Scaffolded spheroids as building blocks for bottom-up cartilage tissue engineering show enhanced bioassembly dynamics.

Due to the capability of cell spheroids (SPH) to assemble into large high cell density constructs, their use as building blocks attracted a lot of attention in the field of biofabrication. Nevertheless, upon maturation, the composition along with the size of such building blocks change, affecting their fusiogenic ability to form a cohesive tissue construct of controllable size. This natural phenomenon remains a limitation for the standardization of spheroid-based therapies in the clinical setting. We recently showed that scaffolded spheroids (S-SPH) can be produced by forming spheroids directly within porous PCL-based microscaffolds fabricated using multiphoton lithography (MPL). In this new study, we compare the bioassembly potential of conventional SPHs versus S-SPHs depending on their degree of maturation. Doublets of both types of building blocks were cultured and their fusiogenicity was compared by measuring the intersphere angle, the length of the fusing spheroid pairs (referred to as doublet length) as well as their spreading behaviour. Finally, the possibility to fabricate macro-sized tissue constructs (i.e. cartilage-like) from both chondrogenic S-SPHs and SPHs was analyzed. This study revealed that, in contrast to conventional SPHs, S-SPHs exhibit robust and stable fusiogenicity, independently from their degree of maturation. In order to understand this behavior, we further analyze the intersection area of doublets, looking at the kinetic of cell migration and at the mechanical stability of the formed tissue using dissection measurements. Our findings indicate that the presence of microscaffolds enhances the ability of spheroids to be used as building blocks for bottom-up tissue engineering, which is an important advantage compared to conventional spheroid-based therapy approaches. STATEMENT OF SIGNIFICANCE: The approach of using SPHs as building blocks for bottom-up tissue engineering offers a variety of advantages. At the same time the self-assembly of large tissues remains challenging due to several intrinsic properties of SPHs, such as for instance the shrinkage of tissues assembled from SPHs, or the reduced fusiogenicity commonly observed with mature SPHs. In this work, we demonstrate the capability of scaffolded spheroids (S-SPH) to fuse and recreate cartilage-like tissue constructs despite their advanced maturation stage. In this regard, the presence of microscaffolds compensates for some of the intrinsic limitations of SPHs and can help to overcome current limitations of spheroid-based tissue engineering.

Embryonic and juvenile snakes (Natrix maura, Linnaeus 1758) compensate for high elevation hypoxia via shifts in cardiovascular physiology and metabolism.

The colonization of novel environments requires a favorable response to conditions never, or rarely, encountered in recent evolutionary history. For example, populations colonizing upslope habitats must cope with lower atmospheric pressure at elevation, and thus reduced oxygen availability. The embryo stage in oviparous organisms is particularly susceptible, given its lack of mobility and limited gas exchange via diffusion through the eggshell and membranes. Especially little is known about responses of Lepidosaurian reptiles to reduced oxygen availability. To test the role of physiological plasticity during early development in response to high elevation hypoxia, we performed a transplant experiment with the viperine snake (Natrix maura, Linnaeus 1758). We maintained gravid females originating from low elevation populations (432 m above sea level [ASL]-normoxia) at both the elevation of origin and high elevation (2877 m ASL-extreme high elevation hypoxia; approximately 72% oxygen availability relative to sea level), then incubated egg clutches at both low and high elevation. Regardless of maternal exposure to hypoxia during gestation, embryos incubated at extreme high elevation exhibited altered developmental trajectories of cardiovascular function and metabolism across the incubation period, including a reduction in late-development egg mass. This physiological response may have contributed to the maintenance of similar incubation duration, hatching success, and hatchling body size compared to embryos incubated at low elevation. Nevertheless, after being maintained in hypoxia, juveniles exhibit reduced carbon dioxide production relative to oxygen consumption, suggesting altered energy pathways compared to juveniles maintained in normoxia. These findings highlight the role of physiological plasticity in maintaining rates of survival and fitness-relevant phenotypes in novel environments.

Temperature-independent telomere lengthening with age in the long-lived human fish (Proteus anguinus).

Despite a number of studies showing a negative relationship between age and telomere length, the universality of this pattern has been recently challenged, mainly in ectothermic animals exhibiting diverse effects of age on telomere shortening. However, data on ectotherms may be strongly affected by the thermal history of the individuals. We thus investigated the age-related changes in relative telomere length in the skin of a small but long-lived amphibian living naturally in a stable thermal environment over its entire life, allowing comparison with other homeothermic animals like birds and mammals. The present data showed a positive relation between telomere length and individual age, independent of sex and body size. A segmented analysis highlighted a breakpoint in the telomere length-age relationship, suggesting that telomere length reached a plateau at the age of 25 years. Further studies focusing on the biology of animals that live much longer than expected based on body mass will contribute to our better understanding of how ageing processes evolved and may also bring innovation for extending human health span.

Plastic responses to warmer climates: a semi-natural experiment on lizard populations.

Facing warming environments, species can exhibit plastic or microevolutionary changes in their thermal physiology to adapt to novel climates. Here, using semi-natural mesocosms, we experimentally investigated over two successive years whether a 2°C-warmer climate produces selective and inter- and intragenerational plastic changes in the thermal traits (preferred temperature and dorsal coloration) of the lizard Zootoca vivipara. In a warmer climate, the dorsal darkness, dorsal contrast, and preferred temperature of adults plastically decreased and covariances between these traits were disrupted. While selection gradients were overall weak, selection gradients for darkness were slightly different between climates and in the opposite direction to plastic changes. Contrary to adults, male juveniles were darker in warmer climates either through plasticity or selection and this effect was strengthened by intergenerational plasticity when juveniles' mothers also experienced warmer climates. While the plastic changes in adult thermal traits alleviate the immediate overheating costs of warming, its opposite direction to selective gradients and to juveniles' phenotypic responses may slow down evolutionary shifts toward phenotypes that are better adapted to future climates. Our study demonstrates the importance of considering inter- and intragenerational plasticity along with selective processes to better understand adaptation and population dynamics in light of climate change.

Investigating the genetic basis of vertebrate dispersal combining RNA-seq, RAD-seq and quantitative genetics.

Although animal dispersal is known to play key roles in ecological and evolutionary processes such as colonization, population extinction and local adaptation, little is known about its genetic basis, particularly in vertebrates. Untapping the genetic basis of dispersal should deepen our understanding of how dispersal behaviour evolves, the molecular mechanisms that regulate it and link it to other phenotypic aspects in order to form the so-called dispersal syndromes. Here, we comprehensively combined quantitative genetics, genome-wide sequencing and transcriptome sequencing to investigate the genetic basis of natal dispersal in a known ecological and evolutionary model of vertebrate dispersal: the common lizard, Zootoca vivipara. Our study supports the heritability of dispersal in semi-natural populations, with less variation attributable to maternal and natal environment effects. In addition, we found an association between natal dispersal and both variation in the carbonic anhydrase (CA10) gene, and in the expression of several genes (TGFB2, SLC6A4, NOS1) involved in central nervous system functioning. These findings suggest that neurotransmitters (serotonin and nitric oxide) are involved in the regulation of dispersal and shaping dispersal syndromes. Several genes from the circadian clock (CRY2, KCTD21) were also differentially expressed between disperser and resident lizards, supporting that the circadian rhythm, known to be involved in long-distance migration in other taxa, might affect dispersal as well. Since neuronal and circadian pathways are relatively well conserved across vertebrates, our results are likely to be generalisable, and we therefore encourage future studies to further investigate the role of these pathways in shaping dispersal in vertebrates.

Hybrid spheroid microscaffolds as modular tissue units to build macro-tissue assemblies for tissue engineering.

Since its inception, tissue engineering and regenerative medicine (TERM) has been relying on either scaffold-based or scaffold-free strategies. Recent reports outlined the possibility of a synergistic, convergence approach, referred to as the third TERM strategy, which could alleviate bottlenecks of the two previous options. This strategy requires the fabrication of highly porous microscaffolds, allowing to create single spheroids within each of them. The resulting tissue units can then be combined and used as modular building blocks for creating tissue constructs through a bottom-up self-assembly. Such strategy can have a significant impact for the future of TERM, but so far, no reports have assessed its feasibility in detail. This work reports a first systematic study, which includes a comparison of the in vitro behavior of tissue units based on adipose derived stem cell spheroids cultured within microscaffolds versus conventional spheroids. We first proved that the presence of the microscaffold neither impairs the cells 'ability to form spheroids nor impacts their viability. Importantly, the fusiogenic and the differentiation potential (i.e. chondrogenesis and osteogenesis), which are important features for cellularized building blocks to be used in TERM, are preserved when spheroids are cultured within microscaffolds. Significant benefits of microscaffold-based tissue units include the enhanced cell retention, the decreased compaction and the better control over the size observed when larger tissue constructs are formed through self-assembly. The proof of concept study presented here demonstrates the great potential offered by those microsize tissue units to be used as building blocks for directed tissue self-assembly. STATEMENT OF SIGNIFICANCE: One of the most exciting and recent advances in tissue engineering and regenerative medicine (TERM) is to combine together multiple micro-size cellularized units, which are able to self-assemble altogether to recreate larger tissue constructs. In this work, we produce such modules by forming single spheroids within highly porous microscaffolds, and study how this new microenvironment impacts on the spheroid's behavior and stemness potential. This work highlights as well that such novel route is enabled by two-photon polymerization, which is an additive manufacturing technique offering high spatial resolution down to 100 nm. These findings provide a first scientific evidence about the utilization of hybrid spheroid microscaffold-based tissue units with great perspective as a modular tool for TERM.

Adaptive Maternal Effects Shape Offspring Phenotype and Survival in Natal Environments.

AbstractMaternal effects can give newborns a head start in life by adjusting natal phenotypes to natal environments, yet their strength and adaptiveness are often difficult to investigate in natural populations. Here, we studied anticipatory maternal effects and their adaptiveness in common lizards in a seminatural experimental system. Specifically, we investigated how maternal environments (i.e., vegetation cover) and maternal phenotype (i.e., activity levels and body length) can shape offspring phenotype. We further studied whether such maternal effects influenced offspring survival in natal environments varying with respect to vegetation cover, conspecific density, and, consequently, maternal fitness. More active females from dense vegetation habitats produced bigger offspring than their less active counterparts, the contrary being true for sparse vegetation habitats. Moreover, females from dense vegetation habitats produced more active offspring and more active offspring survived better in dense vegetation habitats, resulting in greater maternal fitness through maternal effects. These results suggest adaptive anticipatory maternal effects, induced by vegetation structure and mediated by activity levels that may shape early-life prospects in natal environments.

Interplay between biofilm microenvironment and pathogenicity of Pseudomonas aeruginosa in cystic fibrosis lung chronic infection.

Pseudomonas aeruginosa (PA) is a highly, if not the most, versatile microorganism capable of colonizing diverse environments. One of the niches in which PA is able to thrive is the lung of cystic fibrosis (CF) patients. Due to a genetic aberration, the lungs of CF-affected patients exhibit impaired functions, rendering them highly susceptible to bacterial colonization. Once PA attaches to the epithelial surface and transitions to a mucoid phenotype, the infection becomes chronic, and antibiotic treatments become inefficient. Due to the high number of affected people and the severity of this infection, CF-chronic infection is a well-documented disease. Still, numerous aspects of PA CF infection remain unclear. The scientific reports published over the last decades have stressed how PA can adapt to CF microenvironmental conditions and how its surrounding matrix of extracellular polymeric substances (EPS) plays a key role in its pathogenicity. In this context, it is of paramount interest to present the nature of the EPS together with the local CF-biofilm microenvironment. We review how the PA biofilm microenvironment interacts with drugs to contribute to the pathogenicity of CF-lung infection. Understanding why so many drugs are inefficient in treating CF chronic infection while effectively treating planktonic PA is essential to devising better therapeutic targets and drug formulations.

An Antibiotic-Loaded Hydrogel Demonstrates Efficacy as Prophylaxis and Treatment in a Large Animal Model of Orthopaedic Device-Related Infection.

Local antibiotic therapy is increasingly being recognised for its role in preventing and treating orthopaedic device-related infection (ODRI). A bioresorbable, injectable gentamicin-loaded hydrogel has been developed to deliver local antibiotics at the time of surgery with potential for both prevention and treatment of ODRI. In a prophylaxis model, the antibiotic hydrogel was compared with systemic perioperative antibiotic prophylaxis alone in twelve sheep (six per group) at the time of intramedullary (IM) nail insertion to the tibia, which was inoculated with methicillin-sensitive Staphylococcus aureus (MSSA). In a treatment model of single-stage revision surgery, adjunctive antibiotic-loaded hydrogel was compared with systemic antibiotics alone in a single stage revision of MSSA infection associated with a tibia intramedullary nail in eleven sheep (five/six per group). The primary endpoint was quantitative microbiological results of soft tissue, bone and sonicate fluid from explanted hardware at the time of euthanasia. At euthanasia, the control sheep that received no local antibiotics in the prophylaxis model were all culture-positive (median 1x108, range 7x106-3x108 colony forming units, CFU) while only two of six sheep receiving local gentamicin had any culture positive biopsies (median 1x101, range 0 - 1x105 CFU). For the treatment model, sheep receiving only systemic antibiotics were all culture-positive (median 8x105, range 2x103- 9x106 CFU) while only two of six sheep treated with gentamicin-loaded hydrogel had any culture positive biopsies (median 3x102, range 0 - 7x104 CFU). Local gentamicin concentrations measured in extracellular fluid in the tibial canal show a burst release of gentamicin from the hydrogel. Serum gentamicin concentrations peaked in both models at one day post application and were below detection limit thereafter. This study has demonstrated the effective use of a locally delivered antibiotic hydrogel for both the prevention and treatment of ODRI that is superior to that of systemic antibiotics alone. Future studies will endeavour to translate from preclinical to clinical research trials.

Polyoxazoline hydrogels fabricated by stereolithography.

The development of hydrogel materials in additive manufacturing displaying stiff and strong mechanical properties while maintaining high water uptake remains a great challenge. Taking advantage of the versatility of poly(oxazoline) (POx) chemistry and properties, we investigated in this article a new generation of POx hydrogels fabricated by stereolithography (SLA). A large range of photosensitive poly(2-methyl-2-oxazoline) resins were synthesized as hydrogel precursors for SLA photofabrication. Functionalization has been performed by direct di-methacrylation of POx terminal groups (MA2POxn) or by multi-methacrylation of poly(ethyleneimine) (PEI) units resulting from partial POx hydrolysis (MAmPOxn-PEIp). The length and the functionality of these UV-active macro-crosslinkers influence both the mechanical properties and the hydration behavior of the resulting hydrogels. The benefit of the layer-by-layer crosslinking of the POx resin during the vat photopolymerization allowed the fabrication of complex and well-defined 3D objects. The high-definition and high mechanical strength of these copolymers allow the fabrication of stiff and strong 3D hydrogels. The cytocompatibility test of the POx derivatives was conducted in solution and once the cells are encapsulated within 3D hydrogels. Finally, porous 3D scaffolds with gyroid architectures were built which provide opportunities for POx materials in tissue engineering applications.

Impacts of temperature on O2 consumption of the Pyrenean brook newt (Calotriton asper) from populations along an elevational gradient.

Global warming impacts biodiversity worldwide, leading to species' adaptation, migration, or extinction. The population's persistence depends on the maintenance of essential activities, which is notably driven by phenotypic adaptation to local environments. Metabolic rate - that increases with temperature in ectotherms - is a key physiological proxy for the energy available to fuel individuals' activities. Cold-adapted ectotherms can exhibit a higher resting metabolism than warm-adapted ones to maintain functionality at higher elevations or latitudes, known as the metabolic cold-adaptation hypothesis. How climate change will affect metabolism in species inhabiting contrasting climates (cold or warm) is still a debate. Therefore, it is of high interest to assess the pace of metabolic responses to global warming among populations adapted to highly different baseline climatic conditions. Here, we conducted a physiological experiment in the endemic Pyrenean brook newt (Calotriton asper). We measured a proxy of standard metabolic rate (SMR) along a temperature gradient in individuals sampled among 6 populations located from 550 to 2189 m a.s.l. We demonstrated that SMR increased with temperature, but significantly diverged depending on populations' origins. The baseline and the slope of the relationship between SMR and temperature were both higher for high-elevation populations than for low-elevation populations. We discussed the stronger metabolic response observed in high-elevation populations suggesting a drop of performance in essential life activities for these individuals under current climate change. With the increase of metabolism as the climate warms, the metabolic-cold adaptation strategy selected in the past could compromise the sustainability of cold-adapted populations if short-term evolutionary responses do not allow to offset this evolutionary legacy.

Photoprintable Gelatin-graft-Poly(trimethylene carbonate) by Stereolithography for Tissue Engineering Applications.

The stereolithography process is a powerful additive manufacturing technology to fabricate scaffolds for regenerative medicine. Nevertheless, the quest for versatile inks allowing one to produce scaffolds with controlled properties is still unsatisfied. In this original article, we tackle this bottleneck by synthesizing a panel of photoprocessable hybrid copolymers composed of gelatin-graft-poly(trimethylene carbonate)s (Gel-g-PTMCn). We demonstrated that by changing the length of PTMC blocks grafted from gelatin, it is possible to tailor the final properties of the photofabricated objects. We reported here on the synthesis of Gel-g-PTMCn with various lengths of PTMC blocks grafted from gelatin using hydroxy and amino side groups of the constitutive amino acids. Then, the characterization of the resulting hybrid copolymers was fully investigated by quantitative NMR spectroscopy before rendering them photosensitive by methacrylation of the PTMC terminal groups. Homogeneous composition of the photocrosslinked hybrid polymers was demonstrated by EDX spectroscopy and electronic microscopy. To unravel the individual contribution of the PTMC moiety on the hybrid copolymer behavior, water absorption, contact angle measurements, and degradation studies were undertaken. Interestingly, the photocrosslinked materials immersed in water were examined using tensile experiments and displayed a large panel of behavior from hydrogel to elastomer-like depending on the PTMC/gel ratio. Moreover, the absence of cytotoxicity was conducted following the ISO 10993 assay. As a proof of concept, 3D porous objects were successfully fabricated using stereolithography. Those results validate the great potential of this panel of inks for tissue engineering and regenerative medicine.

A Hyaluronic Acid Hydrogel Loaded with Gentamicin and Vancomycin Successfully Eradicates Chronic Methicillin-Resistant Staphylococcus aureus Orthopedic Infection in a Sheep Model.

Implantable orthopedic devices have had an enormously positive impact on human health; however, despite best practice, patients are prone to developing orthopedic device-related infections (ODRI) that have high treatment failure rates. One barrier to the development of improved treatment options is the lack of an animal model that may serve as a robust preclinical assessment of efficacy. We present a clinically relevant large animal model of chronic methicillin-resistant Staphylococcus aureus (MRSA) ODRI that persists despite current clinical practice in medical and surgical treatment at rates equivalent to clinical observations. Furthermore, we showed that an injectable, thermoresponsive, hyaluronic acid-based hydrogel loaded with gentamicin and vancomycin outperforms current clinical practice treatment in this model, eliminating bacteria from all animals. These results confirm that local antibiotic delivery with an injectable hydrogel can dramatically increase treatment success rates beyond current clinical practice, with efficacy proven in a robust animal model.

Enhancing performance of biocarriers facilitated gravity-driven membrane (GDM) reactor for decentralized wastewater treatment: Effect of internal recirculation and membrane packing density.

This study aims to investigate the effect of internal recirculation and membrane packing density on the performance (water quality, membrane performance, and microbial community) of a biocarriers facilitated gravity-driven membrane (GDM) reactor under intermittent aeration condition. The results revealed that the presence of internal recirculation in the GDM reactors could effectively improve water quality (especially increasing nitrogen removal) and membrane performance (especially reducing cake layer resistance) compared to those without internal recirculation. In addition, compared to a high packing density membrane module (1150 m2/m3), a lower packing density membrane module (290 m2/m3) benefited to improve 15% of nitrogen removal and 44% of permeate flux due to the effective aeration scouring effect and less-limited eukaryotic activity, as well as reduce 20% of total treatment cost. In addition, the presence and absence of internal recirculation could lead to dissimilar microbial community compositions of the biofilms in the GAC layers and on the membrane surfaces. However, the membrane packing density could play an insignificant effect on the microbial community compositions of the biofilms in the GDM reactors with internal recirculation.

Reduction in the metabolic levels due to phenotypic plasticity in the Pyrenean newt, Calotriton asper, during cave colonization.

According to theories on cave adaptation, cave organisms are expected to develop a lower metabolic rate compared to surface organisms as an adaptation to food scarcity in the subterranean environments. To test this hypothesis, we compared the oxygen consumption rates of the surface and subterranean populations of a surface-dwelling species, the newt Calotriton asper, occasionally found in caves. In this study, we designed a new experimental setup in which animals with free movement were monitored for several days in a respirometer. First, we measured the metabolic rates of individuals from the surface and subterranean populations, both maintained for eight years in captivity in a natural cave. We then tested individuals from these populations immediately after they were caught and one year later while being maintained in the cave. We found that the surface individuals that acclimated to the cave significantly reduced their oxygen consumption, whereas individuals from the subterranean population maintained in the cave under a light/dark cycle did not significantly modify their metabolic rates. Second, we compared these metabolic rates to those of an obligate subterranean salamander (Proteus anguinus), a surface aquatic Urodel (Ambystoma mexicanum), and a fish species (Gobio occitaniae) as references for surface organisms from different phyla. As predicted, we found differences between the subterranean and surface species, and the metabolic rates of surface and subterranean C. asper populations were between those of the obligate subterranean and surface species. These results suggest that the plasticity of the metabolism observed in surface C. asper was neither directly due to food availability in our experiments nor the light/dark conditions, but due to static temperatures. Moreover, we suggest that this adjustment of the metabolic level at a temperature close to the thermal optimum may further allow individual species to cope with the food limitations of the subterranean environment.

3D Printing of large-scale and highly porous biodegradable tissue engineering scaffolds from poly(trimethylene-carbonate) using two-photon-polymerization.

The introduction of two-photon polymerization (2PP) to the field of tissue engineering and regenerative medicine (TERM) has led to great expectations for the production of scaffolds with an unprecedented degree of complexity and tailorable architecture. Unfortunately, resolution and size are usually mutually exclusive when using 2PP, resulting in a lack of highly-detailed scaffolds with a relevant size for clinical application. Through the combination of using a highly reactive photopolymer and optimizing key printing parameters, we propose for the first time a biodegradable and biocompatible poly(trimethylene-carbonate) (PTMC)-based scaffold of large size (18 × 18 × 0.9 mm) with a volume of 292 mm3 produced using 2PP. This increase in size results in a significant volumetric increase by almost an order of magnitude compared to previously available large-scale structures (Stichel 2010 J. Laser Micro./Nanoeng. 5 209-12). The structure's detailed design resulted in a highly porous scaffold (96%) with excellent cytocompatibility, supporting the attachment, proliferation and differentiation of human adipose-derived mesenchymal stem cells towards their osteogenic and chondrogenic lineages. This work strongly attests that 2PP is becoming a highly suitable technique for producing large-sized scaffolds with a complex architecture. We show as a proof-of-concept that an arrayed design of repetitive units can be produced, but a further perspective will be to print scaffolds with anisotropic features that are more representative of human tissues.

Poly(Aspartic Acid) Functionalized Poly(ϵ-Caprolactone) Microspheres with Enhanced Hydroxyapatite Affinity as Bone Targeting Antibiotic Carriers.

Bone infection is a feared complication for patients with surgically fixed bone fractures and local antibiotic delivery is important in prophylaxis and treatment of these infections. Recent studies indicated that Staphylococcus aureus can penetrate bone tissue through micron-sized canaliculi and evade systemic and currently available local antibiotic treatments. Targeting bacteria within the bone requires highly efficient delivery of antimicrobials to the infected bone tissue. In this work, a biodegradable microsphere carrier loaded with antibiotics and with specific affinity to bone mineral was developed. Two widely used antibiotics, i.e., Gentamicin-dioctyl sulfosuccinate (GM-AOT) and Ciprofloxacin (CF) were embedded in poly(ϵ-caprolactone) (PCL) microspheres fabricated by oil-in-water emulsion techniques with carboxylated poly(vinyl alcohol) (cPVA) as surfactant. The carboxylic acid groups present at the Poly(ϵ-caprolactone)/cPVA (PCL-cPVA) microsphere surface were functionalized with aspartic acid oligomers (ASP) granting bone targeting properties. We report on cPVA synthesis, microsphere formulation, and antibiotic loading of PCL/cPVA-ASP microspheres. Antibiotic loaded PCL/cPVA-ASP microspheres show sustained release of its antibiotic load and can inhibit bacterial growth in vitro for up to 6 days. PCL/cPVA-ASP microspheres show enhanced affinity to mineralized substrates compared to non-functionalized PCL/cPVA microspheres. These findings support further development of these bone targeting antibiotic carriers for potential treatment of persistent bone infections.

High-elevation hypoxia impacts perinatal physiology and performance in a potential montane colonizer.

Climate change is generating range shifts in many organisms, notably along the elevational gradient in mountainous environments. However, moving up in elevation exposes organisms to lower oxygen availability, which may reduce the successful reproduction and development of oviparous organisms. To test this possibility in an upward-colonizing species, we artificially incubated developing embryos of the viperine snake (Natrix maura) using a split-clutch design, in conditions of extreme high elevation (hypoxia at 2877 m above sea level; 72% sea-level equivalent O2 availability) or low elevation (control group; i.e. normoxia at 436 m above sea level). Hatching success did not differ between the two treatments. Embryos developing at extreme high elevation had higher heart rates and hatched earlier, resulting in hatchlings that were smaller in body size and slower swimmers compared to their siblings incubated at lower elevation. Furthermore, post-hatching reciprocal transplant of juveniles showed that snakes which developed at extreme high elevation, when transferred back to low elevation, did not recover full performance compared to their siblings from the low elevation incubation treatment. These results suggest that incubation at extreme high elevation, including the effects of hypoxia, will not prevent oviparous ectotherms from producing viable young, but may pose significant physiological challenges on developing offspring in ovo. These early-life performance limitations imposed by extreme high elevation could have negative consequences on adult phenotypes, including on fitness-related traits.

Multiple glacial refugia and contemporary dispersal shape the genetic structure of an endemic amphibian from the Pyrenees.

Historical factors (colonization scenarios, demographic oscillations) and contemporary processes (population connectivity, current population size) largely contribute to shaping species' present-day genetic diversity and structure. In this study, we use a combination of mitochondrial and nuclear DNA markers to understand the role of Quaternary climatic oscillations and present-day gene flow dynamics in determining the genetic diversity and structure of the newt Calotriton asper (Al. Dugès, 1852), endemic to the Pyrenees. Mitochondrial DNA did not show a clear phylogeographic pattern and presented low levels of variation. In contrast, microsatellites revealed five major genetic lineages with admixture patterns at their boundaries. Approximate Bayesian computation analyses and linear models indicated that the five lineages likely underwent separate evolutionary histories and can be tracked back to distinct glacial refugia. Lineage differentiation started around the Last Glacial Maximum at three focal areas (western, central and eastern Pyrenees) and extended through the end of the Last Glacial Period in the central Pyrenees, where it led to the formation of two more lineages. Our data revealed no evidence of recent dispersal between lineages, whereas borders likely represent zones of secondary contact following expansion from multiple refugia. Finally, we did not find genetic evidence of sex-biased dispersal. This work highlights the importance of integrating past evolutionary processes and present-day gene flow and dispersal dynamics, together with multilocus approaches, to gain insights into what shaped the current genetic attributes of amphibians living in montane habitats.

Thermo-Responsive Antimicrobial Hydrogel for the In-Situ Coating of Mesh Materials for Hernia Repair.

The prophylactic coating of prosthetic mesh materials for hernia repair with antimicrobial compounds is commonly performed before implantation of the mesh in the abdominal wall. We propose a novel alternative, which is a rifampicin-loaded thermo-responsive hydrogel formulation, to be applied on the mesh after its implantation. This formulation becomes a gel in-situ once reached body temperature, allowing an optimal coating of the mesh along with the surrounding tissues. In vitro, the hydrogel cytotoxicity was assessed using rabbit fibroblasts and antimicrobial efficacy was determined against Staphylococcus aureus. An in vivo rabbit model of hernia repair was performed; implanted polypropylene meshes (5 × 2 cm) were challenged with S. aureus (106 CFU), for two study groups-unloaded (n = 4) and 0.1 mg/cm2 rifampicin-loaded hydrogel (n = 8). In vitro, antibacterial activity of the hydrogel lasted for 5 days, without sign of cytotoxicity. Fourteen days after implantation, meshes coated with drug-free hydrogel developed a strong infection and resulted in poor tissue integration. Coating meshes with the rifampicin-loaded hydrogel fully prevented implant infection and permitted an optimal tissue integration. Due to its great performance, this, degradable, thermo-responsive antimicrobial hydrogel could potentially be a strong prophylactic armamentarium to be combined with prosthesis in the surgical field.