Z. morio Hemolymph Relieves E. coli-Induced Mastitis by Inhibiting Inflammatory Response and Repairing the Blood–Milk Barrier

Escherichia coli (E. coli) is a major environmental pathogen causing coliform mastitis, characterized by cell death and mammary tissue damage. Our previous study has shown the antimicrobial effect of Zophobas morio (Z. morio) hemolymph against mastitis pathogens. In this study, we established E. coli-induced cellular and animal models for mastitis, aiming to evaluate the protective effect of Z. morio hemolymph against E. coli-induced mastitis in vivo and in vitro. In mice with E. coli, Z. morio hemolymph attenuated bacterial burden and histopathological impairment, reduced the production of interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α) and the ratio of CD4⁺ T/CD8⁺ T, and increased the production of IL-2 triggered by E. coli. Z. morio hemolymph also enhanced the integrity of the blood-milk barrier in E. coli-induced mastitis. In E. coli-stimulated porcine mammary epithelial cells, Z. morio hemolymph inhibited E. coli-induced inflammatory responses and upregulated tight junction proteins (ZO-1, Claudin-3 and Occludin). Moreover, we found that the anti-inflammatory effect of Z. morio hemolymph was mediated by inhibiting E. coli-induced NLRP3 inflammasome assembly, Caspase-1 activation, and reversing the inhibitory effect of E. coli on autophagy. Besides, Z. morio hemolymph augmented ATG5/ATG16L1-mediated autophagy activation, negatively regulated NLRP3 inflammasome activation. Our results reveal that Z. morio hemolymph alleviates E. coli-induced mastitis via lessening the inflammatory response by regulating the NLRP3 and ATG5/ATG16L1 signaling pathway, as well as repairing the blood-milk barrier.     

Silver Nanoparticles from Oregano Leaves’ Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses

The oregano leaves’ extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl–methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV–Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.     

Within-Host Adaptation of Staphylococcus aureus in a Bovine Mastitis Infection Is Associated with Increased Cytotoxicity

Within-host adaptation is a typical feature of chronic, persistent Staphylococcus aureus infections. Research projects addressing adaptive changes due to bacterial in-host evolution increase our understanding of the pathogen’s strategies to survive and persist for a long time in various hosts such as human and bovine. In this study, we investigated the adaptive processes of S. aureus during chronic, persistent bovine mastitis using a previously isolated isogenic strain pair from a dairy cow with chronic, subclinical mastitis, in which the last variant (host-adapted, Sigma factor SigB-deficient) quickly replaced the initial, dominant variant. The strain pair was cultivated under specific in vitro infection-relevant growth-limiting conditions (iron-depleted RPMI under oxygen limitation). We used a combinatory approach of surfaceomics, molecular spectroscopic fingerprinting and in vitro phenotypic assays. Cellular cytotoxicity assays using red blood cells and bovine mammary epithelial cells (MAC-T) revealed changes towards a more cytotoxic phenotype in the host-adapted isolate with an increased alpha-hemolysin (α-toxin) secretion, suggesting an improved capacity to penetrate and disseminate the udder tissue. Our results foster the hypothesis that within-host evolved SigB-deficiency favours extracellular persistence in S. aureus infections. Here, we provide new insights into one possible adaptive strategy employed by S. aureus during chronic, bovine mastitis, and we emphasise the need to analyse genotype–phenotype associations under different infection-relevant growth conditions.     

Compartmentalized Innate Immune Response of Human Fetal Membranes against Escherichia coli Choriodecidual Infection

An infectious process into the uterine cavity represents a major endangered condition that compromises the immune privilege of the maternal–fetal unit and increases the risk for preterm birth (PTB) and premature rupture of membranes (PROM). Fetal membranes are active secretors of antimicrobial peptides (AMP), which limit bacterial growth, such as Escherichia coli. Nevertheless, the antibacterial responses displayed by chorioamniotic membranes against a choriodecidual E. coli infection have been briefly studied. The objective of this research was to characterize the profile of synthesis, activity, and spatial distribution of a broad panel of AMPs produced by fetal membranes in response to E. coli choriodecidual infection. Term human chorioamniotic membranes were mounted in a two independent compartment model in which the choriodecidual region was infected with live E. coli (1 × 10⁵ CFU/mL). Amnion and choriodecidual AMP tissue levels and TNF-α and IL-1β secretion were measured by the enzyme-linked immunosorbent assay. The passage of bacterium through fetal membranes and their effect on structural continuity was followed for 24 h. Our results showed that E. coli infection caused a progressive mechanical disruption of the chorioamniotic membranes and an activated inflammatory environment. After the challenge, the amnion quickly (2–4 h) induced production of human beta defensins (HBD)-1, HBD-2, and LL-37. Afterwards (8–24 h), the amnion significantly produced HBD-1, HBD-2, HNP-1-3, S100A7, sPLA2, and elafin, whereas the choriodecidua induced LL-37 synthesis. Therefore, we noticed a temporal- and tissue-specific pattern regulation of the synthesis of AMPs by infected fetal membranes. However, fetal membranes were not able to contain the collagen degradation or the bacterial growth and migration despite the battery of produced AMPs, which deeply increases the risk for PTB and PROM. The mixture of recombinant HBDs at low concentrations resulted in increased bactericidal activity compared to each HBD alone in vitro, encouraging further research to study AMP combinations that may offer synergy to control drug-resistant infections in the perinatal period.     

Inactivation of E. coli,S. aureus,and Bacteriophages in Biofilms by Humidified Air Plasma

In this study, humidified air dielectric barrier discharge (DBD) plasma was used to inactivate Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and bacteriophages in biofilms containing DNA, NaCl, carbohydrates, and proteins. The humidified DBD plasma was very effective in the inactivation of microbes in the (≤1.0 μm) biofilms. The number of surviving E. coli, S. aureus, and bacteriophages in the biofilms was strongly dependent on the constituent and thickness of the biofilms and was greatly reduced when the plasma treatment time increased from 5 s to 150 s. Our analysis shows that the UV irradiation was not responsible for the inactivation of microbes in biofilms. The short-lived RONS generated in the humidified air DBD plasma were not directly involved in the inactivation process; however, they recombined or reacted with other species to generate the long-lived RONS. Long-lived RONS diffused into the biofilms to generate very active species, such as ONOOH and OH. This study indicates that the geminated NO₂ and OH pair formed due to the homolysis of ONOOH can cause the synergistic oxidation of various organic molecules in the aqueous solution. Proteins in the biofilm were highly resistant to the inactivation of microbes in biofilms, which is presumably due to the existence of the unstable functional groups in the proteins. The unsaturated fatty acids, cysteine-rich proteins, and sulfur–methyl thioether groups in the proteins were easily oxidized by the geminated NO₂ and OH pair.     

β-Cyclodextrin Inclusion Complex to Improve Physicochemical Properties of Pipemidic Acid: Characterization and Bioactivity Evaluation

The aptitude of cyclodextrins (CDs) to form host-guest complexes has prompted an increase in the development of new drug formulations. In this study, the inclusion complexes of pipemidic acid (HPPA), a therapeutic agent for urinary tract infections, with native β-CD were prepared in solid state by kneading method and confirmed by FT-IR and ¹H NMR. The inclusion complex formation was also characterized in aqueous solution at different pH via UV-Vis titration and phase solubility studies obtaining the stability constant. The 1:1 stoichiometry was established by a Job plot and the inclusion mechanism was clarified using docking experiments. Finally, the antibacterial activity of HPPA and its inclusion complex was tested on P. aeruginosa, E. coli and S. aureus to determine the respective EC_50s and EC_90s. The results showed that the antibacterial activity of HPPA:β-CD against E. coli and S. aureus is higher than that of HPPA. Furthermore, HPPA and HPPA:β-CD, tested on human hepatoblastoma HepG2 and MCF-7 cell lines by MTT assay, exhibited, for the first time, antitumor activities, and the complex revealed a higher activity than that of HPPA. The use of β-CD allows an increase in the aqueous solubility of the drug, its bioavailability and then its bioactivity.     

LncRSPH9-4 Facilitates Meningitic Escherichia coli-Caused Blood–Brain Barrier Disruption via miR-17-5p/MMP3 Axis

Brain microvascular endothelial cells (BMECs) constitute the structural and functional basis for the blood–brain barrier (BBB) and play essential roles in bacterial meningitis. Although the BBB integrity regulation has been under extensive investigation, there is little knowledge regarding the roles of long non-coding RNAs (lncRNAs) in this event. The present study aimed to investigate the roles of one potential lncRNA, lncRSPH9-4, in meningitic E. coli infection of BMECs. LncRSPH9-4 was cytoplasm located and significantly up-regulated in meningitic E. coli-infected hBMECs. Electrical cell-substrate impedance sensing (ECIS) measurement and Western blot assay demonstrated lncRSPH9-4 overexpression in hBMECs mediated the BBB integrity disruption. By RNA-sequencing analysis, 639 mRNAs and 299 miRNAs were significantly differentiated in response to lncRSPH9-4 overexpression. We further found lncRSPH9-4 regulated the permeability in hBMECs by competitively sponging miR-17-5p, thereby increasing MMP3 expression, which targeted the intercellular tight junctions. Here we reported the infection-induced lncRSPH9-4 aggravated disruption of the tight junctions in hBMECs, probably through the miR-17-5p/MMP3 axis. This finding provides new insights into the function of lncRNAs in BBB integrity during meningitic E. coli infection and provides the novel nucleic acid targets for future treatment of bacterial meningitis.     

Expression Profiles and Characteristics of Apple lncRNAs in Roots,Phloem, Leaves,Flowers, and Fruit

LncRNAs impart crucial effects on various biological processes, including biotic stress responses, abiotic stress responses, fertility and development. The apple tree is one of the four major fruit trees in the world. However, lncRNAs’s roles in different tissues of apple are unknown. We identified the lncRNAs in five tissues of apples including the roots, phloem, leaves, flowers, and fruit, and predicted the intricate regulatory networks. A total of 9440 lncRNAs were obtained. LncRNA target prediction revealed 10,628 potential lncRNA–messenger RNA (mRNA) pairs, 9410 pairs functioning in a cis-acting fashion, and 1218 acting in a trans-acting fashion. Functional enrichment analysis showed that the targets were significantly enriched in molecular functions related to photosynthesis-antenna proteins, single-organism metabolic process and glutathione metabolism. Additionally, a total of 88 lncRNAs have various functions related to microRNAs (miRNAs) as miRNA precursors. Interactions between lncRNAs and miRNAs were predicted, 1341 possible interrelations between 187 mdm-miRNAs and 174 lncRNAs (1.84%) were identified. MSTRG.121644.5, MSTRG.121644.8, MSTRG.2929.2, MSTRG.3953.2, MSTRG.63448.2, MSTRG.9870.2, and MSTRG.9870.3 could participate in the functions in roots as competing endogenous RNAs (ceRNAs). MSTRG.11457.2, MSTRG.138614.2, and MSTRG.60895.2 could adopt special functions in the fruit by working with miRNAs. A further analysis showed that different tissues formed special lncRNA–miRNA–mRNA networks. MSTRG.60895.2–mdm-miR393–MD17G1009000 may participate in the anthocyanin metabolism in the fruit. These findings provide a comprehensive view of potential functions for lncRNAs, corresponding target genes, and related lncRNA–miRNA–mRNA networks, which will increase our knowledge of the underlying development mechanism in apple.     

A Novel Nano-Antimicrobial Polymer Engineered with Chitosan Nanoparticles and Bioactive Peptides as Promising Food Biopreservative Effective against Foodborne Pathogen E. coli O157-Caused Epithelial Barrier Dysfunction and Inflammatory Responses

For food quality and safety issues, the emergence of foodborne pathogenic bacteria has further accelerated the spread of antibiotic residues and drug resistance genes. To alleviate the harm caused by bacterial infections, it is necessary to seek novel antimicrobial agents as biopreservatives to prevent microbial spoilage. Nanoantimicrobials have been widely used in the direct treatment of bacterial infections. CNMs, formed by chitosan nanoparticles and peptides, are promising antibiotic alternatives for use as excellent new antibacterial drugs against pathogenic bacteria. Herein, the current study evaluated the function of CNMs in the protection of foodborne pathogen Escherichia coli (E. coli) O157 infection using an intestinal epithelial cell model. Antibacterial activity assays indicated that CNMs exerted excellent bactericidal activity against E. coli O157. Assessment of the cytotoxicity risks toward cells demonstrated that 0.0125–0.02% of CNMs did not cause toxicity, but 0.4% of CNMs caused cytotoxicity. Additionally, CNMs did not induced genotoxicity either. CNMs protected against E. coli O157-induced barrier dysfunction by increasing transepithelial electrical resistance, decreasing lactate dehydrogenase and promoting the protein expression of occludin. CNMs were further found to ameliorate inflammation via modulation of tumor factor α, toll-like receptor 4 and nuclear factor κB (NF-κB) expression via inhibition of mitogen-activated protein kinase and NF-κB activation and improved antioxidant activity. Taken together, CNMs could protect the host against E. coli O157-induced intestinal barrier damage and inflammation, showing that CNMs have great advantages and potential application as novel antimicrobial polymers in the food industry as food biopreservatives, bringing new hope for the treatment of bacterial infections.     

Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.     

Trifluoromethylcinnamanilide Michael Acceptors for Treatment of Resistant Bacterial Infections

A series of thirty-two anilides of 3-(trifluoromethyl)cinnamic acid (series 1) and 4-(trifluoromethyl)cinnamic acid (series 2) was prepared by microwave-assisted synthesis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). All the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. (2E)-3-[3-(Trifluoromethyl)phenyl]-N-[4-(trifluoromethyl)phenyl]prop-2-enamide (1j), (2E)-N-(3,5-dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]prop-2-enamide (1o) and (2E)-N-[3-(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)-phenyl]prop-2-enamide (2i), (2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)phenyl]-prop-2-enamide (2p) showed antistaphylococcal (MICs/MBCs 0.15–5.57 µM) as well as anti-enterococcal (MICs/MBCs 2.34–44.5 µM) activity. The growth of M. marinum was strongly inhibited by compounds 1j and 2p in a MIC range from 0.29 to 2.34 µM, while all the agents of series 1 showed activity against M. smegnatis (MICs ranged from 9.36 to 51.7 µM). The performed docking study demonstrated the ability of the compounds to bind to the active site of the mycobacterial enzyme InhA. The compounds had a significant effect on the inhibition of bacterial respiration, as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity but also bactericidal activity. Preliminary in vitro cytotoxicity screening was assessed using the human monocytic leukemia cell line THP-1 and, except for compound 2p, all effective agents did show insignificant cytotoxic effect. Compound 2p is an interesting anti-invasive agent with dual (cytotoxic and antibacterial) activity, while compounds 1j and 1o are the most interesting purely antibacterial compounds within the prepared molecules.     

Role of Long Noncoding RNAs ZlMSTRG.11348 and UeMSTRG.02678 in Temperature-Dependent Culm Swelling in Zizania latifolia

Temperature influences the physiological processes and ecology of both hosts and endophytes; however, it remains unclear how long noncoding RNAs (lncRNAs) modulate the consequences of temperature-dependent changes in host–pathogen interactions. To explore the role of lncRNAs in culm gall formation induced by the smut fungus Ustilago esculenta in Zizania latifolia, we employed RNA sequencing to identify lncRNAs and their potential cis-targets in Z. latifolia and U. esculenta under different temperatures. In Z. latifolia and U. esculenta, we identified 3194 and 173 lncRNAs as well as 126 and four potential target genes for differentially expressed lncRNAs, respectively. Further function and expression analysis revealed that lncRNA ZlMSTRG.11348 regulates amino acid metabolism in Z. latifolia and lncRNA UeMSTRG.02678 regulates amino acid transport in U. esculenta. The plant defence response was also found to be regulated by lncRNAs and suppressed in Z. latifolia infected with U. esculenta grown at 25 °C, which may result from the expression of effector genes in U. esculenta. Moreover, in Z. latifolia infected with U. esculenta, the expression of genes related to phytohormones was altered under different temperatures. Our results demonstrate that lncRNAs are important components of the regulatory networks in plant-microbe-environment interactions, and may play a part in regulating culm swelling in Z. latifolia plants.     

The Disordered EZH2 Loop: Atomic Level Characterization by 1HN- and 1Hα-Detected NMR Approaches,Interaction with the Long Noncoding HOTAIR RNA

The 96-residue-long loop of EZH2 is proposed to play a role in the interaction with long non-coding RNAs (lncRNAs) and to contribute to EZH2 recruitment to the chromatin. However, molecular details of RNA recognition have not been described so far. Cellular studies have suggested that phosphorylation of the Thr345 residue localized in this loop influences RNA binding; however, no mechanistic explanation has been offered. To address these issues, a systematic NMR study was performed. As the ¹H^N-detected NMR approach presents many challenges under physiological conditions, our earlier developed, as well as improved, ¹H^α-detected experiments were used. As a result of the successful resonance assignment, the obtained chemical shift values indicate the highly disordered nature of the EZH2 loop, with some nascent helical tendency in the Ser407–Ser412 region. Further investigations conducted on the phosphomimetic mutant EZH2^T345D showed that the mutation has only a local effect, and that the loop remains disordered. On the other hand, the mutation influences the cis/trans Pro346 equilibrium. Interactions of both the wild-type and the phosphomimetic mutant with the lncRNA HOTAIR₁₄₀ (1–140 nt) highlight that the Thr367–Ser375 region is affected. This segment does not resemble any of the previously reported RNA-binding motifs, therefore the identified binding region is unique. As no structural changes occur in the EZH2 loop upon RNA binding, we can consider the protein–RNA interaction as a “fuzzy” complex.     

A Novel Strategy for Creating an Antibacterial Surface Using a Highly Efficient Electrospray-Based Method for Silica Deposition

Adhesion of bacteria on biomedical implant surfaces is a prerequisite for biofilm formation, which may increase the chances of infection and chronic inflammation. In this study, we employed a novel electrospray-based technique to develop an antibacterial surface by efficiently depositing silica homogeneously onto polyethylene terephthalate (PET) film to achieve hydrophobic and anti-adhesive properties. We evaluated its potential application in inhibiting bacterial adhesion using both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria. These silica-deposited PET surfaces could provide hydrophobic surfaces with a water contact angle greater than 120° as well as increased surface roughness (root mean square roughness value of 82.50 ± 16.22 nm and average roughness value of 65.15 ± 15.26 nm) that could significantly reduce bacterial adhesion by approximately 66.30% and 64.09% for E. coli and S. aureus, respectively, compared with those on plain PET surfaces. Furthermore, we observed that silica-deposited PET surfaces showed no detrimental effects on cell viability in human dermal fibroblasts, as confirmed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide and live/dead assays. Taken together, such approaches that are easy to synthesize, cost effective, and efficient, and could provide innovative strategies for preventing bacterial adhesion on biomedical implant surfaces in the clinical setting.     

Investigation of antibacterial activity and related mechanism of a ruthenium(II) polypyridyl complex

Metal based drug represents a novel group of antimicrobial agents with potential application for the control of bacterial and fungal infections. In this study, we fabricate ruthenium(II) complex containing the polypyridyl ligands, namely [Ru(phen)₂(tip)] (ClO₄)₂ (RuTh) and carefully investigate its antibacterial activities against both the Gram-negative (G −) bacteria Escherichia coli (E. coli) and the Gram-positive (G +) bacteria Staphylococcus aureus (S. aureus). The RuTh is more toxic to S. aureus than that to E. coli. The antibacterial effects of RuTh are further investigated, revealing specific mechanisms. The results demonstrate that RuTh functions as a bactericide against the E. coli and S. aureus through disrupting bacterial cell wall integrity and its cellular components.