Development and evaluation of RFID-integrated endoscopic clips for laparoscopic surgery marking.

BACKGROUND: As advancements in surgical instruments and techniques continue to evolve, minimally invasive surgery has become increasingly preferred as a means of reducing patient pain and recovery time. However, one major challenge in performing minimally invasive surgery for early gastrointestinal cancer is accurately identifying the location of the lesion. This is particularly difficult when the lesion is confined to the lumen of the intestine and cannot be visually confirmed from the outside during surgery. In such cases, surgeons must rely on CT or endoscopic imaging to locate the lesion. However, if the lesion is difficult to identify with these images or if the surgeon has less experience, it can be challenging to determine its precise location. This can result in an excessive resection margin, deviating from the goal of minimally invasive surgery. To address this challenge, researchers have been studying the development of a marker for identifying the lesion using a radio-frequency identification (RFID) system. One proposed method for clinical application of this detection system is to attach an RFID tag to an endoscopic hemostatic clip and fix it to the intended position, providing a stable marker for the inner wall of the organ. This approach has the potential to improve the accuracy and effectiveness of minimally invasive surgery for early gastrointestinal cancer. METHODS: In the development of a marker for identifying gastrointestinal lesions using a radio-frequency identification (RFID) system, the shape of the clip and suitable materials for attaching the RFID tag were determined through finite element method (FEM) analysis. A prototype of the clip was then fabricated and ex-vivo experiments were conducted using porcine intestine to evaluate the stability of the clip in relation to its position. To further evaluate the performance of the RFID-integrated clip in vivo, the clip was placed in the gastric wall of the stomach of anesthetized porcine using an endoscopic instrument. The clip was then detected using a RFID detector designed for laparoscopic approach. And later, the accuracy of detection was confirmed by incising the lesion. RESULTS: The design and fabrication of a clip with varying thicknesses using STS316 and STS304 stainless steel were accomplished using the results of finite element method analysis. The stability of the clip was evaluated through ex-vivo experiments, showing it to be a viable option. In-vivo experiments were performed on anesthetized porcine, in which the RFID-integrated clip was placed in the gastric wall and detected using a custom-made RFID detector. The resection margin, measured at about 30 mm from the detector position, was accomplished with low error. These findings indicate the feasibility and efficacy of using an RFID-integrated clip as a marker in minimally invasive surgery for the identification of gastrointestinal lesions. CONCLUSIONS: The study evaluated the feasibility of using stainless steel clips for lesion detection in endoscopic surgery using computer-aided engineering analysis and ex-vivo experimentation. Results showed that STS304 was suitable for use while STS316L was not. The ex-vivo experiments revealed that the clip holding force and tissue retention length varied depending on the location of attachment. In-vivo experiments confirmed the accuracy and usefulness of the RFID lesion detection system. However, challenges remain for its use in clinical field, such as ensuring the stability of the clip and the safe attachment of the RFID tag, which requires further research for commercialization.

Rhamnogalacturonan lyase 1 (RGL1), as a suppressor of E3 ubiquitin ligase Arabidopsis thaliana ring zinc finger 1 (AtRZF1), is involved in dehydration response to mediate proline synthesis and pectin rhamnogalacturonan-I composition.

Proline metabolism plays a crucial role in both environmental stress responses and plant growth. However, the specific mechanism by which proline contributes to abiotic stress processes remains to be elucidated. In this study, we utilized atrzf1 (Arabidopsis thaliana ring zinc finger 1) as a parental line for T-DNA tagging mutagenesis and identified a suppressor mutant of atrzf1, designated proline content alterative 31 (pca31). The pca31 mutant suppressed the insensitivity of atrzf1 to dehydration stress during early seedling growth. Using Thermal Asymmetric Interlaced-PCR, we found that the T-DNA of pca31 was inserted into the promoter region of the At2g22620 gene, which encodes the cell wall enzyme rhamnogalacturonan lyase 1 (RGL1). Enzymatic assays indicated that RGL1 exhibited rhamnogalacturonan lyase activity, influencing cell wall pectin composition. The decrease in RGL1 gene expression suppressed the transcriptomic perturbation of the atrzf1 mutant. Silencing of the RGL1 gene in atrzf1 resulted in a sensitive phenotype similar to pca31 under osmotic stress conditions. Treatment with mannitol, salt, hydrogen peroxide, and abscisic acid induced RGL1 expression. Furthermore, we uncovered that RGL1 plays a role in modulating root growth and vascular tissue development. Molecular, physiological, and genetic experiments revealed that the positive modulation of RGL1 during abiotic stress was linked to the AtRZF1 pathway. Taken together, these findings establish that pca31 acts as a suppressor of atrzf1 in abiotic stress responses through proline and cell wall metabolisms.

Exosome-Coated Prussian Blue Nanoparticles for Specific Targeting and Treatment of Glioblastoma.

Glioblastoma is one of the most aggressive and invasive types of brain cancer with a 5-year survival rate of 6.8%. With limited options, patients often have poor quality of life and are moved to palliative care after diagnosis. As a result, there is an extreme need for a novel theranostic method that allows for early diagnosis and noninvasive treatment as current peptide-based delivery standards may have off-target effects. Prussian Blue nanoparticles (PBNPs) have recently been investigated as photoacoustic imaging (PAI) and photothermal ablation agents. However, due to their inability to cross the blood-brain barrier (BBB), their use in glioblastoma treatment is limited. By utilizing a hybrid, biomimetic nanoparticle composed of a PBNP interior and a U-87 cancer cell-derived exosome coating (Exo:PB), we show tumor-specific targeting within the brain and selective thermal therapy potential due to the strong photoconversion abilities. Particle characterization was carried out and showed a complete coating around the PBNPs that contains exosome markers. In vitro cellular uptake patterns are similar to native U-87 exosomes and when exposed to an 808 nm laser, show localized cell death within the specified region. After intravenous injection of Exo:PB into subcutaneously implanted glioblastoma mice, they have shown effective targeting and eradication of tumor volume compared to PEG-coated PBNPs (PEG:PB). Through systemic administration of Exo:PB particles into orthotopic glioblastoma-bearing mice, the PBNP signal was detected in the brain tumor region through PAI. It was seen that Exo:PB had preferential tumor accumulation with less off-targeting compared to the RGD:PB control. Ex vivo analysis validated specific targeting with a direct overlay of Exo:PB with the tumor by both H&E staining and Ki67 labeling. Overall, we have developed a novel biomimetic material that can naturally cross the BBB and act as a theranostic agent for systemic targeting of glioblastoma tissue and photothermal therapeutic effect.

Arabidopsis thaliana ubiquitin-associated protein 2 (AtUAP2) functions as an E4 ubiquitin factor and negatively modulates dehydration stress response.

E4, a ubiquitin (Ub) chain assembly factor and post-translational modification protein, plays a key role in the regulation of multiple cellular functions in plants during biotic or abiotic stress. We have more recently reported that E4 factor AtUAP1 is a negative regulator of the osmotic stress response and enhances the multi-Ub chain assembly of E3 ligase Arabidopsis thaliana RING Zinc Finger 1 (AtRZF1). To further investigate the function of other E4 Ub factors in osmotic stress, we isolated AtUAP2, an AtUAP1 homolog, which interacted with AtRZF1, using pull-down assay and bimolecular fluorescence complementation analysis. AtUAP2, a Ub-associated motif-containing protein, interacts with oligo-Ub5, -Ub6, and -Ub7 chains. The yeast functional complementation experiment revealed that AtUAP2 functions as an E4 Ub factor. In addition, AtUAP2 is localized in the cytoplasm, different from AtUAP1. The activity of AtUAP2 was relatively strongly induced in the leaf tissue of AtUAP2 promoter-ß-glucuronidase transgenic plants by abscisic acid, dehydration, and oxidative stress. atuap2 RNAi lines were more insensitive to osmotic stress condition than wild-type during the early growth of seedlings, whereas the AtUAP2-overexpressing line exhibited relatively more sensitive responses. Analyses of molecular and physiological experiments showed that AtUAP2 could negatively mediate the osmotic stress-induced signaling. Genetic studies showed that AtRZF1 mutation could suppress the dehydration-induced sensitive phenotype of the AtUAP2-overexpressing line, suggesting that AtRZF1 acts genetically downstream of AtUAP2 during osmotic stress. Taken together, our findings show that the AtRZF1-AtUAP2 complex may play important roles in the ubiquitination pathway, which controls the osmotic stress response in Arabidopsis.

Imaging of Indocyanine Green-Human Serum Albumin (ICG-HSA) Complex in Secreted Protein Acidic and Rich in Cysteine (SPARC)-Expressing Glioblastoma.

Glioblastoma is the most common and fatal primary glioma and has a severe prognosis. It is a challenge for neurosurgeons to remove brain tumor tissues completely by resection. Meanwhile, fluorescence-guided surgery (FGS) is a technique used in glioma surgery to enhance the visualization of tumor edges to clarify the extent of tumor resection. Indocyanine green (ICG) is the only FDA-approved NIR fluorescent agent. It non-covalently binds to human serum albumin (HSA). Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein expressed in gliomas and binds to albumin, suggesting that it plays an important role in tumor uptake of the ICG-HSA complex. Here we demonstrate the binding properties of HSA or SPARC to ICG using surface plasmon resonance and saturation binding assay. According to in vitro and in vivo studies, the results showed that the uptake of ICG-HSA complex was higher in SPARC-expressing glioblastoma cell line and tumor region compared with the uptake of free ICG. Here, we visualized the SPARC-dependent uptake of ICG and ICG-HSA complex in U87MG. Our results demonstrated that the ICG-HSA complex is likely to be used as an efficient imaging agent targeting SPARC-expressing tumors, especially glioblastoma.

Isolation and Functional Characterization of Soybean BES1/BZR1 Homolog 3-Like 1 (GmBEH3L1) Associated with Dehydration Sensitivity and Brassinosteroid Signaling in Arabidopsis thaliana

Brassinosteroid (BR) is an important steroid hormone that regulates plant development, abscisic acid (ABA) signaling, and responses to abiotic stress. We previously demonstrated that BEH3 (BES1/BZR1 Homolog 3) of Arabidopsis thaliana regulates dehydration and ABA responses by mediating proline metabolism. Furthermore, BEH3 negatively regulates BR-mediated hypocotyl elongation in dark-grown seedlings. However, the roles of BEH3 ortholog genes in the osmotic stress response of plants have remained largely unknown. Here, GmBEH3L1 (Glycine max BEH3-Like 1), a soybean (G. max) ortholog of the BEH3 gene of A. thaliana, was isolated and functionally characterized. GmBEH3L1 is induced by ABA, dehydration, and drought conditions. The GmBEH3L1-overexpressing transgenic lines (GmBEH3L1-OE/beh3) with the beh3 mutant background have ABA- and dehydration-sensitive phenotypes during early seedling growth, implying that GmBEH3L1 is involved in both osmotic stress and ABA sensitivity as a negative regulator in A. thaliana. Consistent with these results, GmBEH3L1-OE/beh3 complemental lines exhibit decreased expression levels of ABA- or dehydration-inducible genes. Under darkness, GmBEH3L1-OE/beh3 complemental lines display a short hypocotyl length compared to the beh3 mutant, indicating that GmBEH3L1 is linked to BR signaling. Together, our data suggest that GmBEH3L1 participates negatively in ABA and dehydration responses through BR signaling.

Arginine-mediated gut microbiome remodeling promotes host pulmonary immune defense against nontuberculous mycobacterial infection.

Nontuberculous mycobacterial pulmonary diseases (NTM-PDs) are emerging as global health threats with issues of antibiotic resistance. Accumulating evidence suggests that the gut-lung axis may provide novel candidates for host-directed therapeutics against various infectious diseases. However, little is known about the gut-lung axis in the context of host protective immunity to identify new therapeutics for NTM-PDs. This study was performed to identify gut microbes and metabolites capable of conferring pulmonary immunity to NTM-PDs. Using metabolomics analysis of sera from NTM-PD patients and mouse models, we showed that the levels of l-arginine were decreased in sera from NTM-PD patients and NTM-infected mice. Oral administration of l-arginine significantly enhanced pulmonary antimicrobial activities with the expansion of IFN-γ-producing effector T cells and a shift to microbicidal (M1) macrophages in the lungs of NTM-PD model mice. Mice that received fecal microbiota transplants from l-arginine-treated mice showed increased protective host defense in the lungs against NTM-PD, whereas l-arginine-induced pulmonary host defense was attenuated in mice treated with antibiotics. Using 16S rRNA sequencing, we further showed that l-arginine administration resulted in enrichment of the gut microbiota composition with Bifidobacterium species. Notably, oral treatment with either Bifidobacterium pseudolongum or inosine enhanced antimicrobial pulmonary immune defense against NTM infection, even with multidrug-resistant clinical NTM strains. Our findings indicate that l-arginine-induced gut microbiota remodeling with enrichment of B. pseudolongum boosts pulmonary immune defense against NTM infection by driving the protective gut-lung axis in vivo.

Rufomycin Exhibits Dual Effects Against Mycobacterium abscessus Infection by Inducing Host Defense and Antimicrobial Activities.

Nontuberculous mycobacterial pulmonary infection is often aggravated due to antibiotic resistance issues. There is a need for development of new drugs inducing both host immune responses and antimicrobial activities. This study shows that the rufomycins 4/5/6/7 (Rufomycin 4-7), which targets ClpC1 as a subunit of caseinolytic protein complex ClpC1/ClpP1/ClpP2 of mycobacteria, exhibits a dual effect in host innate defense and in vivo antimicrobial activities against a rough morphotype of Mycobacterium abscessus (Mabs-R), a clinically severe morphotype that causes hyperinflammation. Rufomycin 4-7 treatment showed antimicrobial effects against Mabs pulmonary infection in vivo and in macrophages. In addition, Rufomycin 4-7 significantly decreased inflammation, but enhanced the autophagy/lysosomal genes through upregulation of the nuclear translocation of transcription factor EB (TFEB). Furthermore, Rufomycin 4-7 treatment effectively inhibited mitochondrial damage and oxidative stresses in macrophages during Mabs-R infection. Collectively, Rufomycin 4-7-mediated dual effects inducing both antimicrobial activities and host immune defense might confer an advantage to treatment against Mabs-R infection.

Evaluation of Near-infrared Fluorescence-conjugated Peptides for Visualization of Human Epidermal Receptor 2-overexpressed Gastric Cancer.

PURPOSE: A near-infrared (NIR) fluorescence imaging is a promising tool for cancer-specific image guided surgery. Human epidermal receptor 2 (HER2) is one of the candidate markers for gastric cancer. In this study, we aimed to synthesize HER2-specific NIR fluorescence probes and evaluate their applicability in cancer-specific image-guided surgeries using an animal model. MATERIALS AND METHODS: An NIR dye emitting light at 800 nm (IRDye800CW; Li-COR) was conjugated to trastuzumab and an HER2-specific affibody using a click mechanism. HER2 affinity was assessed using surface plasmon resonance. Gastric cancer cell lines (NCI-N87 and SNU-601) were subcutaneously implanted into female BALB/c nu (6-8 weeks old) mice. After intravenous injection of the probes, biodistribution and fluorescence signal intensity were measured using Lumina II (Perkin Elmer) and a laparoscopic NIR camera (InTheSmart). RESULTS: Trastuzumab-IRDye800CW exhibited high affinity for HER2 (KD=2.093(3) pM). Fluorescence signals in the liver and spleen were the highest at 24 hours post injection, while the signal in HER2-positive tumor cells increased until 72 hours, as assessed using the Lumina II system. The signal corresponding to the tumor was visually identified and clearly differentiated from the liver after 72 hours using a laparoscopic NIR camera. Affibody-IRDye800CW also exhibited high affinity for HER2 (KD=4.71 nM); however, the signal was not identified in the tumor, probably owing to rapid renal clearance. CONCLUSIONS: Trastuzumab-IRDye800CW may be used as a potential NIR probe that can be injected 2-3 days before surgery to obtain high HER2-specific signal and contrast. Affibody-based NIR probes may require modifications to enhance mobilization to the tumor site.

Arabidopsis thaliana Ubiquitin-Associated Protein 1 (AtUAP1) Interacts with redundant RING Zinc Finger 1 (AtRZF1) to Negatively Regulate Dehydration Response.

Ubiquitination, one of the most frequently occurring post-translational modifications, is essential for regulating diverse cellular processes in plants during abiotic stress. The E3 ubiquitin (Ub) ligase Arabidopsis thaliana really interesting new gene (RING) zinc finger 1 (AtRZF1) mutation is known to enhance drought tolerance in A. thaliana seedlings. To further investigate the function of AtRZF1 in osmotic stress, we isolated Ub-associated protein 1 (AtUAP1) which interacts with AtRZF1 using a yeast two-hybrid system. AtUAP1, a Ub-associated motif containing protein, increased the amount of Ub-conjugated AtRZF1. Moreover, AtUAP1 RNA interference lines were more tolerant to osmotic stress than wild type, whereas AtUAP1-overexpressing (OX) transgenic lines showed sensitive responses, including cotyledon greening, water loss, proline accumulation and changes in stress-related genes expression, indicating that AtUAP1 could negatively regulate dehydration-mediated signaling. In addition, AtUAP1-green fluorescent protein fusion protein was observed in the nuclei of root cells of transgenic seedlings. Genetic studies showed that the AtRZF1 mutation could rescue the sensitive phenotype of AtUAP1-OX lines in response to osmotic stress, suggesting that AtRZF1 was epistatic to AtUAP1 in dehydration signaling. Taken together, our findings describe a new component in the AtRZF1 ubiquitination pathway which controls the dehydration response in A. thaliana.

BES1/BZR1 Homolog 3 cooperates with E3 ligase AtRZF1 to regulate osmotic stress and brassinosteroid responses in Arabidopsis.

Proline (Pro) metabolism plays important roles in protein synthesis, redox balance, and abiotic stress response. However, it is not known if cross-talk occurs between proline and brassinosteroid (BR) signaling pathways. Here, an Arabidopsis intergenic enhancer double mutant, namely proline content alterative 41 (pca41), was generated by inserting a T-DNA tag in the Arabidopsis thaliana ring zinc finger 1 (atrzf1 ) mutant background. pca41 had a T-DNA inserted at the site of the gene encoding BES1/BZR1 Homolog 3 (BEH3). pca41 has a drought-insensitive phenotype that is stronger than atrzf1 under osmotic stress, including high Pro accumulation and decreased amounts of reactive oxygen species. Analysis of physiological, genetic, and molecular networks revealed that negative regulation of BEH3 during abiotic stress was linked to the BR signaling pathway. Our data also suggest that AtRZF1, an E3 ubiquitin ligase, might control osmotic stress, abscisic acid, and BR responses in a BEH3-dependent manner. Under darkness, pca41 displays a long hypocotyl phenotype, which is similar to atrzf1 and beh3, suggesting that BEH3 acts in the same pathway as AtRZF1. Overexpression of BEH3 results in an osmotic stress-sensitive phenotype, which is reversed by exogenous BR application. Taken together, our results indicate that AtRZF1 and BEH3 may play important roles in the osmotic stress response via ubiquitination and BR signaling.

Efficacy and Safety of Human Serum Albumin-Cisplatin Complex in U87MG Xenograft Mouse Models.

Cisplatin (cis-diamminedichloroplatinum (II), CDDP) is a chemotherapeutic drug widely used against many solid tumors. A pharmacokinetics study found that CDDP can bind to human serum albumin (HSA), which is the most abundant plasma protein in serum. HSA has the advantage of being a nanocarrier and can accumulate in tumors by passive targeting and active targeting mediated by the secreted protein acidic and rich in cysteine (SPARC). In this study, we investigated the possibility of using a CDDP-HSA complex (HSA-CDDP) as a SPARC-mediated therapeutic agent. To investigate the HSA-dependent therapeutic effect of HSA-CDDP, we used two types of U87MG glioma cells that express SPARC differently. HSA-CDDP was highly taken up in SPARC expressing cells and this uptake was enhanced with exogenous SPARC treatment in cells with low expression of SPARC. The cytotoxicity of HSA-CDDP was also higher in SPARC-expressing cells. In the tumor model, HSA-CDDP showed a similar tumor growth and survival rate to CDDP only in SPARC-expressing tumor models. The biosafety test indicated that HSA-CDDP was less nephrotoxic than CDDP, based on blood markers and histopathology examination. Our findings show that HSA-CDDP has the potential to be a novel therapeutic agent for SPARC-expressing tumors, enhancing the tumor targeting effect by HSA and reducing the nephrotoxicity of CDDP.

Conjugation of arginylglycylaspartic acid to human serum albumin decreases the tumor-targeting effect of albumin by hindering its secreted protein acidic and rich in cysteine-mediated accumulation in tumors.

Human serum albumin (HSA) accumulates in tumors by the enhanced permeability and retention (EPR) effect, which is a passive targeting effect in tumors. A recent study showed that secreted protein acidic and rich in cysteine (SPARC), an albumin-binding protein, mediates albumin accumulation in tumors. Arg-Gly-Asp (RGD) is a peptide targeting integrin αvß3, which is highly expressed during tumor angiogenesis. We investigated whether conjugation of RGD to HSA could synergistically enhance tumor targeting. Accumulation of cRGDyK-HSA in integrin αvß3-expressing SK-OV3 cells was observed by confocal microscopy. In SK-OV3 cells overexpressing the albumin binding protein SPARC, cellular uptake of HSA increased, but uptake of cRGDyK-HSA did not. cRGDyK-HSA showed decreased tumor accumulation compared with HSA by positron emission tomography (PET) scanning and biodistribution studies in an SK-OV3 xenograft mouse model. In SK-OV3 tumors, HSA accumulation colocalized with SPARC expression, while cRGDyK-HSA only accumulated in the outer region of the tumor, even though SPARC and integrin αvß3 were expressed within the tumor core. We speculate that cRGDyK conjugation to HSA changes the characteristics of HSA and hinders its tumor-targeting effect. Therefore, HSA should be modified to preserve its native characteristics and enhance the tumor-targeting effects of HSA conjugates.

The Peroxisome Proliferator-Activated Receptor α- Agonist Gemfibrozil Promotes Defense Against Mycobacterium abscessus Infections.

Peroxisome proliferator-activated receptor α (PPARα) shows promising potential to enhance host defenses against Mycobacterium tuberculosis infection. Herein we evaluated the protective effect of PPARα against nontuberculous mycobacterial (NTM) infections. Using a rapidly growing NTM species, Mycobacterium abscessus (Mabc), we found that the intracellular bacterial load and histopathological damage were increased in PPARα-null mice in vivo. In addition, PPARα deficiency led to excessive production of proinflammatory cytokines and chemokines after infection of the lung and macrophages. Notably, administration of gemfibrozil (GEM), a PPARα activator, significantly reduced the in vivo Mabc load and inflammatory response in mice. Transcription factor EB was required for the antimicrobial response against Mabc infection. Collectively, these results suggest that manipulation of PPARα activation has promising potential as a therapeutic strategy for NTM disease.

Secreted protein acidic and rich in cysteine mediates active targeting of human serum albumin in U87MG xenograft mouse models.

Human serum albumin (HSA) is the most abundant plasma protein. The main reason for using HSA as a versatile tool for drug delivery is based on its ability to accumulate in tumors. However, the mechanism of albumin accumulation in tumors is not yet clear. Many researchers using HSA as a drug-carrier have focused on the passive tumor targeting by enhanced permeability and retention (EPR) effect, while other investigators proposed that albumin binding proteins mediate albumin accumulation in tumors. We investigated whether HSA accumulation in tumors is mediated by the EPR effect or by secreted protein acidic and rich in cysteine (SPARC), which is known to be an albumin-binding protein. Methods: To investigate the role of SPARC on HSA accumulation in tumors, we compared HSA uptake in U87MG glioblastoma cells with different SPARC expression. U87MG cells generally express high levels of SPARC and were, therefore, used as SPARC-rich cells. SPARC-less U87MG (U87MG-shSPARC) cells were established by viral-shSPARC transduction. We detected cellular uptake of fluorescence-labeled HSA by confocal microscopy in U87MG and U87MG-shSPARC cells. To demonstrate the mechanism of HSA accumulation in tumors, we injected FNR648-labeled HSA and FITC-labeled dextran in U87MG and U87MG-shSPARC tumor-bearing mice and observed their micro-distribution in tumor tissues. Results: HSA was internalized in cells by binding with SPARC in vitro. HSA accumulation in U87MG glioma was associated with SPARC expression in vivo. FITC-dextran was distributed in U87MG tumors in the vicinity of blood vessels. The distribution of HSA, on the other hand, was observed in the regions remote from blood vessels of U87MG tumor tissues but not in U87MG-shSPARC tumor tissues. Conclusion: Our results demonstrate that the tumor-distribution of HSA is affected not only by the EPR-effect but also by SPARC expression. SPARC enhances HSA accumulation in U87MG glioma and mediates active targeting of HSA in tumors.

Acute and genetic toxicity of GS-E3D, a new pectin lyase-modified red ginseng extract.

Korean red ginseng and its extract have been used as traditional medicines and functional foods in countries worldwide. Pectin lyase-modified red ginseng extract (GS-E3D) was newly developed as a dietary supplement for obesity, diabetes-related renal dysfunction, etc. In this study, the safety of GS-E3D on acute toxicity and genotoxicity was evaluated. For acute study, Sprague-Dawley rats were administrated by oral gavage at a dose of 5000 mg/kg GS-E3D. To evaluate genotoxicity of GS-E3D, we conducted three-battery tests, which are Ames test using Escherichia coli (WP2uvrA pKM101) and Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), chromosomal aberration test -using Chinese hamster lung cells, and micronucleus test using ICR mice. In acute toxicity studies, there were no dead animals or abnormal necropsy findings in the control group and GS-E3D (5000 mg/kg) treated group. GS-E3D did not induce mutagenicity in the bacterial test, chromosomal aberrations in Chinese hamster lung cells and micronuclei in bone marrow cells of mice. Conclusively, the approximate lethal dose of GS-E3D was greater than 5000 mg/kg bw and GS-E3D has no genotoxic potential in the three-battery tests on genotoxicity.

A basic helix-loop-helix 104 (bHLH104) protein functions as a transcriptional repressor for glucose and abscisic acid signaling in Arabidopsis.

Transduction of glucose (Glc) signaling is critical for plant development, metabolism, and stress responses. However, identifying initial Glc sensing and response stimulating mechanisms in plants has been difficult due to dual functions of glucose as energy sources and signaling component. A basic Helix-Loop-Helix 104 (bHLH104) protein is a homolog of bHLH34 previously isolated from Arabidopsis that functions as a transcriptional activator of Glc and abscisic acid (ABA) responses. In this study, we characterized bHLH104 as a transcription factor that binds to the regulatory region of Arabidopsis Plasma membrane Glc-responsive Regulator (AtPGR) gene. The bHLH104 binds to 5'-AANA-3' element of the promoter region of AtPGR in vitro and represses beta-glucuronidase (GUS) activity in AtPGR promoter-GUS transgenic plants. Genetic approaches show that bHLH104 positively regulates Glc and abscisic acid (ABA) response. These results suggest that bHLH104 is involved in Glc- and ABA-mediated signaling pathway. Taken together, these findings provide evidence that bHLH104 is an important transcription regulator in plant-sensitivity to Glc and ABA signaling.

In vivo imaging of activated macrophages by 18F-FEDAC, a TSPO targeting PET ligand, in the use of biologic disease-modifying anti-rheumatic drugs (bDMARDs).

Rheumatoid arthritis (RA) is a chronic disease with systemic inflammation resulting in destruction of multiple articular cartilages and bones. Activated macrophage plays a pivotal role during the disease course and has been one of main targets to inhibit inflammatory reaction of RA by using biological disease-modifying anti-rheumatic drugs (bDMARDs). 18F-FEDAC is one of PET imaging agents targeting TSPO, which is overexpressed in activated macrophages. The aim of this study was to evaluate the roles of 18F-FEDAC PET as an in vivo imaging of activated macrophages on etanercept (ETN), a TNF-antagonist as one of bDMARDs in collagen induced arthritis mice. In RAW 264.7 cells, the expressions of TSPO as well as iNOS and infiltrated nucleus of NF-κB were induced by activation with lipopolysaccharide and interferon-gamma. TSPO expression was slightly attenuated by ETN treatment, not by methotrexate (MTX) as a cytotoxic agent. However, cell uptake of 18F-FEDAC did not show significant changes according to both of the treatments. Similarly in CIA mice, 18F-FEDAC uptake in inflamed paws on PET imaging did not show significant changes during both of the treatments, contrary to the uptake decrease of 18F-FDG, a glucose analog to reflect metabolic or active inflammatory activity. Interestingly, when we divided joints according to the degree of 18F-FEDAC uptake before ETN treatment, the joints of high 18F-FEDAC uptake showed better response to ETN than the joints with low 18F-FEDAC uptakes. In case of 18F-FDG, there was no such kinds of patterns. We can speculate that 18F-FEDAC PET imaging may identify activated macrophage-induced arthritis because that 18F-FEDAC can reflect activated macrophages, which is the therapeutic target of ETN by TNF antagonistic effect. Thus, in vivo imaging using 18F-FEDAC may be used as a predictor of therapeutic effects among those kinds of bDMARDs having anti-inflammatory actions to inhibit activated macrophage.

Multi-laboratory evaluation of 1,3-propane sultone, N-propyl-N-nitrosourea, and mitomycin C in the Pig-a mutation assay in vivo.

The mutagenic potencies of 1,3-propane sultone (PS), N-propyl-N-nitrosourea (PNU), and mitomycin C (MMC) were investigated in three independent laboratories in Korea using the Pig-a assay in vivo. Sprague-Dawley rats were treated with vehicle or test substance on three consecutive days. Blood samples were collected for measuring Pig-a mutant phenotypes (CD59-deficient erythrocytes, RBCCD59-; CD59-deficient reticulocytes, RETCD59-) on days -1, 15, and 29 after the first treatment. In some studies, blood was collected for determining DNA damage (comet assay) on day 3 and measuring micronucleated reticulocytes (MN-RET) on day 4. Treatment with the alkylating agents PS and PNU induced dose-dependent increases in the frequency of RBCCD59- on days 15 and 29, and caused maximum elevations in the frequency of RETCD59- on day 15. Inter-laboratory comparison of the day 29 Pig-a assay data confirmed the mutagenic potencies of PS and PNU, and showed good agreement among the test sites. Treatment with the DNA cross-linker MMC induced increases in the frequencies of RBCCD59- and RETCD59- on days 15 and 29 (all three laboratories). MN-RETs increased significantly in animals treated with PS, PNU, or MMC, but biologically significant increases in DNA damage were observed only with PS and PNU, and not with MMC. The results of this study indicate that the Pig-a assay is a sensitive, reproducible method for evaluating the in vivo mutagenicity of various test substances, in particular, DNA cross-linkers and alkylating agents. Our limited data on integrating the Pig-a assay with the comet and micronucleus assays indicate that a short-term treatment protocol evaluating these three endpoints in a single set of animals may be a robust strategy for evaluating in vivo genotoxicity.

The accessory proteins REEP5 and REEP6 refine CXCR1-mediated cellular responses and lung cancer progression.

Some G-protein-coupled receptors have been reported to require accessory proteins with specificity for proper functional expression. In this study, we found that CXCR1 interacted with REEP5 and REEP6, but CXCR2 did not. Overexpression of REEP5 and REEP6 enhanced IL-8-stimulated cellular responses through CXCR1, whereas depletion of the proteins led to the downregulation of the responses. Although REEPs enhanced the expression of a subset of GPCRs, in the absence of REEP5 and REEP6, CXCR1 was expressed in the plasma membrane, but receptor internalization and intracellular clustering of ß-arrestin2 following IL-8 treatment were impaired, suggesting that REEP5 and REEP6 might be involved in the ligand-stimulated endocytosis of CXCR1 rather than membrane expression, which resulted in strong cellular responses. In A549 lung cancer cells, which endogenously express CXCR1, the depletion of REEP5 and REEP6 significantly reduced growth and invasion by downregulating IL-8-stimulated ERK phosphorylation, actin polymerization and the expression of genes related to metastasis. Furthermore, an in vivo xenograft model showed that proliferation and metastasis of A549 cells lacking REEP5 and REEP6 were markedly decreased compared to the control group. Thus, REEP5 and REEP6 could be novel regulators of G-protein-coupled receptor signaling whose functional mechanisms differ from other accessory proteins.