Brain Immune Cell Infiltration and Serum Metabolomic Characteristics Reveal that Lauric Acid Promotes Immune Cell Infiltration in Brain and Streptococcus suis Meningitis in Mice.

Although naturally Streptococcus suis serotype 2 (SS2) causes meningitis resulting in death or sequela of neurological symptoms in pigs and humans, severely threatening public health in the world, it has been difficult to build up and confirm experimental meningitis mouse models with obvious neurological syndrome for about two decades, which strongly hampers the in-depth study on the control measures and mechanisms of SS2-induced meningitis. In this study, a typical meningitis mouse model of SS2 was successfully established, as confirmed by the behavioral indicators of balance beam test, suspension test, and gait analysis. With bacteria gathering in the brain, distinguishable unique features including meningeal thickening, vacuolization of the Nissl body, brain barrier damage, glial cell activation, and more infiltration of T cells, macrophages, and DCs are observed in SS2 meningitis mice with typical neurological signs. Some meningitis mice were also accompanied by identical nephritis, ophthalmia, and cochlearitis. Investigation of the metabolic features demonstrated the downregulated cholic acid and upregulated 2-hydroxyvaleric acid, tetrahydrocortisone, nicotinic acid, and lauric acid in blood serum of mice and piglets with meningitis. And feeding trials show that lauric acid can promote meningitis by promoting the infiltration of immune cells into brain. These findings demonstrated that infection of ICR (improved castle road) mice with SS2 was able to induce typical meningitis accompanied by immune cell infiltration and lauric acid upregulation. These data provide a basis for the deep study of SS2 meningitis.

Identification and application of an endophytic fungus Arcopilus aureus from Panax notoginseng against crop fungal disease.

Endophytic fungi are important microbial resources for developing novel antibacterial and antifungal drugs to prevent and control crop diseases. Panax notoginseng has been used as a Chinese medicinal herb for a long time, as it has various bioactivities. However, information on endophytic fungi isolated from Panax notoginseng is rare. In this study, an endophytic fungus known as SQGX-6, which was later identified as the golden hair fungus Arcopilus aureus, was isolated from Panax notoginseng. SQGX-6 was extracted using ethyl acetate, and the active components of the fungus were identified using ultra-performance liquid chromatography-mass spectrometry (UHPLC-MS). The antifungal and antioxidant activities of the extract were determined and evaluated in vitro and in vivo. SQGX-6 and its extract inhibited the growth of Corn stalk rot (Fusarium graminearum), Corn southern leaf blight (Helminthosporium maydis), and Tomato gray mold (Botrytis cinerea) in vitro. The free radical scavenging rates for 2,2-Diphenyl-1-pyridinyl hydrazide (DPPH) radical scavenging activity, 3-Ethylbenzothiazoline-6-Sulfonic Acid Radical scavenging (ABTS) activity were also downregulated by the SQGX-6 extract. In vivo, the SQGX-6 extract inhibited the mycelial growth rates of the three aforementioned fungi and downregulated malondialdehyde (MDA) content and upregulated peroxidase (POD) and phenylalanine ammonia-lyase (PAL) content in fruits, leading to significant reduction in damage to cherry tomatoes caused by Botrytis cinerea. UHPLC-MS was performed to identify various active substances, including Alkaloids, Azoles, Benzofurans, Coumarins, Flavonoids, Organic acids, Phenols, and plant growth regulators contained in the extract. These results suggested that the endophytic fungus SQGX-6 of Panax notoginseng and its extract have excellent antifungal and antioxidant activities, and thus, it is an important microbial resource for the developing novel drugs against plant fungal infections.

Enolase of Streptococcus suis serotype 2 promotes biomolecular condensation of ribosomal protein SA for HBMECs apoptosis.

BACKGROUND: Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. RESULTS: Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca2+) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. CONCLUSIONS: Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces' RPSA.

Tracheal epithelial cell-exosome-derived MiR-21-5p inhibits alveolar macrophage pyroptosis to resist pulmonary bacterial infection through PIK3CD-autophagy pathway.

AIMS: Structural cells play an important role in regulating immune cells during infection. Our aim was to determine whether structural porcine tracheal epithelial cells (PTECs) can regulate alveolar macrophages (AMs) to prevent bacterial pneumonia, explore the underlying mechanism(s) and therapeutic target. MATERIALS AND METHODS: Actinobacillus pleuropneumoniae (APP) was used as the model strain for infection studies. Small RNA sequencing was used to identify differentially abundant exosome-derived miRNAs. The role of PTECs exosome-derived miR-21-5p in regulating AMs autophagy, pyroptosis, reactive oxygen species (ROS) was determined using RT-qPCR, western-blotting, flow cytometry, immunohistochemistry. Luciferase reporter assays were conducted to identify potential binding targets of miR-21-5p. The universality of miR-21-5p action on resistance to bacterial pulmonary infection was demonstrated using Klebsiella pneumoniae or Staphylococcus aureus in vitro and in vivo infection models. KEY FINDINGS: MiR-21-5p was enriched in PETCs-derived exosomes, which protected AMs against pulmonary bacterial infection. Mechanistically, miR-21-5p targeted PIK3CD, to promote autophagy of AMs, which reduced the pyroptosis induced by APP infection via inhibiting the over-production of ROS, which in turn suppressed the over-expression of pro-inflammatory cytokines, and increased bacterial clearance. Importantly, the protective effect and mechanism of miR-21-5p were universal as they also occurred upon challenge with Klebsiella pneumoniae and Staphylococcus aureus. SIGNIFICANCE: Our data reveals miR-21-5p can promote pulmonary resistance to bacterial infection by inhibiting pyroptosis of alveolar macrophages through the PIK3CD-autophagy-ROS pathway, suggesting PIK3CD may be a potential therapeutic target for bacterial pneumonia.

O-acetyl-homoserine sulfhydrylase deficient Streptococcus suis serotype 2 strain SC19 becomes an avirulent strain and provides immune protection against homotype infection in mice.

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host's BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.

High-dimensional analysis reveals an immune atlas and novel neutrophil clusters in the lungs of model animals with Actinobacillus pleuropneumoniae-induced pneumonia.

Due to the increase in bacterial resistance, improving the anti-infectious immunity of the host is rapidly becoming a new strategy for the prevention and treatment of bacterial pneumonia. However, the specific lung immune responses and key immune cell subsets involved in bacterial infection are obscure. Actinobacillus pleuropneumoniae (APP) can cause porcine pleuropneumonia, a highly contagious respiratory disease that has caused severe economic losses in the swine industry. Here, using high-dimensional mass cytometry, the major immune cell repertoire in the lungs of mice with APP infection was profiled. Various phenotypically distinct neutrophil subsets and Ly-6C+ inflammatory monocytes/macrophages accumulated post-infection. Moreover, a linear differentiation trajectory from inactivated to activated to apoptotic neutrophils corresponded with the stages of uninfected, onset, and recovery of APP infection. CD14+ neutrophils, which mainly increased in number during the recovery stage of infection, were revealed to have a stronger ability to produce cytokines, especially IL-10 and IL-21, than their CD14- counterparts. Importantly, MHC-II+ neutrophils with antigen-presenting cell features were identified, and their numbers increased in the lung after APP infection. Similar results were further confirmed in the lungs of piglets infected with APP and Klebsiella pneumoniae infection by using a single-cell RNA-seq technique. Additionally, a correlation analysis between cluster composition and the infection process yielded a dynamic and temporally associated immune landscape where key immune clusters, including previously unrecognized ones, marked various stages of infection. Thus, these results reveal the characteristics of key neutrophil clusters and provide a detailed understanding of the immune response to bacterial pneumonia.

Characterisation of Variants of Cyclic di-GMP Turnover Proteins Associated with Semi-Constitutive rdar Morphotype Expression in Commensal and Uropathogenic Escherichia coli Strains.

Expression of rdar (red, dry, and rough) colony morphology-based biofilm formation in Escherichia coli is highly variable. To investigate the molecular mechanisms of semi-constitutive rdar morphotype formation, we compared their cyclic di-GMP turnover protein content and variability to the highly regulated, temperature-dependent morphotype of the historical and modern ST10 isolates E. coli MG1655 and Fec10, respectively. Subsequently, we assessed the effects of cyclic di-GMP turnover protein variants of the EAL phosphodiesterases YcgG and YjcC and the horizontally transferred diguanylate cyclase DgcX on biofilm formation and motility. The two YcgG variants with truncations of the N-terminal CSS signaling domain were oppositely effective in targeting downregulation of rdar biofilm formation compared to the full-length reference protein. Expression of the C-terminal truncated variants YjcCFec67 and YjcCTob1 showed highly diminished apparent phosphodiesterase activity compared to the reference YjcCMG1655. For YjcCFec101, substitution of the C-terminus led to an apparently inactive enzyme. Overexpression of the diguanylate cyclase DgcX contributed to upregulation of cellulose biosynthesis but not to elevated expression of the major biofilm regulator csgD in the "classical" rdar-expressing commensal strain E. coli Fec10. Thus, the c-di-GMP regulating network is highly complex with protein variants displaying substantially different apparent enzymatic activities.

An In-Situ Tester for Extracting Piezoresistive Coefficients.

In this study, an electrostatic force-driven on-chip tester consisting of a mass with four guided cantilever beams was employed to extract the process-related bending stiffness and piezoresistive coefficient in-situ for the first time. The tester was manufactured using the standard bulk silicon piezoresistance process of Peking University, and was tested on-chip without additional handling. In order to reduce the deviation from process effects, the process-related bending stiffness was first extracted as an intermediate value, namely, 3590.74 N/m, which is 1.66% lower than the theoretical value. Then, the value was used to extract the piezoresistive coefficient using a finite element method (FEM) simulation. The extracted piezoresistive coefficient was 9.851 × 10-10 Pa-1, which essentially matched the average piezoresistive coefficient of the computational model based on the doping profile we first proposed. Compared with traditional extraction methods, such as the four-point bending method, this test method is on-chip, achieving automatic loading and precise control of the driving force, so it has high reliability and repeatability. Because the tester is manufactured together with the MEMS device, it has the potential to be used for process quality evaluation and monitoring on MEMS sensor production lines.

Adh Promotes Actinobacillus pleuropneumoniae Survival in Porcine Alveolar Macrophages by Inhibiting CHAC2-Mediated Respiratory Burst and Inflammatory Cytokine Expression.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes porcine pleuropneumonia that seriously endangers pig's health. Adh, located in the head region of trimeric autotransporter adhesion of A. pleuropneumoniae, affects bacterial adhesion and pathogenicity. However, how Adh mediates A. pleuropneumoniae immune invasion is still unclear. Here, we established the A. pleuropneumoniae strain L20 or L20 ΔAdh-infected porcine alveolar macrophages (PAM) model, and applied protein overexpression, RNA interference, qRT-PCR, Western blot and immunoflourescence techniques to dissect the effects of Adh on PAM during A. pleuropneumoniae infection. We found that Adh could increase the A. pleuropneumoniae adhesion and intracellular survival in PAM. Gene chip analysis of piglet lungs further showed that Adh significantly induced cation transport regulatory-like protein 2 (CHAC2) expression, whose overexpression suppressed the phagocytic capacity of PAM. Furthermore, CHAC2 overexpression dramatically increased glutathione (GSH) expression, decreased reactive oxygen species (ROS), and promoted A. pleuropneumoniae survival in PAM, while the knockdown of CHAC2 reversed these phenomena. Meanwhile, CHAC2 silence activated the NOD1/NF-κB pathway, resulting in an increase in IL-1ß, IL-6, and TNF-α expression, whereas this effect was weakened by CHAC2 overexpression and addition of NOD1/NF-κB inhibitor ML130. Moreover, Adh enhanced the secretion of LPS of A. pleuropneumoniae, which regulated the expression of CHAC2 via TLR4. In conclusion, through a LPS-TLR4-CHAC2 pathway, Adh inhibits respiratory burst and inflammatory cytokines expression to promote A. pleuropneumoniae survival in PAM. This finding may provide a novel target for the prevention and treatment of A. pleuropneumoniae.

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment.

BACKGROUND: IFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown. METHODS: BMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics. RESULTS: IFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), ß-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG). CONCLUSIONS: These results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

Metagenomics analysis reveals potential pathways and drivers of piglet gut phage-mediated transfer of ARGs.

The growing prevalence of antibiotic-resistant pathogens has led to a better understanding of the underlying processes that lead to this expansion. Intensive pig farms are considered one of the hotspots for antibiotic resistance gene (ARG) transmission. Phages, as important mobile carriers of ARGs, are widespread in the animal intestine. However, our understanding of phage-associated ARGs in the pig intestine and their underlying drivers is limited. Here, metagenomic sequencing and analysis of viral DNA and total DNA of different intestinal (ileum, cecum and feces) contents in healthy piglets and piglets with diarrhea were separately conducted. We found that phages in piglet ceca are the main repository for ARGs and mobile genetic element (MGE) genes. Phage-associated MGEs are important factors affecting the maintenance and transfer of ARGs. Interestingly, the colocalization of ARGs and MGE genes in piglet gut phages does not appear to be randomly selected but rather related to a specific phage host (Streptococcus). In addition, in the feces of piglets with diarrhea, the abundance of phages carrying ARGs and MGE genes was significantly increased, as was the diversity of polyvalent phages (phages with broad host ranges), which would facilitate the transfection and wider distribution of ARGs in the bacterial community. Moreover, the predicted host spectrum of polyvalent phages in diarrheal feces tended to be potential enteropathogenic genera, which greatly increased the risk of enteropathogens acquiring ARGs. Notably, we also found ARG-homologous genes in the sequences of piglet intestinal mimiviruses, suggesting that the piglet intestinal mimiviruses are a potential repository of ARGs. In conclusion, this study greatly expands our knowledge of the piglet gut microbiome, revealing the underlying mechanisms of maintenance and dissemination of piglet gut ARGs and providing a reference for the prevention and control of ARG pollution in animal husbandry.

Development and application of an indirect ELISA and nested PCR for the epidemiological analysis of Klebsiella pneumoniae among pigs in China.

Introduction: Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques. Methods: In this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed. Results: We showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists. Discussion: In conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae.

Recent advances on the regulation of bacterial biofilm formation by herbal medicines.

Biofilm formation is a fundamental part of life cycles of bacteria which affects various aspects of bacterial-host interactions including the development of drug resistance and chronic infections. In clinical settings, biofilm-related infections are becoming increasingly difficult to treat due to tolerance to antibiotics. Bacterial biofilm formation is regulated by different external and internal factors, among which quorum sensing (QS) signals and nucleotide-based second messengers play important roles. In recent years, different kinds of anti-biofilm agents have been discovered, among which are the Chinese herbal medicines (CHMs). CHMs or traditional Chinese medicines have long been utilized to combat various diseases around the world and many of them have the ability to inhibit, impair or decrease bacterial biofilm formation either through regulation of bacterial QS system or nucleotide-based second messengers. In this review, we describe the research progresses of different chemical classes of CHMs on the regulation of bacterial biofilm formation. Though the molecular mechanisms on the regulation of bacterial biofilm formation by CHMs have not been fully understood and there are still a lot of work that need to be performed, these studies contribute to the development of effective biofilm inhibitors and will provide a novel treatment strategy to control biofilm-related infections.

Therapeutic effects of JLX001 on neuronal necroptosis after cerebral ischemia-reperfusion in rats.

In recent years, more attention has been given to novel patterns of cell death observed during ischemia/reperfusion (I/R). Necroptosis is a regulable secondary cell death pathway; necroptosis is different from traditional forms of cell death, and it is regulated by the RIPK1-RIPK3-MLKL signaling pathway. JLX001 is the double hydrochloride of the natural compound cyclovirobuxine D (CVB-D). Previous studies have confirmed that CVB-D exerts a significant effect on cardiovascular and cerebrovascular diseases and that JLX001 can reduce ischemic brain injury by inhibiting cell apoptosis. For the first time, this project explored the in vivo and in vitro inhibitory effects of the therapeutic administration of JLX001 on the neuronal necroptosis caused by cerebral ischemia-reperfusion injury (CIRI). The middle cerebral artery occlusion reperfusion (MCAO/R) model was used to simulate I/R injury in rats in vivo, and oxygen-glucose deprivation and reperfusion (OGD/R) was used to simulate I/R injury in vitro. After the administration of JLX001, the relative expression of necroptosis-related molecules was measured by ELISA, RT-PCR, HE staining, immunofluorescence and Western blotting. The results showed that JLX001 significantly reduced pathological damage and the cerebral infarction rate in rat brain tissues, and the expression of neuronal necroptosis-related molecules was reduced, suggesting that JLX001 may regulate CIRI through the classic RIPK1-RIPK3-MLKL necroptosis pathway.

LAP3 contributes to IFN-γ-induced arginine depletion and malignant transformation of bovine mammary epithelial cells.

BACKGROUND: IFN-γ has been traditionally recognized as an inflammatory cytokine that involves in inflammation and autoimmune diseases. Previously we have shown that sustained IFN-γ induced malignant transformation of bovine mammary epithelial cells (BMECs) via arginine depletion. However, the molecular mechanism underlying this is still unknown. METHODS: In this study, the amino acids contents in BMECs were quantified by a targeted metabolomics method. The acquisition of differentially expressed genes was mined from RNA-seq dataset and analyzed bioinformatically. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), western blotting, and immunohistochemistry (IHC) assay were performed to detect gene mRNA and protein expression levels. CCK-8 and would healing assays were used to detect cell proliferation and migration abilities, respectively. Cell cycle phase alternations were analyzed by flow cytometry. RESULTS: The targeted metabolomics analysis specifically discovered IFN-γ induced arginine depletion through accelerating arginine catabolism and inhibiting arginine anabolism in BMECs. Transcriptome analysis identified leucine aminopeptidase 3 (LAP3), which was regulated by p38 and ERK MAPKs, to downregulate arginine level through interfering with argininosuccinate synthetase (ASS1) as IFN-γ stimulated. Moreover, LAP3 also contributed to IFN-γ-induced malignant transformation of BMECs by upregulation of HDAC2 (histone deacetylase 2) expression and promotion of cell cycle proteins cyclin A1 and D1 expressions. Arginine supplementation did not affect LAP3 and HDAC2 expressions, but slowed down cell cycle process of malignant BMECs. In clinical samples of patients with breast cancer, LAP3 was confirmed to be upregulated, while ASS1 was downregulated compared with healthy control. CONCLUSIONS: These results demonstrated that LAP3 mediated IFN-γ-induced arginine depletion to malignant transformation of BMECs. Our findings provide a potential therapeutic target for breast cancer both in humans and dairy cows.

Novel strategies to promote resolution of inflammation to treat lower extremity artery disease.

Lower extremity artery disease (LEAD) is a chronic inflammatory disease that occurs when atherosclerotic plaques form in the lower extremities, which may lead to amputation if not manged properly. Given clinical standardcare (pharmacological and surgical) have limited efficacy in LEAD, developing novel strategies to manage LEAD remains an unmet clinical need. Given that active resolution of inflammation is essential to facilitate tissue healing and repair, failure to resolve inflammation may lead to chronic inflammation, dysregulated cellular homeostasis and adverse tissue remodeling. Several studies have demonstrated the importance of the balance between endogenous pro-resolving mediators and pro-inflammatory factors. There is growing evidence to suggest endogenous pro-resolving mediators engage with pro-resolving G-protein-coupled receptors to reduce the initiation and progression of inflammatory responses and to increase therapeutic angiogenesis in LEAD. Here, we highlight the mechanisms and the consequences of resolved inflammation, and the therapeutic potential of endogenous pro-resolving mediators-based strategy for this devastating disease.

Carnosine Synthase 1 Contributes to Interferon Gamma-Induced Arginine Depletion via Mitogen-activated Protein Kinase 11 Signaling in Bovine Mammary Epithelial Cells.

Arginine is one of the host semiessential amino acids with diverse biological activities, and arginine depletion is associated with the incidence of many diseases. Arginine depletion induced by diet-derived interferon gamma (IFN-γ) leads to malignant transformation and impaired milk quality in healthy lactating bovine mammary epithelial cells (BMECs). However, the molecular mechanism of IFN-γ-induced arginine depletion is unclear. In this study, the BMEC cell line, mammary alveolar cells-large T antigen cells (MAC-T), was stimulated with IFN-γ (10 ng/mL) for 24 h, and cellular arginine and ornithine quantified by liquid chromatography-tandem mass spectrometry. Carnosine synthase 1 (CARNS1) was identified from RNA-seq data, CARNS1 knockdown was achieved using an shRNA interfering plasmid. The expression levels of CARNS1, argininosuccinate synthetase 1 (ASS1), mitogen-activated protein kinase 11 (p38 MAPK), and phosphorylated (p)-p38, and their cognate genes, were analyzed by Western blotting and real-time quantitative polymerase chain reaction. The results showed that IFN-γ inhibited the biosynthesis of arginine, but enhanced its catalysis via disruption of key enzymes involved in arginine metabolism. IFN-γ also inhibited the expression of CARNS1, ASS1, and cationic amino acid transporter 1, while activating the expression and phosphorylation of p38. However, knockdown of CARNS1 reduced arginine level and ASS1 expression and block of either the IFN-γ receptor IFN-γ receptor 2 or p38 relieved both the expression of Carnosine synthase 1 (CARNS1) and ASS1. In summary, these results indicate that IFN-γ induced arginine depletion through inhibition of CARNS1 signaling via activation of p38 in BMECs. These findings provide a novel insight for IFN-γ-related disease control strategies in dairy cows.

Oxidative Stress and Antioxidative Therapy in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is clinically characterized by a progressive increase in pulmonary artery pressure, followed by right ventricular hypertrophy and subsequently right heart failure. The underlying mechanism of PAH includes endothelial dysfunction and intimal smooth muscle proliferation. Numerous studies have shown that oxidative stress is critical in the pathophysiology of PAH and involves changes in reactive oxygen species (ROS), reactive nitrogen (RNS), and nitric oxide (NO) signaling pathways. Disrupted ROS and NO signaling pathways cause the proliferation of pulmonary arterial endothelial cells (PAECs) and pulmonary vascular smooth muscle cells (PASMCs), resulting in DNA damage, metabolic abnormalities, and vascular remodeling. Antioxidant treatment has become a main area of research for the treatment of PAH. This review mainly introduces oxidative stress in the pathogenesis of PAH and antioxidative therapies and explains why targeting oxidative stress is a valid strategy for PAH treatment.

Patatin-like phospholipase CapV in Escherichia coli - morphological and physiological effects of one amino acid substitution.

In rod-shaped bacteria, morphological plasticity occurs in response to stress, which blocks cell division to promote filamentation. We demonstrate here that overexpression of the patatin-like phospholipase variant CapVQ329R, but not CapV, causes pronounced sulA-independent pyridoxine-inhibited cell filamentation in the Escherichia coli K-12-derivative MG1655 associated with restriction of flagella production and swimming motility. Conserved amino acids in canonical patatin-like phospholipase A motifs, but not the nucleophilic serine, are required to mediate CapVQ329R phenotypes. Furthermore, CapVQ329R production substantially alters the lipidome and colony morphotype including rdar biofilm formation with modulation of the production of the biofilm activator CsgD, and affects additional bacterial traits such as the efficiency of phage infection and antimicrobial susceptibility. Moreover, genetically diverse commensal and pathogenic E. coli strains and Salmonella typhimurium responded with cell filamentation and modulation in colony morphotype formation to CapVQ329R expression. In conclusion, this work identifies the CapV variant CapVQ329R as a pleiotropic regulator, emphasizes a scaffold function for patatin-like phospholipases, and highlights the impact of the substitution of a single conserved amino acid for protein functionality and alteration of host physiology.

IL-5 enhances the resistance of Actinobacillus pleuropneumoniae infection in mice through maintaining appropriate levels of lung M2, PMN-II and highly effective neutrophil extracellular traps.

Interleukin 5 (IL-5) regulates the maturation, activation, proliferation and function of immune cells, and plays an important role in the inflammatory response induced by an allergy. However, its anti-pathogen effect is poorly understood currently, especially on pneumonia. Here, this study was designed to elucidate the immunological role of IL-5 in the infection of mice with Actinobacillus pleuropneumoniae (APP). We established an acute lung infection model of APP in IL-5 knockout mice (IL-5-/-) and wild-type mice (WT) through nasal infusion or intraperitoneal injection, compared the survival rate, clinical symptoms, lung bacterial load, proportion of various immune cells, immune molecular expression, and neutrophil germicidal ability through flow cytometry, RT-qPCR, ELISA and immunofluorescence. Compared to WT mice, the IL-5-/- mice had a lower survival rate, more severe clinical symptoms, significantly increased bacterial load, and inflammatory cell infiltration in the lung after APP infection. In an uninfected state, IL-5 deficiency decreased the number of M1 interstitial macrophages and CD14- monocytes, while after infection, IL-5 deficiency significantly reduced the M2 alveolar macrophages, and increased PMN-II cells in the lung. Furthermore, the expression of IL-10, IL-4, IL-33, TNF-α, iNOS in the lung was lower in IL-5-/- mice under an uninfected condition, and the secretion of IL-18 was significantly increased after infection. In addition, IL-5 deficiency decreased bactericidal ability by inhibiting the formation of neutrophil extracellular traps (NETs). Collectively, these results provide evidence that IL-5 can enhance the resistance of APP infection, and its anti-infection mechanism, implying new targets and ideas for APP or similar respiratory agents' prevention and treatment.