Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn's disease.

OBJECTIVE: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP ß-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) ß-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP ß-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP ß-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

Third-generation sequencing-selected Scardovia wiggsiae promotes periodontitis progression in mice.

BACKGROUNDS: Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis. OBJECTIVE: This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice. METHODS: Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. RESULTS: Scardovia wiggsiae (S. wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S. wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model. CONCLUSION: Our metagenome results suggested that S. wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S. wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S. wiggsiae and other periodontopathic bacteria are warranted.

Vapor-Induced Pore-Forming Atmospheric-Plasma-Sprayed Zinc-, Strontium-, and Magnesium-Doped Hydroxyapatite Coatings on Titanium Implants Enhance New Bone Formation-An In Vivo and In Vitro Investigation.

OBJECTIVES: Titanium implants are regarded as a promising treatment modality for replacing missing teeth. Osteointegration and antibacterial properties are both desirable characteristics for titanium dental implants. The aim of this study was to create zinc (Zn)-, strontium (Sr)-, and magnesium (Mg)-multidoped hydroxyapatite (HAp) porous coatings, including HAp, Zn-doped HAp, and Zn-Sr-Mg-doped HAp, on titanium discs and implants using the vapor-induced pore-forming atmospheric plasma spraying (VIPF-APS) technique. METHODS: The mRNA and protein levels of osteogenesis-associated genes such as collagen type I alpha 1 chain (COL1A1), decorin (DCN), osteoprotegerin (TNFRSF11B), and osteopontin (SPP1) were examined in human embryonic palatal mesenchymal cells. The antibacterial effects against periodontal bacteria, including Porphyromonas gingivalis and Prevotella nigrescens, were investigated. In addition, a rat animal model was used to evaluate new bone formation via histologic examination and micro-computed tomography (CT). RESULTS: The ZnSrMg-HAp group was the most effective at inducing mRNA and protein expression of TNFRSF11B and SPP1 after 7 days of incubation, and TNFRSF11B and DCN after 11 days of incubation. In addition, both the ZnSrMg-HAp and Zn-HAp groups were effective against P. gingivalis and P. nigrescens. Furthermore, according to both in vitro studies and histologic findings, the ZnSrMg-HAp group exhibited the most prominent osteogenesis and concentrated bone growth along implant threads. SIGNIFICANCE: A porous ZnSrMg-HAp coating using VIPF-APS could serve as a novel technique for coating titanium implant surfaces and preventing further bacterial infection.

The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair.

BACKGROUNDS & AIMS: Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. METHODS: Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial-specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. RESULTS: ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1-deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. CONCLUSIONS: ZO-1 makes critical, tight junction-independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

Experimental Realization of a Fermionic Spin-Momentum Lattice.

We experimentally realize a spin-momentum lattice with a homogeneously trapped Fermi gas. The lattice is created via cyclically rotated atom-laser couplings between three bare atomic spin states, and are such that they form a triangular lattice in a synthetic spin-momentum space. We demonstrate the lattice and explore its dynamics with spin- and momentum-resolved absorption imaging. This platform will provide new opportunities for synthetic spin systems and the engineering of topological bands. In particular, the use of three spin states in two spatial dimensions would allow the simulation of synthetic magnetic fields of high spatial uniformity, which would lead to ultranarrow Chern bands that support robust fractional quantum Hall states.

Current Development and Application of Anaerobic Glycolytic Enzymes in Urothelial Cancer.

Urothelial cancer is a malignant tumor with metastatic ability and high mortality. Malignant tumors of the urinary system include upper tract urothelial cancer and bladder cancer. In addition to typical genetic alterations and epigenetic modifications, metabolism-related events also occur in urothelial cancer. This metabolic reprogramming includes aberrant expression levels of genes, metabolites, and associated networks and pathways. In this review, we summarize the dysfunctions of glycolytic enzymes in urothelial cancer and discuss the relevant phenotype and signal transduction. Moreover, we describe potential prognostic factors and risks to the survival of clinical cancer patients. More importantly, based on several available databases, we explore relationships between glycolytic enzymes and genetic changes or drug responses in urothelial cancer cells. Current advances in glycolysis-based inhibitors and their combinations are also discussed. Combining all of the evidence, we indicate their potential value for further research in basic science and clinical applications.

Inflammation-induced Occludin Downregulation Limits Epithelial Apoptosis by Suppressing Caspase-3 Expression.

BACKGROUND & AIMS: Epithelial tight junctions are compromised in gastrointestinal disease. Processes that contribute to the resulting barrier loss include endocytic occludin removal from the tight junction and reduced occludin expression. Nevertheless, the relatively-normal basal phenotype of occludin knockout (KO) mice has been taken as evidence that occludin does not contribute to gastrointestinal barrier function. We asked whether stress could unmask occludin functions within intestinal epithelia. METHODS: Wildtype (WT), universal and intestinal epithelial-specific occludin KO, and villin-EGFP-occludin transgenic mice as well as WT and occludin knockdown (KD) Caco-2BBe cell monolayers were challenged with DSS, TNBS, staurosporine, 5-FU, or TNF. Occludin and caspase-3 expression were assessed in patient biopsies. RESULTS: Intestinal epithelial occludin loss limited severity of DSS- and TNBS-induced colitis due to epithelial resistance to apoptosis; activation of both intrinsic and extrinsic apoptotic pathways was blocked in occludin KO epithelia. Promoter analysis revealed that occludin enhances CASP3 transcription and, conversely, that occludin downregulation reduces caspase-3 expression. Analysis of biopsies from Crohn's disease and ulcerative colitis patients and normal controls demonstrated that disease-associated occludin downregulation was accompanied by and correlated with reduced caspase-3 expression. In vitro, cytokine-induced occludin downregulation resulted in reduced caspase-3 expression and resistance to intrinsic and extrinsic pathway apoptosis, demonstrating an overall protective effect of inflammation-induced occludin loss. CONCLUSIONS: The tight junction protein occludin regulates apoptosis by enhancing caspase-3 transcription. These data suggest that reduced epithelial caspase-3 expression downstream of occludin downregulation is a previously-unappreciated anti-apoptotic process that contributes to mucosal homeostasis in inflammatory conditions.

The scaffolding protein ZO-1 coordinates actomyosin and epithelial apical specializations in vitro and in vivo.

Polarized epithelia assemble into sheets that compartmentalize organs and generate tissue barriers by integrating apical surfaces into a single, unified structure. This tissue organization is shared across organs, species, and developmental stages. The processes that regulate development and maintenance of apical epithelial surfaces are, however, undefined. Here, using an intestinal epithelial-specific knockout (KO) mouse and cultured epithelial cells, we show that the tight junction scaffolding protein zonula occludens-1 (ZO-1) is essential for development of unified apical surfaces in vivo and in vitro We found that U5 and GuK domains of ZO-1 are necessary for proper apical surface assembly, including organization of microvilli and cortical F-actin; however, direct interactions with F-actin through the ZO-1 actin-binding region (ABR) are not required. ZO-1 lacking the PDZ1 domain, which binds claudins, rescued apical structure in ZO-1-deficient epithelia, but not in cells lacking both ZO-1 and ZO-2, suggesting that heterodimerization with ZO-2 restores PDZ1-dependent ZO-1 interactions that are vital to apical surface organization. Pharmacologic F-actin disruption, myosin II motor inhibition, or dynamin inactivation restored apical epithelial structure in vitro and in vivo, indicating that ZO-1 directs epithelial organization by regulating actomyosin contraction and membrane traffic. We conclude that multiple ZO-1-mediated interactions contribute to coordination of epithelial actomyosin function and genesis of unified apical surfaces.

Distinct cytoprotective roles of pyruvate and ATP by glucose metabolism on epithelial necroptosis and crypt proliferation in ischaemic gut.

KEY POINTS: Intestinal ischaemia causes epithelial death and crypt dysfunction, leading to barrier defects and gut bacteria-derived septic complications. Enteral glucose protects against ischaemic injury; however, the roles played by glucose metabolites such as pyruvate and ATP on epithelial death and crypt dysfunction remain elusive. A novel form of necrotic death that involves the assembly and phosphorylation of receptor interacting protein kinase 1/3 complex was found in ischaemic enterocytes. Pyruvate suppressed epithelial cell death in an ATP-independent manner and failed to maintain crypt function. Conversely, replenishment of ATP partly restored crypt proliferation but had no effect on epithelial necroptosis in ischaemic gut. Our data argue against the traditional view of ATP as the main cytoprotective factor by glucose metabolism, and indicate a novel anti-necroptotic role of glycolytic pyruvate under ischaemic stress. ABSTRACT: Mesenteric ischaemia/reperfusion induces epithelial death in both forms of apoptosis and necrosis, leading to villus denudation and gut barrier damage. It remains unclear whether programmed cell necrosis [i.e. receptor-interacting protein kinase (RIP)-dependent necroptosis] is involved in ischaemic injury. Previous studies have demonstrated that enteral glucose uptake by sodium-glucose transporter 1 ameliorated ischaemia/reperfusion-induced epithelial injury, partly via anti-apoptotic signalling and maintenance of crypt proliferation. Glucose metabolism is generally assumed to be cytoprotective; however, the roles played by glucose metabolites (e.g. pyruvate and ATP) on epithelial cell death and crypt dysfunction remain elusive. The present study aimed to investigate the cytoprotective effects exerted by distinct glycolytic metabolites in ischaemic gut. Wistar rats subjected to mesenteric ischaemia were enterally instilled glucose, pyruvate or liposomal ATP. The results showed that intestinal ischaemia caused RIP1-dependent epithelial necroptosis and villus destruction accompanied by a reduction in crypt proliferation. Enteral glucose uptake decreased epithelial cell death and increased crypt proliferation, and ameliorated mucosal histological damage. Instillation of cell-permeable pyruvate suppressed epithelial cell death in an ATP-independent manner and improved the villus morphology but failed to maintain crypt function. Conversely, the administration of liposomal ATP partly restored crypt proliferation but did not reduce epithelial necroptosis and histopathological injury. Lastly, glucose and pyruvate attenuated mucosal-to-serosal macromolecular flux and prevented enteric bacterial translocation upon blood reperfusion. In conclusion, glucose metabolites protect against ischaemic injury through distinct modes and sites, including inhibition of epithelial necroptosis by pyruvate and the promotion of crypt proliferation by ATP.

Tumor Necrosis Factor α-Dependent Neutrophil Priming Prevents Intestinal Ischemia/Reperfusion-Induced Bacterial Translocation.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) causes barrier impairment and bacterial influx. Protection against I/R injury in sterile organs by hypoxic preconditioning (HPC) had been attributed to erythropoietic and angiogenic responses. Our previous study showed attenuation of intestinal I/R injury by HPC for 21 days in a neutrophil-dependent manner. AIM: To investigate the underlying mechanisms of neutrophil priming by HPC, and explore whether adoptive transfer of primed neutrophils is sufficient to ameliorate intestinal I/R injury. METHODS: Rats raised in normoxia (NM) and HPC for 3 or 7 days were subjected to sham operation or superior mesenteric artery occlusion for I/R challenge. Neutrophils isolated from rats raised in NM or HPC for 21 days were intravenously injected into naïve controls prior to I/R. RESULTS: Similar to the protective effect of HPC-21d, I/R-induced mucosal damage was attenuated by HPC-7d but not by HPC-3d. Naïve rats reconstituted with neutrophils of HPC-21d rats showed increase in intestinal phagocytic infiltration and myeloperoxidase activity, and barrier protection against I/R insult. Elevated free radical production, and higher bactericidal and phagocytic activity were observed in HPC neutrophils compared to NM controls. Moreover, increased serum levels of tumor necrosis factor α (TNFα) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) were seen in HPC rats. Naïve neutrophils incubated with HPC serum or recombinant TNFα, but not CINC-1, exhibited heightened respiratory burst and bactericidal activity. Lastly, neutrophil priming effect was abolished by neutralization of TNFα in HPC serum. CONCLUSIONS: TNFα-primed neutrophils by HPC act as effectors cells for enhancing barrier integrity under gut ischemia.

Commensal bacterial endocytosis in epithelial cells is dependent on myosin light chain kinase-activated brush border fanning by interferon-γ.

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.

Enteric dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistant and commensal bacteria through epithelial transcytosis.

Antibiotic usage promotes intestinal colonization of antibiotic-resistant bacteria. However, whether resistant bacteria gain dominance in enteric microflora or disseminate to extraintestinal viscera remains unclear. Our aim was to investigate temporal diversity changes in microbiota and transepithelial routes of bacterial translocation after antibiotic-resistant enterobacterial colonization. Mice drinking water with or without antibiotics were intragastrically gavaged with ampicillin-resistant (Amp-r) nonpathogenic Escherichia coli (E. coli) and given normal water afterward. The composition and spatial distribution of intestinal bacteria were evaluated using 16S rDNA sequencing and fluorescence in situ hybridization. Bacterial endocytosis in epithelial cells was examined using gentamicin resistance assay and transmission electromicroscopy. Paracellular permeability was assessed by tight junctional immunostaining and measured by tissue conductance and luminal-to-serosal dextran fluxes. Our results showed that antibiotic treatment enabled intestinal colonization and transient dominance of orally acquired Amp-r E. coli in mice. The colonized Amp-r E. coli peaked on day 3 postinoculation and was competed out after 1 wk, as evidenced by the recovery of commensals, such as Escherichia, Bacteroides, Lachnospiraceae, Clostridium, and Lactobacillus. Mucosal penetration and extraintestinal dissemination of exogenous and endogenous enterobacteria were correlated with abnormal epithelial transcytosis but uncoupled with paracellular tight junctional damage. In conclusion, antibiotic-induced enteric dysbiosis predisposes to exogenous infection and causes systemic dissemination of both antibiotic-resistant and commensal enterobacteria through transcytotic routes across epithelial layers. These results may help explain the susceptibility to sepsis in antibiotic-resistant enteric bacterial infection.

The application of electrocoagulation for the conversion of MSWI fly ash into nonhazardous materials.

This research investigated the electrocoagulation of municipal solid waste incineration (MSWI) fly ash at a liquid-to-solid ratio (L/S) of 20:1. The leachate that was obtained from this treatment was recovered for reutilization. Two different anodic electrodes were investigated, and two unit runs were conducted. In Unit I, the optimum anode was chosen, and in Unit II, the optimum anode and the recovered leachate were used to replace deionized water for repeating the same electrocoagulation experiments. The results indicate that the aluminum (Al) anode performed better than the iridium oxide (IrO2) anode. The electrocoagulation technique includes washing with water, changing the composition of the fly ash, and stabilizing the heavy metals in the ash. Washing with water can remove the soluble salts from fly ash, and the fly ash can be converted into Friedel's salt (3CaO·Al2O3·CaCl2·10H2O) under an uniform electric field and the sacrificial release of Al(+3) ions, which stabilizes the toxic heavy metals and brings the composition of the fly ash to within the regulatory limits of the toxicity characteristic leaching procedure (TCLP). Use of the Al anode to manage the MSWI fly ash and the leachate obtained from the electrocoagulation treatment is therefore feasible.

Healthcare 5.0: A secure and distributed network for system informatics in medical surgery.

INTRODUCTION: Health records serve not only as a database of a patient's health history and treatment process but also as a crucial tool for doctors to diagnose and treat patients. However, the storage and sharing of these records are sensitive issues as they involve maintaining patient privacy and ensuring data transparency, security, and interoperability between different parties. Challenges to achieving these goals in the current surgical process can impact the allocation of medical resources and surgical outcomes. METHODS: This article proposes a healthcare 5.0 framework for medical surgery that deploys a secure and distributed network using Blockchain to demonstrate transactions between different parties in the orthopedic surgery process. The proposed network uses the Hyperledger Composer platform for deployment, and a patient-doctor-supplier orthopedic surgery network is designed and implemented to enable the safe sharing of medical records. RESULTS: A benchmarking tool was implemented for analyzing different scenarios of applying blockchain technology to orthopedic surgery. The application of blockchain technology to orthopedic surgery presents a promising solution for data sharing and supply chain management in the field. The integration of blockchain with cloud storage and hybrid encryption ensures secure and efficient storage of Electronic Health Record (EHR) and Personal Health Record (PHR) data. By leveraging the tamper-proof nature of blockchain and addressing concerns regarding centralized data storage, this scenario demonstrates enhanced security, improved access efficiency, and privacy protection in medical data sharing. CONCLUSIONS: The article demonstrates the feasibility of using an IoT-based blockchain network in orthopedic surgery, which can reduce medical errors and improve data interoperability among different parties. This unique application of blockchain enables secure sharing of medical records, ensuring transparency, security, and interoperability. The network design may also be applicable to other surgeries and medical applications in the future.

Oral Lactobacillus zeae exacerbates the pathological manifestation of periodontitis in a mouse model.

INTRODUCTION: The worldwide prevalence of periodontitis is considerably high, and its pathogenic mechanisms must be investigated and understood in order to improve clinical treatment outcomes and reduce the disease prevalence and burden. The exacerbation of the host immune system induced by oral microbial dysbiosis and the subsequent tissue destruction are the hallmarks of the periodontitis. However, the oral bacteria involved in periodontitis are not fully understood. We used the Oxford Nanopore Technologies (ONT) sequencing system to analyze metagenomic information in subgingival dental plaque from periodontitis and non-periodontitis patients. The number of Lactobacillus zeae (L. zeae) in the periodontitis patients was 17.55-fold higher than in the non-periodontitis patients, suggesting that L. zeae is a novel periodontitis-associated pathogen. Although several Lactobacillus species are used in vivo as probiotics to treat periodontitis and compete with Porphyromonas gingivalis (P. gingivalis), the roles of L. zeae in periodontitis progression, and the relationship between L. zeae and P. gingivalis needs to be investigated. METHODS: Both L. zeae and P. gingivalis were inoculated in the ligature-implant site of periodontitis mice. We collected mouse gingival crevicular fluid to analyze inflammatory cytokine secretion using a multiplex assay. Intact or sliced mouse maxilla tissue was used for micro-computed tomography analysis or hematoxylin and eosin staining, immunohistochemistry, and tartrate-resistant acid phosphatase staining to evaluate alveolar bone loss, neutrophil infiltration, and osteoclast activation, respectively. RESULTS: We observed that L. zeae competed with P. gingivalis, and it increased inflammatory cytokine secretion at the ligature-implant site. Similar to P. gingivalis, L. zeae promoted ligature-induced neutrophile infiltration, osteoclast activation, and alveolar bone loss. DISCUSSION: We, therefore, concluded that L. zeae accelerated the progression of periodontitis in the ligature-induced periodontitis mouse model.

Controlled release of Clenbuterol from a hydroxyapatite carrier for the treatment of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is a neurodegenerative disorder, and Aß aggregation is considered to be the central process implicated in its pathogenesis. Current treatments are faced by challenges such as serious side effects and reduced drug bioavailability. In this study, we developed a drug delivery system for intramuscular injection that uses cellular activity to achieve constant and long-term drug release. METHODS: Synthesized mesoporous hydroxyapatite (SHAP) was prepared via co-precipitation, and hydrophobic surface modification using stearic acid was then used to load clenbuterol by physical absorption, thus creating the drug delivery system. Clenbuterol release was achieved through cellular activity, with macrophage uptake triggering lysosome/endosome disruption, cytoplasmic release, extracellular exocytosis, and subsequent systemic circulation. RESULTS: We found that clenbuterol-loaded SHAP enabled sustained release for more than 2 weeks and effectively modulated inflammation, reduced Aß oligomer-induced toxicity, and prevented Aß aggregation. CONCLUSIONS: Our findings suggest that treatment with clenbuterol loaded in this SHAP delivery system could be a promising strategy for treating Alzheimer's disease.

The synthesis and evaluation of thiolated alginate as the barrier to block nutrient absorption on small intestine for body-weight control.

Obesity is the most common health concern all over the world. However, till now, there is no promising way to manage obesity or body-weight control. The aim of the study is to develop an edible gel as a health supplement that temporarily attaches to the mucus of the intestines, forming an absorption barrier to block the nutrients. We modify the alginate with the thiol group as thiolated alginate (TA) that may stay on the mucosa layer for a much longer time to reduce nutrient absorption. In this study, the TA is synthesized successfully and proved a good mucosal adhesion to serve as a barrier for nutrient absorption both in vitro and in vivo. The results of in vivo imaging system (IVIS) show that the synthesized TA can be exiled from the gastrointestinal tract within 24 h. The animal study shows that the TA by daily oral administration can effectively reduce body weight and fat deposition. The biosafety is evaluated in vitro at the cellular level, based on ISO-10993, and further checked by animal study. We do believe that the TA could have a greater potential to be developed into a safe health supplement to manage obesity and for body-weight control.

A combination of all-trans retinoic acid derivative and COX-2 inhibitor has anticancer effects in human pharyngeal carcinoma cells.

Backgrounds and aims: Carcinogenesis is characterized by an unlimited growth of cells exacerbated by Cox-2 overexpression. Cox-2 inhibitors have been proven effective in preventing and treating tumors. In our previous studies, we found that 4-Amino-2-Trifluoromethylphenyl Retinate (ATPR) induces cell apoptosis and inhibits cell proliferation to exhibit anti-cancer properties. The use of ATRA as well as Cox-2 inhibitors in clinical settings can cause adverse reactions. It is unknown what the effects and mechanisms of co-administration of ATPR and Cox-2 inhibitors are. Results: A combination of ATPR and Cox-2 inhibitors, Celecoxib, inhibited pharyngeal cancer cell proliferation in vitro and induced apoptosis. The cell cycle was arrested at G0/G1 by activating P53 and CDNA1. By activating MAPK/JNK pathways, ATPR and Celecoxib led to intrinsic and extrinsic apoptosis in pharyngeal cancer cells. ATPR/Celecoxib combined treatment suppressed tumor growth in the pharyngeal cancer cell-derived xenograft mouse model by increasing the number of apoptotic cells. The expression of the RARA and PTGS2 genes was significantly increased in tumor tissue compared to non-tumor tissue in the clinical analysis of the head and neck squamous cell carcinoma dataset. An association was found between this and the level of intrinsic apoptotic signals. Furthermore, a survival analysis conducted over a period of five years indicated that higher levels of RARA expression were associated with a better clinical outcome. Conclusion: ATPR and celecoxib inhibit the proliferation of cancer cells as well as induce apoptosis. Co-administration of ATPR and Cox-2 inhibitors has the potential to be a novel treatment plan for cancer.

Structural polymorphism of c-di-GMP bound to an EAL domain and in complex with a type II PilZ-domain protein.

Cyclic di-GMP (c-di-GMP) is a novel secondary-messenger molecule that is involved in regulating a plethora of important bacterial activities through binding to an unprecedented array of effectors. Proteins with a canonical PilZ domain that bind c-di-GMP play crucial roles in regulating flagellum-based motility. In contrast, noncanonical type II PilZ domains that do not effectively bind c-di-GMP regulate twitching motility, which is dependent on type IV pili (T4P). Recent data indicate that T4P biogenesis is initiated via the interaction of a noncanonical type II PilZ protein with the GGDEF/EAL-domain protein FimX and the pilus motor protein PilB at high c-di-GMP concentrations. However, the molecular details of such interactions remain to be elucidated. In this manuscript, the first hetero-complex crystal structure between a type II PilZ protein and the EAL domain of the FimX protein (FimX(EAL)) from Xanthomonas campestris pv. campestris (Xcc) in the presence of c-di-GMP is reported. This work reveals two novel conformations of monomeric c-di-GMP in the XccFimX(EAL)-c-di-GMP and XccFimX(EAL)-c-di-GMP-XccPilZ complexes, as well as a unique interaction mode of a type II PilZ domain with FimX(EAL). These findings indicate that c-di-GMP is sufficiently flexible to adjust its conformation to match the corresponding recognition motifs of different cognate effectors. Together, these results represent a first step towards an understanding of how T4P biogenesis is controlled by c-di-GMP at the molecular level and also of the ability of c-di-GMP to bind to a wide variety of effectors.