Aconitate Decarboxylase 1 Deficiency Exacerbates Mouse Colitis Induced by Dextran Sodium Sulfate.

Ulcerative colitis is a complex inflammatory bowel disorder disease that can induce rectal and colonic dysfunction. Although the prevalence of IBD in Western countries is almost 0.5% of the general population, genetic causes are still not fully understood. In a recent discovery, itaconate was found to function as an immune-modulating metabolite in mammalian immune cells, wherein it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. However, the association between the Acod1 (Aconitate decarboxylase 1)-itaconate axis and ulcerative colitis has rarely been studied. To elucidate this, we established a DSS-induced colitis model with Acod1-deficient mice and then measured the mouse body weights, colon lengths, histological changes, and cytokines/chemokines in the colon. We first confirmed the upregulation of Acod1 RNA and protein expression levels in DSS-induced colitis. Then, we found that colitis symptoms, including weight loss, the disease activity index, and colon shortening, were worsened by the depletion of Acod1. In addition, the extent of intestinal epithelial barrier breakdown, the extent of immune cell infiltration, and the expression of proinflammatory cytokines and chemokines in Acod1-deficient mice were higher than those in wild-type mice. Finally, we confirmed that 4-octyl itaconate (4-OI) alleviated DSS-induced colitis in Acod1-deficient mice and decreased the expression of inflammatory cytokines and chemokines. To our knowledge, this study is the first to elucidate the role of the Acod1-itaconate axis in colitis. Our data clearly showed that Acod1 deletion resulted in severe DSS-induced colitis and substantial increases in inflammatory cytokine and chemokine levels. Our results suggest that Acod1 may normally play an important regulatory role in the pathogenesis of colitis, demonstrating the potential for novel therapies using 4-OI.

Comparison of Macrophage Immune Responses and Metabolic Reprogramming in Smooth and Rough Variant Infections of Mycobacterium mucogenicum.

Mycobacterium mucogenicum (Mmuc), a rapidly growing nontuberculous mycobacterium (NTM), can infect humans (posttraumatic wound infections and catheter-related sepsis). Similar to other NTM species, Mmuc exhibits colony morphologies of rough (Mmuc-R) and smooth (Mmuc-S) types. Although there are several case reports on Mmuc infection, no experimental evidence supports that the R-type is more virulent. In addition, the immune response and metabolic reprogramming of Mmuc have not been studied on the basis of morphological characteristics. Thus, a standard ATCC Mmuc strain and two clinical strains were analyzed, and macrophages were generated from mouse bone marrow. Cytokines and cell death were measured by ELISA and FACS, respectively. Mitochondrial respiration and glycolytic changes were measured by XF seahorse. Higher numbers of intracellular bacteria were found in Mmuc-R-infected macrophages than in Mmuc-S-infected macrophages. Additionally, Mmuc-R induced higher levels of the cytokines TNF-α, IL-6, IL-12p40, and IL-10 and induced more BMDM necrotic death. Furthermore, our metabolic data showed marked glycolytic and respiratory differences between the control and each type of Mmuc infection, and changes in these parameters significantly promoted glucose metabolism, extracellular acidification, and oxygen consumption in BMDMs. In conclusion, at least in the strains we tested, Mmuc-R is more virulent, induces a stronger immune response, and shifts bioenergetic metabolism more extensively than the S-type. This study is the first to report differential immune responses and metabolic reprogramming after Mmuc infection and might provide a fundamental basis for additional studies on Mmuc pathogenesis.

Verbascoside-Rich Abeliophyllum distichum Nakai Leaf Extracts Prevent LPS-Induced Preterm Birth Through Inhibiting the Expression of Proinflammatory Cytokines from Macrophages and the Cell Death of Trophoblasts Induced by TNF-α.

Background: Preterm birth is a known leading cause of neonatal mortality and morbidity. The underlying causes of pregnancy-associated complications are numerous, but infection and inflammation are the essential high-risk factors. However, there are no safe and effective preventive drugs that can be applied to pregnant women. Objective: The objectives of the study were to investigate a natural product, Abeliophyllum distichum leaf (ADL) extract, to examine the possibility of preventing preterm birth caused by inflammation. Methods: We used a mouse preterm birth model by intraperitoneally injecting lipopolysaccharides (LPS). ELISA, Western blot, real-time PCR and immunofluorescence staining analyses were performed to confirm the anti-inflammatory efficacy and related mechanisms of the ADL extracts. Cytotoxicity and cell death were measured using Cell Counting Kit-8 (CCK-8) analysis and flow cytometer. Results: A daily administration of ADL extract significantly reduced preterm birth, fetal loss, and fetal growth restriction after an intraperitoneal injection of LPS in mice. The ADL extract prevented the LPS-induced expression of TNF-α in maternal serum and amniotic fluid and attenuated the LPS-induced upregulation of placental proinflammatory genes, including IL-1ß, IL-6, IL-12p40, and TNF-α and the chemokine gene CXCL-1, CCL-2, CCL3, and CCL-4. LPS-treated THP-1 cell-conditioned medium accelerated trophoblast cell death, and TNF-α played an essential role in this effect. The ADL extract reduced LPS-treated THP-1 cell-conditioned medium-induced trophoblast cell death by inhibiting MAPKs and the NF-κB pathway in macrophages. ADL extract prevented exogenous TNF-α-induced increased trophoblast cell death and decreased cell viability. Conclusions: We have demonstrated that the inhibition of LPS-induced inflammation by ADL extract can prevent preterm birth, fetal loss, and fetal growth restriction.

Hippo effector YAP directly regulates the expression of PD-L1 transcripts in EGFR-TKI-resistant lung adenocarcinoma.

Developments of EGFR-TKI and immunotherapy targeting the PD1/PD-L1 pathway are considered most important medical breakthroughs in lung cancer treatment. Nowadays, 3rd generation EGFR TKI is widely used for T790M positive 1st and 2nd EGFR-TKI resistant lung cancer patients. Immunotherapy is powerful option for lung cancer patients without drug targets and chemotherapy resistant patients. It also has changed the concept of conventional anti-cancer therapy in the point of regulating tumor microenvironment. There are many studies linking these two important pathways. Recent studies demonstrated that PD-L1 expression is significantly correlated to the mutation status of EGFR, and activation of EGFR signaling can also induce the expression of PD-L1. However, the real linker between PD-L1 and EGFR signaling remains to be revealed. Our previous study revealed that the Hippo pathway effector YAP confers EGFR-TKI resistance in lung adenocarcinoma, and inhibition of YAP restores sensitivity to EGFR-TKIs. Thus, we examined whether PD-L1 is relevant, in terms of conferring EGFR-TKI resistance and whether YAP directly regulates the expression of PD-L1 in this context. First, we compared the expression levels of PD-L1 and YAP between EGFR-TKI-resistant PC9 cells and the parental PC9 adenocarcinoma cells. The expression levels of both YAP and PD-L1 were markedly higher in the EGFR-TKI-resistant cells compared to the parental cells, suggesting differential expression pattern between two cell types. YAP knockdown significantly decreased the expression of PD-L1 in the EGFR-TKI-resistant cells, while YAP overexpression increased the expression of PD-L1 in the parental PC9 cells. Then, our results revealed that YAP regulates the transcription of PD-L1, and the YAP/TEAD complex binds to the PD-L1 promoter. Surprisingly, knockdown of PD-L1 was sufficient to decrease cell proliferation and wound healing in the EGFR-TKI-resistant PC9 cells. These data suggest a PD1-independent oncogenic function of PD-L1. The Hippo effector YAP plays a crucial role in linking the PD-L1 and EGFR-TKI resistance by directly regulating the expression of PD-L1 in lung cancer. Targeting PD-L1 directly or via YAP could provide an effective therapeutic strategy for EGFR-TKI-resistant lung adenocarcinoma.

Isoegomaketone exhibits potential as a new Mycobacterium abscessus inhibitor.

Although the incidence of Mycobacterium abscessus infection has recently increased significantly, treatment is difficult because this bacterium is resistant to most anti-tuberculosis drugs. In particular, M. abscessus is often resistant to available macrolide antibiotics, so therapeutic options are extremely limited. Hence, there is a pressing demand to create effective drugs or therapeutic regimens for M. abscessus infections. The aim of the investigation was to assess the capability of isoegomaketone (iEMK) as a therapeutic option for treating M. abscessus infections. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of iEMK for both reference and clinically isolated M. abscessus strains. In addition to time-kill and biofilm formation assays, we evaluated iEMK's capability to inhibit M. abscessus growth in macrophages using an intracellular colony counting assay. iEMK inhibited the growth of reference and clinically isolated M. abscessus strains in macrophages and demonstrated effectiveness at lower concentrations against macrophage-infected M. abscessus than when used to treat the bacteria directly. Importantly, iEMK also exhibited anti-biofilm properties and the potential to mitigate macrolide-inducible resistance, underscoring its promise as a standalone or adjunctive therapeutic agent. Overall, our results suggest that further development of iEMK as a clinical drug candidate is promising for inhibiting M. abscessus growth, especially considering its dual action against both planktonic bacteria and biofilms.

Diesel exhaust particle exposure accelerates oxidative DNA damage and cytotoxicity in normal human bronchial epithelial cells through PD-L1.

Diesel exhaust particles (DEPs) are a major cause of cancer progression as well as a variety of acute and chronic diseases. It is well-known that programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that can induce immune escape in tumor cells. However, the function of PD-L1 in bronchial epithelial cells or how PD-L1 relates to cellular oxidation under DEPs-mediated oxidative stress is not well known. In this study, we investigated how PD-L1 affected DEPs-induced oxidative stress and cytotoxicity in human bronchial epithelial (HBE) cells, Beas-2B. DEPs not only induced intracellular reactive oxygen species (ROS) production, but also increased PD-L1 expression in HBE cells. Beas-2B cells overexpressing PD-L1 showed higher levels of ROS production, DNA damage, and apoptosis after DEPs treatment compared to control cells. In particular, the expression of an antioxidant enzyme heme-oxygenase-1 (HO-1) and nuclear translocation and transcriptional activity of Nrf2, a major regulator of HO-1, were lower in Beas-2B overexpressing PD-L1 cells than in control cells. DEPs-induced ROS generation, DNA damage and apoptosis in Beas-2B cells overexpressing PD-L1 were significantly restored by overexpressing HO-1. Collectively, our results suggest that DEPs can increase the expression of PD-L1 in HBE cells and that overexpressing PD-L1 might eventually promote DEPs-induced oxidative DNA damage and apoptosis.

Disseminated infection due to Mycobacterium avium subsp. avium in an Asian elephant (Elephas maximus).

A disseminated infection caused by Mycobacterium avium subspecies avium (MAA) was diagnosed in a 57-yr-old male Asian elephant (Elephas maximus) housed at the Seoul Zoo, Gyeonggi, Republic of Korea. An apparent granulomatous inflammation with central caseous necrosis was evident in the lung sections. To confirm mycobacterial infection, polymerase chain reaction-restriction enzyme polymorphism analysis (PCR-RFLP) of the rpoB and hsp65 genes was performed from multiple organs and cultured bacteria. The PCR-RFLP revealed a M. avium subspecies. MAA was identified by multiplex PCR for detection of IS901 and IS1311. Thus, it is believed that MAA caused the disseminated infection in this case. Although the source of infection was not determined, the elephant may have become infected through contamination of soil and feed by free-living birds infected with MAA. This is the first reported case of disseminated infection due to MAA in a captive elephant in the Republic of Korea.

Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR.

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.

Differentially expressed genes in Mycobacterium tuberculosis H37Rv under mild acidic and hypoxic conditions.

The survival mechanism of dormant tubercle bacilli is unknown; however, accumulating evidence indicates that Mycobacterium tuberculosis can survive and persist in hypoxic and mildly acidic microenvironments. Such conditions are found in the acidic vacuoles of macrophages, which M. tuberculosis is known to target. We used DECAL (differential expression using customized amplification library) to identify the genes expressed under acidic and hypoxic conditions, following the cultivation of M. tuberculosis H37Rv at an acidic pH and/or under hypoxic or anoxic conditions in vitro. Of 960 clones analysed, 144 genes, consisting of 71 induced and 8 repressed genes, were identified by sequencing and divided into functional categories to characterize their cellular roles. In general, the genes induced under acidic and hypoxic conditions were involved in the biosynthesis of secondary metabolites (e.g. pks4), lipid metabolism, energy production (e.g. pckA) and cell wall biogenesis (e.g. Rv0696 and plcB). The combination of genes identified may explain the energy processing and energy storage of M. tuberculosis during latent infection. These findings not only enhance our understanding of the mechanism of dormancy, but they also may be useful in the design of therapeutic tools and vaccines for latent tuberculosis.

Natural course and outcomes of spontaneous isolated celiac artery dissection according to morphological findings on computed tomography angiography: STROBE compliant article.

We aimed to identify natural course and optimal management of spontaneous isolated celiac artery dissection (SICAD) according to morphologic classification determined on computed tomography angiography (CTA), and to investigate the association between symptoms and morphological classification of SICAD.This retrospective observational study included 21 consecutive patients with SICAD from January 2012 to April 2017. Demographic data, clinical features, treatment modalities, follow-up results, and CTA findings including morphologic classification, dissection length, and relative diameter of the true lumen (TLRD) were reviewed. Changes in follow-up CTA were recorded and compared to prior studies to reveal natural course of the disease.The serial changes of SICAD on follow-up CTA according to morphologic classifications were as follows; type I (5/5, no interval change), type IIa (1/1, no interval change), type IIb (1/1, partial remodeling), type IIIa (1/4, complete remodeling; 1/4, partial remodeling; 1/4, no interval change; 1/4, deterioration), type IIIb (4/6, no interval change; 2/6, partial remodeling), and type IV (2/2, no interval change). Thirteen (61.9%) symptomatic and 8 (38.1%) asymptomatic patients were all treated with conservative management with or without antiplatelet and/or anticoagulation therapies. Symptomatic group (SG) more commonly had type IIb, IIIa, IIIb, and IV than asymptomatic group (AG) (SG; 11 patients, AG; 1 patient, P = .002). TLRD in AG was larger than that in SG (SG: 40.5 ±â€Š24.1%, AG: 61.7 ±â€Š7.0%, P = .045).SICAD might be treated by conservative management in stable patients irrespective of the morphologic classification except for with type IV (dissecting aneurysm) and extension of celiac branch who may need an early intervention. Types IIb, IIIa, IIIb, and IV are TLRD are associated with patients' symptoms. Further studies on extended natural course of SICAD with a larger number of subjects are needed to draw a strong conclusion.

Structure, magnetic and ion-exchange properties of self-assembled triaza-copper(II)-oxalate hybrides having nanoscale one-dimensional channel.

The X-ray structure, porous and magnetic property of a self-assembled network 1 is described in detail. The single crystal X-ray analysis provides 1 as a three-dimensional network, which contains two-dimensional permanent ring forming nanoscale one-dimensional channels. The inter-replacement of perchlorate and hexafluorophosphate anions in solid 1 proves its porous structure. There is somewhat strong antiferromagnetic interaction (J = -74.1 cm(-1)) between two copper(II) ions through oxalate bridge and weak antiferromagnetic interaction (J = -5.1 cm(-1) through AEP ligand.

Enhancement of the Hydrogen-Storage Properties of MgH2 by the Addition of Ni, NaAlH4, Ti, and CNT via Reactive Mechanical Grinding.

MgH2 was used as the starting material in this study. A sample with the composition of 84 wt% MgH2 + 10 wt% Ni + 2 wt% NaAlH4 + 2 wt% Ti + 2 wt% CNT (named MgH2-10Ni-2NaAIH4-2Ti-2CNT) was prepared by the reactive mechanical grinding. Hydriding and dehydriding property measurements, X-ray diffraction (XRD) analyses, and microstructural observations were then performed. The activation of the sample was not required. At the first cycle (n = 1), the sample absorbed 2.84 wt% H for 5 min, 3.75 wt% H for 10 min, 4.09 wt% H for 15 min, and 4.17 wt% H for 60 min at 593 K under 12 bar H2. The MgH2-10Ni-2NaAlH4-2Ti-2CNT sample showed quite a high hydriding rate at a relatively low temperature of 423 K under 12 bar H2 (at n = 4), absorbing 2.81 wt% H for 5 min, 3.23 wt% H for 10 min, and 3.56 wt% H for 60 min. The reactive mechanical grinding of MgH2 with Ni, NaAlH4, Ti, and CNT is considered to create defects on the surface and in the interior of MgH2 as well as decrease the particle size of MgH2-

Epigallocatechin-3-gallate-mediated Tollip induction through the 67-kDa laminin receptor negatively regulating TLR4 signaling in endothelial cells.

BACKGROUND: Green tea polyphenol epigallocatechin-3-gallate (EGCG) has the potential to impact a variety of inflammation-related diseases; however, the anti-inflammatory action of EGCG in endothelial cells has not been understood. Recently, we demonstrated that the 67-kDa laminin receptor (67LR) acts as a cell-surface EGCG receptor. AIM: This research was carried out to clarify the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by EGCG in lipopolysaccharide (LPS)-stimulated endothelial cells. RESULTS: RNAi-mediated silencing of 67LR resulted in an abrogation of the inhibitory action of EGCG on the LPS-induced activation of downstream signaling pathways. Also, we found that EGCG induced a rapid upregulation of Toll-interacting protein (Tollip), a negative regulator of TLR signaling, through 67LR in endothelial cells. RNAi-mediated silencing of Tollip impaired the TLR4 signaling inhibitory activity of EGCG. Additionally, silencing of Tollip resulted in an abrogation of the inhibitory action of EGCG on the LPS-induced expressions of cell-associated adhesion molecules, such as ICAM-1 and VCAM-1. CONCLUSION: Taken together, these novel findings provide new insights into an understanding of negative regulatory mechanisms of the TLR4 signaling pathway and effective therapeutic intervention for the treatment of inflammatory disease.

Anti-inflammatory effect of gamma-irradiated genistein through inhibition of NF-κB and MAPK signaling pathway in lipopolysaccharide-induced macrophages.

Genistein was irradiated with γ-irradiation at doses of 0, 10, 30, 50, 100, and 150 kGy. We observed that the decrease in the genistein peak after gamma irradiation was concomitant with the appearance of several new peaks. 150 kGy gamma-irradiated genistein did not exert cytotoxicity in macrophages, and inhibited inducible nitric oxide synthase-mediated nitric oxide production and pro-inflammatory cytokines level, such as tumor necrosis factor-α, interleukin-6 and interleukin-1ß, in lipopolysaccharide (LPS)-induced macrophages. The treatment of LPS-stimulated macrophages with 150 kGy gamma-irradiated genistein resulted in a significant decrease in cyclooxygenase-2 levels, as well as the expression of cell surface molecules, such as CD80 and CD86. Furthermore, we also found that the anti-inflammatory action of 150 kGy gamma-irradiated genistein occurred through an inhibition of mitogen-activated protein kinases (extracellular signal-regulated kinase 1/2, p38 and c-Jun N-terminal kinase) and nuclear factor-κB signaling pathways based on a toll-like receptor 4 in macrophages, which may be speculated that several radiolysis products of genistein transformed by gamma-irradiation induce the inhibition of pro-inflammatory mediators. From these findings, it seems likely that gamma-irradiated genistein could play a potent role in the treatment of inflammatory disease as a value-added product in the medical industry.

Polymerase chain reaction-restriction fragment length polymorphism of the rpoB gene for identification of Mycobacterium avium subsp. paratuberculosis and differentiation of Mycobacterium avium subspecies.

Mycobacterial speciation by polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the rpoB gene was evaluated for identification of Mycobacterium avium subsp. paratuberculosis (MAP) and other Mycobacterium avium complex (MAC) members to the species or subspecies level by comparison with conventional methods including hsp65 sequencing, high-performance liquid chromatography, and PCR for accepted species- or subspecies-specific genomic targets. A total of 185 type and clinical mycobacterial strains from humans, animals, and environments were tested. A 360-bp PCR product was subsequently digested with MspI, HaeIII, and SmaI restriction enzymes. The PRA using SmaI restriction showed a unique digestion pattern for MAP distinguishing it from other MAC members and other Mycobacterium spp. Moreover, HaeIII and MspI restriction of the rpoB gene enabled MAC-species and -subspecies discrimination. The rpoB-PRA using SmaI or MspI and HaeIII restriction of the rpoB gene is a simple, convenient, and reliable confirmatory assay for simultaneous identification of MAP and other MAC members.

Improved sensitivity of diagnosis of tuberculosis in patients in Korea via a cocktail enzyme-linked immunosorbent assay containing the abundantly expressed antigens of the K strain of Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading cause of death from a single infectious agent in Korea. In this study, we compared the proteins present in culture filtrates from Mycobacterium tuberculosis strain K, which is the dominant clinical isolate in Korea, with those present in culture filtrates from M. tuberculosis H37Rv. Several differences in expression were detected between the two strains for those proteins with a molecular mass of <20 kDa. ESAT-6, HSP-X, and CFP-10 were found to be abundantly expressed in the strain K culture filtrates by liquid chromatography-electrospray ionization-time of flight mass spectrometry. The serodiagnostic potentials of recombinant antigens rESAT-6, rHSP-X, and rCFP-10 and two native antigens (Ag85 and PstS1) were evaluated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using sera collected from 46 TB patients with active disease and 46 healthy controls. As for our ELISA results, HSP-X was superior to the other antigens in terms of sensitivity when a single antigen was employed. The results of a receiver operator characteristic analysis revealed that a cocktail ELISA using all five antigens was significantly more sensitive (77.8%) than the use of a single antigen and offered equivalent specificity; moreover, it produced the largest area under the curve (0.91 versus 0.55 to 0.87). Therefore, a cocktail ELISA containing abundantly expressed antigens enhances the sensitivity of a single antigen and can be a useful diagnostic tool for the detection of active TB.

A multiresidue method for the determination of 109 pesticides in rice using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) sample preparation method and gas chromatography/mass spectrometry with temperature control and vacuum concentration.

A rapid, specific and sensitive multiresidue method based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) sample preparation method and gas chromatography with mass spectrometric detection by selected ion monitoring (GC/MS-SIM) has been developed for the routine analysis of 109 pesticides in rice. The method uses one quantification ion and two identification ions. Temperature control during sample preparation helps improve the recovery of thermally labile pesticides such as captan. The method was validated by the analysis of samples spiked at 0.025-0.150 mg/kg in rice matrix. The recoveries of all pesticides were between 80% and 115% with a relative standard deviation of less than 15%. The limit of quantitation (LOQ) for most compounds met the maximum residue limits (MRLs) for pesticides in rice in Korea.