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This paper has measured local traffic density under real driving conditions. The methodology employed is based on the traditional process of image recording. However, the conventional method has suffered from three typical limitations: long processing times, low vehicle detection performance, and inaccurate road length estimates. This study proposed two sequential approaches to resolve the three drawbacks. First, the you-only-look-once (YOLO) algorithm was used to shorten the computation time and enhance vehicle detection accuracy. Second, the vanishing point was identified during digital image processing to adjust the vertical and horizontal curves of roads, thereby calculating accurate road lengths. The proposed framework was applied to an arterial road in Seoul, South Korea. This approach was fast enough to measure local traffic density in real time. The performance of vehicle detection evaluated using a weighted mean average precision (mAP) was high at 88.73%. The accuracy of estimated road lengths evaluated using the Pearson correlation coefficient was also high at 0.97. Some co-benefits expected from the proposed method are also discussed.

Supplementary Figure S1. Loss of NF2 protects mechanical stress-induced cell death. Supplementary Figure S2. TβR1 is sensor of physical stimulation. Supplementary Figure S3. TβR1 kinase activity is required for RKIP and NF2 suppression. Supplementary Figure S4. Loss of NF2 induces unbalanced TβR1 and TβR2 expression. Supplementary Figure S5. TβR1-mediated oncogenic signal such as invasion can be blocked by TβR2. Supplementary Figure S6. NF2 blocks the binding between RKIP and TβR1. Supplementary Figure S7. TβR1 phosphorylates RKIP. Supplementary Figure S8. The effect of TβR1 kinase inhibitors on NF2-deficient cells. Supplementary Figure S9. The effect of several kinds of selective TβR1 inhibitors. Supplementary Figure S10. Gene expression analysis. Supplementary Figure S11. TEW7197 induces HEI-193 differentiation into Schwann cell. Supplementary Figure S12. Histological analysis in NF2 syndrome model mouse. Supplementary Figure S13. Mouse phenotype. Supplementary Figure S14. Effect of TEW7197 treatment on the primary tumor cells isolated from vehicle-treated NF2 syndrome model mouse.

Toxin-antitoxin (TA) systems are growth-controlling genetic elements consisting of an intracellular toxin protein and its cognate antitoxin. TA systems have been spread among microbial genomes through horizontal gene transfer and are now prevalent in most bacterial and archaeal genomes. Under normal growth conditions, antitoxins tightly counteract the activity of the toxins. Upon stresses, antitoxins are inactivated, releasing activated toxins, which induce growth arrest or cell death. In this study, among nine functional TA modules in Bosea sp. PAMC 26642 living in Arctic lichen, we investigated the functionality of BoHigBA2. BohigBA2 is located close to a genomic island and adjacent to flagellar gene clusters. The expression of BohigB2 induced the inhibition of E. coli growth at 37°C, which was more manifest at 18°C, and this growth defect was reversed when BohigA2 was co-expressed, suggesting that this BoHigBA2 module might be an active TA module in Bosea sp. PAMC 26642. Live/dead staining and viable count analyses revealed that the BoHigB2 toxin had a bactericidal effect, causing cell death. Furthermore, we demonstrated that BoHigB2 possessed mRNA-specific ribonuclease activity on various mRNAs and cleaved only mRNAs being translated, which might impede overall translation and consequently lead to cell death. Our study provides the insight to understand the cold adaptation of Bosea sp. PAMC 26642 living in the Arctic.

Abscisic acid (ABA) plays an important role in seed germination, stomatal closure, and seedling growth inhibition in plants. Among downstream genes whose expression levels are regulated by AFA1 (Arabidopsis F-box Protein Hypersensitive to ABA 1), one gene, AtHAD1 upregulated by ABA was selected from Arabidopsis. AtHAD1 was induced by drought and salt stresses as well as by ABA and was found in dry seeds. Its loss-of-function mutants exhibited increased ABA-sensitivity in germination, seedling growth, and stomatal closure. In addition, the mutants displayed a lower water loss rate and higher survival rate under drought stress than the wild-type plants, indicating that a loss of AtHAD1 leads to enhanced drought tolerance. These results show that AtHAD1 has an inhibitory role in ABA response and ABA-mediated drought tolerance. The expression levels of several ABA-responsive genes in athad1 were higher than those in the wild-type under the ABA treatment, suggesting that AtHAD1, as a negative regulator in ABA response, could be associated with the downregulation of the ABA-responsive genes. The phosphatase assay showed that AtHAD1 exhibits phosphatase activity. Monitoring of the subcellular localization of GFP-fused AtHAD1 proteins indicated that AtHAD1 exists in the nucleus and cytoplasm. Overall, this study shows that Arabidopsis HAD1 as an intracellular phosphatase negatively functions in the ABA-mediated cellular responses. This research could serve as a research basis to understand the functional link between ABA signaling and the regulation process of the cellular phosphate level.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry–based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma.Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462–72. ©2018 AACR.

Supplementary Table S5. UBE2N loss resulted in distinct alterations to the signaling landscape, as revealed by Mass spectrometry-based phosphoproteomic analysis

The Supplementary file contains supplementary methods, figures and tables that depict the importance of UBE2N, UBE2V1, and UBE2V2 in melanoma growth and progression.

ABA is a phytohormone involved in diverse plant events such as seed germination and drought response. An F-box protein functions as a substrate receptor of the SCF complex and is responsible for ubiquitination of target proteins, triggering their subsequent degradation mediated by ubiquitin proteasome system. Here, we have isolated a gene named ARABIDOPSIS F-BOX PROTEIN HYPERSENSITIVE TO ABA 1 (AFA1) that was upregulated by ABA. AFA1 interacted with adaptor proteins of the SCF complex, implying its role as a substrate receptor of the complex. Its loss of function mutants, afa1 seedlings, exhibited ABA-hypersensitivity, including delayed germination in the presence of ABA. Moreover, loss of AFA1 led to increased drought tolerance in adult plants. Microarray data with ABA treatments indicated that 129 and 219 genes were upregulated or downregulated, respectively, by more than three times in afa1 relative to the wild type. Among the upregulated genes in afa1, the expression of 28.7% was induced by more than three times in the presence of ABA, while only 9.3% was repressed to the same extent. These data indicate that AFA1 is involved in the downregulation of many ABA-inducible genes, in accordance with the ABA-hypersensitive phenotype of afa1. Epistasis analysis showed that AFA1 could play a role upstream of ABI4 and ABI5 in the ABA signaling for germination inhibition. Collectively, our findings suggest that AFA1 is a novel F-box protein that negatively regulates ABA signaling.

It is generally believed that epitaxial thin films are subject to compressive strain by the nearest layer with a smaller lattice constant or to tensile strain by the nearest layer with a larger lat...

Toxin–antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli. The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNAfMet, implying that degradation of tRNAfMet might trigger growth arrest in E. coli. Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl coenzyme A (succinyl-CoA) was shown to increase the affinity of binding of RamB to its binding sites and enable RamB to bind to RamBS2, which is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1; MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis. Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis, this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis- and trans-acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that a similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis.

Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl coenzyme A (succinyl-CoA) was shown to increase the affinity of binding of RamB to its binding sites and enable RamB to bind to RamBS2, which is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1; MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis. Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis, this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis- and trans-acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that a similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis.

Toxin–antitoxin (TA) systems are genetic modules composed of a toxin interfering with cellular processes and its cognate antitoxin, which counteracts the activity of the toxin. TA modules are widespread in bacterial and archaeal genomes. It has been suggested that TA modules participate in the adaptation of prokaryotes to unfavorable conditions. The Bosea sp. PAMC 26642 used in this study was isolated from the Arctic lichen Stereocaulon sp. There are 12 putative type II TA loci in the genome of Bosea sp. PAMC 26642. Of these, nine functional TA systems have been shown to be toxic in Escherichia coli. The toxin inhibits growth, but this inhibition is reversed when the cognate antitoxin genes are coexpressed, indicating that these putative TA loci were bona fide TA modules. Only the BoVapC1 (AXW83_01405) toxin, a homolog of VapC, showed growth inhibition specific to low temperatures, which was recovered by the coexpression of BoVapB1 (AXW83_01400). Microscopic observation and growth monitoring revealed that the BoVapC1 toxin had bacteriostatic effects on the growth of E. coli and induced morphological changes. Quantitative real time polymerase chain reaction and northern blotting analyses showed that the BoVapC1 toxin had a ribonuclease activity on the initiator tRNA(fMet), implying that degradation of tRNA(fMet) might trigger growth arrest in E. coli. Furthermore, the BoVapBC1 system was found to contribute to survival against prolonged exposure at 4°C. This is the first study to identify the function of TA systems in cold adaptation.

Mesenchymal stem cell (MSC) therapies have been used as cell-based treatments for decades, owing to their anti-inflammatory, immunomodulatory, and regenerative properties. With high expectations, many ongoing clinical trials are investigating the safety and efficacy of MSC therapies to treat arthritic diseases. Studies on osteoarthritis (OA) have shown positive clinical outcomes, with improved joint function, pain level, and quality of life. In addition, few clinical MSC trials conducted on rheumatoid arthritis (RA) patients have also displayed some optimistic outlook. The largely positive outcomes in clinical trials without severe side effects establish MSCs as promising tools for arthritis treatment. However, further research is required to investigate its applicability in clinical settings. This review discusses the most recent advances in clinical studies on MSC therapies for OA and RA.

The partner switching system (PSS) of the SigF regulatory pathway in Mycobacterium smegmatis has been previously demonstrated to include the anti-sigma factor RsbW (MSMEG_1803) and two anti-sigma factor antagonists RsfA and RsfB. In this study, we further characterized two additional RsbW homologs and revealed the distinct roles of three RsbW homologs [RsbW1 (MSMEG_1803), RsbW2 (MSMEG_6129), and RsbW3 (MSMEG_1787)] in the SigF PSS. RsbW1 and RsbW2 serve as the anti-sigma factor of SigF and the protein kinase phosphorylating RsfB, respectively, while RsbW3 functions as an anti-SigF antagonist through its protein interaction with RsbW1. Using relevant mutant strains and RNA sequencing analysis, RsfB was demonstrated to be the major anti-SigF antagonist in M. smegmatis. The phosphorylation state of Ser-63 was shown to determine the functionality of RsfB as an anti-SigF antagonist. RsbW2 was demonstrated to be the only protein kinase that phosphorylates RsfB in M. smegmatis. Phosphorylation of Ser-63 inactivates RsfB to render it unable to interact with RsbW1. Our comparative RNA sequencing analysis of the wild-type strain of M. smegmatis and its isogenic aa3 mutant strain lacking the aa3 cytochrome c oxidase of the respiratory electron transport chain revealed that expression of the SigF regulon is strongly induced under respiration-inhibitory conditions in an RsfB-dependent way.

In prokaryotes, toxin-antitoxin (TA) systems are commonly found. They likely reflect the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by slowing their growth rate. Genomic analysis of the extremophile Deinococcus radiodurans R1 revealed the presence of eight type II TA systems, including the genes dr0417, dr0660, dr1530, dr0690, and dr1807. Expression of these toxin genes led to inhibition of Escherichia coli growth, whereas their antidote antitoxins were able to recover the growth defect. Remarkably, Dr0417 (DrMazF) showed endoribonuclease activity toward rRNAs as well as mRNAs, as determined by in vivo and in vitro RNA cleavage assays, and this activity was inhibited by Dr0416 (DrMazE). It was also found that the expression of dr0416-0417 module is directly regulated by the DrMazE-MazF complex. Furthermore, this TA module was induced under stress conditions and plays an important role in survival. To understand the regulatory mechanism at the molecular level, we determined the first high-resolution structures of DrMazF alone and of the DrMazE-MazF complex. In contrast with the hetero-hexameric state of typical MazE-MazF complexes found in other species, DrMazE-MazF crystal structure consists of a hetero-trimer, with the DNA-binding domain of DrMazE undergoing self-cleavage at the flexible linker loop. Our structure revealed that the unique residue R54 provides an additional positive charge to the substrate-binding pocket of DrMazF, its mutation significantly affects the endonuclease activity. Thus, our work reports the unique structural and biochemical features of the DrMazE-MazF system.

The BipA (BPI-inducible protein A) protein is highly conserved in a large variety of bacteria and belongs to the translational GTPases, based on sequential and structural similarities. Despite its conservation in bacteria, bipA is not essential for cell growth under normal growth conditions. However, at 20°C, deletion of bipA causes not only severe growth defects but also several phenotypic changes such as capsule production, motility, and ribosome assembly, indicating that it has global regulatory properties. Our recent studies revealed that BipA is a novel ribosome-associating GTPase, whose expression is cold-shock-inducible and involved in the incorporation of the ribosomal protein (r-protein) L6. However, the precise mechanism of BipA in 50S ribosomal subunit assembly is not completely understood. In this study, to demonstrate the role of BipA in the 50S ribosomal subunit and possibly to find an interplaying partner(s), a genomic library was constructed and suppressor screening was conducted. Through screening, we found a suppressor gene, rplT, encoding r-protein L20, which is assembled at the early stage of ribosome assembly and negatively regulates its own expression at the translational level. We demonstrated that the exogenous expression of rplT restored the growth of bipA-deleted strain at low temperature by partially recovering the defects in ribosomal RNA processing and ribosome assembly. Our findings suggest that the function of BipA is pivotal for 50S ribosomal subunit biogenesis at a low temperature and imply that BipA and L20 may exert coordinated actions for proper ribosome assembly under cold-shock conditions.

UBE2N is a K63-specific ubiquitin conjugase linked to various immune disorders and cancer. Here, we demonstrate that UBE2N and its partners UBE2V1 and UBE2V2 are highly expressed in malignant melanoma. Silencing of UBE2N and its partners significantly decreased melanoma cell proliferation and subcutaneous tumor growth. This was accompanied by increased expression of E-cadherin, p16, and MC1R and decreased expression of melanoma malignancy markers including SOX10, Nestin, and ABCB5. Mass spectrometry–based phosphoproteomic analysis revealed that UBE2N loss resulted in distinct alterations to the signaling landscape: MEK/ERK signaling was impaired, FRA1 and SOX10 gene regulators were downregulated, and p53 and p16 tumor suppressors were upregulated. Similar to inhibition of UBE2N and MEK, silencing FRA1 decreased SOX10 expression and cell proliferation. Conversely, exogenous expression of active FRA1 increased pMEK and SOX10 expression, and restored anchorage-independent cell growth of cells with UBE2N loss. Systemic delivery of NSC697923, a small-molecule inhibitor of UBE2N, significantly decreased melanoma xenograft growth. These data indicate that UBE2N is a novel regulator of the MEK/FRA1/SOX10 signaling cascade and is indispensable for malignant melanoma growth. Our findings establish the basis for targeting UBE2N as a potential treatment strategy for melanoma. Significance: These findings identify ubiquitin conjugase UBE2N and its variant partners as novel regulators of MAPK signaling and potential therapeutic targets in melanoma. Cancer Res; 78(22); 6462–72. ©2018 AACR.

Neurofibromatosis type 2 (NF2) syndrome is a very rare human genetic disease, and there has been no proper treatment for it until now. In our recent study, it has been reported that the loss of NF2 activates MAPK signaling through reduction of RKIP in a mesothelioma model. Here, we show that loss of NF2 induces reduction of the TGFβ receptor 2 (TβR2) expression, and an overwhelming expression of TGFβ receptor 1 (TβR1) is activated by physical stimuli such as pressure or heavy materials. Activated TβR1 induces the phosphorylation and degradation of RKIP. RKIP reduction consequently results in MAPK activation as well as Snail-mediated p53 suppression and occurrence of EMT in NF2-deficient cells by physical stimuli. Thus, TβR1 kinase inhibitors restore cell differentiation and induce growth suppression in NF2-deficient Schwannoma cell line and MEF. Moreover, TEW7197, a specific TβR1 kinase inhibitor, reduces tumor formation in the NF2-model mouse (Postn-Cre;NF2f/f). Gene expression profiling reveals that TEW7197 treatment induces the expression of lipid metabolism–related gene set, such as NF2-restored cells in HEI-193 (NF2-deficient Schwannoma). Our results indicate that reduction or deletion of TβR2 or NF2 induces the TβR1-mediated oncogenic pathway, and therefore inhibition of the unbalanced TGFβ signaling is a putative strategy for NF2-related cancers (NF2 syndrome and mesothelioma) and TβR2-mutated advanced cancers. Mol Cancer Ther; 17(11); 2271–84. ©2018 AACR.

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have shown promising potential, specifically because of their accessibility and plasticity. Hence, the clinical applicability of iPSCs was investigated in various fields of research. However, only a few iPSC studies pertaining to osteoarthritis (OA) have been performed so far, despite the high prevalence rate of degenerative joint disease. In this review, we discuss some of the most recent applications of iPSCs in disease modeling and the construction of 3D models in various fields, specifically focusing on osteoarthritis and OA-related conditions. Notably, we comprehensively reviewed the successful results of iPSC-derived disease models in recapitulating OA phenotypes for both OA and early-onset OA to encompass their broad etiology. Moreover, the latest publications with protocols that have used iPSCs to construct 3D models in recapitulating various conditions, particularly the OA environment, were further discussed. With the overall optimistic results seen in both fields, iPSCs are expected to be more widely used for OA disease modeling and 3D model construction, which could further expand OA drug screening, risk assessment, and therapeutic capabilities.

Inorganic iron is essential for various metabolic processes, including RNA synthesis, electron transport, and oxygen detoxification in microorganisms. Many bacterial pathogens compete for iron acquisition in diverse environmental condition such as host. Salmonella Typhimurium SL1344 also requires inorganic iron as a cofactor for growth. When a M9 minimal liquid medium was supplemented with ethylenediamine di-o-hydroxyphenylactic acid (EDDA) which acts as an iron-chelating agent, growth of Salmonella Typhimurium SL1344 in the supplemented medium was completely arrested by deficient of useful iron under iron-depleted condition. However, a number of siderophores, which are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria and fungi, were produced for utilization of restricted iron under iron-depleted condition. A M9 minimal liquid medium complemented with human transferrin (hTf)-iron complex turned completely off production of siderophores, but growth of Salmonella Typhimurium SL1344 maintained level similar to compare one complemented with iron (Ⅲ) chloride (FeCl3). This means that human transferrin (hTf)-bound iron can utilize via directly interaction with Salmonella Typhimurium SL1344 without productions of siderophores. Through construction and analysis of negative mutant for utilization of human transferrin (hTf)-bound iron, we confirm that the bacterium can directly use human transferrin (hTf)-bound iron without extracellularly intermediated carriers such as siderophores.

Background The human hair is a potential material for assessing the exposure to environmental contaminants and tracing human mobility. Although various digestion methods have been proposed for determining trace elements in the human hair, there is no consensus about the best method among them yet. Findings We examined five different methods in order to determine the best method yielding the most accurate and precise data of trace elements in the human hair using two certified reference human hairs (IAEA085 and IAEA086) under controlled conditions (temperature, the volume of hydrogen peroxide (H2O2), and the number of digestion). Results showed that a high temperature of 160 °C yields better recovery rates than a low temperature of 70 °C. Furthermore, the additional input of H2O2 increases the recovery rate from 90 to 102%, and the two-time digestion also promotes the recovery rate. Conclusions This study suggests that although the combination of high temperature (160 °C), high volume of H2O2 (0.4 mL), and two-time digestion yields the most accurate and precise data of trace elements in the human hair, the digestion method should be carefully selected depending on the content of organometallic cation.

Objective: The enzyme composition (NRDT50) which includes glucosyl transferase, fructosyl transferase, amylase, glucose oxidase, and catalase can regulate the absorption of glucose into the body by converting the carbohydrates in food to a form of sugar that is not absorbed in the stomach before being decomposed in the small intestine into glucose. The aim of this study was to evaluate the antidiabetic effects of repeated oral administration of NRDT50 in db/db mice. Methods: The 7-week-old db/db mice were divided into 3 groups; control, NRDT50 (300mg/kg/day), and voglibose (0.3mg/kg/day). Mice received a standard diet containing drugs for 1 month. Fasting and postprandial glucose level was measured every week. Mixed meal test, biochemical assays, and fecal microbiota analysis were performed. Results: There were no significant differences in body weight or food intake between the three groups. However, NRDT50 treatment led to a significant reduction in fasting and postprandial blood glucose levels compared to control after 3, 4 weeks. The blood glucose levels during the mixed meal test were significantly lower in NRDT50 group compared to control group. NRDT50 treatment reduced triglyceride level, tend to reduce LDL level, and increased relative Bacteroidetes-Firmicutes ratio. NRDT50 treatment did not demonstrate any negative side effects on biochemical and histopathological examination. Conclusion: NRDT50 is expected to be useful for people who are at risk of hyperglycemia or diabetes and thus need to regulate blood sugar with safe. It may also improve the gut microbiota profile by inducing the production of oligosaccharides in the alimentary tract.

The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg 2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae ; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der -null phenotype. Only K. pneumoniae and S . Typhimurium Der proteins enabled the E. coli der -null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S . Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der -null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE In Escherichia coli , Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli . Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits.

// Jung-Hyun Cho 1, * , Ho-Young Chun 1, * , Jung Suk Lee 2, * , Jee-Hyun Lee 2 , Kyu Jin Cheong 2 , Youn-Sang Jung 1 , Tae-Gyun Woo 1 , Min-Ho Yoon 1 , Ah-Young Oh 1 , So-Mi Kang 1 , Chunghui Lee 3 , Hokeun Sun 3 , Jihwan Hwang 4 , Gyu-Yong Song 2 , Bum-Joon Park 1 1 Department of Molecular Biology, College of Natural Science, Pusan National University, Busan, Korea 2 College of Pharmacy, Chungnam National University, Daejoen, Korea 3 Department of Statistics, College of Natural Science, Pusan National University, Busan, Korea 4 Department of Microbiology, College of Natural Science, Pusan National University, Busan, Korea * These authors equally contribute at this work Correspondence to: Bum-Joon Park, e-mail: bjpark1219@pusan.ac.kr Gyu-Yong Song, e-mail: gysong@cnu.ac.kr Keywords: stress hormone, MTOC, cancer prevention Received: October 06, 2015 Accepted: April 11, 2016 Published: April 27, 2016 ABSTRACT Stress has been suggested as one of important cause of human cancer without molecular biological evidence. Thus, we test the effect of stress-related hormones on cell viability and mitotic fidelity. Similarly to estrogen, stress hormone cortisol and its relative cortisone increase microtubule organizing center (MTOC) number through elevated expression of γ-tubulin and provide the Taxol resistance to human cancer cell lines. However, these effects are achieved by glucocorticoid hormone receptor (GR) but not by estrogen receptor (ER). Since ginsenosides possess steroid-like structure, we hypothesized that it would block the stress or estrogen-induced MTOC amplification and Taxol resistance. Among tested chemicals, rare ginsenoside, CSH1 (Rg6) shows obvious effect on inhibition of MTOC amplification, γ-tubulin induction and Taxol resistance. Comparing to Fulvestant (FST), ER-α specific inhibitor, this chemical can block the cortisol/cortisone-induced MTOC deregulation as well as ER-α signaling. Our results suggest that stress hormone induced tumorigenesis would be achieved by MTOC amplification, and CSH1 would be useful for prevention of stress-hormone or steroid hormone-induced chromosomal instability.

BPI-inducible protein A (BipA) is a conserved ribosome-associated GTPase in bacteria that is structurally similar to other GTPases associated with protein translation, including IF2, EF-Tu, and EF-G. Its binding site on the ribosome appears to overlap those of these translational GTPases. Mutations in the bipA gene cause a variety of phenotypes, including cold and antibiotics sensitivities and decreased pathogenicity, implying that BipA may participate in diverse cellular processes by regulating translation. According to recent studies, a bipA-deletion strain of Escherichia coli displays a ribosome assembly defect at low temperature, suggesting that BipA might be involved in ribosome assembly. To further investigate BipA’s role in ribosome biogenesis, here, we compared and analyzed the ribosomal protein compositions of MG1655 WT and bipA-deletion strains at 20 °C. Aberrant 50S ribosomal subunits (i.e. 44S particles) accumulated in the bipA-deletion strain at 20 °C, and the ribosomal protein L6 was absent in these 44S particles. Furthermore, bipA expression was significantly stimulated at 20 °C, suggesting that it encodes a cold shock–inducible GTPase. Moreover, the transcriptional regulator cAMP receptor protein (CRP) positively promoted bipA expression only at 20 °C. Importantly, GFP and α-glucosidase refolding assays revealed that BipA has chaperone activity. Our findings indicate that BipA is a cold shock–inducible GTPase that participates in 50S ribosomal subunit assembly by incorporating the L6 ribosomal protein into the 44S particle during the assembly.

It is generally assumed that each organism has evolved to possess a unique ribosomal RNA (rRNA) species optimal for its physiological needs. However, some organisms express divergent rRNAs, the functional roles of which remain unknown. Here, we show that ribosomes containing the most variable rRNAs, encoded by the rrnI operon (herein designated as I-ribosomes), direct the preferential translation of a subset of mRNAs in Vibrio vulnificus, enabling the rapid adaptation of bacteria to temperature and nutrient shifts. In addition, genetic and functional analyses of I-ribosomes and target mRNAs suggest that both I-ribosomal subunits are required for the preferential translation of specific mRNAs, the Shine-Dalgarno sequences of which do not play a critical role in I-ribosome binding. This study identifies genome-encoded divergent rRNAs as regulators of gene expression at the ribosome level, providing an additional level of regulation of gene expression in bacteria in response to environmental changes.

Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

The chapter aims to explain the sources of South Korea’s intractable conflict with Japan and China, by focusing on the impacts of ideational factors in international relations and how these have influenced Koreans’ perceptions towards Japan and China. We found that Koreans have generally unfavourable perceptions towards these two countries: they have negative images towards Japan and ambivalent and complex feelings towards China. In particular, deep-seated historical antagonism based on the events of colonial history and repeated disputes over this historical legacy have strongly affected the policy-making of South Korea and generated quite negative impacts on its bilateral relations with both Japan and China.

Here, we report a resistance mechanism that is induced through the modulation of 16S ribosomal RNA (rRNA) processing on the exposure of Escherichia coli cells to aminoglycoside antibiotics. We observed decreased expression levels of RNase G associated with increased RNase III activity on rng mRNA in a subgroup of E. coli isolates that transiently acquired resistance to low levels of kanamycin or streptomycin. Analyses of 16S rRNA from the aminoglycoside-resistant E. coli cells, in addition to mutagenesis studies, demonstrated that the accumulation of 16S rRNA precursors containing 3–8 extra nucleotides at the 5’ terminus, which results from incomplete processing by RNase G, is responsible for the observed aminoglycoside resistance. Chemical protection, mass spectrometry analysis and cell-free translation assays revealed that the ribosomes from rng-deleted E. coli have decreased binding capacity for, and diminished sensitivity to, streptomycin and neomycin, compared with wild-type cells. It was observed that the deletion of rng had similar effects in Salmonella enterica serovar Typhimurium strain SL1344. Our findings suggest that modulation of the endoribonucleolytic activity of RNase III and RNase G constitutes a previously uncharacterized regulatory pathway for adaptive resistance in E. coli and related gram-negative bacteria to aminoglycoside antibiotics.

Toxin–antitoxin (TA) systems, which consist of an intracellular toxin and its antidote (antitoxin), are encoded by ubiquitous genetic modules in prokaryotes. Commonly, the activity of a toxin is inhibited by its antitoxin under normal growth conditions. However, antitoxins are degraded in response to environmental stress, and toxins liberated from antitoxins consequently induce cell death or growth arrest. In free-living prokaryotes, TA systems are often present in large numbers and are considered to be associated with the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by shifting cells to a slow growth rate. Genomic analysis of the human pathogen Mycobacterium tuberculosis H37Rv (Mtb) revealed the presence of a large number of TA systems. Accordingly, we investigated 5 uncharacterized TA systems (Rv2019-Rv2018, Rv3697c-Rv3697A, Rv3180c-Rv3181c, Rv0299-Rv0298, and Rv3749c-Rv3750c) of Mtb. Among these, the expression of the Rv2019 toxin inhibited the growth of Escherichia coli, and Mycobacterium smegmatis and this growth defect was recovered by the expression of the Rv2018 antitoxin. Interestingly, Rv3180c was toxic only in Mycobacterium smegmatis, whose toxicity was neutralized by Rv3181c antitoxin. In vivo and in vitro assays revealed the ribosomal RNA (rRNA) cleavage activity of the Rv2019 toxin. Moreover, mRNAs appeared to be substrates of Rv2019. Therefore, we concluded that the ribonuclease activity of the Rv2019 toxin triggers the growth defect in E. coli and that the Rv2018 antitoxin inhibits the ribonuclease activity of the Rv2019 toxin.

In this work, PZT (52/48) thin films were deposited at substrate temperature ranging from room temperature (RT) to 600°C and with various oxygen percentage in a mixed argon and oxygen gas at substrate temperature of 600°C on LNO/Si by an r.f. magnetron sputtering method. It was found that the crystallinities were enhanced with increasing substrate temperature. Hence, the ferroelectricities and electrical properties were also simultaneously improved. The crystallinities degraded with increasing O2 percentages, hence, the degraded ferroelectricities and relevant electrical properties.

In this work, PZT (52/48) and PZT (30/70) thin films were deposited at a substrate temperature of 600°C with Argon gas pressure ranging from 2.8 × 10−2 mbar to 4.3 × 10−3 mbar by r. f. magnetron sputtering on LNO/Si substrates. Highly (100)-oriented rhombohedral structured PZT (52/48) thin films were formed. We found that the out-of-plane lattice constant increased commensurately with a decrease in Ar pressure, while remnant polarization and dielectric constant increased with decreasing Ar pressure. On the other hand, the PZT (30/70) thin films sputter-deposited on LNO/Si(100) at substrate temperatures of 600°C with various Ar gas pressures formed a tetragonal structure. The fraction of c-domain, hence the remnant polarization, increased with decreasing Ar pressure, while the dielectric constant decreased with decreasing Ar pressure.

Der (double Era-like GTPase) is an essential GTPase consisting of two GTP-binding motifs in tandem followed by a KH-like domain. Der plays a critical role in 50S ribosome maturation at a later biogenesis step. Here, we attempted to identify a protein interacting with Der that modulates its function and regulation. Using a yeast two-hybrid, we discovered that Der interacts with YihI, which activates the GTPase activity of Der. Its overexpression affected cell growth, causing accumulation of rRNA precursors and an aberrant ribosome profile that was similar to that of Der-depleted cells, suggesting that Der and YihI are involved in the 50S ribosome assembly. The yihI deletion strain showed a shorter lag phase than wild-type strain, suggesting that YihI may be a negative regulator for ribosome assembly. We propose that YihI is a GAP (GTPase-activating protein)-like protein that modulates Der function to negatively regulate cell growth at the beginning of exponential growth.