Extracellular Secretion Research Articles

Ellagic acid and its metabolites urolithins A/B ameliorate most common disease phenotypes in cellular and mouse models for lysosomal storage disorders by enhancing extracellular vesicle secretion

Niemann Pick diseases types A (NPDA) and C (NPDC) are lysosomal storage disorders (LSDs) leading to cognitive impairment, neurodegeneration, and early death. NPDA and NPDC have different genetic origins, being caused by mutations in the acid sphingomyelinase (ASM) or the cholesterol transport protein NPC1, respectively. However, they share a common pathological hallmark in the accumulation of lipids in the endolysosomal compartment. Here, we tested the hypothesis that polyphenols reduce lipid overload in NPD cells by enhancing the secretion of extracellular vesicles (ECVs). We show that among the polyphenols tested, the ellagic acid metabolites, urolithin A and B, were the safest and most efficient in increasing ECV secretion. They reduced levels of accumulating lipids and lysosomal size and permeabilization in cultured bone marrow-derived macrophages and neurons from ASMko and NPC1 mutant mice, which mimic NPDA and NPDC, respectively. Moreover, oral treatment with ellagic acid reduced lipid levels, ameliorated lysosomal alterations, and diminished microglia activation in the brain of NPD mice. These results support the therapeutic value of ECV secretion and polyphenols for NPDs, which may also help treat other LSDs characterized by intracellular lipid overload.

Characterizing expression changes in noncoding RNAs during aging and heterochronic parabiosis across mouse tissues

Molecular mechanisms of organismal and cell aging remain incompletely understood. We, therefore, generated a body-wide map of noncoding RNA (ncRNA) expression in aging (16 organs at ten timepoints from 1 to 27 months) and rejuvenated mice. We found molecular aging trajectories are largely tissue-specific except for eight broadly deregulated microRNAs (miRNAs). Their individual abundance mirrors their presence in circulating plasma and extracellular vesicles (EVs) whereas tissue-specific ncRNAs were less present. For miR-29c-3p, we observe the largest correlation with aging in solid organs, plasma and EVs. In mice rejuvenated by heterochronic parabiosis, miR-29c-3p was the most prominent miRNA restored to similar levels found in young liver. miR-29c-3p targets the extracellular matrix and secretion pathways, known to be implicated in aging. We provide a map of organism-wide expression of ncRNAs with aging and rejuvenation and identify a set of broadly deregulated miRNAs, which may function as systemic regulators of aging via plasma and EVs.

Influences of flavonoids from Sedum aizoon L. on biofilm formation of Pseudomonas fragi.

Pseudomonas fragi (P. fragi) is one of the main categories of bacteria responsible for the spoilage of chilled meat. In the processing and preservation of chilled meat, it is easy to form biofilms on the meat, leading to the development of slime on the meat, which becomes a major quality defect. Flavonoids, as one of the critical components of secondary plant metabolites, are receiving increasing attention for their antibacterial activity. Flavonoids in Sedum aizoon L. (FSAL), relying on its prominent antibacterial activity, are of research importance in food preservation and other applications. This article aims to investigate the effect of FSAL on the biofilm formation of P. fragi, to better apply FSAL to the processing and preservation of meat products. The disruption of cellular structure and aggregation properties by FSAL was demonstrated by the observation of the cellular state within the biofilm. The amount of biofilm formation was determined by crystal violet staining, and the content of polysaccharides and proteins in the extracellular wrapped material was determined. It was shown that the experimental concentrations of FSAL (1.0 MIC) was able to inhibit biofilm formation and reduce the main components in the extracellular secretion. The swimming motility assay and the downregulation of flagellin-related genes confirmed that FSAL reduced cell motility and adhesion. The downregulation of cell division genes and the lowering of bacterial metabolic activity suggested that FSAL could hinder bacterial growth and reproduction within P. fragi biofilms. KEY POINTS: • FSAL inhibited the activity of Pseudomonas fragi in the dominant meat strain • The absence of EPS components affected the formation of P. fragi biofilms • P. fragi has reduced adhesion capacity due to impaired flagellin function.

Endosidin 5 disruption of the Golgi Apparatus and extracellular matrix secretion in the unicellular charophyte, Penium margaritaceum.

Endosidins are a group of small molecular weight compounds, first identified by "chemical biology" screening assays, that have been used to target specific components of the endomembrane system. In this study, we employed multiple microscopy-based screening techniques to elucidate effects of Endosidin 5 (ES5) on the Golgi Apparatus and the secretion of extracellular matrix (ECM) components, Penium margaritaceum. These effects were compared with those caused by treatments with brefeldin A and concanamycin A. Penium margaritaceum's extensive Golgi Apparatus and endomembrane system make it an outstanding model organism for screening changes to the endomembrane system. Here we detail changes to the Golgi Apparatus and secretion of ECM material caused by Endosidin 5. Changes to extracellular polymeric substance (EPS) secretion and cell wall expansion were screened using fluorescence microscopy. Confocal laser scanning microscopy, and transmission electron microscopy were used to assess changes to the Golgi Apparatus, the cell wall and the vesicular network. Electron tomography was also performed in order to detail the changes to the Golgi Apparatus. While other endosidins were able to impact EPS secretion and cell wall expansion only ES5 completely inhibited EPS secretion and cell wall expansion over 24h. Short treatments of ES5 resulted in displacement of the Golgi bodies from their typical linear alignment. The number of cisternae decreased per Golgi stack and trans face cisternae in-curled to form distinct elongate circular profiles. Longer treatment resulted in a transformation of the Golgi body to an irregular aggregate of cisternae. These alterations could be reversed by removal of ES5 and returning cells to culture. ES5 alters secretion of ECM material in Penium by affecting the Golgi apparatus and does so in a markedly different way than other endomembrane inhibitors such as Brefeldin A and Concanamycin A.

Advances in Drug Discovery Targeting Lysosomal Membrane Proteins.

Lysosomes are essential organelles of eukaryotic cells and are responsible for various cellular functions, including endocytic degradation, extracellular secretion, and signal transduction. There are dozens of proteins localized to the lysosomal membrane that control the transport of ions and substances across the membrane and are integral to lysosomal function. Mutations or aberrant expression of these proteins trigger a variety of disorders, making them attractive targets for drug development for lysosomal disorder-related diseases. However, breakthroughs in R&D still await a deeper understanding of the underlying mechanisms and processes of how abnormalities in these membrane proteins induce related diseases. In this article, we summarize the current progress, challenges, and prospects for developing therapeutics targeting lysosomal membrane proteins for the treatment of lysosomal-associated diseases.

RAB27B Drives a Cancer Stem Cell Phenotype in NSCLC Cells Through Enhanced Extracellular Vesicle Secretion.

Expression of RAB27B in CSCs leads to elevated levels of EVs that mediate communication between CSCs and BCCs that maintains a stem-like phenotype in NSCLC cells.

Efficient Autotransporter-Mediated Extracellular Secretion of a Heterologous Recombinant Protein by Escherichia coli.

The autotransporter protein secretion system has been used previously to target the secretion of heterologous proteins to the bacterial cell surface and the extracellular milieu at the laboratory scale. The platform is of particular interest for the production of "difficult" recombinant proteins that might cause toxic effects when produced intracellularly. One such protein is IrmA. IrmA is a vaccine candidate that is produced in inclusion bodies requiring refolding. Here, we describe the use and scale-up of the autotransporter system for the secretion of an industrially relevant protein (IrmA). A plasmid expressing IrmA was constructed such that the autotransporter platform could secrete IrmA into the culture supernatant fraction. The autotransporter platform was suitable for the production and purification of IrmA with comparable physical properties to the protein produced in the cytoplasm. The production of IrmA was translated to scale-up protein production conditions resulting in a yield of 29.3 mg/L of IrmA from the culture supernatant, which is consistent with yields of current industrial processes. IMPORTANCE Recombinant protein production is an essential component of the biotechnology sector. Here, we show that the autotransporter platform is a viable method for the recombinant production, secretion, and purification of a "difficult" to produce protein on an industrially relevant scale. Use of the autotransporter platform could reduce the number of downstream processing operations required, thus accelerating the development time and reducing costs for recombinant protein production.

Mechanisms of carbonate precipitation induced by two model bacteria

The investigation of microbially induced calcium carbonate precipitation is essential to understanding CO2 fixation, marine carbonate rock formation, and biogeochemical cycling of life-essential elements. In this study, two model bacteria Bacillus subtilis (possess the cell wall of G+ bacteria) and Escherichia coli (possess the cell wall of G− bacteria) were used as representatives to induce carbonate mineralization. The mineralization ability of the two bacteria and carbonate precipitation mechanisms were studied with the biochemical analysis of bacterial fermentation products, mineral characterization, and biomimetic mineralization experiments. The results showed that both bacteria induced biomineral formation by using the cell wall as a template, but B. subtilis was more capable of inducing mineralization than E. coli. Particularly, the percentage of Ca2+ removal from the culture medium by the former reached 36% in 7 days, which induced the formation of vaterite and calcite, while that of Ca2+ removal by the latter reached 28%, and the resulting carbonate was amorphous calcium carbonate. Biomimetic mineralization experiments showed that protein molecules were the key factors inducing the formation of biological vaterite, while polysaccharides tended to bind small vaterite particles to form large organo-mineral aggregates. By comparing the results of inducing calcium carbonate under the same culture conditions, the two types of bacteria with completely different cell wall structures had significant differences in the ability to induce calcium carbonate synthesis, among which that was stronger by Bacillus subtilis (G+) than by Escherichia coli (G−). It was found that factors such as bacterial cell wall structure, extracellular secretions including carbonic anhydrase, pH value, etc. all affect the microbial-induced carbonate precipitation to varying degrees, providing new insights into the theory of bacterial-induced carbonate mineralization.

Targeting Endogenous Reactive Oxygen Species Removal and Regulating Regenerative Microenvironment at Annulus Fibrosus Defects Promote Tissue Repair

The excessive reactive oxygen species (ROS) level, inflammation, and weak tissue regeneration ability after annulus fibrosus (AF) injury constitute an unfavorable microenvironment for AF repair. AF integrity is crucial for preventing disc herniation after discectomy; however, there is no effective way to repair the AF. Herein, a composite hydrogel integrating properties of antioxidant, anti-inflammation, and recruitment of AF cells is developed through adding mesoporous silica nanoparticles modified by ceria and transforming growth factor β3 (TGF-β3) to the hydrogels. The nanoparticle loaded gelatin methacrylate/hyaluronic acid methacrylate composite hydrogels eliminate ROS and induce anti-inflammatory M2 type macrophage polarization. The released TGF-β3 not only plays a role in recruiting AF cells but is also responsible for promoting extracellular matrix secretion. The composite hydrogels can be solidified in situ in the defect area to effectively repair AF in rats. The strategies targeting endogenous ROS removal and improving the regenerative microenvironment by the nanoparticle-loaded composite hydrogels have potential applications in AF repair and intervertebral disc herniation prevention.

Abstract 2444: RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion

Abstract Non-small cell lung cancer (NSCLC), a major subtype of lung cancer, accounts for ~85% of all lung cancer diagnoses. A sub-population of cells within human NSCLC tumors called cancer stem cells (CSCs) have been found to exhibit distinct properties that contribute to factors associated with poor outcomes in NSCLC patients including metastasis, relapse, and therapeutic resistance. We previously performed RNAseq analysis on bulk cancer cells (BCCs) and CSCs from a panel of primary NSCLC tumors and cell lines to identify signaling mechanisms involved in the tumorigenic behavior of NSCLC CSCs. Our analysis revealed that expression of RAB27B, a small GTPase that plays a critical role in extracellular vesicle (EV) formation and secretion, was significantly upregulated in CSCs when compared to BCCs. Interestingly, RAB27B is overexpressed and predicts patient survival in NSCLC. The goal of this study was to assess the role of RAB27B in the tumorigenic behavior of NSCLC CSCs and determine the mechanism by which RAB27B promotes an aggressive stem-cell like phenotype in NSCLC cells an aspect of RAB27B function that has not been investigated in NSCLC. QPCR and immunoblot analysis was used to compare RAB27B mRNA and protein expression in a panel of NSCLC BCCs and CSCs. Two independent shRNA constructs were used to knockdown (KD) RAB27B in NSCLC CSCs. The effects of RAB27B KD on CSC expansion, transformed growth and invasion were analyzed by clonal expansion, soft agar colony formation and Boyden chamber assays, respectively. Xenograft and tail vein mouse models were used to assess the effects of RAB27B KD on CSC tumor growth and lung colonization. EVs were isolated from the conditioned media of BCCs, CSCs and RAB27B KD CSCs using differential ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblotting according to MISEV guidelines. Our analysis revealed that RAB27B is significantly upregulated in NSCLC CSCs compared to BCCs. RAB27B KD cells exhibited a significant decrease in clonal expansion, transformed growth and invasion when compared to non-target shRNA control CSCs. In vivo studies demonstrated that RAB27B is important for the tumorigenicity of CSCs and promotes tumor cell proliferation, and angiogenesis. EVs were positive for EV-specific markers and negative for an intracellular vesicle marker. CSCs secrete significantly more EVs when compared to BCCs and KD of RAB27B in CSCs led to a decrease in the levels of EVs secreted. Furthermore, delivery of CSC-derived, but not BCC-derived EVs to BCCs induced spheroid growth, clonal expansion, and invasion in BCCs. Taken together, our results indicate that RAB27B is required for maintenance of a highly tumorigenic, cancer-initiating and invasive stem-like cell population in NSCLC and RAB27B is involved in propagating EV-mediated communication from NSCLC CSCs to BCCs. Citation Format: Kayleah M. Meneses, Jennifer A. Lindemann, Prita Pandya, Dania S. Al-Qasrawi, Ryan Argo, Celeste Weems, Danielle J. Beetler, Irene K. Yan, Joy Wolfram, Tushar C. Patel, Verline Justilien. RAB27B drives a cancer stem cell phenotype in non-small cell lung cancer cells through enhanced extracellular vesicle secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2444.

Abstract 2633: Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer

Abstract Zanidatamab zovodotin (ZW49) is an antibody drug conjugate (ADC) comprised of a bispecific anti-HER2 IgG1 antibody (ZW25, zanidatamab) conjugated to a microtubule inhibitor auristatin payload (ZD02044) via a protease cleavable linker. Clinical studies of ZW49 in HER2-expressing advanced solid tumors are currently underway (NCT03821233). We have previously presented data illustrating the rapid internalization of ZW49 and the anti-tumor activity of ZW49 in multiple patient-derived xenograft models originating from breast carcinomas. Here we present ZW49’s ability to induce in vitro hallmarks of immunogenic cell death (ICD) including increased calreticulin exposure, increased high mobility group box-1 (HMGB1) exposure, and increased extracellular ATP secretion in a HER2 dependent manner. Additionally, we present the results from a study evaluating the anti-tumor activity of ZW49 in a panel of patient-derived xenografts of gastric cancer. HER2-positive SK-BR-3 and HER2-negative MDA-MB-468 tumor cell lines were treated with ZW49 and cell surface calreticulin was assessed by flow cytometry. ZW49 treatment of SKBR-3 cells induced a significantly higher percentage of cells staining positive for cell surface calreticulin compared to untreated cells. In contrast, ZW49 treatment of MDA-MB-468 cells resulted in no significant difference in the percentage of cells staining positive for calreticulin compared to untreated cells. ZW49 treated HER2-positive cancer cell lines SK-BR-3 and SK-OV-3 induced higher levels of extracellular ATP compared to untreated cells. In the HER2-negative cell line MDA-MB-468, treatment with ZW49 did not alter levels of extracellular ATP compared to untreated cells. ZW49 induced higher levels of HMGB1 in the HER2-positive cancer cell lines SKBR3 and N87 compared to untreated cells, whereas in the HER2-negative cell line MDA-MB-468, ZW49 induced similar levels of HMGB1 to untreated cells. Anti-tumor activity was observed in 5/7 (71%) PDX models of gastric cancer after a single i.v. dose of 6 mg/kg, including in models with moderate and weak HER2 expression. The strong anti-tumor activity of ZW-49 in vivo, together with its ability to induce ICD and potential adaptive immune responses, support ZW49 as a promising ADC for the treatment of HER2-expressing cancers warranting further investigation, including potential combination with checkpoint inhibitors. Citation Format: Stuart D. Barnscher, Andrea Hernández Rojas, Kevin J. Hamblett, Nichole Escalante. Zanidatamab zovodotin (ZW49) induces hallmarks of immunogenic cell death and is active in patient-derived xenograft models of gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2633.

Abstract 1370: Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer

Abstract Rab27B is a member of the Rab small GTPases that belong to the RAS-like GTPase superfamily. Rab27B has been shown to regulate endosomal trafficking, exosome secretion, and delivery of secretory granules. However, the role of Rab27B in colorectal cancer (CRC) tumor biology is unknown. Using CRC tissue microarrays, a significant upregulation of Rab27B was detected in staged adenocarcinoma compared to normal colon tissue. Interestingly, microsatellite unstable (MSI) tumors showed a higher increase in Rab27B (2.4-fold) than microsatellite stable (MSS) tumors (1.6-fold), consistent with TCGA data. To study the role of Rab27B in CRC, we targeted Rab27B in MSI HCT116 cell line by CRISPR-Cas9 deletion and siRNA knockdown. In both cases, loss of Rab27B resulted in a dysregulation of cellular autophagy and reduction in extracellular vesicle secretion. Rab27B-depleted cells showed an abnormal accumulation of autophagy vesicles and increase in autophagy markers LC3-II and p62, indicating a defect in autophagy flux. To characterize this autophagy defect, we used eGFP-mCherry-LC3 reporter, lysotracker and Cyto-ID staining to identify that autophagy flux is blocked at the autophagosome/lysosome fusion step when Rab27B is lost. To test whether the GTPase activity of Rab27B is needed for the autophagy role, we added back constitutively active and dominant negative GTPase mutants of Rab27B in Rab27B KO cells and observed autophagy phenotype rescue with only wildtype and constitutively active GTPase forms. As autophagy has been shown to have a pro-survival role in tumor growth and stress response, we hypothesized that this defect in autophagy flux resulting from Rab27B deletion will impact cellular stress response and reduce tumor growth. While Rab27B knockout (KO) cells show similar in vitro 2D-growth characteristics as wildtype cells under normal conditions, loss of Rab27B resulted in a 60% reduction in cell viability in response to starvation conditions. Similarly, a 94% reduction in colony formation was observed when grown in soft agar, along with deficiencies in spheroid formation. In vivo, Rab27B KO cells showed a major reduction (p<0.0001) in tumor growth xenografts compared to wildtype HCT116 cells. Taken together, these results demonstrate a new role of Rab27B in the autophagy trafficking process in CRC. Futures studies will focus on further characterizing the relationship among Rab27B overexpression, microsatellite instability, and autophagy in vitro and in vivo using Rab27B knockout mouse model, as well as investigating whether Rab27B can be targeted as a potential new therapeutic strategy. Citation Format: Sahida Afroz, Ranjan Preet, Vikalp Vishwakarma, Dan A. Dixon. Overexpression of the small GTPase Rab27B regulates autophagy flux and contributes to tumor growth in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1370.

Abstract 3937: An intracellular variant of zona pellucida glycoprotein 3 is expressed in cancer

Abstract This study describes the expression of an intracellular variant of the Zona Pellucida glycoprotein 3 (ZP3) that is predominantly expressed in cancer. ZP3 is an important constituent of the Zona Pellucida (ZP), the extracellular layer surrounding oocytes. It is highly expressed in maturing oocytes, but not in other adult human tissues. As ZP3 protein has also been detected in cancer, it may serve as an interesting diagnostic and therapeutic target. Here, we extend the cancer-related ZP3 expression data using immunohistochemistry (IHC) of tumor biopsies, interrogating publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, as well as computational analysis and real-time quantitative PCR (qPCR) of CCLs. IHC data for several cancer types shows abundant ZP3 protein expression, which is confined to the cytoplasm, contradicting the extracellular ZP3 localization in oocytes. An alternative ZP3 mRNA variant, which we term ‘ZP3-Cancer’, is annotated in the NCBI and Ensembl databases, and lacks the genetic information encoding the N-terminal signal peptide that governs extracellular secretion. Analysis of publicly available RNA-seq data of 1339 CCLs indicates that ZP3-Cancer is the dominant variant as compared to ZP3-Oocyte, which was validated by independent computational analysis. Expression of ZP3-Cancer in CCLs was confirmed by qPCR. Publicly available RNAseq data of tumor and normal tissues confirms strongly enhanced expression of ZP3-Cancer in cancer cells. In addition, ZP3-Cancer expression is upregulated in advanced stages and shows a relationship with patient survival in a number of cancer types. The folliculogenesis specific transcription factor FIGLA, which activates transcription of ZP3-Oocyte in maturing oocytes, appears absent in cancer as inferred from publicly available transcriptome data, indicating alternative ZP3-Cancer transcriptional activation mechanisms in this disease. The cancer restricted expression of this ZP3 transcript variant renders it an attractive tumor specific antigen for the development of a therapeutic cancer vaccine, particularly using mRNA technology. Citation Format: Iman J. Schultz, Yvette Zimmerman, Cathy B. Moelans, Marcin Chrusciel, Jan Krijgh, Paul J. van Diest, Ilpo T. Huhtaniemi, Herjan J. Coelingh Bennink. An intracellular variant of zona pellucida glycoprotein 3 is expressed in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3937.

High-throughput spatiotemporal monitoring of single-cell secretions via plasmonic microwell arrays

Methods for the analysis of cell secretions at the single-cell level only provide semiquantitative endpoint readouts. Here we describe a microwell array for the real-time spatiotemporal monitoring of extracellular secretions from hundreds of single cells in parallel. The microwell array incorporates a gold substrate with arrays of nanometric holes functionalized with receptors for a specific analyte, and is illuminated with light spectrally overlapping with the device’s spectrum of extraordinary optical transmission. Spectral shifts in surface plasmon resonance resulting from analyte–receptor bindings around a secreting cell are recorded by a camera as variations in the intensity of the transmitted light while machine-learning-assisted cell tracking eliminates the influence of cell movements. We used the microwell array to characterize the antibody-secretion profiles of hybridoma cells and of a rare subset of antibody-secreting cells sorted from human donor peripheral blood mononuclear cells. High-throughput measurements of spatiotemporal secretory profiles at the single-cell level will aid the study of the physiological mechanisms governing protein secretion.

Plastic leachate exposure drives antibiotic resistance and virulence in marine bacterial communities

Plastic pollution is a serious global problem, with more than 12 million tonnes of plastic waste entering the oceans every year. Plastic debris can have considerable impacts on microbial community structure and functions in marine environments, and has been associated with an enrichment in pathogenic bacteria and antimicrobial resistance (AMR) genes. However, our understanding of these impacts is largely restricted to microbial assemblages on plastic surfaces. It is therefore unclear whether these effects are driven by the surface properties of plastics, providing an additional niche for certain microbes residing in biofilms, and/or chemicals leached from plastics, the effects of which could extend to surrounding planktonic bacteria. Here, we examine the effects of polyvinyl chloride (PVC) plastic leachate exposure on the relative abundance of genes associated with bacterial pathogenicity and AMR within a seawater microcosm community. We show that PVC leachate, in the absence of plastic surfaces, drives an enrichment in AMR and virulence genes. In particular, leachate exposure significantly enriches AMR genes that confer multidrug, aminoglycoside and peptide antibiotic resistance. Additionally, enrichment of genes involved in the extracellular secretion of virulence proteins was observed among pathogens of marine organisms. This study provides the first evidence that chemicals leached from plastic particles alone can enrich genes related to microbial pathogenesis within a bacterial community, expanding our knowledge of the environmental impacts of plastic pollution with potential consequences for human and ecosystem health.

Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation.

Membrane lipids play vital roles in small extracellular vesicle (sEV) biogenesis. However, the function of various lipids in the biogenesis of sEVs is still poorly understood. Phosphoinositolphosphates (PIPs), a group of the most critical lipids in vesicle transport, can undergo rapid conversion in response to a variety of cell signals, which in turn influence the generation of vesicles. Due to the challenge in detecting the low amount of PIP content in biological samples, the function of PIPs in sEVs has been insufficiently investigated. Here, we employed an LC-MS/MS method to detect the levels of PIPs in sEVs. We revealed phosphatidylinositol-4-phosphate (PI4P) was the main PI-monophosphate in macrophage-derived sEVs. The release of sEVs was regulated in a time-dependent manner and correlated with the PI4P level during the lipopolysaccharide (LPS) stimulation. In terms of mechanism, within 10h of LPS treatment, the LPS-induced production of type I interferon inhibited the expression of PIP-5-kinase-1-gamma, which increased the PI4P content on multivesicular bodies (MVBs) and recruited RAB10, member RAS oncogene family, to promote sEV generation. When LPS stimulation was extended to 24h, the heat shock protein family A member 5 (HSPA5) expression level was elevated. PI4P interacted with HSPA5 on the Golgi or endoplasmic reticulum away from MVBs, which disrupted the continuous fast sEV release. In conclusion, the present study demonstrated an inducible sEV release model response to LPS treatment. The inducible release may be due to PI4P regulating the generation of intraluminal vesicles secreted as sEVs.

Microbial community evolution and functional trade-offs of biofilm in odor treatment biofilters

Biofilters inoculated with activated sludge are widely used for odor control in WWTP. In this process, biofilm community evolution plays an important role in the function of reactor and is closely related to reactor performance. However, the trade-offs in biofilm community and bioreactor function during the operation are still unclear. Herein, an artificially constructed biofilter for odorous gas treatment was operated for 105 days to study the trade-offs in the biofilm community and function. Biofilm colonization was found to drive community evolution during the start-up phase (phase 1, days 0–25). Although the removal efficiency of the biofilter was unsatisfactory at this phase, the microbial genera related to quorum sensing and extracellular polymeric substance secretion led to the rapid accumulation of the biofilm (2.3 kg biomass/m3 filter bed /day). During the stable operation phase (phase 2, days 26–80), genera related to target-pollutant degradation showed increases in relative abundance, which accompanied a high removal efficiency and a stable accumulation of biofilm (1.1 kg biomass/m3 filter bed/day). At the clogging phase (phase 3, days 81–105), a sharp decline in the biofilm accumulation rate (0.5 kg biomass/m3 filter bed /day) and fluctuating removal efficiency were observed. The quorum quenching-related genera and quenching genes of signal molecules increased, and competition for resources among species drove the evolution of the community in this phase. The results of this study highlight the trade-offs in biofilm community and functions during the operation of bioreactors, which could help improve bioreactor performance from a biofilm community perspective.

IL13RA2 downregulation in fibroblast promotes keloid fibrosis via JAK/STAT6 activation.

Keloid is considered as a fibro-proliferative disease characterized by chronic inflammation that is induced following skin injury. Deciphering the underlying mechanism of keloid formation is essential for improving treatment outcomes. Here, we found that more macrophages were activated towards M2 subtype in keloid dermis when compared to normal dermis. Western Blot revealed that the level of phosphorylated STAT6, a known inducer of M2 polarization, was higher in keloid fibroblasts as opposed to fibroblasts from normal dermis. Moreover, keloid fibrosis was shown to be positively correlated with the level of phosphorylated STAT6. Further, we identified downregulation of IL13RA2, a 'decoy' receptor of IL13, in keloid fibroblasts compared to fibroblasts from normal dermis. Ectopic expression of IL13RA2 in keloid fibroblasts resulted in inhibition of STAT6 phosphorylation, cell proliferation, migration, invasion, extracellular matrix secretion and myofibroblast marker expression, as well as an increase in apoptosis. Consistently, knockdown of IL13RA2 in normal fibroblasts induced a 'keloidal' status. Furthermore, both in vitro application and intra-tumoral injection of pSTAT6 inhibitor AS1517499 in a PDX keloid-implantation mouse model, resulted in proliferation inhibition, tissue necrosis, apoptosis and myofibroblast marker reduction. Collectively, this study elucidates the key role of IL13RA2 in keloid pathology and inspire further translational research of keloid treatment concerning JAK/STAT6 inhibition.

GPx3 deficiency exacerbates maladaptive RV remodeling in experimental pulmonary artery banding.

The stressed right ventricle (RV) is particularly susceptible to producing and accumulating reactive oxygen species, leading to extracellular matrix deposition and secretion of natriuretic peptides. The role of specific enzymes with anti-oxidative capacity, like Glutathione peroxidase 3 (GPx3), in RV pathogenesis is currently unknown. Here, we employ a murine model of pulmonary artery banding (PAB) to study the role of GPx3 in isolated RV pathology. Compared to WT mice undergoing PAB surgery, GPx3-deficient PAB mice presented with higher RV systolic pressure and higher LV eccentricity indices. PAB-induced changes in Fulton's Index, RV free wall thickness, and RV fractional area change were more pronounced in GPx3-deficient mice compared to WT controls. Adverse RV remodeling was enhanced in GPx3-deficient PAB animals, evidenced by increased RV expression levels of CTGF, TGF-b and ANP. In summary, GPx3 deficiency exacerbates maladaptive RV remodeling and causes signs of RV dysfunction.

Red ginseng dietary fiber promotes probiotic properties of Lactiplantibacillus plantarum and alters bacterial metabolism.

Korean red ginseng has been widely used as an herbal medicine. Red ginseng dietary fiber (RGDF) is a residue of the processed ginseng product but still contains bioactive constituents that can be applied as prebiotics. In this study, we evaluated changes on fermentation profiles and probiotic properties of strains that belong to family Lactobacillaceae with RGDF supplementation. Metabolomic analyses were performed to understand specific mechanisms on the metabolic alteration by RGDF and to discover novel bioactive compounds secreted by the RGDF-supplemented probiotic strain. RGDF supplementation promoted short-chain fatty acid (SCFA) production, carbon source utilization, and gut epithelial adhesion of Lactiplantibacillus plantarum and inhibited attachment of enteropathogens. Intracellular and extracellular metabolome analyses revealed that RGDF induced metabolic alteration, especially associated with central carbon metabolism, and produced RGDF-specific metabolites secreted by L. plantarum, respectively. Specifically, L. plantarum showed decreases in intracellular metabolites of oleic acid, nicotinic acid, uracil, and glyceric acid, while extracellular secretion of several metabolites including oleic acid, 2-hydroxybutanoic acid, hexanol, and butyl acetate increased. RGDF supplementation had distinct effects on L. plantarum metabolism compared with fructooligosaccharide supplementation. These findings present potential applications of RGDF as prebiotics and bioactive compounds produced by RGDF-supplemented L. plantarum as novel postbiotic metabolites for human disease prevention and treatment.