The highly contagious nature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19), requires rapid diagnostic tests to prevent the virus from spreading within hospitals and communities. Reverse transcription followed by the polymerase chain reaction (RT-PCR) test is the gold standard for detecting SARS-CoV-2 infections but is time-consuming, labor-intensive, and restricted to centralized laboratories. There is a growing need to develop and implement point-of-care and rapid tests for SARS-CoV-2 detection to address these limitations. We aimed to evaluate the performance of BioFire Film Array Respiratory Panel 2.1 (BioFire FA-RP2.1) for SARS-CoV-2 detection in a pediatric hospital setting. The BioFire FA-RP2.1 test provides rapid results and can identify several viral and bacterial infections in a single test. This prospective, cross-sectional, diagnostic accuracy study enrolled participants ranging from 0 to 18 years of age, seeking medical consultation for any reason, who had been in contact with individuals confirmed to have COVID-19 or managed at the hospital for medical or surgical reasons. We employed a systematic sampling technique to ensure a representative sample. The study included 339 participants with a median age of 5 years. The BioFire FA-RP2.1 test detected SARS-CoV-2 in 18.6% of cases, while the reference RT-PCR test in 14% of cases. The BioFire FA-RP2.1 sensitivity and specificity for SARS CoV-2 detection were 98% and 94%, respectively. The positive probability coefficient (LR+) was 18. The agreement between the two tests was 0.80. In addition, the BioFire FA-RP2.1 test detected coinfection with two viruses in 7,6% of cases. The BioFire FA-RP2.1 is a reliable solution to meet pediatric healthcare needs and improve prognosis in the post-pandemic era thanks to its friendly interface and rapid testing process. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019, causing a significant public health crisis. Patients initially exhibited symptoms of severe pneumonia Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This is a useful study demonstrating the clinical utility of BioFire Respiratory panel for the detection of SARS-CoV-2. The study is well-performed and the paper is well written.1. The authors should cite more extensively to introduce the pathogenesis and clinical significance based on the following references.Liu BM, Martins TB, Peterson LK, Hill HR. Clinical significance of measuring serum cytokine levels as inflammatory biomarkers in adult and pediatric COVID-19 cases: A review. Cytokine. 2021 Jun;142:155478. doi: 10.1016/j.cyto.2021.155478. Epub 2021 Feb 23. PMID: 33667962; PMCID: PMC7901304.Hu B, Huang S, Yin L. 2020. The cytokine storm and COVID-19. J Med Virol. DOI: 10.1002/jmv.26232.Liu B, Hill HR. Role of Host Immune and Inflammatory Responses in COVID-19 Cases with Underlying Primary Immunodeficiency: A Review. J Interferon Cytokine Res. 2020 Dec;40(12):549-554. doi: 10.1089/jir.2020.0210. PMID: 33337932; PMCID: PMC7757688.2. Please clarify inclusion and exclusion criteria3. Is the statistical analysis two-tailed or one-tailed?4. In the Introduction or Discussion, please discuss the following point based on the following reference: though the BioFire Respiratory panel is a large multiplex PCR panel, it still lacks some important respiratory pathogen to be covered, e.g., Pneumocystis jirovecii, which can be detected otherwise by some other novel molecular assays.Liu B, Totten M, Nematollahi S, Datta K, Memon W, Marimuthu S, Wolf LA, Carroll KC, Zhang SX. Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens. J Mol Diagn. 2020 Dec;22(12):1482-1493. doi: 10.1016/j.jmoldx.2020.10.003. Epub 2020 Oct 15. Erratum in: J Mol Diagn. 2021 Apr;23(4):506. PMID: 33069878.5. In the Introduction or Discussion, please discuss that BioFire Respiratory panel provides some level of commutablity and standardization in different clinical labs as the same platform and panel is being used. According to the following reference, different reverse transcriptase with varied efficiency may cause unreliable results. BioFire Respiratory panel solves this problem as a popular and widely used commercial molecular test. Please discuss this based on this reference.Liu B, Forman M, Valsamakis A. Optimization and evaluation of a novel real-time RT-PCR test for detection of parechovirus in cerebrospinal fluid. J Virol Methods. 2019 Oct;272:113690. doi: 10.1016/j.jviromet.2019.113690. Epub 2019 Jul 5. PMID: 31283959.6. Error: Line 57: "Wuhan province" should be "Wuhan city"**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. 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The PLOS ONE style templates can be found at AnswerWe have carefully reviewed the additional requirements and have made the necessary revisions to ensure that our manuscript complies with PLOS ONE's style requirements, including file naming. We have used the provided PLOS ONE style templates to guide us in this process.2. Thank you for stating in your Funding Statement:https://www.conacyt.gov.py/. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This study was financially supported by Consejo Nacional de Ciencia y Tecnología, CONACYT, Paraguay (grant number PINV 20-287), http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement.Please provide an amended statement that declares *all* the funding or sources of support  received during this study, as detailed online in our guide for authors at Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.AnswerWe provided an amended statement that declares all the funding or sources of support received during this study. We also included the statement 'There was no additional external funding received for this study' in our updated Funding Statement within our cover letter.http://journals.plos.org/plosone/s/data-availability.3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/ plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.AnswerReferences were reviewed and formatted according to the Plos One journal requirements.Reviewer #1:This is a useful study demonstrating the clinical utility of BioFire Respiratory panel for the detection of SARS-CoV-2. The study is well-performed and the paper is well written.Thank you for your thoughtful review and positive feedback regarding our study. We appreciate your valuable comments.1. The authors should cite more extensively to introduce the pathogenesis and clinical significance based on the following references.Liu BM, Martins TB, Peterson LK, Hill HR. Clinical significance of measuring serum cytokine levels as inflammatory biomarkers in adult and pediatric COVID-19 cases: A review. Cytokine. 2021 Jun;142:155478. doi: 10.1016/j.cyto.2021.155478. Epub 2021 Feb 23. PMID: 33667962; PMCID: PMC7901304. Hu B, Huang S, Yin L. 2020. The cytokine storm and COVID-19. J Med Virol. DOI: 10.1002/jmv.26232.Liu B, Hill HR. Role of Host Immune and Inflammatory Responses in COVID-19 Cases with Underlying Primary Immunodeficiency: A Review. J Interferon Cytokine Res. 2020 Dec;40(12):549-554. doi: 10.1089/jir.2020.0210. PMID: 33337932; PMCID: PMC7757688.AnswerWe concur with the significance of introducing the role of cytokines in the pathogenesis of COVID-19 within the introduction section. We have emphasized the great relevance of this matter and integrated the suggested references (lines 66-71).2. Please clarify inclusion and exclusion criteriaAnswerThe inclusion and exclusion criteria were clarified in the text (lines 142-144).3. Is the statistical analysis two-tailed or one-tailed?AnswerAll statistical analyses were conducted using a two-tailed approach. This clarification has been included in the text for clarity (lines 246-247).4. In the Introduction or Discussion, please discuss the following point based on the following reference: though the BioFire Respiratory panel is a large multiplex PCR panel, it still lacks some important respiratory pathogen to be covered, e.g., Pneumocystis jirovecii, which can be detected otherwise by some other novel molecular assays.Liu B, Totten M, Nematollahi S, Datta K, Memon W, Marimuthu S, Wolf LA, Carroll KC, Zhang SX. Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens. J Mol Diagn. 2020 Dec;22(12):1482- 1493. doi: 10.1016/j.jmoldx.2020.10.003. Epub 2020 Oct 15. Erratum in: J Mol Diagn. 2021 Apr;23(4):506. PMID: 33069878.5. In the Introduction or Discussion, please discuss that BioFire Respiratory panel provides some level of commutablity and standardization in different clinical labs as the same platform and panel is being used. According to the following reference, different reverse transcriptase with varied efficiency may cause unreliable results. BioFire Respiratory panel solves this problem as a popular and widely used commercial molecular test. Please discuss this based on this reference.Liu B, Forman M, Valsamakis A. Optimization and evaluation of a novel real-time RT-PCR test for detection of parechovirus in cerebrospinal fluid. J Virol Methods. 2019 Oct;272:113690. doi: 10.1016/j.jviromet.2019.113690. Epub 2019 Jul 5. PMID: 31283959.AnswerIn response to these two comments (4 and 5), we respectfully disagree with the reviewer regarding the relevance of incorporating the suggested references into our study discussion. The first reference pertains to the development of an assay for the detection ofPneumocystis jirovecii in Bronchoalveolar samples, a topic not within the scope of our study, which exclusively evaluates the BioFire Respiratory panel for SARS-CoV-2 detection. Similarly, the second reference concerns parechovirus detection, which is unrelated to the subject of our study.Consequently, we believe these references do not align with the focus of our research and, therefore, cannot be reasonably discussed in our study.6. Error: Line 57: "Wuhan province" should be "Wuhan city"AnswerThe correction has been implemented.Additional clarificationWe would like to clarify that there was a typographical error in our previous statement regarding the BioFire FA-RP2.1 specificity. The accurate specificity for this test is 94%, not 75%. As a result, we have made the necessary revisions to the discussion section (lines 327-330).AttachmentResponse to Reviewers.pdfSubmitted filename: Click here for additional data file. 18 Sep 2023Performance evaluation of Biofire Film Array Respiratory Panel 2.1 for SARS-CoV-2 detection in a pediatric hospital settingPONE-D-23-25121R1Dear Dr. Aquino,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact Kind regards,Benjamin M. Liu, MBBS, PhD, D(ABMM), MB(ASCP)Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 28 Sep 2023PONE-D-23-25121R1 Performance evaluation of Biofire Film Array Respiratory Panel 2.1 for SARS-CoV-2 detection in a pediatric hospital setting Dear Dr. Aquino:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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Salmonella enterica: Typhi, Paratyphi A, Typhimurium, and Enteritidis. We showed previously that a bivalent vaccine targeting S. Typhi and S. Paratyphi A using a Multiple Antigen-Presenting System (MAPS) induced functional antibodies against S. Typhi and S. Paratyphi. In the current study, we describe the preclinical development of a first candidate quadrivalent combination Salmonella vaccine with the potential to cover all four leading invasive Salmonella serotypes. We showed that the quadrivalent Salmonella MAPS vaccine, containing Vi from S. Typhi, O-specific Polysaccharide (OSP) from S. Paratyphi A, S. Enteritidis and S. Typhimurium, combined with the Salmonella-specific protein SseB, elicits robust and functional antibody responses to each of the components of the vaccine. Our data indicates that the application of MAPS technology to the development of vaccines targeting invasive forms of Salmonella is practical and merits additional consideration.Bloodstream infections in low- and middle-income countries (LMICs) are most frequently attributed to invasive Salmonella disease caused by four primary serovars of  Salmonella disease is the most common cause of blood stream infections in low- and middle-income countries (LMICs), but it is much more than just typhoid; it encompasses enteric fever (typhoid and paratyphoid A) and invasive nontyphoidal Salmonella disease. Four key serovars of Salmonella enterica are responsible for a large majority of invasive Salmonella disease: Salmonella enterica serovars Typhi, Paratyphi A, Typhimurium, and Enteritidis. Invasive nontyphoidal Salmonella (iNTS) disease has a high associated case fatality rate and particularly affects infants and immunocompromised individuals in sub-Saharan Africa. Although conjugate vaccines against Salmonella Typhi are licensed and World Health Organization (WHO)-prequalified, there are currently no such vaccines for invasive non-typhoidal Salmonella (iNTS) disease and paratyphoid A. This study outlines the preclinical development of the first candidate quadrivalent combination Salmonella vaccine, aiming to cover all four major invasive Salmonella serotypes. The focus is on generating components against S. Typhimurium and S. Enteritidis to complement the components against S. Typhi and S. Paratyphi A described in our previous report . I. IS. Ental cells C. Rhavi-izations .Salmonella MAPS vaccines. Based on this, we conducted all subsequent studies using Rhavi-SseB as the fusion protein. We conclude that, of the three fusion proteins evaluated, Rhavi-SseB resulted in the highest immune response induced by Salmonella-specific protein as the fusion protein is the ability to provide additional (polysaccharide-independent) protection against infection. To evaluate this possibility, we studied the ability of protein-directed antibodies to kill the organism. Rabbits were immunized twice with 100 µg protein of Rhavi-SseB on a two-week schedule. OPAs were performed with the post two immunizations sera and S. Typhimurium and S. Enteritidis as the target bacteria . However, such a difference was not noted when killing activity against S. Enteritidis was analyzed in the same fashion. These data suggest that a pathogen-specific fusion protein may enhance killing activity of sera from immunized animals for certain organisms.We then tested the function of protein-directed antibodies generated following immunization with MAPS vaccines, using sera from animals immunized with MAPS vaccines made with fusion protein CP1 for comparison. As shown previously, rabbits immunized with SseB-containing MAPS vaccines generated higher antibody concentrations to the polysaccharides than did those with CP1 as fusion protein. To adjust for this, we compared the killing activity in OPA per arbitrary unit of OSP antibody B. For S.S. Typhimurium and Rhavi-SseB as the fusion protein. First, we assessed the production of antibodies against the OSP in rabbits immunized with Rhavi-SseB-S. Typhimurium MAPS construct after the first and second immunizations. After two immunizations, the rabbits generated a robust antibody response to S. Typhimurium OSP  and a quadrivalent MAPS  both using Rhavi-SseB as the fusion protein. To target several clinically relevant We tested the antibody production in rabbits immunized with the bivalent MAPS vaccine. Robust antibody responses were generated against both OSPs, at similar levels to those achieved by each monovalent MAPS vaccine A. These Salmonella species was tested with opsonophagocytic assays for all organisms except S. Paratyphi A, for which we used a bactericidal assay. For the bivalent MAPS vaccine, killing activity against S. Typhimurium and S. Enteritidis was very similar to the killing titers generated with each of the monovalent MAPS vaccines . A livelunteers ,30. A polunteers ,30. S. Tshedding ,32,33. Shigella sonnei (NCT02676895 and NCT03527173) [Another approach is the use of outer membrane vesicles (OMVs), as, for example, the Generalized Modules for Membrane Antigens (GMMA) platform being used by the GSK Vaccines Institute for Global Health (GVGH) ,34. GMMA3527173) ,37,38 anS. Typhimurium and S. Enteritidis OSP are protective in mice [S. Typhimurium that had bactericidal activity [S. Typhimurium and S. Enteritidis protected mice from lethal Salmonella infections [S. Typhimurium and S. Enteritidis. A vaccine based on core and O-specific polysaccharides (OSPs) chemically linked to flagellin protein FliC was immunogenic and protective in mice and has completed a Phase 1 clinical trial (NCT03981952) [Salmonella proteins have been studied for their potential to generate protection from infection. Members of the outer membrane protein family (OmpCDF), siderophores, and proteins from the type III secretion system are immunogenic in mice [Several studies have demonstrated that antibodies against  in mice ,41,42,43activity ,45,46. Tfections ,48,49. T3981952) ,48. As a in mice ,51,52.S. Typhimurium and S. Enteritidis using O-specific polysaccharides from both organisms. We determined that the Salmonella-specific protein SseB was an excellent fusion protein in these constructs (S. Typhimurium (In this work, we designed two MAPS constructs to target nstructs , resultinstructs . We alsohimurium . Our appSalmonella MAPS vaccines and demonstrated that combining MAPS into one immunization can potentially provide protection against multiple organisms. The bivalent MAPS vaccine containing OSPs for S. Typhimurium and S. Enteritidis demonstrated significant antibody production and killing activity, comparable to their monovalent counterparts (S. Typhimurium, S. Enteritidis, S. Typhi Vi, and S. Paratyphi A induced production of significant functional antibodies (Salmonella enterica with two doses of this vaccine. This would eliminate the need for multiple differing immunizations in endemic areas where vaccine compliance may be difficult [Salmonella is feasible and warrants further consideration and confirmation of protection by challenge studies.We designed bivalent and quadrivalent terparts . The quatibodies . The datifficult . OverallS. Typhimurium, S. Enteritidis, and S. Paratyphi A, and conserved Salmonella protein SseB generated functional antibodies against all the components included in the vaccine. The results support further development of the vaccine.Our study showed that a combinational MAPS vaccine containing Vi, OSPs from


Ganoderma lucidum (G. lucidum) has been widely used for its health benefits as an edible and traditional medicinal mushroom for thousands of years in Asian countries. It is currently used as a nutraceutical and functional food owing to its major bioactive compounds, polysaccharides and triterpenoids. G. lucidum exhibits a broad range of hepatoprotective impacts in various liver disorders, such as hepatic cancer, nonalcoholic fatty liver disease (NAFLD), alcohol-induced liver disease, hepatitis B, hepatic fibrosis, and liver injury induced by carbon tetrachloride (CCl4) and α-amanitin. G. lucidum protects the liver through a broad range of mechanisms that include the modulation of liver Phase I and II enzymes, the suppression of β-glucuronidase, antifibrotic and antiviral actions, the regulation of the production of nitric oxide (NO), the maintenance of hepatocellular calcium homeostasis, immunomodulatory activity, and scavenging free radicals. G. lucidum could signify an encouraging approach for the management of various chronic hepatopathies, and its potential mechanisms make it a distinctive agent when used alone or with other drugs and applied as a functional food, nutraceutical supplement, or adjuvant to modern medicine. This review summarizes the hepatoprotective properties of G. lucidum with its various mechanisms of action on different liver ailments. Biologically active substances derived from G. lucidum are still being studied for their potential benefits in treating different liver ailments. G. lucidum is a substantial source of nutritionally and pharmacologically important potential constituents. It has been used as an herbal cure for many years in traditional Japanese and Chinese medicine [G. lucidum can prolong one’s life and improve health. As a source of distinctive bioactive metabolites, which grants it a number of attributes, it has been demonstrated to be efficient in the treatment and management of a variety of diseases [G. lucidum has demonstrated potent anticancer [medicine ,2. The pmedicine . It has diseases . G. luciticancer ,6, anti-ticancer , hepatopticancer , antidiaticancer , cardiopticancer , immunomticancer , antioxiticancer , and antticancer  activititicancer . Accorditicancer ,14.G. lucidum has a variety of hepatoprotective effects on liver illnesses, including alcoholic liver disease [G. lucidum are a promising approach to surmount such challenges and provide a potential natural hepatoprotective agent [The term “liver diseases” is used to describe a broad range of illnesses that prevent or impair the liver’s ability to operate. Recent studies have found that  disease , viral h disease , NAFLD [ disease , fibrosi disease , hepatic disease , CCl4 an disease . The preve agent .G. lucidum, G. lucidum triterpenoids, G. lucidum polysaccharides (GPLS), β-glucans, D-glucans, ganoderic acids, ganoderic acid A, ganoderic acid B, ganoderic acid C1, G. lucidum triterpenoids extracts, hepatic fibrosis, hepatic cancer, hepatitis and hepatitis B. The following phrases were included: “G. lucidum hepatoprotective effects”, “Effect of G. lucidum in hepatic cancer”, “Effect of G. lucidum in hepatic fibroris”, “Effect of G. lucidum in NAFLD”, “Effect of G. lucidum in CCl4 induced hepatic injury”, “Effect of G. lucidum in hepatitis B”, “Effect of G. lucidum in formaldehyde induced hepatic injury”, “Effect of G. lucidum in α-Amanitin induced liver injury” and “Effect of G. lucidum in alcohol-induced hepatic injury”. Articles published in English were chosen to study the hepatoprotective effects of G. lucidum in our literature survey.For the present comprehensive review, we collected evidence through diverse databases that included Science Direct, Saudi Digital Library, Scopus, Google Scholar and PubMed. The following keywords were used: G. lucidum. Several monomers—for instance, galactose, glucose, xylose, mannose, fucose, rhamnose and arabinose—in the composition of G. lucidum polysaccharide have been described as significantly contributing to its antioxidant, immunomodulatory, antitumor, and antibacterial characteristics [G. lucidum, particularly β-glucans, synergistically improve immune system functioning and potentiate immune cells’ anticancer activities and cytokine production, whereas G. lucidum triterpenes inhibit the cancer cells growth, proliferation and invasion [Polysaccharides and triterpenoids are two of the main biologically potent components of eristics ,20. It ieristics . Additioinvasion ,22,23. Ginvasion ,25. The invasion ,27.G. lucidum’s phenolic composition, with phenolic acids emerging as the most significant class. These acids include chlorogenic, gallic, cinnamic, protocatechuic, p-coumaric, p-hydroxybenzoic and p-coumaric acids. Anti-inflammatory, antibacterial, antityrosinase and antioxidant activities have been linked to these compounds [G. lucidum indicates a high potential for its application as a functional food and nutraceutical in various forms, such as tablets, pills and capsules [Triterpenes, which include ganoderic acids (GAs), ganodermic acid, lucinedic acids, ganoderols and lucidones, are the most prevalent terpenes and have been linked to a variety of potential benefits against cancer, inflammation, hepatitis, hypoglycemia, microbes and HIV-1 ,29,30. Nompounds ,32,33. Icapsules ,35.G. lucidum may influence the efficiency of its hepatoprotective effects by reducing the overabundance of free radicals and preserving cells from oxidative stress [G. lucidum water extract prevented hepatic damage in mice subjected to acute hepatic injury induced by cadmium or amanitin [G. lucidum, such as spore oil, sporoderm-breaking spores and spore powder have proven to be effective against hepatic ailments [G. lucidum can be seen in Triterpenoids and polysaccharide-rich e stress . Throughamanitin ,37. Tritamanitin . Differeailments ,39,40,41G. lucidum is widely regarded as a potentially valuable, conventional healing approach for various types of hepatic disorders. G. lucidum is a medicinal mushroom that is also considered an edible mushroom. G. lucidum possesses immunomodulators and anticancer activities through various mechanisms of action, demonstrating profound impacts against various types of cancer and other diseases as well as hepatoprotective activity [G. lucidum have been reported in a wide range of diseases due to its multifaceted mechanism of action. G. lucidum polysaccharide (GLPS) illustrates immunomodulatory characteristics through the enhancing action of the mononuclear phagocyte system and antigen-presenting cells, along with cellular and humoral immunity. β-glucans obtained from G. lucidum are predicted to exhibit an immune reaction via pathogen-associated molecular patterns (PAMPs) [G. lucidum that demonstrate anticancer properties through a variety of mechanisms, including host immune response activation, cytotoxic action on cancer cells, the suppression of angiogenesis, the downregulation of uPA and uPA receptor expression in cancerous cells and the induction of cell differentiation [G. lucidum against different liver disorders can be seen in activity ,42. Immu (PAMPs) . β-gluca (PAMPs) ,45. Addintiation ,47. The G. lucidum could be a good approach to protecting against a wide range of hepatic disorders. The mechanisms of G. lucidum’s hepatoprotective impacts are widely undefined. Nevertheless, substantial evidence suggests a number of mechanisms which include radical-scavenging behavior, antioxidant properties, the modulation of hepatic enzymes, β-glucuronidase inhibition, antiviral and antifibrotic activity, hepatocellular calcium maintenance, homeostasis, the production of nitric oxide and the impacts of immunomodulation. Its molecular mechanisms as well as bioactive constituents should be researched to further enhance the management of hepatic diseases and chemically induced hepatic complications [G. lucidum exhibits against different hepatic disorders are compiled in The use of ications ,48,49,50Fibrosis is a condition defined by the excessive accumulation of extracellular matrix components which can cause organ failure and death. Up to 45% of all fatalities in affluent countries are caused by fibrosis . ThroughG. lucidum extract (GLE) exhibits potential preventive as well as therapeutic results in studies of formaldehyde (FA)-induced liver fibrosis [G. lucidum’s hepatoprotective effectiveness against hepatic fibrosis induced by FA was assessed by measuring aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). ALT is a crucial enzyme for liver fibrosis, which leads to catalyzing the transamination process. Liver fibrosis is directly influenced by the increase in enzyme activity [G. lucidum. The pathogenesis of liver fibrosis is significantly impacted by TNF, IL-1 and IL-6. TNF is primarily a group of pro-inflammatory cytokines that are known to be crucial in causing liver fibrosis. During hepatotoxic fibrosis, the liver releases IL-1, IL-6 and TNF into the blood. When FA was given to the rats, it was noted that FA significantly increased the amounts of these cytokines in their livers. The group that received 100 mg/kg of G. lucidum had significantly lower cytokine levels than those of the control group. These results provided evidence for the hepatoprotective properties of G. lucidum [G. lucidum, through lowering malondialdehyde (MDA) and hydrogen peroxide levels, boosting glutathione (GSH) and antioxidant enzymes, and maintaining normal ranges of nitrite and myeloperoxidase formation in FA-treated rats, exhibits liver-protective effects [fibrosis . G. luciactivity ,66. Thes lucidum . G. luci effects .G. lucidum triterpenes on hepatic fibrosis were evaluated [G. lucidum total triterpene extracts. The ideal hepatoprotective outcome for the total triterpene extract from G. lucidum was found at an amount of 180 mg/kg, based on biological indicators and a liver histopathology investigation [G. lucidum triterpenoids had a potent efficacy against D-GalN-induced hepatic fibrosis. The activity of enzymes that neutralize free radicals, thereby increasing antioxidant potential, may be associated with the hepatoprotective efficacy of G. lucidum triterpenoid extract. Ganoderma of other species was also found to be effective against hepatic fibrosis. Triterpenoids in Ganoderma applanatumm such as ganoapplanic acid A, ganoapplanic acids C and F, and ganoapplaniates D, inhibited the proliferation of hepatic stellate cells (HSCs) [Comprehensive research was conducted on hepatic fibrosis induced by D-galactosamine , and the effects of valuated . The sertigation ,54. Thess (HSCs) .G. lucidum has a number of amazing advantages for lipid metabolism and liver health. It has been found that G. lucidum ethanol extract (high in GAs) exhibits a defensive effect against liver injury induced by alcohol in mice [G. lucidum ethanol extract inhibits anomalous upsurges in total cholesterol (TC), serum triglyceride (TG), low-density lipoprotein cholesterol, ALT and AST. Additionally, GLE diet interventions significantly reduce MDA and lactate dehydrogenase (LDH) levels in the liver and increase catalase (CAT), GSH, alcohol dehydrogenase (ADH) and SOD levels, which all help to combat alcohol-induced oxidative stress [G. lucidum ethanol extract has the potential to be helpful in reducing alcohol-induced liver injury [Due to the fact that alcohol is one of the most popular psychoactive constituents, alcohol abuse and dependency are progressively becoming a crucial issue on a global scale. About 2.5 million people each year die as a result of frequent and excessive alcohol consumption . One of  in mice . In addie stress . In addie stress . In addir injury .In addition, a study found that GA supplementation significantly reduced abnormally elevated liver indices, serum lipid parameters, AST, ALT and lipid accumulation in mice exposed to alcohol . In a spNAFLD is the term used to describe hepatic steatosis that is not linked to increased alcohol intake or other obvious hepatotoxic factors . The metG. lucidum (FYGL), a hyperbranched proteoglycan (a composition of lipophilic protein and hydrophilic polysaccharide) isolated from G. lucidum, inhibits the steatosis caused by palmitic acid (PA) in HepG2 hepatocytes [Effective drugs against NAFLD are needed. Fudan-Yueyang atocytes . FYGL siatocytes . These fGanoderma species, such Ganoderma amboinense, have also demonstrated efficacy against liver disease. This species has shown potential action in liver disorders. According to earlier studies, Ganoderma amboinense polysaccharide (GAP) has protective effects on the liver [However, other he liver . In one he liver . Simultahe liver . Its mulG. lucidum chemical compounds have anticancer activities primarily through multiple pathways, such as host immunomodulators, cytotoxic properties, the induction of metabolizing enzymes, etc. Among the numerous compositions of GLPS and triterpenoids, G. lucidum is being studied extensively due to numerous studies in which it has shown effects on cancer [Cancer has become a growing global public health concern. In developed nations, it is the most common cause of death . G. lucin cancer ,8.Hepatocellular carcinoma (HCC) is one of the world’s most dangerous cancers . ChemoemG. lucidum fruiting body dry extract on peripheral blood lymphocytes and human liver tumor cells (HepG2/C3A) were assessed. It was discovered that fruiting body dry extract exhibited toxic effects to tumor cells, reducing their viability by causing DNA damage and boosting their production of ROS. In contrast, fruiting body dry extract was hazardous to lymphocytes only at high doses and reduced their viability, whereas at low quantities, it improved lymphocyte viability. Furthermore, G. lucidum fruiting body dry extract only caused primary DNA damage at the highest measured dose. As a result, G. lucidum exhibits cytotoxic and genotoxic activity as well as possible anticancer effects on malignant liver cells [G. lucidum spore polysaccharide (GLSP) activates macrophages, controls their polarization and encourages the discharge of numerous inflammatory mediators and cytokines [The effects of er cells . In ordeytokines . Additioytokines . Therefoytokines ,81.Moreover, the anti-invasion impact of GLE on human hepatoma HepG2 cells was assessed . HepG2 c2O2 content, GSH and CAT were restored after treatment with GLE, preserving the enzymes [G. lucidum is a potent antioxidant that significantly inhibits the elevated MDA level and demonstrates substantial free radical scavenging activity [The CCl4 metabolite trichloromethyl free radical is the major cause of hepatotoxicity. It binds with tissue macromolecules and then encourages membrane lipid degradation and finally damages the membrane. It is expected that such progress tends toward lipid peroxidation ,84,85. A enzymes . G. luciactivity ,92.G. lucidum’s effects are depicted in In one study, GLPS was found to have hepatoprotective effects on common carp hepatocyte injury brought on by CCl4. According to the findings, GLPS greatly boosted cell viability, suppressed the elevations of the marker enzymes , and MDA caused by CCl4, and dramatically increased the levels of SOD. The expression of CYP1A and CYP3A was markedly downregulated during the GLPS treatments, along with extrinsic apoptosis and the immunological inflammatory response. GLPS can prevent hepatocyte injury brought on by CCl4 by decreasing lipid peroxidation, increasing the activities of antioxidant enzymes, and suppressing apoptosis and the immunological inflammatory response . AdditioG. lucidum, exhibits hepatoprotective properties against such toxicities [G. lucidum triterpenoids’ hepatoprotective effects on liver damage by α-amanitin (α-AMA) in rats were examined, and significant effects were reported through radical scavenging and antiapoptotic properties. The mice were treated with and monitored by total or individual triterpenoids of G. lucidum, and the triterpenoids’ hepatoprotective impacts were evaluated by comparing them with silibinin (SIL) [G. lucidum’s total triterpenoids decreased death rates by 20–40% and considerably reduced the serum levels of ALT and AST. In addition, triterpenoids and SIL dramatically increased catalase and SOD activity and decreased MDA levels in the mice livers. The treatment with GA-C2 dramatically reduced caspase-3, -8, and -9 activity and significantly suppressed DNA fragmentation. The findings showed that triterpenoids have hepatoprotective effects on liver injury induced by α-AMA, and that these benefits may be caused by their antioxidative radical-scavenging properties as well as their prevention of apoptosis [The majority of deadly mushroom poisonings are brought on by Amanita species; these mushrooms contain amatoxins that cause acute liver failure. A distinguished traditional healing mushroom, xicities . G. luciin (SIL) ,93. The poptosis .Amanita exitialis, followed by the administration of GLE. A reference medicine, SIL, was used to compare GLE’s hepatoprotective activity. The effects of α-AMA included a considerable increase in the serum levels of ALT and AST, as well as a substantial decline in the activities of the antioxidant enzymes’ catalase and SOD. When compared to the α-AMA control group, the treatments with GLE or SIL considerably reduced serum AST and ALT levels, considerably boosted the actions of CAT and SOD, and significantly diminished the MDA content in the liver [The effects of GLE on liver damage by α-AMA were also examined in a similar investigation, as well as potential mechanisms for hepatoprotection linked to radical scavenging activity. Mice were given an injection of α-AMA made from he liver .G. lucidum [G. lucidum at a concentration of 8 µg/mL. The mice were also significantly protected by GAs from liver damage caused by M. bovis BCG and lipopolysaccharide (from Escherichia coli 0127:B8) [G. lucidum was tested for anti-HBV and hepatoprotective action. Radix Sophorae flavescentis aqueous extract, a Chinese herbal medicine, was added to the cultured broth. In vitro, the cultured broth exhibited anti-HBV activity and protected mice from hepatic injury. Additionally, it has been claimed that co-fermenting G. lucidum broth with Radix Sophorae flavescentis aqueous extract results in stronger therapeutic effects than merely combining these two components [Hepatitis is an inflammation of the liver, and the liver is an important organ for filtering blood . It dist lucidum , which e0127:B8) ,95. Moremponents .Faecalibaculum, Lactobacillus, Bifidobacterium and Romboutsia and lowering the level of Helicobacter, GA intervention altered the composition of intestinal microflora, as shown by intestinal microbiota profiling. In addition, liver metabolomic profiling indicated that GA intervention had a notable regulatory influence on liver metabolism after drinking too much alcohol [G. lucidum spores exhibited substantial effects against cancer and altered gut microbiota caused by AOM/DSS. The spores increased goblet cells, MUC2 production and tight junction protein expression. They also significantly enhanced gut barrier function. In addition to reducing the expression of IL-1, iNOS, and COX-2, GLP therapy also prevented macrophage infiltration. Additionally, GLP reduced the inflammatory markers in macrophage RAW264. 7, intestinal NCM460, HT-29 cells and the activation of mitogen-activated protein kinases. These findings suggest that GLP is a viable prebiotic for the management of colorectal cancer [Excessive alcohol intake is one of the foremost causes of intestinal microbial ailments, which have been meticulously linked to the pathogenesis of hepatic diseases . It has  alcohol . Moreovel cancer .G. lucidum was discovered to have a defensive effect on liver injury due to H2O2 stress in mice by enhancing oxidative status. Two different polysaccharides purified from GLP, acidic-glucan (GLPC2) and neutral-glucan (GLPB2), exhibited a stronger hepatoprotective effect against H2O2-induced liver injury in HepG2 cells [2O2-induced HepG2 cells [G. lucidum protects from drug-induced hepatic injury through inhibiting HMGB-1/NF-kB and caspase-3. It modulates oxidative stress and the ensuing cross-talk between the inflammatory and apoptotic cascades, revealing its potential contribution to drug-induced hepatic injury. In addition, the G. lucidum mushroom also exerts hepatoprotective effects against cadmium and tert-butyl hydroperoxide (t-BHP)-induced hepatic injury [The crude polysaccharide of G2 cells . As a prG2 cells . HydrogeG2 cells ,102. In G2 cells . Acute lc injury .G. lucidum is a traditional medicinal and edible mushroom that has a significant role in preserving human health. The demand for this mushroom is rising across the world as a medicinal, nutraceutical and functional food. The efficient hepatoprotective action of the naturally occurring, biologically active compounds confined to G. lucidum is promising in the endeavor to find successful hepatoprotective substances. Novel drugs from natural sources that can be alternatives to synthetic medicine with fewer side effects are needed. As a result, these investigations offer insightful information and a solid foundation for developing new medications to treat hepatic disorders from G. lucidum. Because of the global trend of an increase in the number of people with liver ailments, there is substantial demand for effective medications that can provide potential outcomes through multifaceted mechanisms. Moreover, research and clinical trials are in progress to determine the efficacy of numerous compounds obtained from G. lucidum in support of hepatoprotective activity. Studies and more research could make it easier to create medicinal and nutraceutical formulations that could be used to treat a wide range of diseases, particularly hepatic cancer, hepatitis, hepatic fibrosis and NAFLD. Additional investigations are needed through different parameters to find out the various unrevealed compounds that could be used in further experimental and clinical studies to eradicate hepatic disorders.


In acute lung injury, the lung endothelial barrier is compromised. Loss of endothelial barrier integrity occurs in association with decreased levels of the tight junction protein claudin-5. Restoration of their levels by gene transfection may improve the vascular barrier, but how to limit transfection solely to regions of the lung that are injured is unknown. We hypothesized that thoracic ultrasound in combination with intravenous microbubbles (USMBs) could be used to achieve regional gene transfection in injured lung regions and improve endothelial barrier function. Since air blocks ultrasound energy, insonation of the lung is only achieved in areas of lung injury (edema and atelectasis); healthy lung is spared. Cavitation of the microbubbles achieves local tissue transfection. Here we demonstrate successful USMB-mediated gene transfection in the injured lungs of mice. After thoracic insonation, transfection was confined to the lung and only occurred in the setting of injured  lung. In a mouse model of acute lung injury, we observed downregulation of endogenous claudin-5 and an acute improvement in lung vascular leakage and in oxygenation after claudin-5 overexpression by transfection. The improvement occurred without any impairment of the immune response as measured by pathogen clearance, alveolar cytokines, and lung histology. In conclusion, USMB-mediated transfection targets injured lung regions and is a novel approach to the treatment of lung injury.NEW & NOTEWORTHY Acute respiratory distress syndrome is characterized by spatial heterogeneity, with severely injured lung regions adjacent to relatively normal areas. This makes targeting treatment to the injured regions difficult. Here we use thoracic ultrasound and intravenous microbubbles (USMBs) to direct gene transfection specifically to injured lung regions. Transfection of the tight junction protein claudin-5 improved oxygenation and decreased vascular leakage without impairing innate immunity. These findings suggest that USMB is a novel treatment for ARDS. Acute lung injury is a common diagnosis in critical care units and in its most severe form has a mortality rate approaching 40% . Common Regardless of the cause, most forms of acute lung injury demonstrate heterogeneous involvement of the lungs within a given patient: densely consolidated, edematous, or atelectatic lung is observed adjacent to other lung regions that appear relatively normal . This heRegionally targeted treatment delivered early may be important to prevent further extension with clinical deterioration. We hypothesized that the loss of air in the injured lung could be leveraged to enable targeted and increased delivery of drugs and genetic material using ultrasound and microbubbles . MicrobuWe reasoned that in acute lung injury, ultrasound waves will preferentially penetrate the most damaged areas of the lung, i.e., fluid-filled, or atelectatic regions, leaving more normal (air-filled) areas of the lung unaffected. Ultrasound energy that penetrates severely injured lung regions could then act on circulating microbubbles, enhancing local gene or drug delivery. Since the ultrasound transducer can be focused on the thorax, the effect on other organs is limited, further increasing the specificity of delivery.In this report, we establish the feasibility of this approach (termed USMB) for the delivery of genetic cargo to the injured lungs of animals. We find that USMB can achieve transfection in a highly spatially restricted manner, resulting in functional protein expression in both lung endothelial and epithelial cells only in injured and insonated lungs. We show that USMB-mediated transfection of the tight junction constituent claudin-5 causes decreased vascular leakage and a significant improvement in oxygen saturation in mice with lung injury. This improvement occurs without impairing innate immunity or pathogen clearance. Together, these data indicate that USMB-mediated gene delivery is feasible as a targeted therapeutic approach for acute lung injury.All experimental procedures were approved by the St. Michael’s Hospital Animal Care Committee . C57BL/6J male mice (10–14 wk old) were purchased from Jackson Laboratories; in pilot studies, we observed the efficacy of USMB-mediated transfection in animals of both sexes. Only male mice were used in this study to limit costs.Unless otherwise indicated, a SONOS 5500 (Phillips) ultrasound machine and an S3 phased array transducer were used. The following ultrasound settings were used: transmitter frequency, 1.3 MHz; 67 V; 0.2 W; mechanical index, 0.9; pulse interval, 5 s. Cationic microbubbles for plasmid transfection were prepared as previously reported  of E. coli (OD = 1.0) in a volume of 130 μL; healthy controls received no fluid. Furthermore, 0.5–6 h postinfection (hpi), mice were intravenously administered 10 μg plasmid with 3 × 108 cationic microbubbles in a volume of 300 μL followed immediately by 5 min of ultrasound on each (left and right) side of the chest. Sixteen to twenty-four hours posttreatment, mice were euthanized via cardiac puncture. For luciferase experiments, 24-h post-USMB mice were injected intraperitoneally with 2.25 mg of d-luciferin dissolved in 150 μL of PBS. After 15 min, mice were euthanized and harvested for lung and kidney. Organ luminescence was detected using a Newton 7.0 FT-500 Bioluminescent and Fluorescent Animal Imager. Luminescent signal was analyzed by subtracting background luminescence of control animal organs that did not receive luciferin.For For Evans Blue measurements, mice received thoracic ultrasound using the Vevo 2100 Imaging system  with the MS250 transducer with burst mode activated every 6 s to achieve cavitation. Sixteen hours later mice were injected intravenously with 200 µL of 0.5% Evans Blue in PBS. After 1 h, mice were anesthetized and euthanized via cardiac puncture. Blood, lungs, and bronchoalveolar lavage fluid (BAL) were collected as previously described . Evans B5–1.5 × 106 plaque-forming units (PFUs) of influenza A  and diluted to a final volume of 130 µL as previously described  were used to measure arterial oxygen saturation of infected mice immediately before and 24 h after USMB treatment. Prior to infection, neck hair was shaved so that the small collar could accurately obtain the escribed ], were mFor histological analysis, lungs were fixed for 48 h using 10% neutral-buffered formalin and embedded in paraffin. Furthermore, 5-μm thick slides were prepared. For immunofluorescent staining, we used anti-Tie-2 , anti-aquaporin-5 , and anti-GFP. For hematoxylin and eosin staining, the Leica Autostainer XL was used. Slides were analyzed in a blinded fashion according to consensus criteria .GGATCACTCTCGGCATGGAC, human claudin-5 reverse CCTCTTTGAAGGTTCGGGGG, mouse 18 s forward GTAACCCGTTGAACCCCATT, and mouse 18 s reverse CCATCCAATCGGTAGTAGCG.For qPCR analysis, RNA was extracted from whole lung lysates using TRIzol. cDNA was generated using PowerUp SYBR Green Master Mix (Applied Biosystems). Primers used (5′ to 3′) were: human claudin-5 forward Primary human pulmonary microvascular endothelial cells (PromoCell C-12281) were grown on transwells  or glass coverslips (for immunofluorescent imaging). TEER was measured using Endohm-12 from World Precision Instruments. For imaging, cells were fixed with 4% PFA and counterstained with DAPI. Confocal imaging was performed with a Quorum TIRF/SD microscope.P value was determined by two-tailed t tests. In experiments with more than two groups, data were analyzed by one-way ANOVA and post hoc t tests when appropriate. For the change in oxygen saturation 24 h after USMB, a one-way ANOVA with mixed-effects analysis was used due to unequal sample sizes. Statistical comparisons were completed using GraphPad Prism software .All experiments were repeated a minimum of three times independently; data are presented as means and standard errors of the mean. In experiments with two groups, the E. coli, a common cause of nosocomial pneumonia. Mice rapidly develop hypoxemia, pulmonary edema, and alveolar neutrophilia ; 24 h later, lungs were analyzed by immunofluorescence. We observed GFP protein in treated but not control animals C as wellGiven that vascular leakage to plasma is not required for leukocyte emigration from the circulation during inflammation , we reasE. coli model of lung injury; using a plasmid for human claudin-5 allowed us to design species-specific PCR primers to distinguish the transfected plasmid from the endogenous transcript. Sixteen hours after treatment, we observed significantly increased expression of human claudin-5 in USMB-treated animals .We wanted to verify that the improved oxygenation did not come at the expense of an appropriate immune response to the virus. Transfection of claudin-5 had no effect on pathogen clearance, as viral titers in lung homogenates were unchanged A. CytokiAcute lung injury is a common complication of infection with many viral and bacterial lung pathogens, including influenza A and SARS-CoV-2 . It is aAlthough ultrasound and microbubbles have been used to enhance drug delivery in other organs , the refOur data indicating that transfection of claudin-5 is beneficial demonstrates that the overall strategy of USMB-mediated gene delivery has merit. Preventing vascular leakage regionally may prevent an extension of injury, provided that it does not prevent the action of local immunity. The pathognomonic abnormality in acute lung injury (and ARDS) is increased alveolar-capillary permeability and overexpression of claudin-5 addresses this directly. In vivo transfection using USMB did not aggravate lung injury as measured histologically, nor did it interfere with pathogen clearance or the generation of cytokine response in the lung. Instead, overexpression of claudin-5 in the injured lung significantly improved oxygenation and decreased vascular leakage; these data suggest that tightening of the vascular barrier by molecular means is feasible and does not necessarily compromise the immune response . More geIn this study, we have used plasmids given their ease of production in large quantities and the potential for their enhancement using customized  promoters. The delivery of oligonucleotides such as miRNA and siRNA should also be feasible .Despite our data in mice showing physiological improvement and no worsening of histological injury, USMB in patients with ARDS would require further study to ensure no acute adverse effects . Sufficient tissue penetration of ultrasound may also be an issue in larger animals, although endobronchial ultrasound could in principle permit access to deeper areas of the injured lung. Optimization of USMB for its use in humans and delineation of its potential additional applications is now underway .This study was supported by Collaborative Health Research Program grant from the Canadian Institutes of Health Research  and the Natural Sciences and Engineering Research Council of Canada (523598-18); Operating grant from the CIHR (OV2 170656); Canada Research Chair Program.W.L.L., H.L.P., and R.K. are coinventors on a patent related to this work. W.L.L. is the Chief Scientific Officer and R.K. is the Secretary of a spin-off company related to this work. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.R.S., V.M., R.K., H.L.-P., D.M.H., A.G., and W.L.L. conceived and designed research; R.S., V.M., M.D., A.L., E.L., S.G., and N.K. performed experiments; R.S., V.M., S.G., N.K., D.M.H., A.G., and W.L.L. analyzed data; R.S., D.M.H., A.G., and W.L.L. interpreted results of experiments; R.S. prepared figures; R.S. and W.L.L. drafted manuscript; R.S., L.B., A.G., A.S.S., and W.L.L. edited and revised manuscript; R.S., R.K., H.L.-P., D.M.H., L.B., A.G., A.S.S., and W.L.L. approved final version of manuscript.


E. coli upon desiccation–rehydration stress. In silico analyses suggest that various mechanisms and interactions are responsible for their formation, including LLPS, disordered sequences and aggregation-prone regions. These data support findings that intrinsically disordered proteins and LLPS may contribute to desiccation tolerance not only in eukaryotic cells but also in bacteria. LLPS-driven aggregation may be a strategy used by pathogens to survive antibiotic treatment and desiccation stress in the hospital environment.Liquid–liquid phase separation (LLPS) and the formation of membraneless organelles (MLOs) contribute to the spatiotemporal organization of various physiological processes in the cell. These phenomena have been studied and characterized mainly in eukaryotic cells. However, increasing evidence indicates that LLPS-driven protein condensation may also occur in prokaryotes. Recent studies indicate that aggregates formed during proteotoxic stresses may also play the role of MLOs and increase the fitness of bacteria under stress. The beneficial effect of aggregates may result from the sequestration and protection of proteins against irreversible inactivation or degradation, activation of the protein quality control system and induction of dormancy. The most common stress that bacteria encounter in the natural environment is water loss. Therefore, in this review, we focus on protein aggregates formed in  Increasing evidence has shown that the liquid–liquid phase separation (LLPS) process is crucial to form multiple assemblies within the cell. During LLPS, a homogenous solution of macromolecules is separated into macromolecule-rich liquid droplets and a diluted phase A 1,2]. ,2. 1,2].E. coli upon desiccation–rehydration stress, assuming that water loss and cytoplasm condensation under these conditions may facilitate LLPS and the formation of protective aggregates. Numerous reports have indicated that intrinsically disordered proteins (IDPs) and LLPS contribute to desiccation tolerance in eukaryotes [This review will present background information on LLPS , LLPS-prkaryotes ,23,24,25karyotes ,27. Undekaryotes ,29. AnotLLPS is driven by weak multivalent interactions, including electrostatic interactions, cation–π, π–π and other hydrophobic contacts, oligomerization domains, motif-binding domains, helix–helix interactions and β-zippers ,31,32,33Structural disorder is a common feature of proteins capable of LLPS . AlthougThe proteomes of different species comprise a whole spectrum of conformations from fully structured to completely disordered . The anaThere are multiple examples of condensates formed by LLPS and regulated by PTMs in eukaryotic cells ,36,43. PE. coli proteins that can form liquid droplets (see the next section) are post-translationally modified: SSB and TmaR are phosphorylated [E. coli [PTMs of proteins in bacteria are less abundant and more diverse than in eukaryotic cells . Low stoorylated ,48, and orylated ,50. The [E. coli . HoweverDepending on conditions, condensation may lead to the formation of highly ordered structures such as glassy solids, amyloid fibers or even crystals. In contrast to dynamic liquid droplets, these states are mostly irreversible C. SeveraE. coli membrane also contribute to the differentiation of cell structure [E. coli aggregates formed upon desiccation–rehydration stress  and SBS—the SlmA-binding sequence [FtsZ assembly. FtsZ is a tubulin homolog that assembles into the Z ring at the site of cell division. Monterroso et al. reported that FtsZ from sequence . The preE. coli SSB homotetramer possesses an N-terminal DNA domain containing a single OB-fold, an intrinsically disordered linker (IDL) and a C-terminal protein–protein interaction peptide (CTP) [E. coli SSB forms viscous, liquid-state protein droplets in vitro under physiological concentrations of ions and protein [Single-stranded DNA binding protein (SSB). SSB is another bacterial protein that forms liquid–liquid phase separated coacervates . Each sude (CTP) . SSB for protein . All str protein .E. coli RNA polymerase (RNAP) formed condensates sensitive to hexanediol, which dissolves liquid-like compartments in eukaryotic cells [RNA polymerase clusters. Ladouceur et al. demonstrated that during the transition from lag phase to log phase, ic cells . RNAP clE. coli, HU-A, HU-B and Dps, can form coacervates with different forms of DNA and RNA [E. coli cells [E. coli cells. In addition, Dps is an iron-storage protein with ferroxidase activity. These Dps functions, ferrous ions scavenging and DNA compaction, protect bacteria against various stress factors [HU and Dps. The most abundant nucleoid-binding proteins in  and RNA ,62. It w factors .E. coli exposed to proteotoxic stresses [The proteins described above have been detected as components of aggregates that contain hundreds of proteins isolated from stresses ,19. The Staphylococcus aureus, E. coli and A. baumannii. The peptides were effective against drug-resistant clinical isolates and reduced bacterial load in a murine infection model [Proteostasis  is crucial for optimal cell growth. Intrinsic or environmental stresses such as heat shock, desiccation, antibiotics, oxidative stress or metal ion exposure often disturb proteostasis, which is manifested by protein aggregation ,67,68,69on model ,75.E. coli, the main DnaK-DnaJ-GrpE chaperone system and the GroEL-GroES chaperonin are responsible for ATP-dependent refolding of denatured proteins, whereas proteases  degrade irreversibly damaged proteins [Bacteria have evolved a complex quality-control machinery, comprising molecular chaperones and proteases, to cope with the detrimental effects of stressful conditions and maintain proteostasis ,77. In Eproteins ,77. UndeE. coli cells accumulated mainly at the cell poles [Bacterial populations can diminish toxic effects caused by misfolded proteins by asymmetrical segregation of aggregates during cell division. It has been demonstrated that protein aggregates formed during heat shock and in aging ll poles ,79. Accoll poles . Howeverll poles . It has ll poles ,79. ThisRecent studies indicate that protein aggregates can have protective functions despite the detrimental effects of protein misfolding. This topic will be presented in the next section, with a particular focus on aggregates formed during desiccation–rehydration stress.It is worth noting that aggregates formed upon proteotoxic stresses sequester hundreds of proteins ,18,19, iE. coli exposed to sublethal proteotoxic stresses such as high temperature, peroxide and streptomycin [E. coli [An interesting example of protective aggregates are “memory” inclusions formed in ptomycin   or the lon gene deletion resulting in diminished degradation of misfolded proteins. Moreover, using β-galactosidase as a model enzyme, Wang et al. showed that proteins sequestered in the aggregates may retain their activities [A. baumannii by preserving and protecting proteins. Apart from the direct effects of the aggregates, concomitant upregulation of several molecular chaperones in A. baumannii enabled the maintenance of proteostasis during desiccation, similar to aggresome-accumulating E. coli cells [Aggregates of endogenous proteins were also detected in n stress . It was tivities . Therefoli cells .E. coli proteins and the generation of persister bacteria is another example of protective mechanisms in bacteria exposed to antibiotics [E. coli proteins, was particularly prone to aggregation during the stationary phase and was the main component of the aggregates. Thus, the inhibition of translation and other processes caused by the sequestration of EF-Tu and other essential proteins resulted in dormancy and the generation of persisters. It should be noted that protein aggregation is only one of the mechanisms linked to persister formation. Other factors and processes promoting the generation of persisters are described in several excellent reviews [A correlation between the aggregation of endogenous ibiotics ,86. Persibiotics ,88. Sincibiotics . The foribiotics . The elo reviews ,87,88.E. coli cells increased in correlation with the fraction of insoluble proteins forming “aggresomes”. The aggresomes contained numerous E. coli proteins involved in a range of essential processes. It was proposed that the main cause of protein aggregation was ATP depletion. ATP level reduction diminished the efficiency of ATP-dependent molecular chaperones and proteases responsible for the removal of damaged proteins. The other possibility is that ATP also acts as a hydrotrope and prevents the formation of aggregates [E. coli but also in other Gram-negative bacteria [E. coli mutants, which sustained high ATP levels, led to an increased sensitivity to antibiotics and P1 phage infection. Similar effects, reduced aggresome formation and decreased bacterial fitness, were observed in E. coli cultures supplemented with MOPS. Most importantly, high-resolution optical microscopy revealed that aggresomes were formed through LLPS. As in other MLOs, aggresome components were mobile, underwent turnover, and initial liquid droplets were fused into larger dynamic condensates [Further studies using a single-cell approach  confirmegregates . Pu et agregates . Furtherbacteria . Inhibitdensates .E. coli during desiccation–rehydration stress were enriched in proteins prone to liquid–liquid phase separation [E. coli proteome using the D2P2 platform, which detects disordered sequences [E. coli aggregates. Glycation products were detected mainly in the outer membrane and the soluble protein fraction. We also found that the formation of protein aggregates and glycation products was inhibited by lower concentration (0.2%) of osmolytes: carnosine, glycine betaine and trehalose. Notably, supplementation of E. coli culture with higher osmolyte concentration  enhanced protein aggregation but reduced glycation and increased E. coli survival. Therefore, we concluded that glycation was the main cause of the loss of cell viability, whereas aggregates possibly played a protective function [Our previous studies revealed that protein aggregates formed in paration . Proteinequences . Some diequences . Apart fequences ,92,93, wfunction . It is afunction .http://www.pondr.com, accessed on 20 May 2023), FuzDrop, . Ribosomal proteins were the most abundant components of the aggregates, which contained a total of 547 proteins [For this review, we performed further analyses using different applications and algorithms, including PONDR (May 2023 ), PSPredMay 2023 ) and theproteins . In addiE. coli cells. However, the correlation between the total protein levels and their abundance in the aggregates  was relatively moderate (Spearman’s coefficient of 0.47). As mentioned above, Dps may form coacervates with DNA or RNA in vitro [E. coli cells, Dps coprecipitates with DNA [The high level of these proteins in aggregates may reflect their high abundance in in vitro . In statwith DNA  and can with DNA .E. coli prion-like proteins predicted by the PLAAC algorithm [E. coli IDPs in the DisProt database, of which 31 have been detected in the aggregates  state, separate condensates, oligomers or small aggregates can be recruited into the final aggregate (In summary, these analyses suggest that protein aggregation in ggregate C. It shoggregate  may formStudying liquid phase separation of proteins and MLO formation in bacteria is a big challenge, mainly due to the small size of bacterial cells. However, developing high-resolution microscopy and single-molecule tracking techniques has enabled significant progress in this field in recent years. Information on LLPS-prone proteins and IDPs has been gathered in several rapidly growing databases, and over 100 predictors of disordered sequences have been developed in the last decade . There a


Diabrotica virgifera virgifera, is a highly adaptable insect pest that has evolved resistance to a variety of control strategies, including insecticides. Therefore, it is interesting to examine how housekeeping proteins in WCR have been changed under WCR-controlling strategies. In this study, we focused on one of such proteins in WCR, a ubiquitous enzyme 5'-triphosphate nucleotidohydrolase (dUTPase). In the thymidine synthetic pathway, dUTPase hydrolyzes deoxyuridine triphosphate (dUTP) and supplies the substrate, deoxyuridine monophosphate, for the thymidylate synthase (TS). It decreases the cellular content of uracil, reducing uracil misincorporation into DNA. Suppressing the dUTPase activity, therefore, contributes to thymineless death. In this study, we investigated the enzymatic properties of dUTPase.The Western corn rootworm (WCR), dUTPase gene (DUT) was synthesized with the addition of His-tag corresponding DNA sequence and then cloned and expressed in Escherichia coli, and the protein product was purified. The product of WCR DUT hydrolyzed dUTP and was designated as dUTPase. WCR dUTPase did not hydrolyze dATP, dTTP, dCTP, or dGTP. WCR dUTPase was analyzed via size-exclusion chromatography and exhibited a molecular weight corresponding to that of trimer. The present format can be interpreted as nuclear trimer type. Possible isomers will be examined once transcriptome analyses are conducted.The WCR The online version contains supplementary material available at 10.1186/s13104-023-06618-2. Diabrotica virgifera virgifera LeConte, commonly known as Western corn rootworm (WCR), is a major corn pest in North America . –7. 4–7].DUT) in mice [DUT mRNA in planarian [DUT silencing efficiently killed fruit fly, Drosophila melanogaster, at the early pupal stage [DUT expression resulted in a 100% mortality in red flour beetle, Tribolium castaneum, at the larval stage [DUT dsRNA to neonates for 9 days killed 54% of the larvae and inhibited the growth of 80% of the survivors [Knockout of the dUTPase gene  contained dUTPase gene . The dUTPase protein sequences used in this study are Tribolium castaneum (EFA05862.1), Drosophila melanogaster (Q9V3I1), Saccharomyces cerevisiae (P33317), Homo sapiens (P33316), and E. coli .Genomic sequence  of th residue of the WCR dUTPase to maintain the ability of the subunits to assemble in trimers based on sequence comparisons and 3D structural modeling .Kinetic assay of WCR dUTPase was conducted using the cresol red method according to Larsson et al.   than other eukaryotes. WCR dUTPase exhibited a strict preference for dUTP. However, human [D. melanogaster dUTPases have exhibited slight activities against dTTP and dCTP [D. melanogaster and human dUTPases  and WCR, the C3–OH of 2'-deoxyribose forms a hydrogen bond with Asp102 (106 in WCR dUTPase) and C2 facing Tyr105  [2 of dsDUT revealed only 54% mortality in WCR neonates at 9 days [Previous knockdown of L dsDUT) . In the t 9 days .D. melanogaster and T. castaneum. dUMP-Forming dCTP deaminase is observed in Methanococci but not in the three aforementioned insects. It is noteworthy that while mammals have two types of thymidine kinases , insectTK1-like . Thus, hThe redundant dUMP supply may explain why suppression of dUTPase expression had limited effect on WCR mortality. Independent of the dUMP availability, administration of 5FU , a TS inhibitor commonly used to treat cancer in humans , is expeThis study found that WCR DUT encodes a uracil-specific pyrophosphatase, which could explain the physiological effects of WCR DUT knockdown . Future . [The identification, confirmation, and characterization of possible isoforms of dUTPase are necessary to understand its structure and function. The levels of glycosylation and phosphorylation should also be considered, as these can affect the activity of the protein. Research on dUTPase in humans is ongoing, with the goal of developing new cancer chemotherapeutic agents and malaria treatments. Rácz et al.  have ideA study of dUTPase levels post-knockdown is key to understanding dUTPase inhibitors and pest control potential. TAS-114  is a potAdditional file 1: Fig. S1. Structural modeling. a The monomer model of WCR dUTPase (subunit A). The five conserved motifs (M1-M5), Arg89, N- and C-termini are indicated. b The dUTPase trimer model. Chains B and C are added to the monomer view (a). The stick model represents the key residues in the active site. The boxed area is enlarged in Fig 1c. Method: Structural models were built using SWISS-MODEL [SS-MODEL  with a hSS-MODEL , 29 and SS-MODEL . StructuSS-MODEL .Additional file 2: Fig. S2. The WCR DUT construct sequence after being optimized for codon utilization of E. coli. The DUT construct sequence was cloned into pET-15 using NcoI and XhoI sites. One internal mutation (G → A at the 253th nucleotide) was placed to produce Arg89Lys. After thrombin cleavage (indicated by //), the N-terminal sequence, including the His tag, was removed.Additional file 3: Fig. S3. Peptides identified by mass spectrophotometry. a. Mass spectrum. b. Identified peptides. The full-length of the dUTPase protein construct sequence was identified by MS/MS. Sixty-seven exclusive unique spectra were identified out of the 968 total spectra. Likely deaminated glutamines are highlighted in cyan. All 33 unique peptide fragments are aligned against the dUTPase construct sequence. The numbers after each aligned peptide fragment represent the absolute abundance out of the 968 spectra. Method: The SDS-band was cut [ was cut , 36 and  was cut , 38.


Accumulating evidence suggests that in various systems, not all bidirectional microbiota–host interactions involve direct cell contact. Bacterial extracellular vesicles (BEVs) may be key participants in this interkingdom crosstalk. BEVs mediate microbiota functions by delivering effector molecules that modulate host signaling pathways, thereby facilitating host–microbe interactions. BEV production during infections by both pathogens and probiotics has been observed in various host tissues. Therefore, these vesicles released by microbiota may have the ability to drive or inhibit disease pathogenesis in different systems within the host. Here, we review the current knowledge of BEVs and particularly emphasize their interactions with the host and the pathogenesis of systemic diseases. In 1963, the presence of BEVs was first observed in the cell wall of gram-negative bacteria by electron microscopy, and initial research primarily focused on BEV functions. Initially, they were viewed as cellular debris that occurred after dead cells degraded . NonetheAlthough gram-negative and gram-positive bacteria have different vesicle secretory pathways due to different cell wall structures, as vectors of diverse bioactive compounds, BEVs participate in bacterial intraspecific and interspecific communication and interactions with hosts, including horizontal gene transfer, the killing of competing bacteria, phage neutralization and the delivery of virulence factors to host cells . In addiIn this review, we discuss the key functions of BEVs. Furthermore, we summarize the pathways of BEV synthesis and the mechanisms through which BEVs released by commensal and pathogenic bacteria are recognized by host PRRs to initiate inflammatory responses or mediate immunomodulation. Moreover, we highlight their biological role in microbiota-host interactions, in addition to their role in the pathogenesis of diseases of specific systems, namely, the nervous, digestive, circulatory, respiratory and motor systems.2.The structures of gram-positive bacteria and gram-negative bacteria have obvious differences, as do their released vesicles . Current2.1.The cell wall of gram-negative bacteria consists of an outer membrane, an inner membrane, and a peptidoglycan layer between them. Gram-negative bacteria release the outer membrane and cytoplasmic contents through the formation of a nanosized spherical structure with diameters in the range of 10–300 nm, whose composition is analogous to the outer membrane, so they are called outer membrane vesicles (OMVs). OMVs basically consist of proteins, virulence factors, lipids, and peptidoglycan . Most ofvia cell lysis (Gram-negative bacteria produce OMVs mainly in two ways: (1) through an imbalance in biosynthetic peptidoglycan or when hydrophobic molecules are embedded in the outer membrane, thereby causing cell membrane destabilization, which ultimately leads to the vesiculation of the outer membrane: the reduction of crosslink between peptidoglycan and the outer membrane induces the expansion of the outer membrane; the accumulation of bacterial cell wall peptidoglycan fragments increases peripheral pressure so that the outer membrane is bent to generate OMVs; and the accumulation of molecules that increase membrane curvature such as LPS can trigger membrane swelling ; and (2)ll lysis . In recell lysis . These mll lysis .2.2.Gram-positive bacteria are a class of bacteria surrounded by a plasma membrane and a thick peptidoglycan layer. Due to the lack of an outer membrane and a thick cell wall, they were initially thought to be unable to produce and secrete EVs. In 2007, researchers isolated vesicles from mycobacteria and conducted extensive proteomic studies of their components, confirming that gram-positive bacteria also release EVs . Researcvia encapsulation from the remaining peptidoglycan layer  contents of BEVs enable them to bind to PRRs on the membrane surface and in the cytoplasm of immune cells and nonimmune cells, thereby activating downstream inflammatory signaling pathways ; Table 13.1.3.1.1.via multiple pathways has been demonstrated, allowing their cargo to be detected by PRRs, and subsequently activating a series of signaling pathways. In phagocytic cells, phagocytosis is the major route to internalize OMVs . Clathrin-mediated endocytosis can be utilized by BEVs with diameters ranging from 20 to 250 nm  preferentially enter recipient cells o 250 nm . Toxins ocytosis .3.1.3.Bacterial extracellular vesicles enter host cells with multiple uptake mechanisms among vesicles from different species of bacteria and even among that from the same bacterium. The quantifiable and dynamical assay of uptake pathways will be important in the illustration of bioprocesses that underlie the bacteria-host interactions, but also in the design of BEV-engineered delivery vectors and improvement of their treatment efficiency based on their entry into target cells.3.2.3.2.1.Pseudomonas aeruginosa OMVs triggers the immune response in epithelial cells through the MyD88-dependent TLR4 signaling pathway and promotes the expression of IL-8 in lung epithelial cells  2 signaling and promoted cytokine and chemokine release by epithelial cells  and LL-37 by human gastric epithelial cells /PAMPs carried by BEVs, activate signaling pathways, promote the release of proinflammatory cytokines, and trigger inflammatory responses . For insal cells . DNA, RNal cells . Upon stal cells . These i3.2.2.Although most studies have reported that BEVs activate PRRs on the cell surface, PAMPs carried by BEVs can also be perceived and recognized by PRRs in the cytoplasm of host cells, thereby activating intracellular innate immunity and promoting the assemble assembly of inflammasomes .3.2.2.1.in vivo models, inducing caspase-1-mediated pyroptosis and TNF-α, IL-1β, and IL-18 secretion  or caspase 4/5 (human) . OMVs trproteins . Active proteins . In humat manner .3.2.3.Bacterial extracellular vesicles are potent activators of PRRs in charge of regulating inflammatory responses that are correlated with pathogenesis in systemic diseases . Additio4.In contrast to their parent bacteria, BEVs carry a higher concentration of virulence factors and insulate them during delivery to different organs and vascular-based tissue targets. These properties allow BEVs to travel long distances and access tissues that their parent bacteria cannot reach, strengthening the pathogenic functions of bacteria in both the local microenvironment and distant parts of the body and leading to the occurrence of Alzheimer’s disease (AD), metabolic diseases, cardiovascular disease (CVD), osteoporosis, etc.4.1.4.1.1.Campylobacter jejuni OMVs have been reported to cleave occludin and E-cadherin, promoting intestinal penetrability and paracellular pathways  and neurofibrillary tangles -C3a receptor (C3aR) signaling, ultimately leading to aggravated Aβ pathology  expression in hippocampal neurons while increasing IL-1β expression in the blood  treatment,  effects . In chropocampus . Moreoveerotonin . These r4.2.4.2.1.4.2.1.1.S. mutans MVs can disseminate over long distances, their local exploitation of nutrient substances such as sucrose to produce extracellular polysaccharides (EPS) on the hydroxyapatite surface could facilitate bacterial colonization and biofilm formation associated with cariogenicity , glucan-binding proteins and dextranase (DexA)  . Moreovetabolism .S. mutans MVs, including biofilm quantity  in monocytes  production, and accelerated periodontal bone loss in a periodontitis mouse model /glycogen synthase kinase-3 beta (GSK-3β) signaling pathways , and they can also be applied to the diagnosis and treatment of T2DM and its complications . A signi in mice . Gingipa by OMVs . In thespathways . Moreovepathways . These s4.2.2.2.Akkermansia muciniphila-derived EVs enhanced the expression of tight junction proteins in Caco-2 cells and eventually increased gut barrier integrity in an HFD-induced diabetic model through AMPK activation  activation through TLR4 and subsequent liver inflammation, glycogen synthesis reduction and progression toward steatohepatitis  and induced liver fibrosis  or nonalcoholic steatohepatitis (NASH)-related liver abnormalities. Intragastrically administered feces-derived EVs (fEVs) entered the liver and increased proinflammatory cytokines and chemokines from hepatic sinusoidal endothelial cells te cells . Likewisepatitis . H. pylofibrosis . FurtherAS/STING . Collect4.2.2.4.A. muciniphila EVs could efficiently enhance the regression of activated HSCs  induced regulatory T cells (Tregs), and Bacteroides thetaiotaomicron OMVs also exerted an effect on T-cell functions  and an increase in ATP affinity to H1 histone proteins in chromosomes , and participate in the progression of gastrointestinal cancer.  of time . Intrave6 tumors . These v6 tumors . EVs froal cells . Likewiser cells . H. pyloomosomes . Moreoveomosomes . Therefoomosomes .4.2.3.4.B. fragilis modified the gene expression of TLR2 and TLR4 in epithelial cells and increased the secretion of IL-10 by CD4+ T cells -treated mice  in human umbilical vein endothelial cells, causing endothelial dysfunction , which can trigger apoptosis in T-lymphocytes , invasion (IcmK), persistence and spreading in the lung (fliC)  . Furtherlearance .In summary, by targeting the BEV-related contents involved in the interaction between these vesicles and human lung cells or immune cells, new treatments for pulmonary infections may emerge, such as vaccines or drugs, that protect patients from bacterial invasion.4.5.4.5.1.P. gingivalis, which implied a correlation between BEVs and rheumatoid arthritis (RA)  and IL-6 increase in RA synovial fluid upon interaction with OMVs, promoting signal transduction in infected joints and damaging bone tissues during bacterial infection  . Peptidytis (RA) ,b. Moreonfection . BEVs alodstream . Therefoin vivo model of MAMP-induced inflammatory bone loss in mice was established, and Filifactor alocis EVs triggered systemic bone loss and osteoclastogenesis through TLR2 activation (To investigate the association between BEVs and osteoporosis, an tivation . These s4.5.2.Lactobacillus animalis EVs could access the femoral head and improve trabecular bone microarchitecture (A. muciniphila and the gut microbiota from children can access and accumulate in bone tissues to ameliorate ovariectomy-induced osteoporotic phenotypes by enhancing osteogenic activity and dampening osteoclast formation (Proteus mirabilis OMVs inhibited osteoclast differentiation and caused mitochondria-dependent apoptosis (K. kingae OMVs decreased osteoclastogenesis in a dose-dependent manner and inhibited proinflammatory cytokine production by infected macrophages (In contrast, probiotic-derived vesicles exhibit osteoprotective effects. After oral administration to GC-treated mice, itecture . EVs proormation . Likewispoptosis . In the poptosis . Intrigurophages . Therefo5.In recent decades, our knowledge of the physiological and pathological effects of EVs derived from gram-negative and gram-positive bacteria has improved unprecedentedly. BEVs are now commonly recognized as a delivery system that consolidate bacterial roles in bacterial survival, inflammation and pathogenesis in diverse biological milieu, and bacteria can modulate the biogenesis and content of BEVs in a tailored manner as needed. Moreover, recent advances in BEV science have attempted to address the question of how BEV-host interactions contribute to systemic diseases from different perspectives. To expound the intricate mechanisms underlying the role that BEVs play in infection and anti-infection activities in almost every system, we hope to explore novel therapeutic interventions.YW: Writing – original draft. XL: Supervision, Writing – review & editing. XX: Writing – review & editing, Visualization. CH: Writing – review & editing, Validation. DM: Writing – review & editing.