STING mediates nuclear PD-L1 targeting-induced senescence in cancer cells.

Immune checkpoint molecule programmed death-ligand 1 (PD-L1) is overexpressed in cancer cells and imparts resistance to cancer therapy. Although membrane PD-L1 has been targeted for cancer immune therapy, nuclear PD-L1 was reported to confer cancer resistance. Therefore, it is important to regulate the nuclear PD-L1. The mechanisms underlying the therapeutic efficacy of PD-L1 targeting have not been well-established. Cellular senescence has been considered a pivotal mechanism to prevent cancer progression, and recently, PD-L1 inhibition was shown to be involved in cancer cell senescence. However, the relevance of PD-L1 targeting-induced senescence and the role of stimulator of interferon genes (STING) has not been reported. Therefore, we aimed to identify the role of PD-L1 in cancer progression and how it regulates cancer prevention. In this study, we found that PD-L1 depletion-induced senescence via strong induction of STING expression in mouse melanoma B16-F10 and colon cancer CT26 cells, and in human melanoma A375 and lung cancer A549 cells. Interestingly, nuclear PD-L1 silencing increased STING promoter activity, implying that PD-L1 negatively regulates STING expression via transcriptional modulation. Furthermore, we showed that PD-L1 binds to the STING promoter region, indicating that PD-L1 directly controls STING expression to promote cancer growth. In addition, when we combined PD-L1 silencing with the senescence-inducing chemotherapeutic agent doxorubicin, the effect of PD-L1-targeting was even more powerful. Overall, our findings can contribute to the understanding of the role of PD-L1 in cancer therapy by elucidating a novel mechanism for PD-L1 targeting in cancer cells.

HMGB1 orchestrates STING-mediated senescence via TRIM30α modulation in cancer cells.

Although cellular senescence has emerged as a novel therapeutic concept in cancer, its underlying mechanisms remain unclear. High mobility group box 1 (HMGB1) and stimulator of interferon genes (STING) are involved in senescence. However, their interactions in senescence have not been reported. Therefore, in this study, we investigated the relationships between HMGB1 and STING in senescence in cancer and other cells. In mouse melanoma cells and several other cell lines, doxorubicin treatment induced senescence in an HMGB1-dependent manner. These responses were mediated by STING, and this function of STING was negatively regulated by the E3 ligase tripartite motif protein 30α (TRIM30α). We also found that HMGB1 bound to the TRIM30α promoter and then suppressed its expression by inhibiting its transcription, which enhanced STING-induced senescence. This mechanism was further mediated by signal transducer and activator of transcription 6 (STAT6) and p21. Overall, our findings demonstrated that HMGB1 orchestrated STING-STAT6-p21-mediated senescence by regulating TRIM30α as an alternative anticancer mechanism.

A Study on Herd Immunity of COVID-19 in South Korea: Using a Stochastic Economic-Epidemiological Model.

Vaccination is an effective measure to control the diffusion of infectious disease such as COVID-19. This paper analyzes the basic reproduction number in South Korea which enables us to identify a necessary level of vaccine stockpile to achieve herd immunity. An susceptible-infected-susceptible model is adopted that allows a stochastic diffusion. The result shows that the basic reproduction number of South Korea is approximately 2 which is substantially lower than those of the other regions. The herd immunity calculated from economic-epidemiological model suggests that at least 62% of the susceptible population be vaccinated when COVID-19 vaccine becomes available.

Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEFOprN Efflux Pump in Pseudomonas aeruginosa.

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.

MS2 Labeling of Endogenous Beta-Actin mRNA Does Not Result in Stabilization of Degradation Intermediates.

The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of ß-actin mRNA extracted from the Actb-MBS knock-in and MBS×MCP hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of ß-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.

Recent progress in single-molecule studies of mRNA localization in vivo.

From biogenesis to degradation, mRNA goes through diverse types of regulation and interaction with other biomolecules. Uneven distribution of mRNA transcripts and the diverse isoforms and modifications of mRNA make us wonder how cells manage the complexity and keep the functional integrity for the normal development of cells and organisms. Single-molecule microscopy tools have expanded the scope of RNA research with unprecedented spatiotemporal resolution. In this review, we highlight the recent progress in the methods for labeling mRNA targets and analyzing the quantitative information from fluorescence images of single mRNA molecules. In particular, the MS2 system and its various applications are the main focus of this article. We also review how recent studies have addressed biological questions related to the significance of mRNA localization in vivo. Efforts to visualize the dynamics of single mRNA molecules in live cells will push forward our knowledge on the nature of heterogeneity in RNA sequence, structure, and distribution as well as their molecular function and coordinated interaction with RNA binding proteins.

NAD(+)-specific glutamate dehydrogenase (EC.1.4.1.2) in Streptomyces coelicolor; in vivo characterization and the implication for nutrient-dependent secondary metabolism.

While glutamate and glutamate-rich compounds are widely used for culturing Streptomyces sp., little is known regarding glutamate catabolism at molecular level. Noting the presence of two distinct putative glutamate dehydrogenases (GDH), we constructed knockout mutants of each gene with Streptomyces coelicolor M145 and examined the functionality related to antibiotic production. Out of the two, the sco2999 knockout (ΔgdhB, NAD(+)-specific) showed outstanding effects; it decreased the growth sevenfold but initiated the undecylprodigiosin (RED) production in complex Difco nutrient media which otherwise does not support the production from M145. With glucose supplementation, the growth difference by ΔgdhB disappeared but we could obtain significantly increased actinorhodin (ACT) and RED biosynthesis with the mutant by limiting the glucose content (0.5∼1.0 %, w/v). Complementing the gene to the knockout mutant inhibited the production, confirming its gene specificity. Along with the extended impacts on overall nitrogen metabolism based on the intracellular metabolite analysis and enzyme assays, GdhB and glutamate utilization were shown to interfere with N-acetylglucosamine metabolism and the activity of its associated global transcriptional regulator (DasR). Taken together, GdhB-subjected to the nutritional context-dependent regulation-is proposed as a key member of central nitrogen metabolism to control the secondary metabolism initiation in exploiting the organic nitrogen sources.

Transcriptional analysis of the cell division-related ssg genes in Streptomyces coelicolor reveals direct control of ssgR by AtrA.

SsgA-like proteins are a family of actinomycete-specific regulatory proteins that control cell division and spore maturation in streptomycetes. SsgA and SsgB together activate sporulation-specific cell division by controlling the localization of FtsZ. Here we report the identification of novel regulators that control the transcription of the ssgA-like genes. Transcriptional regulators controlling ssg gene expression were identified using a DNA-affinity capture assay. Supporting transcriptional and DNA binding studies showed that the ssgA activator gene ssgR is controlled by the TetR-family regulator AtrA, while the γ-butyrolactone-responsive AdpA (SCO2792) and SlbR (SCO0608) and the metabolic regulator Rok7B7 (SCO6008) were identified as candidate regulators for the cell division genes ssgA, ssgB and ssgG. Transcription of the cell division gene ssgB depended on the sporulation genes whiA and whiH, while ssgR, ssgA and ssgD were transcribed independently of the whi genes. Our work sheds new light on the mechanisms by which sporulation-specific cell division is controlled in Streptomyces.

NdgR, a common transcriptional activator for methionine and leucine biosynthesis in Streptomyces coelicolor.

We show here that NdgR, a known transcriptional activator of isopropylmalate dehydratase in actinomycetes, may have other targets in the cell. An in-frame deletion mutant of ndgR showed unexpectedly poor growth in defined minimal medium even in the presence of leucine. To our surprise, it was supplementation of cysteine and methionine that corrected the growth. Based on this, we propose that NdgR induces cysteine-methionine biosynthesis. Direct involvement of NdgR in the very last steps of methionine synthesis with methionine synthase (metH) and 5,10-methylenetetrahydrofolate reductase (metF) was examined. From a pulldown assay, it was seen that NdgR was enriched from crude cell lysates with a strong affinity to metH and metF upstream sequences. Direct physical interaction of NdgR with these targets was further examined with a gel mobility shift assay. ndgR, leuC, metH, and metF were inducible in M145 cells upon nutrient downshift from rich to minimal medium but were not induced in the ndgR knockout mutant. Taking these observations together, NdgR-dependent metH-metF expression would account for the abnormal growth phenotype of the ndgR mutant although there may be additional NdgR-dependent genes in the Cys-Met metabolic pathways. As the first transcriptional factor reported for regulating Cys-Met metabolism in Streptomyces, NdgR links two disparate amino acid families, branched-chain amino acids (BCAAs) and sulfur amino acids, at the transcriptional level. Considering that Cys-Met metabolism is connected to mycothiol and one-carbon metabolism, NdgR may have broad physiological impacts.

Transcriptional profiling of gastric epithelial cells infected with wild type or arginase-deficient Helicobacter pylori.

BACKGROUND: Helicobacter pylori causes acute and chronic gastric inflammation induced by proinflammatory cytokines and chemokines secreted by cells of the gastric mucosa, including gastric epithelial cells. Previous studies have demonstrated that the bacterial arginase, RocF, is involved in inhibiting T cell proliferation and CD3ζ expression, suggesting that arginase could be involved in a more general dampening of the immune response, perhaps by down-regulation of certain pro-inflammatory mediators. RESULTS: Global transcriptome analysis was performed on AGS gastric epithelial cells infected for 16 hours with a wild type Helicobacter pylori strain 26695, an arginase mutant (rocF-) or a rocF+ complemented strain. H. pylori infection triggered altered host gene expression in genes involved in cell movement, death/growth/proliferation, and cellular function and maintenance. While the wild type strain stimulates host inflammatory pathways, the rocF- mutant induced significantly more expression of IL-8. The results of the microarray were verified using real-time PCR, and the differential levels of protein expression were confirmed by ELISA and Bioplex analysis. MIP-1B was also significantly secreted by AGS cells after H. pylori rocF- mutant infection, as determined by Bioplex. Even though not explored in this manuscript, the impact that the results presented here may have on the development of gastritis, warrant further research to understand the underlying mechanisms of the relationship between H. pylori RocF and IL-8 induction. CONCLUSIONS: We conclude that H. pylori arginase modulates multiple host signaling and metabolic pathways of infected gastric epithelial cells. Arginase may play a critical role in anti-inflammatory host responses that could contribute to the ability of H. pylori to establish chronic infections.

Helicobacter pylori arginase mutant colonizes arginase II knockout mice.

AIM: To investigate the role of host and bacterial arginases in the colonization of mice by Helicobacter pylori (H. pylori). METHODS: H. pylori produces a very powerful urease that hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Urease is absolutely essential to H. pylori pathogenesis; therefore, the urea substrate must be in ample supply for urease to work efficiently. The urea substrate is most likely provided by arginase activity, which hydrolyzes L-arginine to L-ornithine and urea. Previous work has demonstrated that H. pylori arginase is surprisingly not required for colonization of wild-type mice. Hence, another in vivo source of the critical urea substrate must exist. We hypothesized that the urea source was provided by host arginase II, since this enzyme is expressed in the stomach, and H. pylori has previously been shown to induce the expression of murine gastric arginase II. To test this hypothesis, wild-type and arginase (rocF) mutant H. pylori strain SS1 were inoculated into arginase II knockout mice. RESULTS: Surprisingly, both the wild-type and rocF mutant bacteria still colonized arginase II knockout mice. Moreover, feeding arginase II knockout mice the host arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC), while inhibiting > 50% of the host arginase I activity in several tissues, did not block the ability of the rocF mutant H. pylori to colonize. In contrast, BEC poorly inhibited H. pylori arginase activity. CONCLUSION: The in vivo source for the essential urea utilized by H. pylori urease is neither bacterial arginase nor host arginase II; instead, either residual host arginase I or agmatinase is probably responsible.

Isolation and characterization of rpoS from a pathogenic bacterium, Vibrio vulnificus: role of sigmaS in survival of exponential-phase cells under oxidative stress.

A gene homologous to rpoS was cloned from a fatal human pathogen, Vibrio vulnificus. The functional role of rpoS in V. vulnificus was accessed by using an rpoS knockout mutant strain. This mutant was impaired in terms of the ability to survive under oxidative stress, nutrient starvation, UV irradiation, or acidic conditions. The increased susceptibility of the V. vulnificus mutant in the exponential phase to H2O2 was attributed to the reduced activity of hydroperoxidase I (HPI). Although sigmaS synthesis was induced and HPI activity reached the maximal level in the stationary phase, the mutant in the stationary phase showed the same susceptibility to H2O2 as the wild-type strain in the stationary phase. In addition, HPII activity, which is known to be controlled by sigmaS in Escherichia coli, was not detectable in V. vulnificus strains under the conditions tested. The mutant in the exponential phase complemented with multiple copies of either the rpoS or katG gene of V. vulnificus recovered both resistance to H2O2 and HPI activity compared with the control strain. Expression of the katG gene encoding HPI in V. vulnificus was monitored by using a katG::luxAB transcriptional fusion. The expression of this gene was significantly reduced by deletion of sigmaS in both the early exponential and late stationary phases. Thus, sigmaS is necessary for increased synthesis and activity of HPI, and sigmaS is required for exponentially growing V. vulnificus to develop the ability to survive in the presence of H2O2.