Membrane Barrier Function Research Articles

Diagnostic models for differentiating fatty liver disease of alcohol and non-alcoholic genesis

Introduction. Fatty liver disease is the largest contributor to the burden of chronic liver disease worldwide. Current approaches do not allow sufficient differentiation between alcoholic and non-alcoholic etiology of the process.Aim. Create diagnostic panels including electrical and viscoelastic parameters of erythrocytes to differentiate fatty liver disease of alcoholic and non-alcoholic genesis.Materials and methods. The study included 38 men (47.5 ± 2.9 years) with NAFLD; 31 men with alcoholic fatty liver disease (AFLD) (45.1 ± 3.1 years) according to ultrasound of the abdominal organs, the degree of fibrosis did not exceed F1 (FibroScan® 502). Electrical and viscoelastic parameters of erythrocytes were studied by dielectrophoresis using an electro-optical cell detection system. To determine the parameters of erythrocytes – biomarkers for distinguishing between AFLD and NAFLD, a system of machine learning methods – Random Forest was used.Results. Electrical, viscoelastic parameters of erythrocytes, which are biomarkers for distinguishing between AFLD and NAFLD, were established: cell membrane capacity (p = 1.21E-11), the degree of change in the deformation amplitude at a frequency of 5 x 105 Hz (p = 2.38E-08), cell polarizability at a frequency of 106 Hz (p = 9.38E-08), the speed of erythrocyte movement to the electrodes (p = 4.32E-06), the magnitude of the dipole moment (p = 1.66E-05), relative polarizability (p = 2.35E-05), the index of erythrocyte destruction at a frequency of 5 x 105 Hz (p = 0.016), the position of the crossover frequency (p = 2.13E- 06). The diagnostic model, including five parameters – the position of the crossover frequency, cell polarizability at a frequency of 106 Hz, cell electrical conductivity, membrane capacity, the degree of change in the deformation amplitude at a frequency of 5 x 105 Hz, provided the highest diagnostic accuracy with an AUC of 0.975, a sensitivity of 96.3%, and a specificity of 91.8% in differentiating between AFLD and NAFLD.Conclusion. Thus, systematic exposure to alcohol modifies the structure of erythrocyte membranes, leading to a decrease in the surface charge, the barrier function of membranes, reducing the resistance of cells, their ability to deform, which determines the key role of the identified electrical, viscoelastic parameters of erythrocytes in differentiating between AFLD and NAFLD.

Fission of double-membrane tubes under tension

The division of a cellular compartment culminates with the scission of a highly constricted membrane neck. Scission requires lipid rearrangements, topology changes, and transient formation of nonbilayer intermediate structures driven by curvature stress. Often, a side effect of this stress is pore formation, which may lead to content leakage and thus breaching of the membrane barrier function. In single-membrane systems, leakage is avoided through the formation of a hemifusion (HF) intermediate, whose structure is still a subject of debate. The consequences of curvature stress have not been explored in double-membrane systems, such as the mitochondrion. Here, we combine experimental and theoretical approaches to study neck constriction and scission driven by tension in biomimetic lipid systems, namely single- and double-membrane nanotubes (sNTs and dNTs), respectively. In sNTs, constriction by high tension gives rise to a metastable HF intermediate (seen as stalk or worm-like micelle), whereas poration is universally slower in a simple neck. In dNTs, high membrane tension causes sequential rupture of each membrane. In contrast, low tension leads to the HF of both membranes, which may lead to a leaky fusion pathway, or may progress to further fusion of the two membranes along a number of transformation pathways. These findings provide a new mechanistic basis for fundamental cellular processes.

Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here, we show that early-life thermal stress strongly up-regulates tsp-1, a gene encoding the conserved transmembrane tetraspanin in C. elegans. TSP-1 forms prominent multimers and stable web-like structures critical for membrane barrier functions in adults and during aging. Increased TSP-1 abundance persists even after transient early-life heat stress. Such regulation requires CBP-1, a histone acetyltransferase that facilitates initial tsp-1 transcription. Tetraspanin webs form regular membrane structures and mediate resilience-promoting effects of early-life thermal stress. Gain-of-function TSP-1 confers marked C. elegans longevity extension and thermal resilience in human cells. Together, our results reveal a cellular mechanism by which early-life thermal stress produces long-lasting memory-like impact on organismal resilience and longevity.

The local variation of the Gaussian modulus enables different pathways for fluid lipid vesicle fusion

Viral infections, fertilization, neurotransmission, and many other fundamental biological processes rely on membrane fusion. Straightforward calculations based on the celebrated Canham–Helfrich elastic model predict a large topological energy barrier that prevents the fusion process from being thermally activated. While such high energy is in accordance with the physical barrier function of lipid membranes, it is difficult to reconcile with the biological mechanisms involved in fusion processes. In this work, we use a Ginzburg–Landau type of free energy that recovers the Canham–Helfrich model in the limit of small width-to-vesicle-extension ratio, with the additional ability to handle topological transitions. We show that a local modification of the Gaussian modulus in the merging region both dramatically lowers the elastic energy barrier and substantially changes the minimal energy pathway for fusion, in accordance with experimental evidence. Therefore, we discuss biological examples in which such a modification might play a crucial role.

Comprehensive Analysis of PANoptosis-Related Gene Signature of Ulcerative Colitis.

Accumulating evidence shows that the abnormal increase in the mortality of intestinal epithelial cells (IECs) caused by apoptosis, pyroptosis, and necroptosis is closely related to the function of mucous membrane immunity and barrier function in patients with ulcerative colitis (UC). As a procedural death path that integrates the above-mentioned many deaths, the role of PANoptosis in UC has not been clarified. This study aims to explore the characterization of PANoptosis patterns and determine the potential biomarkers and therapeutic targets. We constructed a PANoptosis gene set and revealed significant activation of PANoptosis in UC patients based on multiple transcriptome profiles of intestinal mucosal biopsies from the GEO database. Comprehensive bioinformatics analysis revealed five key genes (ZBP1, AIM2, CASP1/8, IRF1) of PANoptosome with good diagnostic value and were highly correlated with an increase in pro-inflammatory immune cells and factors. In addition, we established a reliable ceRNA regulatory network of PANoptosis and predicted three potential small-molecule drugs sharing calcium channel blockers that were identified, among which flunarizine exhibited the highest correlation with a high binding affinity to the targets. Finally, we used the DSS-induced colitis model to validate our findings. This study identifies key genes of PANoptosis associated with UC development and hypothesizes that IRF1 as a TF promotes PANoptosome multicomponent expression, activates PANoptosis, and then induces IECs excessive death.

Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity.

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

Сучасні уявлення про значущість забезпечення цинком при інфекційній патології в дітей

Zinc (Zn) is an essential trace element that is critical for growth, development and maintenance of immune function. Zn is an indispensable trace element for humans, which is part of more than 20 metalloenzymes, including DNA and RNA polymerase, phosphatase, carbonic anhydrase and some others. The cycle of cell development requires this trace element. The permeability of cell membranes is also related to Zn. It has antioxidant properties and delays the apoptosis of peripheral cells. The formation of immunity depends on the body’s supply of Zn, and its deficiency causes atrophy of the thymic-lymphatic system. Zn is part of insulin, accelerates the regeneration of the mucous layer of the intestines, increases the activity of enzymes of the brush border of enterocytes, increases the level of secretory antibodies and the intensity of cellular immunity. The provision of this element cannot but affect the course of various infectious pathologies. Purpose - by analyzing modern literature data, to find out the importance of Zn provision in infectious pathologies in children. It is analyzed a review of the literature on the pathogenetic role of Zn in the diagnosis, pathogenesis and treatment of infectious diseases, based on a search for articles in the Science, Medline and PubMed databases published from January 2017 to June 2021. An analysis of current literature has shown that Zn is an important micronutrient involved in the regulation of innate and adaptive immune responses. Zn is necessary to ensure the barrier function of membranes. It is involved in the modulation of the pro-inflammatory response. Zn homeostasis is essential for many aspects of the immune system, including hematopoiesis, cell maturation and differentiation, cell cycle progression, and proper immune cell function. Consequently, Zn deficiency leads to cell-mediated immune dysfunctions. Such dysfunctions lead to deterioration of the body's response to bacterial infection. Zn is known to modulate antiviral immunity. Due to zinc, the production of interferon-α is activated and its antiviral activity is increased. Studies show that during the infectious process there is a decrease in the concentration of zinc in the blood plasma. Such changes, on the one hand, provide the needs of immune protection factors, and on the other hand, reduce the availability of the trace element for pathogens. Conclusions. Zn is one of the important trace elements that plays a leading role in maintaining homeostasis and is an integral component of the pathogenesis of various pathological conditions of infectious origin. The most studied is Zn deficiency in infectious diarrhea in children, but studies have been conducted in developing countries with limited resources. No works studying the prevalence of Zn deficiency in Ukraine were found. No conflict of interests was declared by the authors.

DRUG RESISTANCE: THE STATUS OF THE PROBLEM AND THE SEARCH OF WAYS TO OVERCOME IT (literature review and own research)

The ability of multicellular associates to undergo changes that provide increased resistance to adverse environmental factors determines the development of drug resistance. Over the past decades, it has turned into a complex medical and social problem, which complicates significantly the treatment of countless diseases. In particular, the rapid formation and spread of antibiotic-resistant forms of microorganisms causes the risk of relegating clinical medicine to the pre-antibiotic era. An equally acute problem is the growing resistance of cells of malignant neoplasms to the action of cytostatics as the tumor progresses and during its recurrence. The obvious relevance of these problems for means of counteracting such changes determines the unabated interest in elucidating the molecular and cellular bases of the development of drug resistance. The existence of a certain parallel in the functioning of the cellular societies of biofilms and malignant neoplasms allows us to approach the understanding of the molecular and cellular mechanisms of the development of drug resistance. The role of disruption of the barrier function of the outer cell membranes and the increase in their permeability to extracellular nucleic components is shown in this process. The role of individual components of cellular associates in the formation of drug-resistant, mechanisms of their spread and malignization of surrounding tissues is discussed. Key words: drug resistance, biofilms, malignant neoplasms, cell membranes.

Nanosecond PEF Induces Oxidative Stress and Apoptosis via Proteasomal Activity Inhibition in Gastric Adenocarcinoma Cells with Drug Resistance.

Nanosecond (ns) pulsed electric field (PEF) is a technology in which the application of ultra-short electrical pulses can be used to disrupt the barrier function of cell plasma and internal membranes. Disruptions of the membrane integrity cause a substantial imbalance in cell homeostasis in which oxidative stress is a principal component. In the present study, nsPEF-induced oxidative stress was investigated in two gastric adenocarcinoma cell lines (EPG85-257P and EPG85-257RDB) which differ by their sensitivity to daunorubicin. Cells were exposed to 200 pulses of 10 ns duration, with the amplitude and pulse repetition frequency at 1 kHz, with electric field intensity varying from 12.5 to 50 kV/cm. The electroporation buffer contained either 1 mM or 2 mM calcium chloride. CellMask DeepRed visualized cell plasma permeabilization, Fluo-4 was used to visualize internal calcium ions content, and F-actin was labeled with AlexaFluor®488 for the cytoskeleton. The cellular viability was determined by MTT assay. An alkaline and neutral comet assay was employed to detect apoptotic and necrotic cell death. The luminescent method estimated the modifications in GSSG/GSH redox potential and the imbalance of proteasomal activity (chymotrypsin-, trypsin- and caspase-like). The reactive oxygen species (ROS) level was measured by flow cytometry using dihydroethidium (DHE) dye. Morphological visualization indicated cell shrinkage, affected cell membranes (characteristic bubbles and changed cell shape), and the reorganization of actin fibers with sites of its dense concentration; the effect was more intense with the increasing electric field strength. The most significant decrease in cell viability and GSSG/GSH redox potential was noted at the highest amplitude of 50 kV/cm, and calcium ions amplified this effect. nsPEF, particularly with calcium ions, inhibited proteasomal activities, resulting in increased protein degradation. nsPEF increased the percentage of apoptotic cells and ROS levels. The EPG85-257 RDB cell line, which is resistant to standard chemotherapy, was more sensitive to applied nsPEF protocols. The applied nsPEF method disrupted the metabolism of cancer cells and induced apoptotic cell death. The nsPEF ability to cause apoptosis, oxidative stress, and protein degradation make the nsPEF methodology a suitable alternative to current anticancer pharmacological methods.

Sphingolipid Metabolism and Signaling in Lung Cancer: A Potential Therapeutic Target.

Sphingolipids are important bioactive lipids that not only play an important role in maintaining the barrier function and fluidity of cell membranes but also regulate multiple processes in cancer development by controlling multiple signaling pathways in the signal transduction network. Dysregulation of sphingolipid metabolism is thought to be one of the most important dysregulated pathways in lung cancer, the most prevalent type of cancer in terms of incidence and mortality worldwide. This article focuses on lung cancer, reviewing the important lipids in sphingolipid metabolism and the related enzymes in relation to lung cancer progression and their effects on the tumor microenvironment and discussing their roles in the diagnosis and treatment of lung cancer.

3D-printed bi-layered polymer/hydrogel construct for interfacial tissue regeneration in a canine model

ObjectivesThere are complications in applying regenerative strategies at the interface of hard and soft tissues due to the limited designs of constructs that can accommodate different cell types in different sites. The problem originates from the challenges in the adhesion of dissimilar materials, such as polymers and hydrogels, that can be suitable for regenerating different tissues such as bone and soft tissues. This paper presents a design of a new hybrid construct in which a polymer (polycaprolactone (PCL)) membrane firmly adheres to a layer of hydrogen (gelatin). MethodsPCL membranes with defined size and porosity were fabricated using 3D printing. The gelatin layer was attached to the PCL membranes using the aminolysis procedure. We have examined this construct for the application of Guided Bone Regeneration (GBR) as a typical surgical regenerative procedure of the oral cavity at the interface of bone and soft tissue. Complete in vitro and in vivo investigations on canine tibia bone defects have been performed. Histological analyses for fibrosis morphometric and bone morphometric evaluation, as well as bone-fibrosis histological grading and CBCT imaging, were conducted. ResultsChemical and morphological studies of the membrane proved that gelatin was uniformly attached to the aminolyzed PCL membranes. The in vitro and in vivo studies indicated the membrane's biocompatibility, mechanical stability, and barrier function for the GBR application. Furthermore, in vitro study showed that the membranes could improve osteogenesis and the regeneration of bone defects. The results illustrated that the mean bone density in the membrane groups was about three times more than that of the control group. SignificanceThe fabricated 3D-printed hybrid Gelatin/PCL bi-layered membrane can be a good candidate for interfacial tissue engineering and a promising membrane for GBR procedure.

Lactobacillus plantarum improves LPS-induced Caco2 cell line intestinal barrier damage via cyclic AMP-PKA signaling.

Lactobacillus plantarum (LP) has been shown to exhibit protective effects on intestinal barrier function in septic rats, although the regulatory mechanism has not been established. We determined whether LP imparts such protective effects in a lipopolysaccharide (LPS)-induced Caco2 cell monolayer model and whether cAMP-PKA signaling is the underlying mechanism of action. The cyclic adenosine monophosphate (cAMP) agonist, forskolin (FSK), and the protein kinase A (PKA) inhibitor, HT89, were used to study the protective effect of LP on the destruction of the tight junction (TJ) structure of cells treated with LPS and the corresponding changes in cAMP-PKA signaling. Our experimental results demonstrated that LP promoted the expression of TJ proteins between Caco2 cells after LPS treatment, and increased the electrical barrier detection (TEER) between Caco2 cells. Moreover, transmission electron microscopy (TEM) revealed that the TJ structural integrity of cells treated with LPS + LP was improved compared to cells treated with LPS alone. In addition, our findings were consistent between the FSK and LP intervention group, while HT89 inhibited LP influence. Taken together, our results indicate that LP has an improved protective effect on LPS-induced damage to the monolayer membrane barrier function of Caco2 cells and is regulated by the cAMP-PKA pathway.

Loss of β-Ketoacyl Acyl Carrier Protein Synthase III Activity Restores Multidrug-Resistant Escherichia coli Sensitivity to Previously Ineffective Antibiotics.

ABSTRACTAntibiotic resistance is one of the most prominent threats to modern medicine. In the latest World Health Organization list of bacterial pathogens that urgently require new antibiotics, 9 out of 12 are Gram-negative, with four being of “critical priority.” One crucial barrier restricting antibiotic efficacy against Gram-negative bacteria is their unique cell envelope. While fatty acids are a shared constituent of all structural membrane lipids, their biosynthesis pathway in bacteria is distinct from eukaryotes, making it an attractive target for new antibiotic development that remains less explored. Here, we interrogated the redundant components of the bacterial type II fatty acid synthesis (FAS II) pathway, showing that disrupting FAS II homeostasis in Escherichia coli through deletion of the fabH gene damages the cell envelope of antibiotic-susceptible and antibiotic-resistant clinical isolates. The fabH gene encodes the β-ketoacyl acyl carrier protein synthase III (KAS III), which catalyzes the initial condensation reactions during fatty acid biosynthesis. We show that fabH null mutation potentiated the killing of multidrug-resistant E. coli by a broad panel of previously ineffective antibiotics, despite the presence of relevant antibiotic resistance determinants, for example, carbapenemase kpc2. Enhanced antibiotic sensitivity was additionally demonstrated in the context of eradicating established biofilms and treating established human cell infection in vitro. Our findings showcase the potential of FabH as a promising target that could be further explored in the development of therapies that may repurpose currently ineffective antibiotics or rescue failing last-resort antibiotics against Gram-negative pathogens.IMPORTANCE Gram-negative pathogens are a major concern for global public health due to increasing rates of antibiotic resistance and the lack of new drugs. A major contributing factor toward antibiotic resistance in Gram-negative bacteria is their formidable outer membrane, which acts as a permeability barrier preventing many biologically active antimicrobials from reaching the intracellular targets and thus limiting their efficacy. Fatty acids are the fundamental building blocks of structural membrane lipids, and their synthesis constitutes an attractive antimicrobial target, as it follows distinct pathways in prokaryotes and eukaryotes. Here, we identified a component of fatty acid synthesis, FabH, as a gate-keeper of outer membrane barrier function. Without FabH, Gram-negative bacteria become susceptible to otherwise impermeable antibiotics and are resensitized to killing by last-resort antibiotics. This study supports FabH as a promising target for inhibition in future antimicrobial therapies.

Disruption of the Cytoplasmic Membrane Structure and Barrier Function Underlies the Potent Antiseptic Activity of Octenidine in Gram-Positive Bacteria.

ABSTRACTThe antimicrobial killing mechanism of octenidine (OCT), a well-known antiseptic is poorly understood. We recently reported its interaction with Gram-negative bacteria by insertion of OCT into the outer and cytoplasmic membrane of Escherichia coli, resulting in a chaotic lipid rearrangement and rapid disruption of the cell envelope. Its action primarily disturbs the packing order of the hydrophobic moiety of a lipid, which consequently might result in a cascade of multiple effects at a cellular level. Here, we investigated OCT’s impact on two different Gram-positive bacteria, Enterococcus hirae and Bacillus subtilis, and their respective model membranes. In accordance with our previous results, OCT induced membrane disorder in all investigated model systems. Electron and fluorescence microscopy clearly demonstrated changes in cellular structure and membrane integrity. These changes were accompanied by neutralization of the surface charge in both E. hirae and B. subtilis and membrane disturbances associated with permeabilization. Similar permeabilization and disordering of the lipid bilayer was also observed in model membranes. Furthermore, experiments performed on strongly versus partly anionic membranes showed that the lipid disordering effect induced by OCT is a result of maximized hydrophobic over electrostatic forces without distinct neutralization of the surface charge or discrimination between the lipid head groups. Indeed, mutants lacking specific lipid head groups were also susceptible to OCT to a similar extent as the wild type. The observed unspecific mode of action of OCT underlines its broad antimicrobial profile and renders the development of bacterial resistance to this molecule less likely.IMPORTANCE OCT is a well-established antiseptic molecule routinely used in a large field of clinical applications. Since the spread of antimicrobial resistance has restricted the use of antibiotics worldwide, topically applied antiseptics like OCT, with a broad spectrum of antimicrobial activity and high safety profile, gain increasing importance for effective infection prevention and therapy. To eliminate a wide spectrum of disease-causing microorganisms, a compound’s antiseptic activity should be unspecific or multitarget. Our results demonstrate an unspecific mechanism of action for OCT, which remained largely unknown for years. OCT disturbs the barrier function of a bacterial cell, a function that is absolutely fundamental for survival. Because OCT does not distinguish between lipids, the building blocks of bacterial membranes, its mode of action might be attributed to all bacteria, including (multi)drug-resistant isolates. Our results underpin OCT’s potent antiseptic activity for successful patient outcome.

Measuring the barrier function of skeletal muscle in individuals with muscular dystrophy

A pilot study (NCT:01851447) was conducted to identify predictive biomarkers of decreasing barrier function of skeletal muscle membranes associated with the pathophysiology and clinical severity of fragile sarcolemmal muscular dystrophies. Membrane barrier function was evaluated over five days using plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), and lactate dehydrogenase (LDH) levels, before and after routine morning activities and motor function testing in eleven ambulatory subjects with limb-girdle muscular dystrophy (LGMD).

Epithelial immune regulation of inflammatory airway diseases: Chronicrhinosinusitis with nasal polyps(CRSwNP).

Background: The epithelial immune regulation is an essential and protective feature of the barrier function of the mucous membranes of the airways. Damage to the epithelial barrier can result in chronic inflammatory diseases, such as chronic rhinosinusitis (CRS) or bronchial asthma. Thymic stromal lymphopoietin (TSLP) is a central regulator in the epithelial barrier function and is associated with type 2 (T2) and non-T2 inflammation. Materials and methods: The immunology of chronic rhinosinusitis with polyposis nasi (CRSwNP) was analyzed in a literature search, and the existing evidence was determined through searches in Medline, Pubmed as well as the national and international study and guideline registers and the Cochrane Library. Human studies or studies on human cells that were published between 2010 and 2020 and in which the immune mechanisms of TSLP in T2 and non-T2 inflammation were examined were considered. Results: TSLP is an epithelial cytokine (alarmin) and a central regulator of the immune reaction, especially in the case of chronic airway inflammation. Induction of TSLP is implicated in the pathogenesis of many diseases like CRS and triggers a cascade of subsequent inflammatory reactions. Conclusion: Treatment with TSLP-blocking monoclonal antibodies could therefore open up interesting therapeutic options. The long-term safety and effectiveness of TSLP blockade has yet to be investigated.

DISRUPTION OF THE BARRIER FUNCTION OF CELL MEMBRANES IN THE FORMATION OF MALIGNANT NEOPLASMS AND BIOFILMS

The common features of the functioning of cellular systems of malignant neoplasms and biofilms are considered. Particular attention is paid to the role of the barrier function of cell membranes. The causes of changes in membrane permeability and their functional consequences are discussed. The position on the increase of cell permeability through membranes as a key condition for the formation and development of both types of cellular anomalies is substantiated.