Pseudomonas aeruginosa outer membrane vesicle-packed sRNAs can enter host cells and regulate innate immune responses.

Bacterial outer membrane vesicles (OMVs) can package and deliver virulence factors into host cells, which is an important mechanism mediating host-pathogen interactions. It has been reported that small RNAs (sRNAs) can be packed into OMVs with varying relative abundance, which might affect the function and/or stability of host mRNAs. In this study, we used OptiPrep density gradient ultra-high-speed centrifugation to purify OMVs from Pseudomonas aeruginosa. Next, the sequences and abundance of sRNAs were detected by using Small RNA-Seq. In particular, sRNA4518698, sRNA2316613 and sRNA809738 were the three most abundant sRNAs in OMVs, which are all fragments of P. aeruginosa non-coding RNAs. sRNAs were shielded within the interior of OMVs and remained resistant to external RNase cleavage. The miRanda and RNAhybrid analysis demonstrated that those sRNAs could target a large number of host mRNAs, which were enriched in host immune responses by the functions of GO and KEGG enrichment. Experimentally, we demonstrated that the transfection of synthetic sRNA4518698, sRNA2316613, or sRNA809738 could reduce the expression of innate immune response genes in RAW264.7 cells. Together, we demonstrated that P. aeruginosa OMVs sRNAs can regulate innate immune responses. This study uncovered a mechanism in which the OMVs regulate host responses by transferring bacterial sRNAs.

Visual metaphor of sadness in poetry comics: a socio-cognitive perspective.

Earlier literature on conceptual metaphor studies has extensively examined verbal metaphors of sadness in different text types and with cultural variations. However, there has been by far limited research on the visual metaphor of sadness. Adopting a socio-cognitive perspective, this study investigates the conceptual metaphor of sadness in the exemplary case of Chinese poetry comics drawn by Cai Zhizhong. The findings reveal that (1) BEING SAD IS BEING CONFRONTED WITH NATURAL FORCE and BEING SAD IS BEING PHYSICALLY ISOLATED are the two most frequently occurring visual metaphors across the panels; (2) all the visual metaphors at play can be explained according to the conceptual metaphor theory; (3) SADNESS IS BITTERSWEET FOOD OR DRINK and BEING SAD IS BEING PHYSICALLY ISOLATED are two additional kinds of sadness metaphors identified; and (4) the visual metaphors of sadness with Chinese cultural variations are rooted in mainstream Chinese cultural philosophies in the relevant period of history. The article also discusses the underlying mechanisms of the investigated visual metaphors in the Chinese culture by unveiling three cultural characteristics in the particular context.

Research trends in multimodal metaphor: a bibliometric analysis.

The concept of multimodal metaphor has generated a growing body of literature over the past decades. However, a systemic review of the domain seems to be lacking in relevant literature. This study, therefore, is an attempt to conduct a bibliometric analysis of the field of multimodal metaphor during 1977-2022, with a focus on 397 relevant publications retrieved from the Web of Science Core Collection (WoSCC) with the visualization tool VOSviewer. Some major quantitative findings are: (i) the number of publications in multimodal research began to surge in 2010 upon the seminal work of Forceville's (2009); (ii) USA, China and Spain are the most productive countries; (iii) journals in the field of advertising, communication and linguistics are important sources of publications; and (iv) eleven clusters of keywords are identified, such as "visual metaphor", "persuasion", "pictures", "impact", "multimodal metaphor", "model", etc., representing crucial areas of interests. We also identified, by qualitative observations, three research trends in multimodal metaphor, driven by cognitive linguistic theory, the theory of pragmatics and visual/multimodal rhetoric theory, respectively. Various theoretical perspectives may shed light on possible further research on multimodal metaphor.

Salmonella Induces the cGAS-STING-Dependent Type I Interferon Response in Murine Macrophages by Triggering mtDNA Release.

Salmonella enterica serovar Typhimurium (S. Typhimurium) elicited strong innate immune responses in macrophages. To activate innate immunity, pattern recognition receptors (PRRs) in host cells can recognize highly conserved pathogen-associated molecular patterns (PAMPs). Here, we showed that S. Typhimurium induced a robust type I interferon (IFN) response in murine macrophages. Exposure of macrophages to S. Typhimurium activated a Toll-like receptor 4 (TLR4)-dependent type I IFN response. Next, we showed that type I IFN and IFN-stimulated genes (ISGs) were elicited in a TBK1-IFN-dependent manner. Furthermore, cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) and immune adaptor protein stimulator of interferon genes (STING) were also required for the induction of type I IFN response during infection. Intriguingly, S. Typhimurium infection triggered mitochondrial DNA (mtDNA) release into the cytosol to activate the type I IFN response. In addition, we also showed that bacterial DNA was enriched in cGAS during infection, which may contribute to cGAS activation. Finally, we showed that cGAS and STING deficient mice and cells were more susceptible to S. Typhimurium infection, signifying the critical role of the cGAS-STING pathway in host defense against S. Typhimurium infection. In conclusion, in addition to TLR4-dependent innate immune response, we demonstrated that S. Typhimurium induced the type I IFN response in a cGAS-STING-dependent manner and the S. Typhimurium-induced mtDNA release was important for the induction of type I IFN. This study elucidated a new mechanism by which bacterial pathogen activated the cGAS-STING pathway and also characterized the important role of cGAS-STING during S. Typhimurium infection. IMPORTANCE As one of the most common foodborne transmitted zoonotic pathogens, S. Typhimurium infection causes diarrheal disease in humans and animals. S. Typhimurium infection has been implicated as an inducer for the type I interferon (IFN) response in macrophages, but the mechanisms are not fully understood. In this study, we reported that in addition to TLR4-dependent response, the cytosolic surveillance pathway (CSP) cGAS-STING is also required for the activation of type I IFN response during S. Typhimurium infection. We further showed that the infection of S. Typhimurium triggered mtDNA release into the cytosol, which induces the type I IFN response. In addition, physical interactions between cGAS and S. Typhimurium DNA have been identified in the context of infection. Importantly, we also provided convincing in vivo and in vitro evidence that the cGAS-STING pathway was potently implicated in the host defense against S. Typhimurium infection. Together, we uncovered a mechanism by which type I IFN response is elicited during S. Typhimurium infection in murine macrophages in an mtDNA-cGAS-STING-dependent manner.

Perceiving the poetic world: A corpus-assisted transitivity analysis of poetry comics.

While modern adaptations of Chinese classics have drawn keen scholarly interests lately, the comic adaptation of Chinese traditional poetry remains under-investigated. Extending the previous research on intersemiotic translation and comics, this paper, drawing on the analytical framework of systemic functional semiotics, examines distribution of process types of language in poems in comparison with that in comic images in the exemplary case drawn by Cai Zhizhong, using UAM image as the annotation tool. The comic book formulates a multimodal corpus that consists of 1,097 clauses and 605 images. We have manually analyzed the process type of the poems and their corresponding comic panels. Our quantitative and qualitative results show that there are distinct patterns of process-type distributions between verbal poems and images. Poems have been turned into perceptions, actions, and verbal processes in comic strips, which serve various purposes such as construction of the poet's gaze, relations building, storyline development, dramatization, metaphor visualization, etc. The paper is concluded with discussion on how the intersemiotic translation of poems might produce effects on readers.

In Vivo Detection of Circulating Tumor Cells in High-Risk Non-Metastatic Prostate Cancer Patients Undergoing Radiotherapy.

High-risk non-metastatic prostate cancer (PCa) has the potential to progress into lethal disease. Treatment options are manifold but, given a lack of surrogate biomarkers, it remains unclear which treatment offers the best results. Several studies have reported circulating tumor cells (CTCs) to be a prognostic biomarker in metastatic PCa. However, few reports on CTCs in high-risk non-metastatic PCa are available. Herein, we evaluated CTC detection in high-risk non-metastatic PCa patients using the in vivo CellCollector CANCER01 (DC01) and CellSearch system. CTC counts were analyzed and compared before and after radiotherapy (two sampling time points) in 51 high-risk non-metastatic PCa patients and were further compared according to isolation technique; further, CTC counts were correlated to clinical features. Use of DC01 resulted in a significantly higher percentage of CTC-positive samples compared to CellSearch (33.7% vs. 18.6%; p = 0.024) and yielded significantly higher CTC numbers (range: 0-15 vs. 0-5; p = 0.006). Matched pair analysis of samples between two sampling time points showed no difference in CTC counts determined by both techniques. CTC counts were not correlated with clinicopathological features. In vivo enrichment using DC01 has the potential to detect CTC at a higher efficiency compared to CellSearch, suggesting that CTC is a suitable biomarker in high-risk non-metastatic PCa.

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization.

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell's amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (<10 - >50%) and needs some further attention. This device is not cleared for the use in patients.

In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells.

BACKGROUND: Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS: We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS: In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1-76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS: Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

Multiplex Gene Expression Profiling of In Vivo Isolated Circulating Tumor Cells in High-Risk Prostate Cancer Patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers. For 27 patients, samples were available before and after treatment. We developed highly sensitive multiplex RT-qPCR assays for 14 genes (KRT19, EpCAM, CDH1, HMBS, PSCA, ALDH1A1, PROM1, HPRT1, TWIST1, VIM, CDH2, B2M, PLS3, and PSA), including epithelial markers, stem cell markers, and epithelial-to-mesenchymal-transition (EMT) markers. RESULTS: We observed high heterogeneity in gene expression in the captured CTCs for each patient. At least 1 marker was detected in 74 of 105 patients (70.5%), 2 markers in 45 of 105 (40.9%), and 3 markers in 16 of 105 (15.2%). Epithelial markers were detected in 31 of 105 (29.5%) patients, EMT markers in 46 of 105 (43.8%), and stem cell markers in 15 of 105 (14.3%) patients. EMT-marker positivity was very low before therapy (2 of 27, 7.4%), but it increased after therapy (17 of 27, 63.0%), whereas epithelial markers tended to decrease after therapy (2 of 27, 7.4%) compared with before therapy (13 of 27, 48.1%). At least 2 markers were expressed in 40.9% of patients, whereas the positivity was 19.6% for CellSearch, 38.1% for EPISPOT, and 43.8% for CellCollector-based IF-staining. CONCLUSIONS: The combination of in vivo CTC isolation with downstream RNA analysis is highly promising as a high-throughput, specific, and ultrasensitive approach for multiplex liquid biopsy-based molecular diagnostics.

Biological and Molecular Characterization of Circulating Tumor Cells: A Creative Strategy for Precision Medicine?

Circulating tumor cells (CTCs) are a group of rare cells disseminated from either primary or metastatic tumors into the blood stream. CTCs are considered to be the precursor of cancer metastasis. As a critical component of liquid biopsies, CTCs are a unique tool to understand the formation of metastasis and a valuable source of information on intratumor heterogeneity. Much effort has been invested in technologies for the detection of CTCs because they are rare cells among the vast number of blood cells. Studies in various cancers have repeatedly demonstrated that increased CTC counts prior to or during treatment are significantly associated with poor outcomes. In the new era of precision medicine, the study of CTCs reaches far beyond detection and counting. The rapidly growing field of analytical platforms for rare-cell analysis allows in-depth characterization of CTCs at the bulk cell and single-cell level. Genetic profiling of CTCs may provide an insight into the real-time tumor status, may allow the monitoring and evaluation of treatment response in clinical routine, and may lead to the development of novel therapeutic targets as well.

Catch and Release: rare cell analysis from a functionalised medical wire.

Enumeration and especially molecular characterization of circulating tumour cells (CTCs) holds great promise for cancer management. We tested a modified type of an in vivo enrichment device (Catch&Release) for its ability to bind and detach cancer cells for the purpose of single-cell molecular downstream analysis in vitro. The evaluation showed that single-cell analysis using array comparative genome hybridization (array-CGH) and next generation sequencing (NGS) is feasible. We found array-CGH to be less noisy when whole genome amplification (WGA) was performed with Ampli1 as compared to GenomePlex (DLRS values 0.65 vs. 1.39). Moreover, Ampli1-processed cells allowed detection of smaller aberrations (median 14.0 vs. 49.9 Mb). Single-cell NGS data obtained from Ampli1-processed samples showed the expected non-synonymous mutations (deletion/SNP) according to bulk DNA. We conclude that clinical application of this refined in vivo enrichment device allows CTC enumeration and characterization, thus, representing a promising tool for personalized medicine.

Feedback mechanisms between M2 macrophages and Th17 cells in colorectal cancer patients.

IL-17 and IL-22 are linked to the development of intestinal inflammation and colorectal cancer (CRC). However, the maintenance of IL-17 and IL-22 production, as well as the cell type (Th17) that mediates these cytokines in CRC patients, remains unknown. To examine this, untreated CRC patients and healthy controls were recruited in this study. We first observed that CRC patients contained significantly elevated levels of IL-17- and IL-22-producing CD4+ T cells. The vast majority of IL-22-expressing CD4+ T cells also expressed IL-17. We then found that the production of both IL-17 and IL-22 required support from autologous monocytes, since the depletion of monocytes significantly downregulated IL-17 and IL-22 secretion. Naive T cells from CRC patients did not secrete IL-17 or IL-22 initially, but long-term coculture with autologous monocytes significantly upregulated IL-17 and IL-22 production in an IL-6-dependent manner. Blockade of IL-6 significantly reduced the levels of both IL-17 and IL-22. We then observed that CD163+ M2 macrophages were the main contributor of IL-6. Interestingly, incubation of monocytes with CCR4+CCR6+ Th17 cells resulted in significantly higher levels of CD163+ macrophages as well as higher IL-6 secretion, than incubation with non-Th17 CD4+ T cells. Together, our study discovered a positive feedback mechanism between Th17 and M2 macrophages in CRC patients.

Heat-Induced Fragmentation and Adapter-Assisted Whole Genome Amplification Using GenomePlex® Single-Cell Whole Genome Amplification Kit (WGA4).

Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields > 2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e.g. formalin-fixed paraffin-embedded (FFPE) material] and works well when used for array-comparative genome hybridization (array-CGH).

Using Multiplex PCR for Assessing the Quality of Whole Genome Amplified DNA.

This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).

Low-Volume On-Chip Single-Cell Whole Genome Amplification for Multiple Subsequent Analyses.

Multiple analyses such as DNA profiling, sequencing, or comparative genome hybridization (CGH) done on the single-cell level long for pre-amplification due to the diploid human genome. Isothermal whole genome amplification allows amplification of long DNA templates from single cells. When analysis needs to be performed under rare cell conditions additional care needs to be taken due to the fact that, even after pre-enrichment, few candidate target cells are still dispersed among an overwhelming number of non-target background cells. Here, we describe a protocol where we define a population of candidate target cells based on specific staining. Candidate cells are then isolated by laser microdissection and pressure catapulting (LMPC) and transferred onto a microliter reaction slide. This slide allows monitoring the single-cell isolation process and isothermal whole genome amplification in less than 2 µL. The amplification products obtained from single cells can be forwarded to multiple analyses.

TGF-ß1 induces epigenetic silence of TIP30 to promote tumor metastasis in esophageal carcinoma.

TGF-ß1, a potent EMT (epithelial-mesenchymal transition) inducer present in the tumor microenvironment, is involved in the metastasis and progression of various carcinomas, including esophageal squamous cell carcinoma (ESCC). TIP30 (30kDa HIV-1 Tat interacting protein) is a putative tumor metastasis suppressor. Here, we found TIP30 was decreased in cells undergoing EMT induced by TGF-ß1, an occurrence that was related to promoter hypermethylation. TGF-ß1 induced TIP30 hypermethylation via increasing DNMT1 and DNMT3A expression, which could be restored by TGF-ß antibodies. In our in vitro and in vivo studies, we showed that silence of TIP30 led to EMT, enhanced migrative and invasive abilities of ESCC cells, promoted tumor metastasis in xenografted mice; alternatively, overexpression of TIP30 inhibited TGF-ß1-induced EMT, and metastatic abilities of ESCC cells. Mechanically, TIP30 silencing induced the nuclear translocation and transcriptional activation of ß-catenin in an AKT-dependent manner, which further resulted in the initiation of EMT. Consistently, TIP30 was frequently methylated and downregulated in ESCC patients. Loss of TIP30 correlated with nuclear ß-catenin and aberrant E-cadherin expression. TIP30 was a powerful marker in predicting the prognosis of ESCC. Taken together, our results suggest a novel and critical role of TIP30 involved in TGF-ß1-induced activation of AKT/ß-catenin signaling and ESCC metastasis.

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex).

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻5(= 280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻4(=2800 ELD50). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

[Multiplex fluorescent real-time PCR detection of bovine, goat and sheep derived materials in animal products].

We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.