Benjamin Gantenbein

The phylogeographic importance of the Strait of Gibraltar as a gene flow barrier in terrestrial arthropods: a case study with the scorpion Buthus occitanus as model organism

The phylogenetic relationship between Buthus occitanus populations across the Strait of Gibraltar was investigated using nuclear 18S/ITS-1 DNA sequences and mitochondrial 16S and COI DNA sequences. All analyses showed that the European samples are highly separated from North African samples, and also suggest the existence of three main groups within this species complex, i.e., an European, an Atlas (=Moroccan samples) and a Tell-Atlas group (=Tunisian samples). The European clade was subdivided into three distinct subclades. The application of a previous calibration of the molecular clock of another buthid species suggested that most of the detected mitochondrial DNA lineages including the European lineages are about three times older than the re-opening of the Gibraltar Strait, and consequently, that other and older vicariant events are responsible for the observed phylogeographic structure of this species complex. Concerning the Moroccan samples, a discordance between nuclear and mitochondrial gene markers was observed. The 18S/ITS-1 gene tree could not resolve the phylogenetic relationships among the Moroccan B. occitanus subspecies and the closely related species B. atlantis, whereas mitochondrial genes suggested the co-existence of several old phylogenetic lineages in Morocco. We hypothesized that this difference may be explained by male-biased gene flow and gene conversion at the tandemly repeated 18S/ITS-1 gene regions.

An in vitro organ culturing system for intervertebral disc explants with vertebral endplates : a feasibility study with ovine caudal discs

Study Design. Whole ovine caudal intervertebral discs with vertebral endplates were cultured under uniaxial diurnal loading for 7 days. Objectives. To establish and characterize an organ culture system for intervertebral discs, in which disc cells may be “maintained” in their native three-dimensional environment under load. Summary of Background Data. In vitro culturing of entire discs with preserved biologic and structural integrity would be a useful model to study the effects of nutrition and mechanical loading. Methods. To maintain endplate permeability, sheep were systemically anticoagulated before death and their caudal vasculature was evacuated with saline postmortem. The first 4 caudal discs were explanted with their adjacent endplates and cultured in bioreactors under uniaxial diurnal loading (0.2 MPa for 8 hours and 0.8 MPa for 16 hours) for 4 or 7 days. Solute transport into the center of the disc was measured after 4 days of culture using a low molecular weight fluorescent marker. Cell viability, glycosaminoglycan synthesis rate, and gene expression profile were measured after 7 days of culture and compared with fresh tissue. Results. Fluorescent images showed that solutes could diffuse into the disc under both static and diurnal loading, but penetration through the endplate increased with diurnal loading. Cell viability and glycosaminoglycan synthesis rates remained unchanged after 7 days of culture. Expression of catabolic genes was significantly up-regulated, whereas anabolic genes tended to be down-regulated after 7 days. Conclusions. With this novel preparation and culturing technique, endplate permeability could be maintained, which allowed culturing of intact disc explants with endplates for up to 7 days.

Evidence for recombination in scorpion mitochondrial DNA (Scorpiones: Buthidae)

There has been very little undisputed evidence for recombination in animal mitochondrial DNA (mtDNA) provided so far. Previous unpublished results suggestive of mtDNA recombination in the scorpion family Buthidae, together with cytological evidence for a unique mechanism of mitochondrial fusion in that family, prompted us to investigate this group in more details. First, we sequenced the complete mtDNA genome of Mesobuthus gibbosus, and chose two genes opposing each other (16S and coxI). We then sequenced 150 individuals from the natural populations of four species of Buthidae (Old World genera Buthus and Mesobuthus). We observed strong evidence for widespread recombination through highly significant negative correlations between linkage disequilibrium and physical distance in three out of four species. The evidence is further confirmed when using five other tests for recombination and by the presence of a high amount of homoplasy in phylogenetic trees.

Mesobuthus gibbosus (Scorpiones: Buthidae) on the island of Rhodes — hybridization between Ulysses’ stowaways and native scorpions?

Allozyme data suggest that the Rhodes population of Mesobuthus gibbosus is a hybrid population of recent origin. Namely, it is a mixture between an autochthonous population and an artificially introduced population probably from the Greek mainland. All samples were mainly composed of F1 hybrid genotypes and genotypes either fixed for autochthonous or introduced alleles. Back‐cross hybrid genotypes were very rare. Mitochondrial DNA analysis, in contrast, revealed only one group of closely related haplotypes that are unique for the Rhodes populations, thus suggesting asymmetric introgression of the two marker classes.

Activation of intervertebral disc cells by co-culture with notochordal cells, conditioned medium and hypoxia

BackgroundNotochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen).MethodsPorcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted.ResultsWe provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM.ConclusionsOur results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.

Euscorpius balearicus Caporiacco, 1950, stat. nov. (Scorpiones: Euscorpiidae): molecular (allozymes and mtDNA) and morphological evidence for an endemic Balearic Islands species

Abstract The geographic variation of the circum-Mediterranean scorpion species Euscorpius carpathicus (L.) was traditionally analysed using morphological characters such as trichobothrial patterns, which resulted in the recognition of 23 subspecies; however, the biological reality of these subspecies remains unclear. Here, we focus on populations from the western Mediterranean and provide new molecular evidence that those from the island of Mallorca (Balearic Islands, Spain) represent a highly divergent lineage separate from E. carpathicusfrom the mainland of France (Vaucluse) and Italy (Liguria and Piemonte). This divergence is evidenced by morphological analysis. Moreover, allozyme and mtDNA divergences (about 10%) agree with our hypothesis that the Balearic island populations became isolated from the mainland about 5 Ma BP since the refilling of the Mediterranean Basin and have to be considered autochthonous. This hypothesis is additionally supported by the comparison of the genetic differentiation between artificially transplanted island populations and mainland populations in the congeneric species E. flavicaudis(de Geer). The phylogenetic species concept (PSC) is applied to elevate the subspecies E. carpathicus balearicus Caporiacco, 1950 to species rank. A lectotype is designated for this species.

THE FIRST DNA PHYLOGENY OF FOUR SPECIES OF MESOBUTHUS (SCORPIONES, BUTHIDAE) FROM EURASIA

Abstract The first molecular phylogeny is presented for four species of the scorpion genus Mesobuthus, based on DNA sequences of three gene fragments (two mitochondrial and one nuclear protein coding gene, ∼1 kb). The inferred phylogeny based on a pooled maximum likelihood analysis revealed a clear deep splitting between the “western clade” consisting of M. gibbosus and M. cyprius (Greece/Anatolia, Cyprus) and the “eastern clade” consisting of M. eupeus and M. caucasicus (Anatolia/Central Asia). The species M. caucasicus (recently placed in the genus Olivierus Farzanpay 1987) groups monophyletically within Mesobuthus; thus, the genus Olivierus is synonymized here with Mesobuthus. Sequences of M. eupeus and M. caucasicus sampled mainly from Kazakhstan and Uzbekistan are highly structured, indicating the possible existence of multiple species.

AN EXTREMELY LOW GENETIC DIVERGENCE ACROSS THE RANGE OF EUSCORPIUS ITALICUS (SCORPIONES, EUSCORPIIDAE)

Abstract Little or no genetic divergence is detected using mitochondrial 16S rDNA sequence comparisons across the entire geographic range of the scorpion Euscorpius italicus (Herbst 1800) from Switzerland, Italy, Slovenia, Greece and Turkey. This is consistent with known absence of patterns of allozymes and morphological variation. Euscorpius italicus is found almost exclusively in human habitations. Its sister species, E. naupliensis, exhibits much higher genetic diversity within southern Greece. We suggest that the natural populations of the thermophilic E. italicus underwent a bottleneck during the glaciations, and that its modern range could be a result of dispersal with humans.

A photopolymerized composite hydrogel and surgical implanting tool for a nucleus pulposus replacement

Nucleus pulposus replacements have been subjected to highly controversial discussions over the last 40 years. Their use has not yet resulted in a positive outcome to treat herniated disc or degenerated disc disease. The main reason is that not a single implant or tissue replacement was able to withstand the loads within an intervertebral disc. Here, we report on the development of a photo-polymerizable poly(ethylene glycol)dimethacrylate nano-fibrillated cellulose composite hydrogel which was tuned according to native tissue properties. Using a customized minimally-invasive medical device to inject and photopolymerize the hydrogel insitu, samples were implanted through an incision of 1 mm into an intervertebral disc of a bovine organ model to evaluate their long-term performance. When implanted into the bovine disc model, the composite hydrogel implant was able to significantly re-establish disc height after surgery (p < 0.0025). The height was maintained after 0.5 million loading cycles (p < 0.025). The mechanical resistance of the novel composite hydrogel material combined with the minimally invasive implantation procedure into a bovine disc resulted in a promising functional orthopedic implant for the replacement of the nucleus pulposus.

The BMP2 variant L51P restores the osteogenic differentiation of human mesenchymal stromal cells in the presence of intervertebral disc cells

Spinal fusion is hampered by the presence of remaining intervertebral disc (IVD) tissue and leads to spinal non-union. While the exact mechanism remains unknown, we hypothesise that factors preventing disc ossification, such as antagonists of the bone morphogenetic proteins (BMP), could be responsible for this process. The objective of this study was to investigate spinal non-union using an in vitro human model with a focus on the BMP signalling components and to identify factors contributing to the incomplete and delayed ossification. Human bone marrow-derived mesenchymal stromal cells (MSC) were cocultured with IVD cells in the presence of L51P, a BMP2 variant with osteoinductive potential. The ossification of MSC was evaluated by quantitative reverse transcription polymerase chain reaction (qPCR), alkaline phosphatase (ALP) activity and alizarin red staining. Endogenous expression of major BMP antagonists, namely Gremlin (GREM1), Noggin (NOG) and Chordin (CHRD) was detected in IVD-derived cells, with abundance in nucleus pulposus cells. Osteogenesis of MSC was hindered by IVD cells as shown by reduced alizarin red staining, ALP activity and qPCR. L51P, added to the cocultures, restored mineralisation, blocking the activity of the BMP antagonists secreted by IVD cells. It is possible that the BMP antagonists secreted by IVD cells are responsible for spinal non-unions. The inhibition of BMP antagonists with L51P may result in an efficient and more physiological osteoinduction rather than delivery of exogenous osteogenic factors. Therefore, L51P might represent an attractive therapeutic candidate for bone healing.