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Periodontal disease is one of the most common disorders in the oral cavity of dogs and humans. Periodontitis, the irreversible periodontal disease, arises progressively from gingivitis, the reversible inflammatory condition caused by dental plaque. Although the etiology of periodontitis has been widely studied in humans, it is still insufficient for the etiological studies on periodontitis in dogs. Many studies have reported that human periodontitis-related bacteria are putative pathogens responsible for periodontitis in dogs. However, most of these studies have focused on the appearance of a specific microbiome, and most of the cohort studies have insufficient sample sizes to generalize their results. In the present study, subgingival samples collected from 336 teeth were categorized into three groups at first, based on clinical outcomes (healthy, gingivitis, periodontitis). Subsequently, the periodontitis samples were further divided into three subgroups (early, moderate, and advanced periodontitis) according to the degree of periodontal attachment loss. Healthy and gingivitis were grouped as a reversible group, and the three subgroups were grouped as an irreversible group. To investigate trends of periodontopathic bacteria in the samples of dogs, a quantitative real-time polymerase chain reaction (PCR) was performed for quantification of 11 human periodontopathic bacteria as follows: Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia, Treponema denticola (Td), Fusobacterium nucleatum, Prevotella nigrescens, Prevotella intermedia, Parvimonas micra, Eubacterium nodatum, Campylobacter rectus, and Eikenella corrodens. The PCR results showed that Aa and Pg, the representative periodontopathic bacteria, were not significantly correlated or associated with the periodontitis cases in dogs. However, interestingly, Td was strongly associated with the irreversible periodontal disease in dogs, in that it was the most prevalent bacterium detected from the dog samples. These findings indicate that the presence and numbers of Td could be used as a prognostic biomarker in predicting the irreversible periodontal disease and the disease severity in dogs.

Background Intraoperative hypertension and blood pressure (BP) fluctuation are known to be associated with negative patient outcomes. During robotic lower abdominal surgery, the patient’s abdominal cavity is filled with CO2, and the patient’s head is steeply positioned toward the floor (Trendelenburg position). Pneumoperitoneum and the Trendelenburg position together with physiological alterations during anesthesia, interfere with predicting BP changes. Recently, deep learning using recurrent neural networks (RNN) was shown to be effective in predicting intraoperative BP. A model for predicting BP rise was designed using RNN under special scenarios during robotic laparoscopic surgery and its accuracy was tested. Methods Databases that included adult patients (over 19 years old) undergoing low abdominal da Vinci robotic surgery (ovarian cystectomy, hysterectomy, myomectomy, prostatectomy, and salpingo-oophorectomy) at Soonchunhyang University Bucheon Hospital from October 2018 to March 2021 were used. An RNN-based model was designed using Python3 language with the PyTorch packages. The model was trained to predict whether hypertension (20% increase in the mean BP from baseline) would develop within 10 minutes after pneumoperitoneum. Results Eight distinct datasets were generated and the predictive power was compared. The macro-average F1 scores of the datasets ranged from 68.18% to 72.33%. It took only 3.472 milliseconds to obtain 39 prediction outputs. Conclusions A prediction model using the RNN may predict BP rises during robotic laparoscopic surgery.

Background Although the inter-arm blood pressure (BP) difference has been advocated to be associated with cardiovascular events, the implication of inter-leg BP difference has not been well established. This study was conducted to investigate whether inter-arm and -leg BP differences have prognostic value in patients undergoing percutaneous coronary intervention (PCI). Methods In this prospective study, we consecutively enrolled 667 patients who underwent PCI. Both arm and leg BPs were measured at the day after PCI. The primary outcome was a major adverse cardiovascular event (MACE) including cardiac death, acute coronary syndrome, coronary revascularization, stroke, and hospitalization for heart failure during the follow-up period. Results Mean age was 64.0±11.1 years old, and males were predominant (70.5%). During a mean follow-up period of 3.0 years, MACE occurred in 209 (31.3%) patients. The inter-leg systolic BP difference (ILSBPD) was significantly higher in patients with MACE than those without (9.9±12.3 vs. 7.2±7.5 mmHg, P = 0.004). The inter-arm systolic BP difference was not significantly different between patients with and without MACE (P = 0.403). In multivariable Cox regression analysis, increased ILSBPD was independently associated with the development of MACE (per 5 mmHg; hazard ratio, 1.07; 95% confidence interval, 1.01–1.14). The inter-arm systolic BP difference was not associated with MACE in the multivariable analysis. Conclusion Increased ILSBPD was independently associated with worse cardiovascular outcomes after PCI. As ILSBPD is easy to measure, it may be helpful in the risk stratification of patients undergoing PCI.

BackgroundObesity and metabolic syndrome (MetS) are associated with high risk of cardiac dysfunction and heart failure. We assessed the effect of obesity and metabolic health status on left ventricular (LV) structure and function in subjects without overt heart disease.MethodsIn 789 subjects (58.8±13.0 years, 50.7% males) without overt heart disease, LV morphology and function were compared among 6 groups stratified by body mass index (BMI) (normal weight, overweight and obese) and metabolic health status (meeting ≤1 criterion of MetS excluding waist circumference defined as metabolically healthy; otherwise, metabolically unhealthy).ResultsLV ejection fraction (LVEF) was not different among the 6 groups (P>0.05). However, high BMI and poor metabolic health were associated with poorer global longitudinal strain (GLS), higher LV mass index (LVMI) and higher E/e' (PConclusionsObesity and poor metabolic health status were associated with subclinical decrement in LV systolic and diastolic function, and higher LV mass, but not with LVEF, in subjects without overt heart disease.

Arterial hypotension during the early phase of anesthesia can lead to adverse outcomes such as a prolonged postoperative stay or even death. Predicting hypotension during anesthesia induction is complicated by its diverse causes. We investigated the feasibility of developing a machine-learning model to predict postinduction hypotension. Naïve Bayes, logistic regression, random forest, and artificial neural network models were trained to predict postinduction hypotension, occurring between tracheal intubation and incision, using data for the period from between the start of anesthesia induction and immediately before tracheal intubation obtained from an anesthesia monitor, a drug administration infusion pump, an anesthesia machine, and from patients' demographics, together with preexisting disease information from electronic health records. Among 222 patients, 126 developed postinduction hypotension. The random-forest model showed the best performance, with an area under the receiver operating characteristic curve of 0.842 (95% confidence interval [CI]: 0.736-0.948). This was higher than that for the Naïve Bayes (0.778; 95% CI: 0.65-0.898), logistic regression (0.756; 95% CI: 0.630-0.881), and artificial-neural-network (0.760; 95% CI: 0.640-0.880) models. The most important features affecting the accuracy of machine-learning prediction were a patient's lowest systolic blood pressure, lowest mean blood pressure, and mean systolic blood pressure before tracheal intubation. We found that machine-learning models using data obtained from various anesthesia machines between the start of anesthesia induction and immediately before tracheal intubation can predict hypotension occurring during the period between tracheal intubation and incision.

The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of in vivo model systems suitable for high-throughput screening. In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagents.

Allergic diseases such as atopic dermatitis, rhinitis, asthma, and anaphylaxis are attractive research areas. Tyrosol (2-(4-hydroxyphenyl)ethanol) is a polyphenolic compound with diverse biological activities. In this study, we investigated whether tyrosol has anti-allergic inflammatory effects. Ovalbumin-induced active systemic anaphylaxis and immunoglobulin E-mediated passive cutaneous anaphylaxis models were used for the immediate-type allergic responses. Oral administration of tyrosol reduced the allergic symptoms of hypothermia and pigmentation in both animal models. Mast cells that secrete allergic mediators are key regulators on allergic inflammation. Tyrosol dose-dependently decreased mast cell degranulation and expression of inflammatory cytokines. Intracellular calcium levels and activation of inhibitor of kappa B kinase (IKK) regulate cytokine expression and degranulation. Tyrosol blocked calcium influx and phosphorylation of the IKK complex. To define the molecular target for tyrosol, various signaling proteins involved in mast cell activation such as Lyn, Syk, phosphoinositide 3-kinase (PI3K), and Akt were examined. Our results showed that PI3K could be a molecular target for tyrosol in mast cells. Taken together, these findings indicated that tyrosol has anti-allergic inflammatory effects by inhibiting the degranulation of mast cells and expression of inflammatory cytokines; these effects are mediated via PI3K. Therefore, we expect tyrosol become a potential therapeutic candidate for allergic inflammatory disorders.

Abnormally elevated formation and activation of osteoclasts are primary causes for a majority of skeletal diseases. In this study, we found that KP-A159, a newly synthesized thiazolopyridine derivative, inhibited osteoclast differentiation and function in vitro, and inflammatory bone loss in vivo. KP-A159 did not cause a cytotoxic response in bone marrow macrophages (BMMs), but significantly inhibited the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts induced by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). KP-A159 also dramatically inhibited the expression of marker genes related to osteoclast differentiation, including TRAP (Acp5), cathepsin K (Ctsk), dendritic cell-specific transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Moreover, actin ring and resorption pit formation were inhibited by KP-A159. Analysis of the signaling pathway involved showed that KP-A159 inhibited RANKL-induced activation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase1/2 (MEK1/2). In a mouse inflammatory bone loss model, KP-A159 significantly rescued lipopolysaccharide (LPS)-induced bone loss by suppressing osteoclast numbers. Therefore, KP-A159 targets osteoclasts, and may be a potential candidate compound for prevention and/or treatment of inflammatory bone loss.

Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.

Diallyl sulfide, diallyl disulfide, and daillyl trisulfide (DATS) are major volatile components of garlic oil. In this study, we assessed their relative potency in inducing antioxidant enzyme expression. Among the three organosulfur compounds, DATS was found to be most potent in inducing heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1) in human gastric epithelial (AGS) cells. Furthermore, DATS administration by gavage increased the expression of HO-1 and NQO1 in C57BL/6 mouse stomach. Treatment with DATS increased the accumulation of nuclear factor-erythroid-2-related factor-2 (Nrf2) in the nucleus of cultured AGS cells and in mouse stomach in vivo. The DATS-induced expression of HO-1 and NQO1 was abrogated in the cells transiently transfected with Nrf2-siRNA or in the embryonic fibroblasts from Nrf2-null mice, indicating that Nrf2 is a key mediator of the cytoprotective effects of DATS. Pretreatment of AGS cells with N-acetylcysteine or dithiothreitol attenuated DATS-induced nuclear localization of Nrf2 and the expression of HO-1 and NQO1. Cysteine-151, -273 and -288 of Kelch-like ECH-associated protein-1 (Keap1), a cytosolic repressor of Nrf2, have been considered to act as a redox sensor and play a role in Nrf2 activation. To determine whether DATS could inactivate Keap1 through thiol modification, we established cell lines constitutively expressing wild type-Keap1 or three different mutant constructs in which cysteine-151, -273, or -288 of Keap1 was replaced with serine by retroviral gene transfer. DATS failed to activate Nrf2, and to induce expression of HO-1 and NQO1 only in Keap1-C288S mutant cells. LC-ESI-MS/MS analysis of recombinant Keap1 treated with DATS revealed that the peptide fragment containing Cys288 gained a molecular mass of 72.1 Da equivalent to the molecular weight of mono-allyl mono-sulfide. Taken together, these findings suggest that DATS may directly interact with the Cys288 residue of Keap1, which partly accounts for its ability to induce Nrf2 activation and upregulate defensive gene expression.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the specificity, sensitivity, and accuracy of this technique. In order to obtain reliable gene expression data from RT-qPCR experiments, it is important to utilize optimal reference genes for the normalization of target gene expression under varied experimental conditions. Previously, we developed and validated a novel icv-STZ cynomolgus monkey model for Alzheimer's disease (AD) research. However, in order to enhance the reliability of this disease model, appropriate reference genes must be selected to allow meaningful analysis of the gene expression levels in the icv-STZ cynomolgus monkey brain. In this study, we assessed the expression stability of 9 candidate reference genes in 2 matched-pair brain samples (5 regions) of control cynomolgus monkeys and those who had received intracerebroventricular injection of streptozotocin (icv-STZ). Three well-known analytical programs geNorm, NormFinder, and BestKeeper were used to choose the suitable reference genes from the total sample group, control group, and icv-STZ group. Combination analysis of the 3 different programs clearly indicated that the ideal reference genes are RPS19 and YWHAZ in the total sample group, GAPDH and RPS19 in the control group, and ACTB and GAPDH in the icv-STZ group. Additionally, we validated the normalization accuracy of the most appropriate reference genes (RPS19 and YWHAZ) by comparison with the least stable gene (TBP) using quantification of the APP and MAPT genes in the total sample group. To the best of our knowledge, this research is the first study to identify and validate the appropriate reference genes in cynomolgus monkey brains. These findings provide useful information for future studies involving the expression of target genes in the cynomolgus monkey.

BackgroundIt has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.Methodology/principal findingsRats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.Conclusions/significanceThe results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.

The aim of this study was to investigate whether brachial-ankle pulse wave velocity (baPWV) is associated with the severity of coronary artery disease (CAD) assessed by coronary computed tomography angiography (CCTA), and to evaluate baPWV as a predictor of obstructive CAD on CCTA. A total of 470 patients who underwent both baPWV and CCTA were included. We evaluated stenosis degree and plaque characteristics on CCTA. To estimate the severity of CAD, we calculated the number of segment with plaque (segment involvement score; SIS), stenosis degree-weighted plaque score (segment stenosis score; SSS), and coronary artery calcium score (CACS). The mean baPWV was 1,485 ± 315 cm/s (range, 935-3,175 cm/s). Non-obstructive (stenosis < 50%) and obstructive (stenosis ≥ 50%) CAD was found in 129 patients (27.4%) and 144 (30.6%), respectively. baPWV in patients with obstructive CAD was higher than that of patients with non-obstructive (1,680 ± 396 cm/s versus 1,477 ± 244 cm/s, P < 0.001) or no CAD (1,680 ± 396 cm/s versus ± 196 1,389 cm/s, P < 0.001). baPWV showed significant correlation with SSS (r = 0.429, P < 0.001), SIS (r = 0.395, P < 0.001), CACS (r 0.346, P < 0.001), and the number of segment with non-calcified plaque (r 0.092, P = 0.047), mixed plaque (r = 0.267, P < 0.001), and calcified plaque (r = 0.348, P < 0.001), respectively. The optimal baPWV cut-off value for the detection of obstructive CAD was 1,547 cm/s. baPWV ≥ 1,547 cm/s was independent predictor for the obstructive CAD. In conclusion, baPWV is well correlated with the severity of CAD evaluated by CCTA. baPWV has the potential to predict severity of coronary artery atherosclerosis.

We investigated whether eyedrop vaccination using modified outer membrane vesicles (mOMVs) is effective for protecting against hemolytic uremic syndrome (HUS) caused by enterohemorrhagic E. coli (EHEC) O157:H7 infection. Modified OMVs and waaJ-mOMVs were prepared from cultures of MsbB- and Shiga toxin A subunit (STxA)-deficient EHEC O157:H7 bacteria with or without an additional waaJ mutation. BALB/c mice were immunized by eyedrop mOMVs, waaJ-mOMVs, and mOMVs plus polymyxin B (PMB). Mice were boosted at 2 weeks, and challenged peritoneally with wild-type OMVs (wtOMVs) at 4 weeks. As parameters for evaluation of the OMV-mediated immune protection, serum and mucosal immunoglobulins, body weight change and blood urea nitrogen (BUN)/Creatinin (Cr) were tested, as well as histopathology of renal tissue. In order to confirm the safety of mOMVs for eyedrop use, body weight and ocular histopathological changes were monitored in mice. Modified OMVs having penta-acylated lipid A moiety did not contain STxA subunit proteins but retained non-toxic Shiga toxin B (STxB) subunit. Removal of the polymeric O-antigen of O157 LPS was confirmed in waaJ-mOMVs. The mice group vaccinated with mOMVs elicited greater humoral and mucosal immune responses than did the waaJ-mOMVs and PBS-treated groups. Eyedrop vaccination of mOMVs plus PMB reduced the level of humoral and mucosal immune responses, suggesting that intact O157 LPS antigen can be a critical component for enhancing the immunogenicity of the mOMVs. After challenge, mice vaccinated with mOMVs were protected from a lethal dose of wtOMVs administered intraperitoneally, conversely mice in the PBS control group were not. Collectively, for the first time, EHEC O157-derived mOMV eyedrop vaccine was experimentally evaluated as an efficient and safe means of vaccine development against EHEC O157:H7 infection-associated HUS.