Suppressors of cGAS-STING are downregulated during fin-limb regeneration and aging in aquatic vertebrates.

During the early stages of limb and fin regeneration in aquatic vertebrates (i.e., fishes and amphibians), blastema undergo transcriptional rewiring of innate immune signaling pathways to promote immune cell recruitment. In mammals, a fundamental component of innate immune signaling is the cytosolic DNA sensing pathway, cGAS-STING. However, to what extent the cGAS-STING pathway influences regeneration in aquatic anamniotes is unknown. In jawed vertebrates, negative regulation of cGAS-STING activity is accomplished by suppressors of cytosolic DNA such as Trex1, Pml, and PML-like exon 9 (Plex9) exonucleases. Here, we examine the expression of these suppressors of cGAS-STING, as well as inflammatory genes and cGAS activity during caudal fin and limb regeneration using the spotted gar (Lepisosteus oculatus) and axolotl (Ambystoma mexicanum) model species, and during age-related senescence in zebrafish (Danio rerio). In the regenerative blastema of wounded gar and axolotl, we observe increased inflammatory gene expression, including interferon genes and interleukins 6 and 8. We also observed a decrease in axolotl Trex1 and gar pml expression during the early phases of wound healing which correlates with a dramatic increase in cGAS activity. In contrast, the plex9.1 gene does not change in expression during wound healing in gar. However, we observed decreased expression of plex9.1 in the senescing cardiac tissue of aged zebrafish, where 2'3'-cGAMP levels are elevated. Finally, we demonstrate a similar pattern of Trex1, pml, and plex9.1 gene regulation across species in response to exogenous 2'3'-cGAMP. Thus, during the early stages of limb-fin regeneration, Pml, Trex1, and Plex9.1 exonucleases are downregulated, presumably to allow an evolutionarily ancient cGAS-STING activity to promote inflammation and the recruitment of immune cells.

PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates.

We have uncovered a role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the mammalian PML protein forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish function as cytoplasmic DEDDh exonucleases. In contrast, PML proteins from amniote species localized both to the cytoplasm and formed nuclear bodies. We also identified the PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases. Plex9 proteins resemble TREX1 but are unique from the TREX family and share homology to gar PML. We also characterized the molecular evolution of TREX1 and the first non-mammalian TREX1 homologs in axolotl. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus by enhancing ORF1p degradation through the ubiquitin-proteasome system. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its new role in the assembly of nuclear bodies.

The Use of Small Molecules to Dissect Developmental and Regenerative Processes in Axolotls.

The axolotl provides an interesting model organism to study different biological processes that are of interest to basic biological sciences and biomedical research. Although axolotls have been in labs for close to 160 years, genetic manipulations still represent a major challenge for most labs. The use of small molecules to target specific signaling pathways allows studies to proceed in animals that are difficult to manipulate genetically. This chapter provides a description of how we administer these chemicals to axolotls.

Profiles of Patients with Opioid Use Disorders Presenting a History of Suicidal Ideations and Attempts.

Suicide rates are higher for people with an opioid use disorder, compared to the general population. This study aims to characterize opioid agonist treatment entrants who present a history of suicidal ideations or suicide attempts, according to concurrent comorbidity profiles, in an opioid use disorder treatment facility. A chart review design was used. Data was collected from 202 patient files. Bivariate and multivariate analyses were conducted. In multivariate analysis, patients with a diagnosis or symptoms of a mood disorder were 2.48 [1.01 - 6.11] times more likely to report suicidal ideations and 2.64 [1.05 - 6.62] times more likely to report suicide attempts. Those with a diagnosis or symptoms of an anxiety disorder were 2.41 [1.01 - 5.81] times more likely to report suicidal ideations. Patients who report chronic pain were 2.59 [1.06 - 6.35] times more likely to report suicidal ideations as well. The probability to report suicide attempts was 5.09 [1.16 - 22.4] times higher for those with a confirmed or suspected personality disorder. Clinicians should bear in mind the high suicide rates in people with opioid use disorder, as well as the importance of addressing suicidal risk and providing easy access to mental health and chronic pain treatment as part of the service offer in opioid agonist treatment. Future research should focus on evaluating the effectiveness of treatments aimed at addressing the needs of opioid agonist treatment patients with interrelated mental health and pain comorbidity profiles to reduce risks associated with suicide.

Tgf-ß superfamily and limb regeneration: Tgf-ß to start and Bmp to end.

Axolotls represent a popular model to study how nature solved the problem of regenerating lost appendages in tetrapods. Our work over many years focused on trying to understand how these animals can achieve such a feat and not end up with a scarred up stump. The Tgf-ß superfamily represents an interesting family to target since they are involved in wound healing in adults and pattern formation during development. This family is large and comprises Tgf-ß, Bmps, activins and GDFs. In this review, we present work from us and others on Tgf-ß & Bmps and highlight interesting observations between these two sub-families. Tgf-ß is important for the preparation phase of regeneration and Bmps for the redevelopment phase and they do not overlap with one another. We present novel data showing that the Tgf-ß non-canonical pathway is also not active during redevelopment. Finally, we propose a molecular model to explain how Tgf-ß and Bmps maintain distinct windows of expression during regeneration in axolotls.

Axolotls' and Mices' Oral-Maxillofacial Trephining Wounds Heal Differently.

The Ambystoma maxicanum (axolotl) regenerates strikingly from wounds and amputations. Comparing its healing ability to non-regenerative species such as the mouse should help narrow in on mechanisms to improve human wound healing. Here, the tongue and intermandibular soft tissues of both mice (C57BL/6NCrl) and axolotls were wounded with a 2-2.5 mm punch biopsy. The study aimed to compare the differences between these 2 species following surgical resection with regard to the macroscopic and histological characteristics. These include wound closure times, epithelial wound sealing and thickness as well as acute immune marker myeloperoxidase (MPO) response over 30 days. Post surgery, mice visually showed greater haemorrhage; their wounds immediately collapsed while it took 14 days for the axolotls mandibular void to close. The epithelium sealed the axolotls' wound margins within 24 h with a maximal mean thickness of 0.42 ± 0.13-fold normalized to unwounded skin. In mice, the epithelium separately sealed the ventral and dorsal sides, respectively at 7 and 7-30 days with mean maximal epithelial thicknesses reaching 13 ± 5.6 and 3.0 ± 0.63-fold. Mean MPO-positive cell values peaked in axolotls at 14 ± 1.5-fold between hours 6-12; while in mice, it peaked at 8.7 ± 0.9-fold between hours 24-96. We conclude that axolotls form smaller blood clots, have a faster and thinner epithelial cell migrating front, and a shorter MPO-positive cell response in comparison to mice. These observations may help refine future oral and facial wound-healing research and treatment.

A Collaborative Model to Implement Flexible, Accessible and Efficient Oncogenetic Services for Hereditary Breast and Ovarian Cancer: The C-MOnGene Study.

Medical genetic services are facing an unprecedented demand for counseling and testing for hereditary breast and ovarian cancer (HBOC) in a context of limited resources. To help resolve this issue, a collaborative oncogenetic model was recently developed and implemented at the CHU de Québec-Université Laval; Quebec; Canada. Here, we present the protocol of the C-MOnGene (Collaborative Model in OncoGenetics) study, funded to examine the context in which the model was implemented and document the lessons that can be learned to optimize the delivery of oncogenetic services. Within three years of implementation, the model allowed researchers to double the annual number of patients seen in genetic counseling. The average number of days between genetic counseling and disclosure of test results significantly decreased. Group counseling sessions improved participants' understanding of breast cancer risk and increased knowledge of breast cancer and genetics and a large majority of them reported to be overwhelmingly satisfied with the process. These quality and performance indicators suggest this oncogenetic model offers a flexible, patient-centered and efficient genetic counseling and testing for HBOC. By identifying the critical facilitating factors and barriers, our study will provide an evidence base for organizations interested in transitioning to an oncogenetic model integrated into oncology care; including teams that are not specialized but are trained in genetics.

BMP signaling is essential for sustaining proximo-distal progression in regenerating axolotl limbs.

Amputation of a salamander limb triggers a regeneration process that is perfect. A limited number of genes have been studied in this context and even fewer have been analyzed functionally. In this work, we use the BMP signaling inhibitor LDN193189 on Ambystoma mexicanum to explore the role of BMPs in regeneration. We find that BMP signaling is required for proper expression of various patterning genes and that its inhibition causes major defects in the regenerated limbs. Fgf8 is downregulated when BMP signaling is blocked, but ectopic injection of either human or axolotl protein did not rescue the defects. By administering LDN193189 treatments at different time points during regeneration, we show clearly that limb regeneration progresses in a proximal to distal fashion. This demonstrates that BMPs play a major role in patterning of regenerated limbs and that regeneration is a progressive process like development.

Epithelial to mesenchymal transition is mediated by both TGF-ß canonical and non-canonical signaling during axolotl limb regeneration.

Axolotls have the amazing ability to regenerate. When compared to humans, axolotls display a very fast wound closure, no scarring and are capable to replace lost appendages perfectly. Understanding the signaling mechanism leading to this perfect healing is a key step to help develop regenerative treatments for humans. In this paper, we studied cellular pathways leading to axolotl limb regeneration. We focus on the wound closure phase where keratinocytes migrate to close the lesion site and how epithelial to mesenchymal transitions are involved in this process. We observe a correlation between wound closure and EMT marker expression. Functional analyses using pharmacological inhibitors showed that the TGF-ß/SMAD (canonical) and the TGF-ß/p38/JNK (non-canonical) pathways play a role in the rate to which the keratinocytes can migrate. When we treat the animals with a combination of inhibitors blocking both canonical and non-canonical TGF-ß pathways, it greatly reduced the rate of wound closure and had significant effects on certain known EMT genes.

Endoglin haploinsufficiency is associated with differential regulation of extracellular matrix production during skin fibrosis and cartilage repair in mice.

Transforming growth factor (TGF)-ß is a multifunctional growth factor with potent pro-fibrotic effects. Endoglin is a TGF-ß co-receptor that strongly regulates TGF-ß signaling in a variety of cell types. Although aberrant regulation of TGF-ß signaling is known to play a key role in fibrotic diseases such as scleroderma and impaired cartilage repair, the significance of endoglin function in regulating these processes is poorly understood. Here we examined whether endoglin haploinsufficiency regulates extracellular (ECM) protein expression and fibrotic responses during bleomycin induced skin fibrosis and surgically induced osteoarthritis, using endoglin-heterozygous (Eng+/-) mice and wild-type (Eng+/+) littermates. Skin fibrosis was induced by injecting mice intradermally with bleomycin or vehicle. Osteoarthritis was induced surgically by destabilization of medial meniscus. Dermal thickness, cartilage integrity and ECM protein expression were then determined. Eng+/- mice subjected to bleomycin challenge show a marked decrease in dermal thickness (P < 0.005) and reduced collagen content and decreased collagen I, fibronectin, alpha-smooth muscle actin levels as compared to Eng+/+ mice, both under basal and bleomycin treated conditions. Eng+/- mice undergoing surgically induced osteoarthritis show no differences in the degree of cartilage degradation, as compared to Eng+/+ mice, although chondrocytes isolated from Eng+/- display markedly enhanced collagen II levels. Our findings suggest that endoglin haploinsufficiency in mice ameliorates bleomycin-induced skin fibrosis suggesting that endoglin represents a pro-fibrotic factor in the mouse skin. However, endoglin haploinsufficiency does not protect these mice from surgically indiced cartilage degradation, demonstrating differential regulation of endoglin action during skin and cartilage repair.

Senescence gives insights into the morphogenetic evolution of anamniotes.

Senescence represents a mechanism to avoid undesired cell proliferation that plays a role in tumor suppression, wound healing and embryonic development. In order to gain insight on the evolution of senescence, we looked at its presence in developing axolotls (urodele amphibians) and in zebrafish (teleost fish), which are both anamniotes. Our data indicate that cellular senescence is present in various developing structures in axolotls (pronephros, olfactory epithelium of nerve fascicles, lateral organs, gums) and in zebrafish (epithelium of the yolk sac and in the lower part of the gut). Senescence was particularly associated with transient structures (pronephros in axolotls and yolk sac in zebrafish) suggesting that it may play a role in the elimination of these tissues. Our data supports the notion that cellular senescence evolved early in vertebrate evolution to influence embryonic development.

Activation of Smad2 but not Smad3 is required to mediate TGF-ß signaling during axolotl limb regeneration.

Axolotls are unique among vertebrates in their ability to regenerate tissues, such as limbs, tail and skin. The axolotl limb is the most studied regenerating structure. The process is well characterized morphologically; however, it is not well understood at the molecular level. We demonstrate that TGF-ß1 is highly upregulated during regeneration and that TGF-ß signaling is necessary for the regenerative process. We show that the basement membrane is not prematurely formed in animals treated with the TGF-ß antagonist SB-431542. More importantly, Smad2 and Smad3 are differentially regulated post-translationally during the preparation phase of limb regeneration. Using specific antagonists for Smad2 and Smad3 we demonstrate that Smad2 is responsible for the action of TGF-ß during regeneration, whereas Smad3 is not required. Smad2 target genes (Mmp2 and Mmp9) are inhibited in SB-431542-treated limbs, whereas non-canonical TGF-ß targets (e.g. Mmp13) are unaffected. This is the first study to show that Smad2 and Smad3 are differentially regulated during regeneration and places Smad2 at the heart of TGF-ß signaling supporting the regenerative process.

Oral-Facial Tissue Reconstruction in the Regenerative Axolotl.

Absence of large amounts of orofacial tissues caused by cancerous resections, congenital defects, or trauma results in sequelae such as dysphagia and noticeable scars. Oral-neck tissue regeneration was studied in the axolotl (regenerative amphibian) following a 2.5-mm punch biopsy that simultaneously removed skin, connective tissue, muscle, and cartilage in the tongue and intermandibular region. The untreated wound was studied macroscopically and histologically at 17 different time points ranging from 0 to180 days (N = 120 axolotls). At 12 hr, the wound's surface was smoothened and within 1mm, internal lingual muscular modifications occurred; at the same distance, between days 4-7 lingual muscle degradation was complete. Immunofluorescence indicates complete keratinocytes migration by 48 hr. These cells with epidermal Leydig cells, appearing yellow, lead the chin's deep tissue outgrowth until its closure on the 14th day. Regeneration speeds varied and peaked in time for each tissue, (1) deep chin 84.3 µm/hr from 24 to 96 hr, (2) superficial chin 71.1 µm/hr from 7-14 days, and (3) tongue 86.0 µm/hr between 48 hr and 7 days. Immunofluorescence to Col IV showed basement membrane reconnected between days 30-45 coinciding with the chin's dermal tissue's surface area recovery. New muscle appeared at 21 days and was always preceded by the formation of a collagen bed. Both chin tissues regain all surface area and practically all components while the lingual structure lacks some content but is generally similar to the original. The methodology and high-resolution observations described here are the first of its kind for this animal model and could serve as a basis for future studies in oral and facial regenerative research.

Evaluation of the anesthetic effects of MS222 in the adult Mexican axolotl (Ambystoma mexicanum).

The Mexican axolotl (Ambystoma mexicanum) is a unique research model in several fields of medicine, where surgical and invasive procedures may be required. As yet, little is known about the efficacy of MS222 (tricaine methanesulfonate), which is the most commonly used anesthetic agent in amphibians. The main objectives of this study were to evaluate the anesthetic effects and physiological changes in adult axolotls following a 20-minute immersion bath, containing progressive MS222 concentrations starting at 0.1%. Depth of anesthesia and physiological changes were evaluated every 15 minutes post-MS222 exposure with the following parameters: righting behavior, withdrawal reflex, acetic acid test response, heart rate, and blood oxygen saturation, as well as cloacal and body surface temperatures. A 20-minute exposure in a 0.1% MS222 immersion bath (n=6 animals) had no anesthetic effects on adult axolotls after 20 minutes of exposure. With a 0.2% MS222 solution, all axolotls (n=9) were deeply anesthetized at 15 minutes, and 80% were still unresponsive at 30 minutes postexposure. Blood oxygen saturation and heart rate were slightly, but significantly, increased when compared with the baseline value and remained stable up to recovery. There was no significant increase in surface and cloaca temperatures, compared with baseline. With the 0.4% MS222 solution, the duration of anesthesia lasted for 90 minutes to at least 120 minutes (n=3 animals) and this concentration was deemed too high. In conclusion, a 20-minute immersion bath with 0.2% MS222 may be used for short procedures (15-30 minutes) requiring anesthesia of adult axolotls.

Culture and transfection of axolotl cells.

The use of cells grown in vitro has been instrumental for multiple aspects of biomedical research and especially molecular and cellular biology. The ability to grow cells from multicellular organisms like humans, squids, or salamanders is important to simplify the analyses and experimental designs to help understand the biology of these organisms. The advent of the first cell culture has allowed scientists to tease apart the cellular functions, and in many situations these experiments help understand what is happening in the whole organism. In this chapter, we describe techniques for the culture and genetic manipulation of an established cell line from axolotl, a species widely used for studying epimorphic regeneration.

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing.

Transforming growth factor-ß (TGF-ß) is a multifunctional growth factor involved in all aspects of wound healing. TGF-ß accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol-anchored protein, as a novel TGF-ß coreceptor and inhibitor of TGF-ß signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild-type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin-1α and monocyte chemoattractant protein-1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.

Axolotl as a Model to Study Scarless Wound Healing in Vertebrates: Role of the Transforming Growth Factor Beta Signaling Pathway.

SIGNIFICANCE: The skin is our largest organ, with the primary role of protection against assaults from the outside world. It also suffers frequent damage, from minor scrapes to, more rarely, complete destruction such as in third-degree burns. It is therefore, by its nature, an organ that would benefit tremendously from being able to regenerate itself. RECENT ADVANCES: This review highlights the axolotl, a less well-known model organism capable of scarless wound healing and regeneration. Axolotls are salamanders with unsurpassed healing and regenerative capacities. Understanding how these animals can regenerate their tissues could help identify the pathways that need to be activated or inhibited in humans to improve wound healing. CRITICAL ISSUES: Presently, there are no therapies leading to skin regeneration or scarless wound healing. Various animal models have thus been developed for use in research, such as mice and pigs, to help us understand how wound healing could be improved or stimulated. However, these more common models cannot regenerate and, consequently, cannot direct us toward a solution to regenerate damaged tissues. Axolotls, on the other hand, can regenerate perfectly and therefore may offer avenues to identify molecular targets for therapeutic intervention. FUTURE DIRECTIONS: Identifying signaling pathways regulating tissue regeneration in vertebrate models is important. The use of animals such as axolotls, which hold the secret of full regeneration, will likely play a significant role in helping us achieve scarless wound healing for humans.

Polymerase chain reaction detection of potentially pathogenic free-living amoebae in dental units.

Several genera of amoebae can be found in water from dental units and on the inner surface of waterlines. The presence of bacterial biofilms on these surfaces is thought to favor the proliferation of amoebae. Potentially pathogenic Acanthamoeba and Naegleria spp. may be an infection risk for patients through contact with open surgical sites or aerosolization. A polymerase chain reaction of DNA extracted from pelleted samples showed that Acanthamoeba spp. and Naegleria spp. were present in water from dental units, suction lines, and suction filters at the dental clinic of the Université de Montréal. Acanthamoeba spp. were detected in 24.2% of 66 samples and Naegleria spp. in 3.0%. We discuss the infection risk associated with these results.

In situ semi-quantitative analysis of polluted soils by laser-induced breakdown spectroscopy (LIBS).

Time-saving, low-cost analyses of soil contamination are required to ensure fast and efficient pollution removal and remedial operations. In this work, laser-induced breakdown spectroscopy (LIBS) has been successfully applied to in situ analyses of polluted soils, providing direct semi-quantitative information about the extent of pollution. A field campaign has been carried out in Brittany (France) on a site presenting high levels of heavy metal concentrations. Results on iron as a major component as well as on lead and copper as minor components are reported. Soil samples were dried and prepared as pressed pellets to minimize the effects of moisture and density on the results. LIBS analyses were performed with a Nd:YAG laser operating at 1064 nm, 60 mJ per 10 ns pulse, at a repetition rate of 10 Hz with a diameter of 500 µm on the sample surface. Good correlations were obtained between the LIBS signals and the values of concentrations deduced from inductively coupled plasma atomic emission spectroscopy (ICP-AES). This result proves that LIBS is an efficient method for optimizing sampling operations. Indeed, "LIBS maps" were established directly on-site, providing valuable assistance in optimizing the selection of the most relevant samples for future expensive and time-consuming laboratory analysis and avoiding useless analyses of very similar samples. Finally, it is emphasized that in situ LIBS is not described here as an alternative quantitative analytical method to the usual laboratory measurements but simply as an efficient time-saving tool to optimize sampling operations and to drastically reduce the number of soil samples to be analyzed, thus reducing costs. The detection limits of 200 ppm for lead and 80 ppm for copper reported here are compatible with the thresholds of toxicity; thus, this in situ LIBS campaign was fully validated for these two elements. Consequently, further experiments are planned to extend this study to other chemical elements and other matrices of soils.