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Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease which can be induced by inoculation with activated CD4+ T lymphocytes specific for myelin proteins. It has been postulated that autoreactive Th1-type CD4+ cells are responsible for the lesions, whereas autoreactive Th2-type cells suppress or modulate the disease. However, the mechanisms involved in the development of inflammatory lesions and neurologic deficits in EAE have not been fully described, and probably involve a complex array of mediators and alternative or redundant pathways. To identify additional factors associated with the pathological role of T cells in EAE, we adapted the differential mRNA display technique to fingerprint the transcripts expressed by encephalitogenic and non-encephalitogenic T cells. Using 60 primer combinations, 21 differentially expressed cDNAs were identified. Among them 13 are known sequences, including IL-3 in non-encephalitogenic lymphocytes. Their relevance for the disease process is discussed. [ABSTRACT FROM PUBLISHER]

Acinetobacter baumannii is often highly resistant to multiple antimicrobials, posing a risk of treatment failure, and colistin is a "last resort" for treatment of the bacterial infection. However, colistin resistance is easily developed when the bacteria are exposed to the drug, and a comprehensive analysis of colistin-mediated changes in colistin-susceptible and -resistant A. baumannii is needed. In this study, using an isogenic pair of colistin-susceptible and -resistant A. baumannii isolates, alterations in morphologic and transcriptomic characteristics associated with colistin resistance were revealed. Whole-genome sequencing showed that the resistant isolate harbored a PmrB L208F mutation conferring colistin resistance, and all other single-nucleotide alterations were located in intergenic regions. Using scanning electron microscopy, it was determined that the colistin-resistant mutant had a shorter cell length than the parental isolate, and filamented cells were found when both isolates were exposed to the inhibitory concentration of colistin. When the isolates were treated with inhibitory concentrations of colistin, more than 80% of the genes were upregulated, including genes associated with antioxidative stress response pathways. The results elucidate the morphological difference between the colistin-susceptible and -resistant isolates and different colistin-mediated responses in A. baumannii isolates depending on their susceptibility to this drug.

Early- to mid-season apple cultivars have recently been developed in response to global warming; however, their metabolite compositions remain unclear. Herein, metabolites, such as free sugars, and organic acids and antioxidant activity were determined in 10 new and 14 traditional apple cultivars. Additionally, the phenolic profiles of the apple pulp and peel were characterized by high-resolution mass spectrometry. Major phenolic compounds in apples varied depending on the cultivar and tissue (i.e., peel or pulp). Among the new apple cultivars, Decobell and Tinkerbell, showed high antioxidant activity and contained higher phenolic compound content than other cultivars in the peel and pulp, respectively. Honggeum showed high phenolic content with similar sugar to acid ratio compared to popular traditional cultivars. In addition to antioxidant phenolic contents, metabolite profile information can be used to select apple cultivars for various purposes. For example, Indo can be selected for sweet apple taste because of its higher sugar to acid ratio. This information can be used to select apple cultivars for various purposes. For example, Decobell peel could be used as sources of food supplements and food additives, and Tinkerbell pulp can be utilized for apple juice making according to its metabolite profile., Competing Interests: The authors declare no conflict of interest.

Thermotoga maritima , a deep-branching hyperthermophilic bacterium, expresses an extraordinarily stable Thermotoga maritima acyl carrier protein ( Tm -ACP) that functions as a carrier in the fatty acid synthesis system at near-boiling aqueous environments. Here, to understand the hyperthermal adaptation of Tm -ACP, we investigated the structure and dynamics of Tm -ACP by nuclear magnetic resonance (NMR) spectroscopy. The melting temperature of Tm -ACP (101.4 °C) far exceeds that of other ACPs, owing to extensive ionic interactions and tight hydrophobic packing. The D59 residue, which replaces Pro/Ser of other ACPs, mediates ionic clustering between helices III and IV. This creates a wide pocket entrance to facilitate the accommodation of long acyl chains required for hyperthermal adaptation of the T. maritima cell membrane. Tm -ACP is revealed to be the first ACP that harbor an amide proton hyperprotected against hydrogen/deuterium exchange for I15. The hydrophobic interactions mediated by I15 appear to be the key driving forces of the global folding process of Tm -ACP. Our findings provide insights into the structural basis of the hyperthermal adaptation of ACP, which might have allowed T. maritima to survive in hot ancient oceans.

Listeria monocytogenes is a food-borne pathogen present in various environmental reservoirs. It exhibits resistance and tolerance to antibiotics and sanitizing agents used in several food processing industries. It has been reported that L. monocytogenes chitinase can catalyze hydrolysis of chitin polymeric carbohydrate present in the environment and act as a virulence factor that support its survival in mammalian host cells. By taking advantage of chitinase, L. monocytogenes has both saprophytic and pathogenic lifestyles in the soil and the living host, respectively. The objective of the present study was to determine the involvement of chitin degradation products such as chitooligosaccharides (COS) in biofilm formation of L. monocytogenes. Results showed that different concentrations of COS with various molecular weight enhanced biofilm formation of L. monocytogenes. Such enhancement in biofilm formation contributed to the development of antibiotics resistance and disinfectants tolerance of cells present in the biofilm. The present article also described diverse roles of chitin, chitinase, and degradation of chitin and chitin-like substrates in saprophytic and pathogenic lifestyles of L. monocytogenes. This study offers a new direction for further exploration of the mechanisms of pathogenesis caused by L. monocytogenes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aquatic birds are known to be a reservoir for the most common influenza A viruses (IAVs). In the annual surveillance program, we collected the feces of migratory birds for the detection of IAVs in South Korea in November 2016. A novel reassorted H3N3 avian influenza virus (AIV) containing genes from viruses of wild and domestic birds was identified and named A/aquatic bird/South Korea/sw006/2016(H3N3). The polymerase basic 2 (PB2) and non-structural (NS) genes of this isolate are most closely related to those of wild-bird-origin AIV, while the polymerase basic 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), and matrix (M) genes are most closely related to those of domestic-bird-origin AIV. A/aquatic bird/South Korea/sw006/2016 contains PA, NP, M, and NS genes were most closely related to those of AIV subtype H4 and PB2, PB1, and HA genes that are most closely related to those of AIV subtype H3N8, while the NA gene was most closely related to those of subtype H10, which was recently detected in humans in China. These results suggest that novel reassortment of AIV strains occurred due to interaction between wild and domestic birds. Hence, we emphasize the need for continued surveillance of avian influenza virus in bird populations.

The industrial development and the increase in the use of fossil fuels have been accelerating global warming and climate change, thereby causing more frequent and intense natural disasters than ever before. Since electrical facilities are generally installed outdoors, they are greatly affected by natural disasters, thus accidents related to electrical equipment has been on the rise. In this paper, we present the risk rating associated with climate change by analyzing the statistics of electrical fires, electric shock accidents and electrical equipment accidents caused by domestic climate change. Further, we present a risk rating analysis model for electrical fires on a monthly basis through the data analysis of electrical hazards associated with various regional (metropolitan city) climatic conditions (temperature, humidity), and analyze the accident risk rating for natural disasters related to low and high voltage equipment. Through this risk analysis model for each region and type of equipment, we presented a basic prediction model for electrical hazards. Therefore, it is possible to provide electrical safety services in the future by displaying a risk prediction map of electrical hazards for each region and type of electrical equipment through web sites or smart phone apps using the presented analysis data. Further, efforts should be made to increase the robustness or reliability of electrical equipment in order to prevent electrical accidents caused by natural disasters due to climate change in advance.

Originally annotated as the initiator of fatty acid synthesis (FAS), β-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics., (© 2018 John Wiley & Sons Ltd.)

Pseudin-2 (Ps), isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity and cytotoxicity. To develop antimicrobial peptides with anti-inflammatory activity and low cytotoxicity, we designed Ps analogues with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. We further substituted Gly 11 with Pro (Ps-P analogues) to increase bacterial cell selectivity. Ps analogues retained antimicrobial activity and exhibited reduced cytotoxicity, whereas Ps-P analogues exhibited lower cytotoxicity and antimicrobial activity. Tertiary structures revealed that Ps has a linear α-helix from Leu 2 to Glu 24 , whereas Ps-P has a bend at Pro 11 between two short α-helixes. Using various biophysical experiments, we found that Ps analogues produced much higher membrane depolarization than Ps-P analogues, whereas Ps-P analogues may penetrate bacterial cell membranes. Ps and its analogue Ps-K18 exhibited potent anti-inflammatory activity in LPS-stimulated RAW264.7 and mouse dendritic cells via a mechanism involving the Toll-like receptor 4 (TLR4) pathway. These activities may arise from their direct inhibition of the formation of TLR4-MD-2_LPS complex, implying that amphipathic α-helical structure with an optimum balance between enhanced cationicity and hydrophobicity may be essential for their anti-inflammatory activity. The bent structure provided by Pro substitution plays an important role in enhancing bacterial cell selectivity and cell penetration.

An increase in the prevalence of the drug-resistant Mycobacteria tuberculosis necessitates developing new types of anti-tuberculosis drugs. Here, we found that phloretin, a naturally-occurring flavonoid, has anti-mycobacterial effects on H37Rv, multi-drug-, and extensively drug-resistant clinical isolates, with minimum inhibitory concentrations of 182 and 364 μM, respectively. Since Mycobacteria cause lung inflammation that contributes to tuberculosis pathogenesis, anti-inflammatory effects of phloretin in interferon-γ-stimulated MRC-5 human lung fibroblasts and lipopolysaccharide (LPS)-stimulated dendritic cells were investigated. The release of interleukin (IL)-1β, IL-12, and tumor necrosis factor (TNF)-α was inhibited by phloretin. The mRNA levels of IL-1β, IL-6, IL-12, TNF-α, and matrix metalloproteinase-1, as well as p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, were suppressed. A mouse in vivo study of LPS-stimulated lung inflammation showed that phloretin effectively suppressed the levels of TNF-α, IL-1β, and IL-6 in lung tissue with low cytotoxicity. Phloretin was found to bind M. tuberculosis β-ketoacyl acyl carrier protein synthase III (mtKASIII) with high affinity (7.221 × 10⁷ M -1 ); a binding model showed hydrogen bonding of A-ring 2'-hydroxy and B-ring 4-hydroxy groups of phloretin with Asn261 and Cys122 of mtKASIII, implying that mtKASIII can be a potential target protein. Therefore, phloretin can be a useful dietary natural product with anti-tuberculosis benefits.

To discover potent antibiotics against the Gram-negative bacteria, we performed a structure-activity relationship (SAR) study of YKsa-6, which was the most potent inhibitor of Staphylococcus aureus β-ketoacyl acyl carrier protein III in our previous study. We identified and selected 11 candidates, and finally screened two active compounds, YKab-4 (4-[(3-chloro-4-methylphenyl)aminoiminomethyl]benzene-1,3-diol) and YKab-6 (4-[[3-(trifluoromethyl)phenyl]aminoiminomethyl]phenol) as inhibitors of Acinetobacter baumannii KAS III (abKAS III). They showed potent antimicrobial activities at 2 or 8 μg/mL, specifically against Acinetobacter baumannii and a strong binding affinity for abKAS III. From the homology modeling, we defined the three-dimensional (3D) structure of abKAS III for the first time and found that it had an extra loop region compared with common Gram-negative bacteria derived KAS IIIs. The docking study revealed that the hydroxyl groups of inhibitors formed extensive hydrogen bonds and the complicated hydrophobic and cation-stacking interactions are important to binding with abKAS III. We confirmed that the hydrophobicity of these compounds might be the essential factor for their antimicrobial activities against Gram-negative bacteria as well as their structural rigidity, a cooperative feature for retaining the hydrophobic interactions between abKAS III and its inhibitors. This study may provide an insight developing strategies for potent antibiotics against A. baumannii., (Copyright © 2016. Published by Elsevier Ltd.)

Isorhamnetin (1) is a naturally occurring flavonoid having anticancer and anti-inflammatory properties. The present study demonstrated that 1 had antimycobacterial effects on Mycobacterium tuberculosis H37Rv, multi-drug- and extensively drug-resistant clinical isolates with minimum inhibitory concentrations of 158 and 316 μM, respectively. Mycobacteria mainly affect the lungs, causing an intense local inflammatory response that is critical to the pathogenesis of tuberculosis. We investigated the effects of 1 on interferon (IFN)-γ-stimulated human lung fibroblast MRC-5 cells. Isorhamnetin suppressed the release of tumor necrosis factor (TNF)-α and interleukin (IL)-12. A nontoxic dose of 1 reduced mRNA expression of TNF-α, IL-1β, IL-6, IL-12, and matrix metalloproteinase-1 in IFN-γ-stimulated cells. Isorhamnetin inhibited IFN-γ-mediated stimulation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase and showed high-affinity binding to these kinases (binding constants: 4.46 × 10(6) M(-1) and 7.6 × 10(6) M(-1), respectively). The 4'-hydroxy group and the 3'-methoxy group of the B-ring and the 5-hydroxy group of the A-ring of 1 play key roles in these binding interactions. A mouse in vivo study of lipopolysaccharide-induced lung inflammation revealed that a nontoxic dose of 1 reduced the levels of IL-1β, IL-6, IL-12, and INF-γ in lung tissue. These data provide the first evidence that 1 could be developed as a potent antituberculosis drug.

This study determined the prevalence and toxin profile of Bacillus cereus and Bacillus thuringiensis in doenjang, a fermented soybean food, made using both traditional and commercial methods. The 51 doenjang samples tested were broadly contaminated with B. cereus; in contrast, only one sample was positive for B. thuringiensis. All B. cereus isolates from doenjang were positive for diarrheal toxin genes. The frequencies of nheABC and hblACD in traditional samples were 22.7 and 0%, respectively, whereas 5.1 and 5.1% of B. cereus isolates from commercial samples possessed nheABC and hblACD, respectively. The detection rate of ces gene was 10.8%. The predominant toxin profile among isolates from enterotoxigenic B. cereus in doenjang was profile 4 (entFM-bceT-cytK). The major enterotoxin genes in emetic B. cereus were cytK, entFM, and nheA genes. The B. thuringiensis isolate was of the diarrheagenic type. These results provide a better understanding of the epidemiology of the enterotoxigenic and emetic B. cereus groups in Korean fermented soybean products.

The Mec1/Tel1 kinases (human ATR/ATM) play numerous roles in the DNA replication stress response. Despite the multi-functionality of these kinases, studies of their in vivo action have mostly relied on a few well-established substrates. Here we employed a combined genetic-phosphoproteomic approach to monitor Mec1/Tel1 signaling in a systematic, unbiased, and quantitative manner. Unexpectedly, we find that Mec1 is highly active during normal DNA replication, at levels comparable or higher than Mec1's activation state induced by replication stress. This "replication-correlated" mode of Mec1 action requires the 9-1-1 clamp and the Dna2 lagging-strand factor and is distinguishable from Mec1's action in activating the downstream kinase Rad53. We propose that Mec1/ATR performs key functions during ongoing DNA synthesis that are distinct from their canonical checkpoint role during replication stress., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Vascular endothelial growth factor (VEGF) is an angiogenic protein with neurotrophic and neuroprotective effects. Previously, we reported that triamterene (Trm) inhibits VEGF-amyloid β (Aβ) interactions without affecting other biological activities of VEGF or Aβ [Jeong, K.-W., et al. (2011) Biochemistry 50, 4843-4854]. We further showed that molecular motions in the N-terminal disordered loop region of the heparin-binding domain (HBD) are important for interaction with Trm. To investigate the importance of motion at the C-terminal domain of HBD, we constructed a binding model of HBD with heparin octasaccharide (HOS) based on measurements of chemical shift changes and docking studies. Furthermore, the dynamic properties of the HBD-HOS and HBD-Trm-HOS complexes were assessed by measuring spin relaxation rates. The results showed that the HOS-binding site is composed of two basic clusters consisting of side chains of residues R13, R14, and K15 and residues K30, R35, and R49. When HOS binds, values for the heteronuclear nuclear Overhauser effect near HOS-binding sites increased dramatically. CPMG (Carr-Purcell-Meiboom-Gill sequence) experiments as well as an R2 relaxation experiment were undertaken to understand millisecond time-scale motions in HBD. There is large relaxation dispersion of residues at Trm- and HOS-binding sites in free HBD. C-Terminal residues such as S34, C48, and D51 near the HOS-binding sites continued to exhibit slow conformational motions in the HBD-Trm complex, while those slow motions disappeared in the bound conformation of HBD with HOS. Collectively, our results demonstrate that the inherent structural flexibilities of the C-terminal region of the HBD are important in the heparin binding process and that Trm does not inhibit VEGF-heparin interactions necessary for the biological activities of VEGF.

Multiple sclerosis (MS) lesions in the CNS are characterized by disseminated demyelination with perivascular infiltrates of macrophages, T cells, and B cells. To investigate the origin and characteristics of the B cell population found in MS plaque tissue, we performed molecular studies in 10 MS patients and 4 non-MS control samples. Ig transcripts from the perivascular infiltrated brain lesions were analyzed by complementary-determining region 3 spectratyping to ascertain the B cell heavy chain gene rearrangement repertoire expressed in MS brains. Significant rearrangement diversity and deviation from the normal Ig heavy (H) chain repertoire was observed. The cloning and sequencing of RT-PCR products from families VH1 and VH4 showed a correlation with the profiles obtained by spectratyping. Generally, restricted spectratyping patterns concurred with repetition of in-frame complementary-determining region 3 identical sequences. The analysis of heavy chain variable (VH), diversity (D), and joining (JH) gene segments revealed the increased usage of VH1-69, VH4-34, and VH4-39. Similarly, gene segments from families D2, D3, and JH4 were over-represented. The presence of restricted patterns of rearranged Ig mRNA within the plaque lesion suggests that Ab production in the demyelinating plaque is a local phenomenon and supports the idea that in MS an Ag-driven immune response might be responsible for demyelination.

Interleukin-3 (IL-3) is a cytokine expressed primarily in activated T cells mast cells and natural killer (NK) cells. Interleukin-3 has the capability to stimulate the proliferation and differentiation of multipotential stem cells and has a role in shaping the immune response. In order to better understand the biological role of IL-3, a study of polymorphisms within the human IL-3 gene and its upstream regulatory elements has been conducted. Using a modification of the bidirectional dideoxy fingerprinting method, a group of 88 Caucasoid individuals were screened for polymorphism in the IL-3 enhancer, promoter and gene coding regions. Analysis revealed the presence of several infrequent polymorphisms which may affect IL-3 functionality., (Copyright 1998 Academic Press Limited)

Objective: Analysis of T-cell receptor (TCR) beta-chain gene expression in atherosclerotic lesions of human aorta., Methods: TCR diversity was studied using non-radioactive polymerase chain reaction for quantitative assessment of TCRBV gene transcripts, together with size and sequence analysis of the beta-chain third complementarity-determining region (CDR3). Samples represent a wide range of atheromatous histology, allowing evaluation of the T-cell repertoire at different stages of disease., Results: Diverse TCRBV family usage was observed in the majority of the samples, as the 25 different TCRBV products were detected at levels exceeding background. The data also showed that TCRBV transcripts expressed in the diseased aorta tissue displayed considerable size heterogeneity and no repetition of CDR3 nucleotide motifs., Conclusions: The early presence of T-lymphocytes in the atheromatous blood vessel has been interpreted as an indication of specific immunological reactions operating during the course of the atherosclerotic process. Although a T-cell infiltrate characterized by limited usage of TCRAV genes cannot be excluded the unrestricted usage of TCRBV genes argues against a local T-cell clonal expansion in atherogenesis.

Callithrix jacchus is an outbred New World primate characterized by a naturally occurring bone marrow chimerism, restricted polymorphism at many MHC loci, and unusual susceptibility to viral pathogens, adenocarcinoma, colitis, and, following immunization with myelin antigens, a demyelinating disease of the central nervous system closely resembling human multiple sclerosis. Here we characterize the TCRB repertoire in this species, representing the first such analysis in a New World monkey. Two TCRBC, 13 BJ, 2 BD, and 15 BV genes were identified. Overall, a high degree of similarity with human TCRBV-D-J-C gene sequences was observed, indicating a close phylogenetic relationship. Biased usage in favor of genes from the TCRBC1-BJ1 cluster was present in 77% of sequences, in contrast to preferential usage of BC2-BJ2 genes known to occur in humans and mice. Complementarity-determining region 3 averaged 10 amino acids in length and were diverse. Framework regions of TCRBV genes were extensively conserved. Phylogenetic analysis of TCRBV sequences from different species indicated that TCR genes are highly stable across primates. Thus, a diverse TCRB repertoire is generated in C. jacchus despite the limited polymorphism of class I MHC loci. Extensive homology to human TCR genes, natural chimerism, and susceptibility to inflammatory disorders are characteristics of C. jacchus that create a useful model system for the study of human autoimmunity.

Rasmussen's syndrome is a progressive and intractable form of epilepsy characterized pathologically by focal brain inflammation with large numbers of infiltrating T lymphocytes. To better understand the nature of the T cell response in this disease, we analyzed TCR expression in the brain lesions using PCR for quantitative assessment of TCRBV gene transcripts, together with size and sequence analysis of the third complementarity-determining region (CDR3) of the dominant TCR rearrangements. Restricted (oligoclonal) BV family usage was not observed, as all of the 22 BV PCR products were usually detected at levels exceeding the background. However, significant individual biases in the frequencies of different TCR families was evident. The distinct pattern of BV expression by infiltrating lymphocytes detected in the original PCR screening suggested a specific immune response. The primary structure of the rearranged CDR3 sequences for the BV family expressed at highest level in each sample was studied by size and sequence analysis. The data showed that predominant TCR BV families expressed in diseased brain tissue displayed limited size heterogeneity and extensive repetition of in-frame CDR3 nucleotide motifs. These findings demonstrate that the local immune response in Rasmussen's syndrome includes restricted T cell populations that have likely expanded from a few precursor T cells responding to discrete antigenic epitopes.

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.