Emerging experimental evidence shows that isoflavones could induce cancer cell death by regulating multiple cellular signaling pathways including Akt, NF-κB, MAPK, Wnt, androgen receptor (AR), p53 and Notch signaling, all of which have been found to be deregulated in cancer cells. Therefore, homeostatic regulation of these important cellular signaling pathways by isoflavones could be useful for the activation of cell death signaling, which could result in the induction of apoptosis of both pre-cancerous and/or cancerous cells without affecting normal cells. In this article, we have attempted to summarize the current state-of-our-knowledge regarding the induction of cancer cell death pathways by isoflavones, which is believed to be mediated through the regulation of multiple cellular signaling pathways. The knowledge gained from this article will provide a comprehensive view on the molecular mechanism(s) by which soy isoflavones may exert their effects on the prevention of tumor progression and/or treatment of human malignancies, which would also aid in stimulating further in-depth mechanistic research and foster the initiation of novel clinical trials. Soy isoflavones have been documented as dietary nutrients broadly classified as “natural agents” which plays important roles in reducing the incidence of hormone-related cancers in Asian countries, and have shown inhibitory effects on cancer development and progression  Th. Th78]. The cellular signaling is a complex signal network with positive or negative feedback loops and also regulated by compensatory mechanism. It is important to note that the apoptosis pathway is regulated by multiple cellular signaling. In cancer cells, the deregulations of multi-signaling and subsequent defective apoptosis often exist; therefore, targeting multiple signaling to induce apoptotic cell death by natural compound isoflavone could open a new avenue for cancer prevention and therapy as detailed in the following sections.XL, and activated PARP and caspases, leading to the induction of apoptotic cell death [Isoflavones are mainly found in members of the Leguminosae family. Foods such as soy, lentil, bean, and chickpea are sources of isoflavones; however, soybean is the food that contains abundant amounts of isoflavones. Several epidemiological studies have shown that soy could have protective effects against prostate and other cancers ,87. The ll death ,92,95,962O2 or TNF-α. By immunochemistry and confocal microscopic analysis, we found that isoflavone, genistein inhibited the translocation of NF-κB to the nucleus, suggesting that isoflavone genistein may reduce the NF-κB binding to its target DNA and thereby inhibiting the transcription of its target genes such as Bcl-2, one of the important regulators of apoptosis. Recent studies have also shown that the induction of apoptosis by isoflavone, genistein and biochanin-A was mediated through both NF-κB dependent and independent pathways [It is well known that NF-κB inhibits apoptosis. Therefore, we examined the status of NF-κB activity in isoflavone genistein treated breast, prostate, lung, and pancreatic cancer cells by electrophoresis mobility shift assay (EMSA) during our earlier studies ,93,95,97pathways ,99, suggin vivo effects of isoflavone genistein on NF-κB signaling, we conducted in vivo study [ex-vivo failed to activate NF-κB activity in lymphocytes harvested from these volunteers, while lymphocytes from these volunteers collected prior to soy isoflavone intervention showed activation of NF-κB DNA binding activity upon TNF-α treatment ex-vivo. These results have demonstrated that soy isoflavone supplementation has a protective effect against TNF-α induced NF-κB activation in humans, suggesting that soy isoflavone could exert its cancer chemopreventive activity through the inhibition of NF-κB signaling and induction of apoptosis, which is required for elimination of damaged or pre-cancerous cells in order to maintain normal homeostasis  for the prevention of tumor progression and/or treatment of most human malignancies. A number of phase I and II clinical trials have been conducted to investigate the toxicity and effects of isoflavones in healthy men and in patients with prostate cancer [in vitro, relevant animal model studies in vivo and novel clinical trials are needed in order to better understand the molecular mechanisms of isoflavone action, and to further realize the value of isoflavone and/or its synthetic analogs for the prevention of tumor progression and/or treatment of human malignancies.The data from e cancer . The rese cancer , suggest


The cholinergic and nitrergic phenotypes in human fetal ganglia (inferior) of the glossopharyngeal and vagus nerves were overlooked in basic research. Lack of a positive neuronal NO synthase (nNOS) phenotype in the inferior vagal fetal ganglion was recently suggested to be an individually variable phenotype. Choline acetyltransferase (ChAT) was not evaluated previously in ontogenesis. We aimed to evaluate these phenotypes in human midterm fetuses. Samples from five specimens with gestational ages varying from 4 to 6 months were used. Immunohistochemistry for nNOS, ChAT, neurofilaments, and S100 protein was performed. Neuronal somata were positively stained for nNOS, ChAT and neurofilaments in the inferior glossopharyngeal and vagal ganglia. S100 protein distinctively labelled the satellite glial cells ensheating the respective neurons. In human midterm fetuses vagal and glossopharyngeal inferior ganglia are nitrergic and cholinergic. To evaluate a functional role of these phenotypes in ontogenesis, the specific anatomic circuits should be further checked. Differences in immune labelling should be evaluated by use of similar antibodies from different manufacturers.  The nNOS isoform is required for the thermoregulation and participates in the production of fever in rats [2]. NO may act on vascular smooth muscle, decreasing vascular tone, stimulating thermogenesis in brown fat, and modulating neuroendocrine function [2]. Nitric oxide (NO) is produced in many different cells and is involved in the regulation of physiologic events, such as inflammation, vascular tone, and metabolism. Depending on cell type, NO is formed in an enzymatic reaction catalyzed by one of the three isoforms of NO synthase (NOS). Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutive, and produce small amounts of NO after stimuli that raise intracellular calcium concentrations. Agents such as cytokines express the third isoform, inducible NOS (iNOS), after induction [3]. Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is currently the most specific indicator for the monitoring of the functional state of cholinergic neurones in the peripheral nervous system [4]. However, the ChAT phenotype was not previously investigated in human fetal cranial ganglia.It was shown that neurons in the inferior vagal ganglion (nodose ganglion) are ChAT immunoreactive [5]. Unexpectedly, no nNOS immune reactivity was found in the fetal inferior vagal ganglion [5]. However, nNOS is a marker for non-cholinergic parasympathetic nerves, and nNOS-positive neurons were demonstrated in the human tracheal wall, which is supplied by vagal fibers [6].Neuronal NO synthase immune reactivity in human fetal cranial ganglia was searched in a single study, recently performed . The microscopic slides were analyzed and micrographs were taken and scaled using a Zeiss working station which was described elsewhere .It resulted here that the inferior glossopharyngeal and vagal ganglia are cholinergic, as well as nitrergic. This is a similar phenotype to that identified in cardiac ganglia [12]. Afferent neurons in the nodose ganglia that distribute to the stomach were demonstrated to express nNOS; anterograde-traced vagal endings are, however, nNOS negative, indicating that NOS is not transported peripherally [13]. A subpopulation of enteroendocrine cells in the mucosa were nNOS positive, which were found anatomically in close apposition with mucosal vagal afferent endings. This indicates an inhibitory neuromodulatory role of epithelial NO, which targets a distinctive population of vagal afferences [13]. It was previously shown that microganglia in the carotid body and glossopharyngeal and carotid sinus nerves exhibit NOS- and ChAT-positive immune reactivity, and functionally related NOS-containing neurons are located in the petrosal ganglion [14]. It seemed natural to find a nNOS phenotype in ganglia of cranial nerves known to be related to autonomic functions. Neurons with immune reactivity to NOS were found to be distributed in various mucosae [15]. As resulted from the present study, this could be an ontogenetic feature. The presence of ChAT in the sensory nodose projection to the solitary nucleus in medulla oblongata cannot be ruled out [16].By immunohistochemistry, neurons within the nodose ganglion were found ChAT-positive, suggestive for choline uptake and an endogenous content of acetylcholine [It appears so that the nitrergic and cholinergic neuronal phenotypes in the petrosal and nodose ganglia in human midterm fetuses are positive and able to support different functional mechanisms. However, the corresponding anatomic circuits should be completely evaluated for the specific immune phenotypes involved in neural transmission and function. AcknowledgementsThis study was supported by the Sectoral Operational Programme Human Resources Development (SOP HRD), financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/89/1.5/S/64153 (author #3).  Conflicts of interestThe authors confirm that there are no conflicts of interest.


For any organism, population size, and fluctuations thereof, are of primary importance in determining the forces driving its evolution. This is particularly true for viruses—rapidly evolving entities that form populations with transient and explosive expansions alternating with phases of migration, resulting in strong population bottlenecks and associated founder effects that increase genetic drift. A typical illustration of this pattern is the progression of viral disease within a eukaryotic host, where such demographic fluctuations are a key factor in the emergence of new variants with altered virulence. Viruses initiate replication in one or only a few infection foci, then move through the vasculature to seed secondary infection sites and so invade distant organs and tissues. Founder effects during this within-host colonization might depend on the concentration of infectious units accumulating and circulating in the vasculature, as this represents the infection dose reaching new organs or “territories”. Surprisingly, whether or not the easily measurable circulating (plasma) virus load directly drives the size of population bottlenecks during host colonization has not been documented in animal viruses, while in plants the virus load within the sap has never been estimated. Here, we address this important question by monitoring both the virus concentration flowing in host plant sap, and the number of viral genomes founding the population in each successive new leaf. Our results clearly indicate that the concentration of circulating viruses directly determines the size of bottlenecks, which hence controls founder effects and effective population size during disease progression within a host. Infecting viruses progress within multi-cellular hosts via two distinct mechanisms: cell-to-cell proximal contamination and long-distance migration to remote organs through the vasculature. In distant susceptible organs, it seems logical that the number of initially infected cells, the number of viral genomes entering each of these cells, and thus the number of founders of new viral “colonies”, depends on the concentration of infectious units transported in the vasculature. For any organism, the number of founders colonizing a “virgin territory”, is of prime importance in determining the forces driving its evolution. This is particularly true for viruses where the so-called founder effect is a key factor in the emergence of new variants with altered virulence. It is surprising to note, however, that whether the circulating virus load directly drives the size of viral populations during host colonization remains elusive. By monitoring for the first time the virus concentration flowing in host plant sap, in parallel with the number of viral genome founders in each successive leaf, we provide unequivocal evidence that the concentration of circulating viruses can directly determine the founder effect and effective population size during disease progression in a eucaryotic host. Virus progression within multi-cellular hosts operates via two distinct mechanisms: cell-to-cell proximal contamination and long-distance migration (either as free infectious units or in circulating cells) to colonize new organs and/or tissues. Both animals and plants can be considered as heterogeneous landscapes consisting of an ensemble of very different organs or “territories”, variably distant, and interconnected by a complex vascular system transferring nutrients, metabolic product and wastes, and information. Soon after entry into a healthy host, the vast majority of viruses use this connecting vasculature to travel long distances and expand their populations into virgin territories. In distant susceptible organs, it seems intuitively obvious that the number of initially infected cells, the number of viral genomes entering each of these cells, and thus the number of founders of new viral “colonies”, will depend on the concentration of infectious units transported in the plasma or sap flooding the vasculature. In other words, the virus load in the circulating flux will determine the bottlenecks in a virus population progressing within a host, and hence the effective population size and the pace at which new variants are produced, selected, and emerge. Taking an alternative view, however, one could speculate that the number of “entry points” into various organs of the host might sometimes be extremely limited, and hence the number of founder viral genomes in such territories may always be low or constant, regardless of the circulating virus load. Surprisingly, for both animal and plant viruses, experimental data supporting one or the other of these contrasting scenarios are extremely rare and only fragmentary at best.For animal viruses, many studies have quantified the virus load in the plasma of infected individuals. That this virus load changes drastically during progression of the infection, upon the onset of host defenses or during drug treatments, has been reported for several viruses, e.g. hepatitis B virus HBV, , hepatitCauliflower mosaic virus  bottlenecks at leaf entry. The viral population bottlenecks associated with leaf colonization have been investigated in several plant virus genera—Potyvirus Here, we report the first quantification of a plant virus titer within the vasculature of its host, and monitoring of this important trait as infection progresses. In addition, we demonstrate that changes in sap virus titer correlate with changes in the size of viral population bottlenecks at leaf entry, strongly suggesting that the circulating virus load is a major factor determining the effective size of within-host viral populations.We first conducted a pilot experiment to collect pure phloem sap from severed aphid stylets inserted precisely within sieve tubes, as previously described We next decided to estimate the virus load directly within whole aphids, processed after a 16-hour acquisition period on CaMV-infected turnip plants—a time at which aphids are most often engaged in a phase of sustained sap ingestion from the phloem sieve tubes Myzus persicae seldom reach deep phloem tissues before a feeding time of about 30 min has elapsed The control experiment gave an estimate of what aphids can potentially acquire when feeding superficially only in epidermal and mesophyll cells. Because aphids always conduct test probes in these tissues before settling in the sieve tubes Finally, we also immediately estimated the virus load in cells of leaves used directly for aphid-feeding experiments . InteresWe next assessed whether a higher virus concentration in the circulating sap represents a higher viral dose, which would in turn increase the number of founder viral genomes in newly infected leaves. For this experiment, we conducted a similar longitudinal analysis of infected plants, measuring the size of bottlenecks imposed on CaMV populations at the entry to successive leaf levels. Fifty replicate plants were inoculated with a mixture of two CaMV variants (Mys4 and Mys7) at a 1∶1 ratio, and their relative frequency was monitored in order to evaluate bottleneck sizes upon entry to leaf levels 5, 16 and 21, as described in the Materials and Methods. Briefly, Mys4 and Mys7 are equally competitive in doubly infected plants  and the In contrast, the initial source populations from which viral genomes in leaf levels 16 and 21 originate are much harder to define because many leaves below them are exporting virions. We made the same assumption as in per se.To confirm the observed changes in bottleneck size over time , we arguhyperbolic-logarithmic>2000), confirming the pattern of bottleneck changes shown in We first plotted the variances of marker frequency calculated from the two sets of plants used in Remarkably, the pattern of bottleneck size changes at successive leaf levels  and 3 reThough stemming from different plant sets, the above results together suggest a match between the pattern of the virus concentration within the sap and that of the size of the bottlenecks endured by CaMV populations when initially colonizing leaves. In order to confirm this relationship in a single experiment, we further analyzed DNA samples used in +/−SD 318+/−244 copies/nl) compares with values quantified from whole aphids (Myzus persicae (Sulz.) can individually contain around 25 nl of sap, continuously flowing and rapidly transiting (about 1 hour) from ingestion to excretion. This volume would consistently correspond to a range of 55–452 virus copies per nanoliter of sap, depending on the leaf level  no liquid exudes from the wound e aphids  depends The approximation reported here for the viral sap load is certainly specific to CaMV infecting turnip; however, it represents the first estimation of the load of a plant virus circulating within the vasculature of its host. In addition to the cautionary remark mentioned above, to reliably reflect the virus load in plant sap, the technique used in Tobacco mosaic virus in different leaves  in the first systemically infected leaf, the virus titer in the sap is far too low to saturate all existing entry points, and the limiting factor is thus the availability of virions; (ii) as more leaves become infected and shed virions within the sap, the virus titer increases and relaxes the bottleneck during disease progression, with no apparent limit due to entry points; and finally (iii) the situation reverts later in infection by unknown mechanisms that either halt virus export from infected cells, store virions elsewhere than in the vasculature, or accelerate virion decay in the sap (or a combination thereof), resulting in a drop in the number of circulating virions and thus of the size of bottlenecks. Interestingly, we previously described a very similar pattern of dynamic changes in the MOI of CaMV at different leaf levels, indicating that the CaMV load in the sap probably influences the number of viral genomes entering leaves as well as individual cells within the leaves In the scenario suggested here for CaMV, the most significant bottleneck encountered during the virus life cycle is certainly that imposed by aphid transmission, during which very few genomes might be inoculated, as suggested by studies with other non-circulative viruses Caulimovirus) is a circular dsDNA of approximately 8000 bp (depending on the strain), encoding seven independently translated open reading frames (ORF I-VII) TCTACATATTCCTGATAACTCAACGGTCGTCGACGGAGT or AGTAAGTGCTGTAAGTATAATAAGGATACTTGTCGACAG) between the stop codon of ORF I and the start codon of ORF II. These two clones were named Mys4 and Mys7, respectively, and a real-time PCR protocol was developed for their specific quantification in DNA mixtures. PCR conditions and primers are detailed in the online supporting information , conditions ensuring that the colonies are reproducing clonally. The colonies were transferred to a new cage and new host plants every two weeks, and aphids were always collected at the moment of the transfer, thus at comparable population densities. Turnip plants (Brassica rapa cv. “Just Right”) were maintained in an insect-proof growth chamber under controlled conditions . For all experiments, plantlets at the third leaf stage were inoculated mechanically by rubbing a virus suspension containing 400 ng of virus particles and Carborundum abrasive powder, on the second leaf level. All inoculums were prepared by mixing Mys4 and Mys7 purified virus particles at a 1∶1 ratio. The appearance of new leaves (budding) on the inoculated plants was monitored and noted daily in order to allow leaf sampling at a precise leaf age. Depending on the experiment, either entire leaves or six leaf discs (0.8 cm ø) distributed evenly over the leaf surface were sampled. DNA from each leaf sample was extracted as described Colonies of the aphid species th day of development. At this stage, the sink-to-source transition has not yet occurred and the phloem sap flows into the developing leaves, whereas this flow is inverted after the transition that occurs on approximately the 10th day of leaf development, when each leaf exceeds 1/3 of its final size Twenty replicate plants were inoculated and sampled at four time points. The different time points corresponded to leaf levels 5, 9, 14 and 28. The first systemically infected leaves appeared at leaf level 5, the first visual signs of flowering induction (changes in leaf morphology) appeared irregularly between leaf levels 21 and 28, and the plants slowly entered senescence after leaf level 28. We originally planned to collect also leaf level 21, but our aphid colony unfortunately collapsed at this time and recovered only in time to use leaf level 28. Because of the length of the experiment, different aphid cohorts were used at different dates for different leaf levels, but they all originated from the same clonal rearing, maintained in constant conditions and collected at comparable population densities. It is important to note that the sampling dates were chosen when leaves were in their 5M. persicae generally settle and feed continuously within the sieve tubes during such a long period For each time point, fifty aphids were confined in a “cage” enclosing the defined leaf level (see above) on each of 20 replicate plants. Three groups of five immobile aphids—those most likely feeding—were collected from each leaf after an acquisition period of 10 minutes in epidermal and mesophyll cells th day of development, after the sink-to-source transition, when colonization by viruses imported from the phloem sap is over We estimated the size of the CaMV population bottlenecks during colonization of leaf levels 5, 16 and 21, which appear successively on infected plants. In this experiment, leaves were collected on the 13Two sets of 50 plants were inoculated with a mixture of Mys4 and Mys7 purified virions, on leaf level 2. In both sets, we collected the inoculated leaves just before their death in order to minimally affect  virus exit and migration towards systemically infected leaves. In addition, in one of the sets of plants, we sampled leaf discs on leaf level 5 and let the plants grow until leaf level 16 appeared. Using an approach similar to DNA was extracted from all leaf samples, and the relative frequency of Mys4/Mys7 was estimated using Q-PCR as indicated above. In the first plant set, we determined the variance of Mys4/Mys7 relative frequency among leaves 2 (a), among leaves 5 (b), among pools of leaves below leaf 16 (c), and among leaves 16 (d). In the second plant set, we determined the variance of Mys4/Mys7 relative frequency among pools of leaves below leaf 21 (e), and among leaves 21 (f). Comparing a–b, c–d, and e–f allowed evaluation of the size of the bottlenecks at the entry of leaf levels 5, 16 and 21, respectively, as described In order to rapidly establish the pattern of bottleneck changes in a viral population invading successive leaf levels, we tracked the among-plant variance in relative marker frequency over time. Severe bottlenecks should increase among-plant variance, while mild bottlenecks should affect it only slightly. We expected that if the viral effective population size was constant throughout the infection the among-plant variance would increase monotonically, while temporal variation in the effective size would induce non-monotonicity in the pattern of variation of among-plant variance. To illustrate the patterns that could arise from such temporal variation in the effective population size, we simulated the evolution of a single diallelic neutral locus in a number of unconnected populations, representing the different host plants. All populations had the same effective size at any given time, but their effective size could vary over time. We computed the among-population variance in allele frequencies over time. The results, e.g. th day of development by collecting the entire leaf. The Mys4/Mys7 relative frequency was estimated (by Q-PCR as above) in successive leaf levels on the 40 repeated plants, and the function best describing changes of the Mys4/Mys7 variance was determined as follows. To test whether the empirical results were better explained by monotonic or non-monotonic functions, we fitted phenomenological models whose behaviour a priori fitted that of the simulation results in To experimentally investigate how among-plant variance in relative marker frequency evolved over time, 40 plants were inoculated in parallel and sampled at six different time points, corresponding to leaf levels 2 (inoculated leaf), 5, 10,16, 21 and 27. Each leaf level  was sampled when leaves were in their 13We previously reported two methods for evaluating the size of CaMV population bottlenecks, one based on a simple analysis of changes in the variance of Mys4/Mys7 relative frequency at different leaf levels, and the other using Fst statistics Other statistical analyses used various classical tests, which were all performed with the R (v2.11) package. The nature of the tests and their results are indicated in the text.Text S1Online supporting information. The online (DOC)Click here for additional data file.


This article highlights the current knowledge of mTOR biology and provides new insights into the role of mTOR in different cancers. An active mTOR coordinates a response in cell growth directly through its effects on cell cycle regulators and indirectly by sustaining nutrient supply into the cell through the production of nutrient transporters and also through the promotion of angiogenesis. A primary way that mTOR exerts its regulatory effects on cell proliferation is by controlling the production of cyclin D1. mTOR increases the translation of hypoxia-inducible factor 1 (HIF-1)/HIF-2. The HIF transcription factors drive the expression of hypoxic stress response genes, including angiogenic growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor β (PDGF-β), and transforming growth factor a (TGF-α). mTOR also increases the surface expression of nutrient transporters proteins. An increase in these proteins results in greater uptake of amino acids and other nutrients by the cell leading to adequate nutrient support to abnormal cell growth and survival. There is also emerging evidence that mTOR activation may play a role in promoting cell survival through the activation of antiapoptotic proteins that contribute to tumor progression. Given that the mTOR pathway is deregulated in a number of cancers, it is anticipated that mTOR inhibitors will have broad therapeutic application across many tumor types. Until now, no treatment demonstrated Phase III evidence after disease progression on an initial VEGF-targeted therapy in advanced renal cell carcinoma. Everolimus is the first and only therapy with Phase III evidence after failure of VEGF-targeted therapy. Everolimus is a once-daily, oral inhibitor of mTOR  indicated for the treatment of advanced renal cell carcinoma in patients, whose disease has progressed on or after treatment with VEGF-targeted therapy. The mammalian target of rapamycin (mTOR) pathway is a crucial regulator of cell growth and proliferation and research into this area has revealed that mTOR dysregulation has a key role to play in various cancers. mTOR appears to play a central role in signaling caused by nutrients and mitogens such as growth factors to regulate translation. The understanding of the science behind mTOR’s role as a regulator of many cell processes and its potential as a therapeutic target has opened up treatment possibilities in several types of cancer. This article highlights the current knowledge of mTOR biology and provides new insights into the role of mTOR in different cancers.mTOR is a 290 kDa serine–threonine kinase that regulates both cell growth and cell cycle progression through its ability to integrate signals from nutrient and growth factor stimuli. mTOR, a member of the phosphatidylinositol 3-kinase (PI3K)-kinase-related kinase (PIKK) superfamily, is composed of 2549 amino acids that are grouped into highly conserved domains. The mTOR is an intracellular kinase that controls the production of proteins through effects on the machinery of mRNA translation. These proteins include important components of several processes critical to cell metabolism, cell growth, cell division, and responses to cellular stresses such as hypoxia or DNA damage.2mTOR senses the growth conditions within the cellular environment and helps the cells respond to changes in this environment. An active mTOR coordinates a response in cell growth directly through its effects on cell cycle regulators and indirectly by sustaining nutrient supply into the cell through the production of nutrient transporters and into the cell’s environment through the promotion of angiogenesis. The activation of mTOR signifies a decision point that takes into account the availability of the basic materials required for cell growth  and the growth-regulating signals from other cells and tissues  while monitoring conditions of cellular stress . In this manner, the cell is protected from signals outside the cell to grow and proliferate originating when the supply of nutrients and energy inside the cell are not sufficient to support the effort.Upstream in the growth-promoting pathways are critical molecules that converge on mTOR, which are often deregulated in some manner in cancer. Several mutations found in cancer produce inappropriate signals that activate the mTOR switch, driving the growth and proliferation of the cancer cell.mTOR senses the availability of nutrients  and consolidates this information with growth factor signaling through the PI3K/Akt/tuberous sclerosis complex (TSC) pathway. An activA primary way that mTOR exerts its regulatory effects on cell proliferation is by controlling the production of cyclin D1. Cyclins are proteins that regulate the activity of enzymes called cyclin-dependent kinases (CDKs) through the critical G1–S restriction point of the cell cycle, which in turn regulate the passage of cells. Recently, cyclin D1 has been shown to play a role in gene transcription, cell metabolism, and cell migration.Cyclin D1 overexpression had been associated with a number of cancers including breast cancer, colon cancer, prostate cancer, lymphoma, and melanoma.6Mutations in tumor sclerosis complex (TSC1 or TSC2) can also lead to overactivation of mTOR. This overactivation causes unregulated cell proliferation and multisystem tumors in patients with TSC. Increased mTOR activation, as evidenced by hyperphosphorylation of downstream signaling proteins, has been observed in TSC related lesions.8mTOR plays a key role in angiogenesis, i.e., the formation of new blood vessels to provide oxygen and nutrients to growing and dividing cells. mTOR increases the translation of hypoxia-inducible factor 1 (HIF-1)/hypoxia-inducible factor 2 (HIF- 2). The HIF Increased levels of HIF-1α and HIF-1β have been shown to correlate with increased mortality in a number of tumor types, including cervical cancer, breast cancer, non-small-cell lung cancer, ovarian cancer, head and neck cancer, and gastrointestinal stromal tumors. In additCells have a complex sensing system designed to ensure that they do not undergo periods of growth unless adequate levels of nutrients are available to produce the energy necessary to support that growth. The rapidly expanding field of study dedicated to understanding how living systems obtain and utilize energy is known as bioenergetics.Bioenergetic research has shown that mTOR plays a key role in regulating cell metabolism.13 mTOR iAdditionally, abnormally activated mTOR may give cancer cells a competitive growth advantage by increasing production of the core enzymes necessary for glycolysis, which enables cancer cells to survive and grow even under hypoxic conditions.There are several lines of evidence that mTOR activity plays a role in cell survival. Majority of this research has revealed that mTOR inhibition increases sensitivity to cell death pathways; however, there is also emerging evidence that mTOR activation may play a role in promoting cell survival through the activation of antiapoptotic proteins that contribute to tumor progression.A recent study has shown that mTOR activation of pS6K increases protein levels of survivin, an inhibitor of apoptosis protein (IAP). Survivin blocks extrinisic and intrinsic apoptotic pathways, and survivin expression has been observed in a wide number of tumor types and associated with poor prognosis in cancer .16]16]Thus, overactivation of mTOR due to dysregulation of upstream pathways, leading to abnormal activities in cell progression, angiogenesis, cell metabolism and apoptosis has been implicated in various cancer types.mTOR controls production of HIF-1α, an important protein in RCC, where its unregulated activity is causally related to disease pathogenesis. mTOR reg13Several important molecular changes in NETs involve the mTOR pathway. Increased growth factor signaling, namely, epidermal growth factor (EGF) and insulin-like growth factor (IGF) signaling upstream of mTOR, has been observed frequently in NETs. Also, inmTOR is activated in 60–80% of gastric adenocarcinomas25 and is2629mTOR signaling is critical in the pathogenesis of breast cancer. mTOR signaling may be related to estrogen receptor (ER) activation and adaptive estrogen hypersensitivity. mTOR patmTOR-dependent signaling is active in 25–45% of hepatocellular carcinoma (HCC). Activati37Approximately 85% of Non-Hodgkin lymphoma (NHL) s arise from cells of B-cell lineage. mTOR sigBecause the number of potential defects that can cause inappropriate activation of mTOR is large and one or the other is common to most cancer cells, blocking their effect at the point of convergence is a rational approach. The mTOR protein itself is seldom altered, suggesting that mTOR is a stable target for influencing several important pathways in cancer. FurthermThus far, clinical studies have supported preclinical findings regarding the importance of mTOR in cancer and validate mTOR inhibition as an effective cancer therapy. Given that the mTOR pathway is deregulated in a number of cancers, it is anticipated that mTOR inhibitors will have broad therapeutic application across many tumor types. It is possible that only a subset of cancer patients will have tumors sensitive to mTOR inhibitors as a monotherapy. Based on preclinical findings, mTOR inhibitors may be more efficacious when used in rational combination with other cancer regimens with activities supplemental to and/or influenced by mTOR activity, such as DNA-damaging and hormonal agents, oncogene inhibitors, and other targeted therapies. Thus, mTOR inhibitors may play an important role in the management of cancer. mTOR inhibition has been proven to result in reduced tumor cell growth and proliferation, decreased tumor angiogenesis and inhibition of cell metabolism.Treatment of advanced RCC has evolved rapidly in the last few years. The emergence of multiple new treatments has led to frequent use of sequential VEGF tyrosine kinase inhibitor (TKI) therapy. Until now, no treatment demonstrated Phase III evidence after disease progression on an initial VEGF-targeted therapy. Everolimus is the first and only therapy with Phase III evidence after failure of VEGF-targeted therapy. Everolimus is a once-daily oral inhibitor of mTOR indicated for the treatment of advanced RCC in patients whose disease has progressed on or after treatment with VEGF-targeted therapy.mTOR inhibitors are being investigated in clinical trials in several hematologic, gastrointestinal, genitourinary, and neurologic cancers, NETs, and sarcomas, as well as in breast and lung cancers. It is hoped that with this added knowledge, the potential beneficial effects of inhibiting mTOR signaling in cancer patients will be fully recognized and realized in clinical practice.


However, the transgenic expression of the AvrRpt2  gene in Arabidopsis restores optimal growth of the Pto strain defective in TTSS, thereby linking the effector and auxin  formation has been reported Pto and Arabidopsis has not been emphasized. High levels of cytokinins were found to increase the resistance of plants to some viral pathogens and herbivores PR1 gene was shown as upregulated in the presence of enhanced levels of plant cytokinin, while overexpression of cytokinin oxidase caused its repression in ArabidopsisAtIPT1, 2, 5, and 7) increased Arabidopsis resistance to Pto(AtCKX4) enhanced susceptibility of Arabidopsis to infection by pathogens PR1 against biotrophic infections in plants  Alternaria brassicicola  Noco2. They also implicated the negative regulators (type-A ARRs) of cytokinin signaling as acting in a feedback loop (sid2 mutants), the exogenous application of cytokinin partially inhibited the growth of the pathogen. However in the wild type Arabidopsis plants, the enhanced levels of cytokinin gave rise to more protection than that observed in the sid2 mutants. This strongly suggests that cytokinin signaling enhances the contribution of SA-mediated immunity in hormone disease networks. The SA-dependent response to cytokinin application is not limited to a few marker genes, but seems to be a genome-wide transcriptional reprogramming Pto.In contrast to auxin, the role of cytokinin has not been systematically analyzed. Previously, cytokinins were believed to be instrumental in mediating host susceptibility against fungal biotrophs by generating a green island around the infection zones n plants . PR1 is ack loop . Very reArabidopsis infected by Pto , since a Pto TTSS mutant strain failed to induce this response during infection t-zeatin levels during Pto-Arabidopsis interaction CKX4 gene Pto in Arabidopsis. The genes encoding enzymes of cytokinin biosynthesis were found to be downregulated, while those of cytokinin catabolism were induced Pto and Arabidopsis.The individual roles of auxin and cytokinin vary among plant-pathogen systems d by Pto . Auxin ePto enhances the in planta level of auxin, which attenuates the SA responses and hence decreases resistance against infection by a pathogen. Additionally, auxin downsizes the pool of cytokinin, which could also lead to increased susceptibility. Cytokinin, when exogenously applied or transgenically enhanced, expedites SA responses in the host PtoPto and Arabidopsis interaction is still not well understood. One plausible explanation (We propose a working model for the auxin-cytokinin antagonism : To causlanation  for the lanation  of cytokPto. To address this more closely, we advocate a systems-biology approach to analyze the impact of interactions between auxin and cytokinin in plant immunity Here we highlight the auxin-cytokinin antagonism that occurs as part of a complex hormonal interplay and exerts a critical influence on the core SA-JA/ET plant immunity pathways


Oceanospirillales, Colwellia, Cycloclasticus, Rhodobacterales, Pseudoalteromonas, and methylotrophs were found enriched in the contaminated water column. Presented here is a comprehensive overview of the ecogenomics of microbial degradation of MC-252 oil and gases in the water column and shorelines. We also present some insight into the fate of the dispersant Corexit 9500 that was added to aid in oil dispersion process. Our results show the dispersant was not toxic to the indigenous microbes at concentrations added, and different bacterial species isolated in the aftermath of the spill were able to degrade the various components of Corexit 9500 that included hydrocarbons, glycols, and dioctyl sulfosuccinate.The Deepwater Horizon spill released over 4.1 million barrels of crude oil into the Gulf of Mexico. In an effort to mitigate large oil slicks, the dispersant Corexit 9500 was sprayed onto surface slicks and injected directly at the wellhead at water depth of 1,500 m. Several research groups were involved in investigating the fate of the MC-252 oil using newly advanced molecular tools to elucidate microbial interactions with oil, gases, and dispersant. Microbial community analysis by different research groups revealed that hydrocarbon degrading bacteria belonging to  In April 2010, high-pressure oil and gas caused the Deepwater Horizon drilling rig in the Gulf of Mexico to explode making it the worst oil spill in the United States and the largest marine oil spill in the history of the petroleum industry. MC-252 oil was released from the broken riser pipe at a depth of 1,500 m below surface. Approximately 4.1 million barrels of light crude oil was released into the Gulf waters, of which a significant amount has been accounted for in the cleanup effort  than outside the plume  over background non-plume samples. In addition, genes for carbon metabolism, nitrogen assimilation, sulfate reduction, phosphorus release, metal resistance, and bacteriophage replication were higher in abundance in plume waters, along with several functional genes derived from Oceanospirillales. In absence of an isolate, deep sequencing of community DNA and RNA and single-cell genomics provided greater insights into Oceanospirillales for genes and pathway supregulated by the spill  and aliphatic hydrocarbons in MC-252 oil moved through the water column, it was subjected to chemical and physical partitioning. A recent study combining atmospheric, surface, and subsurface chemical data has stated that the readily soluble hydrocarbon components constituted approximately 70% of the deep plume mass and that the remaining traveled as trapped oil droplets throughout the water column  and low abundance of Colwellia, Cycloclasticus, and Oceanospirillales – which had dominated previously. The authors attributed this change to a residual bloom of methanotrophic activity having occurred in July 2010. 16S rRNA clone libraries identified methanotrophs, methylotrophs, Flavobacteria, and Alphaproteobacteria  to be relatively abundant in plume waters sampled later in September 2010 . DNA based dot blot hybridization using a specific probe detected greater than 105 CFU/g of Vibriovulnificus in tar balls and sands from beaches in Mississippi and Alabama collected from July through October 2010 . This implied that temperature was a major determinant in selection of this group of microorganisms in the plume. Further, laboratory-based incubations with MC-252 oil, Corexit, and water outside the plume also revealed an increase in abundance of Colwelliaceae and Oceanospirillales  spectromicroscopy  inhabited the surface waters with visible oil sheen, and Gammaproteobacteria  were prevalent in samples that had heavy oil layer on top. Several Cyanobacteria, Rhodobacterales, and Rhodospirillales have been associated with oil hydrocarbon degradation , marine Alternaria species dominated in brackish Mobile Bay waters. In addition, OTUs related to Aspergillus, Acremonium, Acarospora, Rhodocollybia, and Rhizopus species were reported in higher abundance in the post oil spill samples. A number of these marine fungal groups have been shown previously to metabolize hydrocarbon compounds . Metagenomic and metatranscriptomic analysis revealed an enrichment of a group of organisms within the Rhodobacterales family corresponding to samples with high total petroleum hydrocarbons .Culture-dependent and genomics-based studies were used to characterize microbial community in oil-contaminated and pristine sand samples collected from Pensacola beach in September 2010. Oil tainteds and harbored a higher microbial count of hydrocarbon degraders compared to pristine samples as corroborated by both Most Probable Number (MPN) and molecular methods. Based on initial DNA fingerprinting analysis, detailed Pyrotag sequencing of SSU rRNA amplicons revealed a significant community shift toward Actinobacteria, Bacteroidetes, and Proteobacteria. Firmicutes like Bacilli and Clostridia were more abundant in September when oil concentrations were lower. Oil concentration also influenced the community function, as the relative abundance of hydrocarbon degrading genes increased significantly when TPH concentrations were high, and decreased when hydrocarbons were low  along with the detection of genes involved in anaerobic metabolism such as sulfate reduction, nitrate reduction, and methanogenesis , also hinted toward occurrence of anaerobic hydrocarbon degradation in salt marsh sediments in coastal Alabama , with both hydrophilic and hydrophobic groups that encourage development of small oil-surfactant micelles , two hydrocarbon degrading isolates Acinetobactervenetianus and Marinobacterhydrocarbon oclasticus were severely affected, however the isolate most similar to Pseudomonaspseudoalcaligenes showed almost as much growth as the control at 1:50 dilution. Perhaps the presence of hexadecane helped alleviate the toxic effect of the dispersant in this case , glycols (40%), and dioctylsulfosuccinate . Not only did Corexit 9500 have no effect on the growth of microbial consortia enriched from the Gulf of Mexico, most of its components were biodegraded over time – the hydrocarbon fraction much more rapidly than the DOSS and glycol compounds  or MC-252 oil (100 ppm) + Corexit 9500 (10 ppm) at 4°C. Sacrificial samples were analyzed for TPH as described previously  was the sole source of carbon.To better understand the effect of the dispersant on this organism, experiments were initiated with active cultures of -252 oil 00 ppm or-252 oil 00 ppm orColwellia strain RC25, some isomers of DOSS were degraded within 19 days (Figure Alcanivorax strain 31 was unable to degrade the DOSS components, but could degrade DPnB and propylene glycol components (Figure We also looked deeper into the biodegradability of the dispersant itself by these two isolates. Corexit 9500 components were analyzed over time in sacrificial samples using methods described previously (Bælum et al., s Figure . AlcanivA systems biology approach closely integrated with chemical and statistical analyses fueled by interest from the scientific community, regulating agencies, and general public led to an unprecedented near real-time understanding of the fate of MC-252 oil degradation in the Gulf of Mexico (Chakraborty et al., 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.


Patients suffering from malignancies have increased complications due to corresponding cardiovascular diseases and risk factor for the development of venous thromboembolism. Epidemiological studies have shown that increased homocysteine plasma concentration (hyperhomocysteinemia) is related to a higher risk of coronary heart disease, stroke, peripheral vascular disease and malignancies. Homocysteine (tHcy) is an intermediate sulfur-containing amino acid produced from methionine during processing of dietary proteins. The plasma homocysteine levels are strongly influenced by diet, as well as by genetic factors. Folic acid, vitamins B6 and B12 are dietary components which influence the plasma homocysteine levels the most. Several studies have found that high blood levels of B vitamins are related to the integrity and function of DNA, and, are at least related to lower concentration of homocysteine. Folate depletion has been found to change DNA methylation and DNA synthesis in both animal and human studies. Because of this critical role of folate, most studies including homocysteine have focused on these two actions.Hyperhomocysteinemia proves to be the most common condition highly associated with both venous and arterial thrombosis in many cancer patients, while the associated pathophysiology has not been precisely established yet. Therefore, of current interest is the possible role of folate metabolism developing into a cancer initiating hyperhomocysteinemia. This review will discuss this possibility. It has been long postulated that the plasma homocysteine concentration is inversely related to the occurrence of cardiovascular and cerebrovascular diseases.Proliferating cells secrete more homocysteine compared to non-proliferating cells. Homecysteine is a sulphur-containing intermediate in the normal metabolism of the essential amino acid methionine present in almost all body cells and mostly 5 to 10% of daily synthesized homocysteine (1.2 mmol/day)12et al., showed that 32% of healthy elderly people aged 65 to 75, and 58% of those over 85 years of age suffer from hyperhomocysteinemia, indicating that hyperhomocysteinemia significantly increases with age17In a healthy population the frequency of moderate hyperhomocysteinemia (12–30 μmol/L) is 5 to 7%, with higher values for men being attributed to gender differences like estrogen presence in women. This is confirmed by the fact that after menopause the blood levels of homocysteine of woman approximate those in men.Via the trans-sulfuration pathway homocysteine is converted into cystathionine to form cysteine by cystathionine-ß-synthase, with vitamin B6 as a co-factor. Another pathway of homocysteine metabolism is the re-methylation pathway, which is connected with the folate metabolic pathway. It involves the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine to form methionine, and eventually S-adenosylmethionine. The methyl transfer from 5-methyl-tetrahydrofolate to homocysteine is catalyzed by methionine-synthase, and requires vitamin B12 as a cofactor . Importa19Moreover, hereditary causes of increased homocysteine blood concentrations exist (hyperhomocysteinemia). Most studies refer to changes in the genes for those enzymes that lead to severe hyperhomocysteinemia, such as the CBS gene (cistation-β syntases) or in the GCT gene (γ cistationase), both coding the trans-sulfuration pathway. Further, mutations do occur in genes coding for enzymes involved in the remethylation pathway and the related folate metabolic pathway. For a homozygous person with a mutation MTHFR 677C => T the enzyme activity is reduced to 35% of the normal.19Hyperhomocysteinemia is frequently associated with folate deficiency and it has been long postulated that the plasma homocysteine concentration is inversely related to the occurrence of cardiovascular disease and venous thrombosis.24Hyperhomocysteinemia is commonly occurring in a wide range of unrelated diseases. For example, in patients with renal failure a strong, positive correlation was observed between homocysteine (tHcy) levels, serum creatinine, and the renal glomerular filtration rate. Rheumatoid arthritis impaired gastric and other disturbances results often in elevated blood tHcy.26In vitro it was shown that some cancer cell lines are incapable of remethylating tHcy and it was recently shown that ovarian cancer cells from patients with elevated tHcy have impaired capacity to remethylate tHcy.31Increased tHcy levels are often found in patients with neoplastic diseases.Patient with malignancies often have an increased risk of venous thromboembolic (VTE) disease38et al.Al-Awadi Many other studies showed that the raised tHcy is related to the cancer itself and to the extent of the disease.2in vitro studies it has been shown that tumour cells and other proliferating cells release homocysteine.,in vitro notion might be extrapolated to the in vivo situation and could explain why hyperhomocysteinemia is observed in patients with various kinds of cancers. Within cellular immune activation, T cells release large amounts of the cytokine IFN-γ, which stimulates human monocyte-derived macrophages and dendritic cells to produce neopterin.Both plasma concentration of homocysteine and neopterin, a catabolic product of guanosine triphosphate-GTP and as such an immune system activation marker, are closely associated and elevated in patients with various types of disease.When different tumours are compared, the frequency of increased neopterin concentration is much lower in patients with breast cancer that it is in patients with other types of cancers.Folate, vitamin B12, and vitamin B6, have a number of biologic roles that make them potentially important in cancer. Within DNA synthesis they function as coenzymes in the synthesis of purines and thymidylate. Diminished levels of these vitamins may result in misincorporation of uracil into DNA, leading to chromosome breaks and disruption of DNA repair.Inadequate body levels of biologically active folate, vitamin B6, and vitamin B12 are primary determinants of high blood homocysteine levels.It has been hypothesized in many epidemiologic studies65A recent animal study demonstrated that a B12-deficient diet, which was of insufficient severity to cause anemia or illness, disturbed normal homeostasis of one-carbon metabolism in the colonic mucosa and resulted in diminished genomic DNA methylation and increased uracil misincorporation in DNA, both of which are purported mechanisms for one-carbon metabolism-related colonic carcinogenesis.66In a large prospective study on health care professionals, high intake of folic acid was found to be significantly correlated with low incidence of colorectal adenomas (polyps).Many countries have implemented mandatory folic acid fortification of flour and grain products to reduce the risk of various diseases, especially neural-tube birth effects. Experimental evidence suggests that high doses of folic acid may enhance growth of cancer cells.72Because the safety of folate might depend on its chemical structure , there is the question of potential adverse effects of circulating unmetabolized folic acid.75Many data support a relationship between hyperhomocysteinemia, low B vitamins concentration and risk for various types of cancer. Defective metabolism of tHcy in carcinogenesis is well documented, but the pathophysiology of this association is not fully understood. Many authors suggest that factors contributing to folate status are not protective against certain type of cancer, so further studies are needed to explore folate studies in human.


AbstractOsteoarthritis is the most common joint disorder and a major cause of disability with a major socio-economic impact. In these circumstances is very important to understand its pathogenesis. Although previous research focused primarily on changes in the articular cartilage, more recent studies have highlighted the importance of the subchondral bone, synovium, menisci, ligaments, periarticular muscles and nerves. Now osteoarthritis is viewed as a multifactorial disease affecting the whole joint.Abbreviations: TNF-α – tumor necrosis factor alpha, IL-1 – interleukin -1, IL-6 – interleukin-6, COMP- cartilage oligomeric matrix protein, BSP - bone sialoprotein, MRI - magnetic resonance imaging, NTx - cross-linked N-telopeptide of type I collagen, CTx – C-telopeptide-cross-linked collagen type I, TGF-β – transforming growth factor beta, MMPs- matrix metaloproteinases, VEGF-vascular endothelial growth factor, bFGF - basic fibroblast growth factor. It is the leading cause of activity limitation and absenteeism among working-age adults and is associated with a significant decline in function among older individuals. In this conditions osteoarthritis is also a substantial economic burden, with annual medical care expenditures of billion dollars in the United States (4). Understanding of changes early in the development of osteoarthritis is important, since these changes could still be reversible, and therefore, preventive treatment could be initiated to slow or reverse further progression of the disease.Osteoarthritis is the most common chronic musculoskeletal disorder. Epidemiological studies estimate around 43 million affected patients in the United States alone and about 15% of the world population . The loss of articular cartilage has been thought to be the primary change, but a combination of cellular changes and biomechanical stresses causes several secondary changes, including subchondral bone remodeling, the formation of osteophytes, the development of bone marrow lesions, change in the synovium, joint capsule, ligaments and periarticular muscles, and meniscal tears and extrusion .Initially, osteoarthritis has been considered to be a disease of articular cartilage, but recent research has indicated that the condition involves the entire joint [Articular cartilage12]. The turnover rate of collagen is relatively slow, whereas proteoglycan turnover is rapid [13]. The normal turnover of this matrix components is mediated by the chondrocytes, which synthetize these components and the proteolytic enzymes responsible for their breakdown. Chondrocytes are, in turn, influenced by a number of factors, including polypeptide growth factors and cytokines, structural and physical stimuli and even the components of the matrix itself [14].Normal adult articular cartilage is made up of extracellular matrix  and chondrocytes [15]. It is not known what initiates the imbalance between the degradation and the repair of cartilage. Trauma causing a microfracture or inflammation causing a slight increase in enzymatic activity may allow the formation of ”wear” particles, which could be then engulfed by resident macrophages [16]. At some point in time, the production of these ”wear” particles overwhelms the ability of the system to eliminate them and they become mediators of inflammation, stimulating the chondrocyte to release degradative enzymes. Molecules from breakdown of collagen and proteoglycan, also taken up by synovial macrophages, cause release of proinflammatory cytokines, like TNFα, IL-1 and IL-6. These cytokines can bind to chondrocyte receptors leading to further release of metalloproteinases and inhibition of type II collagen production, thus increasing cartilage degradation [17]. This disruption of homeostasis results in increased water content and decreased proteoglycan content of the extracellular matrix, weakening of the collagen network due to decreased synthesis of type II collagen and increased breakdown of pre-existing collagen [18]. Furthermore, there is increased apoptosis of chondrocytes.Osteoarthritis result from failure of chondrocytes to maintain homeostasis between synthesis and degradation of these extracellular matrix components [19] and proliferation of chondrocytes in the deeper layers of the cartilage, are able to maintain the integrity of the articular cartilage, but in the end loss of chondrocytes and changes in extracellular matrix predominate and osteoarthritic changes develop.Osteoarthritic cartilage is characterized by an increase in anabolic and catabolic activity. At first, compensatory mechanisms such as increased synthesis of matrix molecules  .It is not yet clear whether changes within subchondral bone precede changes in the articular cartilage or whether they occur in the disease progression, secondary to adaptation processes after changes in the biomechanical properties of the overlying articular cartilage. However, the two processes are closely related, as suggested by the concomitant increase in the levels of cartilage oligomeric matrix protein (COMP) and bone sialoprotein (BSP) in people with early osteoarthritis [Subchondral bone consists of the subchondral bone plate and the underlying trabecular bone and bone marrow space. The subchondral bone plate consists of cortical bone and is separated from the articular cartilage by the zone of calcified cartilage.8]. Bone remodeling includes the cupling of mechanisms that resorb bone and form new bone on a previously resorbed surface, whereas bone modeling is a mechanism that drives changes in the architecture and volume of bone via direct apposition to existing bone surfaces [21]. During the osteoarthritic process all of these mechanisms may be altered at some point in time resulting in subchondral bone structure changes.Subchondral bone properties are modified through the cell mediated process of remodeling and modelling . Thus, there is a progressive increase in the subchondral bone plate thickness, a modification in the architecture of subchondral trabecular bone, formation of new bone at the joint margins - osteophytes [24]. In subsets of patients with osteoarthritis, the indices of bone resorbtion showing loss of trabecular tissue indicated by the increase in cross-linked N-telopeptide of type I collagen (NTx) and C-telopeptide (CTx) [25], suggest a progressive loss of trabecular bone, not specifically of subchondral bone. In later stages, severe remodeling processes take place in particular in areas of advanced cartilage destruction, apart from extensive bone sclerosis (osteoid deposition), significant aseptic bone necrosis is a common feature of late-stage [20]. In areas of total cartilage destruction (the eburnated bone plate), synovial fluid gets access to the bone marrow and presumably leads to the bone cysts frequently seen in late stage disease [20]. Growth factors from the synovial fluid are probably involved in inducing fibrocytic and even chondrometaplastic changes, which lead to the ”cartilage nodules” characteristic for late-stage disease [20]. Changes in the bone include sclerotic changes and the development of bone marrow lesions that can be visualized by magnetic resonance imaging (MRI), and which seem to precede temporally and spatially, bone cysts in the subchondral compartment . Thus osteoarthritic subchondral bone has an increased osteoid collagen matrix and an abnormal mineralization resulting in a hypomineralization of this tissue. Although the subchondral bone tissue is hypomineralized in osteoarthritis, the increase in trabecular number and volume compensates for this situation, thus providing an apparent stiffer structure [6]. With alteration in its properties, subchondral bone may be less able to absorb and dissipate energy, thereby increasing forces transmitted through the joint and predisposing the articular surface to deformation [28]. Subchondral bone attrition may be caused by altered mechanical loading resulting in subchondral remodeling and is associated with concomitant bone marrow lessions [29]. Bone attrition is evaluated at conventional radiography as loss of bone density [30] or, at MRI, as flattening/depression of the articular cortex [31]. MRI studies have demonstrated that these bone lesions themselves are associated with development and worsening of cartilage loss [32].In osteoarthritic subchondral bone, type I of collagen is elevated, but this collagen content is abnormal and this leads to abnormal mineralization. In normal bone, type I collagen is composed of a heterotrimer of α1 and α2 chains at an average ratio of 2.4:1. In osteoarthritic bone tissue this ratio varied between 4:1 and 17:1 , which have phagocytic capacity, in the synovial lining layer [20]. Patients with osteoarthritis experience thickening of the synovial lining cell layer, increased vascularity and inflammatory cell infiltration of the synovial membranes, with the most marked changes occurring in advanced osteoarthritis. Studies of the changes in the synovium that occur at various stages of osteoarthritis have found that the amount of fibrin deposited in the synovial membrane and the degree of leukocyte infiltration are correlated with disease severity [42].It remains unclear whether the morphological changes that occur in the osteoarthritic synovial membrane are primary or whether they are the result of joint inflammation, cartilage degradation and lesions of the subchondral bone [43]. Synoviocytes are able to secrete not only proteolytic enzymes, but also proinflammatory cytokines , which are thought to mediate the progression and pain associated with this disease [44]. Adipokines, such as resistin [45] are also expressed by the synovium during osteoarthritis. Thus, synovial tissue seems to be the main source of adipokines in the osteoarthritic joint [46]. Osteopontin, a cytokine whose increased expression levels have been correlated with disease severity, is expressed in high quantity by synovial tissue in osteoarthritis [47]. The synovium produces some of the chemokines and metalloproteinases that degrade cartilage, even though the cartilage itself produces most of these destructive molecules in a vicious autocrine and paracrine fashion [48]. In turn, cartilage breakdown products, resulting from mechanical or enzymatic destruction, can provoke the release of collagenase and other hydrolytic enzymes from synovial cells and lead to vascular hyperplasia in osteoarthritic synovial membranes [49].Synovial cells and osteoarthritic chondrocytes both produces large quantities of matrix metaloproteinases (MMPs), like MMP-1, MMP-3, MMP-9 and MMP-13 . Then, the blood vessel permeability and up-regulation of adhesion molecules that are seen as part of angiogenesis perpetuates the inflammatory response [47]. Angiogenesis in the synovium is closely associated with chronic synovitis and may occur at all stages of osteoarthritis [51].Synovial neovascularization may be largely driven by synovitis as inflammatory cells such as macrophages can themselves secrete pro-angiogenic factors, but also secrete factors that stimulate other cells, such as endothelial cells and fibroblasts, to produce vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and other factors that further promote angiogenesis [Menisci52]. Degeneration of menisci initiates within the substance of the tissue rather than surface. Tissue fibrillation and disruption is first seen at the inner rim, which spreads to the articular surfaces of the meniscus over time, and progresses to total disruption or loss of meniscus tissue mainly in the avascular zone [53]. Type I collagen content decrease gradually from the surface zone to the middle and the deep zone of osteoarthritic meniscus [54]. Different from type I collagen, the decrease of type II collagen content is severe in the surface zone, and also prominent in the middle and deep zones of osteoarthritic meniscus [54]. In turn, proteoglycan content increase in osteoarthritic menisci when compare to normal menisci [54]. All these intrameniscal changes correlated with peri-meniscal synovitis, calcification not limited to the outer, peripheral portion of the menisci  contribute to meniscal degeneration and reduced meniscal tensile strength. The meniscus is less able to withstand loading and force transmission during normal movements of the joint, further leading to degenerative tears [57]. Meniscal tears are often accompanied by varying degrees of meniscal extrusion [58]. The tear might be a preceding feature of incipient osteoarthritis, and meniscus damage and extrusion often have a key role in the structural progression of the disease [59].Meniscal degeneration is commonly seen in osteoarthritis, where menisci appear torn, fissured, fragmented, macerated or completely destroyed [In conclusion, osteoarthritis is a multifactorial disease of whole joint, with a complex pathomechanism involving interaction between the multiple joint tissue. Knowing of this complex process of producing osteoarthritis is essential for development of new methods of diagnostic and treatment.Disclosures: authores declare no disclosure.


CYC2 lineage of floral symmetry TCP genes, CYC2A and CYC2B, demarcate the adaxial  region of the flower in the characteristic zygomorphic flowers of most Malpighiaceae. Several clades within the family, however, have independently lost their specialized oil bee pollinators and reverted to radial flowers (actinomorphy) like their ancestors. Here, we investigate CYC2 expression associated with four independent reversals to actinomorphy. We demonstrate that these reversals are always associated with alteration of the highly conserved CYC2 expression pattern observed in most New World (NW) Malpighiaceae. In NW Lasiocarpus and Old World (OW) Microsteria, the expression of CYC2-like genes has expanded to include the ventral region of the corolla. Thus, the pattern of gene expression in these species has become radialized, which is comparable to what has been reported in the radial flowered legume clade Cadia. In striking contrast, in NW Psychopterys and OW Sphedamnocarpus, CYC2-like expression is entirely absent or at barely detectable levels. This is more similar to the pattern of CYC2 expression observed in radial flowered Arabidopsis. These results collectively indicate that, regardless of geographic distribution, reversals to similar floral phenotypes in this large tropical angiosperm clade have evolved via different genetic changes from an otherwise highly conserved developmental program.Malpighiaceae possess flowers with a unique bilateral symmetry (zygomorphy), which is a hypothesized adaptation associated with specialization on neotropical oil bee pollinators. Gene expression of two representatives of the  Convergence is an evolutionary process in which similar features reoccur independently across the Tree of Life  and Old World (OW) tropics and subtropics. The zygomorphic  floral morphology of the more than 1000 NW species of this clade is very distinctive and highly conserved, especially with regard to symmetry and pollinator reward Figure . The NW CYCLOIDEA2-like (CYC2) transcription factors of the ECE clade within the TCP gene family have been shown to be critical for establishing floral symmetry and to have been repeatedly recruited to regulate floral zygomorphy in diverse angiosperm lineages , the NW Psychopterys clade  W. R. Anderson and S. Corso), and the NW ptilochaetoid clades (represented by Lasiocarpus sp. and Ptilochaeta nudipes Griseb.). The phylogenetic positions of these CYC2 homologs were inferred using our previously published CYC2 homologs, which sampled broadly across Malpighiaceae, including taxa that are closely related to the additions here ], could not be amplified by PCR, but Southern analyses identified more than one copy of CYC2 -PCR was carried out at several stages of floral development  floral buds and show exceptionally low activity in the NW Psychopterys dipholiphylla, especially at late stages of floral development  ectopic CYC2 expression resulting in phenotypic dorsalization of the entire flower, or (ii) loss of function resulting in phenotypic ventralization. However, we cannot exclude the possibility that CYC2 downstream factors may also play a role for floral radialization. The presence of both patterns underscores the fact that each of these four clades independently transitioned to actinomorphy in response to the loss of their specialist NW oil bee pollinators, traversing distinct genetic trajectories in the process.We observed that the conserved n Figure . These cCYC-dependent program was immediately recognized in early studies of CYC mutants in Antirrhinum , may seem atypical in having broad CYC2 expression, a strikingly similar pattern has been observed in the asterid clade Dipsacales  or tightly restricted to the two dorsal petals   are ventralized, this is difficult to determine without information on other aspects of radial organ identity. In the Plantago case described above, data on the CYC2 loci alone might lead to a conclusion that the flowers are dorsalized, but the complete absence of downstream components of both the dorsal and ventral identity pathways suggests that they are actually lateralized  Figure . When acSphedamnocarpus. This OW genus is comprised of two major clades: an African clade represented by S. puriens, which bears yellow or cream-colored flowers with the altered NW zygomorphy, and a Malagasy clade represented by S. sp. (nov.), which bears white actinomorphic flowers  is associated with complete loss of CYC2A, it seems unlikely that this state represents the ancestral condition . Thus, we can propose a model for the stepwise reversion to actinomorphy in this lineage. First, CYC2B expression was lost in the OW ancestor, most likely in concert with the evolution of the altered form of zygomorphy. This ancestor then gave rise to the African clade with its shifted axis of graded differential CYC2A expression, as well as to the Malagasy clade in which loss of CYC2A facilitated the transition to complete actinomorphy. This is the most parsimonious explanation of the current data, but further sampling of this fascinating lineage will allow our hypothesis to be further tested and refined as needed. Thus, the Sphedamnocarpus clade represents a very nice example of the way in which phylogenetically focused studies of floral gene expression can illuminate otherwise uncertain ancestral character state reconstructions involving important morphological innovations.In addition to illuminating broader mechanisms of morphological evolution, our molecular studies have helped us better understand the small scale character state transitions that gave rise to divergent floral forms across Malpighiaceae, particularly in y Figure , but a mCYC2B function in association with loss of the stereotypical NW zygomorphy, nothing is absolutely certain, as evidenced by the retention of CYC2B in Lasiocarpus. There are alternative genetic solutions to the problem of shifting pollinator availability, even when the morphological outcomes appear convergent. In this way, the Malpighiaceae offer a fascinating context in which to study the intersection between biogeography, pollinator interactions, floral morphology, and genetic evolution. Further studies will continue to build on and expand these models, hopefully providing a clearer picture of the complex evolutionary processes at work during the diversification of the family.Taken together, our study illustrates both the constrained and contingent nature of evolution. While certain patterns are fairly predictable, such as loss of Microsteria sp. and Rhynchophora phillipsonii W. R. Anderson are from Toliara, Madagascar; Philgamia glabrifolia Arènes from Mountain Ibity, Fianarantsoa, Madagascar; Sphedamnocarpus sp.(nov.) from Isalo National Park, Fianarantsoa, Madagascar; Lasiocarpus sp. from the municipio de Huitzuco de los Figueroa, Guerrero, Mexico; and Psychopterys dipholiphylla  W. R. Anderson and S. Corso from the municipio de Taxco, Guerrero, Mexico , Lasiocarpus sp., and Psychopterys dipholiphylla were aligned with a previously available matrix including several ingroup accessions of Malpighiaceae, and the outgroup families, Elatinaceae, Centroplacaceae, and Oxalidaceae, by eye with reference to the translated amino acid sequences using MacClade 4.06 . We sampled from the posterior distribution to calculate clade posterior probabilities following a burn-in of 1000 trees. DNA sequences of the newly acquired CYC2-like genes have been deposited in GenBank, under accession numbers KF514885-KF514893.The newly acquired sequences of CYC2-like genes from Microsteria sp., Rhynchophora phillipsonii, Philgamia glabrifolia, and Sphedamnocarpus sp. (nov.) with restriction enzyme EcoRI, fractionated on 0.8% agarose gels, and blotted onto a positively charged nylon membrane  following the protocol in Zhang et al. , and PgCYC2B of Philgamia glabrifolia in equal molar concentration was used as a template to synthesize our 32P labeled probe to examine Philgamia glabrifolia, and Sphedamnocarpus sp. (nov.); and CYC2 sequences of TmCYC2A and TmCYC2B of Triaspis macropteron, a member of the madagasikarioids and MicroCYC2A of Microsteria sp. to examine Microsteria sp., and Rhynchophora phillipsonii. We previously showed that the number of bands in the EcoRI digest is a reliable indicator of CYC2 copy number   Figure .later® -ICE Kit  The micro-dissected samples were processed using the RNAqueous Micro kit . The details about this method described in Zhang et al. ; medium buds (~40–60% of full size buds); large buds (~70–90% of full size buds); and open flowers. All materials were preserved in cryogenic containers, and were processed in the lab using the RNAqueous kit . Floral organs from medium to late stage samples, ~50–80% of flower bud size just before anthesis, were dissected in the lab from a single bud. These buds were dissected using the RNAg et al. .CYC2. The sequence identity of RT-PCR fragments was further confirmed by sequencing.RT-PCR was performed using locus specific primers Table  to exami-tubulin was used as a control to normalize the qRT-PCR  using the Stratagene Mx3005P QPCR System. Class I βThe 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.


Classical genetic studies aim to understand how genes determine biological processes, physical characteristics, behaviour, and disease by identifying heritable variations in DNA sequence that associate with specific phenotypes. However, organisms ranging from plants to mammals also possess some characteristics that can be inherited in the absence of any causal genetic variant in trans, allowing the paramutated allele to cause a phenotype in the absence of any genetic change Kit tyrosine kinase receptor that obey the standard Mendelian rules of genetic inheritance and confer pigmentation phenotypes, but engineered mutations in which LacZ or GFP are inserted downstream of the Kit transcriptional control element can result in Kit-mutant pigmentation phenotypes that are inherited and transmitted by genetically wild-type progeny LacZKit and GFPKit alleles are associated with transmission of aberrant Kit-related RNA transcripts to the zygote. Direct microinjection of these aberrant Kit RNAs into zygotes is sufficient to induce heritable paramutation-like effects Cdk9 or Sox9, respectively, resulting in cardiac hypertrophy (miR-1) or embryo overgrowth (miR-124) phenotypes that are transmissable through both the male and female germlines for at least three generations Non-Mendelian trans-generational inheritance is linked to epigenetic processes that mediate transmissable changes in gene expression independently of any change in DNA sequence. One specific type of non-Mendelian trans-generational inheritance, paramutation, occurs when one mutant allele induces a heritable epigenetic change in a different allele of the same gene PLOS Genetics, Kiani and colleagues demonstrate that DNMT2, an evolutionary conserved member of the DNA methyltransferase family, is absolutely required for some types of paramutation in mice: remarkably, paramutation is completely abolished on a −/−Dnmt2 background such that the LacZKit allele is no longer paramutagenic, and miR-124 is unable to induce paramutation-like changes in embryo weight Although published examples of paramutation are hugely outweighed by examples of classic Mendelian genetic inheritance, understanding the mechanisms that cause paramutation is likely to inform on the way that organisms can establish new heritable epigenetic states during normal development or perhaps in times of nutritional and environmental stress. In plants, the mechanisms that induce epigenetic changes at paramutated alleles are largely unknown, but RNA intermediates and/or physical interaction between the paramutagenic and paramutated alleles have been proposed to initiate paramutation, and enzymes associated with DNA methylation, histone modification, and small RNAs all contribute to this process −/−Dnmt2 mutants exhibit reduced viability under stress conditions and DNMT2-mediated methylation appears to protect tRNAs from stress-induced cleavage in this species bone fide DNA methyltransferases, little evidence has existed that DNMT2 might be able to generate heritable epigenetic phenotypes. The new finding by Kiani and colleagues firmly places DNMT2 at the heart of the mammalian paramutation pathway, and supports previous data suggesting that RNA molecules play a key role in the paramutation mechanism.Although DNMT2  is a member of the DNA methyltransferase family, and can bind and methylate DNA, it is now more closely associated with methylation of RNA, particularly transfer RNAs (tRNAs) Dnmt2-dependent manner, which raises the possibility that paramutation transmission is dependent on processed tRNAs. A second model would be that other RNA species, possibly small RNAs, depend on Dnmt2 activity for their stability, propagation, or subsequent transmission of the paramutated phenotype (Dnmt2 in paramutation may be related to the observation in flies that DNMT2-dependent methylation protects RNA molecules against endonucleolytic cleavage. However, injection of in vitro–methylated “paramutagenic” miRNAs into −/−Dnmt2 mouse embryos suggests that the role of DNMT2 is not restricted to methylating the injected paramutagenic miRNA, and that the paramutation mechanism involves additional events that we currently know little about. Whatever the case, the dependence of paramutation on Dnmt2 function provides a solid experimental pathway to test cherished hypotheses. This will probably involve massive parallel sequencing of target RNA populations and analysis of RNA modifications in affected and transmitting tissues A number of potential models present themselves on the basis of these unique observations. Kiani and colleagues henotype . The cri


Evidence for a relationship between T4 and T3 and glucose metabolism appeared over 100 years ago when the influence of thyroid hormone excess in the deterioration of glucose metabolism was first noticed. Since then, it has been known that hyperthyroidism is associated with insulin resistance. More recently, hypothyroidism has also been linked to decreased insulin sensitivity. The explanation to this apparent paradox may lie in the differential effects of thyroid hormones at the liver and peripheral tissues level. The purpose of this paper is to explore the effects of thyroid hormones in glucose metabolism and analyze the mechanisms whereby alterations of thyroid hormones lead to insulin resistance.  The blood sugar and the production and consumption of glucose are kept within normal bounds, therefore there is an equilibrium between the glands of internal secretions which reduce the blood sugar (pancreas) and those which raise it ”. Thyroid hormones exert both insulin agonistic and antagonistic actions in different organs. However, this occurs in a fine balance necessary for normal glucose metabolism. Deficit or excess of thyroid hormones can break this equilibrium leading to alterations of carbohydrate metabolism. Overt hyperthyroidism has been related to glucose intolerance and even ketoacidosis. With regards to hypothyroidism, cases of hypoglycemia have been reported in the literature despite the fact that peripheral insulin resistance may be present.The effects of T4 and T3 have a large impact on glucose homeostasis. This concept was acknowledged by Nobel Prize winner Dr. Bernardo Alberto Houssay in his lecture in 1947 “ In the century that has elapsed, since the first observations of uncontrolled glucose metabolism in thyrotoxic diabetic patients , new pat In this paper, we summarize the effects of thyroid hormones in glucose metabolism and its alterations when thyroid dysfunction is present.β2-adrenergic receptor mRNA and repression of inhibitory G protein (Gi) RNA of the adenylate cyclase cascade by T3 were also reported. All these results are in favour of a permissive influence of T3 in the glycogenolytic and gluconeogenic effects of epinephrine and glucagon. Other hepatic gluconeogenic enzymes that have been found to be positively regulated by thyroid hormones include phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that catalyzes the rate-controlling step of gluconeogenesis [Thyroid receptor-mediated effects on gene transcription and translation are key in the regulation of glucose metabolism. According to the results of studies with complementary DNA (cDNA) microarray analysis in mouse liver, this organ is a major target of thyroid hormones. Several genes involved in gluconeogenesis, glycogen metabolism, and insulin signaling that are regulated by thyroid hormones have been identified. In the study by Feng et al. , RNA froogenesis  and pyruogenesis . The cat Another mechanism, whereby thyroid hormones are known to increase hepatic glucose output, is through increased hepatic expression of the glucose transporter GLUT2  as previ It has been previously reported that, despite an expected resistance towards the insulin inhibitory effect on gluconeogenesis, the transcription of several enzymes involved in lipid synthesis or lipid metabolism is increased in hyperinsulinemic, insulin-resistant mice . Furtherα, PPARγ, and alternative oxidase (AOX) expression; increase in PPARδ expression; and stimulation of mitochondrial uncoupling thus preventing and reversing hepatic steatosis in this animal model. Surprisingly, in this study, these lipid-lowering actions not mediated by TR were also observed with T3. As a result of the long time quest for thyroid analogs that possess the favourable actions on metabolism without the unwanted thyroid cardiac effects, an indirect way of learning about T3 action in the different tissues has emerged . The dif To summarize, all these findings have helped to understand that thyroid hormones have insulin antagonistic effects at the liver that lead to an increased glucose hepatic output, via an enhanced rate of gluconeogenesis and glycogenolysis. With regards to lipid metabolism, both lipogenesis and lipolysis are stimulated by T3. However, in the context of insulin resistance, the conversion of glucose into fatty acids together with nonsuppressed gluconeogenesis is simply perpetuating the hyperinsulinemic state. Furthermore, nutritional influences, such as those of high-fat diets, should also be taken into consideration as modifiers of the effects of thyroid hormones on insulin sensitivity.Opposite to what occurs at the liver level, at peripheral tissues, thyroid hormones have been shown to exert some of their actions synergically with insulin. The upregulation of the expression of genes such as GLUT-4  or phospIn skeletal muscle, the main site of insulin-mediated glucose disposal, glucose transporter GLUT4, is induced by T3, revealing that it can increase basal and insulin-stimulated glucose transport in this tissue . Another Liver actions of the naturally occurring thyromimetic analog T2 have been discussed above. However, T2 actions have been also explored in skeletal muscle . In a moμg/d of T3 for 14 days has shown that not only genes with agonistic insulin effects but several others with antagonistic insulin effects are upregulated by treatment with T3 [β2-AR, favouring catecolamine-induced lipolysis and it also downregulates Sterol regulatory element binding protein (SREBP1c), involved in lipogenesis, which may constitute a link between hyperthyroidism and insulin resistance [ The result of cDNA microarray analysis on skeletal muscle of a group of healthy men receiving 75  with T3 , underlysistance . α (Hypoxia-inducible factor 1), a key mediator of glycolysis, increased in response to T3. As the glucose transporter GLUT1, several enzymes of glycolysis, and the lactate exporter SLC16A3 were all also found induced by T3 and are target genes of the transcription factor HIF-1α, the authors postulated that the effect of thyroid hormones on the induction of these genes most probably was indirect and HIF-1α mediated. Furthermore, a new mechanism of thyroid action was unraveled by this group of researchers [in lieu of initiating gene transcription in the nucleus, activates the phosphatidylinositol 3-kinase (PI3K) signaling pathway in the cytosol in order to activate HIF-1α gene expression. Skin fibroblasts have been also used to study thyroid hormone-responsive genes involved in metabolism in human cells. Although they are not as metabolically active as hepatic cells, they are easily obtained and also, thyroid hormone-responsive. In cultured human fibroblasts, Moeller et al.  observedearchers . It was  At the cellular level, thyroid hormones can also increase mitochondrial biogenesis, fatty acid oxidation, and TCA cycle activity . These fIt has been shown that the hypothalamus can modulate endogenous glucose production by using functionally reciprocal sympathetic and parasympathetic autonomic outputs to the liver . Moreoveper se from the insulin resistance present in thyrotoxicosis, the stimulatory action of thyroid hormones in excess on lipolysis and free fatty acids availability can also contribute to increased ketogenesis [Thyrotoxic subjects frequently show impaired glucose tolerance. This is a result of increased glucose turnover with increased glucose absorption through the gastrointestinal tract, postabsorptive hyperglycemia, elevated hepatic glucose output, with elevated fasting and/or postprandial insulin and proinsulin levels, elevated free fatty acid concentrations and elevated peripheral glucose transport and utilization. The literature about this topic is vast and has been previously comprehensively reviewed by Dimitriadis and Raptis . Thyrotoogenesis .Thyrotoxicosis has been reported to increase endogenous glucose production in the liver in the basal state and to decrease hepatic insulin sensitivity in humans . The difThe interpretation of the effects of hyperthyroidism on glucose utilization by peripheral tissues is by far the most complex issue on this topic. On one hand, the rates of glucose uptake in peripheral tissues have been found increased by thyroid hormones, suggesting that glucose utilization is highly increased, specially in skeletal muscle , 41–44.  Although glucose intolerance in hyperthyroidism can be easily explained by hepatic insulin resistance without involvement of peripheral tissues, impaired insulin-stimulated peripheral glucose uptake has also been proven in some studies. By means of the arteriovenous difference technique in the forearm muscles of hyperthyroid subjects after the consumption of a mixed meal, it has been clearly demonstrated that muscle blood flow is increased, masking a defect in insulin-stimulated glucose uptake . Moreoveα) from adipose tissue in hyperthyroidism [Alternative explanations for peripheral insulin resistance in hyperthyroidism include an increased secretion of bioactive mediators (adipokines) such as interleukin 6 (IL6) and tumour necrosis factor a , and muscle carnitine palmitoyl transferase 1β (CPT-1β) mRNA levels, explored by real-time quantitative PCR. Plasma leptin levels were lower and adipose tissue mRNA expression of resistin higher, in the hypothyroid state. Looking for a potential role of leptin in the metabolic consequences of hypothyroidism, leptin was infused, and it was found that glucose disposal was recovered. Increased expression of muscle and adipose tissue carnitine palmitoyl transferases, decreased plasma NEFA levels, and reduced muscle triglyceride content after leptin infusion were interpreted as the mediators of the recovery of the insulin resistant state. Therefore, one possible explanation to decreased insulin responsiveness in hypothyroidism, according to the authors of this study, includes a dysregulation of leptin action at the hypothalamus. Insulin resistance was confirmed in another study of rats with mild hypothyroidism . In this Adipocyte-myocyte crosstalk by adipokines has been reported to play a significant role in skeletal muscle insulin resistance and may partially explain insulin resistance present in hypothyroidism . HoweverCompared to the number of reports about insulin resistance in hyperthyroid patients, there are relatively fewer studies in humans dealing with the effects of hypothyroidism on glucose metabolism. Rochon et al.  measuredDimitriadis et al.  exploredSome negative results, however, in this field have been reported. A previous study in overt hypothyroid patients based on the homeostasis model assessment (HOMA-IR)  showed nWith regards to subclinical hypothyroidism, insulin resistance has been demonstrated in some , 74, 79 Thyroid hormones have a large impact on glucose metabolism. A direct regulation on thyroid responsive genes at the target organ has been described and more recently an indirect effect involving hypothalamic pathways that regulate glucose metabolism via control of the sympathetic nervous system has been reported. Furthermore, thyroid hormone effects can be insulin agonistic, such as demonstrated in muscle or antagonistic such as observed in the liver. In hyperthyroidism, dysregulation of this balance may end in glucose intolerance mainly due to hepatic insulin resistance. In hypothyroidism the results are less evident. However, the available data suggest that insulin resistance is present mainly at the peripheral tissues. Possible explanations hypothesized to explain this phenomenon span from the dysregulation of mitochondrial oxidative metabolism to the reduction of blood flow in muscle and adipose tissue under hypothyroid conditions.