetically dominant and fatal neurodegeneration .Unfortunately, metalloproteases are not always associated to positive events, as their activation or over-activation is often detrimental. As discussed in the workshop, neurodegenerative disorders are a primary example of harmful effect together with metastatic disorders. The role of metalloproteases in these pathologies has been the focus of several laboratories for many years. More recently, general pathological processes such as neuroinflammation have emerged as metalloprotease-regulated events, broadening the implication of these molecules. Thus, it is apparent that we are just observing the tip of the iceberg as we still know little of the physiological relevance of metalloproteases in the brain. Full understanding of their function might unravel how the same protease can have a double edge and might give us some hints on how to turn metalloproteases’ detrimental side into a beneficial activity. Reaching this degree of comprehension requires answering a large list of questions, and we would like to point out some of them, which perhaps represent the most obvious ones  and neurodegenerative (AD and Prion Disease) disorders so far known to be associated to the impaired activity of metalloropeases, it is evident that improving our understanding of metalloprotease biology may have important social benefits, in addition to a scientific relevance. As mentioned above, there is already a considerable effort in exploiting metalloproteases for therapeutics purposes. The task is huge as a protease-targeted therapy will need careful evaluation and it is expected to work only in a tight therapeutic window. Unwanted side effects explained by the modulation of other protease-substrate complexes have to be taken into consideration. Nevertheless, there are considerable expectations that we all hope to foster in the years to come.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.


Oligo-anovulation, hyperandrogenism and insulin resistance characterizes polycystic ovary syndrome (PCOs). Metformin is the oldest insulin sensitizer used in the management of type 2 diabetes mellitus. In PCOs, metformin decreases the serum lipids, androgen and insulin; induces ovulation and regular menstrual cycle; increases the pregnancy rate.  Metformin is a first-line therapy for type 2 diabetes mellitus , and is one of the most commonly prescribed drugs worldwide, indicated in the guidelines issued by the American Diabetes Association and European Association for the Study of Diabetes. As a biguanide agent, metformin lowers both basal and postprandial plasma glucose (PPG) . Metformin works by inhibiting the production of hepatic glucose, reducing intestinal glucose absorption and improving glucose uptake and utilization. Besides lowering the blood glucose level, metformin may have additional health benefits, including weight reduction, lowering plasma lipid levels, and prevention of some vascular complications [3]. Metformin is also used for other indications such as polycystic ovary syndrome (PCOs) [1].Metformin hydrochloride is a white to off-white crystalline compound with a molecular formula of C4H11N5 • HCl and a molecular weight of 165.63. Metformin hydrochloride is freely soluble in water and is practically insoluble in acetone, ether, and chloroform. Metformin hydrochloride tablets for oral administration contain 500 mg, 850 mg or 1000 mg of metformin hydrochloride. Mechanism of ActionMetformin is an antihyperglycemic agent that improves glucose tolerance in patients with type 2 diabetes, lowering both basal and postprandial plasma glucose. Its pharmacologic mechanisms of action are different from other classes of oral antihyperglycemic agents. Metformin decreases hepatic glucose production, decreases intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. Metformin does not produce hypoglycemia in either patients with type 2 diabetes or normal subjects and does not cause hyperinsulinemia. With metformin therapy, insulin secretion remains unchanged while fasting insulin levels and daylong plasma insulin response may actually decrease.PharmacokineticsAbsorption and BioavailabilityGiven under fasting conditions the absolute bioavailability of a metformin hydrochloride 500 mg tablet is approximately 50% to 60%. Studies using single oral doses of metformin hydrochloride tablets of 500 mg to 1500 mg, and 850 mg to 2550 mg, indicate that there is a lack of dose proportionality with increasing doses, which is due to decreased absorption rather than an alteration in elimination. Food decreases the extent of and slightly delays the absorption of metformin.DistributionMetformin is negligibly bound to plasma proteins in contrast to sulfonylureas, which are more than 90% protein bound. Metformin partitions into erythrocytes, most likely as a function of time. At usual clinical doses and dosing schedules of metformin hydrochloride tablets, steady-state plasma concentrations of metformin are reached within 24 to 48 hours and are generally <1 mcg/mL. During controlled clinical trials of metformin, maximum metformin plasma levels do not exceed 5 mcg/mL, even at maximum doses.Metabolism and EliminationIntravenous single-dose studies in normal subjects demonstrate that metformin is excreted unchanged in the urine and does not undergo hepatic metabolism (no metabolites have been identified in humans) or biliary excretion. Renal clearance is approximately 3.5 times greater than creatinine clearance, which indicates that tubular secretion is the major route of metformin elimination. Following the oral administration, approximately 90% of the absorbed drug is eliminated via the renal route within the first 24 hours, with a plasma elimination half-life of approximately 6.2 hours. In blood, the elimination half-life is of approximately 17.6 hours, suggesting that the erythrocyte mass may be a compartment of distribution.4] and is excreted unchanged in the urine, with a half-life of ~5 h [5]. The drug is widely distributed into the body tissues including the intestine, liver, and kidney by organic cation transporters [5]. There is a large interindividual variability in metformin pharmacokinetics as measured by differences in trough steady-state metformin plasma concentration ranging from 54 to 4133 ng/ml [6].Metformin is not metabolized , no known increase in multiple pregnancy rate (unlike that associated with CC) and thus no requirement for the inconvenient and costly monitoring of ovulation induction cycles (that many fertility clinics insist upon for CC), and no concern regarding the long term adverse effects on the ovaries, contrasting with the lingering concern over an increased risk of ovarian cancer seen in some cohort studies of women using CC, particularly serous ovarian cancer [9] and amongst those using long treatment courses [10].Metformin is a very suitable alternative to clomiphene as a first line ovulation induction treatment for non-obese women with anovulatory PCOs. In fact, metformin carries some potential advantages over clomiphene, including no known adverse endometrial effect whilst endometrial thinning could reduce embryo receptivity for some women using CC . Clinical pregnancy rate was significantly higher in women receiving dual therapy versus clomiphene alone.There is a growing evidence base that metformin response is better in women with PCOs who have a lower BMI [13]. The passive diffusion of metformin into cells is limited [14], the main transport is the organic cation transporter 1-3 or multidrug and toxic compound extrusion type transporters which are able to internalize metformin as described in gut, hepatocytes, renal tubular epithelial cells and reproductive tissues [15]. One of the direct effects of metformin identified is to inhibit the activity of the respiratory electron transport chain in mitochondria [16] and to activate the cytoplasmic protein kinase known as AMP-activated protein kinase (AMPK) [17]. AMPK is an important sensor of cellular energy homeostasis and is sensitive to the AMP: ATP ratio . It can be noted that several studies have also demonstrated that metformin might act independently of AMPK [20-22]. A deficiency in ATP activates AMPK leading to increased energy production including glucose and lipid catabolism and an inhibition of energy consuming processes such as protein, fatty acid and cholesterol synthesis.Metformin may accumulate in certain tissues at higher concentrations than in plasma . AMPK has been widely studied in the brain as it is involved in regulating food intake, a function regulated by the hypothalamus [25-28]. The various anorectic signals  reduce AMPK activity whereas the orexigenic signals  increase AMPK activity which permits the regulation of food intake . Metformin inhibits AMPK activity in the hypothalamus and neuropeptide Y neurons in the brain, which explains the appetite suppressing nature of AMPK [31]. In the same region of the brain, the functioning of neurons to kisspeptin and to GnRH also seems to be AMPK dependent. Therefore, AMPK is a key participant in the operation of important reproductive neuron regulators.Fertility is centrally controlled at least in part by gonadotropin-releasing hormone (GnRH) neurons that are located in the hypothalamus. These neurons secrete GnRH, which stimulate the luteinizing hormone (LH) and follicle stimulating hormone (FSH) secretions by the pituitary. GnRH neurons are responsive to numerous stimuli including peripheral metabolic signals such as glucose and leptin [32]. The other potential effects of metformin include an increase in glucose uptake, an increase in insulin signaling, a decrease in fatty acid and triglyceride synthesis and an increase in fatty acid β-oxidation.It works mainly by suppressing excessive hepatic glucose production, through a reduction in gluconeogenesis . Indeed, metformin improves insulin-mediated glucose disposal in women with PCOs [34]. Thus, metformin has become one of the key drugs in the treatment of PCOs.Metformin has insulin-lowering effects by improving insulin sensitivity and, in turn, can decrease circulating androgen levels. In addition, it also plays a critical role in the treatment of PCOs, because women with PCOs are at an increased risk of insulin resistance .The molecular mechanisms underlying the metformin action appear to be complex and remain a topic of considerable debate. However, there is a general agreement that the administration of metformin results in the phosphorylation and activation of AMP-activated protein kinase (AMPK) in the liver, which in turn may lead to diverse pharmacologic effects, including inhibition of glucose and lipid synthesis .Metformin may inhibit hepatic gluconeogenesis in an LKB1-independent and AMPK-independent manner . AMPK is a major cellular regulator of lipid and glucose metabolism.A reduction in gluconeogenesis may occur both ways, in an AMPK-dependent and an AMPK-independent manner. Metformin specifically inhibits complex I of the mitochondrial respiratory chain, suggesting that this inhibition may activate AMPK by increasing the cellular AMP: ATP ratio [SLC2A4) translocation activity [15]. The overall pharmacological effect of AMPK activation in the liver includes the stimulation of fatty acid oxidation with inhibition of cholesterol and triglyceride synthesis. Peripheral effects include the stimulation of fatty acid oxidation and glucose uptake in skeletal muscle as well as a systemic increase in insulin sensitivity [37]. However, the role of metformin in insulin-mediated glucose uptake was debated [39].Finally, activated AMPK results in an increase in glucose uptake in skeletal muscle by increasing the GLUT4 , metformin could change the metabolic capacity of ovarian GCs .Polycystic ovary syndrome (PCOs) is a condition of primary ovulatory dysfunction associated with metabolic disturbances. It was established that the GCs metabolize glucose in normal human ovaries by glycolysis and this results in the production of lactate and pyruvate .Indeed, Purcell et al. have demonstrated blunted insulin-stimulated glucose uptake in vitro by cumulus cells from women with PCOs . The previous researchers have also shown that there is an impaired insulin-stimulated lactate production but from intact insulin-responsive steroid production by follicular GCs from women with PCOs . Previous studies which have cultured GC over 48 h in vitro have demonstrated that insulin-induced glucose uptake by GCs in response to increasing doses of insulin is much lower in GCs from anovulatory patients with PCOs than from women with either normal ovaries or ovulatory polycystic ovaries .Tyrosine phosphorylation of the insulin receptor (IR) activates the whole insulin cascade and results in glucose uptake by the cells. Serine phosphorylation of the IR may inhibit the insulin excessive serine phosphorylation which was attributed to the post-binding defect and insulin resistance observed in these women .Metformin alone decreases the fasting plasma glucose concentration to 4–4.5 mM and HbA1c by 1.5–2.0% in patients with type 2 diabetes mellitus.Fig. 1).The metabolic actions of metformin on cells include increases in insulin sensitivity of responsive tissues, IR number and affinity in skeletal muscle and adipose cells, conversion of glucose to lactate by erythrocytes, tyrosine kinase activity and glucose uptake . Metformin results in a decrease in glucose absorption by gut, plasma glucagon levels, gluconeogenesis and glycogenolysis in the liver . Indeed, the exposure to metformin was shown to lead to increased tyrosine kinase activity and 2-deoxy glucose transport in a dose-dependent manner in rat smooth vascular smooth muscle cells suggesting an enhanced sensitivity to insulin and IGF-1 .It was shown that metformin activates the AMPK cascade, which in turn takes part in the glucose transport into the muscle cell  (


We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems.Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as “clusters” by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (“domains”) for the two receptor species. The  The plasma membrane is the initiation site for signaling pathways that govern cell differentiation, proliferation and survival  are not well suited to explore spatial aspects of signaling, since they assume molecules in a system are well mixed. Stochastic modeling approaches offer greater flexibility to consider effects of membrane topography, receptor clustering and diffusion dynamics on signaling events  to an open (dimer-competent) conformation. The open conformation of ErbB3 is stabilized by ligand binding , this translates into a receptor density of ~159 receptors/μm2 for each receptor. Adjusted for a simulation area of 0.1995 μm2, the total number of receptors is 31 of each receptor species.The model was populated with 50,000 ErbB2 and 50,000 ErbB3 receptors/cell. Since the total area of a cell is 314.16 μmReceptor diffusion occurs in the two dimensional membrane simulation space (x and y direction) through Brownian motion. Receptor jumps in these two directions are calculated using diffusion coefficients generated from SPT data and normally distributed random numbers.As in Pryor et al.  and PryoInput files containing the initial simulation space, receptor locations and ligand concentrations are generated in Matlab. These files are then accessed by a program written in Fortran, which simulates brownian diffusion and molecular interactions between the two receptors. At the end of the simulations, all output files are processed in Matlab for analysis of results. Code is available upon request.It is unknown to what extent different receptors share the same membrane domains, how fluid these domains are over time, and whether activation of receptors alter domain overlap. Therefore, we explored these possibilities through simulations, reporting results as changes in homo- and hetero-dimerization and phosphorylation status. Unlike prior work fit to cells overexpressing ErbB family members  ligand conditions. We then explored how changes in domain characteristics, as well as ligand occupancy, influences dimerization and phosphorylation in this system. The sensitivity of the model to these parameters illustrates that variations in domain characteristics amongst different cell and tissue types are likely unappreciated modulators of signaling by these (and other) receptors.Previous spatial stochastic models have shed insight on the effect of domains on signaling  motion , R01GM104973 (JE and ÁH), and R01HG006876 (JE).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.


Plants have the remarkable ability to establish new cell fates throughout their life cycle, in contrast to most animals that define all cell lineages during embryogenesis. This ability is exemplified during sexual reproduction in flowering plants where novel cell types are generated in floral tissues of the adult plant during sporogenesis, gametogenesis, and embryogenesis. While the molecular and genetic basis of cell specification during sexual reproduction is being studied for a long time, recent works disclosed an unsuspected role of global chromatin organization and its dynamics. In this review, we describe the events of chromatin dynamics during the different phases of sexual reproduction and discuss their possible significance particularly in cell fate establishment. Figure 1). Unlike animals, plants do not set aside a germline lineage during embryogenesis. Instead, the reproductive lineage is established late in development. Cells that will share a meiotic fate and hence initiate a “reproductive lineage” differentiate from and within a somatic tissue in dedicated floral organs of adult plants. Sporogenesis is initiated by the differentiation of spore mother cells (SMCs) that engage somatic cells into a meiotic fate entailing the development of haploid, multicellular gametophytes. The female SMC, also called megaspore mother cell (MMC) differentiates in a subepidermal position in an ovule primordium – composed of the L1-outer layer of cells and the nucellus –; the male SMC differentiates from a mitotic division of the archesporial cell within the sporangium of the anther locule . Gametogenesis is the process by which the gametes are formed within the gametophytes. The male and female gametophytes develop from one haploid spore through a limited number of mitosis and cellularization events that will give rise to highly distinct cell types. A vast majority of flowering plants share the seven-celled type of female gametophyte comprising two gametes – the egg cell and the central cell – and five accessory cells – two synergids and three antipodals. All cells are haploid except for the central cell that inherits two polar nuclei, which following fusion generate a di-haploid maternal genome in the central cell. In contrast, the mature male gametophyte contained in the pollen grain is highly reduced and is composed of one vegetative – accessory – cell and two gametes, the sperm cells . During double fertilization, the egg cell fuses with one sperm to give rise to the diploid zygote, while the central cell is fertilized by the second sperm cell – from the same pollen – to produce the triploid endosperm . Strikingly, although genetically identical the two fertilization products share distinct developmental fates. The totipotent zygote engages into embryogenesis that establishes the basic body plan and the symmetries  of the future seedling; in contrast, the primary endosperm cell engages in a syncytial phase of proliferation, before cellularization, to form an extra-embryonic, nurturing tissue . Male SMCs, also called pollen mother cells (PMCs), or microspore mother cells, differentiate within the sporangium formed in the anther locule. In Arabidopsis, the hypodermal cell in the sporangium enlarges to form the archesporial cell that then divides to generate the primary sporogenous cell toward the inside and the primary parietal cell in the outside. The sporogenous cell undergoes mitosis to give rise to PMCs, while the primary parietal cell divides to form the anther wall comprising the epidermis, the endothecium, the middle layer, and the tapetum .Figure 2A, The first visible signs of SMC differentiation are cellular and nuclear enlargement in the sporogenous tissue. Visible changes in nuclear morphology during MMC differentiation were reported on early drawings or micrographs with clear nuclear and nucleolar enlargement compared to the surrounding nucellar cells . In lighFigure 2C, The events described in the MMC are reminiscent of those observed in mouse primordial germ cells (PGCs) that can be seen as functional equivalent of plant SMCs: mouse PGCs undergo large-scale chromatin reprogramming characterized by chromatin decondensation, DNA demethylation, depletion of linker histone, histone replacement, and extensive erasure of the histone marks such as H3K9me2, H3K9ac, H3K9me3 and H3K27me3 . It would be interesting to determine whether the PMC chromatin undergoes a selective replacement of histone H1 with a male-specific variant, possibly resembling that of mouse testis  is repressed in somatic cells by ACTIN RELATED PROTEIN6 (ARP6), thought to belong to chromatin modulating complexes, possibly via H2A.Z deposition  embryo sacs. Transcriptional profiling of the ago104 mutant suggests that it represses somatic gene expression in a non-cell autonomous way (polar granule component (pgc) represses somatic gene expression in Drosophila germ cells  are potentially mobile sequences within the genome that pose a threat to genome integrity. Epigenetic reprogramming during germline formation in animals, during sporogenesis in plants, is a potential risky window for TE to escape silencing. Both plants and animals have evolved different strategies to restrict TE activity, particularly in the germline .Microgametogenesis begins with an asymmetric and atypical mitosis in the microspore, resulting in the formation of a large vegetative cell engulfing a smaller generative cell in rm cells . The vegArabidopsis, the observed disperse of 180-bp centromeric repeats (180CEN) is possibly caused by the absence of the chromatin remodeler DDM1 (DECREASE IN DNA METHYLATION 1) from the SWI/SNF-family of in this cell . While both cells are devoid of the somatic H3.1 variants, they contain each a specific repertoire of H3.3 variants: the chromatin of the vegetative cell includes a few canonical H3.3 variants (HTR5 and HTR8) and the variant HTR14, while the sperm chromatin contains HTR5 and a sperm-specific variant . Comparatively to somatic cells, the microspore chromatin is devoid of CHH methylation mostly from retrotransposon loci. Gametogenesis entails antagonist changes in the sperm and vegetative cells: while the sperm cells inherit the CHH DNA methylation patterns from the microspore, with more pronounced depletion, the vegetative cells restore CHH methylation at TE loci. In contrast, CG methylation is globally retained, in the sperm cells, but depleted from a subset of TE loci and intergenic regions in the vegetative cell. While compared to that in the sperm cells, CHG methylation is generally higher in the vegetative cell, albeit depleted from the same demethylated CG TE loci . Concomitantly, unequal expression of DNA methyltransferases in the central cell and egg cell – with notably undetectable level of these enzymes in the central cell contrasting with the presence of de novo DNA methyltransferases DRM1/2 in the egg – may contribute to reinforce the epigenetic dimorphism . Similar to that in the male gametes, both of the female gametes are devoid of most of the canonical, somatic H3 variants. The mature egg cell only harbors the H3.3 variant HTR5, while the central cell retains one H3.1 (HTR3) and two H3.3 variants . It was considered that the absence of H3.1 in the egg cell may be caused by the arrested cell cycle before S-phase, as H3.1 incorporation is linked with DNA synthesis  are detected, the central cell keeps barely detectable levels of MET1 and CMT3 and low levels of DRM1/2 . While this model is often taken for granted largely due to inferences made from DNA methylome profiling data in the fertilization products at a relatively late stage of seed development  that act in trans on the sperm cells’ chromatin to reinforce TE silencing via RNA-directed DNA methylation RdDM; . The effLikewise in sperm cells, a control over TE activity in the egg cell would be meaningful. It has been proposed that, similar to the vegetative cell toward the sperm cells, the central cell may play a role in reinforcing TE silencing in the egg cell. This model is inferred from the observation that when the endosperm is derived from a central cell lacking the activity of the DEMETER DNA glycosylase, hypermethylation of TEs is observed, suggesting that those loci are normally demethylated (by DME) and may, likewise the vegetative cell produces TE-derived siRNA. Similarly, trans-silencing of a reporter gene was successfully achieved in the egg by expressing the corresponding amiRNA in the central cell , comfort(MEG) active in the endosperm are demethylated in the central cell via both active and passive mechanisms , FERTILIZATION INDEPENDENT SEEDS2 (FIS2), MEDEA , although the latter can be maternally activated in a DME and DNA-methylation-independent manner  offers another window of opportunity to establish parental imprints. Erasure of DNA methylation at MEG loci for instance may be achieved in the MMC following the eviction of H1 and H2AZ  known fode facto transcriptionally active  is thought to be necessary to epigenetically reprogram the zygotic genome toward a totipotent ability, through priming developmental regulator genes for expression . The casy active , a statey active . Conversy active . Being aEmbryogenesis is a long developmental process progressing along consecutive phases of proliferation, morphogenesis, organogenesis, and maturation. Our knowledge is too scarce to draw a developmental atlas of chromatin dynamics events during those phases; large-scale processes have been mostly reported both immediately following fertilization, on which we will focus below, or at maturation stages .Soon after fertilization, rapid exchanges of gametic H3.3 histone variants occur in the zygote and endosperm and a somatic pattern of H3.3 variant composition is reestablished in the zygote , 2010. TFurthermore, developmental progression of the fertilization products entails additional chromatin dynamics as inferred by the molecular profiles of DNA methylation patterns in the embryo and endosperm in well-developed seeds (6–8 days after pollination). Particularly, the maternal genome of the endosperm undergoes DME-mediated global DNA demethylation, while, comparatively, DNA methylation levels are higher in embryo in all sequence context . This diOverall, while several evidence suggest dynamic reprogramming of the chromatin states (histone and DNA modifications) in the fertilization products, the data remain uneven with distinct developmental stages investigated  and at different resolution levels . Clearly, temporally resolved profiles of histone and DNA methylation patterns in the developing embryo and endosperm starting from soon after fertilization is necessary to elucidate the epigenetic landscape and its dynamics. But for this, daunting technical difficulties remain to be solved to enable massive, and tissue specific nuclei isolation suited for epigenome profiling, particularly from the embryo and endosperm – that are embedded within the maternal seed – at very early stages.Fertilization unites two differentiated and very specialized cells, the egg cell and the sperm cell. To enable embryo development-with the establishment of novel cell types and organ symmetries toward the basic body plan of the future plant – the newly formed zygote must be alleviated from the gametic programs and acquire totipotency. With analogy to animals, it is tempting to speculate that epigenetic reprogramming may occur in the plant zygote toward setting the future transcriptional program. The abrupt replacement of histone H3 variants in the zygote  may enabArabidopsis embryo isolation and bisulfite sequencing from small input fractions offers the realistic possibility in a near future . The possible functions of these events range from epigenetic reprogramming of the genome toward pluri- or totipotency, maintenance of genome integrity, regulation of imprinting but may also functions in immediate cellular tasks at meiosis, mitosis cellularization and patterning in the gametophyte and embryo. In the absence of cell-specific epigenome profiles, however, the impact of chromatin dynamics on epigenetic reprogramming remains largely speculative. Establishing a dogma still requires efforts to overcome the daunting obstacles that obstruct cell-specific epigenome profiling in the reproductive lineage, particularly in the model plant Arabidopsis thaliana. For these experiments, the choice of other model plants  where the gametes are more amenable to mechanical isolation may be judicious. The development of cell-specific nuclei isolation approaches programming of genome expression during those processes. Exciting findings in the past decades uncovered dynamic events of chromatin modifications, DNA methylation, nucleosome remodeling, and small RNA regulation that take place throughout sexual reproduction in flowering plants, particularly during cell fate specification (proaches  may provproaches . Alternaproaches . It enabproaches . The comThe 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.