Charting human development using a multi-endodermal organ atlas and organoid models.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Photomediated Hydro(deutero)acylation of Olefins by Decarboxylative Addition of α-Oxocarboxylic Acids.

Acyl radicals have been generated from the decarboxylation of α-oxocarboxylic acids by using a readily accessible organic pyrimidopteridine photoredox catalyst under ultraviolet-A (UV-A) light irradiation. These reactive acyl radicals were smoothly added to olefins such as styrenes and diverse Michael acceptors, with the assistance of H2O/D2O as hydrogen donors, enabling easy access to a diverse range of ketones/ß-deuterio ketones. A wide range of α-oxocarboxylic acids are compatible with this reaction, which shows a reliable, atom-economical, and eco-friendly protocol. Furthermore, postsynthetic diversifications and applications are presented.

Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model.

The establishment of the small intestinal (SI) lineage during human embryogenesis ensures functional integrity of the intestine after birth. The chromatin dynamics that drive SI lineage formation and regional patterning in humans are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells into SI spheroids with regional specification. Through comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers and enhancer hotspots during the directed differentiation. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or regional patterning. Our ChRO-seq analyses uncover a previously undescribed pattern of enhancer activity and transcription at HOX gene loci underlying SI regional patterning. We also validated this unique HOX dynamics by the analysis of single cell RNA-seq data from human fetal SI. Overall, the results lead to a new proposed working model for the regulatory underpinnings of human SI development, thereby adding a novel dimension to the literature that has relied almost exclusively on non-human models.

Dissecting contributions of representative heavy metal components in PM2.5 to its cytotoxicity.

PM2.5 is a complex pollutant that is a pervasive threat to human health. The health risks and toxicity mechanisms of PM2.5 components must be identified to alleviate the corresponding risks. In this study, a reductionism approach based on model PM2.5 particles was used to investigate the contributions of the most harmful components in PM2.5 to its toxicity. Human liver and kidney cells were used as models. The results showed that Cr(VI) was the most critical toxic component among other components (Pb, As, and benzo[a]pyrene) in human liver and kidney cells. PM2.5-Cr(VI) induced oxidative stress, which led to cytotoxicity by inducing cell cycle arrest in the S-phase in HepG2 and HEK293 cells. The presented findings can provide valuable insights into the toxicity levels of PM2.5 components, which can help clarify the potential health risks from PM2.5 exposure.

[Effect of Astragali Radix-Curcumae Rhizoma compatibility combined with 5-fluorouracil on Th17/Treg balance and tumor-related mRNA and protein expression in orthotopic xenograft model mice of CT26.WT colorectal carcinoma].

Astragali Radix-Curcumae Rhizoma(AR-CR) is a combination commonly used in the clinical treatment of tumors. Based on the T helper 17(Th17)/regulatory T cell(Treg) balance, the present study explored the possible mechanism of AR-CR combined with 5-fluorouracil(5-FU) on the tumor growth of orthotopic xenograft model mice of colorectal carcinoma. Ninety male BALB/c mice were randomly divided into nine groups, i.e., a blank group, a model group, a 5-FU group, high-, medium-, and low-dose AR-CR(2∶1) groups, and high-, medium-, and low-dose AR-CR+5-FU groups, with 10 mice in each group. The orthotopic xenograft model of CT26.WT colorectal carcinoma was induced in mice except those in the blank group. Twenty-four hours after the ope-ration, mice in the blank group and the model group received normal saline by gavage(10 mL·kg~(-1), once per day), and those in the 5-FU group received 5-FU by intraperitoneal injection(25 mg·kg~(-1), once every other day). Mice in the AR-CR groups received AR and CR decoctions by gavage(12, 6, and 3 g·kg~(-1), once a day) and those in the combination groups received AR and CR decoctions and 5-FU(doses and administration methods were the same as above). After intervention for three weeks, all mice were sacrificed and tumor tissues were collected. The tumor mass was weighed and the average tumor weight was calculated. The changing trend of Th17/Treg(%) in the CD4~+T lymphocytes of the spleen tissues of the mice in each group was detected. The mRNA expression in the blood and protein expression in the tumor tissues of transforming growth factor-ß(TGF-ß), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), Smad4, N-cadherin, matrix metalloproteinase-7(MMP-7) were detected. The experimental results revealed that compared with the model group, the groups with drug intervention showed reduced tumor mass(P<0.01), decreased CD4~+IL-17~+ in the spleen tissues to varying degrees(P<0.001), and increased proportion of CD4~+Foxp3~+(P<0.001 or P<0.05), indicating that Th17/Treg maintained dynamic balance, and the effect of the combination groups was predominant. Additionally, the mRNA expression in the blood and protein expression in the tumor tissues of TGF-ß, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 declined to varying degrees in a dose-dependent manner(P<0.01 or P<0.001). The AR-CR combined with 5-FU can inhibit the tumor growth of orthotopic xenograft model mice of CT26.WT colorectal carcinoma. The mechanism may be related to maintenance of Th17/Treg dynamic balance in the body and down-regulation of TGF-ß, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 expression.

Lipidome alterations in human prefrontal cortex during development, aging, and cognitive disorders.

Lipids are essential to brain functions, yet they remain largely unexplored. Here we investigated the lipidome composition of prefrontal cortex gray matter in 396 cognitively healthy individuals with ages spanning 100 years, as well as 67 adult individuals diagnosed with autism (ASD), schizophrenia (SZ), and Down syndrome (DS). Of the 5024 detected lipids, 95% showed significant age-dependent concentration differences clustering into four temporal stages, and resulting in a gradual increase in membrane fluidity in individuals ranging from newborn to nonagenarian. Aging affects 14% of the brain lipidome with late-life changes starting predominantly at 50-55 years of age-a period of general metabolic transition. All three diseases alter the brain lipidome composition, leading-among other things-to a concentration decrease in glycerophospholipid metabolism and endocannabinoid signaling pathways. Lipid concentration decreases in SZ were further linked to genetic variants associated with disease, indicating the relevance of the lipidome changes to disease progression.

Elucidation of the Critical Role of Core Materials in PM2.5-Induced Cytotoxicity by Interrogating Silica- and Carbon-Based Model PM2.5 Particle Libraries.

An insoluble core with adsorbed pollutants constitutes the most toxic part of PM2.5 particles. However, the toxicological difference between carbon and silica cores remains unknown. Here, we employed 32-membered carbon- and silica-based model PM2.5 libraries that each was loaded with four toxic airborne pollutants including Cr(VI), As(III), Pb2+, and BaP in all possible combinations to explore their contributions to cytotoxicity in normal human bronchial cells. The following three crucial findings were revealed: (1) more adsorption of polar pollutants in a silica core (such as Cr(VI), As(III), and Pb2+) and nonpolar ones in a carbon core (such as BaP); (2) about 41% more cell uptake of carbon- than silica-based particles; and (3) about 59% less toxicity in silica- than carbon-based particles when pollutants other than Cr(VI) were loaded. This was reversed after Cr(VI) loading (silica particles were 56% more toxic). The difference maker is that compared to stable silica, carbon particles reduce Cr(VI) to less toxic Cr(III). Our findings highlight the different roles of carbon and silica cores in inducing health risks of PM2.5 particles.

Three-dimensional porous Cu@Cu2O aerogels for direct voltammetric sensing of glucose.

Three-dimensional Cu@Cu2O aerogels with excellent electrocatalytic activity were prepared and used as electrode matrix for constructing novel electrochemical glucose sensors. The aerogels were obtained by adding a fresh solution of NaBH4 into a mixture of CuCl2 and NaOH aqueous solutions under stirring at room temperature. The aerogels were assembled with Cu or Cu2O nanoparticles. The materials show superfine spongy-like structures with large surface-to-volume ratio, numerous active sites and good solubility. The Cu@Cu2O aerogels show highly efficient electrochemical activity toward glucose oxidation with a relatively low-onset potential (0.25 V) in 0.1 M NaOH solution. This non-enzymatic glucose sensor offers a low detection limit of 0.6 µM (S/N = 3), a high sensitivity (195 mA M-1 cm-2), and two wide linear ranges (0.001-5.2 mM, 5.2-17.1 mM) at a working voltage of 0.6 V (vs. Ag/AgCl) in alkaline solution. While in neutral pH values, the respective data are a linear analytical range from 0.1 to 10 mM; a detection limit of 54 µM (S/N = 3) and a sensitivity of 12 mA M-1 cm-2 at scan rate of 100 mV s-1. The sensor possesses high selectivity, good reproducibility and long-time stability. It was utilized to determine glucose levels in (spiked) human serum samples, and satisfactory results were obtained. Graphical abstract Schematic presentation of a glassy carbon electrode modified with 3D porous Cu@Cu2O aerogels. The aerogels were obtained by a reduction reaction at room temperature (Scheme 1A). The aerogel networks were used to develop a highly sensitive electrochemical sensing platform for the detection of glucose (Scheme 1B).

Identification and characterization of functional modules reflecting transcriptome transition during human neuron maturation.

BACKGROUND: Neuron maturation is a critical process in neurogenesis, during which neurons gain their morphological, electrophysiological and molecular characteristics for their functions as the central components of the nervous system. RESULTS: To better understand the molecular changes during this process, we combined the protein-protein interaction network and public single cell RNA-seq data of mature and immature neurons to identify functional modules relevant to the neuron maturation process in humans. Among the 109 functional modules in total, 33 showed significant gene expression level changes (discriminating modules) which participate in varied functions including energy consumption, synaptic functions and housekeeping functions such as translation and splicing. Based on the identified modules, we trained a neuron maturity index (NMI) model for the quantification of maturation states of single neurons or purified bulk neurons. Applied to multiple single neuron transcriptome data sets of neuron development in humans and mice, the NMI model made estimation of neuron maturity states which were significantly correlated with the neuron maturation trajectories in both species, implying the reproducibility and conservation of the identified transcriptome transition. CONCLUSION: We identified 33 discriminating modules whose activities were significantly correlated with single neuron maturity states, which may play important roles in the neuron maturation process.

Construction of cyclobutane-fused tetracyclic skeletons via substrate-dependent EnT-enabled dearomative [2+2] cycloaddition of benzofurans (benzothiophenes)/maleimides.

Herein, a novel and facile organic photosensitizer (thioxanthone)-mediated energy-transfer-enabled (EnT-enabled) dearomative [2+2] cycloaddition of aromatic heterocycles/maleimides for green synthesis of cyclobutane-fused polycyclic skeletons is reported. Mechanistic investigations revealed that different EnT pathways by triplet thioxanthone were initiated when different aromatic heterocycles participated in the reaction, giving the corresponding excited intermediates, which underwent the subsequent intermolecular [2+2] cycloaddition to access the desired highly functionalized cyclobutane-fused polycyclic skeletons.

Auricular therapy for primary dysmenorrhea: A protocol for systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Primary dysmenorrhea (PD) is a common problem among women. It is defined as any degree of perceived cramping pain during menstruation without any evident pathology. Auricular therapy (AT), a widely used alternative medical treatment method as part of traditional Chinese acupuncture, lacks reliable evidence to support its safety and effectiveness for PD. We aimed to conduct a meta-analysis to investigate the efficacy and safety of AT in PD and to investigate possible factors impacting the particular efficacy of AT in PD by meta-regression. METHODS: This protocol followed the PRISMA guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols. The following 9 sources will be searched for randomized control trials of AT for PD: the Cochrane Central Register of Controlled Trials, PubMed, Medline, Embase, Web of Science, Chinese Biomedical Literature Database (CBM), China National Knowledge Infrastructure, Chinese Science and Technology Periodicals (VIP) database and WanFang Database from inception to January 1, 2023. Primary outcomes include visual rating scales and clinical efficacy rates, while secondary outcomes include endocrine hormone indicators related to PD and adverse events. Two reviewers will work independently on study selection, data extraction, and coding, including the risk of bias assessment in the included studies. While conducting a meta-analysis, Review Manager version 5.3 will be employed. Otherwise, a descriptive analysis will be performed. The results will be displayed as a risk ratio with 95% confidence intervals for dichotomous data as well as weight mean difference or standardized mean difference with 95% confidence intervals for continuous data. RESULTS: This study's protocol will investigate the efficacy and safety of AT in the treatment of PD in a systematic way. CONCLUSION: This systematic evaluation will objectively and systematically assess the efficacy and safety of AT in PD based on the available evidence, as well as provides clinicians with evidence to support the treatment of the disease.

Application of fMRI techniques in the study of acupuncture for gynecological diseases: A review.

Acupuncture therapy, as a characteristic of Chinese medical therapy, has a long history and remarkable effect in the treatment of gynecological diseases, and so far, it has formed a complete treatment system, but its efficacy and mechanism of action remain unclear. Functional magnetic resonance imaging, a visual technique, provides an objective basis for the study of acupuncture in the treatment of gynecological diseases. This paper summarizes the current status of acupuncture in the treatment of gynecological diseases and summarizes the progress of functional magnetic resonance imaging research related to acupuncture in the treatment of gynecological diseases in the past 10 years, mainly including the common types of gynecological diseases in acupuncture clinics, and the commonly used acupuncture points. This study is expected to provide literature support for subsequent research on the central mechanisms of acupuncture in the treatment of gynecological diseases.

Siloxane-Modified UV-Curable Castor-Oil-Based Waterborne Polyurethane Superhydrophobic Coatings.

In recent years, superhydrophobic coatings with self-cleaning abilities have attracted considerable attention. In this study, we introduced hydroxyl-terminated polydimethylsiloxane (OH-PDMS) into castor-oil-based waterborne polyurethanes and synthesized silicone-modified castor-oil-based UV-curable waterborne polyurethanes (SCWPU). Further, we identified the optimal amount of OH-PDMS to be added and introduced different amounts of micro- and nanoscale heptadecafluorodecyltrimethoxysilane-modified SiO2 particles (FAS-SiO2) to prepare rough-surface SCWPU coatings with dense micro- and nanostructures, thus realizing waterborne superhydrophobic coatings. The results show that when the OH-PDMS content was 11 wt% and the total addition of FAS-SiO2 particles was 50% (with a 1:1:1 ratio of 100 nm, 1 µm, and 10 nm particles), the coatings exhibited a self-cleaning ability and superhydrophobicity with a contact angle of (152.36 ± 2.29)° and a roll-off angle of (4.9 ± 1.0)°. This castor-oil-based waterborne superhydrophobic coating has great potential for waterproofing, anti-fouling, anti-corrosion, and other applications.

Lactobacillus melliventris promotes hive productivity and immune functionality in Bombus terrestris performance in the greenhouse.

Bumblebees are important pollinators in agricultural ecosystems, but their abundance is declining globally. There is an urgent need to protect bumblebee health and their pollination services. Bumblebees possess specialized gut microbiota with potential to be used as probiotics to help defend at-risk bumblebee populations. However, evidence for probiotic benefits on bumblebees is lacking. Here, we evaluated how supplementation with Lactobacillus melliventris isolated from bumblebee gut affected the colony development of Bombus terrestris. This native strain colonized robustly and persisted long-term in bumblebees, leading to a significantly higher quality of offspring. Subsequently, the tyrosine pathway was upregulated in the brain and fat body, while the Wnt and mTOR pathways of the gut were downregulated. Notably, the field experiment in the greenhouse revealed the supplementation of L. melliventris led to a 2.5-fold increase in the bumblebee survival rate and a more than 10% increase in the number of flowers visited, indicating a better health condition and pollination ability in field conditions. Our study represents a first screening for the potential use of the native gut member, L. melliventris, as probiotic strains in hive supplement for bumblebee breeding, which may be a practical approach to improve immunity and hive health.

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids.

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Acupuncture for Crohn's disease: protocol for a systematic review and meta-analysis of randomized clinical trials.

BACKGROUND: Crohn's disease (CD) is a chronic recurrent gastrointestinal disorder with a high incidence of inflammation due to intestinal inflammation. Acupuncture is commonly used as an alternative therapy for patients with CD. The aim of this study was to design a systematic review and meta-analysis protocol, to provide guidance for the establishment of systematic evaluation and meta-analysis on the efficacy and safety of acupuncture on CD. METHODS: We will search PubMed, the Cochrane Library, Embase, Web of Science, and 4 Chinese databases: China National Knowledge Infrastructure (CNKI), Wanfang database, VIP database, and Chinese Biomedical Database to obtain randomized controlled trials of CD treated with acupuncture from inception to November 5, 2022. Primary outcome include CD symptoms severity and clinical efficacy, secondary outcome indicators include laboratory indicators or inflammatory markers, severity of endoscopic lesions, quality of life, and safety outcomes. We will analyze the data using RevMan V.5.4 software. Two reviewers will assess the risk of bias and study quality by the Cochrane Collaboration Risk of Bias Tool and GRADE methods, respectively. RESULTS: This systematic review and meta-analysis protocol will analyze the efficacy, symptom improvement, quality of life, and safety of acupuncture therapy for CD. CONCLUSION: This protocol outlines the planned scope and methodology of a forthcoming systematic review and meta-analysis to provide guidelines for a rigorous assessment of the efficacy and safety of acupuncture for the treatment of CD.

Pectin oligosaccharides improved lipid metabolism in white adipose tissue of high-fat diet fed mice.

Impacts of pectin oligosaccharide (POS) got from hawthorn fruitage on adiponectin signaling pathway and white adipose metabolism in mice fed with high-fat control. The results showed that POS significantly inhibited the levels of inflammatory cytokines TNF-α and IL-6, and down-regulated expression of CD68. POS dramatically reduced gene expression contents of fatty acid composite concerning enzymes ACC and FAS, as well as TG synthesis-related enzymes SCD-1 and DGAT1 as compared to a high-fat group (HFC). POS dramatically increased expression levels of oxidation-related enzymes of fatty acid ACO, CPT-1, and TG deposition-related enzymes ATGL and HSL as contrast to the high-fat control group. In addition, POS activated adiponectin-mediated AdipoR1/AMPK/PPARα signaling path by upregulating expression levels of AdipoR1, AMPK and PPARα. The results demonstrated that POS can improve lipid metabolism of adipose tissue, and contribute to the creation of functional foods to prevent and treat lipid metabolism disorders. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01109-9.

[Effect of moxibustion combined with acupoint catgut embedding on IL-6/JAK/STAT3 signaling pathway in colonic mucosa of ulcerative colitis rats].

OBJECTIVE: To observe the effect of moxibustion combined with acupoint catgut embedding on the content of interleukin 6 (IL-6), and the expressions of janus activated kinase (JAK), signal transducer and activator of transcription 3 (STAT3) in colonic mucosa of rats with ulcerative colitis (UC), so as to explore its mechanism underlying improvement of UC. METHODS: Thirty SD rats were randomly divided into normal, model, acupoint catgut embedding (ACE), moxibustion and acupoint catgut embedding combined with moxibustion (combination) groups (n=6 rats in each group). The UC model was established by enema of trinitro-benzene-sulfonic acid and ethanol. Moxibustion was applied to bilateral "Tianshu" (ST25), "Dachangshu" (BL25) and "Shangjuxu" (ST37) for 10 min, once daily for 14 days, and ACE applied to the same 3 acupoints, once a week for two weeks. After the treatment, colonic mucosal pathological changes were observed by H.E. staining, the level of IL-6 in colonic mucosa was assayed by ELISA, and the expressions of JAK and STAT3 in colonic mucosa were detected by immunohistochemistry and Western blot. RESULTS: H.E. staining showed severe defect of the colonic mucosal epithelium with infiltration of a large number of inflammatory cells in the model group, which was milder in moxibustion, ACE and combination groups. After modeling, the content of colonic IL-6, and the expression levels of JAK and STAT3 were obviously increased (P<0.01) in the model group relevant to the normal group. Following the intervention, the increase of IL-6 contents, and JAK and STAT3 expressions were reversed (P<0.05, P<0.01) in moxibustion, ACE and combination groups. The therapeutic effects of moxibustion combined with ACE were considerably superior to those of simple ACE and simple moxibustion in down-regulating the levels of JAK and STAT3 expression (P<0.01). CONCLUSION: Acupoint catgut embedding combined with moxibustion can repair the injured colonic mucosa of UC rats, which may be related with its effect in suppressing the activation of IL-6/JAK/STAT3 signaling pathway.

Synergistic effects of carbon nanoparticle-Cr-Pb in PM2.5 cause cell cycle arrest via upregulating a novel lncRNA NONHSAT074301.2 in human bronchial epithelial cells.

Inhalation of carcinogenic PM2.5 particles is a severe threat to all the people in both developing and developed nations. However, which components of PM2.5 and how they perturb human cells to cause various diseases are still not understood. Here, employing a reductionism approach, we revealed that one of the crucial toxic and pathogenic mechanisms of PM2.5 was the blocking of human bronchial cell cycle through upregulation of a novel long non-coding RNA NONHSAT074301.2 by carbon particles with payloads of Cr(VI) and Pb2+. We also discovered that NONHSAT074301.2 is a key regulatory molecule controlling cell cycle arrest at G2/M phase. This work highlights cellular function and molecular signaling events investigations using a 16-membered combinational model PM2.5 library which contain carbon particles carrying four toxic pollutants in all possible combinations at environmental relevant concentrations. This work demonstrates a very powerful methodology to elucidate mechanisms at molecular level and help unlock the "black box" of PM2.5-induced toxicities.