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The mechanistic target of rapamycin (mTOR) is an evolutionary conserved protein with a serine/ threonine kinase activity that regulates cell growth, proliferation, motility, survival, protein synthesis, autophagy and transcription. It is embedded in 2 large protein complexes: mTORC1 and mTORC2. Regulation of specific mTOR pathway functions depends on multiple GTPases, that act either as regulators of mTOR protein complexes, coupling energy availability with mTORC1 activity, or as downstream effectors of both mTORC1 and mTORC2. In this commentary, we highlight the advantages of studying the mTOR pathway in C. elegans, including the subcellular localization of the signaling pathway components and the animal phenotypes following tissue specific protein overexpression or knockdown. One important regulator that is not limited to the mTOR pathway is RHEB. We discuss in vitro and in vivo data suggesting that RHEB can function as an inhibitor of mTOR when not bound to GTP. RHEB-1 itself is regulated by Rab GDP dissociation inhibitor b, which directly binds to ATX-2. We also highlight the roles of these proteins in dietary restriction-depended reduction in animal size and fat content. [ABSTRACT FROM AUTHOR]

Dietary restriction (DR) is a metabolic intervention that extends the lifespan of multiple species, including yeast, flies, nematodes, rodents, and, arguably, rhesus monkeys and humans. Hallmarks of lifelong DR are reductions in body size, fecundity, and fat accumulation, as well as slower development. We have identified atx-2, the Caenorhabditis elegans homolog of the human ATXN2L and ATXN2 genes, as the regulator of these multiple DR phenotypes. Down-regulation of atx-2 increases the body size, cell size, and fat content of dietary-restricted animals and speeds animal development, whereas overexpression of atx-2 is sufficient to reduce the body size and brood size of wild-type animals. atx-2 regulates the mechanistic target of rapamycin (mTOR) pathway, downstream of AMP-activated protein kinase (AMPK) and upstream of ribosomal protein S6 kinase and mTOR complex 1 (TORC1), by its direct associationwith RabGDP dissociation inhibitor β, which likely regulates RHEB shuttling between GDP-bound and GTP-bound forms. Taken together, this work identifies a previously unknown mechanism regulating multiple aspects of DR, as well as unknown regulators of the mTOR pathway. They also extend our understanding of diet-dependent growth retardation, and offers a potential mechanism to treat obesity. [ABSTRACT FROM AUTHOR]

Animals subjected to dietary restriction (DR) have reduced body size, low fecundity, slower development, lower fat content and longer life span. We identified lamin as a regulator of multiple dietary restriction phenotypes. Downregulation of lmn-1, the single Caenorhabditis elegans lamin gene, increased animal size and fat content specifically in DR animals. The LMN-1 protein acts in the mTOR pathway, upstream of RAPTOR and S6 kinase β1 (S6K), a key component of and target of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), respectively. DR excludes the mTORC1 activator RAGC-1 from the nucleus. Downregulation of lmn-1 restores RAGC-1 to the nucleus, a necessary step for the activation of the mTOR pathway. These findings further link lamin to metabolic regulation. [ABSTRACT FROM AUTHOR]

Objective Materials and Methods Results Conclusions To evaluate the efficacy of recombinant psoriasin as a novel treatment for oral candidiasis by eliminating Candida albicans growth on polymethyl methacrylate denture base.Recombinant psoriasin protein was expressed and purified from E. coli, and Candida growth was monitored in vitro with varying concentrations of psoriasin. Subsequently, denture‐base polymethyl methacrylate was immersed in psoriasin's solution or voriconazole, and fungal growth on the acrylic base and in the medium was examined by scanning electron microscopy and optical density, respectively. Cellular viability of HeLa and human gingival fibroblast cells treated with psoriasin was measured by methylene blue assay.The findings reveal an effective antifungal activity of psoriasin, completely inhibiting Candida albicans growth in RPMI at a protein concentration above 400 nM. Immersing the polymethyl methacrylate with 50 μM psoriasin completely eradicates fungal growth. Psoriasin has low cytotoxicity in HeLa cells at a concentration higher than 12 μM and no toxic effect on human gingival fibroblasts.This study marks psoriasin as an effective alternative to conventional antifungal treatments for denture stomatitis and a safe alternative to chemical antifungals in dental medicine and beyond. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides show promise as versatile tools for intracellular delivery of therapeutic agents. Various peptides have originated from natural proteins with antimicrobial activity. We investigated the mammalian cell-penetrating properties of a 16-residue peptide with the sequence GRCRGFRRRCFCTTHC from the C-terminus tail of the Medicago truncatula defensin MtDef4. We evaluated the peptide's ability to penetrate multiple cell types. Our results demonstrate that the peptide efficiently penetrates mammalian cells within minutes and at a micromolar concentration. Moreover, upon N-terminal fusion to the fluorescent protein GFP, the peptide efficiently delivers GFP into the cells. Despite its remarkable cellular permeability, the peptide has only a minor effect on cellular viability, making it a promising candidate for developing a cell-penetrating peptide with potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: Chronological and biological age correlate with DNA methylation levels at specific sites in the genome. Linear combinations of multiple methylation sites, termed epigenetic clocks, can inform us the chronological age and predict multiple health-related outcomes. However, why some sites correlating with lifespan, healthspan, or specific medical conditions remain poorly understood. Kidney fibrosis is the common pathway for chronic kidney disease, which affects 10% of European and US populations. Results: Here we identify epigenetic clocks and methylation sites that correlate with kidney function. Moreover, we identify methylation sites that have a unique methylation signature in the kidney. Methylation levels in majority of these sites correlate with kidney state and function. When kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Interestingly, while the majority of sites are less methylated in the kidney and become more methylated with loss of function, a fraction of the sites are highly methylated in the kidney and become less methylated when kidney function declines. These methylation sites are enriched for specific transcription-factor binding sites. In a large subset of sites, changes in methylation patterns are accompanied by changes in gene expression in kidneys of chronic kidney disease patients. Conclusions: These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. However, this information loss is not random, but guided toward a baseline that is dependent on the genomic loci. Significance statement: DNA methylation at specific sites accurately reflects chronological and biological age. We identify sites that have a unique methylation pattern in the kidney. Methylation levels in the majority of these sites correlate with kidney state and function. Moreover, when kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Thus, the unique methylation signature of the kidney is degraded, and epigenetic information is lost, when kidney disease progresses. These methylation sites are enriched for specific and methylation-sensitive transcription-factor binding sites, and associated genes show disease-dependent changes in expression. These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. [ABSTRACT FROM AUTHOR]

Background: Chronological and biological age correlate with DNA methylation levels at specific sites in the genome. Linear combinations of multiple methylation sites, termed epigenetic clocks, can inform us the chronological age and predict multiple health-related outcomes. However, why some sites correlating with lifespan, healthspan, or specific medical conditions remain poorly understood. Kidney fibrosis is the common pathway for chronic kidney disease, which affects 10% of European and US populations. Results: Here we identify epigenetic clocks and methylation sites that correlate with kidney function. Moreover, we identify methylation sites that have a unique methylation signature in the kidney. Methylation levels in majority of these sites correlate with kidney state and function. When kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Interestingly, while the majority of sites are less methylated in the kidney and become more methylated with loss of function, a fraction of the sites are highly methylated in the kidney and become less methylated when kidney function declines. These methylation sites are enriched for specific transcription-factor binding sites. In a large subset of sites, changes in methylation patterns are accompanied by changes in gene expression in kidneys of chronic kidney disease patients. Conclusions: These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. However, this information loss is not random, but guided toward a baseline that is dependent on the genomic loci. Significance statement: DNA methylation at specific sites accurately reflects chronological and biological age. We identify sites that have a unique methylation pattern in the kidney. Methylation levels in the majority of these sites correlate with kidney state and function. Moreover, when kidney function deteriorates, all of these sites regress toward the common methylation pattern observed in other tissues. Thus, the unique methylation signature of the kidney is degraded, and epigenetic information is lost, when kidney disease progresses. These methylation sites are enriched for specific and methylation-sensitive transcription-factor binding sites, and associated genes show disease-dependent changes in expression. These results support the information theory of aging, and the hypothesis that the unique tissue identity, as captured by methylation patterns, is lost as tissue function declines. [ABSTRACT FROM AUTHOR]

The currently employed tooth extraction methods in dentistry involve mechanical disruption of the periodontal ligament fibers, leading to inevitable trauma to the bundle bone comprising the socket walls. In our previous work, we have shown that a recombinantly expressed truncated version of clostridial collagenase G (ColG) purified from Escherichia coli efficiently reduced the force needed for tooth extraction in an ex-situ porcine jaw model, when injected into the periodontal ligament. Considering that enhanced thermostability often leads to higher enzymatic activity and to set the basis for additional rounds of optimization, we used a computational protein design approach to generate an enzyme to be more thermostable while conserving the key catalytic residues. This process generated a novel collagenase (ColG-variant) harboring sixteen mutations compared to ColG, with a nearly 4℃ increase in melting temperature. Herein, we explored the potential of ColG-variant to further decrease the physical effort required for tooth delivery using our established ex-situ porcine jaw model. An average reduction of 11% was recorded in the force applied to extract roots of mandibular split first and second premolar teeth treated with ColG-variant, relative to those treated with ColG. Our results show for the first time the potential of engineering enzyme properties for dental medicine and further contribute to minimally invasive tooth extraction. [ABSTRACT FROM AUTHOR]

Aim: To reveal the heterogeneity of ex vivo‐cultured human mesenchymal stromal cells derived from either masticatory or lining oral mucosa. Materials and Methods: Cells were retrieved from the lamina propria of the hard palate and alveolar mucosa of three individuals. The analysis of transcriptomic‐level differences was accomplished using single‐cell RNA sequencing. Results: Cluster analysis clearly distinguished between cells from the masticatory and lining oral mucosa, and revealed 11 distinct cell sub‐populations, annotated as fibroblasts, smooth muscle cells or mesenchymal stem cells. Interestingly, cells presenting a mesenchymal stem cell‐like gene expression pattern were predominantly found in masticatory mucosa. Although cells of masticatory mucosa origin were highly enriched for biological processes associated with wound healing, those from the lining oral mucosa were highly enriched for biological processes associated with the regulation of epithelial cells. Conclusions: Our previous work had shown that cells from the lining and masticatory oral mucosae are phenotypically heterogeneous. Here, we extend these findings to show that these changes are not the result of differences in averages but rather represent two distinct cell populations, with mesenchymal stem cells more common in masticatory mucosa. These features may contribute to specific physiological functions and have relevance for potential therapeutic interventions. [ABSTRACT FROM AUTHOR]

Dental calculus is a solid deposit that forms and accumulates on the tooth surface, entrapping oral microorganisms, biomolecules, and other micro-debris found in the oral cavity. A mass spectrometry analysis of its protein content opens a vista into the subject's diet, oral flora, and even some aspects of health, thus providing new insight and expanding our knowledge of archaic cultures. Multiple experimental protocols have been proposed for the optimal extraction of proteins from dental calculus. Herein, we compared various experimental conditions in order to calibrate and validate a protocol for protein extraction. Our results show that a high concentration of acetic acid followed by mechanical crushing and sonication provided the highest protein yield, while acetone precipitation enabled the identification of more distinct proteins. We validated this protocol using archeological samples, identifying human and microbial proteins in specimens from the eighth and seventeenth centuries (approximately 250–1300 years ago). These findings demonstrate that the developed protocol is useful for studying excavated archaeological samples and that it might be utilized to explore the biohistory, dietary habits, and microbiome of archaic populations. [ABSTRACT FROM AUTHOR]

Aims: To compare the gene expression profiles and proliferation rates of fibroblasts from the oral lining and masticatory mucosae. Materials and methods: Primary human fibroblasts were retrieved from the posterior masticatory hard palate and the lining alveolar mucosa of five individuals. The gene expression profile was evaluated using total RNA sequencing. The proliferation rate was determined colorimetrically. Results: Substantial differences in specific gene groups and pathways were observed between fibroblasts from the two tissues. Significantly enriched gene ontology processes were focused on the extracellular components. Lining mucosa fibroblasts exhibited significantly higher expression of the principal structural collagens, cranial neural crest markers, and homeobox genes associated with positional memory. Masticatory mucosa fibroblasts showed greater expression of genes related to transforming growth factor‐β signalling, which may be associated with fibrosis. In addition, they expressed higher levels of the EP2 prostaglandin E2 receptor and Toll‐like receptor 1. Finally, masticatory mucosa fibroblasts exhibited a 10%–30% higher proliferation rate. Conclusions: Fibroblasts from the lining and masticatory oral mucosae are phenotypically heterogeneous, presenting distinct gene expression profiles and proliferation rates. These features may contribute to their specific physiological functions and have relevance for potential therapeutic applications. [ABSTRACT FROM AUTHOR]