Exploring in vivo combinatorial chemo-immunotherapy: Addressing p97 suppression and immune reinvigoration in pancreatic cancer with tumor microenvironment-responsive nanoformulation.

Pancreatic ductal adenocarcinoma (PDAC) has an extremely devastating nature with poor prognosis and increasing incidence, making it a formidable challenge in the global fight against cancer-related mortality. In this innovative preclinical investigation, the VCP/p97 inhibitor CB-5083 (CB), miR-142, a PD-L1 inhibitor, and immunoadjuvant resiquimod (R848; R) were synergistically encapsulated in solid lipid nanoparticles (SLNs). These SLNs demonstrated features of peptides targeting PD-L1, EGFR, and the endoplasmic reticulum, enclosed in a pH-responsive polyglutamic (PGA)-polyethylene glycol (PEG) shell. The homogeneous size and zeta potential of the nanoparticles were stable for 28 days at 4°C. The study substantiated the concurrent modulation of key pathways by the CB, miR, and R-loaded nanoformulation, prominently affecting VCP/Bip/ATF6, PD-L1/TGF-ß/IL-4, -8, -10, and TNF-α/IFN-γ/IL-1, -12/GM-CSF/CCL4 pathways. This adaptable nanoformulation induced durable antitumor immune responses and inhibited Panc-02 tumor growth by enhancing T cell infiltration, dendritic cell maturation, and suppressing Tregs and TAMs in mice bearing Panc-02 tumors. Furthermore, tissue distribution studies, biochemical assays, and histological examinations highlighted enhanced safety with PGA and peptide-modified nanoformulations for CB, miR, and/or R in Panc-02-bearing mice. This versatile nanoformulation allows tailored adjustment of the tumor microenvironment, thereby optimizing the localized delivery of combined therapy. These compelling findings advocate the potential development of a pH-sensitive, three-in-one PGA-PEG nanoformulation that combines a VCP inhibitor, a PD-L1 inhibitor, and an immunoadjuvant for cancer treatment via combinatorial chemo-immunotherapy.

Structural basis and synergism of ATP and Na+ activation in bacterial K+ uptake system KtrAB.

The K+ uptake system KtrAB is essential for bacterial survival in low K+ environments. The activity of KtrAB is regulated by nucleotides and Na+. Previous studies proposed a putative gating mechanism of KtrB regulated by KtrA upon binding to ATP or ADP. However, how Na+ activates KtrAB and the Na+ binding site remain unknown. Here we present the cryo-EM structures of ATP- and ADP-bound KtrAB from Bacillus subtilis (BsKtrAB) both solved at 2.8 Å. A cryo-EM density at the intra-dimer interface of ATP-KtrA was identified as Na+, as supported by X-ray crystallography and ICP-MS. Thermostability assays and functional studies demonstrated that Na+ binding stabilizes the ATP-bound BsKtrAB complex and enhances its K+ flux activity. Comparing ATP- and ADP-BsKtrAB structures suggests that BsKtrB Arg417 and Phe91 serve as a channel gate. The synergism of ATP and Na+ in activating BsKtrAB is likely applicable to Na+-activated K+ channels in central nervous system.

Chlorpyrifos-induced suppression of the antioxidative defense system leads to cytotoxicity and genotoxicity in macrophages.

Chlorpyrifos, widely used for pest control, is known to have various harmful effects, although its toxic effects in macrophages and the mechanisms underlying its toxicity remain unclear. The present study investigated the toxic effects of chlorypyrifos in a macrophage cell line. Here, we found that chlorpyrifos induced cytotoxicity and genotoxicity in RAW264.7 macrophages. Moreover, chlorpyrifos induced intracellular ROS production, subsequently leading to lipid peroxidation. Chlorpyrifos reduced the activation of antioxidative enzymes including superoxide dismutase, catalase, and glutathione peroxidase. Chlorpyrifos upregulated HO-1 expression and activated the Keap1-Nrf2 pathway, as indicated by enhanced Nrf2 phosphorylation and Keap1 degradation. Chlorpyrifos exerted effects on the following in a dose-dependent manner: cytotoxicity, genotoxicity, lipid peroxidation, intracellular ROS production, antioxidative enzyme activity reduction, HO-1 expression, Nrf2 phosphorylation, and Keap1 degradation. Notably, N-acetyl-L-cysteine successfully inhibited chlorpyrifos-induced intracellular ROS generation, cytotoxicity, and genotoxicity. Thus, chlorpyrifos may induce cytotoxicity and genotoxicity by promoting intracellular ROS production and suppressing the antioxidative defense system activation in macrophages.

Vascular endothelial dysfunction induced by 3-bromofluoranthene via MAPK-mediated-NFκB pro-inflammatory pathway and intracellular ROS generation.

3-Bromofluoranthene (3-BrFlu) is the secondary metabolite of fluoranthene, which is classified as a polycyclic aromatic hydrocarbon, through bromination and exists in the fine particulate matter of air pollutants. Endothelial dysfunction plays a critical role in the pathogenesis of cardiovascular and vascular diseases. Little is known about the molecular mechanism of 3-BrFlu on endothelial dysfunction in vivo and in vitro assay. In the present study, 3-BrFlu included concentration-dependent changes in ectopic angiogenesis of the sub-intestinal vein and dilation of the dorsal aorta in zebrafish. Disruption of vascular endothelial integrity and up-regulation of vascular endothelial permeability were also induced by 3-BrFlu in a concentration-dependent manner through pro-inflammatory responses in vascular endothelial cells, namely, SVEC4-10 cells. Generation of pro-inflammatory mediator PGE2 was induced by 3-BrFlu through COX2 expression. Expression of COX2 and generation of pro-inflammatory cytokines, including TNFα and IL-6, were induced by 3-BrFlu through phosphorylation of NF-κB p65, which was mediated by phosphorylation of MAPK, including p38 MAPK, ERK and JNK. Furthermore, generation of intracellular ROS was induced by 3-BrFlu, which is associated with the down-regulated activities of the antioxidant enzyme (AOE), including SOD and catalase. We also found that 3-BrFlu up-regulated expression of the AOE and HO-1 induced by 3-BrFlu through Nrf-2 expression. However, the 3-BrFlu-induced upregulation of AOE and HO-1 expression could not be revised the responses of vascular endothelial dysfunction. In conclusion, 3-BrFlu is a hazardous substance that results in vascular endothelial dysfunction through the MAPK-mediated-NFκB pro-inflammatory pathway and intracellular ROS generation.

Teaghrelin protected dopaminergic neurons in MPTP-induced Parkinson's disease animal model by promoting PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC1-α-mediated mitochondrial biogenesis.

Mitochondrial dysfunction, a common cellular hallmark in both familial and sporadic forms of Parkinson's disease (PD), is assumed to play a significant role in pathologic development and progression of the disease. Teaghrelin, a unique bioactive compound in some oolong tea varieties, has been demonstrated to protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium induced neurotoxicity by binding to the ghrelin receptor to activate the AMPK/SIRT1/PGC-1α pathway. In this study, an animal model was established using a neurotoxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of a prohibited drug, to evaluate the oral efficacy of teaghrelin on PD by monitoring motor dysfunction of mice in open field, pole, and bean walking tests. The results showed that MPTP-induced motor dysfunction of mice was significantly attenuated by teaghrelin supplementation. Tyrosine hydroxylase and dopamine transporter protein were found reduced in the striatum and midbrain of MPTP-treated mice, and significantly mitigated by teaghrelin supplementation. Furthermore, teaghrelin administration enhanced mitophagy and mitochondria biogenesis, which maintained cell homeostasis and prevented the accumulation of αSyn and apoptosis-related proteins. It seemed that teaghrelin protected dopaminergic neurons in MPTP-treated mice by increasing PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC-1α-mediated mitochondria biogenesis, highlighting its potential therapeutic role in maintaining dopaminergic neurons function in PD. Mitochondrial dysfunction, a common cellular hallmark in both familial and sporadic forms of Parkinson's disease (PD), is assumed to play a significant role in pathologic development and progression of the disease. Teaghrelin, a unique bioactive compound in some oolong tea varieties, has been demonstrated to protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium induced neurotoxicity by binding to the ghrelin receptor to activate the AMPK/SIRT1/PGC-1α pathway. In this study, an animal model was established using a neurotoxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of a prohibited drug, to evaluate the oral efficacy of teaghrelin on PD by monitoring motor dysfunction of mice in open field, pole, and bean walking tests. The results showed that MPTP-induced motor dysfunction of mice was significantly attenuated by teaghrelin supplementation. Tyrosine hydroxylase and dopamine transporter protein were found reduced in the striatum and midbrain of MPTP-treated mice, and significantly mitigated by teaghrelin supplementation. Furthermore, teaghrelin administration enhanced mitophagy and mitochondria biogenesis, which maintained cell homeostasis and prevented the accumulation of αSyn and apoptosis-related proteins. It seemed that teaghrelin protected dopaminergic neurons in MPTP-treated mice by increasing PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC-1α-mediated mitochondria biogenesis, highlighting its potential therapeutic role in maintaining dopaminergic neurons function in PD.

Wogonin induces apoptosis in macrophages by exhibiting cytotoxic and genotoxic effects.

Macrophages play an important role in defending the body against invading pathogens. In the face of pathogens, macrophages become activated and release toxic materials that disrupt the pathogens. Macrophage overactivation can lead to severe illness and inflammation. Wogonin has several therapeutic effects, including anti-inflammatory, anticancer, antioxidant, and neuroprotective effects. No studies have investigated the cytotoxic effects of wogonin at concentrations of more than 0.1 mM in RAW264.7 cells. In this study, RAW 264.7 cells were treated with wogonin, which, at concentrations of more than 0.1 mM, had cytotoxic and genotoxic effects in the RAW264.7 cells, leading to apoptosis and necrosis. Further, wogonin at concentrations of more than 0.1 mM induced caspase-3, caspase-8, and caspase-9 activation and mitochondrial dysfunction and death receptor expression. These results suggest that wogonin induces apoptosis through upstream intrinsic and extrinsic pathways by exhibiting cytotoxic and genotoxic effects.

Cyclizine induces cytotoxicity and apoptosis in macrophages through the extrinsic and intrinsic apoptotic pathways.

Cyclizine, an over-the-counter and prescription antihistamine, finds widespread application in the prevention and treatment of motion sickness, encompassing symptoms such as nausea, vomiting, dizziness, along with its effectiveness in managing vertigo. However, the overuse or misuse of cyclizine may lead to hallucinations, confusion, tachycardia, and hypertension. The molecular mechanisms underlying cyclizine-induced cytotoxicity and apoptosis remain unclear. During the 24 h incubation duration, RAW264.7 macrophages were exposed to different concentrations of cyclizine. Cytotoxicity was assessed through the lactate dehydrogenase assay. Flow cytometry employing annexin V-fluorescein isothiocyanate and propidium iodide was utilized to evaluate apoptosis and necrosis. Caspase activity and mitochondrial dysfunction were evaluated through a fluorogenic substrate assay and JC-1 dye, respectively. Flow cytometry employing fluorogenic antibodies was utilized to evaluate the release of cytochrome c and expression of death receptor, including tumor necrosis factor-α receptor and Fas receptor. Western blotting was utilized to evaluate the expression of the Bcl2 and Bad apoptotic regulatory proteins. The findings unveiled from the present study demonstrated that cyclizine exerted a concentration-dependent effect on RAW264.7 macrophages, leading to the induction of cytotoxicity, apoptosis, and necrosis. This compound further activated the intrinsic apoptotic pathway by inducing mitochondrial dysfunction, Bcl2/Bad exchange expression, cytochrome c liberation, and activation of caspases contained caspase 3, 8, and 9. Moreover, the activation of the extrinsic apoptotic pathway was observed as cyclizine induced the upregulation of death receptors and increased caspase activities. Based on our investigations, it can be inferred that cyclizine prompts cytotoxicity and apoptosis in RAW264.7 macrophages in a concentration-dependent manner by triggering both the intrinsic and extrinsic apoptotic pathways.

Resveratrol attenuates advanced glycation end product-induced senescence and inflammation in human gingival fibroblasts.

Background/purpose: The accumulation of advanced glycation end products (AGEs) lead to a series of immune responses such as: increased oxidative stress and inflammation which contribute to the development of diabetic complications and periodontal disease. Resveratrol is a natural compound that has anti-oxidant and anti-inflammatory effects. Studies have found that diabetes-induced periodontitis is mainly caused by oxidative stress, aging and increased inflammation. In view of resveratrol has been proposed to have the ability in anti-oxidant and anti-inflammation in a variety of tissues. However, the role of resveratrol in diabetic periodontitis remains to be investigated. In this study, we aimed to investigate the role of resveratrol in preventing and treating diabetic periodontitis. Materials and methods: First, cell proliferation was measured in AGEs-treated human gingival fibroblast with or without resveratrol. We examined the reactive oxygen species (ROS) generation, senescence-associated beta-galactosidase (SA-ß-gal) and senescence marker p16 in human gingival fibroblasts (HGFs) stimulated with AGEs with or without the treatment of resveratrol. To determine whether resveratrol has the potential to regulate inflammaging which is mediated via the NF-κB signaling pathway and, the expression of p65 and p-IκB were also investigated. Furthermore, the concentration of interleukin (IL)-6 and IL-8 were also measured in AGEs-stimulated HGFs treated with or without resveratrol. Results: ROS generation, cell senescence, and the secretion of IL-6 and IL-8 were significantly upregulated following the treatment of AGEs. However, the administration of resveratrol suppresses the generation of IL-6 and IL-8 and cell senescence via inhibiting NF-κB signaling pathway. Our results revealed that resveratrol inhibits inflammaging by downregulating NF-κB signaling pathway. Conclusion: According to our findings, AGEs increase senescence and the production of proinflammatory cytokines in the gingiva, while the administration of resveratrol impedes inflammaging via suppressing NF-κB signaling pathway.

Quercetin ameliorates advanced glycation end product-induced wound healing impairment and inflammaging in human gingival fibroblasts.

Background/purpose: Diabetes mellitus (DM) and periodontal disease are both prevalent and chronic inflammatory disorders that have significant health impact. Many studies have pointed out that advanced glycation end-products (AGEs) in DM induces inflammaging, which is a pre-aging and hyperinflammatory condition, and it has been linked to a greater likelihood in developing periodontitis. Inflammaging in DM has been shown to be driven by AGEs-induced cell senescence, inflammatory cytokines, and oxidative stress, resulting in the degradation of periodontium. Quercetin has shown abilities to decrease inflammation and oxidative stress in a variety of tissues, however, the effect in diabetic periodontitis remains uncertain. Thus, the aim of this study was to investigate its impacts on inflammaging in diabetic periodontitis. Materials and methods: We examined cell proliferation in human gingival fibroblasts (HGF), wound healing, IL-6 and IL-8 secretions, cellular senescence expression, and the formation of reactive oxygen species (ROS) in response to AGE stimulation with and without Quercetin intervention. Following that, we looked into NF-κß activity to see if Quercetin mediate its effects via this pro-inflammatory signaling. Results: Quercetin at 20 µM and below did not have any impact on HGFs' cell proliferation rate. Quercetin intervention improved the AGEs-impaired wound healing, in addition to the attenuation of AGEs-induced ROS in a dose-dependent pattern. Moreover, Quercetin therapy dose-dependently inhibited AGEs-induced cell senescence activity along with its senescence associated secretion phenotype (SASP) secretions such as IL-6 and IL-8. Western blot analysis indicated that Quercetin was able to reverse the phosphorylation of p65 and Iκß in AGEs-stimulated HGFs, demonstrating it can modulate NF-κß pathway. Conclusion: Accumulation of AGEs can elicit inflammaging in HGFs, as seen by increased pro-inflammatory cytokines, cell senescence expression and oxidative stress. The results proposed that Quercetin is able to ameliorate inflammaging in diabetic periodontitis and improve wound healing via the suppression of NF-κß pathway and hence, may be a promising approach for treatment of diabetes-associated periodontitis.

Association between PM2.5 exposure and risk of Parkinson's disease in individuals with chronic obstructive pulmonary disease in Taiwan: a nested case-control study.

OBJECTIVES: This cohort study investigated the correlation between Parkinson's disease (PD) risk and chronic obstructive pulmonary disease (COPD) risk under particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5) exposure. METHODS: Data from the National Health Research Institutes of Taiwan were used in this study. The Environmental Protection Administration of Taiwan established an air quality monitoring network for monitoring Taiwan's general air quality. COPD was indicated by at least 3 outpatient records and 1 hospitalization for COPD. After the implementation of age, sex, and endpoint matching at a 1:4 ratio, 137 patients and 548 patients were included in the case group and control group, respectively. Based on the 2005 World Health Organization (WHO) standards, monthly air particle concentration data were classified into the following 4 groups in analyses of exposure-response relationships: normal level, and 1.0, 1.5, and 2.0 times the WHO level ([concentration ≥2]×25 µg/m3×number of exposure months). RESULTS: A multivariate logistic regression revealed that the 1.0 and 1.5 WHO level groups did not significantly differ from the normal level group, but the 2.0 WHO level did (odds ratio, 4.091; 95% confidence interval, 1.180 to 14.188; p=0.038). CONCLUSIONS: Elevated PM2.5 concentrations were significantly correlated with an increased risk of PD among patients with COPD. Furthermore, exposure to high PM2.5 levels can further increase the risk of PD.

DHA alleviated hepatic and adipose inflammation with increased adipocyte browning in high-fat diet-induced obese mice.

Obesity is associated with accumulation of inflammatory immune cells in white adipose tissue, whereas thermogenic browning adipose tissue is inhibited. Dietary fatty acids are important nutritional components and several clinical and experimental studies have reported beneficial effects of docosahexaenoic acid (DHA) on obesity-related metabolic changes. In this study, we investigated effects of DHA on hepatic and adipose inflammation and adipocyte browning in high-fat diet-induced obese C57BL/6J mice, and in vitro 3T3-L1 preadipocyte differentiation. Since visceral white adipose tissue has a close link with metabolic abnormality, epididymal adipose tissue represents current target for evaluation. A course of 8-week DHA supplementation improved common phenotypes of obesity, including improvement of insulin resistance, inhibition of macrophage M1 polarization, and preservation of macrophage M2 polarization in hepatic and adipose tissues. Moreover, dysregulated adipokines and impaired thermogenic and browning molecules, considered obesogenic mechanisms, were improved by DHA, along with parallel alleviation of endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and mitochondrial DNA stress-directed innate immunity. During 3T3-L1 preadipocytes differentiation, DHA treatment decreased lipid droplet accumulation and increased the levels of thermogenic, browning, and mitochondrial biogenesis molecules. Our study provides experimental evidence that DHA mitigates obesity-associated inflammation and induces browning of adipose tissue in visceral epididymal adipose tissue. Since obesity is associated with metabolic abnormalities across tissues, our findings indicate that DHA may have potential as part of a dietary intervention to combat obesity.

Cyclizine-induced proinflammatory responses through Akt-NFκB pathway in macrophages.

Cyclizine exhibits sedation and treatment of nausea, vomiting, and motion sickness due to antihistaminic and antimuscarinic effects. Cyclizine has the potential for abuse due to the hallucinogenic and euphoric effect. The response of overdose and illegal abuse of cyclizine includes confusion, tremors, chest pain, ataxia, seizures, and lead to suicide. Macrophage plays the important role in the innate immunity. However, over activation of macrophages results in pro-inflammatory responses in peripheral tissues. In the present study, cyclizine was found to enhanced the generation of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6. We further found that secretion of nitrogen oxide (NO) induced by cyclizine via expression of inducible nitric oxide synthases (iNOS). Cyclizine exhibited parallel stimulation of phosphorylation of nuclear factor-κB (NFκB) p65, and its up-stream factor Akt. These results indicated that the expression of pro-inflammatory cytokines, pro-inflammatory mediators, and adhesion molecules would be induced by cyclizine via activation of Akt-NFκB pathway in macrophages.

NMDA receptor inhibitor MK801 alleviated pro-inflammatory polarization of BV-2 microglia cells.

Microglia have both protective and pathogenic properties, while polarization plays a decisive role in their functional diversity. Apart from being an energetic organelle, mitochondria possess biological capabilities of signaling and immunity involving mitochondrial dynamics. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotransmission, excitatory neurotoxicity and pro-inflammatory properties in a membrane location- and cell context-dependent manner. In this study, we have provided experimental evidence showing that by acting on mitochondrial dynamics, NMDA receptors displayed pro-inflammatory properties, while its non-competitive inhibitor MK801 exhibited anti-inflammatory potential in Lipopolysaccharide (LPS)-challenged BV-2 microglia cells. LPS stimulation increased the protein phosphorylation of cells regarding their NMDA receptor component subunits and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), along with mobilizing intracellular calcium. Additionally, parallel changes occurred in the activation of Transforming Growth Factor-ß (TGF-ß)-Activated Kinase 1 (TAK1), NF-κB p65 and NF-κB DNA binding activity, acquisition of pro-inflammatory M1 polarization and expression of pro-inflammatory cytokines. LPS-treated cells further displayed signs of mitochondrial dysfunction with higher expressions of the active form of Dynamin-Related Protein 1 (Drp1), NADPH Oxidase-2 (NOX2) expression and the generation of DCFDA-/MitoSOX-sensitive Reactive Oxygen Species (ROS). NMDA receptor blockade by MK801, along with CaMKII inhibitor KN93, Drp1 inhibitor Mdivi-1 and antioxidant apocynin alleviated LPS-induced pro-inflammatory changes. Other than the reported CaMKII/TAK1/NF-κB axis, our in vitro study revealed the CaMKII/Drp1/ROS/NF-κB axis being an alternative cascade for shaping pro-inflammatory phenotypes of microglia upon LPS stimulation, and MK801 having the potential for inhibiting microglia activation and any associated inflammatory damages.

Tetramethylpyrazine alleviates mitochondrial abnormality in models of cerebral ischemia and oxygen/glucose deprivation Reoxygenation.

Traditional herbal medicine Ligusticum wallichii Franchat (Chuan Xiong) is frequently prescribed and highly recommended to patients with stroke. Rodent studies have demonstrated the neuroprotective effects of its active component tetramethylpyrazine against post-stroke brain injury and highlighted its role in antioxidant, anti-inflammation, and anti-apoptosis activity. Using permanent cerebral ischemia in rats and oxygen/glucose deprivation and reoxygenation (OGDR) in rat primary neuron/glia cultures, this study sheds light on the role of mitochondria as crucial targets for tetramethylpyrazine neuroprotection. Tetramethylpyrazine protected against injury and alleviated oxidative stress, interleukin-1ß release, and caspase 3 activation both in vivo and in vitro. Reduction of mitochondrial biogenesis- and integrity-related proliferator-activated receptor-gamma coactivator-1 alpha, mitochondrial transcription factor A (TFAM), translocase of outer mitochondrial membrane 20, mitochondrial DNA, and citrate synthase activity, as well as activation of mitochondrial dynamics disruption-related Lon protease, dynamin-related protein 1 (Drp1) phosphorylation, stimulator of interferon genes, TANK-binding kinase 1 phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase phosphorylation, eukaryotic initiation factor 2α phosphorylation, and activating transcription factor 4 were revealed in permanent cerebral ischemia in rats and OGDR in neuron/glia cultures. TMP alleviated those biochemical changes. Our findings suggest that preservation or restoration of mitochondrial dynamics and functional integrity and alleviation of mitochondria-oriented pro-oxidant, pro-inflammatory, and pro-apoptotic cascades are alternative neuroprotective mechanisms of tetramethylpyrazine. Additionally, mitochondrial TFAM and Drp1 as well as endoplasmic reticulum stress could be targeted by TMP to induce neuroprotection. Data of this study provide experimental base to support clinical utility and value of Chuan Xiong towards stroke treatment and highlight an alternative neuroprotective target of tetramethylpyrazine.

Prevention and management of peri-implant disease.

BACKGROUND: As more patients choose dental implants as their primary treatment option to restore edentulous ridges or to replace compromised dentition, preventive strategies for peri-implant diseases and complications have become an important topic. PURPOSE: The aim of the review article is to summarize the current available evidence on the potential risk factors/indicators for peri-implant disease development and then focus on the preventive strategies for peri-implant diseases and conditions. MATERIALS AND METHODS: After reviewing the diagnostic criteria and the etiology of peri-implant diseases and conditions, evidence on the possible associated risk factors/indicators for peri-implant diseases were searched and identified. Recent studies were also surveyed to explore the preventive measures for peri-implant diseases. RESULTS: The possible associated risk factors of peri-implant diseases can be divided into the following categories: patient-specific factors, implant-specific factors, and long-term factors. Patient-specific factors such as history of periodontitis and smoking have been conclusively associated with peri-implant diseases, whereas findings on others, such as diabetes and genetic factors, remain inconclusive. It has been suggested that both implant-specific factors, such as implant position, soft tissue characteristics, and the type of connection used, and long-term factors, such as poor plaque control and a lack of maintenance program, have a strong impact on maintaining the health of a dental implant. Assessment tool for evaluating the risk factors can be a potential preventive measure for peri-implant disease prediction, and it is needed to be properly validated. CONCLUSION: Proper maintenance program for early intervention to control peri-implant diseases at the initial stage and pretreatment assessment of the potential risk factors is the best strategy to prevent implant diseases.

Anti-inflammaging effects of vitamin D in human gingival fibroblasts with advanced glycation end product stimulation.

Background/purpose: :Both periodontal disease and diabetes mellitus (DM) are long-term inflammatory disorders that are highly prevalent and have a significant health impact. Inflammaging, a state of pre-aging and hyperinflammatory state has been acknowledged for its role in DM patients to have heightened risk of periodontitis. Numerous evidences revealed that inflammaging contributed by cell senescence, acceleration of inflammation and oxidative stress participates in the destruction of periodontium in DM. Abilities of vitamin D in suppressing inflammation and oxidative stress have been revealed in a range of tissues, however in DM's gingival cells, the effect remain undefined. Materials and methods: : Under the stimulation of advanced glycation end-products (AGEs), we assessed the cell proliferation in human gingival fibroblast (HGF), IL-6 and IL-8 secretions, cellular senescence expression and generation of reactive oxygen species (ROS) with or without vitamin D intervention. Following that, we examined the expression of Nrf2 and HO-1 to see if vitamin D was able to modulate the anti-oxidant signaling. A knockdown experiment was then conducted to proof the participation of Nrf2 on the secretion of pro-inflammatory IL-6 and IL-8. Results: : Following the treatment of vitamin D, AGEs-elicited IL-6 and IL-8 production and cell senescence were dose-dependently repressed. Moreover, vitamin D attenuated AGEs-induced ROS in a dose-dependent pattern. Results from qRT-PCR demonstrated vitamin D reversed the suppression of Nrf2 and HO-1 induced by AGEs. Our findings revealed that the anti-inflammatory and anti-oxidant effect in vitamin D was mediated via the upregulation of Nrf2 expression. Conclusion: : These data showed that high levels of AGEs in the gingiva lead to inflammaging reflected by increased pro-inflammatory cytokines, cell senescence expression and oxidative stress. Vitamin D supplementation can reduce oxidative stress and inflammation via the upregulation of Nrf2 signaling and hence, may be a potential approach for treatment of diabetes-associated periodontitis.

Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product.

The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-D-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, are reported as the first representative structures of an alkaline α-galactosidase. The structure of AtAkαGal3 comprises three domains: an N-terminal domain with 13 antiparallel ß-strands, a catalytic domain with an (α/ß)8-barrel fold and a C-terminal domain composed of ß-sheets that form two Greek-key motifs. The WW box of the N-terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/ß)8 barrel of the catalytic domain. The C-terminal domain is presumably involved in structural stability. Structures of the D383A mutant in complex with various substrates and products, especially the natural substrate/product stachyose, reveal four complete subsites (-1 to +3) at the catalytic site. A functional loop (residues 329-352) that exists in the alkaline α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-galactosidases, stabilizes the stachyose at the +2 and +3 subsites and extends the catalytic pocket for the transferase mechanism. Considering the similarities in amino-acid sequence, catalytic domain and activity between alkaline α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

Structures of honeybee-infecting Lake Sinai virus reveal domain functions and capsid assembly with dynamic motions.

Understanding the structural diversity of honeybee-infecting viruses is critical to maintain pollinator health and manage the spread of diseases in ecology and agriculture. We determine cryo-EM structures of T = 4 and T = 3 capsids of virus-like particles (VLPs) of Lake Sinai virus (LSV) 2 and delta-N48 LSV1, belonging to tetraviruses, at resolutions of 2.3-2.6 Å in various pH environments. Structural analysis shows that the LSV2 capsid protein (CP) structural features, particularly the protruding domain and C-arm, differ from those of other tetraviruses. The anchor loop on the central ß-barrel domain interacts with the neighboring subunit to stabilize homo-trimeric capsomeres during assembly. Delta-N48 LSV1 CP interacts with ssRNA via the rigid helix α1', α1'-α1 loop, ß-barrel domain, and C-arm. Cryo-EM reconstructions, combined with X-ray crystallographic and small-angle scattering analyses, indicate that pH affects capsid conformations by regulating reversible dynamic particle motions and sizes of LSV2 VLPs. C-arms exist in all LSV2 and delta-N48 LSV1 VLPs across varied pH conditions, indicating that autoproteolysis cleavage is not required for LSV maturation. The observed linear domino-scaffold structures of various lengths, made up of trapezoid-shape capsomeres, provide a basis for icosahedral T = 4 and T = 3 architecture assemblies. These findings advance understanding of honeybee-infecting viruses that can cause Colony Collapse Disorder.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

FeoC from Klebsiella pneumoniae uses its iron sulfur cluster to regulate the GTPase activity of the ferrous iron channel.

Bacteria depend on the ferrous iron transport (Feo) system for the uptake of ferrous iron (Fe2+). The Feo system is crucial for colonization and virulence of pathogens. In γ-proteobacteria, the system consists of FeoA, FeoB, and FeoC. The function of FeoA remains unknown. FeoB likely forms the channel, whose regulation has been suggested to involve its GTPase domain (part of its NFeoB domain). FeoC from Klebsiella pneumonia was found to contain a [4Fe4S] cofactor, whose presence was speculated to enhance the GTPase activity of FeoB (Hsueh, K.-L., et al., J. Bacteriol. 2013 195(20): 4726-34). We present results here that support and extend that hypothesis. We monitored the GTPase activity of FeoB by NMR spectroscopy and found that the presence of 7% FeoC-[4Fe-4S]3+ (the highest level of cofactor achieved in vitro) increased the GTPase rate of NFeoB by 3.6-fold over NFeoB. The effect depends on the oxidation state of the cluster; with reduction of the cluster to [4Fe-4S]2+ the GTPase greatly decreased the GTPase rate. From the effects of point mutations in FeoC on GTPase rates, we conclude that Lys62 and Lys68 on FeoC each contribute to increased GTPase activity on NFeoB. Mutation of Thr37 of NFeoB to Ser nearly abolished the GTPase activity. The GTPase activity of the isolated K. pneumoniae NFeoB-FeoC complex (NFeoBC) was found to be higher in KCl than in NaCl solution. We solved the X-ray structure of the NFeoBC crystallized from KCl and compared it with a prior X-ray structure crystalized from NaCl. We propose a hypothesis, consistent with these results, to explain the factors that influence the GTPase activity. Bacteria may use the oxygen-sensitive cluster as a sensor to up-regulate the gate closing speed.