Search

The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.

Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (AC ext ) can be used in treating AD-related dermatologic symptoms. AC ext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon AC ext treatment. In a mouse model of AD, AC ext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD.

Introduction: Astrocytes are glial-type cells that protect neurons from toxic insults and support neuronal functions and metabolism in a healthy brain. Leveraging these physiological functions, transplantation of astrocytes or their derivatives has emerged as a potential therapeutic approach for neurodegenerative disorders., Methods: To substantiate the clinical application of astrocyte-based therapy, we aimed to prepare human astrocytes with potent therapeutic capacities from human pluripotent stem cells (hPSCs). To that end, we used ventral midbrain patterning during the differentiation of hPSCs into astrocytes, based on the roles of midbrain-specific factors in potentiating glial neurotrophic/anti-inflammatory activity. To assess the therapeutic effects of human midbrain-type astrocytes, we transplanted them into mouse models of Parkinson's disease (PD) and Alzheimer's disease (AD)., Results: Through a comprehensive series of in-vitro and in-vivo experiments, we were able to establish that the midbrain-type astrocytes exhibited the abilities to effectively combat oxidative stress, counter excitotoxic glutamate, and manage pathological protein aggregates. Our strategy for preparing midbrain-type astrocytes yielded promising results, demonstrating the strong therapeutic potential of these cells in various neurotoxic contexts. Particularly noteworthy is their efficacy in PD and AD-specific proteopathic conditions, in which the midbrain-type astrocytes outperformed forebrain-type astrocytes derived by the same organoid-based method., Conclusion: The enhanced functions of the midbrain-type astrocytes extended to their ability to release signaling molecules that inhibited neuronal deterioration and senescence while steering microglial cells away from a pro-inflammatory state. This success was evident in both in-vitro studies using human cells and in-vivo experiments conducted in mouse models of PD and AD. In the end, our human midbrain-type astrocytes demonstrated remarkable effectiveness in alleviating neurodegeneration, neuroinflammation, and the pathologies associated with the accumulation of α-synuclein and Amyloid β proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

The interplay between polyphenols, amines, and metals has broad implications for surface chemistry, biomaterials, energy storage, and environmental science. Traditionally, polyphenol-amine combinations have been recognized for their ability to form adhesive, material-independent thin layers that offer a diverse range of surface functionalities. Herein, we demonstrate that a coating of tannic acid (TA) and polyethyleneimine (PEI) provides an efficient platform for capturing and monitoring metal ions in water. A unique feature of our PEI/TA-coated microbeads is the ' Detec tion-Cap ture ' ( Detec-Ture ) mechanism. The galloyl groups in TA coordinate with Fe(III) ions (capture), initiating their oxidation to gallol-quinone. These oxidized groups subsequently react with PEI amines, leading to the formation of an Fe(II/III)-gallol-PEI network that produces a vivid purple color, thereby enabling visual detection. This mechanism couples metal capture directly with detection, distinguishing our approach from existing studies, which have either solely focused on metal removal or metal detection. The metal capturing capacity of our materials stands at 0.55 mg g -1 , comparable to that of established materials like alginate and wollastonite. The detection sensitivity reaches down to 0.5 ppm. Our findings introduce a novel approach to the utility of metal-polyphenol-amine networks, presenting a new class of materials suited for simultaneous metal ion detection and capture in environmental applications.

Crosslinked hydrophilic polymers with high water absorption rates are known as superabsorbent polymers (SAPs). Most commercial superabsorbent polymers are made with acrylic acid, which is difficult to biodegrade. So, in this investigation, carboxymethyl cellulose (CMC) was utilized as a significant component in the synthesis of polysaccharide-based SAPs. Citric acid (CA) and starch were chosen as crosslinking agents because they are more eco-friendly, non-toxic, and biodegradable than traditional crosslinking agents. FTIR analysis revealed that the superabsorbent polymer product contains a crosslinked structure of CMC and starch with side chains that carry carboxylate functional groups. Superabsorbent weight loss and grafting data were satisfactorily studied using the TGA approach. Under optimum circumstances, the SAP2 water absorbency capacity in distilled water was 287.37 g.g -1 and SAP1 absorbency capacity in a solution containing 0.9 wt% NaCl was 52.18 g.g -1 . Moreover, Schott's pseudo-second-order model was used to determine the kinetic swelling of the superabsorbent. The initial swelling rate of SAPs can be calculated using the Q ∞ data acquired in the following order: SAP2 > SAP1 > SAP3 > SAP4 in distilled water and SAP1 > SAP2 > SAP3 > SAP4 in 0.9 wt% NaCl solution, respectively. The findings suggested that a small amount of citric acid introduced into the SAPs matrix could enhance the swelling rate of SAPs. The results of the cytotoxicity tests show that the extraction liquid of composite hydrogel fibers is less cytotoxic than the positive control. As well, SAP underwent in silico docking investigations on the DNA Gyrase enzyme. As the ligand is a monomer of SAP, it was a long chain of carbohydrate molecules with alcoholic groups, esters groups, and keto groups forms a strong binding interaction with DNA gyrase., Competing Interests: Declaration of competing interest The submission of this work has no potential conflicts of interest. The study was original research that had not yet been published and was not being considered for full or partial publication elsewhere. International ethical standards served as the guide for the study presented in the publication., (Copyright © 2023 Elsevier B.V. All rights reserved.)

This study explores a polyphenolic coacervate, named VATA, formed by poly(vinyl alcohol) (PVA) and tannic acid (TA). Distinct from conventional studies that have focused on the bottom, dense phase of coacervates, this research emphasizes the top, dilute phase, low-viscous coacervate liquid termed liquid-VATA ( l -VATA). Due to TA's capability of intermolecular association as well as adhesiveness, phenomena not typically observed in the upper dilute phase of standard polyelectrolyte-based coacervates are revealed. At first glance, the dilute phase l -VATA coacervate resembles a water-like, low-viscous mixture solution of PVA, TA, and PVA/TA complexes. However, analysis shows that nearly all of the TA molecules associate with PVA chains, forming PVA/TA complexes. Furthermore, supraparticular association was observed between PVA/TA complex nanoparticles upon applying external shear force. A broad survey of shear rate and strain showed that the solution exhibited sequential shear-thickening, followed by shear-thinning behavior. The water-like, low viscosity of l -VATA unexpectedly reveals robust adhesiveness and thus able to lift an entire mouse using just a single human hair strand. Even in cases of failure, no interfacial failure was detected between mouse and human hair. In addition to enabling hair-to-hair bonding, our study also showcases the efficacy of l -VATA in facilitating hair-to-skin adhesion. The results illustrate how the lower viscosity of l -VATA can be exploited for a wide range of industrial and cosmetic applications, allowing the formulation of thin, uniform adhesive layers, something unachievable with the dense, viscous VATA glue. Thus, this study highlights the importance of investigating the top dilute phase of coacervates, shedding light on an area often underestimated compared to the bottom dense phase reported in prevalent coacervate studies., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Agrimonia pilosa Ledeb., an important medicinal herb in traditional East Asian medicine, is primarily used to treat abdominal pain, dysentery, and hemostasis. There are ten other reported species of Agrimonia plants, including Agrimonia coreana Nakai-a naturally growing species in South Korea-and Agrimonia eupatoria Linn. Although recent studies have isolated numerous active constituents and investigated their effects, the medicinal utility of this herb is not yet fully explored. Through patch-clamp recording, a previous study reported that Agrimonia plant extracts inhibit the function of Ca 2+ release-activated Ca 2+ channels (CRACs). Herein, we aimed to identify and isolate the main compounds in A. coreana responsible for CRAC inhibition while assessing the anti-inflammatory effects mediated by this inhibition. We demonstrated for the first time that alphitolic acid isolated from A. coreana has a dose-dependent inhibitory effect on CRAC activity and, thus, an inhibitory effect on intracellular calcium increase. Furthermore, analysis of human CD4 + T cell proliferation via the carboxyfluorescein diacetate succinimidyl ester method revealed that alphitolic acid inhibited T cell proliferation in a concentration-dependent manner. Our findings provide a theoretical basis for the potential therapeutic use of alphitolic acid in the treatment of inflammatory diseases.

Gene therapy and rebalancing therapy have emerged as promising approaches for treating hemophilia A, but there are limitations, such as temporary efficacy due to individual differences. Genome editing for hemophilia has shown long-term therapeutic potential in preclinical trials. However, a cautious approach is necessary because genome editing is irreversible. Therefore, we attempted to induce low-level human factor 8 (hF8) gene knockin (KI) using 244- cis lipid nanoparticles and low-dose adeno-associated virus to minimize side effects and achieve a therapeutic threshold in hemophilia A mice. We selected the serpin family C member 1, SerpinC1 , locus as a target to enable a combined rebalancing strategy with h F8 KI to augment efficacy. This strategy improved blood coagulation activity and reduced hemophilic complications without adverse effects. Furthermore, hemophilic mice with genome editing exhibit enhanced survival for 40 weeks. Here, we demonstrate an effective, safe, and sustainable treatment for hemophilia A. This study provides valuable information to establish safe and long-term genome-editing-mediated treatment strategies for treating hemophilia and other protein-deficient genetic diseases., Competing Interests: Hyukjin Lee received a research grant from ST Pharm, and ST Pharm also has a license for certain intellectual property invented at Ewha Womans University., (© 2023 The Authors.)

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Recently, miraculous therapy approaches involving adeno-associated virus (AAV) for incurable diseases such as spinal muscular atrophy and inherited retinal dysfunction have been introduced. Nonreplicative, nonpathogenic, low rates of chromosome insertional properties and the existence of neutralizing antibodies are main safety reasons why the FDA approved its use in gene delivery. To date, AAV production always results in a mixture of nontherapeutic (empty) and therapeutic (DNA-loaded) full capsids (10-98%). Such existence of empty viral particles inevitably increases viral doses to human. Thus, the rapid monitoring of empty capsids and reducing the empty-to-full ratio are critical in AAV science. However, transmission electron microscopy (TEM) is the primary tool for distinguishing between empty and full capsids, which creates a research bottleneck because of instrument accessibility and technical difficulty. Herein, we demonstrate that atomic force microscopy (AFM) can be an alternative tool to TEM. The simple, noncontact-mode imaging of AAV particles allows the distinct height difference between full capsids (∼22 nm) and empty capsids (∼16 nm). The sphere-to-ellipsoidal morphological distortion observed for empty AAV particles clearly distinguishes them from full AAV particles. Our study indicates that AFM imaging can be an extremely useful, quality-control tool in AAV particle monitoring, which is beneficial for the future development of AAV-based gene therapy.

In this study, we aimed to develop and validate a pretreatment method for separating and analyzing the small amounts of biomarkers contained in topical cream formulations. Analyzing semisolid formulations that contain low concentrations of active ingredients is difficult. Cream formulations containing an aqueous ethanol extract of 0.1% Agrimonia pilosa is an example. Approximately 0.0013% of apigenin-7- O -glucuronide(A7OG) was contained as a biomarker in the cream. To determine the A7OG content present in the cream formulation, liquid-liquid extraction using dichlormethane was applied. In addition, the volume of the distribution liquid was measured using the peak ratios of the indicator component, A7OG, and an internal standard, baicalin. Subsequently, the A7OG content in the cream formulation was calculated. Using this time-saving method, A7OG can be simply analyzed without additional pretreatment steps, such as evaporation and reconstitution. Moreover, the validation results confirmed that this analytical method met all of the criteria. Consequently, A7OG was successfully isolated from the cream, analyzed, and quantified using the developed method.

Radiation techniques are used to modify the physical, chemical and biological properties of polymers. This induces crosslinking and degradation reactions of polymers by utilizing radicals generated through ionizing radiation. However, oxidation products (such as carbonyl) can be formed because oxidation occurs by chain scission in the presence of oxygen. Herein, we demonstrate the gamma-ray irradiation-induced oxidation with and without fluorine using polyethylene, polyvinylidene fluoride and polytetrafluoroethylene under the same conditions. In this study, changes in element-content and chemical-bond structures were analyzed before and after gamma-ray irradiation under air atmosphere. As a result, polytetrafluo-roethylene showed less oxidation and excellent thermal properties after the absorbed dose of 500 kGy. This can be attributed to the generation of stable perfluoroalkylperoxy radicals after gamma ray irradiation in the PTFE structure containing only CF 2 groups, thereby hindering the oxidation reaction.

Ethnopharmacological Relevance: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive., Aims of the Study: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively., Materials and Methods: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR., Results: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 μM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 μM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR., Conclusion: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this study, biodegradable poly(L-lactide-co-ε-caprolactone) (PLCL) and poly(L-co-d,l lactide) (PLDLA) were evaluated using Geant4 (G4EmStandardPhysics_option4) for damage simulation, in order to predict the safety of these biodegradable polymers against gamma ray sterilization. In the PLCL damage model, both chain scission and crosslinking reactions appear to occur at a radiation dose in the range 0-200 kGy, but the chain cleavage reaction is expected to be relatively dominant at high irradiation doses above 500 kGy. On the other hand, the PLDLA damage model predicted that the chain cleavage reaction would prevail at the total irradiation dose (25-500 kGy). To verify the simulation results, the physicochemical changes in the irradiated PLCL and PLDLA films were characterized by GPC (gel permeation chromatography), ATR-FTIR (attenuated total reflection Fourier transform infrared), and DSC (difference scanning calorimetry) analyses. The Geant4 simulation curve for the radiation-induced damage to the molecular weight was consistent with the experimentally obtained results. These results imply that the pre-simulation study can be useful for predicting the optimal irradiation dose and ensuring material safety, particularly for implanted biodegradable materials in radiation processing.

Concerns regarding sustainability have prompted the search of value in the by-products of food manufacturing. Such is the case of the cooking water (CW) of chickpeas, which has shown its potential as a vegan egg white replacement. This study aimed to characterize and compare the CW from three novel legumes (black soybeans, BSB; yellow soybeans, YSB; and small black beans, SBB) obtained from the processing of Korean soybean foods, and the widely used CW from chickpeas (CH), with regard to total polyphenol, total carbohydrate, and protein contents, and further compare their foaming and emulsifying abilities and stabilities. Compositional analysis revealed that all the studied legumes possessed higher values than CH for all parameters. Furthermore, the CW from these legumes exhibited enhanced functional properties, particularly foaming capacity and stability. Taken together, our results suggest that the CW from BSB, YSB, and SBB, sourced from the manufacturing of legume food products, has the potential of being revalorized as a plant-based functional ingredient for vegan product development.

Cancer is a dynamic disease involving constant changes. With these changes, cancer cells become heterogeneous, resulting in varying sensitivity to chemotherapy. The heterogeneity of cancer cells plays a key role in chemotherapy resistance and cancer recurrence. Therefore, for effective treatment, cancer cells need to be analyzed at the single-cell level by monitoring various proteins and investigating their heterogeneity. We propose a microfluidic chip for a single-cell proteomics assay that is capable of analyzing complex cellular signaling systems to reveal the heterogeneity of cancer cells. The single-cell assay chip comprises (i) microchambers ( n = 1376) for manipulating single cancer cells, (ii) micropumps for rapid single-cell lysis, and (iii) barcode immunosensors for detecting nine different secretory and intracellular proteins to reveal the correlation among cancer-related proteins. Using this chip, the single-cell proteomics of a lung cancer cell line, which may be easily masked in bulk analysis, were evaluated. By comparing changes in the level of protein secretion and heterogeneity in response to combinations of four anti-cancer drugs, this study suggests a new method for selecting the best combination of anti-cancer drugs. Subsequent preclinical and clinical trials should enable this platform to become applicable for patient-customized therapies.

Previous studies have investigated the inhibitory effect of BMI-1026 on cyclin-dependent kinase 1 in vitro. However, the molecular mechanisms by which BMI-1026 treatment leads to cancer cell death remain unclear. This study was conducted to investigate the anticancer mechanisms of BMI-1026 on human renal carcinoma Caki cells. BMI-1026 induced apoptosis in association with the cleavage of poly(ADP-ribose) polymerase and pro-caspase-3 and the release of apoptosis-inducing factor and cytochrome c from mitochondria in Caki cells. BMI-1026-induced apoptosis was inhibited by the pan-caspase inhibitor z-VAD-fmk. Furthermore, BMI-1026 downregulated Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP) at the transcriptional level and Mcl-1 (L) and cellular FADD-like IL-1β-converting enzyme inhibitory protein (c-FLIP (L)) at the post-transcriptional level. Interestingly, Mcl-1 (L) and c-FLIP (L), but not Bcl-2 or XIAP, played important roles in BMI-1026-induced Caki cell apoptosis. Although the constitutively active form of Akt did not attenuate BMI-1026-induced apoptosis, blockade of the PI3K/Akt pathway using a subcytotoxic concentration of the PI3K/Akt inhibitor LY294002 enhanced Caki cell apoptosis induced by BMI-1026. Electrophysiological safety was confirmed by determining the cardiotoxicity of BMI-1026 via left ventricular pressure analysis. These results suggest that BMI-1026 is a potent multitarget anticancer agent with electrophysiological safety and should be further investigated.

Background: As one of the main components of mangosteen ( Garcinia mangostana ), a tropical fruit, α -mangostin has been reported to have numerous pharmacological benefits such as anti-cancer, anti-inflammatory, and anti-allergic effects through various mechanisms of action. The effects of α -mangostin on intracellular signaling proteins is well studied, but the effects of α -mangostin on ion channels and its physiological effects in immune cells are unknown. Generation of intracellular calcium signaling is a fundamental step for T cell receptor stimulation. This signaling is mediated not only by the ORAI1 calcium channel, but also by potassium ion channels, which provide the electrical driving forces for generating sufficient calcium ion influx. This study investigated whether α -mangosteen suppress T cell stimulation by inhibiting ORAI1 and two kinds of potassium channels (K v 1.3 and K Ca 3.1), which are normally expressed in human T cells., Methods: This study analyzed the inhibitory effect of α -mangostin on immune cell activity via inhibition of calcium and potassium ion channels expressed in immune cells., Results: α -mangostin inhibited ORAI1 in a concentration-dependent manner, and the IC 50 value was 1.27 ± 1.144 µM. K v 1.3 was suppressed by 41.38 ± 6.191% at 3 µM, and K Ca 3.1 was suppressed by 51.16 ± 5.385% at 3 µM. To measure the inhibition of cytokine secretion by immune cells, Jurkat T cells were stimulated to induce IL-2 secretion, and α -mangostin was found to inhibit it. This study demonstrated the anti-inflammatory effect of α -mangostin, the main component of mangosteen, through the regulation of calcium signals., Competing Interests: The authors declare there are no competing interests., (©2021 Kim et al.)

Protein and peptide therapeutics tend to have a short blood circulation time mainly caused by rapid clearance in kidney, leading to a low therapeutic efficacy. Here, we demonstrate that the antitumor activity of a small-sized protein binder can be significantly enhanced by prolonged blood half-life through site-specific lipidation. An unnatural amino acid was genetically incorporated into a specific site with the highest accessibility in a human interleukin-6 (IL-6)-targeting protein binder with a size of 30.8 kDa, followed by conjugation with palmitic acid using cooper-free click chemistry. The resulting protein binder was shown to have a binding capacity for serum albumin, maintaining a comparable binding affinity for human IL-6 to the native protein binder. The terminal half-life of the lipidated protein binder was estimated to be 10.7 h, whereas the native one had a half-life of 20 min, resulting in a significantly enhanced tumor suppression effect. The present approach can be generally applied to small-sized therapeutic proteins for the elongation of circulation time and increase of bioavailability in blood, consequently enhancing their therapeutic efficacy., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Gardenia jasminoides ( GJ ) is a widely used herbal medicine with antiinflammatory properties, but its effects on the ORAI1 channel, which is important in generating intracellular calcium signaling for T cell activation, remain unknown. In this study, we investigated whether 70% ethanolic GJ extract ( GJ EtOH ) and its subsequent fractions inhibit ORAI1 and determined which constituents contributed to this effect. Whole-cell patch clamp analysis revealed that GJ EtOH (64.7% ± 3.83% inhibition at 0.1 mg/ml) and all its fractions showed inhibitory effects on the ORAI1 channel. Among the GJ fractions, the hexane fraction ( GJ HEX , 66.8% ± 9.95% at 0.1 mg/ml) had the most potent inhibitory effects in hORAI1-hSTIM1 co-transfected HEK293T cells. Chemical constituent analysis revealed that the strong ORAI1 inhibitory effect of GJ HEX was due to linoleic acid, and in other fractions, we found that genipin inhibited ORAI1. Genipin significantly inhibited I ORAI1 and interleukin-2 production in CD3/ CD28-stimulated Jurkat T lymphocytes by 35.9% ± 3.02% and 54.7% ± 1.32% at 30 μM, respectively. Furthermore, the same genipin concentration inhibited the proliferation of human primary CD4+ T lymphocytes stimulated with CD3/CD28 antibodies by 54.9% ± 8.22%, as evaluated by carboxyfluorescein succinimidyl ester assay. Our findings suggest that genipin may be one of the active components of GJ responsible for T cell suppression, which is partially mediated by activation of the ORAI1 channel. This study helps us understand the mechanisms of GJ in the treatment of inflammatory diseases.

Rhinorrhea in allergic rhinitis (AR) is characterized by the secretion of electrolytes in the nasal discharge. The secretion of Cl - and HCO 3 - is mainly regulated by cystic fibrosis transmembrane conductance regulator (CFTR) or via the calciumactivated Cl - channel anoctamin-1 (ANO1) in nasal gland serous cells. Interleukin-4 (IL-4), which is crucial in the development of allergic inflammation, increases the expression and activity of ANO1 by stimulating histamine receptors. In this study, we investigated ANO1 as a potential therapeutic target for rhinorrhea in AR using an ANO1 inhibitor derived from a natural herb. Ethanolic extracts (30%) of Spirodela polyrhiza (SP EtOH ) and its five major flavonoids constituents were prepared. To elucidate whether the activity of human ANO1 (hANO1) was modulated by SP EtOH and its chemical constituents, a patch clamp experiment was performed in hANO1-HEK293T cells. Luteolin, one of the major chemical constituents in SP EtOH , significantly inhibited hANO1 activity in hANO1-HEK293T cells. Further, SP EtOH and luteolin specifically inhibited the calcium-activated chloride current, but not CFTR current in human airway epithelial Calu-3 cells. Calu-3 cells were cultured to confluency on transwell inserts in the presence of IL-4 to measure the electrolyte transport by Ussing chamber. Luteolin also significantly inhibited the ATP-induced increase in electrolyte transport, which was increased in IL-4 sensitized Calu-3 cells. Our findings indicate that SP EtOH- and luteolin may be suitable candidates for the prevention and treatment of allergic rhinitis. SPEtOH- and luteolin-mediated ANO1 regulation provides a basis for the development of novel approaches for the treatment of allergic rhinitis-induced rhinorrhea.

The airway is the major entry route of pathogens due to continuous gas exchange with the environment. In particular, the nasal epithelial layer is the common site of airborne mucotropic virus infections. The inflammatory response to such infections must be tightly controlled due to its non-specific nature. Unrestrained inflammation breaks down the physiological mucosal defense system and leads to secondary bacterial or fungal infections. Chronic rhinosinusitis (CRS) is a prevalent inflammatory disease that compromises quality of life. In spite of its importance in the initiation of inflammation, the role of interferon signaling in nasal airway epithelial cells is largely unknown. We analyzed the expression of interferon signaling genes using clinical lavage specimens and nasal airway epithelial cells collected from CRS patients and controls. Basal expression of IFNAs, IKBKE, STAT1, and some CXC chemokines was elevated in samples from CRS patients. In subsequent in vitro studies, we found IKKε to be the key molecule and augmented CXCL10 secretion. Based on our findings and review of the literature, we hypothesized that high levels of IKKε might induce intractable inflammation via CXCL10. We tested the hypothesis in an animal model and found not only that IKKε induced severe eosinophilic inflammation with CXCL10 over-production, but also that inhibition of IKKε resolved the inflammation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Intracellular calcium signaling is crucial for type 2 helper T cell and mast cell activation, which is essential for allergic inflammation. It is initiated by antigen-mediated receptor stimulation that triggers store-operated calcium entry (SOCE) via ORAI1 calcium channel. Flos Magnoliae (FM) is widely used to treat allergic diseases such as allergic rhinitis and asthma. Although many studies have reported that FM regulates intracellular calcium signaling, research on the exact type of calcium channel modulated by FM is scarce. Therefore, we hypothesized that the anti-allergic effects of FM might result from ORAI1 inhibition in T cells. We investigated whether a 70% ethanolic extract of FM (FM EtOH ) and its constituents inhibit ORAI1 channel activity and subsequent T cell activation. We performed conventional whole-cell patch clamp studies in hSTIM1 and hORAI1-overexpressing HEK293T cells (HEK ORAI1 ) . Intracellular calcium concentration was determined using Fura-2 dye and cytokine production measurement in Jurkat T lymphocytes. FM EtOH (0.03 mg/mL) and its fractions, especially hexane fraction (FM Hex , 0.01 mg/mL), significantly inhibited SOCE and IL-2 cytokine production in Jurkat T lymphocytes. GC/MS analysis showed linoleic acid (LA) as the major component of FM Hex . FM Hex at 0.01 mg/mL (equivalent to 10 μ M LA) inhibited not only SOCE but also IL-2 production, as well as CD3/CD28 receptor co-stimulation induced calcium signaling in Jurkat T lymphocytes. FM EtOH and LA suppressed CD4 + T lymphocyte activation, at least in part, by inhibiting I SOCE . Thus, I SOCE inhibition may be a potential strategy to inhibit immune responses in inflammation.

Myoblast fusion depends on mitochondrial integrity and intracellular Ca 2+ signaling regulated by various ion channels. In this study, we investigated the ionic currents associated with [Ca 2+ ] i regulation in normal and mitochondrial DNA-depleted (ρ0) L6 myoblasts. The ρ0 myoblasts showed impaired myotube formation. The inwardly rectifying K + current (I Kir ) was largely decreased with reduced expression of KIR2.1, whereas the voltage-operated Ca 2+ channel and Ca 2+ -activated K + channel currents were intact. Sustained inhibition of mitochondrial electron transport by antimycin A treatment (24 h) also decreased the I Kir . The ρ0 myoblasts showed depolarized resting membrane potential and higher basal [Ca 2+ ] i . Our results demonstrated the specific downregulation of I Kir by dysfunctional mitochondria. The resultant depolarization and altered Ca 2+ signaling might be associated with impaired myoblast fusion in ρ0 myoblasts., Competing Interests: CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

In this report, the novel and site-specific radioiodination of biomolecules by using aryl diamine and alkyl aldehyde condensation reaction in the presence of a Cu 2+ catalyst under ambient conditions was reported. 125 I-labeled alkyl aldehyde was synthesized using a tin precursor with a high radiochemical yield (72 ± 6%, n = 5) and radiochemical purity (>99%). The utility of the radioiodinated precursor was demonstrated through aryl diamine-installed c[RGDfK(C)] peptide and human serum albumin (HSA). Radioiodinated c[RGDfK(C)] peptide and HSA protein were synthesized with high radiochemical yields and purity. 125 I-HSA protein showed excellent in vivo stability and negligible thyroid uptake as compared with directly radioiodinated HSA by using the tyrosine group. Excellent reaction kinetics and the in vitro and in vivo stabilities of 125 I-labeled alkyl aldehyde have suggested the usefulness of the strategy for the radioiodination of bioactive molecules., Competing Interests: The authors declare no competing financial interest.

Intracellular calcium signaling cascades are integral to early and late allergic responses involving mast cell degranulation and type 2 helper T cell activation, respectively. Both the responses are accompanied by the movement of calcium through the calcium release-activated calcium (CRAC) channel, encoded by the ORAI1 gene. Spirodela polyrhiza (L.) Schleid (SP) has anti-inflammatory and anti-allergic effects, but its effect on calcium signaling has not been reported. This study investigated whether a 30% ethanolic SP extract (SP EtOH ) and its constituents can reduce CRAC currents ([Formula: see text]), and thus inhibit mast cell degranulation and T cell activation. In Jurkat T lymphocytes, we found that 3[Formula: see text]mg/mL SP EtOH inhibited the [Formula: see text] by [Formula: see text]%, whereas one of its constituents vitexin (100[Formula: see text][Formula: see text]M) inhibited the [Formula: see text] by [Formula: see text]%. Furthermore, in the RBL-2H3 mast cell, the [Formula: see text] was inhibited by 3[Formula: see text]mg/mL SP EtOH ([Formula: see text]%) and 100[Formula: see text][Formula: see text]M vitexin ([Formula: see text]%). Investigation of human primary T cell proliferation induced by co-stimulation with antibodies to cluster of differentiation 3 and 28, and of RBL-2H3 mast cell degranulation following IgE-antigen complex stimulation revealed that 100[Formula: see text][Formula: see text]M vitexin inhibited both T-cell proliferation (by [Formula: see text]%) and mast cell degranulation (by [Formula: see text]%). These effects were concentration-dependent, and no cytotoxicity was observed. Our findings suggest that vitexin is a promising candidate compound for the development of therapeutic agents to prevent and treat allergic diseases.

Gamma irradiation is able to affect various structural modification and an increase of the biological properties of biomaterials. This study was conducted to investigate the anti-allergenic effect of γ-irradiated black ginseng extract (BGE) using in vitro and in vivo experiments. IgEantigen complex-induced degranulation was measured in RBL-2H3 mast cells. In addition, an anti-atopic dermatitis (AD) test was carried out by spreading γ-irradiated BGE on the dorsal skin of 2,4-dinitrochlorobenzene (DNCB)-induced BALB/c mice. The content of arginylfructose (AF) of gamma-irradiated BGE was higher than that of BGE. In RBL-2H3 mast cells, γ-irradiated BGE treatments significantly reduced the IgE-antigen complex-induced release of β-hexosaminidase, histamine, intracellular ROS, and Ca2+ influx. A western blot analysis showed that γ-irradiated BGE had an inhibitory activity on the FcεRI-mediated signaling in mast cells. In the DNCB-induced AD model, γ-irradiated BGE significantly alleviated the ADlike skin symptoms and clinical signs. The suppression of AD by γ-irradiated BGE was accompanied by a decrease in the serum level of IgE and IL-4, as well as the number of leukocyte. Gamma-irradiated BGE also suppressed IL-4 and increased IFN-γ in splenocytes. Our data suggests that γ-irradiated BGE may be effective therapeutic agents for the treatment of AD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Flos Magnoliae (FM, Chinese name: Xin-yi) is an oriental medicinal herb commonly used for symptomatic relief from allergic rhinitis, sinusitis, and headache, including in traditional Chinese and Korean medicine formulations. FM inhibits histamine release from mast cells and cytokine secretion from T cells. However, the mechanism of action of FM on the anoctamin-1 (ANO1) ion channel, which is responsible for nasal hypersecretion in allergic rhinitis, has not been elucidated. Therefore, in this study, we investigated the effect of a 30% ethanolic extract of FM (FM EtOH ) and its chemical constituents on ANO1 activity. We used high-performance liquid chromatography analysis to identify five major chemical constituents of FM EtOH : vanillic acid, tiliroside, eudesmin, magnolin, and fargesin. Using a conventional whole-cell patch clamp method, we found that FM EtOH (30, 100, and 300[Formula: see text][Formula: see text]g/mL) and its chemical constituent tiliroside inhibited ANO1 activity in ANO1-overexpressing HEK293T cells. In addition, we found that the treatment of the airway epithelial cell line Calu-3 with interleukin 4 significantly increased Ca[Formula: see text] activated Cl[Formula: see text] current (I CaCC ), but not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride current (I CFTR ). FM EtOH and tiliroside specifically inhibited I CaCC . Thus, in this study, we identified a novel mechanism underlying the alleviation of allergic rhinitis by FM EtOH . Our results indicate that FM EtOH and its chemical constituent tiliroside are promising and potent agents for the prevention and treatment of allergic rhinitis.

Antibody-mimetic proteins are intensively being developed for biomedical applications including tumor imaging and therapy. Among them, repebody is a new class of protein that consists of highly diverse leucine-rich repeat (LRR) modules. Although all possible biomedical applications with repebody are ongoing, it's in vivo biodistribution and excretion pathway has not yet been explored. In this study, hexahistidine (His 6 )-tag bearing repebody (rEgH9) was labeled with [ 99m Tc]-tricarbonyl, and biodistribution was performed following intravenous (I.V.) or intraperitoneal (I.P.) injection. Repebody protein was radiolabeled with high radiolabeling efficiency (>90%) and radiolabeled compound was more than 99% pure after purification. Biodistribution data indicates radiotracer has a rapid clearance from blood and excreted through the kidneys for intravenous (I.V.) injection, but comparatively slow clearance for an intraperitoneal (I.P.) injection. SPECT-CT images were found to be in agreement with biodistribution data, high activity was found inside kidneys. The observed result for rapid blood clearance and renal excretion of repebody (rEgH9) provide useful information for the further development of therapeutic strategy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The integration of a targeted delivery with a tumour-selective agent has been considered an ideal platform for achieving high therapeutic efficacy and negligible side effects in cancer therapy. Here, we present engineered protein nanoparticles comprising a tumour-selective oncolytic protein and a targeting moiety as a new format for the targeted cancer therapy. Apoptin from chicken anaemia virus (CAV) was used as a tumour-selective apoptotic protein. An EGFR-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was employed to play a dual role as a tumour-targeting moiety and a fusion partner for producing apoptin nanoparticles in E. coli, respectively. The repebody was genetically fused to apoptin, and the resulting fusion protein was shown to self-assemble into supramolecular repebody-apoptin nanoparticles with high homogeneity and stability as a soluble form when expressed in E. coli. The repebody-apoptin nanoparticles showed a remarkable anti-tumour activity with negligible side effects in xenograft mice through a cooperative action of the two protein components with distinct functional roles. The repebody-apoptin nanoparticles can be developed as a systemic injectable and tumour-selective therapeutic protein for targeted cancer treatment., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Herein we report the radiosynthesis of a pyridine derived azide prosthetic group for iodine radioisotope labeling of dibenzocyclooctyne (DBCO) conjugated molecules. The radiolabeling of the stannylated precursor 2 was conducted using [(125)I]NaI and chloramine-T to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (72±8%, n=4) and radiochemical purity (>99%). Using (125)I-labeled azide ([(125)I]1), cyclic RGD peptide and near infrared fluorescent molecule were efficiently labeled with modest to good radiochemical yields. The biodistribution study and SPECT/CT images showed that [(125)I]1 underwent rapid renal clearance. These results clearly demonstrated that [(125)I]1 could be used as an useful radiotracer for in vivo pre-targeted imaging as well as efficient in vitro radiolabeling of DBCO containing molecules., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Herein we report an efficient method for iodine radioisotope labeling of cyclooctyne-containing molecules using copper-free click reaction. For this study, radioiodination using the tin precursor 2 was carried out at room temperature to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (85%) and excellent radiochemical purity. Dibenzocyclooctyne (DBCO) containing cRGD peptide and gold nanoparticle were labeled with [(125)I]1 at 37°C for 30min to give triazoles with good radiochemical yields (67-95%). We next carried out tissue biodistribution study of [(125)I]1 in normal ICR mice to investigate the level of organ accumulation which needs to be considered for pre-targeted in vivo imaging. Large amount of [(125)I]1 distributed rapidly in liver and kidney from bloodstream and underwent rapid renal and hepatobiliary clearance. Moreover [(125)I]1 was found to be highly stable (>92%) in mouse serum for 24h. Therefore [(125)I]1 could be used as a potentially useful radiotracer for pre-targeted imaging. Those results clearly indicated that the present radiolabeling method using copper free click reaction would be quite useful for both in vitro and in vivo labeling of DBCO group containing molecules with iodine radioisotopes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background/objectives: Doenjang, Korean traditional fermented soybean paste has been reported to have an anti-obesity effect. Because adipose tissue is considered a major source of inflammatory signals, we investigated the protective effects of Doenjang and steamed soybean on oxidative stress and inflammation in adipose tissue of diet-induced obese mice., Materials/methods: Male C57BL/6J mice were fed a low fat diet (LF), a high-fat diet (HF), or a high-fat containing Doenjang diet (DJ) or a high-fat containing steamed soybean diet (SS) for 11 weeks., Results: Mice fed a DJ diet showed significantly lower body and adipose tissue weights than those in the HF group. Although no significant differences in adipocyte size and number were observed among the HF diet-fed groups, consumption of Doenjang alleviated the incidence of crown-like structures in adipose tissue. Consistently, we observed significantly reduced mRNA levels of oxidative stress markers (heme oxygenase-1 and p40(phox)), pro-inflammatory adipokines (tumor necrosis factor alpha and macrophage chemoattractant protein-1), macrophage markers (CD68 and CD11c), and a fibrosis marker (transforming growth factor beta 1) by Doenjang consumption. Gene expression of anti-inflammatory adipokine, adiponectin was significantly induced in the DJ group and the SS group compared to the HF group. The anti-oxidative stress and anti-inflammatory effects observed in mice fed an SS diet were not as effective as those in mice fed a DJ diet, suggesting that the bioactive compounds produced during fermentation and aging may be involved in the observed health-beneficial effects of Doenjang., Conclusions: Doenjang alleviated oxidative stress and restored the dysregulated expression of adipokine genes caused by excess adiposity. Therefore, Doenjang may ameliorate systemic inflammation and oxidative stress in obesity via inhibition of inflammatory signals of adipose tissue.

Human rhinovirus (HRV)-A and -B is a common cause of upper respiratory tract infections. Recently, a third species, HRV-C, was categorized based on molecular typing studies. The results showed that the HRV-C genome had diverged from that of HRV-A and -B. Despite its late identification, increasing evidence suggests that HRV-C causes more severe pathogenic infections than HRV-A or -B; however, a large amount of epidemiological data is required to confirm this association in different clinical settings. Consequently, a simple and rapid method for identifying HRV-C is required to expedite such epidemiological studies. Here, two degenerate primer sets (HEV and HRVC) were designed based on bioinformatic analyses. The HEV set targeting the fifth IRES domain sequence within the 5'-UTR, which is highly conserved among enteroviruses, was designed to detect all enteroviruses, whereas the HRVC set, which targeted the VP2 coding region, was designed to detect HRV-C alone. Both primer sets were tested against a panel of standard enteroviruses and clinical lavage samples. HEV detected all enteroviruses tested whereas HRVC was specific for HRV-C. Although the primer design strategy was confirmed with a limited number of samples, extensive tests are required to be applied in clinical settings., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: Beta interferon (IFN-β) is involved in a wide range of cellular functions, and its secretion must be tightly controlled to inhibit viral spreading while minimizing cellular damage. Intracellular viral replication triggers cellular signaling cascades leading to the activation of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3) and IRF7 (IRF3/7), which synergistically bind to the IFN-β gene promoter to induce its expression. The mitochondrial antiviral signaling protein (MAVS) is a governing adaptor protein that mediates signaling communications between virus-sensing proteins and transcription factors. The activity of MAVS in the regulation of IFN-β secretion is affected by many cellular factors. However, the mechanism of MAVS-mediated IRF3/7 activation is not completely understood. Here, we identified a highly conserved DLAIS motif at amino acid positions 438 to 442 of MAVS that is indispensable for IRF3/7 activation. Specifically, the L439S and A440R mutations suppress IRF3/7 activation. Pulldown experiments using wild-type and mutant MAVS showed that mindbomb E3 ubiquitin protein ligase 2 (MIB2) binds to the DLAIS motif. Furthermore, the DLAIS motif was found to be critical for MIB2 binding, the ligation of K63-linked ubiquitin to TANK-binding kinase 1, and phosphorylation-mediated IRF3/7 activation. Our results suggest that MIB2 plays a putative role in MAVS-mediated interferon signaling., Importance: Mitochondrial antiviral signaling protein (MAVS) mediates signaling from virus-sensing proteins to transcription factors for the induction of beta interferon. However, the mechanism underlying activation of MAVS-mediated interferon regulatory factors 3 and 7 (IRF3/7) is not completely understood. We found a highly conserved DLAIS motif in MAVS that is indispensable for IRF3/7 activation through TANK-binding kinase 1 (TBK1) and identified it as the binding site for mindbomb E3 ubiquitin protein ligase 2 (MIB2). The mutations that targeted the DLAIS motif abolished MIB2 binding, attenuated the K63-linked ubiquitination of TBK1, and decreased the phosphorylation-mediated activation of IRF3/7., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

IFN regulatory factor 7 (IRF7) is a major regulator of type I (αβ) IFN secretion. A growing body of evidence shows that IRF7 is involved in a wide variety of pathologic conditions in addition to infections; however, the detailed mechanism of IRF7 transactivation remains elusive. Our current knowledge of IRF7 transactivation is based on studies of IRF3, another major regulator of IFN-β secretion. IRF3 and IRF7 are closely related homologs with high sequence similarity in their C-terminal regions, and both proteins are activated by phosphorylation of a specific serine cluster (SC). Nevertheless, the functional domains of the two proteins are arranged in an inverted manner. We generated a model structure of the IRF7 C-terminal region using homology modeling and used it to guide subsequent functional domain studies. The model structure led to the identification of a tripod-helix structure containing the SC. Based on the model and experimental data, we hypothesized that phosphorylation-mediated IRF7 transactivation is controlled by a tripod-helix structure. Inducible IκB kinase binds a tripod-helix structure. Serial phosphorylation of the SC by the kinase liberates C-terminal helix from an inhibitory hydrophobic pocket. A histone acetyltransferase P300 binds the liberated helix. The difference in the P300 binding sites explains why the domain arrangement of IRF7 is inverted relative to that of IRF3., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Hepatitis C virus (HCV) affects 2-3% of the global population. Approximately one-quarter of acute infections cause chronic hepatitis that leads to liver cirrhosis or hepatocellular carcinoma. The major obstacle of current research is the extremely narrow host tropism of HCV. A single HCV strain can replicate in the Huh7 human hepatoma cell line. Huh7 cells can be adapted under selective pressure in vitro to identify host factors that influence viral replication. Here, we extended this strategy to the in vivo condition and generated a series of cell lines by multiple rounds of adaptation in immunocompromised mice. Adaptation increased the cellular resistance to HCV infection. Microarray analyses revealed that the expression levels of several genes were associated with HCV resistance. Notably, up-regulation of the mRNA encoding cysteine-rich secretory protein 3 (CRISP3), a glycoprotein with unknown function that is secreted from multiple exocrine glands, was correlated with HCV resistance. The presence of CRISP3 in the culture medium limited HCV replication at the early phase of infection., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The hepatitis C virus (HCV) is a continuing threat to public health. The systemic administration of interferon alpha with ribavirin is the only currently approved treatment. However, this treatment is associated with a wide spectrum of systemic side effects that limits its effectiveness; thus, there is an urgent need for new treatment modalities. In this study, we describe a novel anti-HCV strategy employing a recombinant transcription factor that we have engineered in such a way that NS3/4a viral protease controls its intracellular localization, thereby restoring interferon secretion specifically in cells infected with HCV. Proof-of-concept experiments validated the strategy, showing that the recombinant transcription factor was triggered to stimulate interferon promoter by NS3/4A and remained inactive in cells without NS3/4a. Using an adenovirus-associated viral vector delivery system, we found that the recombinant transcription factor inhibited HCV replication effectively in vitro in cultured cells.

Coxsackievirus A24 (CVA24) is responsible for acute hemorrhagic conjunctivitis, a highly contagious eye disease for which no prevention or treatment is currently available. We thus assessed the antiviral potential of a small interfering RNA (siRNA) targeting CVA24. HeLa cells with or without four different siRNAs complementary to 2C or 3D genome region, were challenged with various CVA24s. Among several siRNAs, a siRNA targeting the highly conserved genome region called the cis-acting replication element (CVA24-CRE), was the only siRNA that decreased virus replication and subsequent cytotoxicity by both CVA24 variant and clinical isolates. Furthermore, CVA24-CRE had effective antiviral activity against CVA24 in primary human conjunctival cells. In addition, CVA24-CRE was highly resistant to the emergence of genetically altered escape mutants. Collectively, the present study provides evidence that CVA24-CRE targeting a conserved viral genome region had universal, prolonged anti-CVA24 activity. This siRNA may thus hold a potential to act clinically as a novel anti-CVA24 agent.

To enhance the transduction efficiency (TE) of a recombinant adeno-associated virus 2 (rAAV2) in human cancer cells, we examined the combined effects of various chemicals known to influence the rAAV2 transduction process at distinct steps. Among the agents tested were trichostatin A, a histone deacetylase inhibitor, MG-132, a proteosome inhibitor, the genotoxic agents hydroxyurea, aphidicolin, etoposide and camptothecin, and tyrphostin-1, an epidermal growth factor receptor inhibitor. During or after chemical treatment, various human cancer cells were infected with rAAV2 expressing beta-galactosidase. Treatment with hydroxy-urea or etoposide plus tyrphostin-1 dramatically increased the TE in most cell lines. The combination of hydroxyurea plus tyrphostin-1 increased TE to 37.7+/-7.9%, 32.8+/-2.0% and 31.8+/-2.1% in SK-Hep1, HeLa, and HCT116 cells, respectively. In addition, following rAAV2 infection and treatment with hydroxyurea plus tyrphostin-1, long-term transgene expression was observed for up to 6 months, with no damage to the transduced cells. These results indicate that rAAV2 transgene expression can be significantly enhanced by a combination of chemical agents with distinct activity and prolonged gene expression can occur following rAAV2 gene transfer into human cancer cells.

In this study, we report the amelioration of parkinsonian symptoms in rat Parkinson's disease (PD) models, as a result of the expression of glutamate decarboxylase (GAD) 65 with a modified cytomegalovirus (CMV) promoter. The transfer of the gene for gamma-amino butryic acid (GAD), the rate-limiting enzyme in gama-amino butrylic acid (GABA) production, has been investigated as a means to increase inhibitory synaptic activity. Electrophysiological evidence suggests that the transfer of the GAD65 gene to the subthalamic nucleus (STN) can change the excitatory output of this nucleus to inhibitory output. Our in vitro results also demonstrated higher GAD65 expression in cells transfected with the JDK promoter, as compared to cells transfected with the CMV promoter. Also, a rat PD model in which recombinant adeno-associated virus-2 (rAAV2)-JDK-GAD65 was delivered into the STN exhibited significant behavioral improvements, as compared to the saline-injected group. Interestingly, we observed that these behavioral improvements were more obvious in rat PD models in which rAAV2-JDK-GAD65 was injected into the STN than in rat PD models in which rAAV2-CMV-GAD65 was injected into the STN. Moreover, according to electrophysiological data, the rAAV2-JDK-GAD65-injected group exhibited more constant improvements in firing rates than did the rAAV2-CMV-GAD65-injected group. These data indicate that the JDK promoter, when coupled with GAD65 expression, is more effective with regard to parkinsonian symptoms than is the CMV promoter.

Recombinant adeno-associated virus type 2 (rAAV2) viral vector, a non-pathogenic human parvovirus, has recently emerged as a gene transfer vehicle for cancer gene therapy. To utilize rAAV2 properly and safely while carrying out preclinical and clinical studies, it is crucial to exactly titer the virus. We therefore compared biological infectious rAAV2 titers with physical titers of rAAV2 vectors encoding various transgenes with different sized viral genomes. Biological rAAV2 infectivity was assayed by measuring the number of virus particles able to transduce Hela cells using several detection methods, including X-gal staining and immunocytostaining. Physical titers of rAAV2 were determined using a commercially available rAAV2 particle-specific enzyme-linked immunosorbent assay. We found that total rAAV2 particle production was consistent within the limited size variations of the rAAV2 genome, regardless of the difference in transgenes. In contrast, the infectious titer of rAAV2 differed greatly, even for the same viruses, due to variation in the sensitivity of the relevant assays. Thus, the results suggest that both infectious virus titer and total virus particle should be precisely measured for rAAV2 vector utilized in each study.