Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes. Located in the cytoplasm, the main function of the mitochondria is to provide energy for the cell in the form of ATP. Cells use this energy to perform the work necessary for cell survival and function. Moreover, mitochondria are also involved in other cell processes such as cell growth, division, and apoptosis. Pancreatic islets consist of a cluster of 1000–2000 cells and ranging in size between 50–400 μm in diameter. Composing 1-2% of the pancreas mass, each islet consists of at least five different cell types: α-cells (15–20% of islet mass) producing glucagon; β-cells (65–80% of islet mass) producing insulin and amylin; δ-cells (3–10% of islet mass) producing somatostatin; PP cells (3–5% of islet mass) producing pancreatic polypeptide; ε-cells (<1% of islet mass) producing grhelin. Specifically for β-cells, mitochondria play a key role in glucose-stimulated insulin secretion, not only by providing energy in the form of ATP to support insulin secretion, but also by synthesizing anapleurtoic metabolites that can act, both intra- and extramitochondrially, as factors that couple glucose sensing to insulin granule exocytosis. ATP on its own, and possibly modulated by these anapleurotic coupling factors, triggers closure of the ATP-sensitive potassium channel, resulting in membrane depolarization that increases intracellular calcium to cause insulin secretion ) during postisolation culture and nto mice , 83. Sevnto mice , 111.β-cell proliferation, islet secretion, cytoprotection, islet engraftment, and function of the transplanted islets by revascularization and β-cell survival  index. When CsA was withdrawn, in vivo islet graft function was compromised when the patient was maintained with sirolimus [In an irolimus . One cliirolimus .in vivo versus in vitro, short-term versus long-term, cell line versus primary cells, animal versus human, and dosage difference.While a body of evidence seems to support the notion that most immunosuppressants currently used for islet transplant have at least some level of toxicity on islets and affect islet graft function, some conflicting results have been reported. Many contribute these incongruent observations to the selection of experimental models: β-cell destruction [β-cell exhaustion, and glucotoxicity/lipotoxicity [β-cell function. Glucotoxicity involves several transcription factors and is in part mediated by chronic mitochondrial reactive species generation and accumulation. Lipotoxicity is probably mediated by the accumulation of a cytosolic signal derived from the fatty acid esterification pathway. Very few studies have been published that investigate islet grafts in the hepatic portal system due to the lack of good in vitro and in vivo models that can closely mimic this islet graft microenvironment. New alternative sites are currently being investigated including intramuscular, bone marrow, and the peritoneal cavity that could potentially decrease the stress to islets. While this topic is important for improving islet transplantation outcomes it has been previously reviewed in several other papers in greater depth [In addition to immunosuppressant toxicity, the microenvironment of the islet engraftment site within the hepatic portal vein system has also been suggested as one of the main contributing factors associated with the loss of islet mass and function posttransplant. Within moments of islet implantation, a cascade of nonspecific inflammatory events known as instant blood mediated inflammatory reaction (IBMIR) are initiated which include activation of blood-mediated coagulation pathways  and reletruction ; as welltoxicity , 161. Gler depth , 163.Recently, improvements in islet isolation techniques and the selection of optimal immunosuppression regiments have made human islet transplantation a viable clinical avenue in the treatment for T1DM patients. Inconsistent success rates are observed between short-term and long-term graft survival among centers and this could be related to the use of islet products of less than optimal quality and quantity. Mitochondrial functions are potentially compromised in each step of islet isolation and transplant process and subsequently can negatively influence islet engraftment and survival. The evidence presented in this review supports the following hypotheses: (i) mitochondrial dysfunction is a mediator for the onset and progression of islet I/R injury; (ii) potential therapeutics targeting mitochondrial processes, including energy metabolism, cytokine-induced inflammatory reaction, and free radical generation, will be promising in preserving islet function and viability. However, to confirm the role of mitochondrial cytoprotection in the isolated islets for transplant, it will be necessary to demonstrate these results in appropriate animal models and in a large cohort study of islet isolation by multiple centers to correlate clinical transplant outcomes. In addition, it is important to correlate a more sensitive and reliable islet viability and potency assay based on the evaluation of mitochondrial integrity in conjunction with other parameters to prevent the transplantation of a low quality islet preparations. Future efforts are also needed to better understand the underlying mechanisms of immunosuppressant-mediated islet toxicity and to discover a new generation of immunosuppressants that are less damaging to islets. Lastly, it is important to improve the islet transplantation process by decreasing islet loss potentially by finding a more suitable transplantation site in order to achieve consistent transplantation outcomes.


The nirS gene fragments were PCR-amplified, cloned, and sequenced from sediment cores collected at five stations. Sequence analysis of 96–123 nirS clones from each station revealed extensive overall diversity in this estuary, as well as distinct spatial structure in the nirS sequence distributions. Both nirS-based richness and community composition varied among stations, with the most dramatic shifts occurring between low-salinity  and moderate-salinity  sites. For four samples collected in April, the nirS-based richness, nitrate concentrations, and N2-fluxes all decreased in parallel along the salinity gradient from the oligohaline northernmost station to the highest salinity  station near the mouth of the Bay. The vast majority of the 550 nirS sequences were distinct from cultivated denitrifiers, although many were closely related to environmental clones from other coastal and estuarine systems. Interestingly, 8 of the 172 OTUs identified accounted for 42% of the total nirS clones, implying the presence of a few dominant and many rare genotypes, which were distributed in a non-random manner along the salinity gradient of Chesapeake Bay. These data, comprising the largest dataset to investigate nirS clone sequence diversity from an estuarine environment, also provided information that was required for the development of nirS microarrays to investigate the interaction of microbial diversity, environmental gradients, and biogeochemical activity.Chesapeake Bay, the largest estuary in North America, can be characterized as having steep and opposing gradients in salinity and dissolved inorganic nitrogen along the main axis of the Bay. In this study, the diversity of  Due to the critical role of nitrite reductase in the dentrification pathway, the nirK and nirS genes have been most frequently targeted for molecular diversity studies in many environments, including soils  genes have revealed a pattern of ammonia oxidizer diversity correlated with salinity, as well as distinct communities in freshwater and high salinity estuarine environments  catalyzes the first committed step to a gaseous product Zumft, , distingcd1-type nitrite reductase (nirS) sequences in sediments of the Chesapeake Bay. This is the largest estuary in North America, and denitrification is a critical component of the N cycle, which is dominated by sediment N transformations. We have previously examined ammonia-oxidizing (AO) communties in these sediments  and lower Choptank River (CT2) stations, as well as mainstem Chesapeake Bay stations  temperatures  were amplified from pooled sediment DNA extracts using the PCR primers (nirS1F and nirS6R) and conditions described by Braker et al.  using the FastDNA SPIN kit for soil , as described in Francis et al. . nirS ger et al. . ProductnirS nucleotide alignment (of 550 sequences) was used to define operational taxonomic units (OTUs) on the basis of DNA sequence identity. Distance matrices based on this nucleotide alignment were generated using the PAUP software package. To compare the relative nirS richness within each clone library, rarefaction analysis was performed. For this analysis, OTUs were defined as nirS sequence groups in which sequences differed by ≤5% using the furthest neighbor method in the MOTHUR program . Nucleotide sequences were assembled, edited, and aligned using Sequencher™ v.4.2 (GeneCodes Corp.), and translated using MacClade  from the Chesapeake Bay were aligned with representative database sequences (as of July 2012) using ClustalX  was performed in JMP  to the mesohaline lower Choptank station (CT2)  and NO−3 concentration . Oxic conditions were present in the bottom waters of all stations at the time of sediment sampling. Nitrate concentration and salinity were negatively correlated . Cluster analysis of the sites based on normalized values of salinity, nitrate, and ammonium resulted in the formation of two distinct groups , ranging from a high of 172 μmol N m−2h−1 at CB1 to a low of 8 μmol N m−2h−1 at CB3 , despite identical (22 μM) bottom water nitrate concentrations. This difference could be due to greater coupling to nitrification at CT2, where the sediments do not experience seasonal anoxia. In contrast, the sediments at the much deeper CB2 station (18-m vs. 7-m depth at CT2) are exposed to seasonally anoxic conditions and have higher levels of pore water hydrogen sulfide , resulting in an overall database of 550 nirS sequences from the Chesapeake Bay estuary. This represents the most extensive clone library-based sequencing effort, to date, of nirS sequences from any system, let alone an estuary. Since nirK could not be reliably amplified  from all five of these sediment DNA extracts using several primer combinations  and North Bay (CB1) libraries, and the lack of significant curvature after >95 clones suggests that that the diversity of distinct nirS sequences is not yet saturated in these two libraries. By far the lowest richness was observed in the South Bay (CB3) library, while intermediate levels were observed at the two mesohaline stations, CT2 and CB2. Overall, the rarefaction curves illustrate a rather striking trend among these sites spanning the estuarine salinity gradient, in which nirS richness decreased as salinity increased along the estuary  but the lowest and essentially identical levels of richness were detected at the two mesohaline stations, CT2 and CB2, and intermediate levels at CT1 and CB3  had the greatest total number of OTUs that were found exclusively at one site Table . InteresnirS clone library data (Table nirS OTUs within our dataset (172 OTUs) represented only 37 to 48% of the number of OTUs predicted by ACE and Chao1, respectively. Overall, the predicted nirS richness values basically exhibited the same trend from high to low richness along the estuarine gradient that was revealed through rarefaction analysis. The classical ecological diversity indices (Shannon and Simpson's) also supported this trend.Although rarefaction analysis is useful for comparing the relative observed richness among clone libraries, it is not intended to predict the actual community richness  within the original samples  of the total sequences .The number of OTUs shared between sites represents one measure of site similarity Table . CT1 ands Figure . Of thess Figure . The othp = 0.006). The observed clustering of environmental variables and sampling sites suggests a relationship between environmental factors and OTU distribution. However, the rather limited number of samples (5) ultimately limits our statistical power to definitively determine the impact of particular factors on the distribution of nirS-type denitrifier populations. While it is not always feasible (or desirable) to generate massive PCR clone libraries for the extensive number of samples necessary to perform more robust statistical approaches , the extensive nirS dataset described in this study allowed the development of a microarray  Hythe site, located at the head of the River Colne estuary  to sequences from the other site. In the absence of clone library analysis from CT1 in April 2001, comparisons between CB1 and CT1 unavoidably combine temporal and spatial variation. Even less overlap was observed in the While only three CT2 sequences fell into the two “low-salinity” clusters (II and IV), more than half (59 of 111 sequences) of the CT2 sequences fell into three distinct but closely related subclusters within cluster X , Bouvier and Del Giorgio  sedimentary environments. Furthermore, using a nirS microarray, developed using sequences from this study, Bulow et al. (nirS genotypes (OTU1) as well as the major CT2/CB2 sequence type (OTU2) within Cluster X were the most abundant (DNA) and most actively expressed (mRNA) within both CB2 and CB3 sediments. It is worth highlighting that 5 of the 8 most abundant nucleotide-based OTUs identified in the present study . This cluster also included 10 clones from a number of different environments, including sediments from the River Colne estuary, Puget Sound, and Washington continental margin, as well as water column depths within the Baltic Sea and the coastal Arabian Sea oxygen minimum zone. The only cultivated member of this cluster is nirS sequences in this study, comprising 6% of the CT1 clones, and 1–2% of three other libraries, but were not found in the CB1 library. Although Cluster I sequences are quite distinct from most known NirS sequences, there appears to be conservation of key amino acid residues known to be critical for function. For example, Histidine 352 (P. aeruginosa numbering), which serves as a heme-d1 ligand in the active site of cytochrome-cd1 nitrite reductase enzymes, was conserved among Cluster I and all other sequences in this study.The most divergent nitrite reductase sequences obtained in this study, sharing only 35–40% amino acid identity with the nearest cultivated denitrifier sequence, fell into Cluster I along with related sequences from several other marine and sedimentary environments. These distinct sequences represented 2% of the total 550 nirS diversity within Chesapeake Bay estuarine sediments, with the vast majority of 550 sequences falling into numerous novel phylogenetic clusters, lineages, and OTUs, many of which may represent estuarine-specific sequence types. Both the benthic N2 fluxes and nirS gene sequences were non-randomly distributed in relation to the physical/chemical parameters observed across the five estuarine sites. While salinity was most obviously related to the benthic N2 fluxes and observed diversity patterns, covariation of key parameters and the limited number of sampling sites makes it difficult to definitively determine the importance of individual environmental factors in this study. A clear shift in nirS phylogeny and richness occurred between the freshwater and mesohaline stations, where the steepest environmental gradients were also observed. In contrast, the transition from the mesohaline mid-Bay station to the polyhaline South Bay station was less pronounced, with considerable overlap observed in nirS sequence types and fairly comparable richness. Sequences were not evenly distributed among the stations, however, and some dominant nirS genotypes (within clone libraries) were identified, especially at CB3, the most “marine” site. The eight most abundant OTUs accounted for 42% of the total sequences, consistent with the idea that nirS-type denitrifiers exhibit a typical “species” abundance curve, with a few very common types and many rare ones. The dominant nirS genotypes identified here are not obviously affiliated with known denitrifying strains, which implies that we know very little about a group of organisms that are numerically-dominant (and active in gene expression) in this system and ubiquitous in estuarine systems in general. Recent advances in high-throughput sequencing technology will undoubtedly allow future studies to more thoroughly survey the diversity of nirS sequences, and microarray technologies will allow a larger number of samples to be investigated so that interactions with complex environmental factors can be better understood. However, further cultivation and/or metagenomic investigations will ultimately be required to determine the phylogenetic and physiological nature of these estuarine denitrifier groups.This study has revealed extensive and unprecedented 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.


The prevalence of allergic disease has increased dramatically in Western countries over the past few decades. The hygiene hypothesis, whereby reduced exposure to microbial stimuli in early life programs the immune system toward a Th2-type allergic response, is suggested to be a major mechanism to explain this phenomenon in developed populations. Such microbial exposures are recognized to be critical regulators of intestinal microbiota development. Furthermore, intestinal microbiota has an important role in signaling to the developing mucosal immune system. Intestinal dysbiosis has been shown to precede the onset of clinical allergy, possibly through altered immune regulation. Existing treatments for allergic diseases such as eczema, asthma, and food allergy are limited and so the focus has been to identify alternative treatment or preventive strategies. Over the past 10 years, a number of clinical studies have investigated the potential of probiotic bacteria to ameliorate the pathological features of allergic disease. This novel approach has stemmed from numerous data reporting the pleiotropic effects of probiotics that include immunomodulation, restoration of intestinal dysbiosis as well as maintaining epithelial barrier integrity. In this mini-review, the emerging role of probiotics in the prevention and/or treatment of allergic disease are discussed with a focus on the evidence from animal and human studies. Allergic diseases have become a major public health problem over the past few decades. The prevalence of eczema (atopic dermatitis), food allergy, and asthma have all increased dramatically during this time, particularly in Western society. It is believed that between 20 and 30% of individuals living in Western countries suffer at least one form of allergic disease -2 cell immune response to environmental or food antigens  while promoting tolerogenic responses to innocuous antigens, including foods. This is primarily achieved via mucosal antigen sampling by pattern recognition receptors (PRRs) expressed on intestinal epithelial cells and innate immune cells  mice have provided the best evidence for the role of microbiota on the GALT, as these mice have extensive defects in GALT development. A number of studies have shown that GF mice have reduced intraepithelial lymphocytes, Peyer’s patches with impaired germinal center development as well as fewer IgA secreting plasma cells and CD4+ T cells in the lamina propria  define probiotics as “live micro-organisms, which when administered in adequate amounts, confer a health benefit on the host” WHO, . The ideLactobacillus and Bifidobacterium. However, probiotic effects are strain and species-specific, and their biological activity can vary depending on the selected probiotic  or L. casei can modulate the composition of intestinal microbiota of allergic infants by reducing pathogenic bacteria such as clostridia while enhancing or maintaining beneficial bifidobacteria levels in the stool  from allergic patients in vitro experiments as well as animal models. Many studies have examined the use of probiotics in animal models of allergic disease, often with convincing results. Prebiotics, defined as a selectively fermented ingredient that allows specific changes in the composition and/or activity of intestinal microbiota to confer a health benefit on the host  when the newborns were also treated for the first 12 weeks, with reduced clinical symptoms, total plasma IgE levels and enhanced IFN-γ in skin biopsies  was given to mice for 4 weeks during the weaning period . Oral  Curran, . HoweverB. breve M-16V had the most potential due to the ability to improve lung function as well as reduce eosinophil numbers, OVA-specific IgE, IL-4, IL-5, and IL-10 levels in the bronchoalveolar lavage fluid (BALF). Treatment of mice with L. casei plantarum Lcr35 prior to OVA sensitization was found to prevent the development of airway hyperresponsiveness (AHR), BALF eosinophils as well as total serum IgE levels compared to mice treated with Lcr35 after OVA sensitization but before OVA challenge  model of human asthma is frequently used for the examination of probiotic effectiveness. Hougee et al.  revealedLactococcus lactis pre-treatment was shown to reduce clinical symptoms, lower IL-4 and IL-10 levels in mitogen-stimulated mononuclear cell supernatants but lower IgG1/IgG2 and IgE/IgG2 ratios indicating a Th1 bias compared to untreated pigs  was observed to reduce SCORAD to a greater extent than placebo  containing B. breve M-16V and a mixture of galacto- and fructo-oligosaccharides (GOS/FOS) were unable to improve eczema severity compared to placebo although an improved SCORAD was observed for infants with IgE-associated eczema  or ex vivo immune responses  did not show any improvement in lung function or bronchial inflammation although IL-5 levels were reduced (van de Pol et al., L. casei (Giovannini et al., L. gasseri showed significant improvements in clinical symptoms as well as reduced allergic cytokine levels such IL-13 (Chen et al., In contrast to those studies on eczema outcomes, there are limited studies on the effect of probiotic treatment for asthma. In a small study, Prevention of allergic disease remains the greatest challenge for clinicians. While a paucity and evidence – mainly inconclusive – exists for the use of probiotics in the treatment of allergic disease, several clinical trials have been successful in the use of probiotics for the prevention of allergic disease. At present, a total of 14 randomized controlled trials evaluating various probiotics have been reported, mostly involving infants of families with a history of allergic disease (summarized in Table L. rhamnosus LC705, B. breve Bb99, and Propionibacterium freudenreichii+ the prebiotic GOS (Kukkonen et al., L. acidophilus La-5 and B. lactis Bb12 (Dotterud et al., L. reuteri treatment. Two studies using different probiotic mixtures reported a reduction in eczema at 1 year (Kim et al., L. rhamnosus HN001 but not B. animalis subspecies lactis HN013 was able to significantly reduce both eczema and IgE-associated eczema at 2 years. Both of these treatments however had no impact on sensitization status. This supports the view that not all probiotics are the same such that they may elicit unique biological activities. Therefore, selection of probiotic species and strains should be carefully considered in the design of future clinical trials. It is important to note that despite the beneficial effects of probiotics in some of the studies, there were also increased risks of asthma-like symptoms at 2 years in the study by Kopp et al. (Timing and duration of the probiotic intervention appears to be major factor in determining their beneficial effects. Nine of the 14 RCTs involved both a prenatal and postnatal intervention period, while four studies evaluated only postnatal and one study examined a prenatal only approach (Boyle et al., p et al.  and at 7p et al. , suggestL. acidophilus LAVRI-A1 demonstrating an increased risk of both IgE-associated eczema and atopic sensitization at 1 year of age (Taylor et al., L. paracasei F19 (West et al., No beneficial effects on eczema or sensitization were found in three of the four studies using a postnatal treatment approach (Rautava et al., Breastfeeding is one parameter that may be critical in mediating probiotic effects. Infants who were breastfed by mothers treated with probiotics during pregnancy and breastfeeding benefited the most, even when probiotics were not administered directly to them (Dotterud et al., In summary, the use of probiotic bacteria for the prevention and/or treatment of allergic disease have shown promising results to date. However, the validity of these findings need to be confirmed by further randomized controlled trials. In particular, for eczema, the timing of the probiotic intervention appears to be important, with a prenatal component critical for protective effects (Osborn and Sinn, in vitro, preclinical and pilot studies. In addition, there is insufficient evidence that probiotics may be of benefit for the prevention of other allergic conditions. An important limitation of meta-analyses of studies evaluating probiotics for the prevention of allergic diseases is that data have been pooled from studies conducted with a variety of different probiotic combinations. Further clinical studies using the most effective study designs incorporating prenatal/postnatal treatment and selection of appropriate probiotic bacteria are required to validate the findings of earlier studies. Understanding the mechanisms of protection in allergic disease will greatly assist in developing more targeted strategies for the prevention or treatment of allergic disease.Studies suggest a potential role for selected probiotics in the prevention of eczema, especially IgE-associated eczema. The efficacy of probiotics for the treatment of allergic disease however requires further examination. Careful selection of appropriate probiotic bacteria for future studies will be important, which may be aided by 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.


Instead, the study revealed an inverse correlation between inappK in left ventral striatum and alcohol craving scores. To resolve the influx and efflux phases of radiolabeled molecules, we independently estimated the unidirectional blood-brain FDOPA clearance rate (K) and the washout rate of [18F]fluorodopamine and its deaminated metabolites (kloss), and we also calculated the total distribution volume of decarboxylated metabolites and unmetabolized FDOPA as a steady-state index of the dopamine storage capacity (dV) in brain. The craving scores in the 12 alcoholics correlated positively with the rate of loss (lossk) in the left ventral striatum. We conclude that craving is most pronounced in the individuals with relatively rapid dopamine turnover in the left ventral striatum. The blood-brain clearance rate (K), corrected for subsequent loss of radiolabeled molecules from brain, was completely normal throughout the brain of the alcoholics, in whom the volume of distribution (dV) was found to be significantly lower in the left caudate nucleus. The magnitude of dV in the left caudate head was reduced by 43% relative to the 16 controls, consistent with a 58% increase of lossk. We interpret the findings as indicating that a trait for rapid dopamine turnover in the ventral striatum subserves craving and reward-dependence, leading to an acquired state of increased dopamine turnover in the dorsal striatum of detoxified alcoholic patients.A previous study of the DOPA decarboxylase substrate 6-[ The ratio between the enzymatic clearance of FDOPA (K) and the rate of loss (lossk) constitutes a steady-state volume of distribution for the decarboxylated and deaminated metabolites, which reflects the total distribution volume in brain , an index of dopamine storage capacity, as detailed in the earlier publications The measure of the net blood-brain transfer rate of FDOPA obtained in these studies of alcoholic patients (Kinapp) and alcohol craving scores 18F]fluoro-dopamine formed in ventral striatum and subsequent washout of the breakdown products 18F]fluorodopamine washout (lossk) in the left ventral striatum would correlate with the severity of craving in the group of alcoholics, as previously noted for the net blood-brain transfer (Kinapp), and second, that the analysis would reveal increased lossk throughout striatum of detoxified alcoholic patients, relative to an age-matched control group.We predicted that the previous finding of an inverse correlation between the net blood-brain transfer . The control group consisted of the 12 volunteers from the earlier study The mean (± SD) alcohol lifetime alcohol consumption of the patients was 1038±828 kg, and they smoked a mean (± SD) of 17±13 cigarettes per day, yielding a weak trend towards correlation between individual alcohol and cigarette consumption . The lifetime alcohol intake was measured with the Lifetime Drinking History questionnaire 18F]fluoro-L-DOPA (OMFD) were determined in selected samples by reverse-phase high performance liquid chromatography Subjects fasted overnight, and received carbidopa  one hour prior to the PET scan in order to minimize the decarboxylation of FDOPA in peripheral tissues In the present study, dynamic PET sequences were realigned and corrected frame-wise for head motion, and then summed and registered to the Montreal Neurological Institute (MNI) stereotaxic brain, as described previously 1-k2K neurokinetic model. The OMFD curve was regenerated mathematically for subtraction of OMFD contamination. This OMFD subtraction method permits the two hour tomography, which is sufficient for subsequent parameter estimation of the present study. Following this mathematical subtraction of brain OMFD, FDOPA kinetics in brain simplifies to an “inlet-outlet” model K, ml g−1 min−1), and the fractional rate constant for the process of elimination of [18F]fluorodopamine together with its deaminated metabolites from brain . The ratio (K/lossk) is analogous to an effective distribution volume (EDV ml g−1), as defined in dV, ml g−1) equals the sum of the FDOPA plasma volume , the distribution volume of unmetabolized FDOPA in brain , and the decarboxylated metabolite pool, consisting of [18F]fluorodopamine and its deaminated metabolites . In the common MNI stereotaxic space after anatomical standardization, mean dV maps were calculated for the two groups, and difference maps were calculated by subtraction. Voxel-wise statistical analysis was performed using the general linear model (GLM), in order to search for cluster volumes and partial out the effects of smoking rates. Group differences and voxel-wise regression of dV with ACQ scores for the 12 patients were calculated using the GLM implemented in Glim Image, a program developed at the MNI. Then, cluster VOIs were defined directly from the raw t-maps, where the t-value threshold was set arbitrary to form a cluster (t>2.5–2.8), but as high as possible to avoid false positive voxels. The cluster VOIs identified in the dV voxelwise contrast and regression analyses were applied to the OMFD-subtracted dynamic 4D recordings for TAC extraction, and the individual magnitudes of K, lossk and dV were calculated separately from the TACs. The kinetic estimates obtained from the cluster VOIs were tested using the GLM analysis for the contrast between alcoholic and control groups as well as the regression with ACQ scores, treating individual smoking rates as a nuisance variable.To search for brain regions with perturbed [18F]fluorodopamine storage capacity, as revealed by both the mean parametric dV map  and the mean magnitude of dV was reduced by 43% (P = 0.002)  ; there wdV as a function of the ACQ scores revealed a cluster (t >2.5) with inverse correlation in the left ventral striatum  revealed no difference from an age-matched control group, but a highly significant negative correlation between estimates of the net transfer rate (inappK) in the left ventral striatum and individual ACQ scores. In the present study, the regression analysis with the ACQ scores of the alcoholic patients, employing the concurrent and separate estimation of the unidirectional blood-brain clearance (K) and the metabolite washout rate (lossk), revealed a positive correlation with lossk and a negative correlation with dV, but no correlation with the magnitude of the clearance (K) in left ventral striatum, in which no significant group differences of the three kinetic estimates were detected. We conclude that the earlier finding of a negative correlation between inappK and craving reflects the underestimation of the unidirectional clearance of FDOPA (K) in relation to the magnitude of washout of [18F]fluorodopamine and its deaminated metabolites (lossk), which we determined separately from the 120 min PET recordings in the present analysis.Two previous PET studies of dopamine synthesis capacity with FDOPA in alcoholic patients yielded partially disparate results dV and high estimates of lossk in left ventral striatum, predicting a relatively small dopamine pool, characterized by rapid turnover. In functional anatomic terms, the convergence within the ventral striatum of glutamatergic inputs from limbic structures and the dopamine innervation arising in the ventral tegmental region of the mesencephalon is thought to subserve gating of affective signaling from limbic afferents. Given the role of dopamine in incentive salience 18F]fluorodopamine pool and its increased washout in ventral striatum of alcoholic patients may serve to link craving with disinhibition of dopamine turnover.Those patients who experienced the highest craving had relatively low estimates of 11C]raclopride studies of post-synaptic D2/3 receptors revealed reduced binding in striatum of acutely withdrawn alcoholic patients, as compared to healthy control subjects 18F]fallypride 18F]desmethoxyfallypride ([18F]DMFP) 123I]IBZM 123I]-epidepride 11C]dihydrotetrabenazine to the neuronal vesicular monoamine transporter (VMAT2) was reduced in caudate and putamen of seven males with severe chronic alcohol abuse 11C]-methylphenidate found no such reduction 11C]-raclopride binding to psychostimulant challenge in alcoholic patients Chronic alcohol abuse is likely to alter the integrity of dopaminergic pathways in human brain. Indeed, a number of fluorodopamine together with its deaminated metabolites. These results in alcoholic patients stand in clear distinction to our earlier findings of increased K and decreased dV estimates in patients with schizophrenia, a circumstance which we termed “poverty in the midst of plenty” dV in left caudate nucleus of the present alcoholic group exceeds that seen in patients with early Parkinson’s disease The present ion maps  and the ic group , in the dV in the caudate nucleus of abstinent alcoholics, since it may reflect focal degeneration, increased dopamine signaling, or conversely “futile” [18F]fluorodopamine synthesis in a cellular compartment containing monoamine oxidase. However, given the preserved estimates of K, the present findings are more consistent with altered functional state of dopamine innervations, rather than an actual degenerative process. The caudate nucleus is implicated in cognitive performance pertaining to selection of actions and evaluation of outcomes 18F]fluoro-meta-tyrosine As noted in the introduction, there is no simple physiological interpretation of decreased 11C]raclopride studies of healthy pigs, we noted greater nicotine-evoked binding reductions on the left side 11C]raclopride binding changes on the left side 11C]raclopride binding changes on the right ventral striatum of healthy males Significant findings of the present study were asymmetric, as noted in earlier PET studies. For example, in [18F]-fluorodopamine turnover in the alcoholic group. However, the putative transition from a vulnerability trait mediated by greater dopamine turnover in the ventral striatum to an acquired state of elevated dopamine turnover in the dorsal striatum might best be investigated in prospective molecular imaging studies of individuals at risk for alcoholism.There is a significant correlation between sensation-seeking propensity and differences of extracellular dopamine and dopamine receptors in the striatum inappK in the left caudate of two patients In general, comparisons between molecular imaging studies of alcoholism are difficult because of factors such as age, smoking, and other co-morbidities, cumulative alcohol intake, and duration of abstinence. This may explain the neurochemical heterogeneity of type I alcoholics noted in one of the previous PET studies with FDOPA, which reported reduced


Recent studies are reviewed, concerning the in vivo wall stiffness of arteries and arterioles in healthy humans, and how these properties adapt to iterative increments or sustained reductions in local intravascular pressure. A novel technique was used, by which arterial and arteriolar stiffness was determined as changes in arterial diameter and flow, respectively, during graded increments in distending pressure in the blood vessels of an arm or a leg. Pressure-induced increases in diameter and flow were smaller in the lower leg than in the arm, indicating greater stiffness in the arteries/arterioles of the leg. A 5-week period of intermittent intravascular pressure elevations in one arm reduced pressure distension and pressure-induced flow in the brachial artery by about 50 %. Conversely, prolonged reduction of arterial/arteriolar pressure in the lower body by 5 weeks of sustained horizontal bedrest, induced threefold increases of the pressure-distension and pressure-flow responses in a tibial artery. Thus, the wall stiffness of arteries and arterioles are plastic properties that readily adapt to changes in the prevailing local intravascular pressure. The discussion concerns mechanisms underlying changes in local arterial/arteriolar stiffness as well as whether stiffness is altered by changes in myogenic tone and/or wall structure. As regards implications, regulation of local arterial/arteriolar stiffness may facilitate control of arterial pressure in erect posture and conditions of exaggerated intravascular pressure gradients. That increased intravascular pressure leads to increased arteriolar wall stiffness also supports the notion that local pressure loading may constitute a prime mover in the development of vascular changes in hypertension. For decades, it has been recognized that the mechanical properties of peripheral blood vessels play key roles in circulatory control, both in physiological and pathological conditions  load in the head-to-foot direction, individuals with high stiffness in lower-leg arterioles maintain arterial pressure at heart level more efficiently, and hence tolerate substantially higher G loads, than do individuals with low stiffness in the arterioles of the lower legs   during gradually increasing gravitoinertial load in the head-to-foot direction (Eiken et al. That intermittent, moderate pressure elevations in the arm vessels, cumulatively amounting to a mere fraction of the total time period during which such pressure elevations are executed, induce marked stiffness increments in local precapillary vessels (Eiken and Kölegård


Papillomaviruses (PVs) are a group of small, non-encapsulated, species-specific DNA viruses that have been detected in a variety of mammalian and avian species including humans, canines and felines. PVs cause lesions in the skin and mucous membranes of the host and after persistent infection, a subset of PVs can cause tumors such as cervical malignancies and head and neck squamous cell carcinoma in humans. PVs from several species have been isolated and their genomes have been sequenced, thereby increasing our understanding of the mechanism of viral oncogenesis and allowing for the development of molecular assays for the detection of PV infection. In humans, molecular testing for PV DNA is used to identify patients with persistent infections at risk for developing cervical cancer. In felids, PVs have been isolated and sequenced from oral papillomatous lesions of several wild species including bobcats, Asian lions and snow leopards. Since a number of wild felids are endangered, PV associated disease is a concern and there is a need for molecular tools that can be used to further study papillomavirus in these species.E6 and E7 viral oncogenes and amplify two DNA fragments encompassing these genes. We detected viral DNA for E6 and E7 in genomic DNA isolated from saliva, but not in paired blood samples from snow leopards. We verified the identity of these PCR products by restriction digest and DNA sequencing. The sequences of the PCR products were 100% identical to the published UuPV1 genome sequence.We used the sequence of the snow leopard papillomavirus UuPV1 to develop a PCR strategy to amplify viral DNA from samples obtained from captive animals. We designed primer pairs that flank the E6 and E7 oncogenes specifically in the saliva of animals. This assay could be utilized for the molecular investigation of papillomavirus in snow leopards using saliva, thereby allowing the detection of the virus in the anatomical site where oral papillomatous lesions develop during later stages of infection and disease development.We developed a PCR assay to detect papillomavirus in snow leopards and amplified viral DNA encompassing the  E1 and E2), viral shedding (E4), or transformation  . P,5. PE1 aion E5, E and E7  to d to d9] t (HNSCC) , suggestn intact . Therefos Figure . The twoBecause papillomatous lesions occur within the snow leopard oral cavity ,22, we wE6 and E7 primer pairs in amplification reactions using DNA purified from the paired blood and saliva samples .A positive result in a PCR-based assay would indicate presence of the virus in the oral cavity of the animal, as opposed to a serological test which can indicate exposure, but not necessarily current infection. We obtained matched saliva and blood samples from three snow leopards that did not have oral papillomatous lesions: two adult females that were approximately 18 years old (SL 1 and SL 3), and a 7-week old female cub  and two smaller fragments  produced a doublet corresponding to the 288 and 279-bp fragments and a single 127-bp fragment  in a variety of clinical samples ,23. SpecE7 genes . Similarl smears . In somen intact , thus maThe detection of UuPV1 DNA in the saliva of two older snow leopards parallels results from HPV studies that have demonstrated that HPV DNA can be amplified from oral rinses and saliva samples from HNSCC patients and HIV positive individuals ,26-28. BThe utilization of a PCR-based method to detect viral DNA in saliva presents a number of advantages. First, saliva collection is a non-invasive sampling method, thereby circumventing the requirement for anesthesia prior to obtaining a blood sample in captive wild felids. This is particularly important for captive species that are not routinely subjected to chemical restraint, and may only be anesthetized for scheduled physical exams. In these cases, saliva represents a more easily and frequently accessible biological specimen than blood. An additional advantage of testing saliva is that behavioral modification techniques can be used to train snow leopards and other felids to calmly accept an oral swab for DNA collection without chemical restraint. In fact, we were able to collect saliva samples from snow leopards in their enclosure, with minimal disturbance to the animal. Subsequently, animals testing positive for viral DNA can then be scheduled for a more thorough oral examination under sedation. The use of saliva also allows for detection of the virus in the anatomical site where papillomatous lesions develop during later stages of disease progression. Additionally, saliva is extremely stable, and more easily preserved than serum samples, thereby facilitating collection and shipping of samples from different sites. In testing for HPV, it has been demonstrated that PCR-based methods have increased sensitivity as compared to serotesting , and canE6 and E7 DNA in the saliva of snow leopards may have utility in diagnosing infection in animals that have no obvious oral papillomatous lesions, confirming infection in cats with oral papillomas, and detecting infection in animals that have not yet seroconverted. Due to its non-invasiveness and ease of implementation, PCR-based testing of saliva can be incorporated into the routine and regular screening of captive snow leopards, which would greatly facilitate the collection of epidemiological data on UuPV1 infections. Additionally, since papillomatous lesions have been reported to progress to SCC in snow leopards  was used to design PCR primers that amplify DNA fragments encompassing the entire E6 and E7 genes as shown in Figure NM_001009312] was used to design PCR primers that amplify a 503-bp fragment in the coding region of the gene. Primers were designed as described above and used for cross-species amplification of the corresponding region of the snow leopard transferrin receptor gene. Primer sequences are as follows: TfR-Forward 5'-TTTCTTGATATTTGAGTTCATTGTTT-3', TfR-Reverse 5'-AGTAACTGTCGCTGCTTTACTGT-3'The published UuPV1 genome sequence  using the CLC Sequence Viewer 6 software package .The Bp: Basepair; DNA: Deoxyribonucleic acid; HPV: Human papillomavirus; HNSCC: Head and neck squamous cell carcinoma; HIV: Human immunodeficiency virus; OSCC: Oral squamous cell carcinoma; ORF: Open reading frame; PV: Papillomavirus; PCR: Polymerase chain reaction; TfR: Transferrin receptor.E6 and E7 amplicons for sequencing, performed data analysis, prepared figures and drafted the manuscript. EAF participated in the design of the PCR amplification strategy, assisted with PCR amplification and drafted the manuscript. GH performed computational analysis of papillomavirus-host interactions to specifically select E6 and E7 as suitable targets for PCR-based detection of viral DNA. JOJ assisted with the clinical interpretation of oral papillomatous lesions in snow leopards. CE participated in study design and provided clinical interpretation/relevance of experimental results. MCB obtained IACUC approval, coordinated data collection, provided access to snow leopard saliva and blood samples, assisted with data interpretation and drafted the manuscript. KJLI conceived of the study, designed the study, coordinated data collection, designed the PCR amplification strategy, performed data analysis and drafted the manuscript. All authors have read and approved the manuscript.KM conceived of the study, participated in study design, assisted with the collection of saliva and blood samples, purified saliva and blood samples, performed PCR amplification, restriction digests and gel purification of


MicroRNAs (miRNAs) are a class of non-coding RNAs of ∼22 nucleotides in length, and constitute a novel class of gene regulators by imperfect base-pairing to the 3′UTR of protein encoding messenger RNAs. Growing evidence indicates that miRNAs are implicated in several pathological processes in myocardial disease. The past years, we have witnessed several profiling attempts using high-density oligonucleotide array-based approaches to identify the complete miRNA content (miRNOME) in the healthy and diseased mammalian heart. These efforts have demonstrated that the failing heart displays differential expression of several dozens of miRNAs. While the total number of experimentally validated human miRNAs is roughly two thousand, the number of expressed miRNAs in the human myocardium remains elusive. Our objective was to perform an unbiased assay to identify the miRNOME of the human heart, both under physiological and pathophysiological conditions. We used deep sequencing and bioinformatics to annotate and quantify microRNA expression in healthy and diseased human heart (heart failure secondary to hypertrophic or dilated cardiomyopathy). Our results indicate that the human heart expresses >800 miRNAs, the majority of which not being annotated nor described so far and some of which being unique to primate species. Furthermore, >250 miRNAs show differential and etiology-dependent expression in human dilated cardiomyopathy (DCM) or hypertrophic cardiomyopathy (HCM). The human cardiac miRNOME still possesses a large number of miRNAs that remain virtually unexplored. The current study provides a starting point for a more comprehensive understanding of the role of miRNAs in regulating human heart disease. Beginning with the discovery of lin-4 in C. elegans in 1993 In the cardiovascular field, several studies have demonstrated the involvement of miRNAs in a number of physiological and pathological conditions Taking into account that miRNAs can possibly regulate at least 30% of human protein-coding genes, bioinformatics prediction algorithms in conjunction with biologic validation methodologies initially estimated the existence of roughly 35 miRNAs – a number that has kept rising ever since For the reasons mentioned above, miRNA profiling has become of great interest in diverse research areas of biology and medicine, and is mostly defined as a measurement of the relative abundance of a set of miRNAs, ranging from a group of several miRNAs of specific biological relevance to comprehensive profiling of all miRNAs in a specific species. However, evaluating such small RNA molecules implies some intrinsic challenges. Whereas the application of DNA microarray technology to miRNA expression profiling offers advantages in terms of comparative capabilities, miRNA arrays do require a complicated procedure due to the size of miRNAs and the lack of a conserved 3-prime end for easy sample labeling, in conjunction with the fact that they frequently lead to process-related systematic biases. Despite the development of a number of different miRNA array platforms Here, we report the use of large-scale sequencing as a technological RNA discovery platform to: 1) catalog the miRNAs expressed in the non failing and failing heart and 2) to quantify the miRNA content in the non-failing human heart and relative changes in the miRNOME in hearts from patients with dilated cardiomyopathy (DCM) from non-ischemic origin or hearts from patients with familial hypertrophic cardiomyopathy (HCM).Table S1. All DCM patients were treated with a left ventricular assist device  as a bridge to transplantation and were in NYHA class IV at the time of LVAD implantation. The myocardial biopsy at time of LVAD implantation consisted of the LV apical core removed during implantation (diameter was about 1 cm). In patients diagnosed with HCM, biopsies were taken immediately after explantation of the heart. From all biopsies half was immediately frozen in liquid nitrogen and the other half was directly fixed in buffered formalin and embedded in paraffin. Control tissue was taken from the left ventricle of non-used donor hearts. Informed consent to participate in this study was obtained from all patients and individual written permission using standard informed consent procedures and prior approval of the Medical Ethics Committee of the University Medical Center Utrecht, was obtained.In this study, sixteen (16) human subjects were included, from which four (4) were refused donor hearts (controls) and the other twelve (12) were hearts from patients with refractory end-stage heart failure. The heart failure patients were divided in two groups: seven (7) patients that were diagnosed with dilated cardiomyopathy (DCM), and five (5) patients diagnosed with hypertrophic cardiomyopathy (HCM). Characteristics of these patients are summarized in Myocardial samples from the three groups  were used to generate high-titer small RNA libraries by Vertis Biotechnology AG . Briefly, the small RNA fraction from human left ventricular free wall of patients with end-stage heart failure was isolated using the mirVana microRNA isolation kit (Ambion). Sample preparation for parallel sequencing was performed as described http://www.internagenomics.com), as previously described Analysis of primary data was performed using miR-Intess software (Table S2. RNA (1 µg) from mouse or human tissue was isolated with TRIzol reagent (Invitrogen) and reverse-transcribed with the miScript Reverse Transcription Kit (Qiagen). Real-time PCR was performed on 1 ng of cDNA using miScript SYBR Green PCR Kit (Qiagen), on a BioRad iCycler (Biorad). To determine transcript quantity, the relative Ct method was used, where the amount of target normalized to the amount of endogenous control (U6) and relative to the control sample is given by 2–ΔΔCt Primers were designed to detect mouse and human transcripts for 3615376, 5243049 and s2154. Sequences are depicted in nppa), natriuretic peptide B (nppb) and glyceraldehyde 3-phosphate dehydrogenase (gapdh). Sequences are depicted in Table S2. RNA (1 µg) from human cardiac tissue was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) and 10 ng of cDNA were used for the analysis. Real-time PCR was performed on a BioRad iCycler (Biorad) using SYBR Green. Transcript quantities were compared using the relative Ct method, where the amount of target normalized to the amount of endogenous control GAPDH and relative to the control sample is given by 2–ΔΔCt.Primers were designed to detect human transcripts for natriuretic peptide A, (2). For comparison of two experimental groups ANOVA followed by a Student t test was used. Differences were considered significant when p<0.05.The results are presented as mean ± standard error of the mean (s.e.m). Prism software (GraphPad Software) was used to perform all statistical analyses. For the correlation analysis, Reads data were log10-transformed and used to calculate the Pearson’s coefficient of correlation  as well as in pathophysiological (hypertrophic and dilated cardiomyopathy) conditions, massive parallel sequencing technology was performed using an established small RNA cloning approach and 454 Life Sciences sequencing technology, as has been previously described Approximately 22% of the total mapped reads constituted of DNA repeat-related regions. A large fraction (28%) qualified to other non-coding RNA fraction including rRNAs, tRNAs, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs) as well as small interfering RNAs (siRNAs). The sub-fraction of tRNAs was the most prominent one, accounting for 78% of the non-coding RNA fraction. The number of perfectly aligned miRBase annotated miRNAs was 563, accounting for 31% of the total reads , suggesting that read number could be used as an abundance indicator. In addition, increased expression of cardiac stress markers such as nppa and nppb in both the DCM and HCM samples confirm the integrity and relevance of the samples used in this study . The expression pattern of the 10 most abundantly expressed miRNAs in our study for HCM  out of the 141 miRBase miRNAs that have been previously described in other profiling studies, as well as 19 out of 162 miRBase miRNAs that were not, showed potential hypertrophic cardiomyopathy specificity, when compared with the non-failing and dilated cardiomyopathy samples . Although these subsets exhibited extremely low cloning frequencies, in the range of 1–1500, the existence of at least 1 novel miRNA, has-miR-3615376,  in the human heart, both in failing as well as in non-failing situations, rather then a quantitative comparison between those two situations. The resulting miRNA inventory can be used to further investigate potentially important regulators that have been so far overlooked by previous studies.Although deep sequencing is emerging to be the method of choice for miRNA discovery and genome annotation using non-model organisms, profiling inconsistencies for known miRNA levels such as miR-1 and miR-133a, show that microarrays are often a preferable choice for miRNA profiling in model organisms with well-annotated genomes, due to their robust sample processing and analysis pipelines. In fact, while many miRNAs which regulation has been considered a signature of heart failure  Nevertheless, due to the discovery power of high-throughput sequencing, even after applying our robust cutoff, we were able to identify a very considerable set of miRNAs (162) that could potentially be important in cardiovascular disease. Moreover, miRNAs that overlap with other non-coding RNAs such as the large class of tRNAs and the snoRNAs, were readily identifiable and excluded from further analysis, preventing erroneous annotation. Despite the debatable physiological relevance of low abundant miRNAs and their large representation in our study, their existence could prove to become significant in the development of novel-miRNA regulatory pathways. Our analysis also yielded a number of miRNAs that exhibit an etiological (hypertrophic versus dilated cardiomyopathy) specific expression background, which could be used as basis for further research to unveil the various pathways leading to different cardiovascular disease outcome. Finally, a number of novel miRNAs were detected. These novel sequences were missed by conventional analysis probably because they tend to be expressed at low levels in the heart and are, most likely, located within unannotated regions in the genome. Examples of studies where novel miRNAs were identified by parallel sequencing are in embryonic stem cells, leukemia and several types of cancer To our knowledge, this is the first study where a deep sequencing approach was used to identify the complete set of microRNAs expressed (miRnome) in the human (failing) heart. The increased sensitivity of this method, compared to traditional ones, allowed to detect not only novel miRNAs but also the presence of miRNAs that were not previously involved in cardiac disease or neither detected in the heart. In addition, comparative profiling of hearts from patients with dilated cardiomyopathy and hearts from patients with familial hypertrophic cardiomyopathy revealed a pool of etiology-specific microRNAs which can be the basis for further research on the different pathways leading to different cardiovascular disease outcome.Besides a main restraint of our study being the use of limited number of patients with large variation between patients and their characteristics, particularly within the DCM group, we believe that our findings can be used to generate new hypothesis and design new studies to further clarify the role of specific miRNAs, novel or not, in cardiac disease.Table S1Demographic/clinical characteristics and echocardiographic indices of the 16 patients included in the study.(PDF)Click here for additional data file.Table S2Primers sequences used for real-time PCR Analysis(PDF)Click here for additional data file.Table S3p values for DCM versus NF, HCM vs NF and DCM vs HCM are given. Colored columns depict q values  for the different comparisons. MicroRNAs that have been previously involved in cardiac disease are marked in red. MicroRNAs that are differentially expressed between DCM and HCM  are depicted in purple.Quantification  of microRNAs in Non-Failing (NF), Dilated Cardiomyopathy (DCM) and Hypertrophic Cardiomyopathy (HCM) human hearts. (PDF)Click here for additional data file.


Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max after the species diverged. The analysis of Ka/Ks ratios and expression profiles indicated that functional divergence after the gene duplications was restricted. The results suggested that the function and evolution of HMGR gene family were dramatically conserved throughout the plant kingdom.The terpene compounds represent the largest and most diverse class of plant secondary metabolites which are important in plant growth and development. The 3-hydroxy-3-methylglutaryl coenzyme A reductase  is one of the key enzymes contributed to terpene biosynthesis. To better understand the basic characteristics and evolutionary history of the HMGR gene family in plants, a genome-wide analysis of HMGR genes from 20 representative species was carried out. A total of 56 HMGR genes in the 14 land plant genomes were identified, but no genes were found in all 6 algal genomes. The gene structure and protein architecture of all plant HMGR genes were highly conserved. The phylogenetic analysis revealed that the plant HMGRs were derived from one ancestor gene and finally developed into four distinct groups, two in the monocot plants and two in dicot plants. Species-specific gene duplications, caused mainly by segmental duplication, led to the limited expansion of HMGR genes in  Plants produce thousands of secondary metabolites that play important roles in numerous biological processes. Structurally and functionally different terpenes represent the largest and most diverse class of secondary metabolites Arabidopsis thalianaOryza sativaTriticum aestivumGossypium hirsutumSolanum tuberosumCucumis meloHevea brasiliensisIn plant cells, two distinct pathways are responsible for the biosynthesis of terpene compounds, the cytosolic mevalonate pathway (MVA pathway) and the plastidial 2-C-methyl-D-erythritol-4-phosphate pathway (MEP pathway) HMGR protein is comprised of three domains, i.e., the transmembrane domains with changeable number in N-terminal region, the highly divergent linker domain, and the long and conserved catalytic domain in C-terminus. Within the catalytic domain, subdomains have been defined as the small helical N-terminal domain, the large and central L-domain harboring two HMG-CoA binding motifs and an NADP(H) binding motif, and the smallest S-domain harboring an NADP(H) binding motif. The two HMG-CoA binding motifs, EMPVGYVQIP and TTEGCLVA, and two NADP(H) binding motifs, DAMGMNM and GTVGGGT, are functionally important and thus highly conserved in all HMGR proteins Zea mays and Glycine max using published transcriptome data were analyzed as well.Although HMGR genes have been systematically analyzed in Gramineae plants Arabidopsis thaliana HMGR genes http://www.arabidopsis.org) and used as queries to search against other plant genome databases with BlastP and tBlastN programs (default parameters). The 20 completely sequenced genomes of species from unicellular green algae to multicellular higher plants were used in this study (http://pfam.sanger.ac.uk/) to confirm the presence of the conserved domain (PF00368).The amino acid sequences of is study . Subsequhttp://meme.nbcr.net/meme/cgi-bin/meme.cgi). The analysis was performed with a set of parameters as follows: number of repetitions, any; minimum width for each motif, 6; maximum width for each motif, 100; and maximum number of motifs to be found, 5. All obtained motifs were searched in the InterPro database with InterProScan http://gsds.cbi.pku.edu.cn/) The conserved motifs encoded by each HMGR gene were identified using the program of Multiple Em for Motif Elicitation  Multiple sequence alignments of the full-length protein sequences were performed by Clustal X version 2.0 program Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max were collected from the genome annotation data of the corresponding organism, respectively. The chromosomal distribution images of these HMGR genes were generated by MapInspect software according to their starting positions in chromosomes Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max were also investigated. Gene duplication was defined according to (1) the length of aligned sequence cover >80% of the longer gene; and (2) the identity of the aligned regions >80% The locations of HMGR genes in −9 for Zea mays−9 for Populus trichocarpa−9 for Glycine max−6 Mya).The pairwise alignment of HMGR duplicated gene pairs from four plants was performed using Clustal X version 2.0 program Zea mays were obtained from the Plant Expression Database  Glycine max were downloaded from SoyBase (http://soybase.org/soyseq/) The expression profiles of ZmHMGRs and GrHMGRs were clustered using the Cluster 3.0 http://www.arabidopsis.org/).In order to identify HMGR genes in Viridiplantae, the blast searches among the 20 completely sequenced genomes  were carThe Viridiplantae are comprised of two major lineages that split early, i.e., the Chlorophyta  and the Streptophyta  Mesostigma viride contained the HMGR gene Although genome sequence data are currently not available for charophyte algae, which is believed to be the closest relatives of land plants Physcomitrella patens, a species of mosses which is a basal lineage of land plants, contained 3 HMGR genes. The Selaginella moellendorffii, the oldest extant vascular plant belonging to lycophytes, was the fewest HMGR gene family species among the land plants in our survey, which was expected as it has one of the smallest plant genomes known Citrus sinensis and Arabidopsis thaliana contained only two HMGR genes, which was the fewest two species in HMGR gene number among flowering plants we investigated. The other six plants, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Vitis vinifera, Carica papaya, and Brassica rapa, had three HMGR gene members. While the remaining four species had a relative higher number of HMGR genes, 6 HMGRs in Populus trichocarpa, 7 HMGRs in Zea mays, 8 HMGRs in Glycine max, and 9 HMGRs in Gossypium raimondii. The variable size of the HMGR gene family suggested that the gene family underwent species-specific expansion in flowering plants. Furthermore, among these species which underwent the gene expansion, Zea mays belongs to the monocots, and the other three belong to the eudicots, which indicated that the gene expansion occurred both in monocot plants and dicot plants.Additionally, among the land plants, there were 1 to 9 HMGR genes in each species, and most species (10 of 14 species) only had 3 or less HMGR genes. The non-vascular The MEME motif search tool was employed to identify the conserved motifs presented in 56 plant HMGR proteins, and 5 conserved motifs were uncovered . After sA common feature of plant HMGRs is the presence of a transmemebrane region consisting of two separate transmemebrane domains that are linked to the cytoplasmic domain bearing the catalytic center In the protein sequences of plant HMGR, there were four conserved motifs including EMPVGYVQIP, TTEGCLVA, DAMGMNM, and GTVGGGT in the catalytic domain. In this study, the motif 3 represented the two HMG-CoA binding sites (EMPVGYVQIP and TTEGCLVA), the motif 2 represented one of the NADP(H) binding sites (DAMGMNM), and the motif 4 represented the other NADP(H) binding sites (GTVGGGT), respectively . Among tIn order to validate the conservation of residues in the catalytic domain, the sequence logos of the motif 3, motif 2, motif 1 and motif 4 were investigated . It was Analysis of HMGR gene structure for exon/intron organization revealed that the number of introns per gene varied from 1 to 14 . OsHMGR1Regardless of the less variability, the protein architectures and gene structures of plant HMGRs were remarkably conserved, indicating that the molecular characteristics and biological functions of each plant HMGR were highly conserved during evolution. This was not the same as other gene families involved in plant secondary metabolism, which were very much lineage-dependent and varied tremendously among plant taxa To examine the evolutionary relationships of the HMGR genes in Viridiplantae, a neighbor-joining phylogenetic tree  was consGlycine max, which only contained group I HMGR genes. Obviously, the significant expansion of group I genes contributed to the increase of HMGR genes in Glycine max which had up to 8 members. In addition, those HMGR genes from the same species were not equally classified into the two groups. Most of the 8 plant species had more genes in group I than in group II, except for Gossypium raimondii which contained 8 group II genes, but only a single gene in group I. It was indicated that, unlike Glycine max, the increase in HMGR genes in Gossypium raimondii attributed to the remarkable expansion of group II genes.In eudicots, there were 36 HMGR genes from 8 plant species. Group I contained 21 members, and group II contained 15 members. Statistically, 7 out 8 dicot plants contained all two groups of HMGR genes, except for Zea mays that had the largest HMGR gene family in analyzed monocots had five genes in group IV, but two genes in group III.In monocots, all of 4 monocot plants contained the two group genes, group III and IV. As the HMGR genes from dicot plants, the 16 HMGR genes from 4 monocot plants were not evenly distributed in the two groups neither. All investigated monocot plants contained more members in group IV than in group III. Such as Zea mays, Gossypium raimondii, Populus trichocarpa, and Glycine max, underwent considerably more frequent gene duplications, which gave rise to an increase in more members of the HMGR gene family. As we know, the occurrence of most of secondary metabolites and their respective biosynthetic pathways is restricted to specific plants or plant lineages Gossypium raimondii, which contained the largest HMGR gene family (9 genes) in our survey, was known to accumulate a unique group of terpenes included desoxyhemigossypol, hemigossypol, gossypol, hemigossypolone, and the heliocides In general, there were no paralogs of HMGR genes from plants which contained less than or equal to 3 HMGRs, indicating that no gene expansion occurred within the HMGR gene family after the divergence of these plant species. In contrast, four other species, Zea mays were distributed unevenly on 5 chromosomes, and chromosome 4 contained three HMGR genes. In Gossypium raimondii, 9 HMGR genes were located on 5 chromosomes, and one gene cluster contained 4 genes was detected on chromosome 5. While in Populus trichocarpa and Glycine max, the HMGR genes were distributed uniformly, one gene on each chromosome.Based on the coordinate of each HMGR gene on the chromosomes, the chromosomal distribution images of these HMGR genes in four plant species which underwent species-specific gene expansions were generated . The 7 HZea mays, and there were three segmental duplication events in respective genome of Populus trichocarpa and Glycine max, strongly indicating that the segmental duplication was the main cause of the species-specific expansion of HMGR genes in the three species. Particularly, there were up to six duplication events in Gossypium raimondii which had the largest HMGR gene family number in our survey. With three segmental duplications and three tandem duplications, it was suggested that a relatively large extent of species-specific gene expansion was caused by both segmental and tandem duplications after the divergence of Gossypium raimondii.A total of fourteen duplication events which contributed to HMGR gene family expansion in the four plant species were found. Among them, two events as segmental duplications were in Zea mays, Populus trichocarpa and Glycine max were estimated using the Ks as the proxy for time , neofunctionalization (acquisition of novel functions), or subfunctionalization  for time . The Ks Zea mays and Glycine max development were analyzed using the public expression data.To understand the temporal and spatial expression patterns of HMGR genes, their expression profiles during Zea maysThe published microarray data of 60 different tissues and developmental stages of Glycine maxZea mays and Glycine max implied that functional divergence after the gene duplications was restricted. These results indicated that the HMGR gene family was dramatically conserved during plant genome evolution.The expression profiles of HMGR genes in Gossypium raimondii. The functional divergence after the gene duplications was restricted. The findings implied that the HMGR gene family was dramatically conserved during plant evolution, and the HMGR was the committed enzyme for terpene biosynthesis that had essential roles in regulating plant development.The HMGR gene family might be lost in the chlorophyte algae during evolution, but still widespread in land plants. The plant HMGR genes might be derived from one ancestor gene and finally developed into two distinct groups within the monocots and eudicots, respectively. The gene structure and protein architecture of all plant HMGRs were considerable conserved. The HMGR family in four flowering plants underwent species-specific expansions after the species diverged due to the large production for terpene compounds in their respective development. Segmental duplication appeared to be the dominant mechanism for the gene duplication events in three species, whereas segmental duplication and tandem duplication played similar roles in the expansion of the HMGR gene family of Figure S1Distribution of conserved motifs in plant HMGR proteins identified using the MEME search tool. Different motifs are indicated by different colors numbered 1-5. The names of all members of HMGR genes and combined p-values are shown on the left side of the figure, and the positions and sizes of motifs are indicated at the bottom of the figure. The motif 5 is a region including two transmembrane helices. The motif 3, 2, 1 and 4 are located in the catalytic domain of HMGR proteins. Moreover, the N-terminus of the motif 3 is at the start position of catalytic domain in each HMGR protein.(TIF)Click here for additional data file.Figure S2Exon/intron organization of plant HMGR genes. Exons are represented by green boxes and introns by black lines. Names of all plant HMGR genes from different lineages are shown on the left side of the figure.(TIF)Click here for additional data file.Table S1Data sources for the genome sequences used to mine for HMGR genes.(XLSX)Click here for additional data file.Table S2Summary of 56 HMGR genes identified in Viridiplantae.(XLSX)Click here for additional data file.Table S3Ka/Ks analysis and estimated divergence time for the HMGR duplicated genes from Zea mays, Gossypium raimondii, Populus trichocarpa and Glycine max.(XLSX)Click here for additional data file.


Recent evidence indicates that sex hormones may exacerbate autoimmune diseases, particularly in women, by increasing the adjuvant effect of infections. Autoimmune diseases affect approximately 8% of the population, 78% of whom are women. The reasons for the high prevalence in women are unknown, but circumstantial evidence links autoimmune diseases with preceding infections. Animal models of autoimmune diseases have shown that infections can induce autoimmune disease. For example, coxsackievirus B3 (CB3) infection of susceptible mice results in inflammation of the heart (myocarditis) that resembles myocarditis in humans. The same disease can be induced by injecting mice with heart proteins mixed with adjuvant(s), which indicates that an active infection is not necessary for the development of autoimmune disease. We have found that CB3 triggers autoimmune disease in susceptible mice by stimulating elevated levels of proinflammatory cytokines from mast cells during the innate immune response. Sex hormones may further amplify this hyperimmune response to infection in susceptible persons, which leads to an increased prevalence of autoimmune diseases in women. C,Soon after autoimmune diseases were first recognized more than a century ago, researchers began to associate them with viral and bacterial infections. Autoimmune diseases tend to cluster in families and in individuals (a person with one autoimmune disease is more likely to get another), which indicates that common mechanisms are involved in disease susceptibility. Studies of the prevalence of autoimmune disease in monozygotic twins show that genetic as well as environmental factors (such as infection) are necessary for the disease to develop  . MTo address the question of whether autoimmune diseases can be induced by infections, first autoimmunity needs to be defined. Autoimmune disease occurs when a response against a self-antigen(s) involving T cells, B cells, or autoantibodies induces injury systemically or against a particular organ. Understanding of autoimmune diseases is hindered by the fact that some level of autoimmunity, in the form of naturally occurring autoantibodies and self-reactive T and B cells, is present in all normal persons .,,Given those circumstances, how can infections induce autoimmune disease? A mechanism often called on to explain the association of infection with autoimmune disease is "molecular mimicry," that is, antigens (or more properly epitopes) of the microorganism closely resemble self-antigens. The induction of an immune response to the microbial antigen thus results in cross-reaction with self-antigens and induction of autoimmunity  activate the innate immune response in the same pathogen-specific manner when administered with self-antigen; this process results in organ-specific autoimmune disease in animal models , indirect evidence , and circumstantial evidence  should be considered  of the risk of autoimmune disease . Smaller numbers of natural killer cells, B cells, and eosinophils are also present. Both T-cell–mediated and autoantibody-mediated mechanisms have been shown to be important in the development of CB3-induced heart disease in BALB/c mice  have been associated with the same autoimmune disease   . We have,,,Key to understanding the control of susceptibility to autoimmune myocarditis was the finding that adding bacterial lipopolysaccharide (LPS), interleukin (IL)-1β, or tumor necrosis factor (TNF)-α during the innate response to CB3 infection results in the development of the chronic phase of disease in resistant strains of mice ,A and IL-,,,Since the innate immune response is critical in determining the development of adaptive immunity  and sple,,,An elevated IL-4, TNF-α, and IL-1β response is reminiscent of the hypersensitivity reaction of mast cells during allergic responses  (33). We,Even though women's greater susceptibility to autoimmune diseases has been recognized for more than 100 years, only recently has attention focused on this topic  A and mal


Alzheimer's disease (AD) is the most common neurodegenerative disease that causes dementia in the elderly. Patients with AD suffer a gradual deterioration of memory and other cognitive functions, which eventually leads to a complete incapacity and death. A complicated array of molecular events has been implicated in the pathogenesis of AD. The major pathological characteristics of AD brains are the presence of senile plaques, neurofibrillary tangles, and neuronal loss. Growing evidence has demonstrated that oxidative stress is an important factor contributing to the initiation and progression of AD. However, the mechanisms that lead to the disruption of redox balance and the sources of free radicals remain elusive. The excessive reactive oxygen species may be generated from mechanisms such as mitochondria dysfunction and/or aberrant accumulation of transition metals, while the abnormal accumulation of Abeta and tau proteins appears to promote the redox imbalance. The resulted oxidative stress has been implicated in Abeta- or tau-induced neurotoxicity. In addition, evidence has suggested that oxidative stress may augment the production and aggregation of Abeta and facilitate the phosphorylation and polymerization of tau, thus forming a vicious cycle that promotes the initiation and progression of AD. Alzheimer's disease (AD) is the most common neurodegenerative disease that causes dementia in the elderly. It is characterized by the gradual deterioration of memory and other cognitive functions, which eventually leads to a complete incapacity and death of the patients within 3 to 9 years after diagnosis . Increas2-isoprostanes, are also increased in multiple brain regions and cerebrospinal fluid (CSF) of patients with AD or mild cognitive impairment (MCI) [The major pathological characteristics of AD brains are the presence of senile plaques, neurofibrillary tangles (NFTs), and neuronal loss , 6. Senint (MCI) –17. In ant (MCI) . Moreovent (MCI) . ImportaWhile oxidative stress has emerged as one of the important factors in AD pathogenesis, the mechanisms by which the redox balance is altered and the sources of free radicals remain elusive. The present paper reviews the involvement of abnormal accumulation of Abeta and tau proteins in the induction of redox imbalance and the generation of free radicals through mechanisms such as mitochondria dysfunction and/or transition metal homeostasis imbalance and discusses the mechanisms by which oxidative stress promotes Abeta- and tau-mediated neurotoxicity.Abeta is produced via sequential proteolytic cleavages of APP by two membrane-bound proteases, beta-secretase, also known as beta-site APP cleaving enzyme 1 (BACE1), and gamma-secretase, a multiprotein complex consisting of presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer protein 2 (PEN-2) , 22. BACIn vitro experiments using cell models showed that Abeta treatment could increase the levels of hydrogen peroxide and lipid peroxides [A great deal of research has implicated oxidative stress in Abeta-induced neurotoxicity . In vitreroxides . Consisteroxides –35. In heroxides . Consisteroxides –40.In addition to mediating Abeta-induced cytotoxity, numerous studies have suggested that oxidative stress promotes the production of Abeta. It was demonstrated that defects in antioxidant defense system caused elevated oxidative stress and significantly increased Abeta deposition in transgenic mice overexpressing APP mutant , 42, whiStudies on how oxidative stress enhances Abeta production have revealed that oxidative stress decreases the activity of alpha-secretase while promoting the expression and activation of beta- and gamma-secretase, enzymes critical for the generation of Abeta from APP –50. The Alternatively, the augmentation of Abeta production by oxidative stress may be a compensatory reaction to oxidative stress. It was found that neuronal oxidative damage was more pronounced in AD subjects with lesser amounts of Abeta deposition or in AD subjects with a shorter disease duration , 62, andMitochondria are unique organelles that are pivotal for a variety of cellular functions including ATP synthesis, calcium homeostasis, and cell survival and death. Meanwhile, mitochondrial respiratory chain is a major site of ROS production in the cell, and mitochondria are particularly vulnerable to oxidative stress , 71. ExtEvidence suggests that Abeta may directly disrupt mitochondria function and contribute to the deficiency of energy metabolism and neuronal death seen in AD. It was found that Abeta was localized to mitochondria in brains of AD patients and transgenic mice as well as in neuroblastoma cells stably expressing human mutant APP , 75. TheIt is noted that the mitochondria-associated Abeta along with the increase in hydrogen peroxide and decrease in cytochrome oxidase activity was detected prior to the appearance of Abeta plaques, suggesting the defect in mitochondria occurs earlier in the pathogenesis of AD. Therefore, early mitochondrially targeted therapeutic interventions may be effective in delaying the onset and progression of AD , 75.Transition metals such as copper (Cu), zinc (Zn), and iron (Fe) play important catalytic roles in many enzymes and are essential for a broad range of biological processes in human body including brain functions. Both Cu and Zn have been shown to participate in regulating synaptic function. Following NMDA receptor activation, Cu is released from the neuron and regulates neuronal activation by functionally blocking NMDA receptors and limiting calcium entry into the cell . Zn also2+ and Zn2+ can bind to Abeta monomers via three histidine residues  and a tyrosine residue (Tyr10), causing conformational changes of the peptide that promote its aggregation [in vitro data showed that Cu and Zn rapidly induced the aggregation of soluble Abeta peptides [2+ with high affinity, forming a cuproenzyme-like complex [2+ to Cu+ and forming positively charged Abeta radical (Abeta+•) [+ then donates two electrons to oxygen, generating H2O2 [2, the radicalized ·Cu2+ complex may be restored to Abeta·Cu2+ by electron transfer from biological reducing agents such as cholesterol, catecholamines, and vitamin C [2O2 is greater for Abeta42 than Abeta40, correlating with their cytotoxic activity [3+ to Fe2+ and the generation of H2O2 [The presence of transition metals within the amyloid deposits in AD patients indicates that transition metals may directly interact with Abeta –90. Inderegation , 92. Conpeptides , 94. Thepeptides –96. In a complex . ElectroAbeta+•) . Cu+ theing H2O2 , 98, seting H2O2 , 100. Afitamin C . The effactivity . Similar of H2O2 . These dAdditionally, there is a close association between Fe/Cu homeostasis and the production and processing of APP. In SH-SY5Y cells overexpressing the Swedish mutant form of human APP (APPsw), Fe treatment induced the release of Abeta42 , 103. MoStudies have shown that metal transporters such as Zn transporters and divalent metal transporter 1 (DMT1) are increased in the cortex and hippocampus of APP/PS1 transgenic mice; and similar to transition metals, these metal transporters are colocalized with Abeta in senile plaques in the cortex of AD brains , 107. ThAPP, which has a Cu binding site at N-terminal cysteine-rich region , generat2O2 and Zn2+/Cu2+-induced precipitation of synthetic Abeta in vitro and significantly reduce the level of Abeta deposition in brains of Tg2576 transgenic mice [As aberrant metal homeostasis plays an important role in several important aspects of AD pathogenesis including the production and aggregation of Abeta and the oxidative stress mediated by Abeta, AD therapy-targeted metal-Abeta interaction is rapidly emerging as a promising therapeutic option. Various metal chelating compounds have been tested for their efficacy . One of nic mice , 115. Innic mice . Since the generation of ROS is an important factor in AD pathogenesis promoted by the aberrant accumulation of transition metals, natural antioxidants may have protective effects against AD pathogenesis induced by disruption of metal homeostasis . As expeHyperphosphorylated tau protein is the major component of NFT, another hallmark of AD pathology that correlates with neurodegeneration and cognitive decline . AbnormatripleAD), which exhibits both Abeta and tau pathologic features of the disease in the brain of the animal [tripleAD brain samples demonstrated a massive deregulation of 24 proteins, of which one third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system [Although less well studied, evidence has shown that oxidative stress is interlinked with tau pathology. It was also shown that the cells overexpressing tau protein had increased susceptibility against oxidative stress, perhaps due to the depletion of peroxisomes . In a dre animal . Proteomn system . Notablyn system . The effn system . in vitro [in vivo [Growing evidence has also shown that oxidative stress may have a role in the hyperphosphoryaltion and polymerization of tau. The oxidation of fatty acids, which is found to be elevated in AD brains, was reported to facilitate the polymerization of tau, and thus might serve as a possible link between oxidative stress and the formation of the fibrillar pathology in AD . In Tg25in vitro . In hipp[in vivo . Thus, pIn summary, evidence has demonstrated that oxidative stress is inextricably linked with several major pathological processes in AD including Abeta-induced neurotoxicity, tau pathology, mitochondria dysfunction, and metal dyshomeostasis. Excessive ROS may be generated from mitochondria dysfunction and/or aberrant accumulation of transition metals, perhaps caused by a combination of abnormal Abeta accumulation and tau pathology, eventually resulting in oxidative stress. Oxidative stress, which mediates the neurotoxicity induced by abnormal accumulation of Abeta and tau proteins, may augment Abeta production and aggregation as well as facilitate tau phosphorylation and polymerization, further enhancing a variety of neurotoxic events including ROS production, thus forming a vicious cycle that promotes the initiation and progression of AD. Regardless a primary or secondary event, oxidative stress is an important factor contributing to the development of AD. Removal of ROS or prevention of their formation may delay the onset or slow down the progression of AD through multiple mechanisms including, but not limited to, reduction of oxidative stress-mediated neuronal toxicity, inhibition of Abeta production and aggregation, decrease of tau phosphorylation and polymerization, and restoration of mitochondria function and metal homeostasis. Therefore, AD prevention or treatment with natural antioxidant may be an approach that is capable of targeting a number of different molecular events implicated in the pathogenesis of AD.


The efficient transport of blood and lymph relies on competent intraluminal valves that ensure unidirectional fluid flow through the vessels. In the lymphatic vessels, lack of luminal valves causes reflux of lymph and can lead to lymphedema, while dysfunction of venous valves is associated with venous hypertension, varicose veins, and thrombosis that can lead to edema and ulcerations. Despite their clinical importance, the mechanisms that regulate valve formation are poorly understood and have only recently begun to be characterized. Here, we discuss new findings regarding the development of venous and lymphatic valves that indicate the involvement of common molecular mechanisms in regulating valve formation in different vascular beds. The circulatory system is composed of the heart and the blood vessels, which distribute nutrients, hormones, gases, and metabolic waste products in the body, and the lymphatic vessels that ensure that the extravasated fluid and proteins are drained from the tissues and transported back to the blood circulation. The beating of the heart generates pulsatile flow of blood that is pushed through a hierarchical network of arteries, capillaries, and veins, with heart valves controlling the flow of blood through the heart by opening and closing during the contractions of the heart. In addition, contraction of skeletal muscles compresses the walls of deep veins and drives the movement of blood inside the vessels, thus behaving like peripheral muscle pumps . AdditioLike the blood vasculature, the lymphatic system is composed of a hierarchy of vessels with specific features serving their unique functions: the blind-ended lymphatic capillaries  that absorb the interstitial fluid and the collecting lymphatic vessels that transport the lymph to the cardiovascular system (reviewed in ). In birConsistent with their important functions, incompetence of venous valves has clinical implications and is associated with venous hypertension, orthostatic leg swelling, development of varicose veins, and leg ulcerations , 11, 12.Recent advances in ultrasound technology have given insight into the physics of venous valve operation and have delineated the way the movement of the leaflets affects blood flow into four distinct phases of the valve cycle: opening, equilibrium, closing, and closed Fig.  15]. An. An15]. “The bicuspid valve begins as a pair of inconspicuous and more or less transversely placed endothelial ridges inside the vein. By the invasion of the mesenchyme, the elongation and slanting of the ridges, and the formation of a concavity on their leeward face, the anlagen are gradually converted into vulvar sacs. Coincident with such changes, the venous cavity flares outward at the level of these sacs, so aiding in the production of their sinus .”The mature venous and lymphatic valves are typically bicuspid, and are composed of two luminal leaflets that consist of two layers of endothelial cells separated by a defined connective tissue core –26; Fig.a:“The biRecent studies using high-resolution imaging techniques have confirmed Kampmeier’s accurate description of the basic morphogenic events during valve formation. In addition, analyses of genetic mouse models have provided important insight into the molecular and cellular mechanisms regulating morphogenesis of both lymphatic and venous valves. Below, we describe the sequence of developmental events and the molecular mechanisms operating during early onset of valve development, leaflet elongation, and valve maintenance in lymphatic vessels and veins.While it is not fully understood how the process of valve development is initiated, it was recently shown that mechanical forces caused by fluid flow regulate the expression of key molecular regulators and cellular behaviours associated with valve-forming lymphatic endothelial cells . MechaniInterestingly, the early stages of heart valve formation share some common features with lymphatic and venous valve development. In mice, heart valve formation occurs as a result of endocardial cushion formation when signals derived from myocardium induce an endothelial-to-mesenchymal transition of adjacent endothelial cells . ThThe first indication of lymphatic valve development is the appearance and clustering of cells expressing elevated levels of two transcription factors, Prox1 and Foxc2, in defined positions along the vessel , 31, 32;1, 32;3b.Foxc2 deficiency in mice and humans leads to lymphatic valvular aplasia [FOXC2 mutations develop lymphedema [Prox1 deficiency in mice leads to defective lympho-venous valve formation [GATA2 were identified as responsible for Emberger syndrome, an autosomal dominant primary lymphedema that is associated with a predisposition to acute myeloid leukemia (AML) [Upon their induction, Prox1 and Foxc2 control, in response to flow, the expression and activation of the two important downstream regulators of valve formation, Connexin-37 and NFATc1 , 31, 43, aplasia , 31. Conmphedema . Reflux mphedema . While tormation , implyinormation . Inactivia (AML) , as wellia (AML) . HoweverGJC2 (encoding Cx47) was found to be mutated in lymphedema patients [Connexin-37 (Cx37) is highly expressed in lymphatic valve-forming cells and regulates their rearrangement to a ring-like constriction during early stages of valve development, suggesting the importance of gap-junctional communication during this process  appear to have a rounded or cuboidal morphology ; Fig. 4cc4c, f. AThe mature venous valve leaflets are thicker at the sites of attachment to the vessel wall containing condensely packed collagen fibers; however, this deteriorates with age, thus making the valve leaflets more fragile . It has Cnb1 in neonatal mice led to the regression of lymphatic valve leaflets in mice, indicating a continuous requirement for Calcineurin signaling for valve maintenance [At the molecular level, little is known about the mechanisms regulating valve maintenance. Studies on genetic mouse models have indicated important functions for both Integrin-α9 and Ephrin-B2 in the maintenance of venous valves, since conditional ablation of either gene in adult mice led to regression of valve leaflets . Ephrin-ntenance .The first observations on the development of lymphatic and venous valves were made nearly 100 years ago , 29, and


Tumour cells relies mostly on glycolysis to meet their energetic demands thus leading to an overload of lactic acid, which must be exported to the extracellular milieu, through the monocarboxylate transporters (MCTs). Due to their upregulation in cancer, MCTs are currently seen as promising therapeutic targets in cancer. Colorectal carcinoma (CRC) is the second most common type of cancer worldwide, being mainly a disease of industrialized countries. Among the chemotherapeutic agents used in the treatment of CRC, 5-fluorouracil (5-FU) is one of the most efficient, although resistance to 5-FU treatment has been reported. We aimed at understanding the role of MCTs in the glycolytic metabolism of CRC and exploring these transporters as putative therapeutic targets.We performed a detailed characterization of MCT expression in a CRC clinical series and correlated the expression with key metabolic proteins. We also studied the effects of MCT activity inhibition in normal and CRC derived cell lines and evaluated the effect of MCT inhibitors in combination with 5-FU.in vitro assays, we demonstrated that MCT inhibition disrupted tumour cell aggressiveness and potentiated the cytotoxic effect of 5-FU.Our results showed that MCT1 and MCT4, their putative chaperones (CD147 and CD44) and the glucose transporter GLUT1 are overexpressed in CRC samples. Furthermore, we observed that MCT expression is associated with the remaining proteins. In Our results provide novel evidence for the pivotal role of MCTs in CRC maintenance, supporting the exploitation of MCTs as therapeutic targets in CRC.Portuguese Foundation for Science and Technology (FCT). The NCM460 cell line was received by a licensing agreement with INCELL Corporation, San Antonio, TX. The cells were routinely propagated under standard conditions in M3:10™ medium (INCELL).