Ficus presents an opportunity to investigate the ecology of a multi-trophic system that includes parasitoids. Here we estimate the host-plant species specificity of a parasitoid fig wasp genus that attacks the galls of non-pollinating pteromalid and pollinating agaonid fig wasps. We discuss the interactions between parasitoids and the Ficus species present in a forest patch of Uganda in context with populations in Southern Africa. Haplotype networks are inferred to examine intraspecific mitochondrial DNA divergences and phylogenetic approaches used to infer putative species relationships. Taxonomic appraisal and putative species delimitation by molecular and morphological techniques are compared. Results demonstrate that a parasitoid fig wasp population is able to reproduce on at least four Ficus species present in a patch. This suggests that parasitoid fig wasps have relatively broad host-Ficus species ranges compared to fig wasps that oviposit internally. Parasitoid fig wasps did not recruit on all available host plants present in the forest census area and suggests an important ecological consequence in mitigating fitness trade-offs between pollinator and Ficus reproduction. The extent to which parasitoid fig wasps exert influence on the pollination mutualism must consider the fitness consequences imposed by the ability to interact with phenotypes of multiple Ficus and fig wasps species, but not equally across space and time.Parasitoid diversity in terrestrial ecosystems is enormous. However, ecological processes underpinning their evolutionary diversification in association with other trophic groups are still unclear. Specialisation and interdependencies among chalcid wasps that reproduce on  Ficus) that support reproduction of parasitoid fig wasp population's and discuss implications for the pollination mutualism.Underlying mechanisms that maintain the coexistence of widely varying phenotypes within conservative trophic interactions are unclear Ficus species (secondary host) each fig wasp guild is able to reproduce in might also impact selection on one another Ficus to reproduce, a trade-off between the number of ovules galled (which produce wasps that disperse pollen) and those that are not galled (which can be pollinated to produce seeds) must be met Parasitoids have been shown to indirectly influence plant fitness traits via their interaction with herbivores Ficus implies considerable evolutionary history and the relative differences in the strength of species-specificity among them are not fully realised Ficus compared to pteromalid fig wasps Ficus ranges than pollinator and non-pollinator species The form of specialisation fig wasps direct at host species remains a key question because of the implications for the pollination mutualism Ficus preferences Ficus and indicative of host-plant switching Ficus species. For example, the specificity of wasp attraction to two closely related Ficus species has been shown to be less for parasitoids than pollinators Philotrypesis and ApocryptaSycoscapterWatshamiella and ArachoniaSycoryctes have yet to confirm this life history trait, but it is assumed since the behaviour is ubiquitous in the remaining genera. The Sycoryctinae possess extremely long ovipositors for laying eggs inside the fig syconium. Intraspecific variation of the ovipositor length in the sycoryctini Ficus species. Parasitoid fig wasps might use a comparatively wider yet taxonomically constrained spectrum of traits to locate pollinating and non-pollinating fig wasp host species. However, it is not known whether parasitoid specialisation on Ficus is characterised by host switching i.e. different populations reproducing separately on different Ficus species (divergent selection), or, whether a population uses multiple host species. It is difficult to discriminate between the process of host switching and a broadening of host-range based on species-level phylogenetic inference because the underlying genetic mechanisms and phenotypes determining host preference are not well understood.Parasitoid fig wasp speciation has been shown to be a function of host-Ficus species. We use parsimony-based and probabilistic methods to discriminate between within-species and between-species haplotype divergence and infer a phylogeny including a nuclear marker to validate species relationships. We assess the morphological variation among putative species lineages to compare with genetic delimitation approaches. Near-exhaustive sampling of all individual Ficus trees was possible and provided us with an ecological ‘snapshot’ of a fig wasp community located within a patch of primary and mixed forest. Specimens were reared from all trees in the forest that were releasing fig wasps. Variation at the cytochrome oxidase subunit one (COI) and cytochrome b (Cytb) loci for the most commonly occurring parasitoid genus was compared across Ficus species. We tested: i) whether single or multiple populations/species of Arachonia were present in Kibale Forest; ii) their relationship with specimens collected widely over Eastern and Southern Africa; and iii) haplotype structuring according to the Ficus species they were reared from. Our findings indicate that a parasitoid population of Arachonia was able to reproduce in multiple Ficus species in the same forest patch in the period concomitant with the developmental time of a single generation.In this study we test the hypothesis that a parasitoid population is able to reproduce in syconia of more than one a priori criteria for discriminating within and between species-level divergences at the COI and Cytb mtDNA loci. Our COI and Cytb sequence data were collapsed into 92 unique haplotypes (of 145 specimens) with 10 networks. Six of these were represented by a single haplotype. These singleton taxon networks we generated comprised specimens reared from F. sycomorus, F. umbellata, F. ovata, F. sansibarica and F. sur. Reticulations were present in the some of the four remaining major networks. Our results demonstrated several lines of evidence that show haplotype structuring is a function of Ficus host associations and the geographic region from which the specimens were collected. The levels of genetic structuring we uncovered at Kibale appeared independent of the year in which a few specimens were collected. During the month long census over August in Kibale Forest, 116 individual trees were recorded with 11 of them releasing fig wasps; less than 10% of the fig trees , 1 of 2 F. polita (the collection was made from a morpho-type that was near F. umbellata), and 1 of 7 F. sur within Kibale were releasing fig wasps over the sampling period.We generated a haplotype network using statistical parsimony to explore ig trees . For insFicus species they were reared from. The highest concentration of haplotypes (group Ia) was collected from F. ovata, F. chrinidensis, F. polita, and F. artocarpoides. The negligible genetic variance within this group of haplotypes was independent of Ficus species affiliation. The next most genetically similar group (Ib) was collected from only two Ficus species, F. chirindensis and F. artocarpoides. Again, the genetic structuring of the haplotypes within this group was independent of the two Ficus species this population reproduced on. In other words, our results show no evidence that Ficus affiliation of a given population structured the genetic variance of that population. Both haplotype groups Ia and Ib were collected entirely from Kibale Forest during August 2008. The haplotype group Ic included collections from F. ovata, F. artocarpoides, F. sansibarica, F. polita, F. chirindensis, F. sycomorus, and F. sur made in Kibale, elsewhere in Uganda, Zambia, and Kenya. This network included one haplotype found on four species  in Kibale in the 2008 sampling period. Haplotype group Id was collected from F. sur and F. sycomorus, which are not parasitized by groups Ia and Ib. Group Id included haplotypes from Kibale collected one year earlier and another from Zambia two years earlier and all from F. sur and F. sycomorus. The four groups (Ia–Id) of haplotypes are arguably distinct populations or putative genetic Arachonia species. We show that each taxon has host Ficus species ranges >1. The most apparent distinction in Ficus range among the groups is evident in the split between the population parasitizing Ficus from section Sycomorus (subsection Sycomorus) and those from section Galoglychia (subsection Caulocarpae). For instance, haplotype group Id . Haplotype group II is distinct and also comprises specimens from across Southern Africa. There is evidence of cross-Ficus section species ranges from group Ic. However, our Bayesian haplotype tree inference indicated that the two specimens from group Ic on F. sycomorus and F. sur cluster with groups Id and II that are all affiliated with these species of section Sycomorus (see below). The three remaining networks II–IV  were collected from different individuals.Our statistical parsimony analysis showed some ambiguity in haplotype assignment. This largely occurred because of missing characters at flanking ends of some (55 of the 290 COI & Cytb sequences) fragments. These ambiguous connections might signify homoplasies that cannot be assigned a single connection. We used the procedure recommended by Posada and Crandall in group Id  was affiks II–IV  each refa priori designation of populations. The four networks  resulting from our statistical parsimony analyses were nominated as populations and used in the initial AMOVA. A second AMOVA was conducted with network I deconstructed into four populations  in addition to populations II, III, and IV (STF = 0.52 (P<0.001) with variance within and among groups being approximately equivalent (STF = 0.90 (P<0.001). The variance between and within groups was substantially different and indicative of maximally distinct groups of haplotypes, each associated with different sets of Ficus host species.We conducted analysis of molecular variance that requires , and IV . Our iniuivalent . The secP<0.001). The clustering of lineages representative of the population-level branching model were largely concordant with the networks estimated using statistical parsimony. The mixed model likelihood test identified 13 clusters (CI: 12–17) consistent with population-level branching patterns and 19 entities (CI: 18–26) typical of the species-level branching model. The level of Ficus host species conservatism exhibited by Arachonia clades specialising on either section Sycomorus or section Galoglychia evident from the statistical parsimony analysis was concordant with phylogenetic inferences. There were rare exceptions to this within-section conservatism in each of the two major stem clades associated with either section Sycomorus (subsection Sycomorus) or section Galoglychia (subsection Caulocarpe).We used a GMYC likelihood test to estimate which haplotype groups best fit either a coalescent or Yule model of branching. Genetic divergences were estimated using the ultrametric consensus phylogeny implemented under a Bayesian approach . The GMYArachonia haplotypes (Cytb) for this specimen resulted in spurious parsimony networks in this instance. The two major Arachonia clades that we collected from either section Sycomorus or Galoglychia, group as sister-clades with only two instances of host-use paraphyly. The two instances of host-use paraphyly (specimens 12 on F. sur and 131 on F. sycomorus) are well supported in the phylogeny inferred using the COI, Cytb, and EF-1α data set (see below). The specimen 131 is a relatively divergent singleton likely to represent a single species . Negligible branch length differences are evident in clades with haplotypes from groups Ia, Ib, Id, III, and IV. By contrast, posterior probability support for stem clades, sometimes consisting of one or more derived clades, was above 90 in most instances  and colour which varies from dark bluish-black, through green, yellowish-green to brownish-yellow or plain yellow. Five of the morphological species correspond with the haplotypes  that were able to reproduce in either host species of section Sycomorus or Galoglychia and indicative of cryptic species. Morpho-species 7 (group IV), able to reproduce on two host sections, represented the broadest host range for a genetically and morphologically highly similar type.Seven analyses  & S5. Thplotypes . The remplotypes . ArachonArachonia species in Africa have conservative host plant associations among Ficus subsections Sycomorus (section Sycomorus) and Caulocarpe . A population  present within Kibale Forest was able to reproduce in at least four Ficus species, and in one instance, eleven identical haplotypes were collected from these four species. A divergent population of Arachonia species 7 was also associated with a further two species of fig elsewhere in Southern Africa (haplotype group IV). A large majority of haplotypes were associated with a single subsection and use of more than one by a single population was rare. These relationships indicate a high degree of historical host conservatism within Ficus subsections with infrequent horizontal transfer between host-plant subsections and sections.This study demonstrated that Ficus species within section Galoglychia and one species from section Sycomorus. A relatively deep genetic divergence within this distinct morpho-species  was collected from four Ficus species in Kibale Forest. This pattern strongly supports incomplete host-switching and a multiple species host-range. We uncovered five Arachonia species distributed through Southern Africa that were able to reproduce on more than a single species  was collected from five -species  suggests species . We expeFicus subsection and that parasitoids have broader host-ranges than pollinator and possibly non-pollinator galling species. However, this does not imply that all host wasp species are present, or attacked, at the patch scale. Nor must parasitoids have compatible reproductive phenology with all host wasp species that specialize across the subset of their Ficus hosts. The net or effective host wasp range during a given reproductive cycle at a patch need only be the sum of the proportion of host wasp species available from any ‘compatible’ Ficus species in the patch. Host Ficus range and apparent ‘flexibility’ in host wasp species implies that the phenotype's parasitoids interact with in the course of locating a fig and ovipositioning, are quantitatively and qualitatively different than host wasp species. These differences are partly dependent on the form of parasitism, cues for locating hosts, and external and internal oviposition strategies that present different phenotpye interactions. Additionally, immune responses to parasitism common in Hymenoptera Ficus.This study shows that selection for parasitoid fig wasp host-plant species conservatism is concomitant with Fig trees rely exclusively on pollination by a wasp that is technically a seed parasite. Yet, a balance is maintained between pollinator and plant fitness Ficus species within a patch containing host species. Our findings indicate that parasitoids do not recruit to all available figs within a patch  in India; Ficus stupenda Miquel (section Conosycea) in Borneo Ficus benghalensis Linnaeus (section Urostigma) in India Ficus annulata (section Urostigma) in Malaysia Sycoryctes species reported by Compton in Arachonia species Arachonia have a propodeum  that is as long as wide and shaped more like a bowl whereas the Sycorcytes have a transverse propodeum that is thin and narrow (much shorter than wide). See key to Sycoryctinae genera: http://www.figweb.org/Fig_wasps/Pteromalidae/Sycoryctinae/Key/index.htm. In total, 145 separate Arachonia individuals from seven Ficus were sequenced. Eighty-four of these individuals were from Kibale Forest. The specimens were initially designated using Ficus species affiliation and cuticle colour. Voucher specimens were deposited at Iziko South African Museum collection (Cape Town). To maximise DNA sequence variation capture and to compare to known phylogenetic species, Arachonia from previous collections Ficus species.We included the parasitoid fig wasp genus Ficus species. Within 8 hours of being removed from the tree, the syconia collections were placed into rearing canisters. Up to 10 syconia were placed into a sterile cardboard tube with a replaceable and transparent collection jar at one end and sealed. As wasps emerge from the syconia, they migrate toward the jar in to the direction of the light. Wasp fauna were removed from the jar and placed live into >95% ethanol approximately every 8 hours. Rearing canisters produced wasp collections from between 1 and 5 days. Each syconium can potentially contain hundreds of galled ovules. This varies between syconia of different size and wasp presence. Tropical fig species typically have very large syconia. Our collections produced thousands of fig wasps that were sorted using a dissecting microscope at the Museum subsequent to the fieldwork.Fig and wasp larval development takes between three and 20 weeks, with parasitoids arriving and targeting other fig wasp guilds towards the very end of fig development Ficus that produced fig crops in the Kibale Forest National Park in Uganda  over a one-month period in August 2008 before the beginning of the wet-season true. In this regard, our collection represents a snapshot of the fig wasp ecology of Kibale Forest. In order to maximise the rate of encountering the same parasitoid species on several hosts at a local ecological scale, exhaustive sampling over a one month period of all fig trees in a large forest patch that were releasing wasps was conducted. Access to nearly all parts of the forest was possible due to a grid system of tracks created to census primate populations. We located 116 individual Ficus trees  in Kibale Forest National Park situated in Uganda. Of these, we reared wasps from 11 individual trees comprising 9 species that were releasing wasps during the sampling period in August . This dioecious species was abundant and occurred as a small hemi-epiphyte clearly associated with disturbed vegetation. We did not keep accurate locality records of F. asperifolia because it was difficult to count often being found in close proximity to one another not easily discerned as individuals. Arachonia have not been reared from and are unlikely to use this divergent species as a host that typically produce extremely low numbers of divergent wasp fauna. However, we reared a wasp collection from F. asperifolia and confirmed this observation.We collected fig wasps from all species of n August . Only 6 rachonia . Ficus aFicus species we collected  that supports lower densities of Ficus. In order to provide maximal representation of genetic variation, the Arachonia sequence data from the forest patch was augmented with all other sources of available data from specimens collected between 2005 and 2011 from Southern Africa , cytochrome b (Cytb) and nuclear DNA (nDNA) elongation factor one alpha F2 copy (EF-1α) gene regions in one direction only. The head and thorax of single whole fig wasps, preserved in >96% ethanol, were used for DNA extractions and sequencing. A QIAGEN® DNEasy Kit was used for all DNA extractions following the manufacturer's protocol. SuperTherm Taq DNA Polymerase 250U was used in the PCR reactions. Amplifications of mitochondrial DNA were undertaken using the following: 94°C, 30 seconds denaturation; 50°C (mtDNA) or 56°C (nDNA), 1.5 minute annealing; 72°C, 1.5 minute extension for 45 cycles; with a final cycle of 72°C, 7 minute extension. The polymerase enzyme required a 94°C, 3 minutes incubation period for the first cycle only. The PCR mixture was a 25 µl reaction including: 0.2 µl of 5 U/µl of polymerase, 2.5 µl (10 mg/ml) of dNTPs0, 1.0 µl (0.2 pmol/µl) of each primer, an unknown concentration of template DNA, and 2.5 µl 10X buffer and 13.3 µl of distilled water, or 2.0 µl 10X buffer and 13.8 µl of distilled water respectively. Approximately 630 base pairs of the gene for COI was amplified using primer pair sets: COI-070368 forward primer 5′ TTA TCT TTA CCA GTA TTA GC 3′ with COI-070029 reverse primer 5′ AAT GTT GAG GGA AAA ATG T(CT) 3′ Cytb gene fragment were amplified using Cytb-070330 forward primer 5′ CTA CCA TGA GGA CAA ATA TC 3′ with Cytb-070326 reverse primer 5′ (AG)GA AT(TA) GAT CG(TA) A(AG)A AT(TA) GC 3′ EF-1α gene fragment was amplified using EF1a-080588 forward primer 5′-GGT CTT GGA CAA ACT GAA GG-3′ (McLeish unpublished) with EF1a-073534 reverse primer 5′-TTG TC(AG) GT(TG) GG(CT) CTG CT(TG) GG-3′. Sequences were aligned by eye against pre-existing sequence data of other sycoryctine fig wasps. Positions with ambiguous sites were coded with IUPAC symbols. Sequences were submitted to GenBank under the accession numbers JQ838891 to JQ838998 and JQ839017 to JQ839124  JQ839124 .COI and Cytb mtDNA sequence data (together 1032 bp's) to estimate genealogical associations in relation to Ficus species ranges. Ranked uncorrected-p and K2P distributions of COI genetic distances were estimated to provide a barcoding reference frame of divergences represented in the study  preceeds the connection of haplotypes into a network differing by increasing numbers of single site changes CO1 and Cytb mtDNA sequence data to generate haplotype networks with a 95% connection limit probability under statistical parsimony. Gaps were treated as missing and no connection limit step priors were set. To estimate genetic differentiation amongst haplotype groups, STF and P-values (0.05 significance level) were estimated using analyses of molecular variance (AMOVA) implemented in Arlequin version 3.0 STF coefficient is the proportion of the genetic variance within a subpopulation (S) relative to the total genetic variance (T). A high STF (closer to 1 than 0) implies substantial differentiation among groups and was expected under the hypothesis of populations representing putative species. The P-value of each test is the proportion of permutations resulting in an STF value larger or equal to the observed proportions. We estimated a gamma distribution prior of 0.5 using Modeltest version 3.0 STF and P-values.As bifurcating trees can violate assumptions of intraspecific genetic relationships because some individuals are identical, network approaches account for both intra- and interspecific processes. We inferred haplotype networks using he study . We usedhttp://R-Forge.R-project.org). An ultrametric tree reconstruction was generated using a strict molecular clock with gamma distributed invariant sites, GTR substitution prior, empirically estimated base pair frequencies, and unlinked codon positions implemented in BEAST v.1.4.8 The Generalised Mixed Yule Coalescent (GMYC) approach Arachonia specimens was inferred using the mtDNA sequence data to identify (exclusivity) putative population-level clades. A second inference was conducted to infer a phylogeny using a subset of 51 Arachonia specimens with the inclusion of the EF-1α nuclear DNA marker. The second analysis comprised taxa having unique Ficus species associations and without multiple exemplars of the same Ficus association of the same inferred population. A Bayesian approach was implemented using MrBayes 3.1.1 st, 2nd, and 3rd codon positions with both mtDNA and mtDNA-nDNA datasets. Substitution model priors are explained in As haplotypes are grouped according to similarities, bifurcating trees cannot always represent intraspecific relationships. However, the inherent low divergences associated between conspecifics will cause them to cluster and is useful for detecting like types. Poor statistical support (hard polytomies) at nodes within population clusters is expected. Phylogenetic inference is useful in assessing “exclusivity” of populations as monophyletic clades Species delimitation using the molecular approaches was assessed with appraisal of morphological variation among and within putative species. Specimens were dried from ethanol, point mounted, and examined using a Wild stereo microscope. Images were produced using the EntoVision multi-stacking imaging system. This system included a Leica M16 zoom lens attached to a JVC KY-75U 3-CCD digital video camera that fed image data to a notebook computer. The program Cartograph 5.6.0 was then used to merge an image series into a single in focus image. Lighting was achieved using techniques summarized in Figure S1Ultrametric phylogeny of Arachonia haplotypes inferred using a strict molecular clock implemented in BEAST. Red clades fall within the neutral coalescent model for intraspecific branching. The shift from branching under the Yule pure birth model was estimated using a mixed model likelihood test (P<0.001) called the generalized mixed Yule coalescent (GMYC) implemented using SPLITS.(TIF)Click here for additional data file.Figure S2Bayesian consensus haplotype phylogeny of Arachonia. The phylogeny was inferred using COI, and Cytb gene fragments and shows posterior probabilities above 90%.(TIF)Click here for additional data file.Figure S3Bayesian consensus phylogeny of the genus Arachonia. The phylogeny was inferred using COI, Cytb, and EF-1α gene fragments and showing posterior probabilities above 90%. Terminal taxa are shown as the isolate code followed by the Ficus species the specimen was collected from.(TIF)Click here for additional data file.Figure S4Arachonia species, lateral habitus. A: species 1; B: species 2; C: species 3; D: species 4.(TIF)Click here for additional data file.Figure S5Arachonia species, lateral habitus. A: species 5; B: species 6; C: species 7; D: species 4 (repeated for direct comparison with the similar species 7).(TIF)Click here for additional data file.Figure S6Ranked pair-wise uncorrected p and K2P COI distances for all specimens sequenced. Dashed lined indicate either the first instance of an interspecific pair-wise association or an intergeneric association. Note that both cases occur together in the distribution after the second dashed line.(TIF)Click here for additional data file.Table S1Arachonia specimens, voucher codes, location and DNA sequence assession numbers.(DOC)Click here for additional data file.Table S2An inventory of potential host fig wasp species specialising on the Ficus species from which Arachonia were collected in this study. The literature suggests that Sycoryctinae target pollinators mostly and other non-pollinator species only infrequently Galoglychia. Arachonia species might also be attacking the Sycophaginae belonging to section Sycomorus in addition to the pollinators. The ratio of parasitoid fig wasp genera to other pteromalids and the agaonids is potentially 2∶1 for each Ficus species from which collections were made in Kibale. However, relatively smaller parasitoid population sizes and differences in species diversity might instead be a clue to fundamentally different evolutionary diversification processes Ficus. The trophic interactions indicate the Agaoninae and Sychophaginae as prey species of the Sycoryctinae, but did not recognise those between the Otitesellinae and Sycoecinae. Infrequent interactions with the Otitesellinae have been observed (DOC)Click here for additional data file.Table S3Morphological delimitation of Arachonia species collected in this study showing their haplotype affinities.(DOC)Click here for additional data file.


Previous evidence indicates that duodenal lipid sensing engages gut-brain neurocircuits to determine food intake and hepatic glucose production, but a potential role for gut-brain communication in the control of energy expenditure remains to be determined. Here, we tested the hypothesis that duodenal lipid sensing activates a gut–brain–brown adipose tissue neuraxis to regulate thermogenesis. We demonstrate that direct administration of lipids into the duodenum increases brown fat temperature. Co-infusion of the local anesthetic tetracaine with duodenal lipids abolished the lipid-induced increase in brown fat temperature. Systemic administration of the CCKA receptor antagonist devazepide blocked the ability of duodenal lipids to increase brown fat thermogenesis. Parenchymal administration of the N-methyl-d-aspartate receptor blocker MK-801 directly into the caudomedial nucleus of the solitary tract also abolished duodenal lipid-induced activation of brown fat thermogenesis. These findings establish that duodenal lipid sensing activates a gut–brain–brown fat axis to determine brown fat temperature, and thereby reveal a previously unappreciated pathway that regulates thermogenesis. The energetic relevance of brown adipose tissue (BAT) thermogenesis in adult human physiology has recently found new support after the publication of three independent studies consistently demonstrating the presence of metabolically-active BAT in adult humans using tomography technologies Intraduodenal administration of lipids inhibits food intake in both rodents and humans Interestingly, the NTS also innervates the BAT Sprague Dawley rats  were housed in individual cages with wire mesh bottoms and maintained in a temperature-controlled room under a standard 12 h/12 h light/dark cycle with ad libitum access to water and standard chow unless specifically indicated. Animals were handled daily and trained to be connected to the injection systems for the 5 days preceding the experiments. All experimental protocols were approved by the Institute for Animal Studies of the Albert Einstein College of Medicine.Intralipid 20%  was dissolved in saline (50∶50) to a 1 kcal/ml emulsion. The topical anesthetic tetracaine-HCl (Sigma) was dissolved to a 5% solution in saline and used at a final concentration of 360 µg/ml, consistent with doses used to block intestinal extrinsic neural afferent activity and the feeding inhibitory effects of duodenal lipid infusions Rats were overnight food deprived before surgical procedures that were performed under ketamine/xylazine anesthesia. Rats underwent duodenal cannulation, as previously described An additional group of rats was also stereotaxically implanted with a bilateral steel guide cannula (Plastics One) positioned 2 mm above the caudomedial nucleus of the solitary tract . Beveled stainless steel injectors (33 gauge) extending 2.0 mm from the tip of the guide cannulas were used for injections. Accurate cannula placement was confirmed by consumption of more than 1.5 g of chow within the 60 minutes following a parenchymal injection of 24 µg of 5-thio-D-glucose (Sigma) in 100 nl of aCSF per side All injections were performed in a crossover manner and at least 4 days elapsed between each injection. Rats were fasted overnight, attached to a freely moving dual channel swivel assembly (Instech) mounted on their home cages, 2 hours before the beginning of the injections. Home cages were located atop ER-4000 receivers (Philips Respironics), and BAT temperature was monitored during the 2 h preceding and the 2 h following the beginning of the duodenal infusion, in a temperature-controlled room maintained at 72°F. On four separate test days, rats received a 30 min intraduodenal (ID) infusion of one four infusates: 5 ml saline, 5 ml saline +1.8 mg tetracaine-HCl, 5 ml IL 10%, or 5 ml IL 10% +1.8 mg tetracaine-HCl. In the DVZ experiment, rats received an intraperitoneal (ip) injection of 350 µl of saline or 1 mg/kg DVZ 15 min prior to an ID infusion of 5 ml saline or IL 10%. In the MK801 experiments, rats received a 2 min bilateral NTS injection of 100 nl aCSF or MK801 (0.1 nmol/side) 15 min prior to an ID infusion of 5 ml saline or IL 10%.All data, presented as means ± SEM, were analyzed using GraphPad Prism 5. For all statistical tests, an α risk of 5% was used. Data were analyzed using a mixed model ANOVA for repeated measurements and post-hoc tests were performed using Bonferroni correction.Intraduodenal saline infusion in overnight fasted rats minimally affected BAT temperature, which non-significantly decreased by 0.47±0.24°C during the 2 h following the beginning of the infusion . In contWe then asked whether activation of intestinal sensory fibers was implicated in the thermogenic effect induced by duodenal lipids using the local anesthetic tetracaine. Duodenal tetracaine blocks the ability of nutrients to suppress food intake and hepatic glucose production, without having any effect on food intake or glucose homeostasis when administered alone Results from multiple studies support the idea that CCKA receptors on gut vagal afferents play critical roles in relaying duodenal lipid signals important in the negative feedback control food intake and glucose homeostasis N-methyl-d-aspartate (NMDA) receptors have been localized to vagal afferent terminals in the hindbrain NTS Taken together, these data support the suggestion that duodenal lipid sensing activates a neurocircuit engaging CCKA receptors on vagal sensory fibers and their glutamatergic projections in the NTS to regulate BAT temperature, revealing a role for a gut-brain-BAT axis in the control of thermogenesis.The present data are consistent with previous findings demonstrating that duodenal lipid infusions in awake behaving rats at doses much lower than those employed in the present study, delivered at comparable rates (4.5 vs 5 ml/30 min), elicit robust c-fos activation in NTS at the level of the AP We cannot rule out the possibility that the gut luminal exposure to tetracaine may non-specifically modulate local gut neural function in ways not related to the vagal afferent signaling of duodenal lipid availability. For example, as tetracaine is an anesthetic, it might be anticipated to affect intrinsic enteric gut neurons that determine local gut motility. Furthermore, local luminal distribution of lipid infusion may have been affected by tetracaine. However, doses two times greater than those used in the present study have been shown to induce minimal changes in duodenal, colonic or gastric motility, and have no effect on acetylcholine-induced contractions of the isolated rat duodenum, colon or stomach It is also important to consider that we do not directly demonstrate that duodenal tetracaine blocks the ability of lipid to activate the proposed NTS component of the gut-brainstem BAT circuit. For example, gut tetracaine would be predicted to reduce the NTS c-fos response to duodenal lipids under these circumstances. However, damage to the gut afferent vagus nerve has been shown to block the ability of duodenal lipid to stimulate NTS c-fos The present data do not directly address the degree to which CCKA receptors specifically within gut vagal afferents are responsible for the ability of duodenal lipids to increase BAT thermogenesis. Prior studies demonstrated that intestinal infusions of MK329 were sufficient to block the ability of duodenal CCK to inhibit hepatic glucose production Based on the ability of the CCKA receptor antagonist devazepide to block duodenal lipid induced temperature elevations, it is reasonable to anticipate that other nutrient secretagogues of CCK, such as proteins and amino acids, would also stimulate BAT thermogenesis via this pathway. Conversely, nutrients that are poor plasma CCK secretagogues, such as carbohydrates Vagal afferent fibers innervating the proximal small intestine, as well as their brainstem terminations at the caudomedial level of the nucleus of the solitary tract, express both NR1 and NR2 NMDA receptor subunits The present effects of NMDA receptor blockade may not be not strictly limited to the stereotaxically targeted medial NTS through the rostrocaudal extent of the are postrema (AP). However, we have recently demonstrated that labeled tracer injections targeting the mNTS, delivered at equivalent rates and volumes (100 nl/2 min), were confined to the medial NTS at the level of the AP, and did not extend into the subjacent dorsal motor vagal nucleus (DMX) at this level, nor do they impinge on the DMX at more rostral or caudal levels of the brainstem The molecular and neuroanatomical pathways linking NTS glutamatergic stimulation to BAT thermogenesis remain unclear, but GABAergic modulation of brainstem glutamatergic transmission may be important. Disinhibition of medial NTS neuronal activity using GABA antagonists blocks the BAT thermogenic response to cold challenges, and NTS GABA agonist administration facilitates BAT thermogenesis via sympathetic premotor neurons in the raphe pallidus In summary, the present data identify a novel role for gut nutrient sensing in the control of BAT thermogenesis, a potentially important contributor to overall energy balance. This gut-brain-BAT circuit identifies multiple peripheral and central neuroendocrine nodes where pharmacological or nutrient intervention may reveal new molecular therapeutic targets that modulate energy expenditure in obesity and related metabolic diseases.


Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P = 0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents.Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new “multidimensional” capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional  Glioblastoma Multiforme (GBM) is the most common and deadly  adult central nervous system malignancy. Recent evidence demonstrates that a subset of GBM tumor cells harbor an undifferentiated stem cell-like phenotype and are refractory to conventional chemotherapeutic agents To overcome the technical limitations of 2D-PAGE described above and to gain deeper insight into the potential TSC protein markers, we utilized a new method of “multidimensional” capillary isoelectric focusing nano-reversed-phase liquid chromatography with tandem mass spectroscopy (CIEF-nRPLC-MS) to compare the proteomes of primary TSC cultures before and after induction of differentiation with ciliary neurotrophic factor (CNTF) Using CIEF-nRPLC-MS, we isolated a number of TSC membrane proteins that showed at least a 3 log-fold difference in expression after differentiation. Among these, both α and β isoforms of the proteolipid, neuronatin (NNAT), were found to be consistently expressed in TSCs but non-existent within their differentiated counterparts. Given the putative involvement of NNAT with the embryological development of the CNS, as well as reports of its association with more aggressive phenotypes in medulloblastoma Our findings indicate that NNAT is endogenously expressed at low levels in normal human brain tissue, but becomes over expressed in a subset of primary GBM tumors. When transfected into U87 glioma cells, both isoforms of NNAT were associated with significant increases in cellular proliferation. Furthermore, high levels of NNAT expression were correlated to significantly shorter overall survival in a separate cohort of GBM patients. The molecular mechanisms underlying this effect have yet to be elucidated, but given NNAT's expression pattern, it may play a specific role in the pathogenesis or maintenance of primary GBM TSCs.TSCs were established from human GBM tissue. Cultured cells were maintained in an undifferentiated state, confirmed by positive immunocytochemical staining for the neural stem cell markers Nestin and Sox2. Differentiation of TSCs was induced by treatment with CNTF and demonstrated by loss of Nestin and Sox2 expression, as well as concomitant gain of GFAP expression as previously described Both undifferentiated and CNTF-treated differentiated glioma TSCs were analyzed using CIEF-nRPLC-MS/MS. Peptide identification was based on 3 runs of a single tissue sample and was limited by high-mass-accuracy (60 ppm) and high-confidence  hits to fully tryptic proteins. Expression differences between the undifferentiated and the differentiated TSC proteomes yielded 174 membrane proteins. Among those proteins, both NNATα and NNATβ were identified to have greater than 3-log fold levels in TSCs prior to differentiation with near undetectable levels found in the differentiated cells.To assess the efficiency and specificity of a commercial antibody derived against NNAT, we used western blotting to probe for NNAT in brain tumor derived cell lines (U87 and U251), normal brain tissue and tumor resection specimens . NNAT exAfter validating the NNAT antibody, we sought to assess the prevalence of NNAT expression within resected GBM samples. We initially evaluated 13 GBMs and identified NNAT expression through western blot analysis . We founThe absence of NNAT expression within lower grade astrocytomas (Grade II and III) prompted us to question whether its expression is limited to primary de novo GBMs. Subsequently, we assessed for over expression of EGFR, a finding strongly associated with primary GBMS, among NNAT positive tumors and found all of them to be strongly positive for EGFR over expression . We thenA summary of patient characteristics in the survival analysis is detailed in The high expression of NNAT limited to a subset of primary GBMs indicates its potential functional impact in tumor growth and cell proliferation. Prior studies have found NNAT to be a potent stimulator of cellular proliferation in medulloblastoma cells The impact of NNAT on tumor cell growth was first assessed through the MTT proliferation assay , in whicUsing our high throughput proteomic technique, CIEF-nRPLC-MS, we identified a number of novel differentially expressed membrane proteins in GBM TSCs versus their differentiated counterparts. NNAT was identified and selected for further investigation due to its previously described role in embryologic CNS development as well as its association with more aggressive phenotypes of medulloblastoma Current knowledge regarding the functional role of NNAT is still limited. Structurally, NNAT belongs to the proteolipid family of proteins and is thought to be membrane associated due to having an N-terminus that is heavily enriched with hydrophobic amino acids. Multiple studies examining other cell types have found NNAT localization to be robust not only on the cell membrane, but also in the cytoplasm as well Previous studies in medulloblastoma show that expression of both NNAT isoforms is associated with increased proliferation and anchorage independent growth, but that the presence of NNATα is the primary determinate of cytologic response in vitro proliferation  treated at the University of Regensburg Medical Center were investigated. Mean age of the patients was 59.5 years , the median Karnofsy Performance Score was 90, . All patients underwent initial craniotomy for tumor resection. After resection, patients received combined adjuvant treatment consisting of external beam radiation and chemotherapy with temozolomide according to the Stupp regimen Primary TSC cultures from freshly procured human GBM tumors were obtained and expanded as previously described Samples of TSCs prior to and after treatment with CNTF were harvested for proteomic profiling by Calibrant Biosystems , as previously described Microdissected tissue and cell pellets were collected and lysed in T-PER lysis buffer (Thermo), sonicated, and centrifuged. The quantity of protein was determined using a colorimetric Bio-Rad Protein Assay Kit (Bio-Rad). Proteins were separated on NuPAGE 4 to 12% Gradient Bis-Tris gels (Invitrogen) and transferred to PVDF membranes. Membranes were blocked in 5% skim milk in TBS with 0.05% Tween-20 and probed with primary antibody. Visualization was carried out through a species specific horseradish peroxidase conjugated secondary antibody system paired with the Pierce SuperSignal West Pico enhanced chemilumescent substrate .Antibodies used in this study: EGFR , GFAP , Nestin , NNAT , β-Actin , Sox2 .Cells or tissue slices were fixed in Histochoice H108  and incubated in 0.3% Triton X-100/TBS before being blocked with 5% BSA/TBS and subsequently labeled with anti-NNAT  antibodies overnight. For immunofluorescence studies, visualization utilized a species specific Alexa Fluor conjugated secondary antibody (Sigma) followed by nuclear counterstaining with Hoechst 33342 (Invitrogen) before examination using a Zeiss LSM510 confocal microscope. Immunohistochemistry studies utilized a species specific horseradish peroxidase conjugated secondary antibody paired with the VECTASTAIN ABC System  for colorimetric detection. The specimens were then examined using a Leica DM LB light microscope. Grading of immunohistochemical staining was performed by three observers who were blinded to the clinical outcome of the patients. The rating of the immunohistochemical staining was performed by using a four-grade, semiquantitative scale . High NNAT expression was defined by an average rating higher than grade 3 for each patient.5′TTTCTCGACCACCCACCTAC; Reverse – 5′CGGCAATCGGAATAGCAC), Exon 2, Exon 3. Sequencing was subsequently performed at the NINDS DNA Sequencing Facility and results were aligned and analyzed using the ClustalW2 program available online.DNA extraction from human GBM tumors was performed using the Qiagen DNeasy Blood and Tissue Kit . Genomic DNA was utilized as a template for polymerase chain reaction targeting the 3 coding regions of NNAT. The primers are as follow: Exon 1 ( Forward – 7 U87 MG cells with lipofectamine 2000 reagent (Invitrogen). Media was replaced on the fifth day with selection media containing 400 µg/mL G418 and the lines were maintained in selection media for 2 weeks. Stable clones were isolated through three rounds of fluorescence-activated cell sorting (FACS) as previously described The full-length human cDNA of the NNATα and NNATβ isoforms were inserted into pCMV6 mammalian expression vectors. To distinguish the two transcriptional variants, NNATα was fused with an EGFP tag, whereas NNATβ was fused with a DsRed tag. Twenty-four µg of NNATα-EGFP or NNATβ-DsRed vector was delivered into 1.0×10To visualize cell cycle changes, stable transfected U87 MG cells were labeled with 20 µM EdU (Invitrogen) for 4 hours. Cells were then labeled using Click-iT EdU HCS assay kit with either Alexa Fluor 488 or pacific blue labeling. DNA was counterstained with DRAQ5 . Cells were analyzed using an Image Stream X flow cytometry system (Amnis). IDEAS software was used to acquire and quantify fluorescent signal intensities and to graph the data as bivariate dot density plots for cell cycle determination.Comparative significance testing of continuous variables was performed using the students t-test with a significance threshold defined at P<0.05. For the clinical analysis, survival time was measured from the date of resection until death of the patients. Survival rate was calculated by the Kaplan–Meier method and comparisons of survival between the different groups were estimated by generalized Wilcoxon test for univariate analysis. For multivariate analysis, Cox's proportional hazards model (forward stepwise procedure) was used. In addition to NNAT expression, age, Karnofsky score, and the extent of resection were analyzed as potential prognostic factors.


Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are facultative Gram-negative bacilli that cause melioidosis and glanders, respectively. The inhalation of these pathogens can cause lethal disease and death in humans. We sought to compare the pathogenesis of and host responses to BP and BM through contact with human primary ATII cells and monocytes-derived macrophages (MDM). We hypothesized that because BP and BM induce different disease outcomes, each pathogen would induce distinct, unique host immune responses from resident pulmonary cells. Our findings showed that BP adhered readily to ATII cells compared to BM. BP, but not BM, was rapidly internalized by macrophages where it replicated to high numbers. Further, BP-induced significantly higher levels of pro-inflammatory cytokine secretion from ATII cells  and macrophages  at 6 h post-infection compared to BM (p < 0.05). Interestingly, BM-induced the anti-inflammatory cytokine, IL-10, in ATII cells and macrophages at 6 h post-infection, with delayed induction of inflammatory cytokines at 24 h post-infection. Because BP is flagellated and produces LPS, we confirmed that it stimulated both Toll-like receptor (TLR) 4 and TLR5 via NF-κb activation while the non-flagellated BM stimulated only TLR4. These data show the differences in BP and BM pathogenicity in the lung when infecting human ATII cells and macrophages and demonstrate the ability of these pathogens to elicit distinct immune responses from resident lung cells which may open new targets for therapeutic intervention to fight against these pathogens.Alveolar type II pneumocytes (ATII) and alveolar macrophages (AM) play a crucial role in the lung's innate immune response.  Burkholderia pseudomallei (BP) and Burkholderia mallei (BM) are facultative-intracellular Gram-negative bacilli that cause melioidosis and glanders, respectively  encoding genes responsible for environmental adaptation and bacterial survival including the type III secretion system, capsule, and flagellum  encodes for genes involved in exopolysaccharide capsule, LPS, type IV pili, and type III and IV secretion systems . Pulmonary defenses have evolved such that the physiological positioning of resident alveolar cells are able to clear and/or fight against harmful particles. For example, alveolar macrophages (AM) are considered the first line of defense against inhaled pathogens. Successful pathogenic bacteria have developed mechanisms to subvert host defenses. Murine studies with BP showed macrophage invasion without activation of inducible nitric oxide synthase responsible for production of nitric oxide crucial to host attacks on intracellular bacteria  via TLR4, however, these studies did not examine the intact live pathogen. The interaction of BM with primary human macrophages is less studied and poorly understood.Although both pathogens share some antigenicity and virulence-associated similarities their disease outcomes are quite unique. While both Burkholderia is ATII cells, that represent approximately 4–5% of the cells in the alveolar space but comprises 15% of all the cells in the lung  China 7  and Burkholderia pseudomallei (BP) China 3  were obtained from BEI Resources  and propagated overnight at 37°C in Luria-Bertani (LB) broth  from frozen stocks. Bacterial titers were determined using a Bel-Art—KLETT™ Colorimeter  and confirmed by plating serial dilutions on LB agar plates. Both BM and BP are CDC classified category A select agent thus they were used in UTMB's certified BSL3 facility as per CDC and Federal regulations.Primary human type II alveolar epithelial (ATII) cells  were seeded on BioCoat Growth Factor Reduced Matrigel®-coated transwell filters  and maintained using Bronchial Epithelial Medium enhanced with the Bullet kit supplements provided by the manufacturer  supplemented with 10% charcoal-stripped FBS  and 10 ng/mL of keratinocyte growth factor . ATII cells were grown to confluence (4–5 days) then used for experiments. Before each experiment, the A549 or ATII cells were washed 2X with appropriate medium containing no serum or antibiotics. The serum-free medium used during experimentation ensured no interference by serum proteins and did not adversely affect the health or metabolic activity of the cells.Human monocytes were isolated from peripheral blood mononuclear cells obtained from healthy donors with full approval of the UTMB IRB. Monocytes were isolated using a Hypaque-Ficoll  density gradient (800 × g at 21°C) followed by selection using a human monocyte enrichment kit without cd16 depletion (Stemcell #19058) and Easysep magnet (Stemcell #18000) counterflow centrifugal elutriation . The purified monocytes were cultured in RPMI 1640, 10% FBS supplemented with GM-CSF (100 ng/mL) for 7 days to ensure differentiation into macrophages as previously described  were infected with BM or BP at an MOI of 100:1. Un-stimulated cells were used as negative controls. At 6 h post-infection, supernatants were collected from ATII cells and MDM then frozen at −80°C until analysis. At the same time point, parallel ATII cells or MDM cultures were washed with 2 mL of PBS then incubated for 30 min with serum-free medium containing 50 ug/mL of gentamicin (Sigma). ATII cells and MDM were washed then resuspended in serum-free medium containing 5 ug/mL of gentamicin and incubated an additional 14 h at 37°C/5% CO2. Supernatants were collected at 20 h post-infection then 6 and 20 h supernatants were analyzed via individual human ELISA kits (Pierce-Endogen) for the following cytokines: IL-6, IL-8, IL-10, and/or TNF-α.ATII cells or MDM . Samples were visualized using a Hitachi S4700 field emission scanning electron microscope  at 2 kV (UTMB Electron Microscopy Laboratory Facility).ATII cells grown on transwell filters were exposed apically to BM and BP to adhere to ATII cells was performed as previously described  were stimulated with BP or BM (MOI 100:1). To examine phagocytosis and intracellular replication of BM and BP, at 1 h post-infection, macrophages were washed with 2 mL of PBS then incubated for 30 min with serum-free medium containing 50 ug/mL of gentamicin (Sigma) to kill extracellular bacteria  were used as positive controls that included LPS (TLR4) and flagellin (TLR5). Medium alone and HEK293 cells with no agonist served as negative controls. The cells were incubated for 6 h then medium was aspirated and replaced with fresh DMEM with antibiotics and incubated overnight at 37°C and 5% CO2. Collected Supernatants were heat inactivated for 30 min at 65°C. SEAP activity in each sample was quantified  were analyzed for the amount of protein in each sample by the Bradford assay . The supernatants were boiled in loading buffer  then 40 μg of protein was loaded on an 8–16% Tris-glycine gradient gel . Electrophoresed proteins were transferred to a nitrocellulose membrane (Novex) and membranes were blocked with 10% nonfat dried milk for 30 min prior to incubation with rabbit polyclonal anti-(FliC) flagellin  at a dilution of 1:1000 overnight. Blots were washed, followed by the addition of peroxidase-conjugated anti-rabbit immunoglobulin G  at a dilution of 1:10,000 for 3 h. Blots were washed and then incubated for 1 min in enhanced chemiluminescence reagents . Processed blots were placed on X-ray film (Kodak®) for empirically optimized exposures.Western blots were performed as we described previously  using Prism software . Differences were noted to be statistically significant when the p value was < 0.05.Numerical results are presented herein as mean ± SEM of two or three-independent experiments containing replicate experimental conditions per each experiment. Statistically analysis of numerical data was completed by Student's BM or BP and ATII cells during early stages of a pulmonary infection. To remain biologically and physiologically relevant, ATII cells were grown on transwell filters to allow polarization before being infected apically with BM or BP. Four hours post-infection, scanning electron microscopy (SEM) was used to visualize interactions between the bacteria and the ATII cells. Figure BM and BP's contact with the ATII cells. BM contacted the apical surface of ATII cells but did not appear to readily adhere to the ATII cells at 4 h post-infection as such the pathogen could be seen floating free in the medium . At the later time point it was observed that BM provoked responses. Specifically, secretion of IL-6 and TNF-α from MDM and IL-6 and IL-8 from ATII cells increased at 20 h post-infection  in the early stages of infection then transitioned into a traditional immune response.These data demonstrate that BM and BP interacted with MDM and ATII cells to induce cytokines, we sought to determine the invasion capabilities and intracellular replication of BM and BP in these host cells. To this end, MDM and ATII cells were infected with BM or BP (MOI 100:1) for 1 h to determine phagocytosis of the pathogens and at 3 h post-infection to assess intracellular replication. Cells were lysed at each time point, plated then the bacterial CFU were quantified. Several observations were made from these data results. First, BP was more readily phagocytosed at 1 h post-infection by hMDM , which served as positive controls. Our results showed that both BP and BM-induced NF-κb activation via TLR4 stimulation that was equivalent to purified LPS and significantly higher than flagellin or un-stimulated controls  induced by macrophages and ATII cells in the early stages of infection. Sim et al.  between these findings may be explained by the fact that our studies used primary human monocytes-derived macrophages in suspension while Brett et al. (Cheng et al., BM. As a result of our findings, we may hypothesize that the lack of inflammatory cytokine secretion but increased secretion of IL-10 by macrophages and ATII cells is a mechanism evolved by BM to avoid detection by the host. This evasion by BM would allow the pathogen time to adapt to the intracellular environment and replicate before the host detects it and is able to mount an effective, timely immune response.While the intact flagella structure contributes to the adherence and invasion of a pathogen to host cells, it is the primary protein component of the flagellum structure, known as flagellin, that is now recognized as a major virulence factor of Gram-negative organisms (Eaves-Pyles et al., m et al.  showed tt et al.  found thBM and BP compared to ATII cells. Additionally, although BP readily adhered to ATII cells, there was minimal invasion of and replication in ATII cells by both pathogens. Other studies have shown that BP adhere, invade, and replicate to adequate numbers in transformed ATII cell lines (Whitlock et al., Burkholderia pathogenesis using primary human alveolar type II cells in a physiological relevant ex vivo model. While ATII cells are not professional immune cells, they contribute significantly to the pulmonary immune defenses against inhaled invaders via cytokine secretion and surfactant A and D (Crapo et al., BM and BP, however, they are not a reservoir of invasion and intracellular replication for these pathogens, which may open new avenues of the host innate immune response for therapeutic treatment of melioidosis and glanders.Because alveolar macrophages are at the forefront of the host's immune defenses, it was not surprising that macrophages secreted higher levels of cytokines overall in the response to BM and BP pathogenesis regarding their interaction with primary human ATII cells and macrophages. BM and BP are similar in various aspects of their pathogenesis they produce different disease outcomes. Our study demonstrates that each of these pathogens has several unique strain specific characteristics is revealed in their interactions with host cells to induce varied host immune responses. By delineating host immune responses to BM and BP we can better understand the pathogenesis of these pathogens and develop new directions for therapeutic intervention and vaccines.In conclusion, we have shown various distinct differences between 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.


Dramatic advances in recent decades in understanding the genetics of Friedreich ataxia (FRDA)—a GAA triplet expansion causing greatly reduced expression of the mitochondrial protein frataxin—have thus far yielded no therapeutic dividend, since there remain no effective treatments that prevent or even slow the inevitable progressive disability in affected individuals. Clinical interventions that restore frataxin expression are attractive therapeutic approaches, as, in theory, it may be possible to re-establish normal function in frataxin deficient cells if frataxin levels are increased above a specific threshold. With this in mind several drugs and cytokines have been tested for their ability to increase frataxin levels. Cell transplantation strategies may provide an alternative approach to this therapeutic aim, and may also offer more widespread cellular protective roles in FRDA. Here we show a direct link between frataxin expression in fibroblasts derived from FRDA patients with both decreased expression of hydrogen peroxide scavenging enzymes and increased sensitivity to hydrogen peroxide-mediated toxicity. We demonstrate that normal human mesenchymal stem cells (MSCs) induce both an increase in frataxin gene and protein expression in FRDA fibroblasts via secretion of soluble factors. Finally, we show that exposure to factors produced by human MSCs increases resistance to hydrogen peroxide-mediated toxicity in FRDA fibroblasts through, at least in part, restoring the expression of the hydrogen peroxide scavenging enzymes catalase and glutathione peroxidase 1. These findings suggest, for the first time, that stem cells may increase frataxin levels in FRDA and transplantation of MSCs may offer an effective treatment for these patients. FXN gene  is the commonest autosomal recessive ataxic condition, affecting around 1 in 50,000 of the population in vitro and in vivoCurrently there are no effective treatments to prevent the progression of FRDA. Major therapeutic strategies in FRDA include development of agents to protect against oxidative damage and mitochondrial respiratory chain defects or agents that increase cellular frataxin expression, since cells lacking frataxin can be rescued through frataxin expression Administration of MSCs is an attractive therapeutic option for FRDA for several reasons: cells are easily isolated from various anatomical sources, have a versatile growth and differentiation potential, and, in addition, the immunosuppressive properties of MSCs make it probable that allogeneic as well as autologous cell therapy could be considered. If human MSC transplantation is to become a viable therapeutic approach for FRDA, a precise understanding of the effects of MSCs and their secreted factors on cells derived from FRDA patients is required.Here we have performed a series of experiments showing that human MSCs increase both frataxin gene and protein expression in fibroblasts derived from patients with FRDA via secretion of soluble factors. We show a direct link between frataxin expression in fibroblasts derived from FRDA patients with both decreased expression of hydrogen peroxide scavenging enzymes and increased sensitivity to hydrogen peroxide-mediated toxicity. Furthermore, we have demonstrated that exposure to factors produced by MSCs increase resistance to hydrogen peroxide mediated toxicity in these fibroblasts through, at least in part, restoring the expression of the hydrogen peroxide scavenging enzymes catalase and glutathione peroxidase 1. These findings suggest that MSCs may have potential as therapeutic agents for FRDA and, in time, transplantation of bone marrow-derived MSCs may offer an effective cellular therapy in these patients.Primary fibroblast cells, derived from skin biopsies of Caucasian FRDA and control patients, were obtained from the Coreill cell repositories . Fibroblasts were grown in DMEM with 10% foetal calf serum (FCS) , and 100 U/ml penicillin and 100 µg/ml streptomycin . All experiments were on fibroblast cultures between 8–12 passages.5 cells were then transfected with 2.0 µg GFP-tagged ORF clone of human frataxin (FXN)  according to manufactures instructions. The nucleofector programme U-23 was chosen for high transfection efficiency. Cells were then re-suspend in 2ml DMEM/10% FCS and cultured within one well of a 6-well plate. Fluorescence microscopy was used to visualise GFP-tagged frataxin within FXN transfected fibroblasts. For a mock-treated control, distilled water was used to replace the vector expression system.Transfection of fibroblasts was carried out using Amaxa Nucleofector kits for primary cells . Prior to nucleofection, confluent fibroblast cultures trypsinized with trypsin-EDTA. Proteolysis by trypsin was stopped with the addition of DMEM supplemented with 10% FCS. 3×10Bone marrow samples were obtained by an orthopaedic surgeon at Southmead Hospital, Bristol, UK with informed written consent and hospital ethic committee approval by the North Bristol NHS trust research ethics committee. Bone marrow was taken at the time of total hip replacement surgery from the femoral shaft and placed into a sterile 50ml tube containing 1000 I.U heparin. Patients with a history of malignancy, immune disorders or rheumatoid arthritis were excluded from the study. Femoral shaft bone marrow donors were healthy apart from osteoarthritis, and were not receiving drugs known to be associated with myelosuppression or bone-marrow failure.Femoral shaft marrow samples were broken up with a scalpel and washed with Dulbecco's Modified Eagles Medium (DMEM)  until remaining material (bone) looked white at the bottom of the 50 ml tube. All washings were pipetted into a new 50 ml tube and kept for centrifugation. The suspension was centrifuged and re-suspended in DMEM and overlaid onto an equal volume of Lymphoprep  and centrifuged at 600 g for 35 minutes at room temperature to separate the mononuclear cells (MNC) from neutrophils and red cells. The MNC layer was harvested and washed twice in DMEM.2) containing 10 ml of MSC medium were seeded with 1×107 cells for primary culture. Flasks were incubated at 37°C in a humidified atmosphere containing 5% CO2 and fed every week with MSC medium by half medium exchange to remove non-adherent hematopoietic cells until the adherent fibroblast-like MSCs reached approximately 70% confluence. On reaching confluence the adherent cells were re-suspended using 0.25% trypsin  and re-seeded at 2.25×105 cells per (75 cm2) flask into first passage. Cultures were then incubated, fed every week with MSC medium by half medium exchange, and again trypsinised, a cell count taken and re-seeded at 2.25×105 cells per flask (75 cm2).Isolated MNCs were centrifuged and re-suspended in MSC medium consisting of DMEM with 10% foetal calf serum (FCS) selected for the growth of MSCs , and 100 U/ml penicillin and 100 µg/ml streptomycin . Vented flasks Mesenchymal stem cell-conditioned medium, derived from MSCs at varying passages, was analyzed by ELISA using the Human Erythropoietin ELISA kit  according to the manufacturer's instructions. All samples were analyzed in triplicate.5 cells were added to lysis solution plus DNase (5 minutes) before the addition of stop solution (2 minutes). All RNA samples were quantified using a Qubit Fluorometer and Quant-iT RNA assay kit  according to manufacturers' instructions to ensure equal loading of RNA samples. Samples were stored at −80°C prior to use. To synthesize cDNA, 100 ng of extracted RNA was added to the reverse transcription buffer and RT enzyme mix, placed in a thermal cycler, and incubated at 37°C for 1 hour and 95°C for 5 minutes. RT-PCR was performed using the StepOnePlus Real-Time PCR System  with Assay-on-demand Gene Expression Products for FXN  or 18S rRNA  using 10 ng of cDNA in a total volume of 20 µl. Reactions were run at 50°C for 2 minutes; 95°C for 10 minutes; and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. All samples were analyzed in triplicate. The relative gene expression  of FXN was calculated using the 2-ΔΔCt method, and the mean taken for each group. 18S rRNA was used as the reference ‘housekeeping’ gene.Fibroblast cultures were washed in DMEM, trypsinised and resuspended in PBS. RNA was extracted and cDNA produced using the Taqman gene expression cells-Ct-kit  according to the manufacturer's instructions. 10Fibroblasts cultured in a 6-well plate were lysed using Beadlyte cell signalling universal lysis buffer . The Qubit Fluorometer and Quant-iT protein assay kit  was then used to quantify the concentration of total protein within each cell lysate sample according to manufacturers' instructions to ensure equal loading of cell lysates. Lysates were heated to 95°C for five minutes with Laemmli 2x sample buffer  and run on Tris-HCl 10–20% ready gels . After transfer to a nitrocellulose membrane  and blocking in Tris-buffered saline/5% bovine serum albumin, membranes were incubated overnight in primary antibody at 4°C . Antibodies used were mouse anti-human frataxin (this antibody recognizes a band migrating at approximately 18 kDa corresponding to the mature Frataxin protein isoform (aa 56–210)) , mouse anti-GAPDH , rabbit anti-catalase , rabbit anti-glutathione peroxidase 1 (GPX1) . Immunoreactivity was detected using secondary anti-rabbit or anti-mouse horseradish peroxidase conjugated antibodies   and specific protein expression patterns were visualized by chemiluminescence using an Amersham ECL Plus Western Blotting Detection System . Densitometic analysis of western blot bands was performed using ImageJ software .Frataxin levels in fibroblasts-derived from patients with FRDA were measured using MitoSciences frataxin dipstick (MSF31)  assays according to manufacturers' instructions. Briefly, 10 µg of fibroblast protein in 25 µL of extraction buffer was mixed with 25 µL blocking buffer and added to individual wells on a 96-well plate with gold-conjugated monoclonal antibody at the bottom of each well. Dipsticks were then inserted into the wells and frataxin within the each sample was immunocaptured onto designated capture zones on the dipstick. Densitometic analysis of capture zones on developed dipsticks were quantified using ImageJ software .in vitro culture, cells were exposed to experimental conditions. Medium was removed from all wells and cells were washed twice in DMEM. Minimal medium and/or MSC conditioned medium was added to appropriate wells with/without the addition of 3-AminoTriazole . After a further 24 hours media was removed from all wells and replaced with minimal medium containing hydrogen peroxide (600 µM)  with/without the addition of 3-AminoTriazole , catalase from human erythrocytes , glutathione peroxidase from human erythrocytes   for the given timed cell survival experiment (data not shown)). Hydrogen peroxide levels were detectable in the media for approximately 5 hours, therefore at 6 hours post addition of hydrogen peroxide, evaluation of fibroblast cell survival was carried out using the 3--2,5-diphenyltetrazolium bromide (MTT) cell viability assay (96-well plates). For assessment of hydrogen peroxide cytotoxicity in FXN-transfected cells, fibroblasts were cultured in DMEM/10%FCS for a total of 48 hours after seeding into wells prior to varying concentrations hydrogen peroxide for 6 hours as above.Confluent fibroblast cultures were washed in DMEM, trypsinised and cultured in 96-well plates  in DMEM/10%FCS. At 24 hours Briefly, the supernatant was removed and replaced with HBSS/10%FCS containing 1 mg/ml MTT . Cells were then incubated for 1 hour at 37°C and subsequently the HBSS/10%FCS/MTT solution was removed by inverting the plate and the plate was left to dry. DMSO  was then added to each well and the absorbance of the solution in each well read in a plate reader at 540 nm. Cell survival was assessed as a ratio of the amount of formazan production relative to the respective control. In all cases, control cultures grown throughout the experimental period in base ‘minimal’ medium were analyzed and values for the experimental conditions divided by this value, in order to standardize results between experiments.post hoc Bonferroni testing between groups where appropriate. Paired or unpaired t-tests were used for analysis of normally distributed data. Values are expressed as the mean +/− SEM from at least three independent experiments, taking p<0.05 to represent statistical significance.Statistical comparisons were analyzed using one way ANOVA with in vitro and frataxin expression investigated using western blotting and RT-PCR techniques.Primary fibroblast cultures derived from skin biopsies of FRDA and healthy control patients were established see . FibroblWestern blot analysis was used to detect the cellular protein expression of frataxin, and to verify the characteristic decrease in frataxin expression within cells derived from patients with FRDA. Detection using a anti-human frataxin (exon 4) IgG2 antibody revealed a frataxin protein band at approximately 18kDa for both healthy control and FRDA cell lysates which corresponds to the predicted molecular weight of frataxin . Image aHaving shown a significantly reduced frataxin expression in fibroblasts derived from patients with FRDA, we next wished to determine the effects of MSC-conditioned medium on frataxin expression by these cells. Fibroblast cells were cultured for 24 hours before exposure to experimental conditions. Using immunoblotting techniques, after 24 hours post exposure to MSC-conditioned medium, frataxin expression in fibroblasts derived from patients with FRDA  and healA frataxin lateral flow dipstick immunoassay was also used to investigate the MSC-conditioned medium induced increase in frataxin expression in fibroblast derived from patients with FRDA. After 24 hours post exposure to MSC-conditioned medium, frataxin expression in fibroblasts derived from patients with FRDA was detected . Image aTo gain a further understanding into the mechanisms by which MSC-conditioned medium promotes an increase in frataxin expression, we investigated frataxin expression at the genomic level using RT-PCR. At 2, 6 and 24 hours frataxin mRNA levels from both FRDA patients  and healTo determine if MSCs secrete erythropoietin, we performed an erythropoietin ELISA on four different MSC-conditioned medium samples. Using this quantitative method, no detectable amount of erythropoietin was observed in any of the samples tested (data not shown).We next determined the effects of MSC-conditioned medium on expression of the hydrogen peroxide scavenging enzymes, catalase and glutathione peroxidase 1, in fibroblasts derived from FRDA patients and healthy controls. Fibroblast cells were cultured for 24 hours before exposure to experimental conditions. Using immunoblotting techniques and image analysis using ImageJ software, it was calculated that both catalase and glutathione peroxidase 1 expression in fibroblasts derived from patients with FRDA was significantly lower in expression when compared to the age matched healthy controls  (p<0.05)FXN with approximately 76% (SE +/− 2) of the fibroblasts expressing of GFP 48 hours following nucleofection , no significant increase in glutathione peroxidase 1 expression was evident (p>0.05)   . FXN-tra(p>0.05) . GAPDH eFXN-transfection in fibroblasts-derived from patients with FRDA resulted in a significant increase in cell survival when compared to cells exposed to minimal medium alone at identical concentrations of hydrogen peroxide (p<0.05)  . Followi(p<0.05) . The addBoth the addition of catalase or glutathione peroxidase (derived from human erythrocytes) at the time of hydrogen peroxide exposure, led to an increase in FRDA fibroblast cell survival when compared to hydrogen peroxide alone (p<0.05) .Friedreich ataxia is an incurable, progressive neurodegenerative condition characterized by multiple symptoms including gait and limb ataxia, dysarthria and an array of systemic complications including diabetes mellitus, scoliosis and hypertrophic cardiomyopathy which is the principle cause of death Heterozygous carriers of the GAA expansion, who are asymptomatic, have approximately 50% of normal frataxin expression in vitroIn this study, we have performed a series of experiments investigating the effects human MSCs have on both frataxin gene and protein expression in fibroblasts derived from patients with FRDA and healthy controls. We show that human MSCs, via the secretion of soluble factors, are able to increase frataxin mRNA expression promoting a prolonged increase in frataxin protein expression in fibroblasts derived from patients with FRDA. Increases in frataxin mRNA expression over a 24 hour exposure to MSC-condition medium were also evident within our healthy control group although no significant differences in frataxin protein expression were observed. This may however be associated with the relatively low sample numbers in this group. It is believed that GAA repeats in FRDA patients block transcription via several proposed mechanisms including impeding the progress of RNA polymerase FXN-transfection has on the expression of these intracellular hydrogen peroxide scavenging enzymes. Firstly it was shown that there was a deficiency in both catalase and glutathione peroxidase 1 in patients with FRDA when compared to healthy controls. Furthermore, FXN-transfection or human MSCs, via the secretion of soluble factors, are able to increase catalase and/or glutathione peroxidase 1 protein levels in fibroblasts derived from patients with FRDA. Exposure to MSC-conditioned media or FXN-transfection did not result in any significant increases in enzymes expression in healthy control cells. These results are therapeutically interesting as administration of enzymes that scavenge hydrogen peroxide, such as catalase, or the addition of hydrogen peroxide scavenging enzyme mimetics may normalize the antioxidant activity and suppress the deleterious phenotypes associated with frataxin deficiency Oxidative damage and inhibition of mitochondrial function may be key determinants of cellular damage and the pathogenesis of FRDA. Cells deficient of frataxin are shown to have a greater sensitivity to oxidative stress 3kinase/Akt and p38 MAPkinase pathways FXN-transfection, in addition to increasing catalase expression, also rescues cells from hydrogen peroxide exposure. Both the addition of exogenous catalase or glutathione peroxidase rescue FRDA fibroblasts against hydrogen peroxide toxicity. Furthermore, the presence of a catalase inhibitor abrogates the increase in FRDA fibroblast survival, caused by exposure to MSC-conditioned medium during insult by hydrogen peroxide. Overall these results suggest a direct link between frataxin, levels of catalase expression and sensitivity to hydrogen peroxide mediated toxicity. We hypothesize therefore that an increase in frataxin, catalase and/or glutathione peroxidase 1 levels in fibroblasts derived from patients with FRDA after exposure to MSC-conditioned medium results in, at least in part, this rescue effect. Therapeutically, MSCs may be able to rescue normal function and normalize antioxidant activity in frataxin defective cells by increasing frataxin levels above a specific threshold at which point patients' with FRDA become asymptomatic.We have confirmed observations made by others demonstrating that fibroblasts from healthy controls were more resistant to hydrogen peroxide-mediated oxidative stress compared to FRDA fibroblasts Currently there are no effective treatments in preventing the progression of FRDA. Data in this study demonstrates for the first time that MSCs secrete factors that increase cellular frataxin levels in frataxin-deficient fibroblasts and protect those cells from oxidative damage. Increasing evidence suggests a major protective role of MSCs is their capacity to secrete a diverse range of potentially trophic, paracrine and protective factors, in addition to anti-oxidant actions, thereby promoting an environment conducive to cellular protection and support diseased cell survival


Arterial pressure was not different between freely moving Cx45fl/fl and Cx45fl/fl:Nestin-Cre mice during day or night. We conclude that Cx45 is physiologically expressed in VSM, but not in EC in murine arterioles. However, Cx45 is dispensable for the conduction of vasomotor responses along these arterioles. Possibly, other Cx functionally replace the lack of Cx45 in VSM. The reported role of Cx45 in renin secretion does not seem to alter arterial pressure in freely moving mice.Connexins (Cx) form gap junctions and allow the coordination of cellular behaviour. In vessels, expression of Cx40, Cx37, and Cx43 is well established and specifically Cx40 serves important functions in endothelial cells. In contrast, expression and physiological functions of Cx45 is unclear although its expression has been suggested in vascular smooth muscle (VSM). Therefore, we studied expression and function of Cx45 in vessels using different mice models allowing to identify and delete Cx45. Smooth muscle cell (SMC)-specific deletion was achieved by the Cre/loxP system using Cre-recombinase driven by a Nestin promoter. Deletion of Cx45 leads concomitantly to the expression of enhanced green fluorescence protein (EGFP) in these mice. Conduction of vasomotor responses was studied in cremasteric arterioles using intravital microscopy and arterial pressure was measured telemetrically. Cx45 is transcriptionally expressed in VSM as detected by EGFP expression in SMC-specific Cx45-deficient mice (Cx45fl/fl:Nestin-Cre) but not in endothelial cells (Cx45fl/fl:TIE2-Cre). Moreover, EGFP was located at VSM cell borders in arterioles of transgenic mice carrying an EGFP-tagged Cx45. Expectedly, arteriolar conduction of dilations evoked by the endothelium-dependent agonist acetylcholine were not different between Cx45fl/fl:Nestin-Cre mice and controls carrying homozygously a floxed Cx45 gene (Cx45fl/fl). Surprisingly, the amplitude of locally initiated endothelium-independent constrictions (K Gap junctions enable fast signalling between adjacent cells und thus connect individual cells into a functional unit or syncitium. Such signalling is also pivotal in vessels in the microcirculation since thereby vasomotor signals may communicate along the vascular wall, which lead to the coordination of vascular cell behaviour and synchronous vasoconstriction or vasodilation. Arterioles in microcirculatory networks cover substantial distances and thus only concordant diameter changes along their overall length efficiently modulate vascular resistance thereby adjusting organ perfusion to its demand in a wide dynamic range. The functional coupling can be investigated by brief locally confined application of certain vasoactive compounds to elicit a local vasomotor response. The response is not confined to the site of stimulation but travels to remote up- and downstream sites without measurable time delay and is therefore termed conducted response Gap junction channels are composed of connexin (Cx) protein subunits that provide conduits of low electrical resistance between cells Cx43 and Cx45 are suggestedly expressed in vascular smooth cells of cerebral arteries, the aorta and other large conducting arteries We hypothesized that Cx45 is expressed in vascular smooth muscle throughout the circulation, connects smooth muscle cells homocellularly, and is required to support the conduction of vasomotor responses along the smooth muscle layer. To test these hypotheses, we examined Cx45 expression using mice carrying a Cx45 tagged with enhanced green fluorescence protein (EGFP) The investigation conformed to the guide for the care and use of laboratory animals published by the US National Institutes of Health, was performed in accordance with the German law for animal welfare and was approved by local authorities . All surgery was performed under anesthesia (see below), and all efforts were made to minimize suffering. Cell-specific deletion of Cx45 was achieved using mice that harbor a floxed Cx45 gene (Cx45fl). The deletion of Cx45 accomplished by Cre-recombinase activity leads concomitantly to the expression of the reporter enhanced green fluorescent protein (EGFP) + 143, K+ 5, Ca2+ 2.5, Mg2+ 1.2, Cl− 128, HCO3− 25, SO42− 1.2, and H2PO4− 1.2) gassed with 5% CO2 and 95% N2 (pO2∼30 and pCO2∼40 mm Hg). The microcirculation was studied using a microscope  mounted on a movable platform and equipped with a charge-coupled video camera. One to three second-order arterioles in the central region of the tissue were studied in each animal. Microscopic images were displayed on a monitor (700-fold magnification) and recorded on videotape for later measurement of luminal diameter after digitization (resolution after digitization ∼1 µm).Anesthesia was induced by intraperitoneal injection of midazolam (5 mg/kg), fentanyl (0.05 mg/kg), and medetomidine (0.5 mg/kg) followed by continuous infusion (jugular vein). The animals were intubated by tracheotomy to ensure airway patency and their esophageal temperature maintained at 37°C. The cremaster muscle was prepared for intravital microscopy as described Conduction of vasomotor responses was studied by focally confined stimulation using a glass micropipette (tip 1–2 µm) placed in close proximity to the arteriolar wall through which vasoactive compounds  were ejected by a short pressure pulse  In separate animals, arterial pressure was measured by radiotelemetry. Mice were anesthetized using isoflurane (1 to 1.5%) and surgically implanted with microminiaturized radiotelemeters . The catheter was introduced into the left carotid artery and advanced to the aortic arch as described For whole-mount immunolabeling mice were anesthetized, the cremaster muscle prepared, removed shortly after killing the animal, and pinned flat in dishes covered with silicone . Other tissues  were harvested afterwards Vessel diameter changes were normalized to the maximal possible response:Tr is the diameter after treatment, DCo the control diameter before treatment, and DM the maximal possible diameter that is the maximally dilated diameter for dilations or the minimal luminal diameter (zero) for constrictions. To characterize the temporal characteristics of the responses the ‘time to peak’  and the ‘response duration’  were calculated. Comparisons within groups were performed using paired t tests, and corrected according to Bonferroni. Data between groups were compared with ANOVA, followed by post hoc analysis of the means. Data are presented as mean±SEM. Differences were considered significant at a corrected error probability of P<0.05.where DWe used two different mouse models to verify the expression of Cx45 in vascular cells and to circumvent problems associated with antibody specificity. The first model consisted of animals which harbor a floxed Cx45 gene (Cx45fl). In these mice, cell-specific deletion of Cx45 accomplished by Cre-recombinase activity leads concomitantly to the expression of the reporter enhanced green fluorescent protein (EGFP) Vascular smooth muscle cells were also strongly labelled with EGFP in Cx45fl/fl:Nestin-Cre mice in larger vessels including the gracilis artery which feeds a skeletal muscle  includinThe second model studied consisted of mice expressing a transgenic Cx45 tagged with the sequence for EGFP under the endogenous Cx45 promotor A total of 41 arterioles were studied in 20 mice. Resting diameter of untreated arterioles  and maximal diameter  as measured during superfusion of sodium nitroprusside, adenosine, and acetylcholine (each 30 µmol/L) were not significantly different between genotypes. From these values the arteriolar resting tone was calculated as the fraction of resting from maximal diameter. The resting tone was also similar in both groups of mice  indicating a similar contractile state of the arterioles at rest.+ solution, the vasodilator adenosine, and the endothelium-dependent agonist acetylcholine (ACh). Responses to K+ and adenosine conduct most likely along the smooth muscle whereas ACh dilations conduct in a Cx40-dependent manner along the endothelial cell layer + solution (3 mol/L) induced a rapid constriction at the local site in control mice (Cx45fl/fl). A comparable local constriction was evoked by K+ stimulation in mice lacking Cx45 in vascular smooth muscle . Neither the amplitude nor the duration of the dilation in response to adenosine was different at any site in mice lacking Cx45 in vascular smooth muscle from the respective control animals. In a different experimental series, adenosine was examined after inhibition of nitric oxide synthase and cyclooxygenase . In this series, more vigorous stimulation using adenosine evoked a local dilation with a maximal amplitude of 81±6% in Cx45fl/fl and of 70±7% in Cx45fl/fl:Nestin-Cre mice . The dilation conducted to remote upstream sites and was not different in both genotypes at a distance of 1.2 mm .Microapplication of the vasodilator adenosine (10 mmol/L) initiated a transient local dilation (response duration 43±4 s) with a maximal amplitude of 41±5% that was attained within 13±2 s in Cx45fl/fl mice. The amplitude and the temporal behaviour of the locally evoked response were similar in Cx45fl/fl:Nestin-Cre mice . In bothP = 0.05 vs. control) as well as the time to this maximum  and the response duration  which is most likely due to a less efficient stimulation. However, the conduction of the dilation to remote sites was not different in mice devoid of Cx45 in vascular smooth muscle from the respective control animals as verified by a similar maximal amplitude of the dilation at these sites . The temporal behaviour is depicted in Additionally, the endothelium-dependent dilator ACh was studied. Local stimulation of the arterioles using ACh evoked a local dilation with a maximal amplitude of 58±3% that was attained within 11±1 s and lasted for 40±4 s in Cx45fl/fl mice which was conducted to remote sites with nearly unaltered amplitude . The maxArterial pressure was measured by telemetry in awake, freely moving mice in 8 animals of each genotype which were of a similar age . Six days after implantation arterial pressure and heart rate were not different between the genotypes . After aThe present study demonstrates that Cx45 is expressed in smooth muscle cells of arterioles and larger conducting vessels  whereas Cx45 seems to be not expressed in the largest arteries (carotid artery and aorta). Moreover, smooth muscle cells in venules and pericytes surrounding capillaries exhibit Cx45 promotor activity, but endothelial cells of arterioles and venules lack this activity. Although immunolabelling cannot verify colocalisation of different Cx in gap junction plaques, our data do not provide any evidence of colocalisation of Cx45 with endothelial connexins, namely Cx37 and Cx40. Despite convincing evidence for the expression of Cx45 in smooth muscle cells in arterioles we were unable to identify a functional phenotype related to smooth muscle specific deletion of Cx45. Neither the conduction of vasomotor responses along the arteriolar wall in the microcirculation nor the arterial pressure in freely moving conscious mice were altered which suggests that Cx45 is not a crucial gap junctional component in vascular smooth muscle cells and in the regulation of arterial pressure.lacZ was expressed in visceral as well as in vascular smooth muscle, for example in large conducting arteries (aorta) and the renal circulation In contrast to Cx37, Cx40, and Cx43, the expression of Cx45 in the vascular system has been addressed only in a limited number of studies mostly in the rat. Its expression was demonstrated by immunostaining and western blotting in smooth muscle cells of small and larger arteries in the cerebral circulation of the rat +-channels in smooth muscle cells +-solution 2+-dependent K+-channels (KCa3.1) in vivo not the main dilatory mechanism Our functional experiments failed to document a crucial role for Cx45 in the conduction of locally initiated vasomotor responses along arterioles in the microcirculation. The vascular wall comprises two distinct pathways along which signals are conducted depending on the cell type that is initially engaged by the vasoactive substance lacZ expression controlled by the Cx45 promoter Previously, Hanner and coworkers reported an elevated arterial pressure in anesthetized mice deficient for Cx45 in smooth muscle cells which was associated with enhanced plasma renin levels The global deletion of Cx45 and its lethality was associated with vascular malformations