Comparison of the cytokines levels in aqueous humor in vitrectomized eyes versus non-vitrectomized eyes with diabetic macular edema.

BACKGROUND: This study was conducted to compare concentrations of VEGF family growth factors, inflammation-related factors, and adhesion molecules in the aqueous humor of eyes with diabetic macular edema (DME), with and without prior vitrectomy. METHODS: A total of 31 eyes were included, 11 with DME that had undergone vitrectomy, 9 with DME but without vitrectomy, and 11 from age-related cataract patients as controls. The concentrations of cytokines including TNF-α, IL-6, IL-8, IP-10, MCP-1, IFN-γ, MIP-1 α, MIP-1 ß, PECAM-1, MIF, VCAM-1, ICAM-1, PIGF were quantified using Luminex Human Discovery Assay. Central macular thickness (CMT) values of all eyes were measured using optical coherence tomography (OCT). RESULTS: (1) Vitrectomized DME eyes exhibited significantly higher levels of IL-6 and IL-8 compared to non-vitrectomized eyes (P < 0.05). (2) In vitrectomized group, after Benjamini-Hochberg correction, there was a significant positive correlation between the levels of VEGF and PlGF (rs = 0.855, P < 0.05), as well as the levels of TNF-α and IFN-γ (rs = 0.858, P < 0.05). In non-vitrectomized group, significant positive correlations were found between VEGF and PlGF levels after correcting for multiple comparisons (rs = 0.9, P < 0.05). (3) In non-vitrectomized group, the concentrations of VEGF and PlGF in aqueous humor were significantly positively correlated with CMT values (rs = 0.95, P < 0.05; rs = 0.9, P < 0.05, respectively). CONCLUSIONS: The concentrations of IL-6 and IL-8 in the aqueous humor were significantly higher in vitrectomized DME eyes compared to nonvitrectomized DME eyes and the levels of VEGF were similar in the two groups, suggesting that inflammation after vitrectomy may be a key factor in the occurrence and development of DME.

Worm-Based Diagnosis Combining Microfluidics toward Early Cancer Screening.

Early cancer diagnosis increases therapy efficiency and saves huge medical costs. Traditional blood-based cancer markers and endoscopy procedures demonstrate limited capability in the diagnosis. Reliable, non-invasive, and cost-effective methods are in high demand across the world. Worm-based diagnosis, utilizing the chemosensory neuronal system of C. elegans, emerges as a non-invasive approach for early cancer diagnosis with high sensitivity. It facilitates effectiveness in large-scale cancer screening for the foreseeable future. Here, we review the progress of a unique route of early cancer diagnosis based on the chemosensory neuronal system of C. elegans. We first introduce the basic procedures of the chemotaxis assay of C. elegans: synchronization, behavior assay, immobilization, and counting. Then, we review the progress of each procedure and the various cancer types for which this method has achieved early diagnosis. For each procedure, we list examples of microfluidics technologies that have improved the automation, throughput, and efficiency of each step or module. Finally, we envision that microfluidics technologies combined with the chemotaxis assay of C. elegans can lead to an automated, cost-effective, non-invasive early cancer screening technology, with the development of more mature microfluidic modules as well as systematic integration of functional modules.

Inhibition of Anaplastic Lymphoma Kinase Protects From Ischemic Stroke.

BACKGROUND: Ischemic stroke is often accompanied by oxidative stress and inflammatory response, both of which work synergistically to exacerbate the disruption of the blood-brain barrier and ischemic brain injury. ALK (anaplastic lymphoma kinase), a cancer-associated receptor tyrosine kinase, was found to play a role in oxidative stress and inflammation. In this study, we investigated the role of ALK inhibition in a murine model of ischemic stroke. METHODS: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery in mice with a filament. The ALK inhibitor alectinib was administered following the stroke. ALOX15 (arachidonic acid 15-lipoxygenase) was overexpressed by adenovirus injection. The immunohistochemistry, Western blot, oxidative stress, inflammation, blood-brain barrier leakage, infarct volume, and functional outcomes were determined. RESULTS: We found that the expression of ALK was markedly increased in the neurovascular unit after cerebral ischemia. Treatment with the ALK inhibitor alectinib reduced the accumulation of reactive oxygen species, lipid peroxidation, and oxidative DNA, increased the vascular levels of antioxidant enzymes, inactivated the vascular NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome pathway, and reduced vascular inflammation (ICAM-1 [intercellular adhesion molecule-1] and MCP-1 [monocyte chemoattractant protein-1]) after ischemia. Moreover, alectinib reduced the loss of cerebrovascular integrity and blood-brain barrier damage, consequently decreasing brain infarction and neurological deficits. Furthermore, alectinib reduced stroke-evoked ALOX15 expression, whereas virus-mediated overexpression of ALOX15 abolished alectinib-dependent inhibition of oxidative stress and vascular inflammation, blood-brain barrier protection, and neuroprotection, suggesting the protective effects of alectinib for stroke may involve ALOX15. CONCLUSIONS: Our findings demonstrated that alectinib protects from stroke by regulating ischemic signaling cascades and suggest that ALK may be a novel therapeutic target for ischemic stroke.

DeepCBS: shedding light on the impact of mutations occurring at CTCF binding sites.

CTCF-mediated chromatin loops create insulated neighborhoods that constrain promoter-enhancer interactions, serving as a unit of gene regulation. Disruption of the CTCF binding sites (CBS) will lead to the destruction of insulated neighborhoods, which in turn can cause dysregulation of the contained genes. In a recent study, it is found that CTCF/cohesin binding sites are a major mutational hotspot in the cancer genome. Mutations can affect CTCF binding, causing the disruption of insulated neighborhoods. And our analysis reveals a significant enrichment of well-known proto-oncogenes in insulated neighborhoods with mutations specifically occurring in anchor regions. It can be assumed that some mutations disrupt CTCF binding, leading to the disruption of insulated neighborhoods and subsequent activation of proto-oncogenes within these insulated neighborhoods. To explore the consequences of such mutations, we develop DeepCBS, a computational tool capable of analyzing mutations at CTCF binding sites, predicting their influence on insulated neighborhoods, and investigating the potential activation of proto-oncogenes. Futhermore, DeepCBS is applied to somatic mutation data of liver cancer. As a result, 87 mutations that disrupt CTCF binding sites are identified, which leads to the identification of 237 disrupted insulated neighborhoods containing a total of 135 genes. Integrative analysis of gene expression differences in liver cancer further highlights three genes: ARHGEF39, UBE2C and DQX1. Among them, ARHGEF39 and UBE2C have been reported in the literature as potential oncogenes involved in the development of liver cancer. The results indicate that DQX1 may be a potential oncogene in liver cancer and may contribute to tumor immune escape. In conclusion, DeepCBS is a promising method to analyze impacts of mutations occurring at CTCF binding sites on the insulator function of CTCF, with potential extensions to shed light on the effects of mutations on other functions of CTCF.

MACC: a visual interactive knowledgebase of metabolite-associated cell communications.

Metabolite-associated cell communications play critical roles in maintaining the normal biological function of human through coordinating cells, organs and physiological systems. Though substantial information of MACCs has been continuously reported, no relevant database has become available so far. To address this gap, we here developed the first knowledgebase (MACC), to comprehensively describe human metabolite-associated cell communications through curation of experimental literatures. MACC currently contains: (a) 4206 carefully curated metabolite-associated cell communications pairs involving 244 human endogenous metabolites and reported biological effects in vivo and in vitro; (b) 226 comprehensive cell subtypes and 296 disease states, such as cancers, autoimmune diseases, and pathogenic infections; (c) 4508 metabolite-related enzymes and transporters, involving 542 pathways; (d) an interactive tool with user-friendly interface to visualize networks of multiple metabolite-cell interactions. (e) overall expression landscape of metabolite-associated gene sets derived from over 1500 single-cell expression profiles to infer metabolites variations across different cells in the sample. Also, MACC enables cross-links to well-known databases, such as HMDB, DrugBank, TTD and PubMed etc. In complement to ligand-receptor databases, MACC may give new perspectives of alternative communication between cells via metabolite secretion and adsorption, together with the resulting biological functions. MACC is publicly accessible at: http://macc.badd-cao.net/.

An immune cell atlas reveals the dynamics of human macrophage specification during prenatal development.

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.

New genetic and epigenetic insights into the chemokine system: the latest discoveries aiding progression toward precision medicine.

Over the past thirty years, the importance of chemokines and their seven-transmembrane G protein-coupled receptors (GPCRs) has been increasingly recognized. Chemokine interactions with receptors trigger signaling pathway activity to form a network fundamental to diverse immune processes, including host homeostasis and responses to disease. Genetic and nongenetic regulation of both the expression and structure of chemokines and receptors conveys chemokine functional heterogeneity. Imbalances and defects in the system contribute to the pathogenesis of a variety of diseases, including cancer, immune and inflammatory diseases, and metabolic and neurological disorders, which render the system a focus of studies aiming to discover therapies and important biomarkers. The integrated view of chemokine biology underpinning divergence and plasticity has provided insights into immune dysfunction in disease states, including, among others, coronavirus disease 2019 (COVID-19). In this review, by reporting the latest advances in chemokine biology and results from analyses of a plethora of sequencing-based datasets, we outline recent advances in the understanding of the genetic variations and nongenetic heterogeneity of chemokines and receptors and provide an updated view of their contribution to the pathophysiological network, focusing on chemokine-mediated inflammation and cancer. Clarification of the molecular basis of dynamic chemokine-receptor interactions will help advance the understanding of chemokine biology to achieve precision medicine application in the clinic.

Competitive adsorption of lead and cadmium on soil aggregate at micro-interfaces: Multi-surface modeling and spectroscopic studies.

Aggregates are the basic structural units of soils and play a crucial role in metal migration and transformation. Combined contamination of lead (Pb) and cadmium (Cd) is common in site soils, and the two metals may compete for the same adsorption sites and affect their environmental behavior. Herein, the adsorption behavior of Pb and Cd on aggregates of two soils and contributions of soil components in single and competitive systems were studied by combining cultivation experiments, batch adsorption, multi-surface models (MSMs), and spectroscopic techniques. The results demonstrated that < 2 µm size aggregate was the dominant sink for Pb and Cd competitive adsorption in both soils. Compared with Pb, the adsorption capacity and behavior of Cd were affected greatly under competition. MSMs prediction revealed that soil organic matter (SOM) contributed the most to Cd and Pb adsorption on aggregates (> 68.4%), but the dominant competitive effect occurred on different sites for Cd adsorption (primarily on SOM) and Pb adsorption (primarily on clay minerals). Further, 2 mM Pb coexistence caused 5.9 - 9.8% of soil Cd conversion to unstable species (Cd(OH)2). Thus, the competitive effect of Pb on Cd adsorption cannot be ignored in soils with high content of SOM and fine aggregates.

Chang qing formula ameliorates colitis-associated colorectal cancer via suppressing IL-17/NF-κB/STAT3 pathway in mice as revealed by network pharmacology study.

Colitis-associated colorectal cancer (CAC) is a specific type of colorectal cancer (CRC) with high mortality and morbidity, the chronic inflammation in the intestinal mucosal is the characteristic of CAC. Chang Qing formula (CQF) is a Chinese herbal formula used clinically for the treatment of CAC with remarkable clinical efficacy, but its mechanism remains unclear. In the present work, Combined network pharmacology and transcriptomics were used to analyze the potential active ingredients and elucidate molecular mechanism of CQF in treating CAC. Firstly, the constituents migrating to blood of CQF were analyzed and identified by UPLC-Q-TOF-MS/MS, and core genes and pathways were screened by network pharmacology analysis. Encyclopedia of Genes and Genomes (KEGG) analysis showed that the IL-17 signaling pathway involved in CAC may be closely associated with the potential mechanismof action of CQF. Subsequently, the results from animal studies indicated that CQF profoundly reduced tumor numbers and tumor size in AOM/DSS mice. The RNA-seq data was analysed utilizing Ingenuity Pathway Analysis (IPA), and the results supported the idea that CQF exerts a tumour-suppressive effect via the IL-17 signalling pathway. Further studies demonstrated that CQF significantly reduced IL-17A levels, which in turn inhibited NF-κB/IL-6/STAT3 signaling cascade, suppressed MMP9 expression and promoted tumor cell apoptosis. In conclusion, the current study demonstrated that CQF remarkably improved inflammatory tumor microenvironment, and hindered the transformation of inflammation into cancer. These findings may help to design future strategies for the treatment of CAC.

It started with a Cys: Spontaneous cysteine modification during cryo-EM grid preparation.

Advances in single particle cryo-EM data collection and processing have seen a significant rise in its use. However, the influences of the environment generated through grid preparation, by for example interactions of proteins with the air-water interface are poorly understood and can be a major hurdle in structure determination by cryo-EM. Initial interactions of proteins with the air-water interface occur quickly and proteins can adopt preferred orientation or partially unfold within hundreds of milliseconds. It has also been shown previously that thin-film layers create hydroxyl radicals. To investigate the potential this might have in cryo-EM sample preparation, we studied two proteins, HSPD1, and beta-galactosidase, and show that cysteine residues are modified in a time-dependent manner. In the case of both HSPD1 and beta-galactosidase, this putative oxidation is linked to partial protein unfolding, as well as more subtle structural changes. We show these modifications can be alleviated through increasing the speed of grid preparation, the addition of DTT, or by sequestering away from the AWI using continuous support films. We speculate that the modification is oxidation by reactive oxygen species which are formed and act at the air-water interface. Finally, we show grid preparation on a millisecond timescale outruns cysteine modification, showing that the reaction timescale is in the range of 100s to 1,000s milliseconds and offering an alternative approach to prevent spontaneous cysteine modification and its consequences during cryo-EM grid preparation.

Novel insights into probabilistic health risk and source apportionment based on bioaccessible potentially toxic elements around an abandoned e-waste dismantling site.

The study of potentially toxic element (PTE) hazards around e-waste recycling areas has attracted increasing attention but does not consider elemental bioaccessibility. Here, the respiratory and oral bioaccessibilities were incorporated into probabilistic health risk evaluation and source contribution apportionment. The results showed that soil Cd yielded the highest respiratory and oral bioaccessibility, whereas Cr in soils and vegetables attained the lowest oral bioaccessibility. When incorporating metal bioaccessibility into health risk assessment, a 48.3%-55.7% overestimation of non-cancer and cancer risks can be avoided relative to the risk assessment based on the total concentrations of PTEs. More importantly, priority control metals were misidentified without consideration of bioaccessibility. Cadmium, As, and Cr were screened as the priority metal(loid)s for targeted risk control based on the total PTEs, whereas Cd, Zn, and Cu were the priority metal(loid)s based on the bioaccessible PTEs. Furthermore, source apportionment revealed that >50% of oral bioaccessible Cd, Cu, Ni, Pb, and Zn in farmland were contributed by e-waste dismantling activities, whereas bioaccessible As and Cr mainly originated from agrochemical applications and natural sources, respectively. This study emphasizes the refinement of risk estimation and source apportionment through metal bioaccessibility adjustment, which facilitates the realistic assessment of adverse health effects in humans and the precise identification of high-risk sources.

Greenhouse gas emission benefits of adopting new energy vehicles in Suzhou City, China: A case study.

The promotion of new energy in light-duty vehicles (LDVs) is considered as an effective approach for achieving low-carbon road transport targets. In this study, life cycle assessment was performed for five typical vehicle models in Suzhou City (fourth largest LDV stock in China): internal combustion engine vehicle (ICEV), hybrid electric vehicle (HEV), plug-in electric vehicle (PHEV), battery electric vehicle (BEV) and hydrogen fuel cell vehicle (HFCV). Their energy consumption, and greenhouse gas (GHG) and air pollutant emissions during vehicle and fuel cycles in 2020 were examined using the Greenhouse gases, Regulated Emissions, and Energy Use in Transportation (GREET) model. GHG emission reduction potential of LDV fleet was projected under various scenarios for 2021-2040. The results showed that BEVs exhibited advantages for replacing ICEVs over HEVs, PHEVs and HFCVs, taking into account China's road electrification policy. The GHG emission intensity of BEVs in 2040 was estimated to be 19-34% of ICEVs in 2020, with a deep decarbonized electricity mix and improved vehicle efficiency. For the aggressive Sustainable Development Scenario, the GHG emissions of LDVs would peak before 2026, ahead of China's target by 2030, and the ~ 100% share of EVs in 2040 would result in a lower GHG emissions, equivalent to the 2010 level. It highlights the importance of early action, green electricity mix, and public transport development in reducing GHG emissions of large LDV fleet.

Parkinsonism and dysautonomia with anti-CV2/CRMP5 associated paraneoplastic neurological syndromes mimicking multiple system atrophy: a case report.

BACKGROUND: Paraneoplastic neurological syndromes (PNSs) are broad-spectrum disorders that can affect any part of the nervous system varying in core symptoms. Onconeural antibodies, including Hu, Yo, Ri, anti-CV2, amphiphysin, Ma2, and Tr are well-characterized and commonly used for the diagnosis of definite PNS. Generally, anti-CV2 antibodies have usually been associated with cerebellar ataxia, chorea, peripheral and autonomic neuropathies, myelopathy, optic neuritis, and retinitis. However, Parkinsonism has not been reported as the core symptom in patients with anti-CV2 antibodies. CASE PRESENTATION: We report a patient with anti-CV2 antibody manifested as Parkinsonism and autonomic dysfunction, which may lead to the diagnosis of multiple system atrophy with predominant Parkinsonism (MSA-P). A lumbar puncture examination was undergone to find a positive anti-CV2 antibody in cerebrospinal fluid. PET-CT showed no tumor. Immunotherapy was adopted and the symptoms were relieved for 5 months. However, with no evidence of tumor, he died after 8 months. CONCLUSIONS: Our findings indicate that PNS with anti-CV2 antibody can be shown as MSA-P mimic. Considering that MSA is a neurodegenerative disease with a poor prognosis, screening for other treatable or controllable factors like PNS presented in this case is necessary when encountering a rapid progressive MSA-mimic patient.

Biological effects of exosome derived from Cal27 on normal human gingival fibroblasts.

OBJECTIVES: The proliferation, migration capacity, and expression of activation-related proteins of NHGFs+Cal27-exo were determined by coculturing Cal27 exosome (Cal27-exo) with normal human gingival fibroblasts (NHGFs) to explore the effects of Cal27-exo on the activation and biological behavior of NHGFs. METHODS: Cal27-exo was extracted using supercentrifugation, and exosomes were identified using Western blot, transmission electron microscopy (TEM), and particle size detection. Cal27-exo was cocultured with NHGFs to detect the uptake of Cal27-exo by NHGFs, and the proliferation and migration capacity of NHGFs+Cal27-exo were detected using CCK8 and wound healing tests, respectively. The expression levels of NHGF activation-related proteins, i.e., matrix metalloproteinase-9 (MMP-9), fibroblast-activating protein (FAP), alpha smooth muscle actin (αSMA), and transforming growth factor-ß (TGF-ß), were detected using real-time quantitative polymerase chain reaction (qRT-PCR). RESULTS: Cal27-exo was extracted u-sing supercentrifugation, and Western blot showed the positive expression levels of Alix and CD63. TEM showed that Cal27-exo had a circular double-layer vesicle. The particle size was between 30 and 150 nm. Cal27-exo labeled with PKH67 entered NHGFs after the coculture method. The wound healing test showed that the migration capacity of NHGFs+Cal27-exo was stronger after the scratch compared with that of NHGFs. CCK8 results showed that the proliferation activity of NHGFs+Cal27-exo was enhanced. qRT-PCR results showed that the MMP-9 levels of NHGFs+Cal27-exo were upregulated, whereas the TGF-ß and αSMA mRNA levels of NHGFs+Cal27-exo were downregulated (P<0.05). CONCLUSIONS: The proliferation and migration ability of NHGFs+Cal27-exo are enhanced, and the mRNA expression of related proteins is changed. Cal27-exo can activate NHGFs, which suggests that Cal27-exo has potential significance in tumor invasion and metastasis.

Investigation of genes associated with petal variations between diploid and autotetraploid in Chinese cabbage (Brassica rapa L. ssp. pekinensis) by RNA-seq and sRNA-seq.

Polyploidy promotes morphological, physiological, and reproductive diversity in plants. The imminent effect of chromosome doubling in plants is the enlargement of organs such as flowers and fruits, which increases the commercial value of crops. Flowering plays a vital role in the growth and development of angiosperms. Here, we prepared an isolated microspore culture of 'FT', a doubled haploid (DH) line of Chinese cabbage (Brassica rapa L. ssp. pekinensis), and obtained diploid and autotetraploid plants with the same genetic background. Compared with diploids, the autotetraploids were characterized by large floral organs, dark petals, delayed flowering, and reduced fertility. The indole-3-acetic acid (IAA) and jasmonic acid (JA) levels in autotetraploid petals were significantly higher and the abscisic acid (ABA) level was significantly lower than those in the diploid petals. The lutein level in autotetraploid petals was nearly two times higher than that in the diploid petals. A comparative transcriptome analysis revealed 14,412 differentially expressed genes (DEGs) between the diploids and autotetraploids, and they were enriched in 117 Gene Ontology terms and 110 Kyoto Encyclopedia of Genes and Genomes pathways. We detected 231 DEGs related to phytohormone signal transduction and 29 DEGs involved in carotenoid biosynthesis. An miRNA-target mRNA analysis showed that 32 DEGs regulated by 16 DEMs were associated with flowering timing (BraA03000336, BraA09004319, and BraA09000515), petal development (BraA05002408, BraA01004006, BraA09004069, and BraA04000966), flower opening (BraA07000350), and pollen development (BraA01000720, BraA09005727, and BraA01000253). This study provides information to help elucidate the molecular mechanisms underlying phenotypic variations induced by autopolyploidy in Chinese cabbage.

Mechanism and significance of apoptosis of the immortalized human oral mucosal epithelial cells established by Lentivirus-mediated hTERT.

During the transition from human oral mucosal epithelial cells (HOMEC) to oral squamous cell carcinoma cells (Cal27), the cells must have undergone a precancerous state. To explore the malignant rule of HOMEC, plv-HOMEC was used as a model cell for the precancerous state to investigate plv-HOMEC's apoptosis by comparing human oral mucosal epithelial cells established by Lentivirus-mediated hTERT (plv-HOMEC) with HOMEC and human Cal27. The lentiviral particles overexpressing hTERT were packaged and transfected into primary HOMEC to obtain plv-HOMEC. Expression levels of NF-κB were detected in the cytoplasm and nucleus of Cal27, plv-HOMEC and HOMEC. The level of intracellular reactive oxygen species was measured to verify the endoplasmic reticulum pathway, cytochrome C expression was detected to verify the mitochondrial pathway, and FasL gene expression was detected to verify the death receptor apoptosis pathway. The total expression of NF-κB in plv-HOMEC increased, mainly due to the greater nuclear import of NF-κB, but it was still much lower than Cal27. The endoplasmic reticulum apoptosis pathway of plv-HOMEC was not significantly affected, and there were no significant differences between them and the HOMEC cells; the mitochondrial apoptosis pathway of plv-HOMEC was inhibited, and the expression of Cyt C was very close to that of Cal27, indicating that the characteristics of plv-HOMEC are so familiar with cancer cells; the death receptor apoptosis pathway of plv-HOMEC was also inhibited, and in this apoptotic pathway, plv-HOMEC were more similar to cancer cells than to HOMEC cells. The present data suggest that NF-κB nucleation may increase in the early stage of healthy cells' carcinogenesis, followed by inhibition of the mitochondrial pathway and the death receptor apoptotic pathway.

Study on the Role of Salivary Flora and NF-κB Inflammatory Signal Pathway in Oral Lichen Planus.

Oral lichen planus (OLP) is an inflammatory disease. It is believed that infection and immune dysfunction play a key role in its pathogenesis, but the specific mechanism of action remains unclear. The 16s rRNA high-throughput sequencing technique was used to analyze the microbial flora structure in the saliva of OLP patients and healthy controls. The relative abundance of Derxia, Haemophilus, and Pseudomonas in the saliva of the OLP group was lower than that of the healthy control group, but there was no significant difference in the overall structure of the microbial population. In addition, we measured the protein expression levels of toll-like receptor 4 (TLR4) and nuclear factor-kappab p65 (NF-κB p65) in the tissues of OLP patients, and found that there was a significant increase and positive correlation between them (r = 0.907, P = 0.034). The expression levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in the OLP group were consistent with those of NF-κB p65. Therefore, we believe that changes in the composition ratio of microbialflora break the original balance state of flora, promote the occurrence of immune inflammatory reaction, and then lead to the generation or aggravation of OLP disease. This discovery provides new ideas for further research on OLP initiation and immune regulation mechanism.

Changes of DNA repair gene methylation in blood of chronic fluorosis patients and rats.

To investigate the relationship between DNA repair gene methylation and chronic coal-burning fluorosis. The methylation rates of O6-methylguanine-DNA- methyltransferase gene MGMT, a DNA repair gene and mismatch repair gene MutL homolog 1 (MLH1) were analysed by methylation of specific PCR (MSP), and the levels of mRNA in the blood of the chronic fluorosis rats and the patients in the region of endemic coal-burning fluorosis were determined by real-time PCR. The levels of mRNA and protein of MGMT and MLH1 in the liver tissue of the chronic fluorosis rats were determined by real-time PCR and Western blot respectively. The results showed an increased methylation of the MGMT and MLH1 genes in the blood of the patients in the fluorosis region that correlated positively with the severity of fluorosis. The mRNA levels of MGMT and MLH1 genes from the patients in fluorosis region were lower than those of a control group, and also showed a positive correlation with the severity of fluorosis. Both the protein and mRNA levels of MGMT and MLH1 genes from the blood of rats and liver tissue in a fluoride-treated group were lower than those of a control non-fluoride treated group. These results indicate that the degree of methylation of MGMT and MLH1 genes is altered in fluorosis disease, the resulting changed expression of these repair genes may play a role in the liver damage caused by fluoride.

Changes in endogenous phytohormones regulated by microRNA-target mRNAs contribute to the development of Dwarf Autotetraploid Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Polyploidization is considered as the major force that drives plant species evolution and biodiversity. The leaves of Chinese cabbage, an important vegetable crop valued for its nutritional quality, constitute the main edible organ. In this study, we found that autotetraploid Chinese cabbage (Brassica rapa ssp. pekinensis) generated from a doubled haploid (DH) line via isolated microspore culture exhibits a dwarf phenotype, along with thick leaves and delayed flowering. Abscisic acid (ABA) and brassinosteroid (BR) levels were significantly lower in autotetraploids compared to DHs. Comparative transcriptome analysis was performed to examine the gene regulatory network. A total of 13,225 differentially expressed genes (DEGs) were detected. Further microRNA (miRNA) analysis identified 102 DEGs that correspond to 35 differentially expressed miRNAs (DEMs). Subsequent screening of these 102 genes identified 13 key genes with 12 corresponding differentially expressed miRNAs that are related to leaf development and dwarfism. These 13 genes are involved in the regulation of various processes, including BR synthesis (dwarfing), plant growth, flowering time delay, ABA pathway-related growth and metabolism, leaf morphology and development, and cell extension. Two dwarfing-related genes (BraA01000252 and BraA05004386) regulated by two miRNAs (novel_15 and novel_54) were determined to be downregulated, indicating their possible role in leaf thickness and dwarfism in autotetraploid plants. We also propose two possible miRNA-dependent regulatory pathways that contribute to trait formation in autotetraploid Chinese cabbage. These results provide a theoretical basis for further work involving Chinese cabbage varieties by inducing polyploidy.

A missense mutation of plastid RPS4 is associated with chlorophyll deficiency in Chinese cabbage (Brassica campestris ssp. pekinensis).

BACKGROUND: Plastome mutants are ideal resources for elucidating the functions of plastid genes. Numerous studies have been conducted for the function of plastid genes in barley and tobacco; however, related information is limited in Chinese cabbage. RESULTS: A chlorophyll-deficient mutant of Chinese cabbage that was derived by ethyl methanesulfonate treatment on isolated microspores showed uniformly pale green inner leaves and slow growth compared with that shown by the wild type "Fukuda 50' ('FT'). Genetic analysis revealed that cdm was cytoplasmically inherited. Physiological and ultrastructural analyses of cdm showed impaired photosynthesis and abnormal chloroplast development. Utilizing next generation sequencing, the complete plastomes of cdm and 'FT' were respectively re-mapped to the reference genome of Chinese cabbage, and an A-to-C base substitution with a mutation ratio higher than 99% was detected. The missense mutation of plastid ribosomal protein S4 led to valine substitution for glycine at residue 193. The expression level of rps4 was analyzed using quantitative real-time PCR and found lower in than in 'FT'. RNA gel-blot assays showed that the abundance of mature 23S rRNA, 16S rRNA, 5S rRNA, and 4.5S rRNA significantly decreased and that the processing of 23S, 16S rRNA, and 4.5S rRNA was seriously impaired, affecting the ribosomal function in cdm. CONCLUSIONS: These findings indicated that cdm was a plastome mutant and that chlorophyll deficiency might be due to an A-to-C base substitution of the plastome-encoded rps4 that impaired the rRNA processing and affected the ribosomal function.