Chemotaxis Pathway Research Articles

Inflammatory Endotype-Associated Airway Resistome in Chronic Obstructive Pulmonary Disease.

ABSTRACTAntimicrobial resistance is a global concern in chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD). The collection of antibiotic resistance genes or resistome in human airways may underlie the resistance. COPD is heterogeneous, and understanding the airway resistome in relation to patient phenotype and endotype may inform precision antibiotic therapy. Here, we characterized the airway resistome for 94 COPD participants at stable disease. Among all demographic and clinical factors, patient inflammatory endotype was associated with the airway resistome. There were distinct resistome profiles between patients with neutrophilic or eosinophilic inflammation, two primary inflammatory endotypes in COPD. For neutrophil-predominant COPD, the resistome was dominated by multidrug resistance genes. For eosinophil-predominant COPD, the resistome was diverse, with an increased portion of patients showing a macrolide-high resistome. The differential antimicrobial resistance pattern was validated by sputum culture and in vitro antimicrobial susceptibility testing. Ralstonia and Pseudomonas were the top contributors to the neutrophil-associated resistome, whereas Campylobacter and Aggregatibacter contributed most to the eosinophil-associated resistome. Multiomic analyses revealed specific host pathways and inflammatory mediators associated with the resistome. The arachidonic acid metabolic pathway and matrix metallopeptidase 8 (MMP-8) exhibited the strongest associations with the neutrophil-associated resistome, whereas the eosinophil chemotaxis pathway and interleukin-13 (IL-13) showed the greatest associations with the eosinophil-associated resistome. These results highlight a previously unrecognized link between inflammation and the airway resistome and suggest the need for considering patient inflammatory subtype in decision-making about antibiotic use in COPD and broader chronic respiratory diseases.IMPORTANCE Antibiotics are commonly prescribed for both acute and long-term prophylactic treatment in chronic airway disorders, such as chronic obstructive pulmonary disease (COPD), and the rapid growth of antibiotic resistance is alarming globally. The airway harbors a diverse collection of microorganisms known as microbiota, which serve as a reservoir for antibiotic resistance genes or the resistome. A comprehensive understanding of the airway resistome in relation to patient clinical and biological factors may help inform decisions to select appropriate antibiotics for clinical therapies. By deep multiomic profiling and in vitro phenotypic testing, we showed that inflammatory endotype, the underlying pattern of airway inflammation, was most strongly associated with the airway resistome in COPD patients. There were distinct resistome profiles between neutrophil-predominant and eosinophil-predominant COPD that were associated with different bacterial species, host pathways, and inflammatory markers, highlighting the need of considering patient inflammatory status in COPD antibiotic management.

CX3CR1-Expressing Myeloid Cells Regulate Host-Helminth Interaction and Lung Inflammation.

Many helminth life cycles, including hookworm, involve a mandatory lung phase, where myeloid and granulocyte subsets interact with the helminth and respond to infection-induced lung injury. To evaluate these innate subsets in Nippostrongylus brasiliensis infection, reporter mice for myeloid cells (CX3CR1GFP ) and granulocytes (PGRPdsRED ) are employed. Nippostrongylus infection induces lung infiltration of reporter cells, including CX3CR1+ myeloid cells and PGRP+ eosinophils. Strikingly, CX3CR1GFP/GFP mice, which are deficient in CX3CR1, are protected from Nippostrongylus infection with reduced weight loss, lung leukocyte infiltration, and worm burden compared to CX3CR1+/+ mice. This protective effect is specific for CX3CR1 as CCR2-deficient mice do not exhibit reduced worm burdens. Nippostrongylus co-culture with lung Ly6C+ monocytes or CD11c+ cells demonstrates that CX3CR1GFP/GFP monocytes secrete more pro-inflammatory cytokines and actively bind the parasites causing reduced motility. RNA sequencing of Ly6C+ or CD11c+ cells shows Nippostrongylus-induced gene expression changes, particularly in monocytes, associated with inflammation, chemotaxis, and extracellular matrix remodeling pathways. Analysis reveals cytotoxic and adhesion molecules as potential effectors against the parasite, such as Gzma and Gzmb, which are elevated in CX3CR1GFP/GFP monocytes. These studies validate a dual innate cell reporter for lung helminth infection and demonstrate that CX3CR1 impairs monocyte-helminth interaction.

Hyperactive neutrophil chemotaxis contributes to anti-tumor necrosis factor-α treatment resistance in inflammatory bowel disease.

Background and AimAnti‐tumor necrosis factor‐α (anti‐TNF‐α) agents have been used for inflammatory bowel disease; however, it has up to 30% nonresponse rate. Identifying molecular pathways and finding reliable diagnostic biomarkers for patient response to anti‐TNF‐α treatment are needed.MethodsPublicly available transcriptomic data from inflammatory bowel disease patients receiving anti‐TNF‐α therapy were systemically collected and integrated. In silico flow cytometry approaches and Metascape were applied to evaluate immune cell populations and to perform gene enrichment analysis, respectively. Genes identified within enrichment pathways validated in neutrophils were tracked in an anti‐TNF‐α‐treated animal model (with lipopolysaccharide‐induced inflammation). The receiver operating characteristic curve was applied to all genes to identify the best prediction biomarkers.ResultsA total of 449 samples were retrieved from control, baseline, and after primary anti‐TNF‐α therapy or placebo. No statistically significant differences were observed between anti‐TNF‐α treatment responders and nonresponders at baseline in immune microenvironment scores. Neutrophil, endothelial cell, and B‐cell populations were higher in baseline nonresponders, and chemotaxis pathways may contribute to the treatment resistance. Genes related to chemotaxis pathways were significantly upregulated in lipopolysaccharide‐induced neutrophils, but no statistically significant changes were observed in neutrophils treated with anti‐TNF‐α. Interleukin 13 receptor subunit alpha 2 (IL13RA2) is the best predictor (receiver operating characteristic curve: 80.7%, 95% confidence interval: 73.8–87.5%), with a sensitivity of 68.13% and specificity of 84.93%, and significantly higher in nonresponders compared with responders (P < 0.0001).ConclusionsHyperactive neutrophil chemotaxis influences responses to anti‐TNF‐α treatment, and IL13RA2 is a potential biomarker to predict anti‐TNF‐α treatment response.

Effect of Different Initial Fermentation pH on Exopolysaccharides Produced by Pseudoalteromonas agarivorans Hao 2018 and Identification of Key Genes Involved in Exopolysaccharide Synthesis via Transcriptome Analysis.

Exopolysaccharides (EPSs) are carbohydrate polymers produced and secreted by microorganisms. In a changing marine environment, EPS secretion can reduce damage from external environmental disturbances to microorganisms. Meanwhile, EPSs have promising application prospects in the fields of food, cosmetics, and pharmaceuticals. Changes in external environmental pH have been shown to affect the synthesis of EPSs in microorganisms. In this study, we analyzed the effects of different initial fermentation pHs on the production, monosaccharide composition, and antioxidant activity of the EPSs of Pseudoalteromonas agarivorans Hao 2018. In addition, the transcriptome sequence of P. agarivorans Hao 2018 under different initial fermentation pH levels was determined. GO and KEGG analyses showed that the differentially expressed genes were concentrated in the two-component regulatory system and bacterial chemotaxis pathways. We further identified the expression of key genes involved in EPS synthesis during pH changes. In particular, the expression of genes encoding the glucose/galactose MFS transporter, phosphomannomutase, and mannose-1-phosphate guanylyltransferase was upregulated when the environmental pH increased, thus promoting EPS synthesis. This study not only contributes to elucidating the environmental adaptation mechanisms of P. agarivorans, but also provides important theoretical guidance for the directed development of new products using biologically active polysaccharides.

Transcriptomics and proteomics revealed the psychrotolerant and antibiotic-resistant mechanisms of strain Pseudomonas psychrophila RNC-1 capable of assimilatory nitrate reduction and aerobic denitrification

Aerobic denitrification has been proved to be profoundly affected by temperature and antibiotics, but little is known about how aerobic denitrifiers respond to temperature and antibiotic stress. In this study, the nitrate reduction performance and the intracellular metabolism by a psychrotolerant aerobic denitrifying bacteria, named Pseudomonas psychrophila RNC-1, were systematically investigated at different temperatures (10 °C, 20 °C, 30 °C) and different sulfamethoxazole (SMX) concentrations (0 mg/L, 0.1 mg/L, 0.5 mg/L, 1.0 mg/L, and 5.0 mg/L). The results showed that strain RNC-1 performed satisfactory nitrate removal at 10 °C and 20 °C, but its growth was significantly inhibited at 30 °C. Nitrate removal by strain RNC-1 was slightly promoted in the presence of 0.5 mg/L SMX, whereas it was significantly suppressed with 5.0 mg/L SMX. Nitrogen balance analysis indicated that assimilatory nitrate reduction and dissimilatory aerobic denitrification jointly dominated in the nitrate removal process of strain RNC-1, in which the inhibition effected on assimilation process was much higher than that on the aerobic denitrification process under SMX exposure. Further transcriptomics and proteomics analysis revealed that the psychrotolerant mechanism of strain RNC-1 could be attributed to the up-regulation of RNA translation, energy metabolism, ABC transporters and the over-expression of cold shock proteins, while the down-regulation of oxidative phosphorylation pathway was the primary reason for the deteriorative cell growth at 30 °C. The promotion of nitrate reduction with 0.5 mg/L SMX was related to the up-regulation of amino acid metabolism pathways, while the down-regulation of folate cycle, glycolysis/gluconeogenesis and bacterial chemotaxis pathways were responsible for the inhibition effect at 5.0 mg/L SMX. This work provides a mechanistic understanding of the metabolic adaption of strain RNC-1 under different stress, which is of significance for its application in nitrogen contaminated wastewater treatment processes.

Interactions between Salmonella Enteritidis and food processing facility isolate Bacillus paramycoides B5 in dual-species biofilms

Foodborne pathogens tend to live in mixed-species biofilms with resident flora in the food industry, likely posing a higher risk of pathogen contamination due to the strong biofilm formation and disinfectant resistance. The behavior of Salmonella Enteritidis (SE) in the presence of the food-processing environmental bacterium Bacillus paramycoides B5 (Bp5) during dual-species biofilm formation were investigated by Illumina RNA-seq transcriptome analysis combined with phenotype validation. The results showed that SE initial adhesion was significantly enhanced with large microcolony formation when cocultured with Bp5. The cocultures formed more extracellular polymeric substances compared to SE mono-species. Transcriptome analysis showed that a total of 433 differentially expressed genes (DEGs) of SE were present in dual-species biofilms. Functional annotation revealed that the genes involved in bacterial chemotaxis, flagellar assembly, and two-component system pathways were significantly upregulated. Genes involved in amino acid biosynthesis and metabolism, oxidative phosphorylation, and the citrate cycle were downregulated. Selected genes belonging to SE biofilm formation and environmental resistance were significantly upregulated with RT-qPCR validation. Flagellar-mediated motility was enhanced. This study explored the underlying mechanisms of enhanced biofilm formation and environmental resistance of SE in dual-species biofilms, which may help reveal or resolve SE persistence in food processing environments.

Ag NCs as a potent antibiofilm agent against pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii and drug-resistant Bacillus subtilis by affecting chemotaxis and flagellar assembly pathway genes.

Biofilm infections are highly resistant to commercial antibiotics. Therefore, developing a potent agent against such drug-resistant bacterial infections is highly desirable. Here, we synthesized positively charged silver nanoclusters (Ag NCs) with a diameter of <2 nm, which were found to be very effective antibacterial and antibiofilm agents against tetracycline-resistant Bacillus subtilis and most importantly multidrug-resistant pathogenic strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Ag NCs were able to both prevent and eradicate the biofilm formation very effectively. The antibiofilm activity can be significantly increased with α-amylase and/or DNase which degrade the structural components of biofilms. The antibiofilm activity of antibiotics gets considerably lowered due to poor penetration and the acidic microenvironment of biofilms. However, the potency of antibiotics gets significantly increased when applied with Ag NCs. Finally, RNA seq-based analysis has demonstrated that the biofilm degradation was likely due to the regulation of bacterial chemotaxis and flagellar assembly pathway genes by Ag NCs.

Phylogenetic Analysis with Prediction of Cofactor or Ligand Binding for Pseudomonas aeruginosa PAS and Cache Domains.

ABSTRACTPAS domains are omnipresent building blocks of multidomain proteins in all domains of life. Bacteria possess a variety of PAS domains in intracellular proteins and the related Cache domains in periplasmic or extracellular proteins. PAS and Cache domains are predominant in sensory systems, often carry cofactors or bind ligands, and serve as dimerization domains in protein association. To aid our understanding of the wide distribution of these domains, we analyzed the proteome of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 in silico. The ability of this bacterium to survive under different environmental conditions, to switch between planktonic and sessile/biofilm lifestyle, or to evade stresses, notably involves c-di-GMP regulatory proteins or depends on sensory pathways involving multidomain proteins that possess PAS or Cache domains. Maximum likelihood phylogeny was used to group PAS and Cache domains on the basis of amino acid sequence. Conservation of cofactor- or ligand-coordinating amino acids aided by structure-based comparison was used to inform function. The resulting classification presented here includes PAS domains that are candidate binders of carboxylic acids, amino acids, fatty acids, flavin adenine dinucleotide (FAD), 4-hydroxycinnamic acid, and heme. These predictions are put in context to previously described phenotypic data, often generated from deletion mutants. The analysis predicts novel functions for sensory proteins and sheds light on functional diversification in a large set of proteins with similar architecture.IMPORTANCE To adjust to a variety of life conditions, bacteria typically use multidomain proteins, where the modular structure allows functional differentiation. Proteins responding to environmental cues and regulating physiological responses are found in chemotaxis pathways that respond to a wide range of stimuli to affect movement. Environmental cues also regulate intracellular levels of cyclic-di-GMP, a universal bacterial secondary messenger that is a key determinant of bacterial lifestyle and virulence. We study Pseudomonas aeruginosa, an organism known to colonize a broad range of environments that can switch lifestyle between the sessile biofilm and the planktonic swimming form. We have investigated the PAS and Cache domains, of which we identified 101 in 70 Pseudomonas aeruginosa PAO1 proteins, and have grouped these by phylogeny with domains of known structure. The resulting data set integrates sequence analysis and structure prediction to infer ligand or cofactor binding. With this data set, functional predictions for PAS and Cache domain-containing proteins are made.

Escherichia coli chemotaxis is information limited.

Organisms acquire and use information from their environment to guide their behaviour. However, it is unclear whether this information quantitatively limits their behavioural performance. Here, we relate information to the ability of Escherichia coli to navigate up chemical gradients, the behaviour known as chemotaxis. First, we derive a theoretical limit on the speed with which cells climb gradients, given the rate at which they acquire information. Next, we measure cells' gradient-climbing speeds and the rate of information acquisition by their chemotaxis signaling pathway. We find that E. coli make behavioural decisions with much less than the one bit required to determine whether they are swimming up-gradient. Some of this information is irrelevant to gradient climbing, and some is lost in communication to behaviour. Despite these limitations, E. coli climb gradients at speeds within a factor of two of the theoretical bound. Thus, information can limit the performance of an organism, and sensory-motor pathways may have evolved to efficiently use information acquired from the environment.

Involvement of the Heat Shock Protein HtpG of Salmonella Typhimurium in Infection and Proliferation in Hosts.

Salmonella Typhimurium is a common pathogen infecting the gastrointestinal tract of humans and animals, causing host gastroenteritis and typhoid fever. Heat shock protein (HtpG) as a molecular chaperone is involved in the various cellular processes of bacteria, especially under environmental stress. However, the potential association of HtpG with S. Typhimurium infection remains unknown. In this study, we clarified that HtpG could also play a role as an effector in S. Typhimurium infection. RNA-seq indicated that the flagellar assembly pathway, infection pathway, and chemotaxis pathway genes of S. Typhimurium were downregulated after the mutation of HtpG, which resulted in compromises of S. Typhimurium motility, biofilm formation, adhesion, invasion, and inflammation-inducing ability. In addition, HtpG recombinant protein was capable of promoting the proliferation of S. Typhimurium in host cells and the resultant inflammation. Collectively, our results illustrated an important role of HtpG in S. Typhimurium infection.

Integrated plasma proteomics and lung transcriptomics reveal novel biomarkers in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal lung disease with a significant unmet medical need. Development of transformational therapies for IPF is challenging in part to due to lack of robust predictive biomarkers of prognosis and treatment response. Importantly, circulating biomarkers of IPF are limited and none are in clinical use.MethodsWe previously reported dysregulated pathways and new disease biomarkers in advanced IPF through RNA sequencing of lung tissues from a cohort of transplant-stage IPF patients (n = 36) in comparison to normal healthy donors (n = 19) and patients with acute lung injury (n = 11). Here we performed proteomic profiling of matching plasma samples from these cohorts through the Somascan-1300 SomaLogics platform.ResultsComparative analyses of lung transcriptomic and plasma proteomic signatures identified a set of 34 differentially expressed analytes (fold change (FC) ≥ ± 1.5, false discovery ratio (FDR) ≤ 0.1) in IPF samples compared to healthy controls. IPF samples showed strong enrichment of chemotaxis, tumor infiltration and mast cell migration pathways and downregulated extracellular matrix (ECM) degradation. Mucosal (CCL25 and CCL28) and Th2 (CCL17 and CCL22) chemokines were markedly upregulated in IPF and highly correlated within the subjects. The mast cell maturation chemokine, CXCL12, was also upregulated in IPF plasma (fold change 1.92, FDR 0.006) and significantly correlated (Pearson r = − 0.38, p = 0.022) to lung function (%predicted FVC), with a concomitant increase in the mast cell Tryptase, TPSB2. Markers of collagen III and VI degradation (C3M and C6M) were significantly downregulated (C3M p < 0.001 and C6M p < 0.0001 IPF vs control) and correlated, Pearson r = 0.77) in advanced IPF consistent with altered ECM homeostasis.ConclusionsOur study identifies a panel of tissue and circulating biomarkers with clinical utility in IPF that can be validated in future studies across larger cohorts.

The contributions of fliG gene to the pathogenicity of Pseudomonas plecoglossicida and pathogen-host interactions with Epinephelus coioides

Pseudomonas plecoglossicida is a Gram-negative aerobic rod-shaped bacterium with polar flagella. It is the causative agent of visceral white spot disease in cultured fish, resulting in serious economic losses. In our previous study, RNA sequencing showed that the expression of the fliG gene in P. plecoglossicida is significantly up-regulated during infection of orange-spotted grouper (Epinephelus coioides). In this study, four P. plecoglossicida RNA interference (RNAi) mutants were successfully constructed by linking four short hairpin RNAs (shRNAs), which target different sites of the fliG gene, to pCM130/tac, respectively. The mRNA expression levels of the fliG gene in P. plecoglossicida were significantly decreased in four mutants. The shRNA-335 mutant (fliG-RNAi strain) showed the best silencing efficiency (88.2%) and was thus chosen for further analysis. Electron microscopy indicated that the flagella of the fliG-RNAi strain of P. plecoglossicida were shorter and finer than those of the wild type strain. The fliG-RNAi strain also showed significantly decreased mobility, chemotaxis, adhesion, and biofilm formation. Furthermore, compared with wild type strain infection, E. coioides infected with the fliG-RNAi strain exhibited a 0.5-d delay in the time of first death and 55% reduction in accumulated mortality, as well as milder splenic symptoms. RNAi of the fliG gene significantly affected the transcriptomes of both pathogen and host in the infected spleens of E. coioides. KEGG analysis revealed that the flagellar assembly pathway, bacterial chemotaxis pathway, and starch and sucrose metabolism pathway were significantly enriched in the pathogen at 3 days post infection (dpi). In contrast, the complement and coagulation cascade pathway and antigen processing and presentation pathway were significantly enriched in the host at 3 dpi. More immune-related pathways were enriched at 5 dpi and more differentially expressed genes were found in the complement and coagulation cascade and antigen processing and presentation pathways. Cytokine-cytokine receptor interaction, hematopoietic cell lineage, and IgA-producing intestinal immune network pathways were significantly enriched in the host at 5 dpi. These results indicate that fliG is an important virulence gene of P. plecoglossicida and contributes to the pathogenicity of P. plecoglossicida as well as pathogen-host interactions with E. coioides.

Insights from label free-based proteomic analysis into inhibitory effects ε-Poly-lysine against Vibrio parahaemolyticus

Vibrio parahaemolyticus is one of the most common pathogenic bacteria that pose a threat to human health. The purpose of this study was to investigate antibacterial mechanisms of ε-poly-lysine (ε-PL) against V. parahaemolyticus using a lable free-based proteomic analysis. The differentially expressed proteins (DEPs) were subjected to bioinformatics analysis. The results indicated that a total of 196 DEPs, including 118 up-regulated and 78 down-regulated, were identified in the ε-PL-treated cells compared with control group. Upon Go functional enrichment, 13, 9, and 8 specific Go terms in biological processes, molecular functions and cellular components were identified, respectively. KEGG pathways analysis indicated that the DEPs were mainly involved in bacterial chemotaxis, RNA transport and two-component system, which were significantly enriched (P < 0.05). In PPI analysis, Che R and Che V, both involved in bacterial chemotaxis and RNA transport pathways, are closely related to other DEPs. Therefore, the down-regulation of Che R and Che V in ε-PL-treated cells resulted in the reduction or even loss of bacterial adaptability, and they were the critical action sites of ε-PL to inactivate V. parahaemolyticus.

Hfq, a global regulator contributes to the virulence of Vibrio splendidus AJ01

Hfq acts as an RNA-binding protein that ubiquitously exists in bacteria and contributes to global regulation of a wide range of gene expression. Vibrio splendidus is an important pathogen of sea cucumber Apostichopus japonicus, therefore, an hfq gene mutant strain, Δhfq, was constructed to explore its regulation on the virulence of V. splendidus. Our result showed that both the growth and virulence of Δhfq were reduced. To further reveal the pathways regulated by Hfq, the difference in transcriptomics between wild type and Δhfq was analyzed. Results showed that the expression of more than 1/8 of the total genes was affected by Hfq. KEGG analysis showed that the differentially expressed genes in Δhfq were enriched mainly in the bacterial secretion system, quorum sensing system, bacterial biofilm formation and flagellar assembly system, which were commonly related to virulence. Six genes in chemotaxis pathway (cheA, cheB, cheR, cheV, cheY and mcp) and twelve genes in flagellar assembly pathway (flgK, flgF, flgI, fliD, fliC, flgE, flgD, motY, flgG, flgB, flgC and fliS) were significantly down-regulated in Δhfq. Consistent with the transcriptomic analysis, the flagellum of Δhfq was almost undetectable under transmission electron microscopy. The main virulence-related factors of adhesion ability, biofilm formation and swimming motility that were regulated by chemotaxis and flagellar assembly were all attenuated in Δhfq. In conclusion, Hfq contributes to the virulence of V. splendidus AJ01 mainly through its positive regulation of adhesion, biofilm formation, and swimming motility via flagellar assembly system and chemotaxis system.

High-throughput screening of circRNAs reveals novel mechanisms of tuberous sclerosis complex-related renal angiomyolipoma

ObjectiveTuberous sclerosis complex (TSC) is a rare autosomal dominant disease characterized by lesions throughout the body. Our previous study showed the abnormal up-regulation of miRNAs plays an important part in the pathogenesis of TSC-related renal angiomyolipoma (TSC-RAML). circRNAs were known as important regulators of miRNA, but little is known about the circRNAs in TSC-RAMLs.MethodsMicroarray chips and RNA sequencing were used to identify the circRNAs and mRNAs that were differently expressed between the TSC-RAML and normal kidney tissue. A competitive endogenous RNA (ceRNA) regulatory network was constructed to reveal the regulation of miRNAs and mRNAs by the circRNAs. The biological functions of circRNA and mRNA were analyzed by pathway analysis. Microenvironmental cell types were estimated with the MCP-counter package.ResultsWe identified 491 differentially expressed circRNAs (DECs) and 212 differentially expressed genes (DEGs), and 6 DECs were further confirmed by q-PCR. A ceRNA regulatory network which included 6 DECs, 5 miRNAs, and 63 mRNAs was established. Lipid biosynthetic process was significantly up-regulated in TSC-RAML, and the humoral immune response and the leukocyte chemotaxis pathway were found to be down-regulated. Fibroblasts are enriched in TSC-RAML, and the up-regulation of circRNA_000799 and circRNA_025332 may be significantly correlated to the infiltration of the fibroblasts.ConclusioncircRNAs may regulate the lipid metabolism of TSC-RAML by regulation of the miRNAs. Fibroblasts are enriched in TSC-RAMLs, and the population of fibroblast may be related to the alteration of circRNAs of TSC-RAML. Lipid metabolism in fibroblasts is a potential treatment target for TSC-RAML.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Hepatic toxicity of fluorene-9-bisphenol (BHPF) on CD-1 mice

Fluorene-9-bisphenol (BHPF), a substitute for bisphenol A (BPA), has been widely used in the synthesis of polyester polymers. Studies have reported multiple BHPF toxicities but its effect on the liver remains unknown. In this study, we performed short-term and subchronic toxicity tests, as well as primary hepatocyte experiments, to investigate the hepatic toxicity of BHPF using CD-1 mice. And microarray was used to analyze the changes of global gene expression in the liver of mice treated with BHPF. The results showed that the liver coefficient and the activities of serum aminotransferases were obviously elevated by BHPF at doses of 27.8 mg/kg body weight (bw)/day or higher in mice treated for 10 days. Histological analysis showed obvious changes, including narrowed hepatic sinuses, dilated central vein, leucocyte infiltration, and cytoplasmic vacuolation, in the livers of mice treated with BHPF at dosages of 2 mg/kg bw/3-day and higher for 36 days. Microarray analyses revealed 2623 differentially expressed genes (DEGs) in the livers of mice treated with 50 mg/kg bw/day of BHPF for 3 days, which could be enriched in GO terms of T cell activation, leukocyte migration, and leukocyte chemotaxis and KEGG pathways of natural killer cell-mediated cytotoxicity and autoimmune thyroid disease. The top 10 hub DEGs, including LTF and MMP8, were observed in the protein-protein interaction network obtained via STRING database analysis, and are proposed as potential biomarkers for liver injury studies. Primary hepatocyte experiments demonstrated the hepatotoxicity of BHPF at concentrations of 10−6 M and higher. This study indicates that BHPF could cause liver injury at relatively low levels, suggesting that the risk of human BHPF exposure should be of concern.

Protein expression and purification of G-protein coupled receptor kinase 6 (GRK6), toward structure-based drug design and discovery for multiple myeloma

Human G-protein coupled receptor kinase 6 (GRK6) belongs to the GRK4 kinase subfamily of the G protein-coupled receptor kinase family which comprises of GRK1, GRK2, and GRK4. These kinases phosphorylate ligand-activated G-protein coupled receptors (GPCRs), driving heterotrimeric G protein coupling, desensitization of GPCR, and β-arrestin recruitment. This reaction series mediates cellular signal pathways for cell survival, proliferation, migration and chemotaxis. GRK6 is a kinase target in multiple myeloma since it is highly expressed in myeloma cells compared to epithelial cells and has a significant role in mediating the chemotactic responses of T and B-lymphocytes. To support structure-based drug design, we describe three human GRK6 constructs, GRK6, GRK6His/EK, and GRK6His/TEV, designed for protein expression in Spodoptera frugiperda Sf9 insect cells. The first construct did not contain any purification tag whereas the other two constructs contained the His10 affinity tag, which increased purification yields. We report here that all three constructs of GRK6 were overexpressed in Sf9 insect cells and purified to homogeneity at levels that were suitable for co-crystallization of GRK6 with potential inhibitors. The yields of purified GRK6, GRK6His/EK, and GRK6His/TEV were 0.3 mg, 0.8 mg and 0.7 mg per liter of cell culture, respectively. In addition, we have shown that GRK6His/TEV with the His10 tag removed was highly homogeneous and monodisperse as observed by dynamic light scattering measurement and actively folded as exhibited by circular dichroism spectroscopy. The described methods will support the structure-based development of additional therapeutics against multiple myeloma.

Multiple CheY Proteins Control Surface-Associated Lifestyles of Azospirillum brasilense

Bacterial chemotaxis is the directed movement of motile bacteria in gradients of chemoeffectors. This behavior is mediated by dedicated signal transduction pathways that couple environment sensing with changes in the direction of rotation of flagellar motors to ultimately affect the motility pattern. Azospirillum brasilense uses two distinct chemotaxis pathways, named Che1 and Che4, and four different response regulators (CheY1, CheY4, CheY6, and CheY7) to control the swimming pattern during chemotaxis. Each of the CheY homologs was shown to differentially affect the rotational bias of the polar flagellum and chemotaxis. The role, if any, of these CheY homologs in swarming, which depends on a distinct lateral flagella system or in attachment is not known. Here, we characterize CheY homologs’ roles in swimming, swarming, and attachment to abiotic and biotic (wheat roots) surfaces and biofilm formation. We show that while strains lacking CheY1 and CheY6 are still able to navigate air gradients, strains lacking CheY4 and CheY7 are chemotaxis null. Expansion of swarming colonies in the presence of gradients requires chemotaxis. The induction of swarming depends on CheY4 and CheY7, but the cells’ organization as dense clusters in productive swarms appear to depend on functional CheYs but not chemotaxis per se. Similarly, functional CheY homologs but not chemotaxis, contribute to attachment to both abiotic and root surfaces as well as to biofilm formation, although these effects are likely dependent on additional cell surface properties such as adhesiveness. Collectively, our data highlight distinct roles for multiple CheY homologs and for chemotaxis on swarming and attachment to surfaces.

Decreased Treg Cell and TCR Expansion Are Involved in Long-Lasting Graves’ Disease

Graves’ disease (GD) is a T cell-mediated organ-specific autoimmune disorder. GD patients who have taken anti-thyroid drugs (ATDs) for more than 5 years with positive anti-thyroid stimulating hormone receptor autoantibodies value were defined as persistent GD (pGD). To develop novel immunotherapies for pGD, we investigated the role of T cells in the long-lasting phase of GD. Clinical characteristics were compared between the pGD and newly diagnosed GD (nGD) (N = 20 respectively). Flow cytometric analysis was utilized to determine the proportions of Treg and Th17 cells (pGD, N = 12; nGD, N = 14). T cell receptor sequencing (TCR-seq) and RNA sequencing (RNA-seq) were also performed (pGD, N = 13; nGD, N = 20). Flow cytometric analysis identified lower proportions of Th17 and Treg cells in pGD than in nGD (P = 0.0306 and P = 0.0223). TCR-seq analysis revealed a lower diversity (P = 0.0025) in pGD. Specifically, marked clonal expansion, represented by an increased percentage of top V-J recombination, was observed in pGD patients. Interestingly, pGD patients showed more public T cell clonotypes than nGD patients (2,741 versus 966). Meanwhile, RNA-seq analysis revealed upregulation of the inflammation and chemotaxis pathways in pGD. Specifically, the expression of pro-inflammatory and chemotactic genes (IL1B, IL13, IL8, and CCL4) was increased in pGD, whereas Th17 and Treg cells associated genes (RORC, CARD9, STAT5A, and SATB1) decreased in pGD. Additionally, TCR diversity was negatively correlated with the expression of pro-inflammatory or chemotactic genes (FASLG, IL18R1, CCL24, and CCL14). These results indicated that Treg dysregulation and the expansion of pathogenic T cell clones might be involved in the long-lasting phase of GD via upregulating chemotaxis or inflammation response. To improve the treatment of pGD patients, ATDs combined therapies, especially those aimed at improving Treg cell frequencies or targeting specific expanded pathogenic TCR clones, are worth exploring in the future.