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ObjectiveThis study aimed to histologically compare periodontal regeneration of one-wall intrabony defects treated with open flap debridement, β-tricalcium phosphate (β-TCP), and carbonate apatite (CO3Ap) in dogs.MethodsThe mandibular third premolars of four beagle dogs were extracted. Twelve weeks after the extraction, a one-wall bone defect of 4 mm × 5 mm (mesio-distal width × depth) was created on the distal side of the mandibular second premolar and mesial side of the fourth premolar. Each defect was randomly allocated to open flap debridement (control group), periodontal regeneration utilizing β-TCP, or CO3Ap. Eight weeks after the surgery, histologic and histometric analyses were performed.ResultsNo ankylosis, infection, or acute inflammation was observed at any of the experimental sites. Newly formed bone and cementum were observed in all experimental groups. The mineral apposition rate of the alveolar bone crest was higher in the CO3Ap group than in the control and β-TCP groups. The ratio of the new bone area was significantly higher in the CO3Ap group than in the control group (P

Pandemic and endemic strains of Vibrio cholerae arise from toxigenic conversion by the CTXφ bacteriophage, a process by which CTXφ infects nontoxigenic strains of V. cholerae. CTXφ encodes the cholera toxin, an enterotoxin responsible for the watery diarrhea associated with cholera infections. Despite the critical role of CTXφ during infections, signals that affect CTXφ-driven toxigenic conversion or expression of the CTXφ-encoded cholera toxin remain poorly characterized, particularly in the context of the gut mucosa. Here, we identify mucin polymers as potent regulators of CTXφ-driven pathogenicity in V. cholerae. Our results indicate that mucin-associated O-glycans block toxigenic conversion by CTXφ and suppress the expression of CTXφ-related virulence factors, including the toxin co-regulated pilus and cholera toxin, by interfering with the TcpP/ToxR/ToxT virulence pathway. By synthesizing individual mucin glycan structures de novo, we identify the Core 2 motif as the critical structure governing this virulence attenuation. Overall, our results highlight a novel mechanism by which mucins and their associated O-glycan structures affect CTXφ-mediated evolution and pathogenicity of V. cholerae, underscoring the potential regulatory power housed within mucus.

Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast-to-hyphal transition of Candida albicans, a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O-glycans. We isolated and cataloged >100 mucin O-glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa. Using synthetic O-glycans, we identified three structures (core 1, core 1 + fucose and core 2 + galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O-glycan pool. Overall, this work identifies mucin O-glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.

The SARS-CoV-2 betacoronavirus uses its highly glycosylated trimeric Spike protein to bind to the cell surface receptor angiotensin converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. We utilized glycomics-informed glycoproteomics to characterize site-specific microheterogeneity of glycosylation for a recombinant trimer Spike mimetic immunogen and for a soluble version of human ACE2. We combined this information with bioinformatics analyses of natural variants and with existing 3D structures of both glycoproteins to generate molecular dynamics simulations of each glycoprotein both alone and interacting with one another. Our results highlight roles for glycans in sterically masking polypeptide epitopes and directly modulating Spike-ACE2 interactions. Furthermore, our results illustrate the impact of viral evolution and divergence on Spike glycosylation, as well as the influence of natural variants on ACE2 receptor glycosylation. Taken together, these data can facilitate immunogen design to achieve antibody neutralization and inform therapeutic strategies to inhibit viral infection.

Sialyltransferases are a family of 20 gene products in mice and humans that transfer sialic acid from its activated precursor, CMP-sialic acid, to the terminus of glycoprotein and glycolipid acceptors. ST3Gal-II (coded by the St3gal2 gene) transfers sialic acid preferentially to the three positions of galactose on the Galβ1-3GalNAc terminus of gangliosides GM1 and GD1b to synthesize GD1a and GT1b, respectively. Mice with a targeted disruption of St3gal2 unexpectedly displayed late-onset obesity and insulin resistance. At 3 months of age, St3gal2-null mice were the same weight as their wild type (WT) counterparts, but by 13 months on standard chow they were visibly obese, 22% heavier and with 37% greater fat/lean ratio than WT mice. St3gal2-null mice became hyperglycemic and displayed impaired glucose tolerance by 9 months of age. They had sharply reduced insulin responsiveness despite equivalent pancreatic islet morphology. Analyses of insulin receptor (IR) tyrosine kinase substrate IRS-1 and downstream target Akt revealed decreased insulin-induced phosphorylation in adipose tissue but not liver or skeletal muscle of St3gal2-null mice. Thin-layer chromatography and mass spectrometry revealed altered ganglioside profiles in the adipose tissue of St3gal2-null mice compared to WT littermates. Metabolically, St3gal2-null mice display a reduced respiratory exchange ratio compared to WT mice, indicating a preference for lipid oxidation as an energy source. Despite their altered metabolism, St3gal2-null mice were hyperactive. We conclude that altered ganglioside expression in adipose tissue results in diminished IR sensitivity and late-onset obesity.

The cAMP‐PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP‐PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)‐based biosensor spPKA‐KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA‐KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA‐KTR1.0 translocates between the nucleus and cytoplasm in a cAMP‐PKA pathway‐dependent manner, indicating that the spPKA‐KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP‐PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA‐KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP‐PKA pathway in fission yeast cells with higher temporal resolution. Take‐away: spPKA‐KTR1.0 allows visualization of PKA activity at the single‐cell levelLive‐cell imaging reveals the transient decrease in PKA activity after M‐phaseOptogenetics allows simultaneous visualization and manipulation of PKA activity [ABSTRACT FROM AUTHOR]

O -GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O -GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O -GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O -GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O -GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O -GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O -GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 μg of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O -GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O -GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases. [ABSTRACT FROM AUTHOR]

Purpose/objectives: Tooth carvings are evaluated subjectively. However, subjective evaluations are associated with low intra‐ and inter‐evaluator reliability in providing feedback for the fabrication of better tooth carvings. Therefore, this study aimed to analyze the relationship between subjective evaluation and the morphological characteristics of tooth carvings and their morphological characteristics using the feedback method in the low‐scoring group. Methods: Between April 2013 and September 2021, 120 maxillary left first molar carvings fabricated by undergraduate students were subjectively evaluated by an experienced instructor using a five‐point scale. The tooth carvings were scanned to obtain three‐dimensional shape data and divided into two groups, the high‐ and low‐scoring groups, for principal component analysis. Homologous models with dimensions matching those of the average model were created, and principal component analysis was performed to evaluate the morphological characteristics of the tooth. Results: Principal component analysis indicated that an objective evaluation was possible using homologous model. On increasing the subjective evaluation, the occlusal inclination angle and the cervical region of the tooth became steeper, and the shape of the occlusal surface resembled a parallelogram. In addition, large morphological differences were observed in the position of the cervical region of the tooth, height of contour, and shape of the occlusal surface in the low‐scoring group, whereas no such difference was observed in the high‐scoring group. Conclusion: Objective evaluation of tooth carving was possible using homologous model. The evaluation of tooth morphological characteristics could be effective in providing feedback to undergraduate students. [ABSTRACT FROM AUTHOR]

Background: Bone morphogenetic protein-2 (bmp-2) has a high potential to induce bone tissue formation in skeletal muscles. We developed a bone induction system in skeletal muscles using the bmp-2 gene through in vivo electroporation. Natural bone tissues with skeletal muscles can be considered potential candidates for biomaterials. However, our previous system using plate-type electrodes did not achieve a 100% success rate in inducing bone tissues in skeletal muscles. In this study, we aimed to enhance the efficiency of bone tissue formation in skeletal muscles by using a non-viral bmp-2 gene expression plasmid vector (pCAGGS-bmp-2) and needle-type electrodes. Methods: We injected the bmp-2 gene with pCAGGS-bmp-2 into the skeletal muscles of rats' legs and immediately placed needle-type electrodes there. Skeletal tissues were then observed on the 21st day after gene transfer using soft X-ray and histological analyses. Results: The use of needle-type electrodes resulted in a 100% success rate in inducing bone tissues in skeletal muscles. In contrast, the plate-type electrodes only exhibited a 33% success rate. Thus, needle-type electrodes can be more efficient and reliable for transferring the bmp-2 gene to skeletal muscles, making them potential biomaterials for repairing bone defects. [ABSTRACT FROM AUTHOR]