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Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf-MEK-ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions.

Online learning is implemented in response to emergency remote teaching during global pandemic. We conducted a survey on Life Sciences undergraduate students on their preferences on mode of lesson delivery, mode of learning and learning activities. Students across different study years responded in a similar order ranking blended learning delivery as highly favorable. The survey gathered insightful understandings on Life Sciences undergraduates' learning preferences on online learning which inform future purposeful learning design taking student's preferences into consideration., (© 2023 The Author. Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Osteoclasts (OCs) are clinically important cells that resorb bone matrix. Accelerated bone destruction by OCs is closely linked to the development of metabolic bone diseases. In this study, we screened novel chemical inhibitors targeting OC differentiation to identify drug candidates for metabolic bone diseases. We identified that 1,3-dibenzyl-5-fluorouracil, also named OCI-101, is a novel inhibitor of osteoclastogenesis. The formation of multinucleated OCs is reduced by treatment with OCI-101 in a dose-dependent manner. OCI-101 inhibited the expression of OC markers via downregulation of receptor activator of NF-κB ligand and M-CSF signaling pathways. Finally, we showed that OCI-101 prevents ovariectomy-induced bone loss by suppressing OC differentiation in mice. Hence, these results demonstrated that OCI-101 is a good drug candidate for treating metabolic bone diseases., Competing Interests: Conflict of Interest: The authors declare no potential conflicts of interest., (Copyright © 2022. The Korean Association of Immunologists.)

Diverticulitis in the pediatric population is a very rare cause of abdominal pain. When present in the cecum or ascending colon, it is often incorrectly diagnosed preoperatively as acute appendicitis. This is especially true in Western countries where right-sided diverticulitis is less common. Here we detail a case of a pediatric patient with complicated congenital cecal diverticulitis and review the literature on pertinent management. An extensive work up with imaging and endoscopy was completed and definitive surgical treatment with diverticulectomy an appendectomy was performed. As the incidence of diverticular disease in younger individuals increases, right sided diverticulitis is worthy of consideration on the differential diagnosis.

Radiotherapy (RT) resistance is a major cause of treatment failure in cancers that use definitive RT as their primary treatment modality. This study identifies the cancer/testis (CT) antigen G antigen (GAGE) as a mediator of radio resistance in cervical cancers. Elevated GAGE expression positively associates with de novo RT resistance in clinical samples. GAGE, specifically the GAGE12 protein variant, confers RT resistance through synemin-dependent chromatin localization, promoting the association of histone deacetylase 1/2 (HDAC1/2) to its inhibitor actin. This cumulates to elevated histone 3 lysine 56 acetylation (H3K56Ac) levels, increased chromatin accessibility, and improved DNA repair efficiency. Molecular or pharmacological disruption of the GAGE-associated complex restores radiosensitivity. Molecularly, this study demonstrates the role of GAGE in the regulation of chromatin dynamics. Clinically, this study puts forward the utility of GAGE as a pre-screening biomarker to identify poor responders at initial diagnosis and the therapeutic potential of agents that target GAGE and its associated complex in combination with radiotherapy to improve outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aims: Current treatment options for ovarian clear cell carcinoma (OCCC) are limited to combination of platinum-based and other cytotoxic agents to which patients respond poorly due to intrinsic chemoresistance. There is therefore an urgent need to develop alternative therapeutic strategies for OCCC. Results: Cysteine deprivation suppresses OCCC growth in vitro and in vivo with no apparent toxicity. Modes of cell death induced by cysteine deprivation in OCCC are determined by their innate metabolic profiles. Cysteine-deprived glycolytic OCCC is abolished primarily by oxidative stress-dependent necrosis and ferroptosis, which can otherwise be prevented by pretreatment with antioxidative agents. Meanwhile, OCCC that relies on mitochondria respiration for its bioenergetics is suppressed through apoptosis, which can otherwise be averted by pretreatment with cysteine precursor alone, but not with antioxidative agents. Cysteine deprivation induces apoptosis in respiring OCCC by limiting iron-sulfur (Fe-S) cluster synthesis in the mitochondria, without which electron transport chain may be disrupted. Respiring OCCC responds to Fe-S cluster deficit by increasing iron influx into the mitochondria, which leads to iron overload, mitochondria damage, and eventual cell death. Innovation/Conclusion: This study demonstrates the importance of cysteine availability in OCCC that is for its antioxidative property and its less appreciated role in mitochondria respiration. Regardless of OCCC metabolic profiles, cysteine deprivation abolishes both glycolytic and respiring OCCC growth in vitro and in vivo . Conclusion: This study highlights the therapeutic potential of cysteine deprivation for OCCC.

The use of online platform to conduct teaching and learning activities has becoming a new norm for teachers and students during this COVID-19 outbreak. In this commentary, we share our experiences of using online conferencing platform to promote active and interactive learning among students. We also suggest approaches that teachers can use to design their teaching and learning activities by employing reciprocal teaching as a way to engage students online instead of using the platform for didactic teaching by the teacher. The proposed strategy is applicable and transferable to other domains., (© 2020 International Union of Biochemistry and Molecular Biology.)

A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus , respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, P rtxA Phased hypersensitivity resulting from DNase I cleavage of the P rtxA regulatory region suggests that Lrp probably induces DNA bending in P rtxA Lrp activates P rtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to P rtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses P rtxA , and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of P rtxA , which presumably alters the DNA conformation in P rtxA and thus represses rtxA Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA , thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection. IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host., (Copyright © 2020 Lee et al.)

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter P vvhBA , which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of P vvhBA , whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the P vvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of P vvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection., (© 2020 Choi et al.)

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa , has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.

Increasing antibiotic resistance has led to the development of new strategies to combat bacterial infection. Anti-virulence strategies that impair virulence of bacterial pathogens are one of the novel approaches with less selective pressure for developing resistance than traditional strategies that impede viability. In this study, a small molecule CM14 [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] that inhibits the activity of HlyU, a transcriptional regulator essential for the virulence of the fulminating human pathogen Vibrio vulnificus, has been identified. Without affecting bacterial growth or triggering the host cell death, CM14 reduces HlyU-dependent expression of virulence genes in V. vulnificus. In addition to the decreased hemolysis of human erythrocytes, CM14 impedes host cell rounding and lysis caused by V. vulnificus. Notably, CM14 significantly enhances survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. Biochemical, mass spectrometric, and mutational analyses revealed that CM14 inhibits HlyU from binding to target DNA by covalently modifying Cys30. Remarkably, CM14 decreases the expression of various virulence genes of other Vibrio species and thus attenuates their virulence phenotypes. Together, this molecule could be an anti-virulence agent against HlyU-harboring Vibrio species with a low selective pressure for the emergence of resistance.

Slow and continuous release of H 2 S by GYY4137 has previously been demonstrated to kill cancer cells by increasing glycolysis and impairing anion exchanger and sodium/proton exchanger activity. This action is specific for cancer cells. The resulting lactate overproduction and defective pH homeostasis bring about intracellular acidification-induced cancer cell death. The present study investigated the potency of H 2 S released by GYY4137 against invasive and radio- as well as chemo-resistant cancers, known to be glycolytically active. We characterized and utilized cancer cell line pairs of various organ origins, based on their aggressive behaviors, and assessed their response to GYY4137. We compared glycolytic activity, via lactate production, and intracellular pH of each cancer cell line pair after exposure to H 2 S. Invasive and therapy resistant cancers, collectively termed aggressive cancers, are receptive to H 2 S-mediated cytotoxicity, albeit at a higher concentration of GYY4137 donor. While lactate production was enhanced, intracellular pH of aggressive cancers was only modestly decreased. Inherently, the magnitude of intracellular pH decrease is a key determinant for cancer cell sensitivity to H 2 S. We demonstrated the utility of coupling GYY4137 with either simvastatin, known to inhibit monocarboxylate transporter 4 (MCT4), or metformin, to further boost glycolysis, in bringing about cell death for aggressive cancers. Simvastatin inhibiting lactate extrusion thence contained excess lactate induced by GYY4137 within intracellular compartment. In contrast, the combined exposure to both GYY4137 and metformin overwhelms cancer cells with lactate over-production exceeding its expulsion rate. Together, GYY4137 and simvastatin or metformin synergize to induce intracellular hyper-acidification-mediated cancer cell death.

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA , encoding a putative phospholipase, Vv PlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that Vv PlpA is a phospholipase A 2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that Vv PlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that Vv PlpA is a type II secretion system-dependent secretory phospholipase A 2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Aurora kinases, Aurora A (AURKA), Aurora B (AURKB), and Aurora C (AURKC), are serine/threonine kinases required for the control of mitosis (AURKA and AURKB) or meiosis (AURKC). Several Aurora kinase inhibitors are being investigated as novel anticancer therapeutics. Recent studies demonstrated that AURKC activation contributes to breast cancer cell transformation. Therefore, AURKC is both a promising marker and therapeutic target for breast cancer; however, its signaling network has not been fully characterized. Using translocation-based cellular assays, we identified IκBα as a binding partner of AURKC, and found that AURKC phosphorylates IκBα at Ser32, thereby activating it. In silico modeling and computational analyses revealed a small-molecule inhibitor (AKCI) that blocked the AURKC-IκBα interaction and exerted antitumor activity in MDA-MB-231 breast cancer cells. Specifically, AKCI induced G2/M cell-cycle arrest through modulation of the p53/p21/CDC2/cyclin B1 pathways. In addition, the drug significantly inhibited MDA-MB-231 cell migration and invasion, as well as decreasing colony formation and tumor growth. Via its interaction with IκBα, AURKC indirectly induced NF-κB activation; accordingly, AKCI decreased PMA-induced activation of NF-κB. Thus, the small-molecule inhibitor AKCI represents a first step towards developing targeted inhibitors of AURKC protein binding, which may lead to further advances in the treatment of breast cancer., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Herein, we present a simple readout of the binding between a chemical drug and its target proteins in the cytoplasm by using a two-step bioorthogonal labeling method combined with spatially-localized expression of proteins. Dasatinib was modified with trans-cyclooctene (TCO), and its cytoplasmic target kinases were expressed in intracellular compartments, such as endosomes and F-actins. After bioorthogonal labeling, the colocalization between Dasatinib and its target proteins was observed in intracellular compartments.

The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H2O2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H2O2 exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H2O2 with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO3H) by high H2O2 in vitro and in vivo, where the modification deterred the transcription of prx2 The DNA binding preferences of OxyR25CA-C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the -35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H2O2 sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: To investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer., Methods: The inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis., Results: The abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed., Conclusion: Major ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.

Nuclear factor-[Formula: see text]B (NF-[Formula: see text]B)/Rel transcription factors are best known for their central roles in promoting cell survival in cancer. NF-[Formula: see text]B antagonizes tumor necrosis factor (TNF)-[Formula: see text]-induced apoptosis through a process involving attenuation of the c-Jun-N-terminal kinase (JNK). However, the role of JNK activation in apoptosis induced by negative regulation of NF-[Formula: see text]B is not completely understood. We found that allergen-removed Rhus verniciflua Stokes (aRVS) extract-mediated NF-[Formula: see text]B inhibition induces apoptosis in SKOV-3 ovarian cancer cells via the serial activation of caspases and SKOV-3 cells are most specifically suppressed by aRVS. Here, we show that in addition to activating caspases, aRVS extract negatively modulates the TNF-[Formula: see text]-mediated I[Formula: see text]B/NF-[Formula: see text]B pathway to promote JNK activation, which results in apoptosis. When the cytokine TNF-[Formula: see text] binds to the TNF receptor, I[Formula: see text]B dissociates from NF-[Formula: see text]B. As a result, the active NF-[Formula: see text]B translocates to the nucleus. aRVS extract (0.5[Formula: see text]mg/ml) clearly prevented NF-[Formula: see text]B from mobilizing to the nucleus, resulting in the upregulation of JNK phosphorylation. This subsequently increased Bax activation, leading to marked aRVS-induced apoptosis, whereas the JNK inhibitor SP600125 in aRVS extract treated SKOV-3 cells strongly inhibited Bax. Bax subfamily proteins induced apoptosis through caspase-3. Thus, these results indicate that aRVS extract contains components that inhibit NF-[Formula: see text]B signaling to upregulate JNK activation in ovarian cancer cells and support the potential of aRVS as a therapeutic agent for ovarian cancer.

Hydrogen sulfide (H2S) donors including organosulfur compounds (OSC), inorganic sulfide salts, and synthetic compounds are useful tools in studies to elucidate the effects of H2S in cancer biology. Studies using such donors have shown the ability of H2S to suppress tumor growth both in vitro and in vivo, with some of them suggesting the selectivity of its cytotoxic effects to cancer cells. In addition to promoting cancer cell death, H2S donors were also found to inhibit cancer angiogenesis and metastasis. The underlying mechanisms for the anticancer activities of H2S involve (1) cell signaling pathways, such as MAPK and STAT; (2) cell cycle regulation; (3) microRNAs regulation; and (4) cancer metabolism and pH regulation. Altogether, compiling evidences have demonstrated the great potential of using H2S donors as anticancer agents. Nevertheless, the application and development of H2S for therapy are still facing challenges as identification of molecular targets of H2S awaits further investigation.

Herein, we report the development of two fluorescent probes for the highly selective and sensitive detection of H2S. The probes take advantage of a Cu(II)-cyclen complex, which acts as a reaction center for H2S and as a quencher of BODIPY (boron-dipyrromethene)-based fluorophores with emissions at 765 and 680 nm, respectively. These non-fluorescent probes could only be turned on by the addition of H2 S, and not by other potentially interfering biomolecules, including reactive oxygen species, cysteine, and glutathione. In a chemical system, both probes detected H2S with a detection limit of 80 nM. The probes were successfully used for the endogenous detection of H2S in HEK 293 cells, for measuring the H2S-release activity of dietary organosulfides in MCF-7 cells, and for the in vivo imaging of H2S in mice., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background & Aims: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice., Methods: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells., Results: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice., Conclusions: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Many disparate studies have reported the ambiguous role of hydrogen sulfide (H2 S) in cell survival. The present study investigated the effect of H2 S on the viability of cancer and non-cancer cells., Experimental Approach: Cancer and non-cancer cells were exposed to H2 S [using sodium hydrosulfide (NaHS) and GYY4137] and cell viability was examined by crystal violet assay. We then examined cancer cellular glycolysis by in vitro enzymatic assays and pH regulator activity. Lastly, intracellular pH (pHi ) was determined by ratiometric pHi measurement using BCECF staining., Key Results: Continuous, but not a single, exposure to H2 S decreased cell survival more effectively in cancer cells, as compared to non-cancer cells. Slow H2 S-releasing donor, GYY4137, significantly increased glycolysis, leading to overproduction of lactate. H2 S also decreased anion exchanger and sodium/proton exchanger activity. The combination of increased metabolic acid production and defective pH regulation resulted in an uncontrolled intracellular acidification, leading to cancer cell death. In contrast, no significant intracellular acidification or cell death was observed in non-cancer cells., Conclusions and Implications: Low and continuous exposure to H2 S targets metabolic processes and pH homeostasis in cancer cells, potentially serving as a novel and selective anti-cancer strategy., (© 2014 The British Pharmacological Society.)

Hydrogen sulfide (H2S) has been implicated to exhibit antioxidative properties in many models. CSE (cystathionine γ-lyase) is an important enzyme responsible for endogenous H2S production in mammalian systems, but little is known about the modulation of endogenous H2S production and its antioxidative activity. We found that inhibiting CSE activity with PAG (propargylglycine) or silencing CSE expression using an siRNA approach resulted in a greater reduction in cell viability under exposure to the oxidizing agent hydrogen peroxide (H2O2). Cellular oxidative stress also increased significantly upon PAG inhibition or CSE knockdown. Further experiments using an activity-null Y60A mutant, a hyperactive E339A mutant and a control E349A mutant demonstrated that modulation of CSE catalytic activity altered its antioxidative activity. The increased sensitivity towards H2O2-induced cytotoxicity in CSE-siRNA-transfected cells was associated with a decreased glutathione concentration (GSH) and glutathione ratio (GSH/GSSG). Incubation of cells with exogenous H2S increased the GSH concentration and GSH/GSSG ratio. Moreover, exogenous H2S preserved the cellular glutathione status under BSO (buthionine sulfoximine)-induced glutathione depletion. Taken together, the results of the present study provide molecular insights into the antioxidative activity of CSE and highlights the importance of the CSE/H2S system in maintaining cellular glutathione status.

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, and notorious for resistance to chemoradiotherapy. MicroRNAs (miRNAs) are significantly involved in the initiation and progression of numerous cancers; however, the role of miRNAs in recurrence of tumors remains unknown. Here we tried to identify novel miRNAs that are differentially expressed in recurrent GBM. Tissue samples were obtained from patients with primary and recurrent GBM treated with chemoradiotherapy, and the expression changes of miRNAs were measured by microarray. A total of 318 miRNAs were expressed in the GBM patients. The expression of 43 miRNAs were significantly altered at least 2-fold in primary and recurrent GBMs. Bioinformatic analysis revealed that the differentially expressed miRNAs and their putative target genes were mainly involved in cell death, cellular development, and cellular growth and proliferation, which are the key regulators for stem cells. Pathway analysis supported that the miRNAs may regulate signaling associated with induction and maintenance of cancer and stem cell, such as p53, ErbB1, Notch, Wnt, and TGF-β signaling pathways. These data suggest that, in recurrent GBM, growth factor and anti-apoptotic signalings for cancer cell growth and proliferation are regulated by miRNAs. Our findings will aid future research in understanding the pathophysiology of recurrent GBM and identifying diagnostic markers and/or therapeutic targets for recurrence of GBM.

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT-ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress-induced, nine proteins with inflammatory stress-induced, and 14 proteins with endoplasmic reticulum stress-induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c-Jun amino-terminal kinase-dependent manner. In addition, three proteins, 14-3-3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD-95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lactobacillus casei extract (LBX) has been reported to prevent gastric cancer, but the underlying mechanism remains unclear. The proliferation and cell death of gastric cancer KATO3 cells were examined after treatment with LBX for various times and at various doses. LBX inhibited the growth of gastric cancer cells and induced apoptosis by inactivating NF-κB promoter activity. Apoptosis induced by LBX, however, is not directly associated with the intrinsic mitochondrial pathway. Immunoblot analysis revealed that LBX decreased the expressions of NF-κB and IκB. The reduced NF-κB levels led to the decreased phosphorylation of mTOR signaling components, such as PI3K, Akt, and (p70)S6 kinase. These results showed for the first time that LBX induced apoptosis in gastric cancer cells by inhibiting NF-κB and mTOR-mediated signaling.

Alternanthera mosaic virus (AltMV) triple gene block 3 (TGB3) protein is involved in viral movement. AltMV TGB3 subcellular localization was previously shown to be distinct from that of Potato virus X (PVX) TGB3, and a chloroplast binding domain identified; veinal necrosis and chloroplast vesiculation were observed in Nicotiana benthamiana when AltMV TGB3 was over-expressed from PVX. Plants with over-expressed TGB3 showed more lethal damage under dark conditions than under light. Yeast-two-hybrid analysis and bimolecular fluorescence complementation (BiFC) reveal that Arabidopsis thaliana PsbO1 has strong interactions with TGB3; N. benthamiana PsbO (NbPsbO) also showed obvious interaction signals with TGB3 through BiFC. These results demonstrate an important role for TGB3 in virus cell-to-cell movement and virus-host plant interactions. The Photosystem II oxygen-evolving complex protein PsbO interaction with TGB3 is presumed to have a crucial role in symptom development and lethal damage under dark conditions. In order to further examine interactions between AtPsbO1, NbPsbO, and TGB3, and to identify the binding domain(s) in TGB3 protein, BiFC assays were performed between AtPsbO1 or NbPsbO and various mutants of TGB3. Interactions with C-terminally deleted TGB3 were significantly weaker than those with wild-type TGB3, and both N-terminally deleted TGB3 and a TGB3 mutant previously shown to lose chloroplast interactions failed to interact detectably with PsbO in BiFC. To gain additional information about TGB3 interactions in AltMV-susceptible plants, we cloned 12 natural AltMV TGB3 sequence variants into a PVX expression vector to examine differences in symptom development in N. benthamiana. Symptom differences were observed on PVX over-expression, with all AltMV TGB3 variants showing more severe symptoms than the WT PVX control, but without obvious correlation to sequence differences.

Calcium is an essential plant macronutrient that has unique structural and signaling roles related to tip-burn disorder in Brassica spp. crops. For two types of cabbage inbred lines, tip-burn susceptible and resistant, we measured and compared major macronutrient cations, including Ca(2+), in leaves. In both lines, Ca(2+), Mg(2+), Na(+), and K(+), accumulated more in leaf base than in leaf apex. Ca(2+) and K(+) were >2 times more abundant in the tip-burn resistant line, while Na(+) was higher in the susceptible line. Ca(2+) differences between the two lines resulted from differential accumulation of calcium into cell vacuoles. We profiled major vacuolar Ca(2+) transporters, in both cabbage lines, by growth time and intercellular Ca(2+) concentration. Expression pattern of several Ca(2+) transporter genes differed between tip-burn susceptible and resistant lines by growth time points. We also identified promoter regions of the major Ca(2+) vacuole transporter genes, CAX1, ACA4, and ACA11, which displayed hormonal, light and defense-related cis-acting regulatory elements. Finally, transporter genes in the two cabbage lines responded differently to abiotic stresses, demonstrating diversity in gene regulation among orthologous genes.

Mast cells are involved in allergic responses, protection against pathogens and autoimmune diseases. Dexamethasone (Dex) and other glucocorticoids suppress FcεRI-mediated release of inflammatory mediators from mast cells. The inhibition mechanisms were mainly investigated on the downstream signaling of Fc receptor activations. Here, we addressed the effects of Dex on Fc receptor expressions in rat mast cell line RBL-2H3. We measured mRNA levels of Fc receptors by real-time PCR. As expected, Dex decreased the mRNA levels of activating Fc receptor for IgE (FcεR) I and increased the mRNA levels of the inhibitory Fc receptor for IgG FcγRIIb. Interestingly, Dex stimulated transcriptions of other activating receptors such as Fc receptors for IgG (FcγR) I and FcγRIII. To investigate the mechanisms underlying transcriptional regulation, we employed a transcription inhibitor actinomycin D and a translation inhibitor cycloheximide. The inhibition of protein synthesis without Dex treatment enhanced FcγRI and FcγRIII mRNA levels potently, while FcεRI and FcγRIIb were minimally affected. Next, we examined expressions of the Fc receptors on cell surfaces by the flow cytometric method. Only FcγRIIb protein expression was significantly enhanced by Dex treatment, while FcγRI, FcγRIII and FcεRI expression levels were marginally changed. Our data showed, for the first time, that Dex regulates Fc receptor expressions resulting in augmentation of the inhibitory receptor FcγRIIb.

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4's nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.

The current standard of treatment against tuberculosis consists of a cocktail of first-line drugs, including isoniazid and pyrazinamide. Although these drugs are known to be bactericidal, contribution of host cell responses in the context of antimycobacterial chemotherapy, if any, remains unknown. We demonstrate that isoniazid and pyrazinamide promote autophagy activation and phagosomal maturation in Mycobacterium tuberculosis (Mtb)-infected host cells. Treatment of Mtb-infected macrophages with isoniazid or pyrazinamide caused significant activation of cellular and mitochondrial reactive oxygen species and autophagy, which was triggered by bacterial hydroxyl radical generation. Mycobacterium marinum-infected autophagy-defective, atg7 mutant Drosophila exhibited decreased survival rates, which could not be rescued by antimycobacterial treatment, indicating that autophagy is required for effective antimycobacterial drug action in vivo. Moreover, activation of autophagy by antibiotic treatment dampened Mtb-induced proinflammatory responses in macrophages. Together, these findings underscore the importance of host autophagy in orchestrating successful antimicrobial responses to mycobacteria during chemotherapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Microtubules are a component of the cytoskeleton and are important for maintaining cell structure and providing platforms for intracellular transport in diverse cellular processes. Microtubule plus-end tracking proteins (+TIPs), a structurally and functionally diverse group of proteins, are specifically accumulated in the microtubule plus end and regulate dynamic microtubule behavior. We characterized the +TIPs, Clip1, p150(glued), Clasp1, Lis1 and Stim1, in Xenopus laevis and report their expression patterns during embryogenesis in this paper. All the five +TIP genes are maternally expressed and have similar expression patterns during Xenopus embryo development. The expression of +TIPs is localized in the animal hemisphere and ectoderm region at early stages of embryonic development. As development progresses to later stages, the ectodermal expression of +TIPs persists in head and neural tube structures. Clasp1, p150(glued) and Lis1 in particular are specifically expressed in the cranial nerves. Importantly, +TIPs are also expressed in the involuting mesoderm during gastrulation. This is the first study of developmental expression patterns of +TIPs, and our analysis provides insight that could serve as the basis for future research of microtubules in vertebrate development, cell movements during gastrulation and neurogenesis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Targeted disruption of STAT3 function has proven to be a useful cancer therapeutic approach by inducing apoptotic cell death. Cucurbitacin is currently under development as a small molecule of STAT3 inhibitor to trigger cell death in many cancers. Here, we systematically studied the molecular mechanisms underlying cucurbitacin-induced cell death, in particular the involvement of autophagy. Treatment with cucurbitacin resulted in non-apoptotic cell death in a caspase-independent manner. Notably, cucurbitacin enhanced excessive conversion of lipidated LC3 (LC3-II) and accumulation of autophagosomes in many cell types. Such autophagy and cell death induced by cucurbitacin were independent of its ability to inhibit STAT3 function, but mainly mediated by enhanced production of mitochondrial-derived reactive oxygen species (ROS), and subsequently activation of extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK). Interestingly, both the autophagy inhibitor wortmannin and knockdown of Atg5 or Beclin 1 failed to rescue the cells from cucurbitacin-induced cell death, as suppression of autophagy induced the mode of cell death to shift from autophagic cell death to caspase-dependent apoptosis. Thus the present study provides new insights into the molecular mechanisms underlying cucurbitacin-mediated cell death and supports cucurbitacin as a potential anti-cancer drug through modulating the balance between autophagic and apoptotic modes of cell death.

The ubiquitin hybrid genes Uba80 and Uba52 encode ubiquitin (Ub), which is fused to the ribosomal proteins S27a (RPS27a) and L40 (RPL40), respectively. Here, we show that these genes are preferentially over-expressed during hepatoma cell apoptosis. Experiments using the tet-inducible transgenic system revealed that over-expression of the ubiquitin hybrid genes sensitized the cells to apoptosis. Further analysis suggested that Ub, and not RPS27a or RPL40, was associated with apoptotic cell death. Cleavage-resistant mutation analysis revealed that the N-terminal portion and the last two amino acids (GG) of Ub are critical for cleavage at the junction between the two protein moieties. An apoptogenic stimulus enhances the nuclear targeting and aggregation of Ub in the nucleus, resulting in histone H2A deubiquitylation followed by abnormal ubiquitylation of the nuclear envelope and the lamina. These events accompany the apoptotic nuclear morphology in the late stage of apoptosis. Each fused RP is localized in the nucleoli. These results suggest a role for Ub hybrid proteins in the altered nuclear dynamics of Ub during tumor cell apoptosis induced by apoptogenic stimuli.

Administration of mountain ginseng (MG) extract can restore advanced cancer to a normal state. To elucidate the mechanism by which MG extract prevents the progression of lung cancer, the processes of proliferation and death of lung cancer cells (A549) were examined after treatment with MG extract. Butanol-extracted MG (BX-MG) showed a high inhibitory effect (IC(50) = 2 mg/ml) by attenuating proliferation and inducing apoptosis in lung cancer cells. By HPLC-UV analysis of BX-MG, ginsenosides, Rb1 was identified as the most abundant ginsenoside, followed by Rg1, Re, Rc and Rb2. BX-MG induced caspase-3 dependent apoptosis by inhibiting NF-κB. In addition, BX-MG activated p53 and p21, resulting in the attenuated proliferation of A549 cells. Reduced activity of the NF-κB promoter and increased activity of the p53 promoter indicate that BX-MG regulates apoptosis at the level of transcription in lung cancer cells. Furthermore, BX-MG blocked the nuclear translocation of RelA and the associated reduction in surviving. These results suggest that BX-MG inhibits lung cancer cell growth by activating tumor suppressors and inhibiting nuclear translocation of NF-κB.

The slow-releasing hydrogen sulfide (H₂S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H₂S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H₂S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H₂S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G₂/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100-300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H₂S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H₂S donors should be investigated further as potential anti-cancer agents.

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

Autophagy and vitamin D3-mediated innate immunity have been shown to confer protection against infection with intracellular Mycobacterium tuberculosis. Here, we show that these two antimycobacterial defenses are physiologically linked via a regulatory function of human cathelicidin (hCAP-18/LL-37), a member of the cathelicidin family of antimicrobial proteins. We show that 1,25-dihydroxyvitamin D3 (1,25D3), the active form of vitamin D, induced autophagy in human monocytes via cathelicidin, which activated transcription of the autophagy-related genes Beclin-1 and Atg5. 1,25D3 also induced the colocalization of mycobacterial phagosomes with autophagosomes in human macrophages in a cathelicidin-dependent manner. Furthermore, the antimycobacterial activity in human macrophages mediated by physiological levels of 1,25D3 required autophagy and cathelicidin. These results indicate that human cathelicidin, a protein that has direct antimicrobial activity, also serves as a mediator of vitamin D3-induced autophagy.

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.

Evaluating the toxicity of nanoparticles is an integral aspect of basic and applied sciences, because imaging applications using traditional organic fluorophores are limited by properties such as photobleaching, spectral overlaps, and operational difficulties. This study investigated the toxicity of nanoparticles and their biological mechanisms. We found that nanoparticles, quantum dots (QDs), considerably activated the production of tumor necrosis factor (TNF)-alpha and CXC-chemokine ligand (CXCL) 8 through reactive oxygen species (ROS)- and mitogen-activated protein kinases (MAPKs)-dependent mechanisms in human primary monocytes. Nanoparticles elicited a robust activation of intracellular ROS, phosphorylation of p47phox, and nicotinamide adenine dinucleotide phosphate oxidase activities. Blockade of ROS generation with antioxidants significantly abrogated the QD-mediated TNF-alpha and CXCL8 expression in monocytes. The induced ROS generation subsequently led to the activation of MAPKs, which were crucial for mRNA and protein expression of TNF-alpha and CXCL8. Furthermore, confocal and electron microscopy analyses showed that internalized QDs were trapped in cytoplasmic vesicles and compartmentalized inside lysosomes. Finally, several repeated intravenous injections of QDs caused an increase in neutrophil infiltration in the lung tissues in vivo. These results provide novel insights into the QD-mediated chemokine induction and inflammatory toxic responses in vitro and in vivo.

Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn196 and Asn344 respectively. The glycosylation of Asn344 is critical for the folding process; however, glycosylation of Asn196 is not. Interestingly, the N196-glycan (glycosylation of Asn196) remains in an immature form (Glc1Man9GlcNAc2). The mutation of Asn196 to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

Gp91(phox)/NADPH oxidase (NOX) 2 is the main catalytic component of NOX, which mediates the phagocytic killing of ingested pathogens via the production of reactive oxygen species (ROS). However, Mycobacterium tuberculosis (Mtb) is relatively resistant to the microbicidal effects of ROS. Thus, the exact roles of NOX2 in the innate immune control against Mtb infection are not fully resolved. In this study, we show that NOX2 is essential for TLR2-dependent inflammatory responses and 1,25-dihydroxyvitamin D(3) (1,25D(3))-mediated antimicrobial activity against Mtb via cathelicidin expression. NOX2-null macrophages prominently abrogated Mtb-induced ROS production and inflammatory signaling activation in a TLR2-dependent manner. Mtb triggered a physical association between NOX2 and TLR2. In addition, the knockdown of NOX2 inhibited 1,25D(3)-triggered antimicrobial activity against viable Mtb through the modulation of cathelicidin expression in human macrophages. Treatment of NOX2 knocked down cells with cathelicidin restored the 1,25D(3)-induced antimicrobial effect, suggesting that the NOX2-dependent induction of cathelicidin in macrophages is part of a defensive strategy against Mtb. Furthermore, cathelicidin expression was required for the Mtb-induced release of ROS and the production of proinflammatory cytokines/chemokines, indicating a positive circuit of inflammation in response to Mtb. Our data collectively demonstrate a novel regulatory mechanism for TLR2-dependent innate responses to Mtb involving crosstalk between NOX2 and TLR2 and the expression of cathelicidin.

In this paper, we report a simple method for spatially confining Bacillus subtilis (BS) spores into semi-three dimensional, non-biofouling microwells by using biospecific (such as biotin-streptavidin) interactions. Non-biofouling poly(ethylene glycol) (PEG)-based microwells were fabricated by employing a process of capillary molding on a glass slide. The biospecific interactions between biotinylated BS spores and streptavidin led to the selective deposition of BS spores onto the bottom of the microwells of which presented streptavidin. The viability of the patterned spores was confirmed by the induction of germination. Bacterial spores were found to maintain extreme robustness until they were exposed to favorable conditions. This work suggests that the use of bacterial spore-based sensors would increase the shelf-life (such as long-term storage and stability) of cell-based sensors.

Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H(2)O(2). The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor kappaB and mitogen-activated protein kinase (MAPK), in Prx II-deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47(phox). Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II-deficient mice than in wild-type mice. Intravenous injection of Prx II-deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II-deficient cells, which suggests that intracellular H(2)O(2) is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.