Increased Cell Permeability Research Articles

Peptide Nucleic Acids Containing Cationic/Amino-Alkyl Modified Bases Promote Enhanced Hybridization Kinetics and Thermodynamics with Single-Strand DNA.

Peptide nucleic acids (PNAs) are antisense molecules with excellent polynucleotide hybridization properties; they are resistant to nuclease degradation but often have poor cell permeability leading to moderate cellular activity and limited clinical results. The addition of cationic substitutions (positive charges) to PNA molecules greatly increases cell permeability. In this report, we describe the synthesis and polynucleotide hybridization properties of a novel cationic/amino-alkyl nucleotide base-modified PNA (OPNA). This study was designed to quantitate the effect the cationic/amino-alkyl nucleotide base modification had on the kinetic and thermodynamic properties of OPNA-DNA hybridization using surface plasmon resonance and UV thermal melt studies. Kinetic studies reveal a favorable 10-30 fold increase in affinity for a single cationic modification on the base of an adenine, cytosine, or guanidine OPNA sequence compared to the nonmodified PNA strand. The increase in affinity is correlated directly with a favorable decrease in the dissociation rate constant and increase in the association rate constant. Introducing additional amino-alkyl base modifications further favors a decrease in the dissociation rate (3-10-fold per amino-alkyl). The thermodynamics driving the OPNA hybridization is promoted by an additional favorable -80 kJ/mol enthalpy of binding for a single amino-alkyl modification compared to the PNA strand. This increase in enthalpy is consistent with an ion-ion interaction with the DNA strand. These kinetic and thermodynamic hybridization studies reveal for the first time that this type of cationic/amino-alkyl base-modified PNA has favorable hybridization properties suitable for development as an antisense oligomer.

Purification and antimicrobial mechanism of a novel bacteriocin produced by Lactiplantibacillus plantarum FB-2

Lactiplantibacillus plantarum, a major producer of bacteriocins, is generally considered a safe microorganism. In this study, Plantaricin FB-2 was extracted from L. plantarum FB-2 and studied for antimicrobial mechanism against Staphylococcus aureus. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of Plantaricin FB-2 is 1–3.3 kDa. Plantaricin FB-2 is non-hemolytic and its minimum inhibitory concentration against S. aureus ATCC6538 is about 256 μg/mL. Plantaricin FB-2 exerts the antibacterial effect by destroying the structure of the bacterial cell membrane, increasing cell permeability, promoting the leakage of cytoplasmic contents (proteins, DNA, K+, Ca2+, etc.), and increasing intracellular reactive oxygen species levels. This study provides the theoretical basis for the development of L. plantarum produced Plantaricin FB-2 as a new antibacterial agent.

Green synthesis of silver nanoparticles using banana flower extract, and their antibacterial activity

Silver nanoparticles (AgNPs) were synthesised using banana flower extract (BFE) as a reducing and stabilising agent. Spherical, well-dispersed, and stable AgNPs were formed and characterised by ultraviolet-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), and zeta potential. The in vitro antimicrobial properties of AgNPs against Staphylococcus aureus and Escherichia coli were then investigated. The minimum inhibitory concentration (MIC) of AgNPs against S. aureus and E. coli were 32 and 16 μg/mL, respectively. E. coli was more sensitive to AgNPs than S. aureus due to differences in cell wall structures of the bacteria. Regarding the bactericidal mechanisms of AgNPs, an increase in cell permeability and a distinctive deformation in cellular morphology was observed. The antibacterial effect decreased with the addition of the antioxidant N-acetyl-l-cysteine (NAC) which acted as ROS scavenger. In summary, the antibacterial mechanism was likely a combination of cell membrane damage and ROS induction.

Activatable Fluorescence-Encoded Nanoprobes Enable Simple Multiplexed RNA Imaging in Live Cells.

Benefiting from superior programmable performance and flexible design of DNA technologies, a variety of single-molecule RNA fluorescence imaging methodologies have been reported. However, the multiplexing capability is restricted owing to the spectral overlap of fluorophores. To overcome this limitation, some inspiring multiplex imaging strategies have been developed, but in practice, it remains challenging to achieve convenient and rapid imaging in live cells due to complex designs and additional pretreatments to increase cell permeability. Here, we report an activatable fluorescence-encoded nanoprobe (AFENP) strategy, through which fluorescence-encoded functional modules for qualitative analysis and activated nucleic acid assemblies functional modules for quantitative testing enable simple multiplexed RNA imaging in single live cells. As a proof of principle, by two distinguishable fluorophores (fluorescein and rhodamine B) and their seven distinctly differentiated intensity levels, self-assembled AFENP enables simplified and quick simultaneous in situ detection and imaging of seven types of targets in live single cells because the fluorescent quantitative signal is activated only in the presence of target avoiding the washing procedures and additional pretreatment to increase cell permeability is undesired. We expect that this practical single-cell analysis platform will be adopted for multiple gene expression analysis and imaging in live cells on account of its simplicity and multiplex capability.

Smart Nanomaterials in Cancer Theranostics: Challenges and Opportunities.

Cancer is ranked as the second leading cause of death globally. Traditional cancer therapies including chemotherapy are flawed, with off-target and on-target toxicities on the normal cells, requiring newer strategies to improve cell selective targeting. The application of nanomaterial has been extensively studied and explored as chemical biology tools in cancer theranostics. It shows greater applications toward stability, biocompatibility, and increased cell permeability, resulting in precise targeting, and mitigating the shortcomings of traditional cancer therapies. The nanoplatform offers an exciting opportunity to gain targeting strategies and multifunctionality. The advent of nanotechnology, in particular the development of smart nanomaterials, has transformed cancer diagnosis and treatment. The large surface area of nanoparticles is enough to encapsulate many molecules and the ability to functionalize with various biosubstrates such as DNA, RNA, aptamers, and antibodies, which helps in theranostic action. Comparatively, biologically derived nanomaterials perceive advantages over the nanomaterials produced by conventional methods in terms of economy, ease of production, and reduced toxicity. The present review summarizes various techniques in cancer theranostics and emphasizes the applications of smart nanomaterials (such as organic nanoparticles (NPs), inorganic NPs, and carbon-based NPs). We also critically discussed the advantages and challenges impeding their translation in cancer treatment and diagnostic applications. This review concludes that the use of smart nanomaterials could significantly improve cancer theranostics and will facilitate new dimensions for tumor detection and therapy.

Nitric Oxide Production in mixed cultures of infected rats with Neospora caninum and treated with Jataí Honey (Tetragonisca angustula)

Neosporosis is a disease caused by Neospora caninum sp (JP Dubey), an intracellular parasite that affects many animal species, fostering abortions and neurological disorders. Recent studies on Neospora caninum have shown that glial cells have been considered a model of in vitro infections of these protozoa. Honey has been used since ancient times for its anti-inflammatory and antimicrobial properties. The present study aimed to evaluate the in vitro reactivity of glial cells (astrocytes and microglia) infected with N. caninum treated with Jataí honey at 1% (Tetragonisca angustula). After 72 hours from infection, mitochondrial metabolism, dehydrogenase lactate activity (LDH), nitric oxide production (NO) and the number of parasites were checked. Cultures treated with honey at 1% have shown mitochondrial metabolism and cell permeability increase. There was an increase in nitrite production, possible indicating a microglial activation. Nitric Oxide values found in this study may indicate an immune response against Neospora caninum tachyzoites, since such increase may contribute to reduce the number of tachyzoites. So, our results suggest that honey had a protective effect over astrocytes and microglia cultures infected with Neospora caninum.

Tandem mass tag-based proteomics technology provides insights into multi-targeted mechanism of peptide MOp2 from Moringa oleifera seeds against Staphylococcus aureus

Our previous study proved that the peptide MOp2 from Moringa oleifera seeds exhibited membrane-damaging effects on Staphylococcus aureus. In this study, TMT-based proteomics technology was mainly used to investigate the antimicrobial mechanism of MOp2 against S. aureus. A total of 541 differentially expressed proteins (DEPs) were identified in S. aureus treated with MOp2, among which 256 and 285 DEPs were upregulated and downregulated, respectively. They were mainly involved in inositol phosphate metabolism, S. aureus infection, citrate cycle (TCA cycle), and phosphotransferase system (PTS) and acted as ABC transporters and ribosomes. Moreover, the decreasing AKP activity indicated that MOp2 affected cell wall biosynthesis, and excessive ROS accumulation caused apoptosis, resulting in DNA fragmentation, nuclear morphological changes, and phosphatidylserine externalization. Additionally, molecular docking showed that MOp2 could interact with eight DEPs, including ProC, QoxB, SOD2, DnaK, GroEL, RplY, AcpS, and FabG, indicating that MOp2 might act on these molecular targets, leading to increased cell permeability, oxidative damage, impaired protein synthesis, cell wall synthesis obstruction, and energy metabolism disorder. Overall, these findings provided new insights into the multi-targeted mechanism of MOp2 against S. aureus, and could provide a theoretical basis for its application as a novel food preservative and antibiotic substitute.

Lactate transport and metabolism in rumen epithelial cells in SARA condition

Lactate, a main product of rumen microbial fermentation from dietary starch, has been recognised as an important mediator of rumen microbiota regulation in rumen acidosis. However, the mechanism of lactate transport and metabolism in the rumen remains unclear. This study aimed to evaluate the significance of the absorption, transport, and metabolism of lactic acid in the pH and LPS-induced subacute ruminal acidosis (SARA) model of rumen epithelial cells (RECs). In the current study, RECs were obtained from two adult Saanen goats (44.5 ± 4.6 kg BW) and using Lipopolysaccharide (LPS) and low pH to co-stimulate RECs to construct a microenvironment under SARA conditions, to further analyse the lactate transport and metabolism by RECs. The results showed that the LPS and low pH could significantly reduce cell viability by 29.36% (p < 0.05), induce inflammation in RECs, destroy tight junctions between RECs, and increase cell permeability. In addition, stimulation leads to changes in cellular metabolic activity and inhibits the expression of lactate transporters (p < 0.05), promoting an increased risk of lactate accumulation in the intracellular and intestinal lumen. This study hopefully provides a better understanding of lactate metabolic regulation and new perspectives for the nutritional prevention and treatment of SARA. HIGHLIGHTS Lactic acid is an important fermentation product in the rumen of ruminants, and its transport and metabolic regulation mechanisms are still unclear. To understand transport uptake in the rumen epithelium, a SARA model was therefore used. Low pH and high LPS trigger epithelial inflammation, increasing the risk of lactic acid entering the bloodstream.

Structural Optimization and Anticancer Activity of Polo-like Kinase 1 (Plk1) Polo-Box Domain (PBD) Inhibitors and Their Prodrugs.

Polo-like kinase 1 (Plk1), a mitotic kinase whose activity is widely upregulated in various human cancers, is considered an attractive target for anticancer drug discovery. Aside from the kinase domain, the C-terminal noncatalytic polo-box domain (PBD), which mediates the interaction with the enzyme's binding targets or substrates, has emerged as an alternative target for developing a new class of inhibitors. Various reported small molecule PBD inhibitors exhibit poor cellular efficacy and/or selectivity. Here, we report structure-activity relationship (SAR) studies on triazoloquinazolinone-derived inhibitors, such as 43 (a 1-thioxo-2,4-dihydrothieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one) that effectively block Plk1, but not Plk2 and Plk3 PBDs, with improved affinity and drug-like properties. The range of prodrug moieties needed for thiol group masking of the active drugs has been expanded to increase cell permeability and mechanism-based cancer cell (L363 and HeLa) death. For example, a 5-thio-1-methyl-4-nitroimidazolyl prodrug 80, derived from 43, showed an improved cellular potency (GI50 4.1 μM). As expected, 80 effectively blocked Plk1 from localizing to centrosomes and kinetochores and consequently induced potent mitotic block and apoptotic cell death. Another prodrug 78 containing 9-fluorophenyl in place of the thiophene-containing heterocycle in 80 also induced a comparable degree of anti-Plk1 PBD effect. However, orally administered 78 was rapidly converted in the bloodstream to parent drug 15, which was shown be relatively stable toward in vivo oxidation due to its 9-fluorophenyl group in comparison to unsubstituted phenyl. Further derivatization of these inhibitors, particularly to improve the systemic prodrug stability, could lead to a new class of therapeutics against Plk1-addicted cancers.

Changes in physicochemical property and its potential regulatory mechanism for the formation of water core of the Fuji apples post‐harvesting

SummaryThis study analysed the factors for influencing the occurrence of water core in apples and proposed a potential metabolism following its post‐harvesting. By comparing the ordinary pulp and the region within the water core in different post‐harvesting stages of the Fuji apples, although the occurrence of water core did not significantly change the appearance of the apples, its interior firmness decreased with increased cell permeability. Furthermore, fructose content decreased, and sorbitol content increased rapidly accompanied by a reduction of sorbitol dehydrogenase activity at the site of water core. The cell morphology also demonstrated that the cells became denser and the blue aggregates formed. It indicated that the cell walls were saturated with locally highly accumulated sorbitol, followed by visualised transparent spots. In summary, the occurrence of water core during the post‐harvesting of Fuji apples was found to be related to the increased sorbitol content and the corresponding decreased dehydrogenase activity.

Ultrasound stimulated production of exopolysaccharide with anti-UV radiation activity by increasing cell permeability of Paenibacillus polymyxa

Exopolysaccharide (EPS) produced by Paenibacillus polymyxa PYQ1 may be a promising agent used in the cosmetic field for skin care because of its protective benefits against short-wave ultraviolet (UVC, 100–280 nm) radiation, which induces cytotoxicity in HaCaT cells. We attempted to use low-intensity ultrasound to enhance its fermentation, and the yield of EPS increased by 24 % under the optimum ultrasonic treatment (90 W, 45 s in 9 h). Results of further experiments show that low-intensity ultrasound will kill a few bacterial cells. Therefore, the remaining bacterial cells have more space and more nutrients, growing faster and producing EPS more efficiently. The recoverable physical damage caused by ultrasound increased the cell permeability, and for the first time, we found that low-intensity ultrasound up-regulated the expression of membrane transporters involved with the transport of sucrose and polysaccharides. Owing to these changes, the utilization of carbon sources was accelerated, and the transport of EPS was promoted to the outside of bacterial cells such that the production of EPS was enhanced.

A ferrocene-containing analogue of the MCU inhibitor Ru265 with increased cell permeability

An analogue of the mitochondrial calcium uniporter (MCU) inhibitor Ru265 containing axial ferrocenecarboxylate ligands is reported. This new complex exhibits enhanced cellular uptake compared to the parent compound Ru265.

Ceramide induces pyroptosis through TXNIP/NLRP3/GSDMD pathway in HUVECs

BackgroundPyroptosis of endothelial cells is a new cause of endothelial dysfunction in multiple diseases. Ceramide acts as a potential bioactive mediator of inflammation and increases vascular endothelial permeability in many diseases, whether it can aggravate vascular endothelial injury by inducing cell pyroptosis remains unknown. This study was established to explore the effects of C8-ceramide (C8-Cer) on human umbilical vein vascular endothelial cells (HUVECs) and its possible underlying mechanism.MethodsHUVECs were exposed to various concentrations of C8-Cer for 12 h, 24 h, 48 h. The cell survival rate was measured using the cell counting kit-8 assay. Western blotting and Real-time polymerase chain reaction (RT-PCR) were used to detect the pyroptosis-releated protein and mRNA expressions, respectively. Caspase-1 activity assay was used to detect caspase-1 activity. Hoechst 33342/propidium iodide double staining and flow cytometry were adopted to measure positive staining of cells. Lactate dehydrogenase release assay and enzyme-linked immunosorbent assay were adopted to measure leakage of cellular contents. FITC method was used to detect the permeability of endothelial cells. ROS fluorescence intensity were detected by flow cytometry.ResultsThe viability of HUVECs decreased gradually with the increase in ceramide concentration and time. Ceramide upregulated the expression of thioredoxin interacting protein (TXNIP), NLRP3, GSDMD, GSDMD-NT, caspase-1 and Casp1 p20 at the protein and mRNA level in a dose-dependent manner. It also enhanced the PI uptake in HUVECs and upregulated caspase-1 activity. Moreover, it promoted the release of lactate dehydrogenase, interleukin-1β, and interleukin-18. Meanwhile, we found that ceramide led to increased vascular permeability. The inhibitor of NLRP3 inflammasome assembly, MCC950, was able to disrupt the aforementioned positive loop, thus alleviating vascular endothelial cell damage. Interestingly, inhibition of TXNIP either chemically using verapamil or genetically using small interfering RNA (siRNA) can effectively inhibit ceramide-induced pyroptosis and improved cell permeability. In addition, ceramide stimulated reactive oxygen species (ROS) generation. The pretreatment of antioxidant N-acetylcysteine (NAC), ROS scavenger, blocked the expression of pyroptosis markers induced by C8-cer in HUVECs.ConclusionThe current study demonstrated that C8-Cer could aggravate vascular endothelial cell damage and increased cell permeability by inducing cell pyroptosis. The results documented that the ROS-dependent TXNIP/NLRP3/GSDMD signalling pathway plays an essential role in the ceramide-induced pyroptosis in HUVECs.

Flow electrochemical inactivation of waterborne bacterial endospores

Waterborne pathogens have the risk of spreading waterborne diseases and even pandemics. Some Gram-positive bacteria can form endospores, the hardiest known life form that can withstand heat, radiation, and chemicals. Electrochemical inactivation may offer a promising solution, but is hindered by low inactivation efficiencies resulting from limitation of electrode/endospores interaction in terms of electrochemical reaction selectivity and mass transfer. Herein, these issues were addressed through modifying selectivity of active species formation using electroactive ceramic membrane with high oxygen evolution potential, improving mass transfer property by flow-through operation. In this way, inactivation (6.0-log) of Bacillus atrophaeus endospores was achieved. Theoretical and experimental results demonstrated synergistic inactivation to occur through fragmentation of coat via interfacial electron transfer and electro-produced transient radicals (•OH primarily, •Cl and Cl2•– secondarily), thereby increasing cell permeability to facilitate penetration of electro-produced persistent active chlorine for subsequent rupture of intracellular structures. Numbering-up electrode module strategy was proposed to scale up the system, achieving average 5.3-log inactivation of pathogenic Bacillus anthracis endospores for 30 days. This study demonstrates a proof-of-concept manner for effective inactivation of waterborne bacterial endospores, which may provide an appealing strategy for wide-range applications like water disinfection, bio-safety control and defense against biological warfare.

Mifepristone increases AQP1 mRNA expression, angiogenesis, and cell permeability through the ERK MAPK pathway.

The purpose of this study was to investigate the mechanism of mifepristone serves as an anti-implantation contraceptive drug on aquaporins 1 (AQP1) expression. Human umbilical vein endothelial cells (HUVECs) were used to detect the effects of different concentrations of mifepristone (0, 0.065, 0.2, and 1μmol/L) on the activity of angiogenesis and AQP1 expression. The expression of AQP1 was tested by the real-time PCR. The angiogenesis and penetration function of HUVECs was investigated by Matrigel lumen formation and trans-well assay, respectively. The expression of AQP1, angiogenesis and cell permeability were significantly higher than control groups in HUVECs treatment with mifepristone at 1μmol/L for 12h. Estrogen and progesterone decreased the up-regulation of AQP1 and cell permeability, not angiogenesis, induced by mifepristone. Mifepristone increased protein levels of p-ERK, not p-p38 or p-JNK, and pre-treatment with ERK MAPK-specific inhibitor significantly inhibited the up-regulation of AQP1 mRNA expression, angiogenesis and cell permeability induced by mifepristone. si-AQP1 significantly reduced the up-regulation of angiogenesis, cell permeability and p-ERK/ERK ratio expression induced by mifepristone treatment. Overexpression of AQP1 enhanced the increase of expression ratio of p-ERK/ERK induced by mifepristone. Low-dose mifepristone increased cell permeability, angiogenesis and AQP1 expression, which was involved in MAPK pathways. This provides new insights into the molecular mechanism of mifepristone serves as an anti-implantation contraceptive drug.

The Complement System Mediates COVID-19 Endothelial Cell Injury, Modifying Cell Permeability

Background Coronavirus Disease 2019 (COVID-19) is a (SARS-CoV-2)-mediated respiratory disease. The severity of COVID-19 has been classified as mild, moderate, severe and critical per the WHO-China Joint Mission. Among a total of 72 314 case records from the Chinese Center for Disease Control and Prevention, approximately 81% of COVID-19 cases were defined as mild, 14% of as severe, and 5% as critical. The overall hospital mortality of COVID-19 cases is approximately 15-20%, but it is up to 40-49% among critical cases requiring intensive care unit admission. The complement system, is a key component of the innate immune response, consists of more than 50 soluble and membrane-bound proteins that provide defense against microbes and mediate inflammatory responses. The activation of the complement system involves a series of enzymatic transformations, where C3b acts as a mediator. The final step of the complement cascade is the formation of a membrane attack complex (MAC, C5b-9) on the plasma membrane (PM) of the cells. Several pathologies including thrombotic, inflammatory, autoimmune and age-related diseases are associated with abnormal complement activation. There is evidence linking the activation of the complement system and endothelial injury to organ damage and complications that increase the risk of mortality in COVID-19. We hypothesize that endothelial cell injury resulting from complement activation contributes to COVID-19-associated vascular injury and organ damage. Aims To identify differences in complement activation associated with mild and severe forms of COVID-19 To determine the role of differential complement activation by COVID-19 patient sera in mediating endothelial cell damage. Methodology Clinical information, and sera from SARS-CoV-2 positive patients with mild and severe COVID-19 were obtained from the Canadian COVID-19 Prospective Cohort Study (CANCOV) to identify complement activation and endothelial cell (EC) injury. Complement activation was determined using a previously established protocol (Noris et al., 2017). Briefly, primary human umbilical vein endothelial cells (HUVECs) were treated with adenosine diphosphate (ADP), followed by treatment with healthy control or COVID-19 patient serum. To confirm the biological activity of complement, we measured the deposition of C3b and C5b-9 on the plasma membrane of HUVECs via immunofluorescence (IF) on cells exposed to healthy or patient sera. In addition, a permeability assay using a transwell model was used to measure the changes in permeability induced by healthy or patient sera via complement activation. Briefly, HUVECs were seeded on the top part of the insert, allowing them to form a monolayer. Monolayer integrity was assessed by imaging cells stained with Hoechst (nuclear stain) and phalloidin (cytoskeleton stain) on the day of the experiment, as well as before and after incubation with healthy or patient sera. Results We were able to detect complement activation/deposition when exposing the cells to COVID-19 sera. As for C3b deposition, cells exposed to patient sera showed no difference compared to those treated with sera from healthy donors. However, we observed increased C5b-9 deposition on the PM of cells treated with patient sera compared to those treated with sera from healthy donors, with further increased deposition when cells are treated with deceased patient sera. We observed a trend demonstrating increased permeability when cells are treated with deceased COVID-19 patient sera compared to healthy donors. Cellular permeability is significantly lower in those cells treated sera from patients with mild or severe COVID-19. Conclusion There is a differential biological activity of complement on endothelial cells depending on the severity of COVID-19, this complement activation leads to an increase in cell permeability. While more research needs to be done, these results contribute to a better understanding of the consequences of complement activation on endothelium in COVID-19.

Whole transcriptome sequencing analysis of synergistic combinations of plant-based antimicrobials and zinc oxide nanoparticles against Campylobacter jejuni

The emergence of antibiotic resistance among animal farms impels the development of novel antimicrobials or strategies for agri-food production. The combinational use of agents to achieve a synergistic antimicrobial effect provides many advantages such as dosage reduction, shortened treatment time, and avoidance of antimicrobial resistance. In this study, we evaluated the killing efficacy of single agent or combinational use of three antimicrobials, including cinnamon oil, encapsulated curcumin and zinc oxide nanoparticles (ZnO NPs), against a leading foodborne pathogen Campylobacter jejuni. We then investigated the antimicrobial mechanism using whole transcriptome sequencing analysis (RNA-Seq). The single-agent treatment of cinnamon oil, encapsulated curcumin, or ZnO NPs showed a significant antimicrobial effect against C. jejuni by generating more than 8-log reduction within 3 h. The transcriptional signatures of C. jejuni in response to these agents varied, indicating that these agents shared distinct mechanisms of action and were likely to generate synergistic effects. Cinnamon oil affected the integrity of cell membrane, which might lead to an increase in cell permeability. Encapsulated curcumin and ZnO NPs disrupted bacterial outer membranes and cell membranes against the same membrane protein targets. The combinational use of these agents showed synergistic antimicrobial effects and distinct mechanisms of action compared to single treatment. The combination of cinnamon oil and encapsulated curcumin provoked the expression of cellular signaling, but repressed the chemotaxis-associated genes. The antimicrobial resistance associated genes showed a low expression level in the combination of encapsulated curcumin and ZnO NPs. The tri-combination treatment systematically overexpressed genes involved in the amino acid synthesis, protein translation, and membrane protein synthesis. This study provides new insights in combating Campylobacter with minimizing the development of antimicrobial resistance in long-term usage.

Caveolin-1 accelerates hypoxia-induced endothelial dysfunction in high-altitude cerebral edema

BackgroundHigh-altitude cerebral edema (HACE) is a serious and potentially fatal brain injury that is caused by acute hypobaric hypoxia (HH) exposure. Vasogenic edema is the main pathological factor of this condition. Hypoxia-induced disruptions of tight junctions in the endothelium trigger blood‒brain barrier (BBB) damage and induce vasogenic edema. Nuclear respiratory factor 1 (NRF1) acts as a major regulator of hypoxia-induced endothelial cell injury, and caveolin-1 (CAV-1) is upregulated as its downstream gene in hypoxic endothelial cells. This study aimed to investigate whether CAV-1 is involved in HACE progression and the underlying mechanism.MethodsC57BL/6 mice were exposed to HH (7600 m above sea level) for 24 h, and BBB injury was assessed by brain water content, Evans blue staining and FITC-dextran leakage. Immunofluorescence, transmission electron microscope, transendothelial electrical resistance (TEER), transcytosis assays, and western blotting were performed to confirm the role and underlying mechanism of CAV-1 in the disruption of tight junctions and BBB permeability. Mice or bEnd.3 cells were pretreated with MβCD, a specific blocker of CAV-1, and the effect of CAV-1 on claudin-5 internalization under hypoxic conditions was detected by immunofluorescence, western blotting, and TEER. The expression of NRF1 was knocked down, and the regulation of CAV-1 by NRF1 under hypoxic conditions was examined by qPCR, western blotting, and immunofluorescence.ResultsThe BBB was severely damaged and was accompanied by a significant loss of vascular tight junction proteins in HACE mice. CAV-1 was significantly upregulated in endothelial cells, and claudin-5 explicitly colocalized with CAV-1. During the in vitro experiments, hypoxia increased cell permeability, CAV-1 expression, and claudin-5 internalization and downregulated tight junction proteins. Simultaneously, hypoxia induced the upregulation of CAV-1 by activating NRF1. Blocking CAV-1-mediated intracellular transport improved the integrity of TJs in hypoxic endothelial cells and effectively inhibited the increase in BBB permeability and brain water content in HH animals.ConclusionsHypoxia upregulated CAV-1 transcription via the activation of NRF1 in endothelial cells, thus inducing the internalization and autophagic degradation of claudin-5. These effects lead to the destruction of the BBB and trigger HACE. Therefore, CAV-1 may be a potential therapeutic target for HACE.E5r9nDKChFa7F_nrUkRwh3Video abstract

Cigarette smoke promotes inflammasome-independent activation of caspase-1 and -4 leading to gasdermin D cleavage in human macrophages.

Mechanisms and consequences of gasdermin D (GSDMD) activation in cigarette smoke (CS)-associated inflammation and lung disease are unknown. GSDMD is a downstream effector of caspase-1, -8, and -4. Upon cleavage, GSDMD generates pores into cell membranes. Different degrees of GSDMD activation are associated with a range of physiological outputs ranging from cell hyperactivation to pyroptosis. We have previously reported that in human monocyte-derived macrophages CS extract (CSE) inhibits the NLRP3 inflammasome and shifts the response to lipopolysaccharide (LPS) towards the TLR4-TRIF axis leading to activation of caspase-8, which, in turn, activates caspase-1. In the present work, we investigated whether other ASC-dependent inflammasomes could be involved in caspase activation by CSE and whether caspase activation led to GSDMD cleavage and other downstream effects. Presented results demonstrate that CSE promoted ASC-independent activation of caspase-1 leading to GSDMD cleavage and increased cell permeability, in the absence of cell death. GSDMD cleavage was strongly enhanced upon stimulation with LPS+CSE, suggesting a synergistic effect between the two stimuli. Noteworthy, CSE promoted LPS internalization leading to caspase-4 activation, thus contributing to increased GSDMD cleavage. Caspase-dependent GSDMD cleavage was associated with mitochondrial superoxide generation. Increased cleaved GSDMD was found in lung macrophages of smokers compared to ex-smokers and non-smoking controls. Our findings revealed that ASC-independent activation of caspase-1, -4, and -8 and GSDMD cleavage upon exposure to CS may contribute to macrophage dysfunction and feed the chronic inflammation observed in the smokers' lung.

Para-chloro-meta-xylenol reshaped the fates of antibiotic resistance genes during sludge fermentation: Insights of cell membrane permeability, bacterial structure and biological pathways

The occurrence of para-chloro-meta-xylenol (PCMX, as largely consumed antimicrobial chemicals) in waste activated sludge (WAS) would pose environmental risks for WAS utilization. This study revealed that PCMX principally prompted the abundances and diversity of antibiotic resistance genes (ARGs), particularly for the multidrug- genes (i.e., acrB and mexW), and reshaped the resistance mechanism categories during WAS fermentation process. The genotype and phenotype results indicated that PCMX upregulated abundances of transposase and increased cell permeability via disrupting WAS structure, which further facilitated the horizontal transfer of ARGs. The network and correlation analysis among ARGs, mobile genetic elements (MGEs) and genera (i.e., Sphingopyxis and Pseudoxanthomonas) verified that PCMX enriched the potential ARGs hosts associated with multidrug resistance mechanism. Also, PCMX upregulated the genes involved in ARGs-associated metabolic pathways, such as two-component (i.e., phoP and vcaM) and quorum sensing systems (i.e., lasR and cciR), which determined the ARGs proliferation via multidrug efflux pump and outer membrane proteins, and facilitated the recognition between ARGs hosts. Variance partitioning analysis (VPA) implied that the shift of microbial community contributed predominantly to the dissemination of ARGs. These findings unveiled the environmental behaviors and risks of exogenous pollutants in WAS with insightful understanding, which could guide the WAS utilization for resource recovery.