Fusobacterium nucleatum putatively affects the alveoli by disrupting the alveolar epithelial cell tight junction, enlarging the alveolar space, and increasing paracellular permeability.

Fusobacterium nucleatum (Fn) is abundant in the human oral cavity and has been associated with periodontal disease, which in-turn has been linked to respiratory disease development. Tight junctions (TJs) line the airway and alveoli surfaces serving as a first line of defense against multiple pathogens. Fn has already been linked to respiratory diseases, however, how Fn affects the alveolar TJ was not fully elucidated. Here, we designed and analyzed a TJ network, grew Fn cells and inoculated it in vitro (16HBE and primary cells) and in vivo (mice lung), measured transepithelial electrical resistance, performed RT-PCR, checked for in vitro cell and mice lung permeability, and determined air space size through morphometric measurements. We found that Fn can potentially affect TJs proteins that are directly exposed to the alveolar surface. Additionally, Fn could possibly cause neutrophil accumulation and an increase in alveolar space. Moreover, Fn putatively may cause an increase in paracellular permeability in the alveoli.

Structural patterns of SARS-CoV-2 variants of concern (alpha, beta, gamma, delta) spike protein are influenced by variant-specific amino acid mutations: A computational study with implications on viral evolution.

SARS-CoV-2 (SARS2) regularly mutates resulting to variants of concern (VOC) which have higher virulence and transmissibility rates while concurrently evading available therapeutic strategies. This highlights the importance of amino acid mutations occurring in the SARS2 spike protein structure since it may affect virus biology. However, this was never fully elucidated. Here, network analysis was performed based on the COVID-19 genomic epidemiology network between December 2019-July 2021. Representative SARS2 VOC spike protein models were generated and quality checked, protein model superimposition was done, and common contact based on contact mapping was established. Throughout this study, we found that: (1) certain individual variant-specific amino acid mutations can affect the spike protein structural pattern; (2) certain individual variant-specific amino acid mutations had no affect on the spike protein structural pattern; and (3) certain combination of variant-specific amino acids are putatively epistatic mutations that can potentially influence the VOC spike protein structural pattern. This manuscript was submitted as part of a theme issue on "Modelling COVID-19 and Preparedness for Future Pandemics".

Network analysis of the autophagy biochemical network in relation to various autophagy-targeted proteins found among SARS-CoV-2 variants of concern.

Autophagy is an important cellular process that triggers a coordinated action involving multiple individual proteins and protein complexes while SARS-CoV-2 (SARS2) was found to both hinder autophagy to evade host defense and utilize autophagy for viral replication. Interestingly, the possible significant stages of the autophagy biochemical network in relation to the corresponding autophagy-targeted SARS2 proteins from the different variants of concern (VOC) were never established. In this study, we performed the following: autophagy biochemical network design and centrality analyses; generated autophagy-targeted SARS2 protein models; and superimposed protein models for structural comparison. We identified 2 significant biochemical pathways (one starts from the ULK complex and the other starts from the PI3P complex) within the autophagy biochemical network. Similarly, we determined that the autophagy-targeted SARS2 proteins (Nsp15, M, ORF7a, ORF3a, and E) are structurally conserved throughout the different SARS2 VOC suggesting that the function of each protein is preserved during SARS2 evolution. Interestingly, among the autophagy-targeted SARS2 proteins, the M protein coincides with the 2 significant biochemical pathways we identified within the autophagy biochemical network. In this regard, we propose that the SARS2 M protein is the main determinant that would influence autophagy outcome in regard to SARS2 infection.

Porphyromonas gingivalis Mfa1 fimbria putatively binds to TLR2 and induces both IL-6 and IL-8 production in human bronchial epithelial cells.

Porphyromonas gingivalis (Pg) a major periodontal pathogen involved in periodontal disease development and progression. Moreover, Pg has two fimbriae surface proteins (FimA and Mfa1) that are genetically distinct and make-up the fimbrial shaft which in-turn form crucial attachment to oral bacteria and multiple host cells. However, unlike FimA, Mfa1 attachment to non-periodontal cells has not been fully elucidated. Considering Pg-associated periodontal disease contributes to pulmonary disease development, we investigated whether Mfa1 can functionally interact with human bronchial epithelial cells and, likewise, trigger a functional response. Initially, we simulated molecular docking and performed both luciferase and neutralization assays to confirm Mfa1-related functional interaction. Subsequently, we treated BEAS-2B cells with purified Mfa1 and performed cytokine quantification through real time-PCR and ELISA to establish Mfa1-related functional response. We found that both Mfa1-TLR2 and Mfa1-TLR4 docking is possible, however, only Mfa1-TLR2 showed a functional interaction. Additionally, we observed that both IL-8 and IL-6 gene expression and protein levels were induced confirming Mfa1-related functional response. Taken together, we propose that BEAS-2B human bronchial epithelial cells are able to recognize Pg Mfa1 and induce both IL-8 and IL-6 inflammatory responses.

Structural Insights on the SARS-CoV-2 Variants of Concern Spike Glycoprotein: A Computational Study With Possible Clinical Implications.

Coronavirus disease 2019 (COVID-19) pandemic has been attributed to SARS-CoV-2 (SARS2) and, consequently, SARS2 has evolved into multiple SARS2 variants driving subsequent waves of infections. In particular, variants of concern (VOC) were identified to have both increased transmissibility and virulence ascribable to mutational changes occurring within the spike protein resulting to modifications in the protein structural orientation which in-turn may affect viral pathogenesis. However, this was never fully elucidated. Here, we generated spike models of endemic HCoVs (HCoV 229E, HCoV OC43, HCoV NL63, HCoV HKU1, SARS CoV, MERS CoV), original SARS2, and VOC (alpha, beta, gamma, delta). Model quality check, structural superimposition, and structural comparison based on RMSD values, TM scores, and contact mapping were all performed. We found that: 1) structural comparison between the original SARS2 and VOC whole spike protein model have minor structural differences (TM > 0.98); 2) the whole VOC spike models putatively have higher structural similarity (TM > 0.70) to spike models from endemic HCoVs coming from the same phylogenetic cluster; 3) original SARS2 S1-CTD and S1-NTD models are structurally comparable to VOC S1-CTD (TM = 1.0) and S1-NTD (TM > 0.96); and 4) endemic HCoV S1-CTD and S1-NTD models are structurally comparable to VOC S1-CTD (TM > 0.70) and S1-NTD (TM > 0.70) models belonging to the same phylogenetic cluster. Overall, we propose that structural similarities (possibly ascribable to similar conformational epitopes) may help determine immune cross-reactivity, whereas, structural differences (possibly associated with varying conformational epitopes) may lead to viral infection (either reinfection or breakthrough infection).

S-PRG Filler Eluate Induces Oxidative Stress in Oral Microorganism: Suppression of Growth and Pathogenicity, and Possible Clinical Application.

Controlling the oral microbial flora is putatively thought to prevent not only oral diseases, but also systemic diseases caused by oral diseases. This study establishes the antibacterial effect of the novel bioactive substance "S-PRG filler" on oral bacteria. We examined the state of oxidative stress caused by the six types of ions released in eluate from the S-PRG filler in oral bacterial cells. Moreover, we investigated the effects of these ions on the growth and pathogenicity of Gram-positive and Gram-negative bacteria. We found that the released ions affected SOD amount and hydrogen peroxide in bacterial cells insinuating oxidative stress occurrence. In bacterial culture, growth inhibition was observed depending on the ion concentration in the medium. Additionally, released ions suppressed Streptococcus mutans adhesion to hydroxyapatite, S. oralis neuraminidase activity, and Porphyromonas gingivalis hemagglutination and gingipain activity in a concentration-dependent manner. From these results, it was suggested that the ions released from the S-PRG filler may suppress the growth and pathogenicity of the oral bacterial flora. This bioactive material is potentially useful to prevent the onset of diseases inside and outside of the oral cavity, which in turn may have possible applications for oral care and QOL improvement.

Insights on the Structural Variations of the Furin-Like Cleavage Site Found Among the December 2019-July 2020 SARS-CoV-2 Spike Glycoprotein: A Computational Study Linking Viral Evolution and Infection.

The SARS-CoV-2 (SARS2) is the cause of the coronavirus disease 2019 (COVID-19) pandemic. One unique structural feature of the SARS2 spike protein is the presence of a furin-like cleavage site (FLC) which is associated with both viral pathogenesis and host tropism. Specifically, SARS2 spike protein binds to the host ACE-2 receptor which in-turn is cleaved by furin proteases at the FLC site, suggesting that SARS2 FLC structural variations may have an impact on viral infectivity. However, this has not yet been fully elucidated. This study designed and analyzed a COVID-19 genomic epidemiology network for December 2019 to July 2020, and subsequently generated and analyzed representative SARS2 spike protein models from significant node clusters within the network. To distinguish possible structural variations, a model quality assessment was performed before further protein model analyses and superimposition of the protein models, particularly in both the receptor-binding domain (RBD) and FLC. Mutant spike models were generated with the unique 681PRRA684 amino acid sequence found within the deleted FLC. We found 9 SARS2 FLC structural patterns that could potentially correspond to nine node clusters encompassing various countries found within the COVID-19 genomic epidemiology network. Similarly, we associated this with the rapid evolution of the SARS2 genome. Furthermore, we observed that either in the presence or absence of the unique 681PRRA684 amino acid sequence no structural changes occurred within the SARS2 RBD, which we believe would mean that the SARS2 FLC has no structural influence on SARS2 RBD and may explain why host tropism was maintained.

Porphyromonas gingivalis gingipains potentially affect MUC5AC gene expression and protein levels in respiratory epithelial cells.

Porphyromonas gingivalis (Pg) is a periodontopathic pathogen that may affect MUC5AC-related mucus hypersecretion along airway epithelial cells. Here, we attempted to establish whether Pg virulence factors (lipopolysaccharide, FimA fimbriae, gingipains) affect MUC5AC in immortalized and primary bronchial cells. We report that MUC5AC gene expression and protein levels are affected by Pg culture supernatant, but not by lipopolysaccharide or FimA fimbriae. Cells treated with either Pg single (Kgp or Rgp) or double (Kgp/Rgp) mutants had altered levels of MUC5AC gene expression and protein levels, and MUC5AC staining of double mutant-treated mouse lung cells showed that MUC5AC protein levels were unaffected. Taken together, we propose that Pg gingipains may be the primary virulence factor that influences both MUC5AC gene expression and protein levels.

Heat-Killed Fusobacterium nucleatum Triggers Varying Heme-Related Inflammatory and Stress Responses Depending on Primary Human Respiratory Epithelial Cell Type.

Fusobacterium nucleatum (Fn) is generally an opportunistic oral pathogen that adheres to mammalian mucosal sites, triggering a host inflammatory response. In general, Fn is normally found within the human oral cavity; however, it was previously reported that Fn is a risk factor for certain respiratory diseases. Surprisingly, this was never fully elucidated. Here, we investigated the virulence potential of heat-killed Fn on primary human tracheal, bronchial, and alveolar epithelial cells. In this study, we measured the secretion of inflammatory- (IL-8 and IL-6), stress- (total heme and hydrogen peroxide), and cell death-related (caspase-1 and caspase-3) signals. We established that the inflammatory response mechanism varies in each epithelial cell type: (1) along tracheal cells, possible Fn adherence would trigger increased heme secretion and regulated inflammatory response; (2) along bronchial cells, potential Fn adherence would simultaneously initiate an increase in secreted H2O2 and inflammatory response (ascribable to decreased secreted heme amounts); and (3) along alveolar cells, putative Fn adherence would instigate the increased secretion of inflammatory responses attributable to a decrease in secreted heme levels. Moreover, regardless of the epithelial cell-specific inflammatory mechanism, we believe these are putative, not harmful. Taken together, we propose that any potential Fn-driven inflammation along the respiratory tract would be initiated by differing epithelial cell-specific inflammatory mechanisms that are collectively dependent on secreted heme.

Structural insights into the potential changes in receptor binding site found in the 1998-2018 influenza B/Yamagata hemagglutinin: A putative correlation between receptor binding site structural variability and seasonal infection.

Influenza B virus has two distinct lineages (Victoria and Yamagata) and are associated with seasonal influenza epidemics that cause respiratory illness. Influenza B hemagglutinin (HA) is a major surface glycoprotein with the receptor-binding site (RBS) primarily involved in viral pathogenesis. Generally, influenza B exclusively infects the human population which would insinuate that the structural variability of the influenza B HA RBS rarely changes. However, to our knowledge, the potential impact of variations in the influenza B HA RBS structural variability was not fully elucidated. Throughout this study, we generated models from the transitioning (evolving viral lineage) 1998-2018 influenza B/Yamagata HA, verified the quality of each HA model, performed HA RBS structural variability measurements, superimposed varying HA models for comparison, and designed a phylogenetic tree network for further analyses. We found that measurements of the transitioning HA RBS structural variability were generally maintained and, similarly, measurements of the altered (years that differed from the evolving viral lineage, specifically 2003, 2007, 2017) HA RBS structural variability differed from the transitioning HA RBS. Moreover, we observed that the altered HA RBS structural variability favored the formation of a putative Y202-H191 hydrogen bond which we postulate may increase structural stability, thereby, allowing for a winter infection of the virus. Furthermore, we established that changes in HA RBS structural variability does not influence viral evolution, but putatively seasonal infection.

Structural Comparison of the SARS CoV 2 Spike Protein Relative to Other Human-Infecting Coronaviruses.

Coronaviruses (CoV) are enveloped positive-stranded RNA viruses and, historically, there are seven known human-infecting CoVs with varying degrees of virulence. CoV attachment to the host is the first step of viral pathogenesis and mainly relies on the spike glycoprotein located on the viral surface. Among the human-infecting CoVs, only the infection of SARS CoV 2 (SARS2) among humans resulted to a pandemic which would suggest that the protein structural conformation of SARS2 spike protein is distinct as compared to other human-infecting CoVs. Surprisingly, the possible differences and similarities in the protein structural conformation between the various human-infecting CoV spike proteins have not been fully elucidated. In this study, we utilized a computational approach to generate models and analyze the seven human-infecting CoV spike proteins, namely: HCoV 229E, HCoV OC43, HCoV NL63, HCoV HKU1, SARS CoV, MERS CoV, and SARS2. Model quality assessment of all CoV models generated, structural superimposition of the whole protein model and selected S1 domains (S1-CTD and S1-NTD), and structural comparison based on RMSD values, Tm scores, and contact mapping were all performed. We found that the structural orientation of S1-CTD is a potential structural feature associated to both the CoV phylogenetic cluster and lineage. Moreover, we observed that spike models in the same phylogenetic cluster or lineage could potentially have similar protein structure. Additionally, we established that there are potentially three distinct S1-CTD orientation (Pattern I, Pattern II, Pattern III) among the human-infecting CoVs. Furthermore, we postulate that human-infecting CoVs in the same phylogenetic cluster may have similar S1-CTD and S1-NTD structural orientation. Taken together, we propose that the SARS2 spike S1-CTD follows a Pattern III orientation which has a higher degree of similarity with SARS1 and some degree of similarity with both OC43 and HKU1 which coincidentally are in the same phylogenetic cluster and lineage, whereas, the SARS2 spike S1-NTD has some degree of similarity among human-infecting CoVs that are either in the same phylogenetic cluster or lineage.

Structural significance of residues 158-160 in the H3N2 hemagglutnin globular head: A computational study with implications in viral evolution and infection.

Influenza A H3N2 has been linked to annual outbreaks within the human population attributable to continuous structural changes. H3N2 HA contains well identified antigenic sites and receptor-binding sites (RBS) that are possibly correlated to viral evolution and infection. However, the structural significance of amino acid residues associated with both viral evolution and infection were not fully demonstrated. Throughout this study, we generated and analyzed H3N2 HA models that represented the clade 3C.2 population (comprised of clades 3C.2, 3C.2a, and 3C.21 from the transitioning 2014-2018 H3N2 strains) and 3C.3a (from the 2016 H3N2 strain). Model quality estimation, structural analyses and superimposition, and network analytics of H3N2 HA1 evolution were performed. We found that the structural properties of residues 158-160 could influence the overall HA backbone. More specifically, amino acid substitutions at residues 159-160 affected the amino acid orientation at residue 158, thereby, causing the overall HA backbone structure to vary. Our results were consistent with 1968-2018 HA1 evolution. Taken together, we propose that our results would highlight the structural significance of residues 158-160 in HA1 for both antigenic drift and RBS.

Ions released from a S-PRG filler induces oxidative stress in Candida albicans inhibiting its growth and pathogenicity.

Candida albicans causes opportunistic fungal infections usually hidden among more dominant bacteria and does not exhibit high pathogenicity in vivo. Among the elderly, due to reduced host resistance to pathogens attributable to immunoscenesence, oral candidiasis is more likely to develop often leading to systemic candidiasis. Surface pre-reacted glass ionomer filler (S-PRG filler) is an ion-releasing functional bioactive glass that can release and recharge six ions which in turn strengthens tooth structure, inhibits demineralization arising from dental caries, and suppresses dental plaque accumulation. However, its effects on C. albicans have never been elucidated. Here, we evaluated the effects of ion released from S-PRG filler on C. albicans. Results show that extraction liquids containing released ions (ELIS) decreased the amount of hydrogen peroxide and catalase activity in C. albicans. Moreover, ELIS presence was found to affect C. albicans: (1) suppression of fungal growth and biofilm formation, (2) prevent adherence to denture base resin, (3) inhibit dimorphism conversion, and (4) hinder the capability to produce secreted aspartyl proteinase. Taken together, our findings suggest that ELIS induces oxidative stress in C. albicans and suppresses its growth and pathogenicity. In this regard, we propose that ELIS has the potential to be clinically used to help prevent the onset and inhibition of oral candidiasis among the elderly population.

Gingival Periodontal Disease (PD) Level-Butyric Acid Affects the Systemic Blood and Brain Organ: Insights Into the Systemic Inflammation of Periodontal Disease.

Butyric acid (BA) is produced by periodontopathic bacterial pathogens and contributes to periodontal disease (PD) induction. Moreover, PD has been associated with detrimental effects which subsequently may lead to systemic disease (SD) development affecting certain organs. Surprisingly, the potential systemic manifestations and organ-localized effects of BA have never been elucidated. Here, we simulated BA-based oral infection among young (20-week-old) rats and isolated blood cytosol to determine BA effects on stress network-related signals [total heme, hydrogen peroxide (H2O2), catalase (CAT), glutathione reductase (GR), free fatty acid (FFA), NADP/NADPH], inflammation-associated signals [caspases (CASP12 and CASP1), IL-1ß, TNF-α, metallomatrix proteinase-9 (MMP-9), and toll-like receptor-2 (TLR2)], and neurological blood biomarkers [presenilin (PS1 and PS2) and amyloid precursor protein (APP)]. Similarly, we extracted the brain from both control and BA-treated rats, isolated the major regions (hippocampus, pineal gland, hypothalamus, cerebrum, and cerebellum), and, subsequently, measured stress network-related signals [oxidative stress: total heme, NADPH, H2O2, GR, and FFA; ER stress: GADD153, calcium, CASP1, and CASP3] and a brain neurodegenerative biomarker (Tau). In the blood, we found that BA was no longer detectable. Nevertheless, oxidative stress and inflammation were induced. Interestingly, amounts of representative inflammatory signals (CASP12, CASP1, IL-1ß, and TNF-α) decreased while MMP-9 levels increased which we believe would suggest that inflammation was MMP-9-modulated and would serve as an alternative inflammatory mechanism. Similarly, TLR2 activity was increased which would insinuate that neurological blood biomarkers (APP, PS1, and PS2) were likewise affected. In the brain, BA was not detected, however, we found that both oxidative and ER stresses were likewise altered in all brain regions. Interestingly, tau protein amounts were significantly affected in the cerebellar and hippocampal regions which coincidentally are the major brain regions affected in several neurological disorders. Taken together, we propose that gingival BA can potentially cause systemic inflammation ascribable to prolonged systemic manifestations in the blood and localized detrimental effects within the brain organ.

Network analytics approach towards identifying potential antivirulence drug targets within the Staphylococcus aureus staphyloxanthin biosynthetic network.

Staphylococcus aureus is associated with several clinically significant infections among humans and infections associated with antibiotic-resistant strains are growing in frequency. Antivirulence strategies shift the target of drugs from bacterial growth to the infection process resulting to milder evolutionary pressure for the development of bacterial resistant strains. Staphyloxanthin (STX) is a yellowish-orange carotenoid pigment synthesized by S. aureus and this carotenoid functions as an important virulence factor for the bacteria. In this study, we elucidated whether network analytics can be used as a viable tool to identify significant components in the STX biosynthetic network which in-turn could serve as possible antivirulence drug targets. For confirmation, we correlated our results to known drugs that were able to inhibit STX biosynthesis. Throughout this study, we established that crtN(1) activity and 4,4'-diaponeurosporene amounts are significant components in the STX biosynthetic network and, moreover, network analytics can aid in identifying antivirulence drug targets within the STX biosynthetic network. Similarly, we found that network analytics is capable of identifying multiple potential targets simultaneously. Taken together, we propose that an effective antivirulence drug against S. aureus STX biosynthesis would involve targeting crtN(1) activity, 4,4'-diaponeurosporene levels, or both components.

Various cellular stress components change as the rat ages: An insight into the putative overall age-related cellular stress network.

Cellular stress is mainly comprised of oxidative, nitrosative, and endoplasmic reticulum stresses and has long been correlated to the ageing process. Surprisingly, the age-related difference among the various components in each independent stress pathway and the possible significance of these components in relation to the overall cellular stress network remain to be clearly elucidated. In this study, we obtained blood from ageing rats upon reaching 20-, 40-, and 72-wk.-old. Subsequently, we measured representative cellular stress-linked biomolecules (H2O2, glutathione reductase, heme, NADPH, NADP, nitric oxide, GADD153) and cell signals [substance P (SP), free fatty acid, calcium, NF-κB] in either or both blood serum and cytosol. Subsequently, network analysis of the overall cellular stress network was performed. Our results show that there are changes affecting stress-linked biomolecules and cell signals as the rat ages. Additionally, based on our network analysis data, we postulate that NADPH, H2O2, GADD153, and SP are the key components and the interactions between these components are central to the overall age-related cellular stress network in the rat blood. Thus, we propose that the main pathway affecting the overall age-related cellular stress network in the rat blood would entail NADPH-related oxidative stress (involving H2O2) triggering GADD153 activation leading to SP induction which in-turn affects other cell signals.

Structural comparison among the 2013-2017 avian influenza A H5N6 hemagglutinin proteins: A computational study with epidemiological implications.

Avian influenza viruses easily spread allowing viral re-assortment to simply occur which in-turn increases the potential for a pandemic. A novel 2013 H5N6 influenza strain was detected among the avian population and was reported to continuously evolve, however, this was never structurally demonstrated. Here, we elucidated the putative structural evolution of the novel H5N6 influenza strain. Throughout this study, we analyzed 2013-2017 H5N6 HA protein models. Model quality was first verified before further analyses and structural comparison was made using superimposition. We found that Leu was inserted at position 1291 among the 2013-2015 models while Leu was not inserted among the 2016-2017 models. Moreover, presence of Leu at position 1291 shifts residue E1261 by 159.6° affecting nearby residues which may explain the difference between the 2013-2015 and 2016-2017 HA structural groups. Similarly, we believe that our results would support the hypothesis that the current H5N6 strain is still continuously evolving.

Periodontal disease level-butyric acid putatively contributes to the ageing blood: A proposed link between periodontal diseases and the ageing process.

Periodontal diseases are partly attributable to periodontopathic bacteria found in the host, whereas, butyric acid (BA) is a common secondary metabolite produced by periodontopathic bacterial pathogens. BA has been linked to oxidative stress induction while oxidative stress has long been associated with the ageing process. However, the possible link between BA-induced oxidative stress and the ageing process has never been elucidated. Here, we attempted to show the possible role of periodontal diseaselevel-BA (PDL-BA) in influencing the rat blood ageing process. We injected PDL-BA into the young rat gingiva and, after 24h, heart blood extraction was performed. Blood obtained from PDL-BA-treated young rats was compared to untreated young and middle-aged rats. We found that cytosolic, but not mitochondrial, heme was affected 24h post-injection. In addition, we observed that PDL-BA treatment altered blood NOX activation, NADPH-related oxidative stress components (H2O2 and GR), calcium homeostasis, cell death signals (CASP3 and CASP1), and age-related markers (SIRT1 and mTOR) in young rats, with some components more closely mimicking levels found in middle-aged rats. In this regard, we propose that PDL-BA may play a role in contributing to the rat blood ageing process.

Varying butyric acid amounts induce different stress- and cell death-related signals in nerve growth factor-treated PC12 cells: implications in neuropathic pain absence during periodontal disease progression.

Neuropathic pain is absent from the early stages of periodontal disease possibly due to neurite retraction. Butyric acid (BA) is a periodontopathic metabolite that activates several stress-related signals and, likewise, induce neurite retraction. Neuronal cell death is associated to neurite retraction which would suggest that BA-induced neurite retraction is ascribable to neuronal cell death. However, the underlying mechanism of BA-related cell death signaling remains unknown. In this study, we exposed NGF-treated PC12 cells to varying BA concentrations [0 (control), 0.5, 1.0, 5.0 mM] and determined selected stress-related (H2O2, glutathione reductase, calcium (Ca(2+)), plasma membrane Ca(2+) ATPase (PMCA), and GADD153/CHOPS) and cell death-associated (extrinsic: FasL, TNF-α, TWEAK, and TRAIL; intrinsic: cytochrome C (CytC), NF-kB, CASP8, CASP9, CASP10, and CASP3) signals. Similarly, we confirmed cell death execution by chromatin condensation. Our results showed that low (0.5 mM) and high (1.0 and 5.0 mM) BA levels differ in stress and cell death signaling. Moreover, at periodontal disease-level BA concentration (5 mM), we observed that only FasL amounts were affected and occurred concurrently with chromatin condensation insinuating that cells have fully committed to neurodegeneration. Thus, we believe that both stress and cell death signaling in NGF-treated PC12 cells are affected differently depending on BA concentration. In a periodontal disease scenario, we hypothesize that during the early stages, low BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurite non-proliferation, whereas, during the later stages, high BA amounts accumulate resulting to both stress- and cell death-related signals that favor neurodegeneration. More importantly, we propose that neuropathic pain absence at any stage of periodontal disease progression is ascribable to BA accumulation regardless of amount.

Periodontal disease level-butyric acid amounts locally administered in the rat gingival mucosa induce ER stress in the systemic blood.

Periodontal diseases have long been postulated to contribute to systemic diseases and, likewise, it has been proposed that periodontal disease treatment may ameliorate certain systemic diseases. Short-chain fatty acids (SCFA) are major secondary metabolites produced by oral anaerobic bacteria and, among the SCFAs, butyric acid (BA) in high amounts contribute to periodontal disease development. Periodontal disease level-butyric acid (PDL-BA) is found among patients suffering from periodontal disease and has previously shown to induce oxidative stress, whereas, oxidative stress is correlated to endoplasmic reticulum (ER) stress. This would imply that PDL-BA may likewise stimulate ER stress, however, this was never elucidated. A better understanding of the correlation between PDL-BA and systemic ER stress stimulation could shed light on the possible systemic effects of PDL-BA-related periodontal diseases. Here, PDL-BA was injected into the gingival mucosa and the systemic blood obtained from the rat jugular was collected at 0, 15, 60, and 180 min post-injection. Collected blood samples were purified and only the blood cytosol was used throughout this study. Subsequently, we measured blood cytosolic GADD153, Ca(2+), representative apoptotic and inflammatory caspases, and NF-κB amounts. We found that PDL-BA presence increased blood cytosolic GADD153 and Ca(2+) amounts. Moreover, we observed that blood cytosolic caspases and NF-κB were activated only at 60 and 180 min post-injection in the rat gingival mucosa. This suggests that PDL-BA administered through the gingival mucosa may influence the systemic blood via ER stress stimulation and, moreover, prolonged PDL-BA retention in the gingival mucosa may play a significant role in ER stress-related caspase and NF-κB activation. In a periodontal disease scenario, we propose that PDL-BA-related ER stress stimulation leading to the simultaneous activation of apoptosis and inflammation may contribute to periodontal disease pathogenesis.