Particulate matter (PM) is a major and the most harmful component of urban air pollution, which may adversely affect human health. PM exposure has been associated with several human diseases, notably respiratory and cardiovascular diseases. In particular, recent evidence suggests that exposure to biomass-derived PM associates with airway inflammation and can aggravate asthma and other allergic diseases. Defective or excess responsiveness in the immune system regulates distinct pathologies, such as infections, hypersensitivity, and malignancies. Therefore, PM-induced modulation of the immune system is crucial for understanding how it causes these diseases and highlighting key molecular mechanisms that can mitigate the underlying pathologies. Emerging evidence has revealed that immune responses to biomass-derived PM exposure are closely associated with the risk of diverse hypersensitivity disorders, including asthma, allergic rhinitis, atopic dermatitis, and allergen sensitization. Moreover, immunological alteration by PM accounts for increased susceptibility to infectious diseases, such as tuberculosis and coronavirus disease-2019 (COVID-19). Evidence-based understanding of the immunological effects of PM and the molecular machinery would provide novel insights into clinical interventions or prevention against acute and chronic environmental disorders induced by biomass-derived PM. Particulate matter (PM) is a term used to describe a mixture of pollutants, containing a complex mixture of smoke, dust, and other solid particles, as well as liquid droplets, present in the air . PM varyIt has been well known that biomass fuel combustion, as well as wildland and agricultural fires, exhale large quantities of harmful air pollutants to both ambient and indoor air, which is associated with increased incidences of respiratory disease-related morbidity and mortality ,19. ExpoMany previous studies addressed the health effects of different types of PM exposure ,29,30,3110, PM2.5, and PM0.1, according to mean particulate size fractions. Based on “aerodynamic equivalent diameter” (AED), PM is categorized into three classes, including coarse particles or PM10 (ranging from 2.5 to 10 μm); fine particles or PM2.5 ; ultrafine particles or PM0.1  [10) include abraded soil, dust from road and construction sites, and oil combustion products with bio-aerosols, such as fungi, bacteria, pollen, and endotoxins. Particles that are derived from direct emissions owing to combustion processes of gasoline, oil, diesel, wood burning, and coal burning are primary sources of fine (PM2.5) and ultrafine particles (PM0.1) [2.5) and coarse (PM10) particles are described in PM is a mixture of solid and liquid particles suspended in the air, varying in size, number, surface area, chemical composition, and sources. Among the various methods of PM classification, the aerodynamic diameter is one appropriate criterion to define transportability in the atmosphere and inhaling ability through the respiratory tract. PM is categorized as PM 0.1 μm) ,35. Rega 0.1 μm) . General 0.1 μm) ,37,38. T (PM0.1) . The woo (PM0.1) ,40,41. T2.5 contains high levels of transition metals associated with increased potential of oxidative stress in the tissue. Moreover, PAHs, richly distributed in UFP (PM0.1), can generate oxidative stress, whereas their levels are low in both coarse (PM10) and fine (PM2.5) particles [10 settles in the upper respiratory tract and is substantially associated with several diseases, including cardiovascular diseases [2.5 induces vascular inflammation, leading to a potentially increased risk of atherosclerotic cardiovascular complications [2.5 exposure led to a substantial increase in interleukin 4 (IL-4) and IL-13 levels and a decrease in interferon γ (IFN-γ) in the myocardium of rats [PMarticles . Generaldiseases ,43, cancdiseases ,44,45, tdiseases ,47, and diseases ,49. Furtications . In addi of rats . Taken t0.2 and PM2.0 particles are deposited in the acinar region of the lung, some of them can be released into circulation with time and can be found in the kidney and liver [0.2 and PM2.0 particles to blood circulation and extrapulmonary organs might be mediated by particle-internalizing macrophages [10 can activate the NLRP3 inflammasome, subsequently facilitating the release of a mature form of IL-1β and IL-18, whose intracellular expression was induced by NF-κB signaling [2.5 can activate TLR4 and the NLRP3 inflammasome in cooperation with mitogen-activated protein kinase (MAPK) pathways in monocytes [10 exposure [When biomass burning-derived ultrafine and fine particles enter into the airway through inhalation from ambient air, they can reach the alveolar tissue. Compared to most deposited particles, ultrafine particles can cross the airway-blood barrier and enter the pulmonary and systemic circulation ,53,54. And liver . The welnd liver ,57,58. Hrophages . Followirophages , thus dirophages . Throughrophages ,62,63,64rophages . TLRs arrophages ,66,67. Trophages ,68. TLRsrophages ,71,72,73onocytes . IL-1β conocytes ,75. Addionocytes ,77,78. Texposure . Therefo+T cells, also known as T helper cells (Th cells), are key components of the adaptive immune system. The Th cells can be divided into three types, such as Th type-1 (Th1), Th type-2 (Th2), and Th type-17 (Th17) cells, displaying unique cytokine secretion phenotypes with distinct functional characteristics [10 [Herein, we focused on the relationship between adaptive immunity and exposure to PM by reviewing recent studies with mutual interests. Currently, most studies have revealed that T lymphocyte-mediated adaptive immune responses are associated with the pathophysiology following PM exposure. CD4eristics . After teristics . Th2 celeristics ,82. Seveeristics ,83,84. Leristics . Additiotics [10 ,86,87, w2.5-exposed rats [PM can also affect Th2 cytokine profiles. For example, diesel extract particles (DEP) favor Th2-type responses, whereas black carbon particles upregulate both Th1 and Th2 cytokine production . Moreovesed rats . An incr2.5 disrupts the balance of Th1/Th2 cells. Numerous studies have revealed that this imbalance is induced at the transcriptional level, as well as by various cytokines [2.5 and Th2 differentiation process [As stated above, PMytokines ,98. AMs ytokines . Hence, ytokines . Reporteytokines ,102,103.ytokines ,63. Addi process ,63. PM a process . Several process ,106,107.2.5 was administered only during the sensitization period with house dust mite (HDM) as the allergen. The results showed that PM acted as an adjuvant by promoting inflammation and allergic sensitization, whereas other groups failed to induce important inflammatory responses. Another notable result was that the PAH content in PM promoted Th17-responses and led to the activation of the aryl hydrocarbon receptor (AhR). PM exposure stimulated the expression of Th17 cytokines, including IL-17 and IL-22, as well as that of retinoic acid-related orphan receptor gamma t (RORγt), the Th17 lineage determining transcription factor. Following allergen sensitization, the stimulated expression of AhR in DCs promoted T lymphocyte activation and differentiation into Th17 cells. Furthermore, PM induced allergic sensitization via Th2 associated inflammation [Reportedly, the effect of PM as an adjuvant has been assessed on the adaptive response . In thisammation . Th17 suSome reports have demonstrated that PM exposure suppressed T cell-mediated cellular immune responses ,60,78, wFurthermore, accumulated evidence has revealed that PM can deteriorate the host immune system by activating regulatory T cells and IL-10. In vivo experiments in neonatal mice showed that environmentally persistent free radicals significantly increased the levels of pulmonary regulatory T cells and immunosuppressive cytokine IL-10 following influenza infection ,111. ConPM has been well known to affect public health and is considered an economic burden, especially in developing countries. Several scientific studies have confirmed that the pathophysiology of this process is associated with adaptive immunity ,62,63,96For microbes to establish infection in the lower respiratory tract, they must first avoid the innate immune system, composed of the epithelial cell barrier, antimicrobial molecules in mucus, AMs, neutrophils, natural killer cells, and DCs. AMs detect pathogen-associated molecular patterns via TLRs. Through TLR activation, a pathogen-triggered immune reaction cascade is stimulated ,33. This2.5 induces M1 macrophage-dependent inflammation by increasing secretions of IFN-γ, and IL-17 and IL-21 in CD4+T cells, indicating activation or enrichment of Th1 and Th17 responses, respectively [2.5 particles promotes M1 polarization through TLR4-activated JNK and p38 pathways [2.5 reprograms cellular polarization toward M2 macrophage-involved adverse outcomes, including sustained inflammation and emphysematous lesions. Moreover, chronic exposure to PM2.5 downregulates the expression of histone deacetylase 2, which is crucial for deleterious airway remodeling, facilitating COPD [Macrophages act as the first line of defense in recognizing tissue injury in response to PM or microorganisms. Macrophages are mainly involved in the process of phagocytosis, and eradication of bacteria, as well as other harmful organisms . In geneectively . Mechanipathways . In conting COPD . DependiMycobacterium (M.) tuberculosis infection induces M1 polarization, which secretes the proinflammatory mediators, such as TNF-α, IL-6, IL-12, CCL5, and chemokine C-C motif ligand (CXCL18) [During bacterial infection, it is recognized by their PRRs like TLRs activates macrophages to produce M1-like proinflammatory cytokines, such as TNF-α, IL-1, IL-6, IL-12, and NO that can eliminate the invading bacterial pathogens . Some ba(CXCL18) . However(CXCL18) .M. tuberculosis, via the downregulation of TLR-dependent cytokines [2.5 interfered with human anti-mycobacterial immunity [M. tuberculosis-induced TLRs 3, 4, 7, and 10, as well as NF-κB-induced expression of chemokines and cytokines, including IFN-γ, TNF-α, IL-1β, and IL-6, which play an important role in the control of M. tuberculosis. In vitro and in vivo M. tuberculosis infections promoted epithelial cells in the airway, provoking anti-mycobacterial reactions, including the release of cytokines, chemokines, and antimicrobial peptides. A549 cells, a type II alveolar epithelial cell line, generated IL-8 and MCP-1, which attract neutrophils, T cells, basophils, and monocytes, in the early stages of M. tuberculosis infection [M. tuberculosis strains. Human BD-2 and 3 are tiny cationic peptides that are affiliated with a family of antimicrobial peptides. As they possess antimicrobial characteristics and modulate the immune response, human BD-2 and 3 play key roles in the early control of M. tuberculosis infection by reducing the pulmonary bacterial load [A potential link between long-term exposure to suspended PM and increased rates of pulmonary tuberculosis (TB) has been reported ,123,124.ytokines . More spimmunity ,46. Foll2.5 and T cell functions in human PBMCs, it has been observed that urban PM may negatively affect the mechanisms of M. tuberculosis-specific human T cell functions [2.5 reduced the capacity of PBMCs to restrict the growth of M. tuberculosis and downregulated the expression of the early activation marker, CD69, during infection on CD3+T cells. Furthermore, these findings suggest that PM2.5 exposure decreased the production of IFN-γ in CD3+, TNF-α in CD3+, and CD14+ PBMC, which were infected by M. tuberculosis, as well as the M. tuberculosis-induced expression of T-box transcription factor  [2.5 exposure increased the expression levels of anti-inflammatory cytokines, such as IL-10 in CD3+ and CD14+ PBMCs. These data revealed that PM2.5 damaged crucial anti-mycobacterial T cell immune functions [M. tuberculosis in human PBMCs obtained from IFN-γ release assay (IGRA+) and IGRA− healthy study subjects. This study was performed in Iztapalapa, a highly populated tuberculosis-endemic municipality of Mexico City with elevated outdoor air pollution levels, and researchers collected monthly PM2.5 and PM10 from rainy, cold-dry, and warm-dry seasons to evaluate whether seasonal changes affect the human immune response to M. tuberculosis. This result corroborated previous research findings, demonstrating that, except for PM2.5 from the cold-dry season, pre-exposure to all seasonal PM reduced M. tuberculosis phagocytosis by PBMC. Additionally, M. tuberculosis-induced IFN-γ production was suppressed in PM2.5 and PM10 pre-exposed PBMC from IGRA+ subjects. Hence, both pre-exposure to PM10 and PM2.5 resulted in a greater loss of M. tuberculosis growth control [M. tuberculosis, the epidemiological evidence in populations with high tuberculosis prevalence should be explored, including the Korean peninsula, who also present a high risk of PM exposure.On investigating PMunctions ,125. Exp; T-bet) ,125. Not control  As there10 suppressed respiratory tract inflammatory responses to the respiratory syncytial virus (RSV), a frequent cause of viral pneumonia in infants and the elderly [A previous study has demonstrated that high doses of PM elderly . Therefo elderly . Severe  elderly . In part elderly . Like ot elderly . In addi elderly ,132. Mor elderly . Long-te elderly . HoweverExposure to air pollution increases the risk of asthma, allergic rhinitis, atopic dermatitis (AD), and allergen sensitization. The effects of air pollution exposure can be displayed differently depending on the timing of exposure, individual sensitivities, and simultaneous exposure to allergens.10 and PM2.5) increased the exacerbation of asthma, as well as the number of emergency cases [Previous studies have suggested that PM can increase hyperresponsiveness in the respiratory system. Diseases representing respiratory allergies include allergic rhinitis and asthma. For instance, urban levels of air pollution can induce pulmonary hyperresponsiveness in rats; however, the effect was reversed under the nonpolluted condition . Moreovecy cases . Althougcy cases . It needcy cases . AdditioPM can enhance the immune response to specific allergens and increase IgE production. In a previous study, exposure to DEP increased IgE production in the nasal mucosa . DEP expAir pollutants, such as PM, can enhance allergen sensitization by carrying aeroallergens into the airway. Following the introduction of aeroallergens into the airway, airway epithelial cells promote oxidative stress and inflammation, increasing the antigenicity of allergens ,140,141.10 in the first year of life increased the incidence of asthma at 12 years of age [2.5 exposure tend to display an increased incidence of asthma at seven years of age [It is well known that early-life exposure to fine PM in the air is associated with adult infant respiratory diseases, such as pneumonia and childhood asthma, with additional evidence suggesting that UFPs may affect childhood respiratory disease. PM can exacerbate respiratory allergic diseases, such as asthma and allergic rhinitis. In clinical evaluations, exposure to PMs of age . Moreoves of age . Howevers of age ,145.10 than in the control.Several epidemiological studies have reported that living in places with high levels of air pollutants from birth increases the risk of allergic disease ,144,146.2.5 exposure in both the prenatal and postnatal periods induced later development of asthma. However, another study suggests that there is no significant correlation between the incidence of asthma or wheezing and exposure to PM10 and nitrogen dioxide (NO2) [2 decreased from the first year of life to the eleventh year of life; the mean exposure to PM10 reportedly decreased.Asthma is characterized by variable airway obstruction, hyperresponsivity, and airway inflammation. In addition, asthma is a representative disease associated with hyperresponsiveness. However, the contribution of PM to asthma development remains inconsistent. Many birth cohort studies have been conducted. For example, in a cohort study in Asia, children with increased exposure to PM during gestational weeks 6–22 or the postnatal period demonstrated a higher incidence of asthma . In thisde (NO2) , with th2¯), hydrogen peroxide (H2O2), and hydroxyl radical ∙OH [PM causes oxidative damages in the pulmonary system . The maiical ∙OH . Among Rical ∙OH . PM can ical ∙OH , which aical ∙OH ,154,155.ical ∙OH . AhR-XREical ∙OH ,158,159.ical ∙OH ,160.GSTP1), which is involved in the metabolism of the antioxidant glutathione following air pollutant exposure [Following exposure to air pollution, such as fine dust, the immune response to oxidative damage is altered in airway epithelial cells, with inhibited phagocytic action inducing various inflammatory reactions to increase inflammatory mediators, and reduced inflammatory damage in the airway ,13,33. Iexposure . When thexposure .Increased expression of inflammatory mediators in the airway following exposure to air pollution causes an imbalance in immune responses and increases the likelihood of developing allergic diseases ,163,164.In addition to PM, various other matters are known to promote and exacerbate asthma; however, the pathogenesis of asthma induced by these compounds remains unclear. Airway macrophages could react quickly to the inhaled PM as an initial barrier. In patients with severe asthma, airway macrophages are unable to perform their functions, including the phagocytosis of inhaled PM. Th2 cells are essential for IgE isotype switching in B lymphocytes, which may stimulate allergic inflammatory responses, as well as airway hyperresponsiveness ,168. FurAir pollution increases the permeability of airway epithelial cells and increases sensitization by increasing the exposure of immune cells or antigenicity of external allergens ,172. AltEnvironmental exposure during pregnancy or early childbirth is closely linked to the development of allergic diseases, and in recent years, the mechanism has been gradually revealed in the development of welfare genetics. Welfare genetic changes are those in which the gene itself does not change owing to environmental exposure, but changes are observed in gene expression and activity. Changes in gene expression due to environmental exposure can occur over life, but it is postulated that effects are further pronounced in babies and infants. Studies have investigated fetal exposure to harmful environments, reporting that changes in the asthma risk may be substantiated by, for example, exposure to car exhaust and passive smoking in the womb, along with the risk of developing allergic diseases . ExposurIn genetically predisposed individuals, allergic diseases are susceptible to interaction with environmental factors. In particular, the inflammatory regulatory response in allergic diseases is presumed to involve gene regulation of the relevant immune system component; however, it has been recently noted that miRNAs affect the expression of these genes. miRNAs are extremely small noncoding RNAs, only 21–25 bases in size, that regulate gene expression by binding to mainly specific mRNAs, which inhibit or promote protein synthesis of target genes . ReporteIn this review, we systemically discussed the immune alteration-associated disease outcomes with cellular and molecular evidence in response to PM, including biomass-derived pollutants. Epidemiological and experimental studies have demonstrated that all particles are not equally toxic, but cause different adverse health effects. Generally, the particulate size is the deterministic factor of translocation and actions in the airway mucosa and other tissues. Although most fine particles deposits in the air-blood barrier tissues, UFPs, including some components of WSP, can easily migrate to different regions of the body, leading to local and systemic immunological responses. Although the migration of most fine particulates to the circulation is limited, PM-derived or endogenously produced ROS and inflammatory cytokines can mediate acute and chronic distress, including infection and hypersensitivity during PM exposure.2.5 induces M1 macrophage-dependent inflammation by increasing the secretions of Th1 and Th17 cytokines through PRR-activated signaling, while chronic exposure to PM2.5 can reprogram cellular polarization toward M2 macrophage-involved sustained inflammation, fibrosis, and deleterious airway remodeling. In terms of host responses to infection, PRR-activated macrophages produce M1-like proinflammatory cytokines, destroying the invading pathogens. However, some bacterial pathogens tend to prevent M1-like polarization, facilitating M2-like transition during the chronic inflammation and fibrosis in association with polarization toward Th2-lined immune response to PM exposure.Despite controversies about whether PM can either stimulate or inhibit adaptive immune responses, PM-induced cellular, and molecular alterations can contribute to outcomes of human diseases, including infection, hypersensitivity, and chronic diseases depending on chemical composition or size of the PM and exposure regimes . Exposure to PM, including WSP, alters the macrophage activity through the pattern-recognition signaling, leading to the production of proinflammatory cytokines and activation of innate immune cells in the airway and circulation. Depending on the exposure regimes, differentially activated macrophages play crucial roles in disease development and progression via specific molecular pathways. In particular, exposure to PMHowever, there are still a few questions that need to be addressed for a more comprehensive understanding. First, the mechanistic implications of the immune response and mode of action of different PM mixtures have not been fully elucidated with comparative investigations for diverse exposure regimes. In addition to individual chemical-specific toxicity, integrative effects of PM mixture on human immunity-related disease processes need to be further investigated. In particular, clinical evaluation of simultaneous exposure to the emerging microbes and air pollutants are increasing in interest. Epidemic investigation in the population under the biomass-associated air pollution would provide crucial insights to predict unexpected risks in an emerging microbe-abundant society. Second, target cellular molecules linked with immune dysregulation mediated by PM actions remain elusive. The identification of target molecules and their mechanistic network would help identify new strategies to inhibit the adversary health effects of biomass-derived PM, including WSP. The present review provides insights into novel immunotherapeutic interventions against PM-induced acute and chronic environmental disorders. Moreover, it is warranted to address climate change- or deforesting-associated PM exposure and health for the sustainable planet.


Adenophora triphylla (A. triphylla) is an important oriental herb belonging to the Campanulaceae family. A. triphylla complete chloroplast genome is composed of 239,431 bp, which form a large single-copy region , a small single-copy region , and 2 inverted repeats . There are 108 genes annotated, including 74 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Phylogeny indicates that A. triphylla was belong to Adenophora genus as a sister group and most closely related to Adenophora divaricate. Adenophora triphylla is a perennial herb, which is mainly distributed in China, Japan, Korea, and Russia. It is a flowering plant in the family Campanulaceae that is one of the 62 species of Adenophora  and deposited under the NIHHS as voucher number JA2019017.Adenophora triphylla chloroplast genome  as a reference sequence. The mapped 19.4 Gbp reads were performed self-correction, and de novo assembly was conducted using a Canu 2.0 program  and manual curation by Artemis annotation tool , and has a circular structure with a large single-copy (LSC) region of 1,78,906 bp, a small single-copy (SSC) region of 55,819 bp, and a pair of 2,353 bp inverted repeats (IRa/IRb). A total of 108 genes were predicted in the genome, including 74 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Overall GC contents of chloroplast genome is 37.71%. Another A. triphylla chloroplast genome (MG764158) had already been published in the NCBI database. However, the size of the previously assembled and our assembled chloroplast genome was quite different, at 154,223 and 2,39,431 bp, respectively. This discrepancy in genome size was assumed to be due to the various repetitive sequences even though same species.Campanulaceae family species, including the 2 outgroups. Bayesian inference (BI) phylogenetic analysis was constructed based on multiple alignment of 68 protein-coding sequences by MrBayes 3.2.7 .


As the most dominant cell type in the skin, keratinocytes play critical roles in wound repair not only as structural cells but also exerting important immune functions. This review focuses on the communications between keratinocytes and immune cells in wound healing, which are mediated by various cytokines, chemokines, and extracellular vesicles. Keratinocytes can also directly interact with T cells via antigen presentation. Moreover, keratinocytes produce antimicrobial peptides that can directly kill the invading pathogens and contribute to wound repair in many aspects. We also reviewed the epigenetic mechanisms known to regulate keratinocyte immune functions, including histone modifications, non-protein-coding RNAs , and chromatin dynamics. Lastly, we summarized the current evidence on the dysregulated immune functions of keratinocytes in chronic nonhealing wounds. Based on their crucial immune functions in skin wound healing, we propose that keratinocytes significantly contribute to the pathogenesis of chronic wound inflammation. We hope this review will trigger an interest in investigating the immune roles of keratinocytes in chronic wound pathology, which may open up new avenues for developing innovative wound treatments. As the outermost barrier of our body, the skin is subjected to daily assaults from the external environment. Efficient wound repair is vital for survival, and that is achieved by the delicate orchestration of different cellular and molecular events in a temporal sequence. The wound healing process has been characterized as a series of distinct but partially overlapping phases, i.e., inflammation, proliferation, and remodeling . As the Recently, the importance of nonhematopoietic, structural cells for mammalian immunity has been highlighted . With reKeratinocytes have active crosstalk with immune cells during wound repair, which is mediated by secreted signal proteins  . PAMPs, TLR activation is a critical element in initiating and amplifying inflammation after skin injury . As a maOf note, TLR3 activation in the skin epithelium is essential for the normal immune response after injury . TLR3 reIn addition to TLRs, RIG-I-like receptors (RLRs) are important cytosolic nucleic acid sensors in keratinocytes. When bound to dsRNA or dsDNA, they trigger an antiviral defense by activating type I IFN production ,29. TypeWhereas cGAS, RIG-1, and MDA5 all contribute to type I IFN-response in keratinocytes, AIM2 detects cytosolic DNA and triggers IL-1β release in keratinocytes. A strong epidermal upregulation of AIM2 is associated with acute and chronic skin inflammatory conditions, such as psoriasis ,35. AIM2By secreting cytokines and chemokines, keratinocytes can recruit, activate, and regulate immune cells. On the other hand, keratinocytes also sense wound microenvironmental cues, including the signals from immune cells and respond with changed cellular functions . The keyKeratinocytes express receptors of TNF-α and IFN-γ. Stimulation with these cytokines induces the keratinocyte expression of a broad range of pro-inflammatory genes, such as CXCL5, CXCL8 , and intercellular adhesion molecule 1  ,39. AddiIL-1α and IL-1β are important pro-inflammatory cytokines produced by many cell types, including keratinocytes, which constitutively express IL-1α, and to a less extent IL-1β. IL-1 expression is quickly increased upon skin injury, and IL-1α binding to the IL-1 type I receptor induces keratinocytes to express CCL20 to attract immune cells ,42. IL-1IL-6 has various effects on different cells to promote wound healing. Upon skin injury, the expression of IL-6 and its receptor is increased in keratinocytes . The IL-IL-17 and IL-22 are mainly produced by immune cells, and they have a pivotal role in immunoregulation and host defense. IL-17 signaling is mediated through heterodimeric IL-17 receptor A/C (IL-17RA/C) and A/D (IL-17RA/D) to induce IL-23 and AMP expression in keratinocytes, and upon skin injury, keratinocytes upregulate IL-17RA ,44. IL-2The healthy human epidermis has been shown to express chemokine receptors C-C chemokine receptor (CCR)1, CCR3, CCR4, CCR6, CCR10, C-X-C Motif Chemokine Receptor (CXCR)1, CXCR2, CXCR3, and CXCR4 . For fivFor wound healing, it is critical to coordinate the activities of different cell players accurately. The active crosstalk between keratinocytes and immune cells that has been detected through various stages of wound repair is one such example .Upon injury, keratinocytes produce pro-inflammatory cytokines  and chemokines , which play an important role in the recruitment and activation of neutrophils and macrophages at the wound site . NeutropMonocytes infiltrate to the wound site after neutrophils and differentiate into macrophages with a pro-inflammatory phenotype (M1 macrophages), removing cellular debris, microbes, and apoptotic neutrophils . SimilarLCs constitute a dendritic cell population of antigen-presenting cells that play a central role in maintaining immune homeostasis in the skin . Keratin+ TRM cells are not only present in the dermis but are also located in tight contact with keratinocytes, particularly within the hair follicle epithelium. Keratinocyte-secreted factors, such as interleukins and chemokines, are necessary for TRM cell homeostasis and recruitment upon infection [+ TRM cells secrete IFN-γ to modulate the chemokine synthesis of keratinocytes—for instance, the induction of CXCL9 and CXCL10 [+ TRM cells signal through keratinocytes to provide active immune protection. For example, normal skin flora Staphylococcus epidermidis induces CD8+ T cell accumulation and the production of IL-17 and IFN-γ in the mouse epidermis. These CD8+ T cells preferentially localize to the basal epidermis or close to the epithelial layer and express CD103 and CD69, which are cell surface markers for skin TRM cells [S. epidermidis-activated CD8+ T cells express several keratinocyte mitogens, such as amphiregulin (AREG), fibroblast growth factor 2 (FGF2), and TGFB1 [S. epidermidis [TRM cells are a noncirculating population of T cells that reside in the tissue, providing immunosurveillance and a rapid response against pathogen invasion . CD8+ TRnfection ,64,65. Od CXCL10 . CD8+ TRRM cells . IL-17 aRM cells , which hnd TGFB1 . In linedermidis .DETCs are a unique subset of γδ T cells in mice that reside in tight contact with keratinocytes and react rapidly to epidermal injury . In wounCells can extend their communication with other cells by delivering bioactive cargo, such as proteins, lipids, DNA, and RNA, packed in specific membrane-enclosed vesicles called extracellular vesicles (EVs) . EVs areKeratinocyte EV secretion is induced by various stimuli, such as hypoxia, irradiation, and starvation. Increasing evidence depicts that keratinocyte-derived EVs play a role in the wound healing process ,80. KeraA study by Zhou et al. shows that exosome secretion is significantly induced after injury . Wound mThe administration of exosomes derived from mesenchymal stem cells (MSCs) or adipose tissue stem cells has been shown to improve wound healing by promoting fibroblast proliferation and migration, collagen synthesis, and angiogenesis ,88,89,90There is a growing interest in utilizing EVs as a therapeutic tool to treat nonhealing wounds. Genetic engineering or the pre-treatment of EV donor cells or direct drug loading into exosomes are possible strategies in creating EV-based therapies. A phase 1/2A clinical trial is ongoing to assess the safety and efficacy of MSC-derived exosomes to treat dystrophic epidermolysis bullosa, which is a genetic and severe skin blistering disorder (NCT04173650). Moreover, several studies have shown the pro-healing effects of exosomes in animal models with impaired healing capacity ,92,93,94+ keratinocytes were found to control the homeostatic type 1 response to the microbiota [In addition to producing cytokines and EVs, keratinocytes can also function as atypical antigen-presenting cells (APC) to interact with T lymphocytes in an antigen-specific manner. MHC-II expression has been detected in keratinocytes from a wide variety of skin diseases, such as lupus erythematosus, vitiligo, lichen planus, cutaneous T-cell lymphoma, various infectious dermatoses, allergic contact dermatitis, granulomatous dermatoses, lichen sclerosis, erythema nodosum , and psocrobiota . In addicrobiota . Interescrobiota .S. epidermidis exhibits an anti-inflammatory effect on keratinocytes and restricts excessive inflammatory response upon tissue injury [S. epidermidis also secrets lipopeptide 78 (LP78), which inhibits TLR3-mediated skin inflammation and promotes wound healing. In contrast, another lipopeptide (LP01) from these bacteria activates TLR2/CD36-p38 mitogen-activated protein kinase (MAPK) to enhance antimicrobial defense against pathogenic infections [Skin microbiota plays a vital role in many aspects of human health . Its impe injury . S. epidfections ,109. A dEscherichia coli induces psoriasin (S100A7) secretion by keratinocytes, while S. aureus stimulates keratinocytes to produce β-defensins, and these AMPs protect the skin from infections [S. epidermidis produces AMPs to selectively kill and prevent pathogenic bacteria S. aureus colonization [In addition to cytokines and chemokines, the activated keratinocytes also produce large amounts of AMPs, such as β-defensins, cathelicidins, S100 proteins , and RNase 7, which are positively charged amphiphilic molecules and can kill various microbial pathogens directly . For insfections ,112,113.nization . Moreovenization .S. aureus is observed in perforin-2 overexpressing keratinocytes [AMPs are expressed through wound healing stages and contribute to wound repair in many aspects ,117. As inocytes , suggestinocytes .To carry out the immune functions discussed above, keratinocytes need to regulate their gene expression accurately and efficiently in response to various external stimuli. In this regard, epigenetic mechanisms have gained increasing attention in understanding gene expression regulation during wound healing . The epiHistones are the critical proteins involved in DNA assembly into chromatin, and their post-transcriptional modifications have a fundamental impact on chromatin structure and gene expression . SeveralThe Jumonji domain containing-3 (JMJD3) is an epigenetic regulator that specifically catalyzes methyl groups’ removal from lysine residues on histone tails, releasing a repressive epigenetic mark (H3K27me3) and activating gene expression. JMJD3 has been found to be upregulated in wound-edge keratinocytes. In cooperation with NF-κB, JMJD3 activates the expression of several inflammatory genes, such as IL-1β, IL-6, CCL20, and TNF-α, by demethylating their gene promoters in scratch-wounded keratinocytes in culture . MoreoveAIM2, allowing a rapid transcription of AIM2 and its downstream effector genes upon secondary stimulus, i.e., skin injury [“Trained immunity” (TI) or “innate immune memory” is a novel concept describing the capacity of an organism to develop increased responsiveness to secondary stimuli independent of adaptive immunity . TI was n injury .In the human genome, the majority of the transcriptional output is constituted by the RNAs that lack protein-coding capacity ,131. IntMiRNAs have been shown to regulate many different cellular processes spanning the entire wound healing process ,135,136.Moreover, the expression of some miRNAs is controlled by inflammation signals activated upon skin injury, and in turn, these miRNAs can regulate keratinocyte proliferation and migration . The stiFollowing an inflammatory stimulus, several lncRNAs show altered expression, and a growing number of these lncRNAs have been functionally characterized in mediating immune responses . HoweverChronic wounds are defined as cutaneous wounds that fail to heal within three months . This diAs the primary cell type in the epidermis, keratinocytes produce large amounts of cytokines to initiate and regulate inflammation, including IL-1, -6, -7, -8, -10, -12, -15, -18, and -20, and TNF-α . AlthougThe expression of TLR1, TLR2, TLR4, TLR6, TLR9, and MyD88 has been found to be significantly upregulated in the wounds of diabetic patients compared with nondiabetic wounds ,165. We The expression of AMPs is dysregulated in chronic wounds. For example, in the wound-edge epidermis of venous ulcers, psoriasin (S100A7) and hBD-2 expression was strongly induced, whereas no RNase 7 or LL-37 was detected there in comparison with the intact skin . S100A8 Chronic wounds are frequently colonized by bacteria living in a biofilm, which may also contain other microorganisms, such as fungi or viruses. These microbial cells have increased resistance to antimicrobial treatment, as they are physically protected from the host immune attack by a self-produced structured polymer matrix, which contributes to a further influx of immune cells and prolonged inflammation . KeratinMultiple miRNAs are dysregulated in human chronic wounds and contribute to delayed wound healing through regulating keratinocyte immune functions, e.g., miR-132, miR-146, miR-34a/c, miR-19a/b, miR-20a, miR-203, miR-21, and miR-130 ,150 Tab. The miRAs the most dominant cell type in our skin, keratinocytes play critical roles in skin wound repair, not only as structural cells, but also exerting important immune functions. Upon skin injury, keratinocytes are at the frontline of defense in innate immunity. At the early phase of wound healing, under the stimulation of injury and invading microorganisms, keratinocytes release large amounts of cytokines, chemokines, AMPs, and extracellular vesicles, which recruit and activate immune cells, as well as kill pathogens directly. Keratinocytes not only initiate and intensify the immune response, they also have the mechanisms to restrict and reduce inflammation, which is important for helping the wound healing process to enter the proliferative phase. Keratinocytes sense different wound microenvironmental cues and change their states  and immune functions accordingly. The keratinocyte‒immune cell crosstalk loops are often dysregulated in chronic wounds, which are “stuck” in a chronic inflammation status: on the one hand, the inflammation is persistent, damages normal tissue, and hinders wound healing; on the other hand, it is ineffective to combat infection and to activate the signals needed for the following proliferative phases. Therefore, how to disrupt this pathological inflammatory circuit and reactivate the healing program has become the prime concern when treating chronic wounds.Although impaired immune functions of keratinocytes have been reported in chronic wounds, their relative contribution to chronic wound pathology remains elusive. Based on their crucial immune functions in the skin, we postulate that keratinocytes are unlikely to be a sole victim in this case. They may make a significant contribution to the pathogenesis of chronic wound inflammation. However, there is not enough evidence to endorse this idea, as most previous studies on chronic wound inflammation focused on immune cells such as neutrophils and macrophages. Another intriguing question is whether the correction of keratinocytes’ immune dysfunction may help recover the immune balance in the wound microenvironment and restart the suspended healing program. A recent study highlighted the vital role of structural cells, e.g., epithelium, endothelium, and fibroblasts, in regulating the organ-specific immune response . As an e


HOXA2, IKZF1, KLF2, XBP1, EGR2, ETV7, BACH2, CBFA2T3, HLF, and NFE2. TFs including BACH2, CBFA2T3, EGR2, ETV7, NFE2, and XBP1, and their target genes were significantly enriched in signaling by interleukins. BACH2 target genes were enriched in estrogen receptor- (ESR-) mediated signaling and nongenomic estrogen signaling. Furthermore, to clarify the functional roles of immune cells on the UC pathogenesis, we estimated the immune cell proportions in all the samples. The accumulated effector CD8 and reduced proportion of naïve CD4 might be responsible for the adaptive immune response in UC. The accumulation of plasma in UC might be associated with increased gut permeability. In summary, we present a systematic study of the TFs by analyzing the DETFs, their regulating target genes and pathways, and immune cells. These findings might improve our understanding of the TFs in the pathogenesis of UC.Ulcerative colitis (UC) is one of the inflammatory bowel diseases (IBD) characterized by occurrence in the rectum and sigmoid colon of young adults. However, the functional roles of transcription factors (TFs) and their regulating target genes and pathways are not fully known in ulcerative colitis (UC). In this study, we collected gene expression data to identify differentially expressed TFs (DETFs). We found that differentially expressed genes (DEGs) were significantly enriched in the target genes of  Ulcerative colitis (UC) is one of the inflammatory bowel diseases (IBD) with symptoms such as abdominal pain, fever, malnourishment, fatigue, and weight loss . UC is cWith the advances in high-throughput technologies, a growing number of studies have been carried out to investigate the expression of some genes and proteins in the pathogenesis and molecular mechanism of UC. Specifically, the copy number variations (CNVs) in mitochondrial DNA have been identified as the predictor of UC-associated colorectal x`cancer by CNV arrays . MoreoveIn addition, the transcription factors (TFs), a series of molecules involved in regulating gene expression, have been emerged as key regulators in several diseases , 9. HeatThe gene expression data were collected from the Gene Expression Omnibus (GEO) database with accession GSE128682, and the sample collection was described in an earlier study . The coup value of 0.05 were used to determine the DEGs for each comparison.The count-based expression data was used for the differential expression analysis (DEA). R/Bioconductor DESeq2  was empln > 2000) were excluded in the enrichment analysis. The enrichment analysis was implemented in the R clusterProfiler package with enricher function [The Fisher's exact test was used to identify the transcription factors (TFs) and pathways enriched by the DEGs. The DEGs with a significant correlation with their TFs were selected for this analysis and TFs with a large number of target genes (function .https://bioconductor.riken.jp/packages/3.4/bioc/html/scater.html), which was used for the CIBERSORT analysis.The immune cell proportion was estimated by CIBERSORT, which used the gene expression profiles and immune cell-specific genes to characterize the cell composition of complex tissues . The cout test, and multiple-sample comparison was tested by analysis of variance (ANOVA). The Spearman correlation analysis was used to evaluate the correlation of two variables. Symbols of ∗, ∗∗, ∗∗∗, and ∗∗∗∗ indicate the statistical significances of 0.05, 0.01, 0.001, and 0.0001, respectively.The two-sample comparison was tested by Wilcoxon rank-sum test or n = 56) and UC vs. R (n = 46) had greater numbers of differentially expressed transcription factors (TFs) than that of R vs. N (n = 6) , 14 remission (R), and 16 healthy controls (N). With the three groups of mucosal biopsies, we compared one with the other two groups, respectively. UC had significantly different expression profiles as compared with R and N groups, with 3,202 and 2,517 differentially expressed genes (DEGs) in UC vs. N and UC vs. R (Supplementary Table  (n = 6) . These rTotally, we identified 72 TFs significantly differentially expressed between the three groups , we conducted coexpression analysis of the 72 DETFs. Notably, four categories of DETFs  could be identified by the coexpression analysis . FurtherHOXA2, IKZF1, KLF2, XBP1, EGR2, ETV7, BACH2, CBFA2T3, HLF, and NFE2 , and HLF and HOXA2 were downregulated in UC (N = R > UC or N > R > UC)  . It shoued in UC , and theed in UC , suggestBACH2, CBFA2T3, EGR2, ETV7, IKZF1, NFE2, and XBP1 were significantly enriched in the pathways , and plasmacytoid dendritic cells (pDC) were decreased in UC, while effector CD8 and plasma were increased in UC compared with R and N groups . Particud plasma , indicatTranscription factors (TFs) are key proteins involved in regulating gene transcription in cells. However, the functional roles of TFs and their regulating target genes and pathways are still little known in ulcerative colitis (UC).In the present study, we collected gene expression data of mucosal biopsies from 14 UC, 14 remission (R), and 16 healthy controls (N), and identified DEGs in the three groups, of which, 72 were identified as differentially expressed TFs (DETFs). Furthermore, the coexpression analysis of the DETFs revealed three categories of TFs, which were upregulated in UC (N = R < UC), upregulated in R (N < R > UC), and downregulated in UC (N = R > UC).HOXA2, IKZF1, KLF2, XBP1, EGR2, ETV7, BACH2, CBFA2T3, HLF, and NFE2. As BACH2 and NFE2 proteins had similar protein structure [As the function of DETFs could result in dysregulation of their target genes, we found that DEGs were significantly enriched in the target genes of tructure , they hatructure  protein-tructure , 24.IL6, IL18RAP, IL11, STAT5B, and CSF3 were involved in the signaling by interleukins. The interleukins and receptors were frequently reported to promote the inflammatory phenotype in UC [IL11 and IL18RAP were identified as susceptibility loci in UC [BACH2, including AKT3, GNGT2, MMP7, and MMP9, were involved in ESR-mediated signaling and nongenomic estrogen signaling. As patients with UC have a higher risk for colorectal carcinoma (CRC) development [Among the TF target genes, inflammatory factors such as pe in UC –28. Notaci in UC , 30. Furelopment  and the elopment , we then+ might be associated with the upregulation of BACH2 and EGR2 [Furthermore, to clarify the functional roles of immune cells on the UC pathogenesis, we estimated the immune cell proportions in all the samples. The accumulated effector CD8 and reduced proportion of naïve CD4 might be responsible for the adaptive immune response in UC, showing consistency with the previous study . Notablyand EGR2 , 35. Theand EGR2 .In summary, we present a systematic study of the TFs by analyzing the DETFs, their regulating target genes and pathways, and immune cells. These findings might improve our understanding of the TFs in the pathogenesis of UC.


Desulfobulbus oligotrophicus strain Prop6 is a sulfate-reducing, propionate-oxidizing deltabacterota from sewage sludge. Desulfobulbus species are found in anoxic environments, in animal microbiota, and some produce the neurotoxin methylmercury. The 3.1-Mbp D. oligotrophicus genome sequence enables studies of diverse environmental adaptations and the evolutionary genomics of mercury methylation mechanisms. Desulfobulbus oligotrophicus Prop6 is a sulfate-reducing, propionate-oxidizing Deltabacterota (formerly Deltaproteobacteria) strain from sewage sludge. Desulfobulbus species are found in anoxic environments, in animal microbiota, and some produce the neurotoxin methylmercury. The 3.1-Mbp D. oligotrophicus genome sequence enables studies of diverse environmental adaptations and the evolutionary genomics of mercury methylation mechanisms. Desulfobulbus is a genus of sulfate-reducing Deltabacterota (formerly Deltaproteobacteria), originally described based on D. propionicus strain lpr3, isolated from freshwater mud , obtained from the DSMZ  was grown for 7 days at 35°C in medium DSM194. Genomic DNA was isolated using proteinase K-SDS digestion, followed by phenol-chloroform extraction as detailed in reference N50 length, 143,894 nucleotides [nt]) and 478,204 subreads  were used in the assembly, resulting in a final polished contig 3,102,012 nt long, with a 1,236-fold mean coverage, a quality value (QV) of 93, and a G+C content of 52.4%. To determine if the chromosome of D. oligotrophicus is circular, we designed a pair of oligonucleotides  to amplify outwards from the 5′ and 3′ ends of the contig. Sanger sequencing of the resulting 1.4-kbp PCR product and read mapping using Geneious Prime 2020  genome sequence has been deposited in GenBank under the accession number CP054140. The version described in this paper is the first version, CP054140.1. The PacBio reads have been deposited in the SRA under the accession number SRX9754829.The


Inflammasomes, intracellular, multimeric protein complexes, are assembled when damage signals stimulate nucleotide-binding oligomerization domain receptors (NLRs). Several inflammasomes have been reported, including the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), NLRP1, NLRP7, ice protease-activating factor (IPAF), absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4). Among these inflammasomes, the NLRP3 inflammasome is the most well-studied in terms of structure and function. Unlike other inflammasomes that can only be activated by a finite number of pathogenic microorganisms, the NLRP3 inflammasome can be activated by the imbalance of the internal environment and a large number of metabolites. The biochemical function of NLRP3 inflammasome is to activate cysteine-requiring aspartate proteinase-1 (caspase-1), which converts pro-IL-1β and pro-IL-18 into their active forms, namely, IL-1β and IL-18, which are then released into the extracellular space. The well-established, classic role of NLRP3 inflammasome has been implicated in many disorders. In this review, we discuss the current understanding of NLRP3 inflammasome and its critical role in gynecological disorders and obstetrical complications. Thevia the nuclear factor Kappa-B (NF-κB)-dependent pathway -NLRP3 interaction, inducing mitochondrial and lysosomal damage, and increasing the production of ROS  or damaged cell membranes leads to the activation of phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) into DAG and inositol 1,4,5-triphosphate (IP3) via PLC-mediated PIP2 hydrolysis. Finally, IP3 binds to its receptor, IP3R, on ER membranes, which triggers Ca2+ release  An ATP-gated ion channel named purinergic P2RX7 triggers the efflux of Ktiporter , 54. K+ n of ROS , 56. (2) release –59. Ca2+diolipin . (3) Mitdiolipin . (4) Cryammasome , 16, 29.+ efflux  family, is a serine/threonine kinase and is involved in the regulation of the cell cycle and NLRP3 inflammasome . The inf+ efflux . The hig+ efflux . Schmid-+ efflux . Likewis+ efflux . Several+ efflux , 69.3+, AL3+, and R-568, and the PLC agonist m-3M3FBS activate NLRP3 inflammasome by promoting Ca2+ mobilization , inhibitors for the constituents of NLRP3 inflammasome  and direct inhibitors of NLRP3 protein  . In addiIncreasing numbers of studies indicate that NLRP3 inflammasome is the critical modulator in various inflammatory conditions. When cells and tissues are damaged or dying, DAMPs are released and activate NLRP3 inflammasome and various immune cells, including neutrophils, dendritic cells (DCs) and macrophages . Then, tIn type 2 diabetes, chronic hyperglycemia activates NLRP3 inflammasome by promoting the generation of ROS; then, IL-1β induces the dysfunction and destruction of pancreatic islet β cells . In goutvia the myeloid differentiation factor 88 (MyD88)-dependent pathway. Subsequently, the signalosome activates transforming growth factor-β-activated kinase 1 (TAK1) via autophosphorylation. TAK1 then phosphorylates IkB kinases (IKKs). The phosphorylated IKKs induce the phosphorylation of inhibitor of nuclear factor kappa-B kinase (IκB), and IκB dissociates from the inactive P65- NF-κB -IκB trimer. The activated P65-NF-κB dimer translocate into the nucleus and triggers the expression, synthesis and release of various proinflammatory cytokines. Finally, persistent inflammation contributes to the malignant transformation of normal cervical cells and the establishment of a tumor microenvironment , preterm labor, FGR, RPL, PE, intrauterine fetal death, and NHIE –11. The ironment –76. Addiironment . For preNLRP3 inflammasome is closely associated with various gynecological diseases, especially in gynecological-oncology conditions , 78. AccCervical cancer (CC) is an extraordinarily common cancer in women, second only to breast cancer . StudiesIn a study using LPS-stimulated human CC cells, human SiHa and Caski cells , the mRNA and protein levels of inflammasome components, such as NLRP3, pro-IL-1β, IL-1β, and caspase-1, was significantly increased. When immunosuppressor CD200Fc was added, in addition to IL-1β, caspase-1, NLRP3, ASC, the protein levels of TLR4 and P65, and the translocation of P65 to the nucleus were significantly decreased in a dose-dependent manner, suggesting the crucial role of CD200Fc in the modulation of TLR4- NF-κB and NLRP3 inflammasome pathway. However, in HeLa cells  and C33A cells , no such changes were observed . In HeLaThe fetus and placenta, as a semiallograft, express not only maternal antigens but also paternal antigens . TherefoPreterm birth is defined as delivery occurring before 37 weeks of gestation. Preterm birth is the primary cause of neonatal morbidity and mortality worldwide, and the incidence is reported to be 10% or higher . As a syIn a study of women with preterm births (N=37), membranes with acute chorioamnionitis had increased levels of HMGB1, caspase-1, IL-18, and IL-1β compared with those from women who underwent normal labor .In LPS-induced C57BL/6 mice preterm birth model, the mRNA and protein levels of NLRP3, caspase-1, IL-18, and IL-1β in the fetal membrane and basal decidua were markedly elevated as compared with those of the phosphate-buffered saline (PBS)-treated control mice, suggesting that NLRP3 inflammasome may play a critical role in proinflammatory changes. Moreover, when mice undergoing LPS-induced preterm birth were treated with the inflammasome inhibitor MCC950, the time interval from the LPS injection to delivery was prolonged, and the preterm birth rate was reduced by 30%. Additionally, the neonatal mortality rate was decreased by approximately 30% . These fFGR is defined as a fetal weight below the tenth percentile of normal fetal weights . ExcessiIn the MSU-induced FGR rodent model, the prevalence of FGR was increased in a dose-dependent manner. 20% of pups developed growth restriction when the pregnant rat was treated with MSU 250 mcg/Kg. When pregnant rats were treated with MSU 500 or 1000 mcg/Kg, over 80% fetal growth restriction was observed in comparison to the PBS treated controls. Additionally, the growth restriction induced by MSU was reversed by a caspase-1 inhibitor or IL-1β inhibitor .It is speculated that in pathological pregnancies, increases in various DAMPs from the maternal-fetal interface, such as increased levels of MSU and HMGB1, trigger the activation of the NLRP3 inflammasome, which in turn increases IL-1β. Increased IL-1β may induce defective spiral artery remodeling, aberrant uteroplacental hemodynamics, decreased system A amino acid transporter activity, cytotrophoblast cell apoptosis, and decreased syncytialization. All of these pathological changes may conspire to the development of FGR , 99, 100RPL, defined as two or more consecutive clinical pregnancy losses prior to 20 weeks of gestation, is the most common pregnancy complication, and approximately 5–15% of all pregnancies can be affected . Of all In a study of endometrial tissues derived from women with RPL (N=30), the endometrial expression of NLRP3, caspase-1, IL-18, and IL-1β was significantly increased compared with that of normal fertile women . In anotIt is generally accepted that the establishment and maintenance of a successful pregnancy rely on a balance between pro- and anti-inflammatory responses at the maternal-fetal interface. Additionally, endometrial cells convert into a specific phenotype that is suitable for embryo implantation, subsequent placenta and fetal development, and spiral artery invasion, which often requires inflammatory cytokines . Women wPE, characterized by the presence of increased blood pressure (BP>140/90 mmHg) and proteinuria (>300 mg/24 h) after 20 weeks of gestation, is usually accompanied by headache, nausea, vomiting, and epigastric discomfort , 109. PEIn a study of women with PE (N=20), Ingrid C Weel et al. reported that the placental expression of NLRP3, IL-1β, and caspase-1 was significantly increased in the placenta from PE women compared to that from normotensive pregnant women, indicating that NLRP3 inflammasome may participate in the development of PE .In a mice model, C57BL/6 mice were injected with extracellular vesicles (EV), which induces the accumulation of activated platelets in the placental bed and activates the NLRP3 inflammasome. EV-injection induced PE-like phenotypes with renal dysfunction and elevated blood pressure. In addition, the expression of NLRP3, caspase-1, and IL-1β was increased in the placentas of EV-injected mice compared to those of control mice. Additionally, when NLRP3/caspase-1 knockout mice or mice given NLRP3 inflammasome inhibitors received the same treatment, the PE-like phenotype did not appear .NHIE is associated with 23–25% of neonatal death worldwide and results in long term sequelae if affected neonates survive , 115. MaActivated microglial cells release proinflammatory cytokines and ROS, and microglial inflammation is reported to be regulated by the NLRP3 inflammasome . In an eIn a cord blood study of women undergoing cesarean section and vaginal delivery, IL-1β level was significantly lower in elective cesarean section cases as compared with those of emergency cesarean section and vaginal delivery, suggesting cord blood IL-1β level may determine high-risk babies for perinatal hypoxic stress . The NLR+ efflux, Ca2+ mobilization, ROS production, and lysosomal rupture hypotheses, have been proposed to explain inflammasome activation, a widely accepted theory has not yet emerged. In addition, NLRP3 inflammasome has been indicated to play an important role in the pathogenesis of various gynecological and obstetrical diseases. Therefore, NLRP3 inflammasome might be an attractive therapeutic and diagnosis target NLRP3 inflammasome-related disorders. However, clinical studies of NLRP3 inflammasome in normal pregnant women, women with gynecological and obstetrical complications, and neonates with NHIE are significantly limited. Further clinical studies and studies to investigate the feasibility and safety of targeted therapy are needed.In this review, the components, assembly, and activation of NLRP3 inflammasome are discussed. Although multiple hypotheses, such as the KAll authors who participated in the work are listed and agreed for publication. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China (82071650) and (82001641).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival at diagnosis of 16–20 months. Metabolism represents a new attractive therapeutic target; however, due to high intratumoral heterogeneity, the application of metabolic drugs in GBM is challenging. We characterized the basal bioenergetic metabolism and antiproliferative potential of metformin (MF), dichloroacetate (DCA), sodium oxamate (SOD) and diazo-5-oxo-L-norleucine (DON) in three distinct glioma stem cells (GSCs) , as well as U87MG. GBM27, a highly oxidative cell line, was the most resistant to all treatments, except DON. GBM18 and GBM38, Warburg-like GSCs, were sensitive to MF and DCA, respectively. Resistance to DON was not correlated with basal metabolic phenotypes. In combinatory experiments, radiomimetic bleomycin exhibited therapeutically relevant synergistic effects with MF, DCA and DON in GBM27 and DON in all other cell lines. MF and DCA shifted the metabolism of treated cells towards glycolysis or oxidation, respectively. DON consistently decreased total ATP production. Our study highlights the need for a better characterization of GBM from a metabolic perspective. Metabolic therapy should focus on both glycolytic and oxidative subpopulations of GSCs. Glioblastoma (GBM) is the most common, heterogeneous and aggressive primary brain tumor in adults  ,2,3. TheStandard treatment of GBM consists of maximally safe surgical resection, followed by radiotherapy and chemotherapy, usually in the form of temozolomide (TMZ). Despite decades of extensive research and advancements in therapeutics, such as tumor treating fields (TTF), prognosis remains extremely poor, with a median overall survival of 20.9 months . GBM hasSetting aside the uncertainty behind the origin of cancer ,12, one The main challenge facing GBM management is the eradication of all malignant cells, including those able to survive drastic changes in the tumor microenvironment and toxic interventions. For this reason, GBM presents as a unique model to study bioenergetic alterations, as both aerobic glycolysis and oxidative phosphorylation (OXPHOS) have been described in high grade gliomas ,29,30. FTo accurately model this disease in vitro, it has been proposed that GBM stem cells (GSCs) have a remarkable proliferative ability, sufficient to drive tumor maintenance, recurrence and therapeutic resistance ,36,37,38To help us illuminate this question, we performed a Gene Set Variation Analysis (GSVA)  for canoTo verify these observations in vitro, we analyzed the basal metabolic phenotype of three tissue-derived and molecularly distinct GSCs , in addition to traditional established cell line U87MG. As metabolic plasticity is being touted as a distinctive feature of GSCs, we wanted to explore antiproliferative responses to metabolic inhibitors and their correlation with basal bioenergetics. High resistance to MF  and DCA (glycolytic modulator) was detected in GBM27, a distinctively oxidative cell line. GBM38 displayed Warburg-like properties, with higher sensitivity to DCA. Responses to DON (glutaminase inhibitor) varied between cell lines, without a clear correlation with basal metabolic phenotypes. Subsequently, we combined promising drug candidates with bleomycin, a radiomimetic drug that causes single-strand and double-strand DNA breaks ,45. SyneHere, we propose that strategic targeting of dysregulated bioenergetic pathways, after an initial assessment of the metabolic phenotypes coexisting within a tumor, could become a valuable stratification and therapeutic tool, improving the efficacy of adjuvant metabolic therapy.2 at 37 °C, in a media containing, as a base, DMEM/F-12 , supplemented with: Non Essential Amino Acids , HEPES , D-Glucose , BSA-FV , Sodium Pyruvate , L-Glutamine , Antibiotic-Antimycotic , N1 Supplement , Hydrocortisone , Tri-iodothyronine , EGF , bFGF  and Heparin .GSCs were originally isolated from surgical human GBM specimens, as described by our group in . The GSC2 5%.U87MG was purchased from ATCC, Rockville, MD, USA and cultured in DMEM/F-12  supplemented with 10% fetal bovine serum (FBS) and 2% penicillin-streptomycin (PS). Cells were maintained at 37 °C in humidified atmosphere air, COHuman mesenchymal stem cells (hMSCs) (a gift from Dr. Carmen Escobedo Lucea) were cultured in DMEM, high glucose, GlutaMAX , supplemented with a final concentration of 20% FBS and 1% P/S. All hMSCs experiments were performed in the first five passages from isolation.1,1-Dimethylbiguanide hydrochloride (D150959), sodium oxamate (O2751), sodium dichloroacetate (347795) and 6-Diazo-5-oxo-L-norleucine (D2141) were purchased from Sigma-Aldrich. Bleomycin sulfate (HY-17565) was acquired from MedChemExpress, Monmouth Junction, NJ, USA.The sensitivity to different metabolic drugs was assessed using [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) containing solution from Promega, Madison, WI, USA . Briefly, single-cell suspensions of GSCs were plated in a 96-well plate, 3000 cells/well, and allowed to grow and form spheres for 72 h. U87MG were seeded at 3000 cells/well and allowed to grow for 24 h. Cultures were then treated with their respective culture media (control cells) or increasing concentrations of each drug for 0 h, 24 h, 48 h or 72 h. At each timepoint, MTS reactant was added, incubated at 37 °C for 2 to 4 h and absorbance was measured at 490 nm/630 nm, using a Varioskan Flash  or a Sunrise Absorbance Reader . For IC50 calculations, corrected absorbance was transformed, normalized and extrapolated in GraphPad Prism version 8.0.1, using the logarithmic variable slope equation:In specific dose experiments, hMSCs were seeded at 6000 cells/well and allowed to grow for 72 h. Fresh cell culture media was then added to control wells and dissolved treatments to experimental wells. Cells were treated for 72 h before MTS read-out.Combinatory studies were performed in the same manner as single-drug assays. After seeding and cell-specific recovery/attachment intervals, combined treatments were added in the following final concentrations: IC50 for drug A alone; IC50 for drug B alone; full dose IC50 for drug A + drug B; IC50(A + B)/2; IC50(A + B)/8. Experimentally, drug “A” was one of the metabolic inhibitors , whereas drug “B” was the radiomimetic bleomycin. CompuSyn software (version 1.0), based on the Chou-Talalay method, was employed to determine the interaction between the drugs ,48. Thisβ-actin and GAPDH were used as housekeeping genes. For relativization and comparison with a non-tumoral control, we compared our samples with a pool of retrotranscribed RNA from brain tissue obtained from epileptic patients, provided courtesy of Hospital Universitario y Politécnico La Fe .For RT-qRT-PCR, total RNA was isolated from cell pellets using NZYol , following the manufacturer’s recommendations. For chronological parity with other experiments, GSCs were seeded in 6-well plates at a density of 90000 cells/well, allowed to grow for 72 h, fresh cell culture media was added (1:1) and pellets were collected after 72 h; the same protocol was applicable to U87MG, but fresh cell culture media was added after 24 h from seeding. Purity of RNA was assessed based on 260/280 and 260/230 ratios using a Thermo Scientific NanoDrop 2000/2000c. RNA was retrotranscribed to cDNA . Resulting samples were amplified with specific primers  in a CFXAll primary and secondary antibodies were purchased from commercial sources, listed as follows: AMPKα Antibody , phospho-AMPKα (Thr172) , Anti-Pyruvate Dehydrogenase E1-alpha subunit antibody , Anti-PDHA1 (phospho S293) antibody , β-Actin , α-Tubulin . The secondary antibodies for horseradish peroxidase (HRP) detection were anti-rabbit IgG  and anti-mouse IgG .Centrifuged and pelleted U87MG and GSCs were resuspended in 100 μL of radioimmunoprecipitation buffer [RIPA; 100 mM Tris-HCl (pH 8.5), 200 mM NaCl, 5 mM EDTA and 0.2% SDS, with phosphatase and a protease inhibitor cocktail and stored at −80 °C for a minimum of 24 h. Samples were then centrifuged at 13,200 RPM for 20 min at 4 °C; protein-containing supernatant was conserved.Total protein concentration was determined using Bio-Rad Protein Assay according to the manufacturer instructions; after corresponding incubation, absorbance was read at 595 nm.In phosphorylation experiments, treatments dissolved at 1:1 concentration in serum-free medium were added 3–4 days after seeding GSCs, and 24 h in the case of U87MG. U87MG cells were washed twice with PBS and serum-deprived for 1 h prior to sample collection. Protein was subsequently recovered at the indicated timepoints .Western blotting experiments were performed adapting the protocol from Mahmood et al. . BrieflyExperiments were performed in an XFp 8-well microplate using the Seahorse XFp Analyzer . Briefly, GSCs were seeded at a density of 10,000 cells/well and allowed to grow for 72 h, in wells previously coated with 20 µL of Collagen Type IV at 20 µg/mL . U87MG cells were seeded at 6000 cells/well and allowed to grow for 24 h. Metabolic drugs were added to the treatment wells and fresh media was added to the control wells. After 72 h, the original media was carefully pipetted out of each well into a centrifuge tube without disturbing the attached cells; then, Seahorse XF DMEM medium, pH 7.4  was used to wash, pipetted out and centrifuged with the original media at 1000 rpm for 5 min at 25 °C. After centrifugation, supernatant was aspirated from each tube, conserving only the cell pellet, resuspended in Seahorse medium and added back to respective wells.We then followed the standard protocol for Standard XF Real-Time ATP Rate Assay, as described in the Seahorse XF Real-Time ATP Rate Assay User Guide . Seahorse XF technology measures two key parameters of cellular bioenergetics: oxygen consumption rate  and extracellular acidification rate  ,51. Resugliovis.bioinfo.cnio.es) [Affymetrix (HG-U133A) normalized gene expression datasets of GBM and non-tumor tissue samples from TCGA were downloaded from GlioVis repository (cnio.es) . As IDH cnio.es) . The remcnio.es) . Four dicnio.es) : KEGG (ocnio.es) .p values < 0.05 were considered as statistically significant. For all figures, p values were expressed according to GraphPad 8 NEJM p-value style.: p > 0.05 (ns); p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).Statistical analysis was performed using a 2-tailed Student t test (when comparing two groups) and One-Way ANOVA (three or more groups). Data are presented as means ± standard deviation and calculated using the software package GraphPad Prism version 8.0.1 for Windows, GraphPad Software, San Diego, California USA. RT-qRT-PCR expression data was graphed and analyzed directly in CFX Maestro 1.1 software, version 4.1.2433.1219 (Bio-Rad Laboratories). Molecular heterogeneity is a key feature of GBM, with clinical and therapeutic repercussions. To better understand if the vast molecular landscape of GBM could be reduced into a manageable number of metabolic categories, we explored the TCGA expression databases using a GSVA approach. Filtering for canonical gene sets of glycolytic and oxidative pathways, Warburg-like phenotypes were enriched in the mesenchymal subgroup, whereas functional mitochondrial metabolism predominated in healthy tissues a. BetweeSubsequently, to examine the differences in bioenergetic metabolism in phenotypically and molecularly distinct gliomas in vitro, we determined OCR and ECAR of our set of GSCs and U87MG c. We canTaken together, our data suggest that a high degree of metabolic variability is present between our GSCs, and their ATP production rates are faster than those of U87MG. GSCs and U87MG maintain a stable, basal, metabolic profile, and seem to be able to shift, to some extent, between aerobic glycolysis and OXPHOS to meet their bioenergetic needs.At the outset, in order to determine the optimal doses to be used in future experiments, we exposed our GSCs  and the U87MG cell line to escalating concentrations of selected metabolic drugs. Inhibitory curves for all time points are presented in After conducting these experiments, we ascertained maximum inhibitory effects and reliable trends in the viability data at 72 h; therefore, for every cell line, IC50 at 72 h was considered as the optimal inhibitory concentration.As we can appreciate in In summary, GBM18 was the most sensitive to MF, GBM38 to DCA and U87MG to DON; on the other hand, GBM27 was the most resistant to MF and DCA, while GBM18 required the highest doses of DON. A very high resistance towards SOD, as well as low variability in responses, was observed across all cell lines, so this drug was discarded from further assays.To further investigate the relative sensitivity/resistance profiles of each cell line to our selection of metabolic drugs, we aimed to evaluate their basal genetic expression profiles a.MF acts through inhibition of the electron transport chain (ETC) complex I, increasing the ADP/ATP ratio, but its primary downstream target is the activation of AMPK (phosphorylation of Thr172 of AMPKα1), which ultimately leads to mammalian target of rapamycin (mTOR) inhibition. We therefore evaluated expression of PRKAA1 and PRKAA2  and the mTOR gene. Although PRKAA2 was not expressed in our samples, PRKAA1 was significantly upregulated in GBM27 (9-fold relative to control) and, at similar levels (approximately 3 to 4-fold), in GBM18, GBM38 and U87MG. We also found the highest relative expression levels of mTOR in GBM27 and GBM38, but differences did not reach statistical significance. Additionally, we analyzed the phosphorylation of AMPKα to investigate the biological effects of MF b. We obsNext, we studied DCA activity in our GBM cell lines. Altough we analyzed all PDK subunits, PDK2 and PDK4 were not expressed, with major differences detected primarily in PDK3 expression, exceptionally upregulated in GBM27, consistent with sensitivity profiles to DCA. At the protein level, the catalytic subunit PDH-E1α has three major phosphorylation sites, with site 1 (Ser-293) being the most frequent and efficient target, sufficient to fully inhibit PDH activity ,60. WithWhen we evaluated GLS genes, our analysis revealed no detectable amplification of GLS2; therefore, we focused on GLS1 as a potential predictor for DON’s antiproliferative effects. Relativized to epilepsy control, GLS1 was significantly upregulated in GBM18, neutral in GBM27/U87MG and downregulated in GBM38. Higher GLS1 expression correlated with the relative resistance against DON in GBM18, but comparatively lower expression in GBM38 was not associated with lower doses.After completing this set of experiments, we questioned whether our cell lines would respond favorably to bleomycin, a radiomimetic/DNA-targeting drug.The mechanism of action and IC50-72h concentrations for bleomycin are presented in As with any other form of treatment, the success of metabolic therapies could be limited by toxicity to healthy cells. Therefore, we investigated whether all IC50 concentrations determined thus far could be a realistic goal in vitro, exploring their effects on non-tumoral hMSCs b. MF/DCAOne way to solve the challenge of non-specific damage to healthy cells is to exploit coexisting weaknesses of tumoral cells in combinatory strategies. Since GBM27 doses of MF/DCA were also affecting the viability of normal cells, we explored the possibility of dose-reduction attributable to synergistic effects with radiomimetic bleomycin. After individually confirming the validity of each calculated 72h-IC50, we performed combinatory studies to determine the existence of synergy, additive or antagonistic effects. Using the Chou-Talalay theorem, the Combination Index (CI) and the Dose Reduction Index (DRI) were calculated for each drug combination.As shown in All final reports with complete datasets, including Median-Effect Plot, CI Plot, Logarithmic CI Plot, DRI, Isobologram and Sequential Deletion Analysis , are included in Our results describe a wide variety of combinatory effects depending on the cell subtype and Fa level. Next, To conclude, In summary, for GBM27, synergism with radiomimetic bleomycin was observed for all metabolic inhibitors at therapeutically relevant Fa = 0.9. Combining metabolic inhibitors and bleomycin could be leveraged to reduce dosing requirements of oxidative-like GSC subtypes.Using Seahorse XF technology, we determined total ATP production and ratios of mitoATP/glycoATP production under metabolically treated conditions.As shown in Normalized values of OCR/Proton Efflux Rate (PER) in real-time after each drug injection of the Seahorse XF protocol are provided in In conclusion, pre-treatment with metabolic drugs could become a valuable tool to characterize the bioenergetic phenotypes of surviving, resistant fraction of cells, priming them for further treatments. To our knowledge, this is the first time chronic metabolic treatments (>72 h) with DCA and DON have been characterized in GSCs. Despite methodological differences, our results also confirm the bioenergetic changes after low-dose MF in GSCs  and DCA Cancer metabolism has recently regained interest as a promising therapeutic target, coinciding with the development of standardized techniques for real-time assessment of bioenergetics. Given high intratumoral molecular and metabolic heterogeneity, it is necessary to target glycolytic, glutaminolytic and oxidative phenotypes; for this purpose, primary GSCs cultured from human surgical samples are an excellent model .In this paper, we describe how metabolic modulators  could be leveraged to inhibit GSCs proliferation, demonstrating that distinct, stable, metabolic phenotypes contrast in their response to these treatments. A comparison between untreated and metabolically treated GSCs populations is presented here for the first time, leading us to hypothesize that inhibiting metabolic pathways might not only kill malignant cells, but also turn them vulnerable to successive targeted treatments (“metabolic priming”).Consistent with our results, GSCs have been characterized by ample metabolic heterogeneity, exhibiting both glycolytic/oxidative characteristics ,65,66. SKi PDK2 < PDK1 ≃ PDK4 < PDK3). As PDK1 was equally distributed, and PDK2/PDK4 were undetectable in our samples, variances in dose-response profiles were likely related to differences in PDK3. SOD treatment was equally inefficacious in all tested cell lines, but we suspect this was partly related to poor cell membrane permeability of this compound [Regarding our selection of metabolic inhibitors, GBM27 was the most resistant to all drugs, with the exception of DON. One of the reasons for this might be owed to GBM27’s characteristically oxidative metabolism (up to 50% oxidative) and slower proliferation rates. Metabolic flexibility may grant a survival advantage even if mitochondrial energy production is targeted via MF or glycolysis via DCA. GBM18 and GBM38, on the other hand, were significantly more sensitive to MF; this would be consistent with their Warburg-like metabolic phenotypes. At the protein level, our results are consistent with previous research, where 10 mM MF did not significantly increase phospho-Thr172 at 6 h in U87MG . Next, wcompound ,75,76. Dcompound ,78. Conccompound ,80. In ocompound . With DOThe heterogeneity of cancer could also be managed by combinatorial strategies, focusing on multiple molecular pathways to achieve compounded effects. Even though stand-alone metabolic modulation could become a possibility in the future, a more realistic and approachable goal is to design safe and effective combinations together with well-established chemotherapeutics and radiotherapy. Here, we show that non-tumoral hMSCs are not affected by low/intermediate doses of MF, DCA, DON or bleomycin. Concentrations of MF and DCA corresponding to GBM27, however, decreased viability by more than half in hMSCs, revealing that oxidative cancer cells would require a different management strategy: combinatory therapies could be such an option.To the extent of our knowledge, ours is the first report of the combinatorial effects in GSCs of the radiomimetic drug bleomycin together with MF, DCA and DON. Bleomycin acts by inducing single and double strand DNA breaks, causing apoptosis and cell cycle arrest in early G2 phase ; it is FAs far as we know, sustained metabolic changes in GSCs after extended treatment with DCA and DON had not been properly illustrated until now. In terms of OCR/ECAR, all metabolic modulators exerted biologically coherent responses, especially well-defined for MF and DCA. In the case of MF, as would be expected, surviving cells shifted to a primarily glycolytic behavior, with a very low percentage of mitoATP generation; these results are consistent with Seahorse XF assays in established glioma cell lines and colon cancer CSCs . The metFrom a translational medicine perspective, avoiding the risk of toxicity associated with high concentrations of MF might be feasible, as even very low doses of this drug revealed antiproliferative effects and radiotherapy enhancement ,93. FurtWe believe the origin of cancer is complex, with involvement of both oncogenic signaling and metabolic reprogramming ,107. FutIn summary, we show that molecularly distinct GSCs show a high degree of heterogeneity, both in basal metabolic phenotypes and in response to metabolic inhibitors. Subpopulations of GSCs presented both highly glycolytic and highly oxidative characteristics. Stratification of patients according to coexisting metabolic phenotypes could be advanced into clinical tools for improved metabolic therapies. In highly oxidative GSCs , there is potential for synergistic effects with radiomimetic bleomycin. Inhibition of glutaminolysis (via DON) is also an attractive therapeutic target, as synergy was described regardless of basal metabolism. Application of metabolic drugs produces stable changes in the bioenergetic states of GSCs, which could be leveraged as a form of metabolic priming.


According to the search results in database and phylogenetic trees generated in the review, the species of the genera Podostroma and Monosporascus would probably be great sources for producing macrocyclic trichothecenes. Moreover, we propose that the macrocyclic trichothecene roridin E could be formed via acylation or esterification of the long side chain linked with C-4 to the hydroxyl group at C-15, and vice versa. More assays and evidences are needed to support this hypothesis, which would promote the verification of the proposed pathway.Macrocyclic trichothecenes are an important group of trichothecenes bearing a large ring. Despite the fact that many of trichothecenes are of concern in agriculture, food contamination, health care and building protection, the macrocyclic ones are becoming the research hotspot because of their diversity in structure and biologic activity. Several researchers have declared that macrocyclic trichothecenes have great potential to be developed as antitumor agents, due to the plenty of their compounds and bioactivities. In this review we summarize the newly discovered macrocyclic trichothecenes and their bioactivities over the last decade, as well as identifications of genes  Trichothecenes are a family of sesquiterpenoid mycotoxins produced by multiple genera of fungi, including plant and insect pathogens, and they are of great concern because they are toxic to animals and humans and frequently detected in cereal crops, especially in wheat, barley, maize and oats ,2,3,4. IFusarium, the biosynthetic pathway of the representative trichothecenes, e.g., DON, nivalenol and T-2 toxin, have been fully elucidated [TRI) cluster [Myrothecium sp. and Trichoderma sp., the tri4-encoded cytochrome P450 monooxygenase catalyzes oxygenation of trichodiene at three carbons , but Tri4 of Fusarium catalyzes four carbons of trichodiene (combined with C-3) [Fusarium species [TRI genes per cluster also vary in different fungi [TRI cluster of Fusarium sporotrichioides contains 12 genes related to trichothecene biosynthesis, but the cluster of Trichoderma arundinaceum just contains 5 genes [T. arundinaceum, e.g., tri5 initiating biosynthesis of trichothecene, are located in other loci [Myrothecium and Stachybotrys species, the TRI genes at other loci were paralogs of genes in the main cluster [The biochemistry, bioactivity and biosynthesis of simple trichothecenes have been studied extensively ,12. In Fucidated ,12,13. Aucidated ,12. Most cluster ,15,16,17 cluster ,18. For ith C-3) ,20,21. Pith C-3) . Likewis species ,23,24. Tnt fungi ,18,25. T 5 genes . The oth cluster .TRI genes; however, the genetic bases for macrocyclic trichothecenes, namely Type D trichothecenes, are not completely elucidated, especially about the formation of the macrocycle, which is composed of polyketide- and isoprenoid-derived moieties [tri17 and tri18 were presumed to be involved in macrocycle formation [tri17 and tri18, and putative biosynthetic pathway. This review provides a new insight into the biosynthetic mechanism of macrocycle in macrocyclic trichothecene.The genetic bases for most simple trichothecenes have been elucidated by functional analyses of moieties ,26,27. GFusarium, Isaria, Microcyclospora, Myrothecium, Peltaster, Spicellum, Stachybotrys, Trichoderma, Trichothecium, Cephalosporium, Cylindrocarpon, Memnoniella, Phomopsis, Verticimonosporium etc. [Myrothecium [Podostroma [Stachybotrys [Calcarisporium [Cercophora [Cylindrocarpon [Dendrodochium [Phomopsis [Verticinimonosporium [Baccharis, belonging to the Asteraceae family, is the only plant genus that can produce trichothecenes [Trichothecenes can be isolated from many fungal genera, such as ium etc. ,10,28. Mothecium ,30,31, Pdostroma , Stachybhybotrys , Calcariisporium , Cercophrcophora , Cylindrrocarpon , Dendrododochium , Phomopshomopsis , and Verosporium . It is wthecenes . The macthecenes ,42. The Myrothecium , and the others with a side chain of 14-carbon atoms could be recognized as roridins or satratoxins. It was reported that roridin may be the precursor of the respective verrucarin, because the C2-side chain at C-6′ of roridin might be cleaved by oxidation to form verrucarin [1–11 and 23 have a standard tricyclic core, namely EPT; however, compounds 12–20 lack the epoxide at C-12, C-13. Verrucarin Y (1) and verrucarin Z (2) were isolated from M. roridum M10 by Muhammad et al. [1) shares the same structure with epiroridin acid (3) [2-side chain at C-6′, implying that epiroridin acid (3) may be the homologue of verrucarin Y (1) [2) has an epoxide ring at C-2′, C-3′ instead of the olefinic bond of verrucarin Y (1) at the same place, and the number of epoxide rings always has a significant impact on their biological activities [Myrothecium (compounds 5–18), such as mytoxin, satratoxin, vertisporin, roritoxin, and myrothecine, the names of which generally indicated the genus that this kind of toxins were firstly isolated from [9, 10, 15) [10) [11) [18) [11) has an additional epoxide ring at C-6′, C-12′. Due to the absence of the epoxide at C-12, C-13, compounds 12–20 belong to 10,13-cyclotrichothecane derivatives [21) and satratoxin I (22) discovered from Podostroma cornu-damae, a deadly poisonous mushroom, are not complete macrolides, because the macrocyclic ester bridge between C-4 and C-15 are not closed; however, they might be transformed into roridin E and satratoxin H, respectively, through a one-step esterification [23) is the only macrocyclic trichothecene isolated from Stachybotrys during the past decade [Stachybotrys fungi are not great source of trichothecenes, and most of them have been discovered before [Myrothecium and Podostroma, especially the latter.New macrocyclic trichothecenes are constantly being discovered. Here, the novel macrocyclic trichothecenes isolated over the past decade are reviewed. There were 23 compounds related to macrocyclic trichothecene newly discovered between 2010 and 2019, and most of them were isolated from othecium . Only thhybotrys . Four corrucarin ,43. Compd et al. . Verrucaacid (3) , except in Y (1) . Verrucativities . Severalted from . The sec 10, 15) ,44. Rori15) [10) , 6′,12′-10) [11) , and 2′,11) [18)  have theivatives . Generalivatives ,47,48. Rfication . Chartart decade . This cod before . Search Macrocyclic trichothecenes have been shown to possess diverse biological activities, such as antibiotic , antifun12–20, are not likely to show cytotoxicity with a nanomolar level. Secondly, an increase of the additional epoxide groups on the macrolide ring remarkably promote toxicity [11), bearing an epoxide ring at C-6′, C-12′, showed quite strong cytotoxicity towards the KB and NCI-H187 cancer cell lines [3) compared to epiroridin [17) enhanced bioactivity [6, 9, 14–17 were evaluated for their in vitro cytotoxicities against K562 and SW1116, and the relatively large differences in IC50 values suggested that the slight changes of the substituent group have obvious effects on the biological selectivity of macrocyclic trichothecenes against cancer cell lines [21) and satratoxin I (22) without macrocyclic ring were almost inactive against cancer cell lines  showed a similar activity against Mucor miehei at a concentration of 50 μg/disk when compared with the positive drug nystatin [4) and myrothecin A (19) exhibited bioactivities against plant pathogenic fungi Rhizoctonia solani and Fusarium oxysporum [Cochliobolus miyabeanus [5) and 6′,12′-epoxymyrotoxin A (11) exhibited strong antimalarial activity against Plasmodium falciparum with 86.1% (1.84 nM) and 98.85% (2 nM) parasite inhibition, respectively [There are some macrocyclic trichothecenes displaying other biological activities, such as antifungal and antimalarial activities. Verrucarin Z  was confirmed to be essential for synthesis of the polyketide side chain of harzianum A  reported in 2019 [21 could be generated by attaching a free 6,7-dihydroxy-2,4-octadienoate to the hydroxyl group at C-5′ of verrol. Then, 21 could be sequentially converted to roridin E through acylation or esterification. Similar processes may happen to roridin L-2 and satratoxin I (22) [22 could be created through hydrolyzing at C-15 of 8, or attaching a free 5-hydroxy-3-methylpent-2-enoic acid to the end of octadienoate fused to trichoverrols. Until now, there is no obvious evidence showing how 21, 22 or roridin L-2 is formed, and whether the conversion between roridin E and 21 or roridin L-2, between 8 and 22 is reversible. The puzzle may be explained by identifying the specific undiscovered gene accounting for construction of the macrocycle, or knocking out gene tri18 in Myrothecium, Podostroma, and Stachybotrys, all of which are able to produce macrocyclic trichothecenes. Since gene tri18 in Ta37 accounted for esterification of octa-2,4,6-trienedioyl, deletion of it in macrocyclic trichothecene-producing strains may cut off the formation of macrocycle, resulting in an accumulation of verrol, or roridin F (21) and their derivatives. If final products do contain 21 and its derivatives, it suggests that 21 could be generated from verrol, meaning that there is a second putative pathway to form macrocyclic trichothecenes. In addition, isolated from Myrothecium [Podostroma [Although not all genes involved in the biosynthesis of macrocyclic trichothecene have been identified in detail, there are still a few studies proposing several possible biosynthetic pathways of macrocyclic trichothecenes ,69,70,71ygenated ,43. Simiran ring . The hyp in 2019  and rori in 2019 ,73 seem n I (22) . Compoundostroma , trichovMyrothecium, Podostroma and Stachybotrys. Meanwhile, summary review in bioactivity studies revealed that the small changes between structurally similar compounds isolated from different species can result in a considerable alteration on bioactivity and/or selectivity against cancer cell lines [tri17 and tri18 genes in Trichoderma has contributed significantly to insights into the evolutionary history of macrocyclic trichothecene biosynthesis. In terms of phylogenetic trees generated in the review and the presence of the highly similar genes tri17 and tri18 in Monosporascus, it was suggested that these species would probably be a source of macrocyclic trichothecenes. Furthermore, based on several newly isolated intermediates, we propose that there is a second biosynthetic pathway of macrocyclic trichothecene. An identification assay of the genes responsible for constructing the macrocycle in macrocyclic trichothecene-producing strains (maybe tri18 or other unidentified gene), will be critical to more precisely clarify the biosynthesis mechanism of potential anticancer compounds.Trichothecenes have been studied for more than 40 years. Initially, trichothecenes were well known and studied as the important contamination sources of grains ,74, builll lines ,59, whic