Oxidative stress-mediated proapoptosis signaling: A novel theory on the mechanism underlying the pathogenesis of burning mouth syndrome.

BACKGROUND: Burning mouth syndrome (BMS) is a chronic oral pain disorder characterized by a generalized burning sensation in the oral mucosa without apparent medical or dental causes. Despite various hypotheses proposed to explain BMS pathogenesis, a clear understanding of the cellular-level events and associated histologic and molecular findings is lacking. Advancing our understanding of BMS pathogenesis could facilitate the development of more targeted therapeutic interventions. TYPES OF STUDIES REVIEWED: The authors conducted an extensive literature search and review of cellular mechanisms, focusing on evidence-based data that support a comprehensive hypothesis for BMS pathogenesis. The authors explored novel and detailed mechanisms that may account for the characteristic features of BMS. RESULTS: The authors proposed that BMS symptoms arise from the uncontrolled activation of proapoptotic transmembrane calcium permeable channels expressed in intraoral mucosal nerve fibers. Elevated levels of reactive oxygen species or dysfunctional antiapoptosis pathways may lead to uncontrolled oxidative stress-mediated apoptosis signaling, resulting in upregulation of transmembrane transient receptor potential vanilloid type 1 and P2X 3 calcium channels in nociceptive fibers. Activation of these channels can cause nerve terminal depolarization, leading to generation of action potentials that are centrally interpreted as pain. CONCLUSIONS AND PRACTICAL IMPLICATIONS: The authors present a novel hypothesis for BMS pathogenesis, highlighting the role of proapoptotic transmembrane calcium permeable channels and oxidative stress-mediated apoptosis signaling in the development of BMS symptoms. Understanding these underlying mechanisms could provide new insights into the development of targeted therapeutic interventions for BMS. Additional research is warranted to validate this hypothesis and explore potential avenues for effective management of BMS.

Oocyte Aging: A Multifactorial Phenomenon in A Unique Cell.

The oocyte is considered to be the largest cell in mammalian species. Women hoping to become pregnant face a ticking biological clock. This is becoming increasingly challenging as an increase in life expectancy is accompanied by the tendency to conceive at older ages. With advancing maternal age, the fertilized egg will exhibit lower quality and developmental competence, which contributes to increased chances of miscarriage due to several causes such as aneuploidy, oxidative stress, epigenetics, or metabolic disorders. In particular, heterochromatin in oocytes and with it, the DNA methylation landscape undergoes changes. Further, obesity is a well-known and ever-increasing global problem as it is associated with several metabolic disorders. More importantly, both obesity and aging negatively affect female reproduction. However, among women, there is immense variability in age-related decline of oocytes' quantity, developmental competence, or quality. Herein, the relevance of obesity and DNA-methylation will be discussed as these aspects have a tremendous effect on female fertility, and it is a topic of continuous and widespread interest that has yet to be fully addressed for the mammalian oocyte.

Epigenetic aging of mammalian gametes.

The process of aging refers to physiological changes that occur to an organism as time progresses and involves changes to DNA, proteins, metabolism, cells, and organs. Like the rest of the cells in the body, gametes age, and it is well established that there is a decline in reproductive capabilities in females and males with aging. One of the major pathways known to be involved in aging is epigenetic changes. The epigenome is the multitude of chemical modifications performed on DNA and chromatin that affect the ability of chromatin to be transcribed. In this review, we explore the effects of aging on female and male gametes with a focus on the epigenetic changes that occur in gametes throughout aging. Quality decline in oocytes occurs at a relatively early age. Epigenetic changes constitute an important part of oocyte aging. DNA methylation is reduced with age, along with reduced expression of DNA methyltransferases (DNMTs). Histone deacetylases (HDAC) expression is also reduced, and a loss of heterochromatin marks occurs with age. As a consequence of heterochromatin loss, retrotransposon expression is elevated, and aged oocytes suffer from DNA damage. In sperm, aging affects sperm number, motility and fecundity, and epigenetic changes may constitute a part of this process. 5 methyl-cytosine (5mC) methylation is elevated in sperm from aged men, but methylation on Long interspersed nuclear elements (LINE) elements is reduced. Di and trimethylation of histone 3 lysine 9 (H3K9me2/3) is reduced in sperm from aged men and trimethylation of histone 3 lysine 27 (H3K27me3) is elevated. The protamine makeup of sperm from aged men is also changed, with reduced protamine expression and a misbalanced ratio between protamine proteins protamine P1 and protamine P2. The study of epigenetic reproductive aging is recently gaining interest. The current status of the field suggests that many aspects of gamete epigenetic aging are still open for investigation. The clinical applications of these investigations have far-reaching consequences for fertility and sociological human behavior.

Overexpressed kinetochore genes are used by cancer cells as genome destabilizers and transformation catalysts.

Cancer cells have an altered transcriptome, which contributes to their abnormal behavior. Many tumors have high levels of kinetochore genes, which play important roles in genome stability. This overexpression could be utilized to destabilize cancer cell genomes, however this has not been proven specifically. We investigated the link between kinetochore gene overexpression, chromosomal number variations (CNVs) and genomic instability. Data on RNA expression and CNV from 12 different cancer types were evaluated using information theory. In all cancer types, we looked at the relationship between RNA expression and CNVs. Kinetochore gene expression was found to be substantially linked with CNV levels. In all cancer types, with the exception of thyroid cancer, highly expressed kinetochore genes were enriched in the most dominant cancer-specific co-expression subnetworks characterizing the largest patient subgroups. Except for thyroid cancer, kinetochore inner protein CENPA was among the transcripts most strongly associated with CNV values in all cancer types studied, with significantly higher expression levels in patients with high CNVs than in patients with low CNVs. CENPA function was investigated further in cell models by transfecting genomically stable (HCT116) and unstable (MCF7 and HT29) cancer cell lines using CENPA overexpression vectors. This overexpression increased the number of abnormal cell divisions in the stable cancer cell line HCT116 and, to a lesser extent, in the unstable cell lines MCF7 and HT29. Overexpression improved anchorage-independent growth properties of all cell lines. Our findings suggest that overexpression of kinetochore genes in general, and CENPA in particular, can cause genomic instability and cancer progression.

Topical tretinoin treatment for burning mouth syndrome: a pilot study.

OBJECTIVES: Burning mouth syndrome is an intraoral chronic pain condition characterized by a moderate to severe sensation of burning from the oral mucosa. No clinical signs are found and there is no efficient treatment. METHOD AND MATERIALS: This pilot study included 10 women that were resistant to other previous treatments or noncompliant to systemic medications. Patients were asked to apply tretinoin gel 0.05% on their tongues twice daily for 14 days. Treatment effectiveness was assessed by completing a pre-study psychologic questionnaire and recording a daily wellbeing and pain log. RESULTS: Significant pain-score decrease in 50% of the patients (delta numerical rating score -3.15 ± 3.02, P value = .005) was recorded. This finding was in concordance with the verbal statements including major quality-of-life improvement (P value = .05), without any treatment positive or negative predictive factors. CONCLUSIONS: Topical tretinoin exhibits potential efficacy in patients with treatment resistant burning mouth syndrome and may also be used as a primary treatment modality.

Lef1 ablation alleviates cartilage mineralization following posttraumatic osteoarthritis induction.

Cartilage mineralization is a tightly controlled process, imperative for skeletal growth and fracture repair. However, in osteoarthritis (OA), cartilage mineralization may impact the joint range of motion, inflict pain, and increase chances for joint effusion. Here we attempt to understand the link between inflammation and cartilage mineralization by targeting Sirtuin 1 (SIRT1) and lymphoid enhancer binding factor 1 (LEF1), both reported to have contrasting effects on cartilage. We find that inflammatory-dependent cleavage of SIRT1 or its cartilage-specific genetic ablation, directly enhanced LEF1 expression accompanied by a catabolic response. Applying a posttraumatic OA (PTOA) model to cartilage-specific Sirt1 nulls displayed severe OA, which was accompanied by synovitis, meniscal mineralization, and osteophyte formation of the lateral joint compartment. Alternatively, cartilage-specific Lef1 nulls presented reduced lateral mineralization, OA severity, and local pain. Differential gene expression analysis revealed that Lef1 ablation reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Toll-like receptor (Tlr) pathways, while enhancing SRY-Box transcription factor 9 (Sox9) and cartilaginous extracellular matrix genes. The results support a link between inflammation and Lef1-dependent cartilage mineralization, mediated by the inactivation of Sirt1. By ablating Lef1 in a PTOA model, the structural and pain-related phenotypes of OA were reduced, in part, by preventing cartilage mineralization of the lateral joint compartment, partially manifested by meniscal tissue mineralization. Overall, these data provide a molecular axis to link between inflammation and cartilage in a PTOA model.

Loss of heterochromatin and retrotransposon silencing as determinants in oocyte aging.

Mammalian oocyte quality reduces with age. We show that prior to the occurrence of significant aneuploidy (9M in mouse), heterochromatin histone marks are lost, and oocyte maturation is impaired. This loss occurs in both constitutive and facultative heterochromatin marks but not in euchromatic active marks. We show that heterochromatin loss with age also occurs in human prophase I-arrested oocytes. Moreover, heterochromatin loss is accompanied in mouse oocytes by an increase in RNA processing and associated with an elevation in L1 and IAP retrotransposon expression and in DNA damage and DNA repair proteins nuclear localization. Artificial inhibition of the heterochromatin machinery in young oocytes causes an elevation in retrotransposon expression and oocyte maturation defects. Inhibiting retrotransposon reverse-transcriptase through azidothymidine (AZT) treatment in older oocytes partially rescues their maturation defects and activity of the DNA repair machinery. Moreover, activating the heterochromatin machinery via treatment with the SIRT1 activating molecule SRT-1720, or overexpression of Sirt1 or Ezh2 via plasmid electroporation into older oocytes causes an upregulation in constitutive heterochromatin, downregulation of retrotransposon expression, and elevated maturation rates. Collectively, our work demonstrates a significant process in oocyte aging, characterized by the loss of heterochromatin-associated chromatin marks and activation of specific retrotransposons, which cause DNA damage and impair oocyte maturation.

Pre-pubertal oocytes harbor altered histone modifications and chromatin configuration.

Pre-pubertal oocytes are still dormant. They are arrested in a GV state and do not undergo meiotic divisions naturally. A multitude of molecular pathways are changed and triggered upon initiation of puberty. It is not yet clear which epigenetic events occur in oocytes upon pubertal transition, and how significant these epigenetic events may be. We evaluated epigenetic marker levels in mouse pre-pubertal and post-pubertal female oocytes. In addition, we evaluated H3K9me2 levels in human oocytes collected from fertility preservation patients, comparing the levels between pre-pubertal patients and post-pubertal patients. The chromatin structure shows a lower number of chromocenters in mouse post-pubertal oocytes in comparison to pre-pubertal oocytes. All heterochromatin marker levels checked (H3K9me2, H3K27me3, H4K20me1) significantly rise across the pubertal transition. Euchromatin markers vary in their behavior. While H3K4me3 levels rise with the pubertal transition, H3K27Ac levels decrease with the pubertal transition. Treatment with SRT1720 [histone deacetylase (HDAC) activator] or overexpression of heterochromatin factors does not lead to increased heterochromatin in pre-pubertal oocytes. However, treatment of pre-pubertal oocytes with follicle-stimulating hormone (FSH) for 24 h - changes their chromatin structure to a post-pubertal configuration, lowers the number of chromocenters and elevates their histone methylation levels, showing that hormones play a key role in chromatin regulation of pubertal transition. Our work shows that pubertal transition leads to reorganization of oocyte chromatin and elevation of histone methylation levels, thus advancing oocyte developmental phenotype. These results provide the basis for finding conditions for in-vitro maturation of pre-pubertal oocytes, mainly needed to artificially mature oocytes of young cancer survivors for fertility preservation purposes.

Age-Dependent in vitro Maturation Efficacy of Human Oocytes - Is There an Optimal Age?

In vitro maturation of oocytes from antral follicles seen during tissue harvesting is a fertility preservation technique with potential advantages over ovarian tissue cryopreservation (OTC), as mature frozen and later thawed oocyte used for fertilization poses decreased risk of malignant cells re-seeding, as compared to ovarian tissue implantation. We previously demonstrated that in vitro maturation (IVM) performed following OTC in fertility preservation patients, even in pre-menarche girls, yields a fair amount of oocytes available for IVM and freezing for future use. We conducted a retrospective cohort study, evaluating IVM outcomes in chemotherapy naïve patients referred for fertility preservation by OTC that had oocyte collected from the medium with attempted IVM. A total of 133 chemotherapy naïve patients aged 1-35 years were included in the study. The primary outcome was IVM rate in the different age groups - pre-menarche (1-5 and ≥6 years), post-menarche (menarche-17 years), young adults (18-24 years) and adults (25-29 and 30-35 years). We demonstrate a gradual increase in mean IVM rate in the age groups from 1 to 25 years [4.6% (1-5 years), 23.8% (6 years to menarche), and 28.4% (menarche to 17 years)], with a peak of 38.3% in the 18-24 years group, followed by a decrease in the 25-29 years group (19.3%), down to a very low IVM rate (8.9%) in the 30-35 years group. A significant difference in IVM rates was noted between the age extremes - the very young (1-5 years) and the oldest (30-35 years) groups, as compared with the 18-24-year group (p < 0.001). Importantly, number of oocytes matured, percent of patients with matured oocytes, and overall maturation rate differed significantly (p < 0.001). Our finding of extremely low success rates in those very young (under 6 years) and older (≥30 years) patients suggests that oocytes retrieved during OTC prior to chemotherapy have an optimal window of age that shows higher success rates, suggesting that oocytes may have an inherent tendency toward better maturation in those age groups.

Endurance of extremely prolonged nutrient prevention across kingdoms of life.

Numerous observations demonstrate that microorganisms can survive very long periods of nutrient deprivation and starvation. Moreover, it is evident that prolonged periods of starvation are a feature of many habitats, and many cells in all kingdoms of life are found in prolonged starvation conditions. Bacteria exhibit a range of responses to long-term starvation. These include genetic adaptations such as the long-term stationary phase and the growth advantage in stationary phase phenotypes characterized by mutations in stress-signaling genes and elevated mutation rates. Here, we suggest using the term "endurance of prolonged nutrient prevention" (EPNP phase), to describe this phase, which was also recently described in eukaryotes. Here, we review this literature and describe the current knowledge about the adaptations to very long-term starvation conditions in bacteria and eukaryotes, its conceptual and structural conservation across all kingdoms of life, and point out possible directions that merit further research.

A Mouse Model for Studying the Development of Apical Periodontitis with Age.

Older age is associated with reduced immune function. Our aim was to study how age affects the development of apical periodontitis (AP). AP was induced in two age groups of mice (young vs. adult). Histological samples were stained by Hematoxylin Eosin, Brown and Brenn, and Tartrate-Resistant Acid Phosphatase. In addition, the samples were scanned by Micro-Computerized-Tomography (micro-CT) to evaluate apical constriction and periapical lesion size. Cell density in the periapical region was computationally assessed. Moreover, lesion immune cell populations were characterized by flow cytometry and immunofluorescence. The young group presented more canals with necrotic radicular pulp compared to the adults. There was no difference in bacteria location in the canals between the groups. Apical constriction size was larger in the young mice compared to the adults. The periapical cell density was higher in the young group, while the dominant immune cells in the lesions were neutrophils, which also exhibited the highest young/adult ratio. Immunofluorescence demonstrated neutrophils in the lesion. More osteoclasts were present in the lesions of the young mice, in correlation to the higher volume of bone resorption in this group. Overall, we conclude that the immune reaction to AP stimuli was attenuated in the adult mice compared to the young.

Centromeres are dismantled by foundational meiotic proteins Spo11 and Rec8.

Meiotic processes are potentially dangerous to genome stability and could be disastrous if activated in proliferative cells. Here we show that two key meiosis-defining proteins, the topoisomerase Spo11 (which forms double-strand breaks) and the meiotic cohesin Rec8, can dismantle centromeres. This dismantlement is normally observable only in mutant cells that lack the telomere bouquet, which provides a nuclear microdomain conducive to centromere reassembly1; however, overexpression of Spo11 or Rec8 leads to levels of centromere dismantlement that cannot be countered by the bouquet. Specific nucleosome remodelling factors mediate centromere dismantlement by Spo11 and Rec8. Ectopic expression of either protein in proliferating cells leads to the loss of mitotic kinetochores in both fission yeast and human cells. Hence, while centromeric chromatin has been characterized as extraordinarily stable, Spo11 and Rec8 challenge this stability and may jeopardize kinetochores in cancers that express meiotic proteins.

Cause and effect in epigenetics - where lies the truth, and how can experiments reveal it?: Epigenetic self-reinforcing loops obscure causation in cancer and aging.

Epigenetic changes are implicated in aging and cancer. Sometimes, it is clear whether the causing agent of the condition is a genetic factor or epigenetic. In other cases, the causative factor is unclear, and could be either genetic or epigenetic. Is there a general role for epigenetic changes in cancer and aging? Here, I present the paradigm of causative roles executed by epigenetic changes. I discuss cases with clear roles of the epigenome in cancer and aging, and other cases showing involvement of other factors. I also present the possibility that sometimes causality is difficult to assign because of the presence of self-reinforcing loops in epigenetic regulation. Such loops hinder the identification of the causative factor. I provide an experimental framework by which the role of the epigenome can be examined in a better setting and where the presence of such loops could be investigated in more detail.

Colon Cancer-Associated Fusobacterium nucleatum May Originate From the Oral Cavity and Reach Colon Tumors via the Circulatory System.

Fusobacterium nucleatum is a common oral bacterium that is enriched in colorectal adenomas and adenocarcinomas (CRC). In humans, high fusobacterial CRC abundance is associated with chemoresistance and poor prognosis. In animal models, fusobacteria accelerate CRC progression. Targeting F. nucleatum may reduce fusobacteria cancer progression and therefore determining the origin of CRC F. nucleatum and the route by which it reaches colon tumors is of biologic and therapeutic importance. Arbitrarily primed PCR performed previously on matched same-patients CRC and saliva F. nucleatum isolates, suggested that CRC F. nucleatum may originate from the oral cavity. However, the origin of CRC fusobacteria as well as the route of their arrival to the tumor have not been well-established. Herein, we performed and analyzed whole genome sequencing of paired, same-patient oral, and CRC F. nucleatum isolates and confirmed that CRC-fusobacteria originate from the oral microbial reservoir. Oral fusobacteria may translocate to CRC by descending via the digestive tract or using the hematogenous route during frequent transient bacteremia caused by chewing, daily hygiene activities, or dental procedures. Using the orthotropic CT26 mouse model we previously showed that IV injected F. nucleatum colonize CRC. Here, we compared CRC colonization by gavage vs. intravenous inoculated F. nucleatum in the MC38 and CT26 mouse orthotropic CRC models. Under the tested conditions, hematogenous fusobacteria were more successful in CRC colonization than gavaged ones. Our results therefore provide evidence that the hematogenous route may be the preferred way by which oral fusobacteria reach colon tumors.

Isolation and Characterization of Live Yeast Cells from Ancient Clay Vessels.

Ancient fermented food has been studied mainly based on residue analysis and recipes and reconstruction attempts were performed using modern domesticated yeast. Furthermore, microorganisms which participated in fermentation were studied using ancient-DNA techniques. In a recent paper, we presented a novel approach based on the hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. Here we present a pipeline for isolation of yeast from clay vessels uncovered in archeological sites and transferred to the microbiology lab where they can be isolated and characterized. This method opens new avenues for experimental archeology and enables attempts to recreate ancient food and beverages using the original microorganisms.

Inducing Apical Periodontitis in Mice.

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Eukaryotic Adaptation to Years-Long Starvation Resembles that of Bacteria.

The Growth Advantage in Stationary Phase (GASP) phenomenon, described in bacteria, reflects the genetic adaptation of bacteria to stress, including starvation, for a long time. Unlike in stationary phase where no cell division occurs, GASP harbors active cell division, concurrent with genetic adaptation. Here we show that GASP occurs also in eukaryotes. Two strains of Saccharomyces cerevisiae (Sc404 and Sc424) have been isolated from 2-year-old sealed bottles of beer. These strains presented advantage in survival and growth over the parent during stress. The differences between the strains are irreversible and therefore genetic in origin rather than epigenetic. Direct competition assays show that Sc404 and Sc424 outcompete the parent in direct competition. DNA sequencing shows changes of the genome: the TOR complexes are mutated, and DNA repair gene mutations confer a mutator phenotype. The differences between the strains are reflected in a difference in taste between beers brewed from them.

Isolation and Characterization of Live Yeast Cells from Ancient Vessels as a Tool in Bio-Archaeology.

Ancient fermented food has been studied based on recipes, residue analysis, and ancient-DNA techniques and reconstructed using modern domesticated yeast. Here, we present a novel approach based on our hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. We developed a pipeline of yeast isolation from clay vessels and screened for yeast cells in beverage-related and non-beverage-related ancient vessels and sediments from several archaeological sites. We found that yeast cells could be successfully isolated specifically from clay containers of fermented beverages. The findings that genotypically the isolated yeasts are similar to those found in traditional African beverages and phenotypically they grow similar to modern beer-producing yeast strongly suggest that they are descendants of the original fermenting yeast. These results demonstrate that modern microorganisms can serve as a new tool in bio-archaeology research.IMPORTANCE So far, most of the study of ancient organisms has been based mainly on the analysis of ancient DNA. Here we show that it is possible to isolate and study microorganisms-yeast in this case-from ancient pottery vessels used for fermentation. We demonstrate that it is highly likely that these cells are descendants of the original yeast strains that participated in the fermentation process and were absorbed into the clay matrix of the pottery vessels. Moreover, we characterized the isolated yeast strains, their genomes, and the beer they produced. These results open new and exciting avenues in the study of domesticated microorganisms and contribute significantly to the fields of bio- and experimental archaeology that aim to reconstruct ancient artifacts and products.