The role of globins in cardiovascular physiology.

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.

cGAS-STING drives ageing-related inflammation and neurodegeneration.

Low-grade inflammation is a hallmark of old age and a central driver of ageing-associated impairment and disease1. Multiple factors can contribute to ageing-associated inflammation2; however, the molecular pathways that transduce aberrant inflammatory signalling and their impact in natural ageing remain unclear. Here we show that the cGAS-STING signalling pathway, which mediates immune sensing of DNA3, is a critical driver of chronic inflammation and functional decline during ageing. Blockade of STING suppresses the inflammatory phenotypes of senescent human cells and tissues, attenuates ageing-related inflammation in multiple peripheral organs and the brain in mice, and leads to an improvement in tissue function. Focusing on the ageing brain, we reveal that activation of STING triggers reactive microglial transcriptional states, neurodegeneration and cognitive decline. Cytosolic DNA released from perturbed mitochondria elicits cGAS activity in old microglia, defining a mechanism by which cGAS-STING signalling is engaged in the ageing brain. Single-nucleus RNA-sequencing analysis of microglia and hippocampi of a cGAS gain-of-function mouse model demonstrates that engagement of cGAS in microglia is sufficient to direct ageing-associated transcriptional microglial states leading to bystander cell inflammation, neurotoxicity and impaired memory capacity. Our findings establish the cGAS-STING pathway as a driver of ageing-related inflammation in peripheral organs and the brain, and reveal blockade of cGAS-STING signalling as a potential strategy to halt neurodegenerative processes during old age.

Climate-land-use interactions shape tropical mountain biodiversity and ecosystem functions.

Agriculture and the exploitation of natural resources have transformed tropical mountain ecosystems across the world, and the consequences of these transformations for biodiversity and ecosystem functioning are largely unknown1-3. Conclusions that are derived from studies in non-mountainous areas are not suitable for predicting the effects of land-use changes on tropical mountains because the climatic environment rapidly changes with elevation, which may mitigate or amplify the effects of land use4,5. It is of key importance to understand how the interplay of climate and land use constrains biodiversity and ecosystem functions to determine the consequences of global change for mountain ecosystems. Here we show that the interacting effects of climate and land use reshape elevational trends in biodiversity and ecosystem functions on Africa's largest mountain, Mount Kilimanjaro (Tanzania). We find that increasing land-use intensity causes larger losses of plant and animal species richness in the arid lowlands than in humid submontane and montane zones. Increases in land-use intensity are associated with significant changes in the composition of plant, animal and microorganism communities; stronger modifications of plant and animal communities occur in arid and humid ecosystems, respectively. Temperature, precipitation and land use jointly modulate soil properties, nutrient turnover, greenhouse gas emissions, plant biomass and productivity, as well as animal interactions. Our data suggest that the response of ecosystem functions to land-use intensity depends strongly on climate; more-severe changes in ecosystem functioning occur in the arid lowlands and the cold montane zone. Interactions between climate and land use explained-on average-54% of the variation in species richness, species composition and ecosystem functions, whereas only 30% of variation was related to single drivers. Our study reveals that climate can modulate the effects of land use on biodiversity and ecosystem functioning, and points to a lowered resistance of ecosystems in climatically challenging environments to ongoing land-use changes in tropical mountainous regions.

Inter- and intraspecific phytochemical variation correlate with epiphytic flower and leaf bacterial communities.

Microbes associated with flowers and leaves affect plant health and fitness and modify the chemical phenotypes of plants with consequences for interactions of plants with their environment. However, the drivers of bacterial communities colonizing above-ground parts of grassland plants in the field remain largely unknown. We therefore examined the relationships between phytochemistry and the epiphytic bacterial community composition of flowers and leaves of Ranunculus acris and Trifolium pratense. On 252 plant individuals, we characterized primary and specialized metabolites, that is, surface sugars, volatile organic compounds (VOCs), and metabolic fingerprints, as well as epiphytic flower and leaf bacterial communities. The genomic potential of bacterial colonizers concerning metabolic capacities was assessed using bacterial reference genomes. Phytochemical composition displayed pronounced variation within and between plant species and organs, which explained part of the variation in bacterial community composition. Correlation network analysis suggests strain-specific correlations with metabolites. Analysis of bacterial reference genomes revealed taxon-specific metabolic capabilities that corresponded with genes involved in glycolysis and adaptation to osmotic stress. Our results show relationships between phytochemistry and the flower and leaf bacterial microbiomes suggesting that plants provide chemical niches for distinct bacterial communities. In turn, bacteria may induce alterations in the plants' chemical phenotype. Thus, our study may stimulate further research on the mechanisms of trait-based community assembly in epiphytic bacteria.

Plants, pollinators and their interactions under global ecological change: The role of pollen DNA metabarcoding.

Anthropogenic activities are triggering global changes in the environment, causing entire communities of plants, pollinators and their interactions to restructure, and ultimately leading to species declines. To understand the mechanisms behind community shifts and declines, as well as monitoring and managing impacts, a global effort must be made to characterize plant-pollinator communities in detail, across different habitat types, latitudes, elevations, and levels and types of disturbances. Generating data of this scale will only be feasible with rapid, high-throughput methods. Pollen DNA metabarcoding provides advantages in throughput, efficiency and taxonomic resolution over traditional methods, such as microscopic pollen identification and visual observation of plant-pollinator interactions. This makes it ideal for understanding complex ecological networks and their responses to change. Pollen DNA metabarcoding is currently being applied to assess plant-pollinator interactions, survey ecosystem change and model the spatiotemporal distribution of allergenic pollen. Where samples are available from past collections, pollen DNA metabarcoding has been used to compare contemporary and past ecosystems. New avenues of research are possible with the expansion of pollen DNA metabarcoding to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for understanding the evolutionary and ecological processes that support biodiversity, and predicting and responding to the impacts of change.

From Alien Species to Alien Communities: Host- and Habitat-Associated Microbiomes in an Alien Amphibian.

Alien species can host diverse microbial communities. These associated microbiomes may be important in the invasion process and their analysis requires a holistic community-based approach. We analysed the skin and gut microbiome of Eleutherodactylus johnstonei from native range populations in St Lucia and exotic range populations in Guadeloupe, Colombia, and European greenhouses along with their respective environmental microbial reservoir through a 16S metabarcoding approach. We show that amphibian-associated and environmental microbial communities can be considered as meta-communities that interact in the assembly process. High proportions of bacteria can disperse between frogs and environment, while respective abundances are rather determined by niche effects driven by the microbial community source and spatial environmental properties. Environmental transmissions appeared to have higher relevance for skin than for gut microbiome composition and variation. We encourage further experimental studies to assess the implications of turnover in amphibian-associated microbial communities and potentially invasive microbiota in the context of invasion success and impacts. Within this novel framework of "nested invasions," (meta-)community ecology thinking can complement and widen the traditional perspective on biological invasions.

Force behaviour of elastic chains during a simulated gap closure in extraction therapy cases.

OBJECTIVE: Tooth movement with elastic chains requires defined force magnitudes. This study assessed the force behaviour of different elastic chains at different configurations of gap width. METHODS: Self-ligating brackets of teeth 5 & 6 and 2 & 3 were bonded to two movable aluminium plates. The plates were positioned on a joint basis with varying distances of 0.5, 2.0, 4.0, 6.0, and 8.0 mm. Reset forces of open and closed chains from four different manufacturers were investigated in four different configurations. Configurations differed in either having an additional intermediate ring within the gap (#1, #3) and/or having intermediate rings between teeth adjacent to the gap (#1, #2), or by no intermediate rings (#4). Forces were measured with a universal testing machine. The results were statistically analysed using U-test, H-test and (if applicable) post-hoc tests with a significance level of .05. RESULTS: Configurations #1 and #3, and #2 and #4 formed homogenous subgroups (P < .001). Initial forces in configuration #4 were significantly higher than in configuration #3 (P = .029). Initial forces in closed chains were significantly higher than for open chains (P = .029). CONCLUSIONS: Intermediate chain rings adjacent to the gap are not required to modulate the force. In contrast, leaving a ring unapplied in the tooth gap can help modulate the force. Open thermoset chains with an additional ring within the gap (#3) seem to produce suitable initial forces for a gap closure of 4 mm. With a residual gap width of <2 mm, open thermoset chains and closed thermoset chains (#4) seem suitable.

First experimental evidence for active farming in ambrosia beetles and strong heredity of garden microbiomes.

Fungal cultivation is a defining feature for advanced agriculture in fungus-farming ants and termites. In a third supposedly fungus-farming group, wood-colonizing ambrosia beetles, an experimental proof for the effectiveness of beetle activity for selective promotion of their food fungi over others is lacking and farming has only been assumed based on observations of social and hygienic behaviours. Here, we experimentally removed mothers and their offspring from young nests of the fruit-tree pinhole borer, Xyleborinus saxesenii. By amplicon sequencing of bacterial and fungal communities of nests with and without beetles we could show that beetles are indeed able to actively shift symbiont communities. Although being consumed, the Raffaelea food fungi were more abundant when beetles were present while a weed fungus (Chaetomium sp.) as well as overall bacterial diversity were reduced in comparison to nests without beetles. Core symbiont communities were generally of low diversity and there were strong signs for vertical transmission not only for the cultivars, but also for secondary symbionts. Our findings verify the existence of active farming, even though the exact mechanisms underlying the selective promotion and/or suppression of symbionts need further investigation.

Phylogenetic relatedness of food plants reveals highest insect herbivore specialization at intermediate temperatures along a broad climatic gradient.

The composition and richness of herbivore and plant assemblages change along climatic gradients, but knowledge about associated shifts in specialization is scarce and lacks controlling for the abundance and phylogeny of interaction partners. Thus, we aimed to test whether the specialization of phytophagous insects in insect-plant interaction networks decreases toward cold habitats as predicted by the 'altitude niche-breadth hypothesis' to forecast possible consequences of interaction rewiring under climate change. We used a non-invasive, standardized metabarcoding approach to reconstruct dietary relationships of Orthoptera species as a major insect herbivore taxon along a broad temperature gradient (~12°C) in Southern Germany. Based on Orthoptera surveys, feeding observations, collection of fecal pellets from >3,000 individuals of 54 species, and parallel vegetation surveys on 41 grassland sites, we quantified plant resource availability and its use by herbivores. Herbivore assemblages were richer in species and individuals at sites with high summer temperatures, while plant richness peaked at intermediate temperatures. Corresponding interaction networks were most specialized in warm habitats. Considering phylogenetic relationships of plant resources, however, the specialization pattern was not linear but peaked at intermediate temperatures, mediated by herbivores feeding on a narrow range of phylogenetically related resources. Our study provides empirical evidence of resource specialization of insect herbivores along a climatic gradient, demonstrating that resource phylogeny, availability, and temperature interactively shape the specialization of herbivore assemblages. Instead of low specialization levels only in cold, harsh habitats, our results suggest increased generalist feeding due to intraspecific changes and compositional differences at both ends of the microclimatic gradient. We conclude that this nonlinear change of phylogeny-based resource specialization questions predictions derived from the 'altitude-niche breadth hypothesis' and highlights the currently limited understanding of how plant-herbivore interactions will change under future climatic conditions.

BCdatabaser: on-the-fly reference database creation for (meta-)barcoding.

SUMMARY: DNA barcoding and meta-barcoding have become irreplaceable in research and applications, where identification of taxa alone or within a mixture, respectively, becomes relevant. Pioneering studies were in the microbiological context, yet nowadays also plants and animals become targeted. Given the variety of markers used, formatting requirements for classifiers and constant growth of primary databases, there is a need for dedicated reference database creation. We developed a web and command-line interface to generate such on-the-fly for any applicable marker and taxonomic group with optional filtering, formatting and restriction specific for (meta-)barcoding purposes. Also, databases optionally receive a DOI, making them well-documented with meta-data, publicly sharable and citable. AVAILABILITY AND IMPLEMENTATION: source code: https://www.github.com/molbiodiv/bcdatabaser, webservice: https://bcdatabaser.molecular.eco, documentation: https://molbiodiv.github.io/bcdatabaser.

Heterocellular Contact Can Dictate Arterial Function.

RATIONALE: Resistance arteries and conduit arteries rely on different relative contributions of endothelial-derived hyperpolarization versus nitric oxide to achieve dilatory heterocellular signaling. Anatomically, resistance arteries use myoendothelial junctions (MEJs), endothelial cell projections that make contact with smooth muscle cells. Conduit arteries have very few to no MEJs. OBJECTIVE: Determine if the presence of MEJs in conduit arteries can alter heterocellular signaling. METHODS AND RESULTS: We previously demonstrated that PAI-1 (plasminogen activator inhibitor-1) can regulate formation of MEJs. Thus, we applied pluronic gel containing PAI-1 directly to conduit arteries (carotid arteries) to determine if this could induce formation of MEJs. We found a significant increase in endothelial cell projections resembling MEJs that correlated with increased biocytin dye transfer from endothelial cells to smooth muscle cells. Next, we used pressure myography to investigate whether these structural changes were accompanied by a functional change in vasodilatory signaling. Interestingly, PAI-1-treated carotids underwent a switch from a conduit to resistance artery vasodilatory profile via diminished nitric oxide signaling and increased endothelial-derived hyperpolarization signaling in response to the endothelium-dependent agonists acetylcholine and NS309. After PAI-1 application, we also found a significant increase in carotid expression of endothelial alpha globin, a protein predominantly expressed in resistance arteries. Carotids from mice with PAI-1, but lacking alpha globin (Hba1-/-), demonstrated that l-nitro-arginine methyl ester, an inhibitor of nitric oxide signaling, was able to prevent arterial relaxation. CONCLUSIONS: The presence or absence of MEJs is an important determinant for influencing heterocellular communication in the arterial wall. In particular, alpha globin expression, induced within newly formed endothelial cell projections, may influence the balance between endothelial-derived hyperpolarization and nitric oxide-mediated vasodilation.

Preservation methods of honey bee-collected pollen are not a source of bias in ITS2 metabarcoding.

Pollen metabarcoding is emerging as a powerful tool for ecological research and offers unprecedented scale in citizen science projects for environmental monitoring via honey bees. Biases in metabarcoding can be introduced at any stage of sample processing and preservation is at the forefront of the pipeline. While in metabarcoding studies pollen has been preserved at - 20 °C (FRZ), this is not the best method for citizen scientists. Herein, we compared this method with ethanol (EtOH), silica gel (SG) and room temperature (RT) for preservation of pollen collected from hives in Austria and Denmark. After ~ 4 months of storage, DNAs were extracted with a food kit, and their quality and concentration measured. Most DNA extracts exhibited 260/280 absorbance ratios close to the optimal 1.8, with RT samples from Austria performing slightly worse than FRZ and SG samples (P < 0.027). Statistical differences were also detected for DNA concentration, with EtOH samples producing lower yields than RT and FRZ samples in both countries and SG in Austria (P < 0.042). Yet, qualitative and quantitative assessments of floral composition obtained using high-throughput sequencing with the ITS2 barcode gave non-significant effects of preservation methods on richness, relative abundance and Shannon diversity, in both countries. While freezing and ethanol are commonly employed for archiving tissue for molecular applications, desiccation is cheaper and easier to use regarding both storage and transportation. Since SG is less dependent on ambient humidity and less prone to contamination than RT, we recommend SG for preserving pollen for metabarcoding. SG is straightforward for laymen to use and hence robust for widespread application in citizen science studies.

Constitutive SRC-mediated phosphorylation of pannexin 1 at tyrosine 198 occurs at the plasma membrane.

Pannexin 1 (PANX1)-mediated ATP release in vascular smooth muscle coordinates α1-adrenergic receptor (α1-AR) vasoconstriction and blood pressure homeostasis. We recently identified amino acids 198-200 (YLK) on the PANX1 intracellular loop that are critical for α1-AR-mediated vasoconstriction and PANX1 channel function. We report herein that the YLK motif is contained within an SRC homology 2 domain and is directly phosphorylated by SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) at Tyr198 We demonstrate that PANX1-mediated ATP release occurs independently of intracellular calcium but is sensitive to SRC family kinase (SFK) inhibition, suggestive of channel regulation by tyrosine phosphorylation. Using a PANX1 Tyr198-specific antibody, SFK inhibitors, SRC knockdown, temperature-dependent SRC cells, and kinase assays, we found that PANX1-mediated ATP release and vasoconstriction involves constitutive phosphorylation of PANX1 Tyr198 by SRC. We specifically detected SRC-mediated Tyr198 phosphorylation at the plasma membrane and observed that it is not enhanced or induced by α1-AR activation. Last, we show that PANX1 immunostaining is enriched in the smooth muscle layer of arteries from hypertensive humans and that Tyr198 phosphorylation is detectable in these samples, indicative of a role for membrane-associated PANX1 in small arteries of hypertensive humans. Our discovery adds insight into the regulation of PANX1 by post-translational modifications and connects a significant purinergic vasoconstriction pathway with a previously identified, yet unexplored, tyrosine kinase-based α1-AR constriction mechanism. This work implicates SRC-mediated PANX1 function in normal vascular hemodynamics and suggests that Tyr198-phosphorylated PANX1 is involved in hypertensive vascular pathology.

CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees.

Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.

Honey bee waggle dance communication increases diversity of pollen diets in intensively managed agricultural landscapes.

The benefits of honey bee dance communication for colony performance in different resource environments are still not well understood. Here, we test the hypothesis that directional dance communication enables honey bee colonies to maintain a diverse pollen diet, especially in landscapes with low resource diversity. To test this hypothesis, we placed 24 Apis mellifera L. colonies with either intact or experimentally disrupted dance communication in eight agricultural landscapes that differed in the diversity of flowering plants and in the dominance of mass-flowering crops. Pollen from incoming foragers was collected and identified via DNA metabarcoding. Disrupting dance communication affected the way the diversity of honey bee pollen diets was impacted by the dominance of mass-flowering crops in available flower resources (p = .04). With increasing dominance of mass-flowering crops in resource environments, foragers of colonies with intact communication foraged on an increasing proportion of available plant genera (p = .01). This was not the case for colonies with disrupted dance communication (p = .5). We conclude that the honey bee dance communication benefits pollen foraging on diverse plant resources and thereby contributes to high quality nutrition in environments with low-resource diversity.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

Sex-Dependent Regulation of Aromatase-Mediated Synaptic Plasticity in the Basolateral Amygdala.

The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17ß-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17ß-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.

Physical contact between mesenchymal stem cells and endothelial precursors induces distinct signatures with relevance to the very early phase of regeneration.

Multipotent adult stem cells/precursor cells, especially of the mesenchymal and endothelial lineage, may have great potential for bone tissue engineering. Although their potential is highly recognized, not much is known about the underlying molecular mechanisms that initiate the regeneration process, connect osteogenesis, and angiogenesis and, finally, orchestrate renewal of bone tissue. Our study addressed these questions by generating two in vitro cell culture models to examine the changes in the global gene expression patterns of endothelial precursor cells and mesenchymal stem cells after 24 hours of either humoral (conditioned medium) or direct cell-cell interaction (co-culture). Endothelial precursor cells were isolated from human buffy coat and mesenchymal stem cells from the bone marrow of the femoral head. The comparison of the treated and control cells by microarray analyses revealed in total more than 1500 regulated genes, which were analyzed for their affiliation to angiogenesis and osteogenesis. Expression array analyses at the RNA and protein level revealed data with respect to regulated genes, pathways and targets that may represent a valid basis for further dissection of the systems biology of regeneration processes. It may also be helpful for the reconstitution of the natural composition of a regenerative microenvironment when targeting tissue regeneration both in vitro and in situ.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

BMP-2 gene activated muscle tissue fragments for osteochondral defect regeneration in the rabbit knee.

BACKGROUND: Previously published data indicate that BMP-2 gene activated muscle tissue grafts can repair large bone defects in rats. This innovative abbreviated ex vivo gene therapy is appealing because it does not require elaborative and time-consuming extraction and expansion of cells. Hence, in the present study, we evaluated the potential of this expedited tissue engineering approach for regenerating osteochondral defects in rabbits. METHODS: Autologous muscle tissue grafts from female White New Zealand rabbits were directly transduced with an adenoviral BMP-2 vector or remained unmodified. Osteochondral defects in the medial condyle of rabbit knees were treated with either BMP-2 activated muscle tissue implants or unmodified muscle tissue or remained empty. After 13 weeks, repair of osteochondral defects was examined by biomechanical indentation testing and by histology/imunohistochemistry applying an extended O'Driscoll scoring system and histomorphometry. RESULTS: Biomechanical investigations revealed a trend towards slightly improved mechanical properties of the group receiving BMP-2 activated muscle tissue compared to unmodified muscle treatment and empty defect controls. However, a statistically significant difference was noted only between BMP-2 muscle and unmodified muscle treatment. Also, histological evaluation resulted in slightly higher histological scores and improved collagen I/II ratio without statistical significance in the BMP-2 treatment group. Histomorphometry indicated enhanced repair of subchondral bone after treatment with BMP-2 muscle, with a significantly larger bone area compared to untreated defects. CONCLUSIONS: Gene activated muscle tissue grafts showed potential for osteochondral defect repair. There is room for improvement via the use of appropriate growth factor combinations.