An Overview of 24 Years of Molecular Phylogenetic Studies in Phallales (Basidiomycota) With Notes on Systematics, Geographic Distribution, Lifestyle, and Edibility.

The order Phallales (Basidiomycota) is represented by gasteroid fungi with expanded and sequestrate basidiomata, known as stinkhorns and false truffles. In phalloids, the first DNA sequence was published in 1997, and after that, some studies aimed to resolve phylogenetic conflicts and propose new species based on DNA markers; however, the number of families and genera in the order still generates controversies among researchers. Thus, this work aims to provide an overview of Phallales diversity represented by selected DNA markers available in public databases. We retrieved Phallales sequences from DNA databases (GenBank and UNITE) of seven markers: ITS (internal transcribed spacer), nuc-LSU (nuclear large subunit rDNA), nuc-SSU (nuclear small subunit rDNA), mt-SSU (mitochondrial small subunit rDNA), ATP6 (ATPase subunit 6), RPB2 (nuclear protein-coding second largest subunit of RNA polymerase), and TEF1-α (translation elongation factor subunit 1α). To compose our final dataset, all ITS sequences retrieved were subjected to BLASTn searches to identify additional ITS sequences not classified as Phallales. Phylogenetic analyses based on Bayesian and maximum likelihood approaches using single and combined markers were conducted. All ITS sequences were clustered with a cutoff of 98% in order to maximize the number of species hypotheses. The geographic origin of sequences was retrieved, as well as additional information on species lifestyle and edibility. We obtained a total of 1,149 sequences, representing 664 individuals. Sequences of 41 individuals were unidentified at genus level and were assigned to five distinct families. We recognize seven families and 22 genera in Phallales, although the delimitation of some genera must be further revisited in order to recognize only monophyletic groups. Many inconsistencies in species identification are discussed, and the positioning of genera in each family is shown. The clustering revealed 118 species hypotheses, meaning that approximately 20% of all described species in Phallales have DNA sequences available. Information related to geographic distribution represents 462 individuals distributed in 46 countries on all continents, except Antarctica. Most genera are saprotrophic with only one putative ectomycorrhizal genus, and 2.1% of the legitimate specific names recognized in Phallales are confirmed edible species. Great progress in the molecular analyses of phalloids has already been made over these years, but it is still necessary to solve some taxonomic inconsistencies, mainly at genus level, and generate new data to expand knowledge of the group.

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle.

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

The effects of alveolar decortications on orthodontic tooth movement and bone remodelling in rats.

Objectives: Alveolar decortication (AD) is a minimally invasive procedure that can be performed in the orthodontic office as an intervention to accelerate tooth movement. There is a gap in the literature evaluating the earlier and delayed responses after AD using lighter orthodontic forces in a rat model. Therefore, the aim of this study was to determine the effects of AD in the amount of orthodontic tooth movement and on alveolar bone remodelling in a rodent model, after 7 or 14 days. Materials and methods: A total of 32 15-week-old male Wistar rats were used in four treatment groups: (1) orthodontic spring only (7 days), (2) orthodontic spring only + AD (7 days), (3) orthodontic spring only (14 days), and (4) orthodontic spring only + AD (14 days). A closed coil nickel-titanium spring delivering 8-10 g of force was used to move the molar mesially. Alveolar decortication was done using a high speed, quarter round bur adjacent to the left first maxillary molar, on the palatal alveolar bone. At each endpoint, rats were sacrificed and microfocus computed tomography and histological analysis were performed. Results: The spring + AD group presented with a significant increase in the rate of tooth movement when compared with spring only group, 7 and 14 days after the beginning of the experiments. In addition, the spring + AD group had a significant decrease in bone volume and tissue density and a significant increase in the trabecular spacing and the number of osteoclasts at 7 and 14 days. Furthermore, a fibrous tissue was found to replace the alveolar bone in the spring + AD group at day 14. Conclusion: Alveolar decortications enhanced bone remodelling around the tooth movement region and could be used as an adjunct surgical procedure to accelerate the rate of tooth movement.

PTH [1-34] induced differentiation and mineralization of mandibular condylar cartilage.

Intermittent Parathyroid Hormone (I-PTH) is the only FDA approved anabolic drug therapy available for the treatment of osteoporosis in males and postmenopausal females. The effects of I-PTH on the chondrogenic lineage of the mandibular condylar cartilage (MCC) are not well understood. To investigate the role of I-PTH on the MCC and subchondral bone, we carried out our studies using 4 to 5 week old triple transgenic mice (Col1a1XCol2a1XCol10a1). The experimental group was injected with PTH (80 µg/kg) daily for 2 weeks, while control group was injected with saline. Our histology showed that the I-PTH treatment led to an increased number of cells expressing Col1a1, Col2a1 and Col10a1. Additionally, there was an increase in cellular proliferation, increased proteoglycan distribution, increased cartilage thickness, increased TRAP activity, and mineralization. Immunohistochemical staining showed increased expression of pSMAD158 and VEGF in the MCC and subchondral bone. Furthermore our microCT data showed that I-PTH treatment led to an increased bone volume fraction, tissue density and trabecular thickness, with a decrease in trabecular spacing. Morphometric measurements showed increased mandibular length and condyle head length following I-PTH treatment. In conclusion, our study suggests that I-PTH plays a critical role in cellular proliferation, proteoglycan distribution, and mineralization of the MCC.

Cellular and Matrix Response of the Mandibular Condylar Cartilage to Botulinum Toxin.

OBJECTIVES: To evaluate the cellular and matrix effects of botulinum toxin type A (Botox) on mandibular condylar cartilage (MCC) and subchondral bone. MATERIALS AND METHODS: Botox (0.3 unit) was injected into the right masseter of 5-week-old transgenic mice (Col10a1-RFPcherry) at day 1. Left side masseter was used as intra-animal control. The following bone labels were intraperitoneally injected: calcein at day 7, alizarin red at day 14 and calcein at day 21. In addition, EdU was injected 48 and 24 hours before sacrifice. Mice were sacrificed 30 days after Botox injection. Experimental and control side mandibles were dissected and examined by x-ray imaging and micro-CT. Subsequently, MCC along with the subchondral bone was sectioned and stained with tartrate resistant acid phosphatase (TRAP), EdU, TUNEL, alkaline phosphatase, toluidine blue and safranin O. In addition, we performed immunohistochemistry for pSMAD and VEGF. RESULTS: Bone volume fraction, tissue density and trabecular thickness were significantly decreased on the right side of the subchondral bone and mineralized cartilage (Botox was injected) when compared to the left side. There was no significant difference in the mandibular length and condylar head length; however, the condylar width was significantly decreased after Botox injection. Our histology showed decreased numbers of Col10a1 expressing cells, decreased cell proliferation and increased cell apoptosis in the subchondral bone and mandibular condylar cartilage, decreased TRAP activity and mineralization of Botox injected side cartilage and subchondral bone. Furthermore, we observed reduced proteoglycan and glycosaminoglycan distribution and decreased expression of pSMAD 1/5/8 and VEGF in the MCC of the Botox injected side in comparison to control side. CONCLUSION: Injection of Botox in masseter muscle leads to decreased mineralization and matrix deposition, reduced chondrocyte proliferation and differentiation and increased cell apoptosis in the MCC and subchondral bone.

The Effect of Altered Loading on Mandibular Condylar Cartilage.

OBJECTIVE: The purpose of this study was to delineate the cellular, mechanical and morphometric effects of altered loading on the mandibular condylar cartilage (MCC) and subchondral bone. We hypothesized that altered loading will induce differentiation of cells by accelerating the lineage progression of the MCC. MATERIALS AND METHODS: Four-week-old male Dkk3 XCol2A1XCol10A1 mice were randomly divided into two groups: (1) Loaded-Altered loading of MCC was induced by forced mouth opening using a custom-made spring; (2) Control-served as an unloaded group. Mice were euthanized and flow cytometery based cell analysis, micro-CT, gene expression analysis, histology and morphometric measurements were done to assess the response. RESULTS: Our flow cytometery data showed that altered loading resulted in a significant increase in a number of Col2a1-positive (blue) and Col10a1-positive (red) expressing cells. The gene expression analysis showed significant increase in expression of BMP2, Col10a1 and Sox 9 in the altered loading group. There was a significant increase in the bone volume fraction and trabecular thickness, but a decrease in the trabecular spacing of the subchondral bone with the altered loading. Morphometric measurements revealed increased mandibular length, increased condylar length and increased cartilage width with altered loading. Our histology showed increased mineralization/calcification of the MCC with 5 days of loading. An unexpected observation was an increase in expression of tartrate resistant acid phosphatase activity in the fibrocartilaginous region with loading. CONCLUSION: Altered loading leads to mineralization of fibrocartilage and drives the lineage towards differentiation/maturation.