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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs.

BACKGROUND: Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas) in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. RESULTS: The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration) but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor) in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. CONCLUSION: The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has described in other tetrapods. Overexpression of BMP-2 did not cause the formation of extra digits, which is consistent with the hypothesis that it is not the secondary signal of sonic hedgehog. However, it did cause the formation of hypomorphic limbs as a result of increased cellular condensation and apoptosis. Taken together, these results suggest that BMP-2 does not have a direct role in patterning regenerating limbs but may be important to trigger condensation prior to ossification and to mediate apoptosis.

Skin wound healing in axolotls: a scarless process.

Urodele amphibians, such as the axolotl (Ambystoma mexicanum), have the unique faculty among vertebrates to regenerate lost appendages (limbs and tail) and other body parts (apex of the heart, forebrain and jaw) after amputation. Interestingly, axolotls never seem to form scar tissue at the site of amputation once regeneration is completed. Before now, very few studies were directly focused on the description of the events happening during wound healing after a skin injury in salamanders. In this paper, we directly investigated skin wound healing after excisional wounding which removed the epidermis, dermis and basement membrane in the axolotl. Axolotls were wounded with a 1.5-mm skin biopsy punch. Results show rapid re-epithelialization of the wound within 8 hrs after wounding. Histological analysis of wound healing confirmed the absence of tissue fibrosis throughout the process and shows that skin integrity is re-established by 90 days after wounding. Results also reveal the absence of neutrophils in the wound area, suggestive of a lack of or low inflammatory response. The expression of proteins central to wound healing seemed different than in mammals as α-smooth muscle actin was absent and transforming growth factor ß-1 was only transiently expressed during wound healing in the axolotl. Finally, subcutaneous injections of bleomycin were performed to verify whether the induction of scar tissue was possible in axolotls. Surprisingly, results show that axolotls are not resistant to bleomycin-induced tissue fibrosis, but the resulting scar tissue does not seem to contain significant amounts of collagen.

An analysis of immediate serial recall performance in a macaque.

There has been considerable research into the ability of nonhuman primates to process sequential information, a topic that is of interest in part because of the extensive involvement of sequence processing in human language use. Surprisingly, no previous study has unambiguously tested the ability of nonhuman primates to encode and immediately reproduce a novel temporal sequence of perceptual events, the ability tapped in the immediate serial recall (ISR) task extensively studied in humans. We report here the performance of a rhesus macaque on a spatial ISR task, closely resembling tasks widely used in human memory research. Detailed analysis of the monkey's recall performance indicates a number of important parallels with human ISR, consistent with the idea that a single mechanism for short-term serial order memory may be shared across species.

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.

Analysis of the expression and function of Wnt-5a and Wnt-5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs.

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.

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.

Urodele p53 tolerates amino acid changes found in p53 variants linked to human cancer.

BACKGROUND: Urodele amphibians like the axolotl are unique among vertebrates in their ability to regenerate and their resistance to develop cancers. It is unknown whether these traits are linked at the molecular level. RESULTS: Blocking p53 signaling in axolotls using the p53 inhibitor, pifithrin-alpha, inhibited limb regeneration and the expression of p53 target genes such as Mdm2 and Gadd45, suggesting a link between tumor suppression and regeneration. To understand this relationship we cloned the p53 gene from axolotl. When comparing its sequence with p53 from other organisms, and more specifically human we observed multiple amino acids changes found in human tumors. Phylogenetic analysis of p53 protein sequences from various species is in general agreement with standard vertebrate phylogeny; however, both mice-like rodents and teleost fishes are fast evolving. This leads to long branch attraction resulting in an artefactual basal emergence of these groups in the phylogenetic tree. It is tempting to assume a correlation between certain life style traits (e.g. lifespan) and the evolutionary rate of the corresponding p53 sequences. Functional assays of the axolotl p53 in human or axolotl cells using p53 promoter reporters demonstrated a temperature sensitivity (ts), which was further confirmed by performing colony assays at 37 degrees C. In addition, axolotl p53 was capable of efficient transactivation at the Hmd2 promoter but has moderate activity at the p21 promoter. Endogenous axolotl p53 was activated following UV irradiation (100 j/m2) or treatment with an alkylating agent as measured using serine 15 phosphorylation and the expression of the endogenous p53 target Gadd45. CONCLUSION: Urodele p53 may play a role in regeneration and has evolved to contain multiple amino acid changes predicted to render the human protein defective in tumor suppression. Some of these mutations were probably selected to maintain p53 activity at low temperature. However, other significant changes in the axolotl proteins may play more subtle roles on p53 functions, including DNA binding and promoter specificity and could represent useful adaptations to ensure p53 activity and tumor suppression in animals able to regenerate or subject to large variations in oxygen levels or temperature.