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International audience; Background: Nanofat grafting (NG) is a simple and cost-effective method of lipoaspirates with inter-syringe passages, to produce stromal vascular fraction (SVF) and isolate adipose-derived stem cells (ASCs). This represents a tremendous interest in the future clinical needs of tissue engineering. In this study, we optimized the NG technique to increase the yield of ASC extractions. Methods: We analyzed three groups of SVF obtained by 20, 30, and 40 inter-syringe passages. The control group was an SVF obtained by enzymatic digestion with Celase. We studied their cell composition by flow cytometry, observed their architecture by confocal microscopy, and observed immunomodulatory properties of the ASCs from each of the SVFs by measuring inflammatory markers of macrophages obtained by an ASC monocyte co-culture. Results: We have established the first cell mapping of the stromal vascular fraction of adipose tissue. The results showed that SVF obtained by 20 inter-syringe passages contains more statistically significant total cells, more cells expressing the ASC phenotype, more endothelial cells, and produces more CFU-F than the SVF obtained by 30 and 40 passages and by enzymatic digestion. Confocal microscopy showed the presence of residual adipocytes in SVF obtained by inter-syringe passages but not by enzymatic digestion. The functional study indicates an orientation toward a more anti-inflammatory profile and homogenization of their immunomodulatory properties. Conclusion: This study places mechanically dissociated SVF in the center of approaches to easily extract ASCs and a wide variety and number of other progenitor cells, immediately available in a clinical setting to provide both the amount and quality of cells required for decellularized tissues.

Mechanically processed stromal vascular fraction (mSVF) is a promising source for regenerative purposes. To study the in vivo fate of the mSVF, we herein used a vascularized tissue engineering chamber that insulates the target mSVF from the surrounding environment. In contrast to previous models, we propose an arteriovenous (AV) shunt between saphenous vessels in rats without a venous graft. Mechanical SVF was processed from the fat pads of male Sprague Dawley rats, mixed with a fibrin hydrogel and implanted into an inguinal tissue engineering chamber. An arteriovenous shunt was established between saphenous artery and vein. On the contralateral side, an mSVF-fibrin hydrogel mix without vascular axis served as a non-vascularized control. After two and six weeks, rats were sacrificed for further analysis. Mechanical SVF showed significant numbers of mesenchymal stromal cells. Vascularized mSVF explants gained weight over time. Perilipin and CD31 expression were significantly higher in the mSVF explants after six weeks while no difference in DAPI positive cells, collagen deposition and FABP4 expression was observed. Morphologically, no differentiated adipocytes but a dense cell-rich tissue with perilipin-positive cells was found after six weeks. The phosphorylation of ERK1/2 was significantly enhanced after six weeks while Akt activation remained unaltered. Finally, mSVF explants stably expressed and released VEGF, bFGF and TGFb. Vascularized mSVF is able to proliferate and express adipocyte-specific markers. The AV shunt model is a valuable refinement of currently existing AV loop models in the rat which contributes to the fundamental 3R principles of animal research.

BACKGROUND AND AIMS: Under a widely accepted model of pollinator-driven speciation, geographic variation in pollinator assemblage drives floral divergence and automatically causes reproductive isolation. Yet it is unclear whether divergent floral adaptation initially confers strong reproductive isolation, or whether that comes at later stages of speciation and requires other forms of reproductive isolation. This study uses a pair of recently diverged, interfertile and parapatric species in the genus Clarkia to determine whether adaptation to hawkmoths, a novel pollinator functional group, would automatically confer floral isolation upon sympatric contact. METHODS: First, genetically based differences in floral traits between C. breweri and C. concinna that would be maintained upon migration are quantified. Then scenarios of experimental sympatry are constructed in which arrays of flowers are exposed to the novel pollinator, the hawkmoth Hyles lineata, and pollinator preference and heterospecific pollen transfer are assessed. Source populations from across the ranges of each species are used to understand how geographic variation in floral traits within species may affect floral isolation. KEY RESULTS: Although H. lineata has never been observed visiting C. concinna in the wild, it regularly moves between species in experimental floral arrays. Hawkmoth preference and heterospecific pollen transfer vary both among moths and among geographic source locations of C. concinna. The strength of floral isolation in this system is related to variation in flower size, especially hypanthium tube width, and nectar reward among C. concinna forms. CONCLUSIONS: Although C. breweri has adopted a novel hawkmoth pollination system, both ethological and mechanical floral isolation by hawkmoths are incomplete and vary according to the specific phenotype of the C. concinna source population. The results suggest that strong floral isolation is not automatically conferred by a pollinator shift and may require additional evolution of deterrent floral traits and habitat isolation that reduces the immediate spatial co-occurrence of young species.

Background and aims – Aframomum (61 species) is a tropical African genus of the family Zingiberaceae (~53 genera) of perennial rhizomatous herbs most often growing in the forest understorey. The family Zingiberaceae is known for a high diversity in floral morphology and pollination systems in Asia, however, almost nothing is known in Africa. In this study we explore the floral diversity in Aframomum and the potential of this diversity to mediate isolation as driving force for species divergence and maintenance in Aframomum in contrast to other postulated drivers such as allopatry and habitat differentiation. Methods and key results – We conducted a survey of floral morphology in eighteen species across the genus identifying five floral types and their morphological adaptations to different pollinators. Another 38 species were subsequently attributed to one of these types based on monographic descriptions and photos. Differences in floral types were based on slight changes in the relative length and position of dorsal petal, labellum and the complex of anther and style. The mapping of floral types onto an existing phylogenetic tree indicated their repeated independent evolution. The preponderate presence of one rather uniform floral type in about 60% of all taxa (Trumpet type) suggests a low number of inferred pollinator shifts (30%). Conclusion – The low number of inferred shifts in floral types alone cannot alone explain the diversification of this genus. Here the often small non-overlapping distribution ranges and narrow habitat preferences of species and the phylogenetic distance of sympatric species suggest the additional importance of allopatry and phylogenetic incompatibility as isolation mechanisms in maintaining species boundaries in this genus.

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede.