Vacuolar proton pumping pyrophosphatase (H(+)-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PP(i) hydrolysis. A fluorescent probe, fluorescein 5'-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H(+)-PPase. The enzymatic activity and its associated H(+) translocation of vacuolar H(+)-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H(+)-PPase. Changes in K(m) but not V(max) values of vacuolar H(+)-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H(+)-PPase could be protected by its physiological substrate, Mg(2+)-PP(i). These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H(+)-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region.

Gap junctions between astrocytes as well as between astrocytes and oligodendrocytes in rat brain were immunohistochemically labelled with a monoclonal and an affinity-purified polyclonal antibody generated against connexin-26. By light microscopy, the immunolabelling patterns obtained were, with a few exceptions, remarkably similar to previously described distribution patterns of the gap junctional protein connexin-43, which is expressed by astrocytes and is localized at astrocytic gap junctions. By electron microscopy, immunoreactivity with these two anti-connexin-26 antibodies was restricted to astrocytes; inter-astrocytic gap junctional membranes were symmetrically labelled, heterologous oligo-astrocytic junctional membranes were asymmetrically labelled only on the astrocyte side and oligo-oligodendrocyte junctions were unlabelled. Two additional anti-connexin-26 antibodies that were found to produce punctate labelling in leptomeninges and liver failed to do so in brain parenchyma, consistent with reports indicating the absence of authentic connexin-26 in this tissue. Antibodies that labelled astrocytic gap junctions exhibited no cross-reaction with connexin-43 or connexin-32, as demonstrated by western blotting, but recognized liver connexin-26 as well as several brain proteins, including an approximately 32000 mol. wt protein that did not correspond to connexin-32 and a 26000 mol. wt protein that co-migrated with liver connexin-26. These results suggest that connexin-26, or more likely a protein having sequence homology with connexin-26, is targeted to astrocytic gap junctions and raise the possibility of the existence of connexins that may be co-expressed with connexin-43 in most, but perhaps not all, astrocytes.

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, β5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/β-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Osteoarthritis (OA) is the most common degenerative joint disorder. Multipotential stromal cells (MSCs) have a crucial role in joint repair, but how OA severity affects their characteristics remains unknown. Knee OA provides a good model to study this, as osteochondral damage is commonly more severe in the medial weight-bearing compartment compared to lateral side of the joint. This study utilised in vitro functional assays, cell sorting, gene expression and immunohistochemistry to compare MSCs from medial and lateral OA femoral condyles. Despite greater cartilage loss and bone sclerosis in medial condyles, there was no significant differences in MSC numbers, growth rates or surface phenotype. Culture-expanded and freshly-purified medial-condyle MSCs expressed higher levels of several ossification-related genes. Using CD271-staining to identify MSCs, their presence and co-localisation with TRAP-positive chondroclasts was noted in the vascular channels breaching the osteochondral junction in lateral condyles. In medial condyles, MSCs were additionally found in small cavities within the sclerotic plate. These data indicate subchondral MSCs may be involved in OA progression by participating in cartilage destruction, calcification and sclerotic plate formation and that they remain abundant in severe disease. Biological or biomechanical modulation of these MSCs may be a new strategy towards cartilage and bone restoration in knee OA.

Fourth generation polyamidoamine dendrimer (PAMAM, G4) modified with fluorescein units (F) at the periphery and Pt nanoparticles stabilized by L-ascorbate were prepared. These dendrimers modified with hydrophobic fluorescein were used to achieve self-assembling structures, giving rise to the formation of nanoaggregates in water. The photoactive fluorescein units were mainly used as photosensitizer units in the process of the catalytic photoreduction of water propitiated by light. Complementarily, Pt-ascorbate nanoparticles acted as the active sites to generate H2. Importantly, the study of the functional, optical, surface potential and morphological properties of the photosensitized dendrimer aggregates at different irradiation times allowed for insights to be gained into the behavior of these systems. Thus, the resultant photosensitized PAMAM-fluorescein (G4-F) nanoaggregates (NG) were conveniently applied to light-driven water photoreduction along with sodium L-ascorbate and methyl viologen as the sacrificial reagent and electron relay agent, respectively. Notably, these aggregates exhibited appropriate stability and catalytic activity over time for hydrogen production. Additionally, in order to propose a potential use of these types of systems, the in situ generated H2 was able to reduce a certain amount of methylene blue (MB). Finally, theoretical electronic analyses provided insights into the possible excited states of the fluorescein molecules that could intervene in the global mechanism of H2 generation.

Various genotoxic carcinogens ubiquitously present in the human environment or respective reactive metabolites form adducts in DNA and proteins, which can be used as biomarkers of internal exposure. For example, the mass spectrometric determination of Val adducts at the N-termini of hemoglobin (Hb) peptide chains after cleavage by an Edman degradation has a long tradition in occupational medicine. We developed a novel isotope-dilution UHPLC-MS/MS method for the simultaneous quantification of Val adducts of eight genotoxic substances in Hb after cleavage with fluorescein-5-isothiocyanate (FIRE procedure™). The following adducts were included [sources in square brackets]: N-(2,3-dihydroxypropyl)-Val [glycidol], N-(2-carbamoylethyl)-Val [acrylamide], N-(2-carbamoyl-2-hydroxyethyl)-Val [glycidamide], N-((furan-2-yl)methyl)-Val [furfuryl alcohol], N-(trans-isoestragole-3'-yl)-Val [estragole/anethole], N-(3-ketopentyl)-Val [1-penten-3-one], N-(3-ketooctanyl)-Val [1-octene-3-one], and N-benzyl-Val [benzyl chloride], each of which was quantified with a specific isotope-labeled standard. The limits of quantification were between 0.014 and 3.6 pmol/g Hb (using 35 mg Hb per analysis); other validation parameters were satisfactory according to guidelines of the U.S. Food and Drug Administration. The quantification in erythrocyte samples of human adults (proof of principle) showed that the median levels of Hb adducts of acrylamide, glycidamide, and glycidol were found to be significantly lower in six non-smokers (25.9, 12.2, and 4.7 pmol/g Hb, respectively) compared to those of six smokers (69.0, 44.2, and 8.6 pmol/g Hb, respectively). In summary, the method surpasses former techniques of Hb adduct quantification due to its simplicity, sensitivity, and accuracy. It can be extended continuously with other Hb adducts and will be used in epidemiological studies on internal exposure to carcinogens.

Real-time monitoring of dissolved oxygen (DO) and pH is of great significance for understanding cellular metabolism. Herein, a dual optical pH/O2 sensing membrane was prepared by the electrospinning method. Cellulose acetate (CA) and poly(ε-caprolactone) (PCL) nanofiber membrane blended with platinum (II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTFPP) was used as the DO sensing matrix, upon which electrospun nanofiber membrane of chitosan (CS) coupled with fluorescein 5-isothiocyanate (FITC) was used as the pH sensing matrix. The electrospun sensing film prepared from biocompatible biomaterials presented good response to a wide range of DO concentrations and physiological pH. We used it to monitor the exracellular acidification and oxygen consumption levels of cells and bacteria. This sensing film can provide a luminescence signal change as the DO and pH change in the growth microenvironment. Due to its advantages of good biocompatibility and high stability, we believe that the dual functional film has a high value in the field of biotechnology research.

Dielectrophoresis (DEP), a precision nonlinear electrokinetic tool utilized within microfluidic devices, can induce bioparticle polarization that manifests as motion in the electric field; this phenomenon has been leveraged for phenotypic cellular and biomolecular detection, making DEP invaluable for diagnostic applications. As device operation times lengthen, reproducibility and precision decrease, which has been postulated to be caused by ion gradients within the supporting electrolyte medium. This research focuses on characterizing pH gradients above, at, and below the electrode charging frequency (0.2-1.4 times charging frequency) in an aqueous electrolyte solution in order to extend the parameter space for which microdevice-imposed artifacts on cells in clinical diagnostic devices have been characterized. The nonlinear alternating current (AC) electric fields (0.07 Vpp/μm) required for DEP were generated via planar T-shaped and star-shaped microelectrodes overlaid by a 70 μm high microfluidic chamber. The experiments were designed to quantify pH changes temporally and spatially in the two microelectrode geometries. In parallel, a 50 nm hafnium oxide (HfO2) thin film on the microelectrodes was tested to provide insights into the role of Faradaic surface reactions on the pH. Electric field simulations were conducted to provide insights into the gradient shape within the microelectrode geometries. Frequency dependence was also examined to ascertain ion electromigration effects above, at, and below the electrode charging frequency. The results revealed Faradaic reactions above, at, and below the electrode charging frequency. Comparison experiments further demonstrated that pH changes caused by Faradaic reactions increased inversely with frequency and were more pronounced in the star-shaped geometry. Finally, HfO2 films demonstrated frequency-dependent properties, impeding Faradaic reactions.

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. Such inclusions have variably been described as amorphous aggregates or more structured deposits having an amyloid structure. Following the observations that bacterial inclusion bodies generally consist of amyloid aggregates, we have overexpressed full-length TDP-43 and C-terminal TDP-43 in E. coli, purified the resulting full-length and C-terminal TDP-43 containing inclusion bodies (FL and Ct TDP-43 IBs) and subjected them to biophysical analyses to assess their structure/morphology. We show that both FL and Ct TDP-43 aggregates contained in the bacterial IBs do not bind amyloid dyes such as thioflavin T and Congo red, possess a disordered secondary structure, as inferred using circular dichroism and infrared spectroscopies, and are susceptible to proteinase K digestion, thus possessing none of the hallmarks for amyloid. Moreover, atomic force microscopy revealed an irregular structure for both types of TDP-43 IBs and confirmed the absence of amyloid-like species after proteinase K treatment. Cell biology experiments showed that FL TDP-43 IBs were able to impair the viability of cultured neuroblastoma cells when added to their extracellular medium and, more markedly, when transfected into their cytosol, where they are at least in part ubiquitinated and phosphorylated. These data reveal an inherently high propensity of TDP-43 to form amorphous aggregates, which possess, however, an inherently high ability to cause cell dysfunction. This indicates that a gain of toxic function caused by TDP-43 deposits is effective in TDP-43 pathologies, in addition to possible loss of function mechanisms originating from the cellular mistrafficking of the protein.

Persistent infection with the obligate intracellular pathogen Chlamydophila pneumoniae has been implicated in the pathogenesis of many chronic diseases, but its mechanism remains unclear. Many pathogens have been found to modulate cellular apoptosis in order to survive and multiply. Chlamydial species were shown to both induce and inhibit host cell apoptosis depending on the experimental conditions. We utilized in vitro models of acute and long-term continuous (LTC) infection with the same cell line (HEp-2) and chlamydial isolate (TW-183) used in both models. Host cell apoptosis in infected and uninfected cells was assessed by fluorescence microscopy and flow cytometry. While acute infection induced apoptosis 72 h post-infection, LTC-infected cells had low rates of apoptosis and showed resistance to different exogenous inducers of apoptosis (sorbitol, serum withdrawal, hydrogen peroxide) when compared to uninfected cells. Chronicity of infection appears to be a critical factor in the modulation of host cell apoptosis by C. pneumoniae. Induction of apoptosis may help to propagate the infection, while inhibition of apoptosis could help protect the organism in chronic infection.

To reveal if coagulopathies relate to the course of COVID-19, we examined 255 patients with moderate and severe COVID-19, receiving anticoagulants and immunosuppressive drugs. Coagulopathy manifested predominantly as hypercoagulability that correlated directly with systemic inflammation, disease severity, comorbidities, and mortality risk. The prolonged clotting tests in about ¼ of cases were associated with high levels of C-reactive protein and antiphospholipid antibodies, which impeded coagulation in vitro. Contraction of blood clots was hindered in about ½ of patients, especially in severe and fatal cases, and correlated directly with prothrombotic parameters. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with platelet dysfunction. Clots with impaired contraction were porous, had a low content of compressed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin, suggesting that the uncompacted intravital clots are more obstructive but patients could also be prone to bleeding. The absence of consumption coagulopathy suggests the predominance of local and/or regional microthrombosis rather than disseminated intravascular coagulation. The results obtained (i) confirm the importance of hemostatic disorders in COVID-19 and their relation to systemic inflammation; (ii) justify monitoring of hemostasis, including the kinetics of blood clot contraction; (iii) substantiate the active prophylaxis of thrombotic complications in COVID-19.

Various liquid biopsy methods have been developed for the non-invasive and early detection of diseases. In particular, the detection of circulating tumor cells (CTCs) and cancer-associated fibroblasts (CAFs) in blood has been receiving a great deal of attention. We have been developing systems and materials to facilitate such liquid biopsies. In this study, we further developed glass filters (with various patterns of holes, pitches, and non-adhesive coating) that can capture CTCs, but not white blood cells. We optimized the glass filters to capture CTCs, and demonstrated that they could be used to detect CTCs from lung cancer patients. We also used the optimized glass filters for detecting CAFs. Additionally, we further developed a system for visualizing the captured cells on the glass filters. Finally, we demonstrated that we could directly culture the captured cells on the glass filters. Based on these results, our high-performance glass filters appear to be useful for capturing and culturing CTCs and CAFs for further examinations.

Novel therapeutic approaches combining immune-checkpoint inhibitors are needed to improve clinical outcomes for patients with cancer. Lymphocyte-activation gene 3 (LAG-3) is an immune-checkpoint molecule that inhibits T-cell activity and antitumor immune responses, acting through an independent mechanism from that of programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Here, we describe the development and preclinical characterization of relatlimab, a human antibody that binds to human LAG-3 with high affinity and specificity to block the interaction of LAG-3 with the ligands MHC II and fibrinogen-like protein-1, and to reverse LAG-3-mediated inhibition of T-cell function in vitro. Consistent with previous reports, in mouse models, the combined blockade of LAG-3 and PD-1 with surrogate antibodies resulted in enhanced antitumor activity greater than the individual blockade of either receptor. In toxicity studies in cynomolgus monkeys, relatlimab was generally well tolerated when combined with nivolumab. These results are consistent with findings from the RELATIVITY-047 phase II/III trial showing that relatlimab combined with nivolumab is a well-tolerated regimen that demonstrates superior progression-free survival compared with nivolumab monotherapy in patients with unresectable or metastatic melanoma.

Anticancer regimens have been substantially enriched through monoclonal antibodies targeting immune checkpoints, programmed cell death-1/programmed cell death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen-4. Inconsistent clinical efficacy after solo immunotherapy may be compensated by nanotechnology-driven combination therapy. We loaded human serum albumin (HSA) nanoparticles with paclitaxel (PTX) via nanoparticle albumin-bound technology and pooled them with anti-PD-L1 monoclonal antibody through a pH-sensitive linker for targeting and immune response activation. Our tests demonstrated satisfactory preparation of paclitaxel-loaded, PD-L1-targeted albumin nanoparticles (PD-L1/PTX@HSA). They had small particle size (~200 nm) and polydispersity index (~0.12) and successfully incorporated each constituent. Relative to normal physiological pH, the formulation exhibited higher drug-release profiles favoring cancer cell-targeted release at low pH. Modifying nanoparticles with programmed cell death-ligand 1 increased cancer cell internalization in vitro and tumor accumulation in vivo in comparison with non-PD-L1-modified nanoparticles. PD-L1/PTX@HSA constructed by nanoparticle albumin-bound technology displayed successful tumor inhibition efficacy both in vitro and in vivo. There was successful effector T-cell infiltration, immunosuppressive programmed cell death-ligand 1, and regulatory T-cell suppression because of cytotoxic T-lymphocyte antigen-4 synergy. Moreover, PD-L1/PTX@HSA had low organ toxicity. Hence, the anti-tumor immune responses of PD-L1/PTX@HSA combined with chemotherapy and cytotoxic T-lymphocyte antigen-4 is a potential anti-tumor strategy for improving quantitative and qualitative clinical efficacy.

Pericyte coverage on the endothelial tubes leads to the formation of a mature and stable microvessel system, which is critical for brain repair after intracerebral hemorrhage (ICH). We report herein that thrombin promotes pericyte coverage by activating Tie2 and the downstream signaling pathway PI3K/Akt in a rat model of ICH. ICH was induced by injection of autologous blood with or without thrombin inhibitor hirudin. Rats were treated with thrombin alone or in combination with a Tie2 inhibitor. The expression of total- and phospho-Tie2, PI3K and phospho-Akt, blood perfusion, pericyte coverage, IgG extravasation, neuron survival and neurological deficits were evaluated by western blot, fluorescein-5-isothiocyanate-dextran staining, immunohistochemistry, Nissl staining and modified neurological severity scores respectively. Induction of ICH resulted in increased phosphorylation of Tie2 on endothelial cells and pericyte coverage, better formation of integral and functional microvessels, more surviving neurons and accelerated motor function recovery, all of which were significantly attenuated by hirudin at 7 and 14 days after ICH induction. Furthermore, thrombin increased phosphorylation of Tie2 and Akt, expression of PI3K, and pericyte coverage, which were however reversed by pharmacological inhibition of Tie2. Our results demonstrated that thrombin promotes pericyte coverage on microvessels following ICH by enhancing activation of Tie2, in which the downstream PI3K/Akt signaling pathway might be involved.

The study evaluates cellulose nanocrystals (CNCs) as nanocarriers for targeted, intracellular delivery of molecular agents. CNCs were labeled with fluorescein-5'-isothiocyanate as an imaging agent and conjugated to folic acid (FA) as a targeting ligand. The CNC conjugates were characterized by UV-vis spectroscopy, ζ-potential analysis, dynamic light scattering, and atomic force microscopy. Cellular binding/uptake of the FA-conjugated CNCs by KB and MDA-MB-468 cells was quantified with cellular uptake assays. Internalization of the particles was confirmed by confocal microscopy. Uptake mechanisms were determined by inhibition studies with chlorpromazine and genistein. Binding affinity was qualitatively assessed with a free folate inhibition assay. Both KB and MDA-MB-468 cells exhibited significant and folate-receptor specific binding/uptake of FA-conjugated CNCs. Clathrin-mediated endocytosis was a significant uptake mechanism in both cell types, whereas caveolae-mediated endocytosis only played a significant role in MDA-MB-468 cells. Uptake inhibition of FA-conjugated CNCs by KB cells required high concentrations (>1 mM) of free FA. The observed FR-specific internalization of FA-conjugated CNCs by FR-positive cancer cells and tumors and their remarkable high affinity for the FR demonstrate the great potential of CNCs as novel nanocarriers for imaging agents and chemotherapeutics in the early detection and treatment of cancer.

Blood-brain-barrier (BBB) disruption is an important pathological characteristic of ischemic stroke (IS) and mainly results from dysfunction of brain vascular endothelial cells and tight junctions. Zebularine is a novel inhibitor of DNA methyltransferase (DNMT). Here, we assessed its effects on BBB disruption in IS. Firstly, we reported that Zebularine maintained BBB integrity in middle cerebral artery occlusion (MCAO) mice by increasing the expressions of zona occludens-1 (ZO-1) and vascular endothelial (VE)-cadherin. Importantly, we found that Zebularine reduced the production of pro-inflammatory cytokines, attenuated brain edema, and improved neurological deficits. In in vitro experiments, the bEnd.3 brain endothelial cells were exposed to oxygen and glucose deprivation/reoxygenation (OGD/R), and the protective effects of Zebularine were assessed. Our findings demonstrated that Zebularine prevented OGD/R-induced cytotoxicity by reducing the release of lactate dehydrogenase (LDH). Additionally, Zebularine protected bEnd.3 cells against OGD/R-induced hyper-permeability and reduction of trans-endothelial electrical resistance (TEER). Notably, we found that treatment with Zebularine activated the Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway by increasing the phosphorylation of adenosine monophosphate-activated protein kinase α (AMPKα). Blockage of AMPKα using its specific inhibitor compound C abolished the beneficial effects of Zebularine in mitigating endothelial hyper-permeability by reducing the expressions of ZO-1 and VE-cadherin. These findings suggest that the protective effects of Zebularine against OGD/R-induced endothelial hyper-permeability are mediated by the activation of AMPKα. In conclusion, our study sheds light on the potential application of Zebularine in the treatment of IS.

Neutrophil elastase (NE) functions as a host defense factor; however, excessive NE activity can potentially destroy human tissues. Although NE activity is positively correlated to gingival crevicular fluid and clinical attachment loss in periodontitis, the underlying mechanisms by which NE aggravates periodontitis remain elusive. In this study, we investigated how NE induces periodontitis severity and whether NE inhibitors were efficacious in periodontitis treatment. In a ligature-induced murine model of periodontitis, neutrophil recruitment, NE activity, and periodontal bone loss were increased in the periodontal tissue. Local administration of an NE inhibitor significantly decreased NE activity in periodontal tissue and attenuated periodontal bone loss. Furthermore, the transcription of proinflammatory cytokines in the gingiva, which was significantly upregulated in the model of periodontitis, was significantly downregulated by NE inhibitor injection. An in vitro study demonstrated that NE cleaved cell adhesion molecules, such as desmoglein 1, occludin, and E-cadherin, and induced exfoliation of the epithelial keratinous layer in three-dimensional human oral epithelial tissue models. The permeability of fluorescein-5-isothiocyanate-dextran or periodontal pathogen was significantly increased by NE treatment in the human gingival epithelial monolayer. These findings suggest that NE induces the disruption of the gingival epithelial barrier and bacterial invasion in periodontal tissues, aggravating periodontitis.

It has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection.