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.

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.

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.

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.

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.

NME1L Negatively Regulates IGF1-Dependent Proliferation of Breast Cancer Cells.

Non-metastatic cells 1 (NME1) or nm23 is the first metastasis suppressor gene discovered. It functions through various enzymatic activities and interacts with many intracellular proteins. The NME1 gene encodes two splicing variants, NME1 and NME1L. Most studies have focused on NME1 because of its abundance in cells. We previously reported NME1L-mediated suppression of NF-κB signaling by interacting with and inhibiting IKKß. In this study, we demonstrated that NME1L, but not NME1, mediated inhibition of cell proliferation, although both NME1 and NME1L were involved in suppressing metastasis. A reporter gene assay was performed to determine the growth signaling pathway regulated by NME1L but none of the growth factors tested could induce an NF-κB-dependent luciferase expression except TNFα. Interestingly, SRE-reporter gene activation by IGF1 was significantly downregulated, along with reduction of ERK phosphorylation in NME1L expressing cells, compared to vector or NME1 expressing cells. NME1L directly interacted with KSR1, which is a scaffold for Raf-1, MEK, and ERK, that regulates ERK activation. Hence, NME1L plays a crucial role in regulation of cell proliferation by inhibiting IGF1-stimulated ERK phosphorylation through N-terminal 25 amino acid-mediated interaction with KSR1.

A splicing variant of NME1 negatively regulates NF-κB signaling and inhibits cancer metastasis by interacting with IKKß.

IKKß functions as a principal upstream activator of the canonical NF-κB pathway by phosphorylating IκB, leading to its proteasomal degradation. Because IKKß is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKß-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKß. Exogenous expression of NME1L resulted in a potent decrease in TNFα-stimulated NF-κB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-κB. NME1L down-regulates IKKß signaling by blocking IKKß-mediated IκB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.

Soluble overexpression and purification of bioactive human CCL2 in E. coli by maltose-binding protein.

Human chemokine (C-C motif) ligand 2 (hCCL2) is a small cytokine in the CC chemokine family that attracts monocytes, memory T lymphocytes, and natural killer cells to the site of tissue injury- or infection-induced inflammation. hCCL2 has been implicated in the pathogeneses of diseases characterized by monocytic infiltrates, including psoriasis, rheumatoid arthritis, atherosclerosis, multiple sclerosis, and insulin-resistant diabetes. The prokaryotic overexpression of hCCL2 has been investigated previously in an attempt to develop biomedical applications for this factor, but this has been hampered by protein misfolding and aggregation into inclusion bodies. In our present study, we screened 7 protein tags-Trx, GST, MBP, NusA, His8, PDI, and PDIb'a'-for their ability to allow the soluble overexpression of hCCL2. Three tags-MBP, His8, and PDI-solubilized more than half of the expressed hCCL2 fusion proteins. Lowering the expression temperature to 18 °C significantly further improved the solubility of all fusion proteins. MBP was chosen for further study based on its solubility, expression level, ease of purification, and tag size. MBP-CCL2 was purified using conventional chromatography and cleaved using TEV or Factor Xa proteases. Biological activity was assessed using luciferase and cell migration assays. Factor Xa-cleaved hCCL2 was found to be active and TEV-cleaved hCCL2 showed relatively less activity. This is probably because the additional glycine residues present at the N-terminus of hCCL2 following TEV digestion interfere with the binding of hCCL2 to its receptor.

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.

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.

Evolutionarily conserved residues at glucagon-like peptide-1 (GLP-1) receptor core confer ligand-induced receptor activation.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) play important roles in insulin secretion through their receptors, GLP1R and GIPR. Although GLP-1 and GIP are attractive candidates for treatment of type 2 diabetes and obesity, little is known regarding the molecular interaction of these peptides with the heptahelical core domain of their receptors. These core domains are important not only for specific ligand binding but also for ligand-induced receptor activation. Here, using chimeric and point-mutated GLP1R/GIPR, we determined that evolutionarily conserved amino acid residues such as Ile(196) at transmembrane helix 2, Leu(232) and Met(233) at extracellular loop 1, and Asn(302) at extracellular loop 2 of GLP1R are responsible for interaction with ligand and receptor activation. Application of chimeric GLP-1/GIP peptides together with molecular modeling suggests that His(1) of GLP-1 interacts with Asn(302) of GLP1R and that Thr(7) of GLP-1 has close contact with a binding pocket formed by Ile(196), Leu(232), and Met(233) of GLP1R. This study may provide critical clues for the development of peptide and/or nonpeptide agonists acting at GLP1R.

CXCL14 enhances proliferation and migration of NCI-H460 human lung cancer cells overexpressing the glycoproteins containing heparan sulfate or sialic acid.

CXCL14 is a chemokine family member that is involved in various cellular responses in addition to immune cell activation. Although constitutive CXCL14 expression in normal epithelial cells may help protect against infection by activating immune systems, its expression in cancer cells has raised controversy regarding its possible role in tumorigenesis. However, the underlying mechanisms for this disparity remain unknown. Investigation of cellular CXCL14 binding properties might increase our understanding of the peptide's roles in tumorigenesis. In the present study, we found that CXCL14 binds to various cell types. Interestingly, binding to NCI-H460 cells was prevented by heparan sulfate and N-acetyl neuraminic acid. Next, we examined effect of CXCL14 binding in NCI-H460 and NCI-H23. CXCL14 enhanced proliferation and migration in NCI-H460 but had no effect on NCI-H23. A reporter gene assay with various transcription factor response elements revealed that only nuclear factor-κB (NF-κB) signaling was activated by CXCL14 in NCI-H460 cells, which was blocked by BAPTA-AM, TPCA-1, and brefeldin A. Exogenous expression of some glycoproteins such as syndecan-4, podoplanin, and CD43 in these cells enhanced CXCL14 binding and NF-κB activity. Collectively, these results demonstrate that CXCL14 binding to glycoproteins harboring heparan sulfate proteoglycans and sialic acids leads proliferation and migration of some cancer cells.

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.

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.