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Clothianidin (CL) is a neonicotinoid insecticide widely used in crop protection against insect pests. However, its effects on photosynthesis remain largely unknown. Here, by investigating the influence of CL at the concentrations of 22 and 110 μg/L on the primary processes of photosynthesis, membrane fluidity and structural changes of pea chloroplasts, we located several primary binding sites of this pesticide. Similar dynamics were observed for both concentrations. However, statistically significant differences were only found at 110 μg/L for all methods used. The light saturated rate of linear electron flow decreased mainly due to the disturbance of electron flow on the acceptor side of photosystem II (PSII) associated with the appearance of QB‐nonreducing centers and empty QB binding sites of PSII. The functioning of the donor side of PSII, the activity of photosystem I (PSI) and the maximum quantum yield of PSII photochemistry (Fv/Fm) were not found to be significantly altered. Increased membrane fluidity and structural alterations of the thylakoid membrane led to a decrease in the development of the proton gradient ΔрН and membrane energization processes. [ABSTRACT FROM AUTHOR]

The study of cell membrane structures under microgravity is crucial for understanding the inherent physiological and adaptive mechanisms relevant to overcoming challenges in human space travel and gaining deeper insight into the membrane-protein interactions at reduced gravity. However, the membrane dynamics under microgravity conditions is not unraveled yet. Moreover, the complexity of cells poses significant challenges when investigating the effects of microgravity on individual components, including cell membranes. Giant Unilamellar Vesicles (GUVs) serve as valuable cell-mimicking models and act as artificial cells, providing insights into the biophysics of membrane architecture. Herein, we have elucidated the membrane dynamics of artificial cells under simulated microgravity conditions. GUVs were synthesized in the size range of 20 ± 2.1 μm and their morphological changes were examined under simulated microgravity conditions using a random positioning machine. We observed that the well-defined spherical GUVs were transfigured and deformed into elongated structures under microgravity conditions. The membrane fluidity of GUVs increased sevenfold under microgravity conditions compared to GUVs under normal gravity conditions at 48 h. It is also noted that there is a reduction in the membrane microviscosity. The study sheds light on the membrane mechanics under microgravity conditions and contributes valuable insights to the broader understanding of membrane responses to microgravity and its implications for space exploration and biomedical applications. [ABSTRACT FROM AUTHOR]

Polydiacetylene (PDA) is a kind of photopolymerizable polymer, which exhibits a unique color transition in response to external stimuli such as heat, pH, and solvent. PDAs are attractive as eye‐detection stimulus sensors with excellent time performance; however, the sensitivity of PDAs should be improved. Considering the biological membrane‐like structure of diacetylene (DA) vesicles, their modification by incorporating membrane lipids (e.g., diacylphosphocholine, PC) can be used to control the membrane fluidity, and consequently molecular ordering of DAs in the vesicle. Inspired by biological membrane systems, lipid vesicles are employed as platforms to generate PDA, and essential factors that influence the sensitivity of PDA are investigated. By lowering the polymerization temperature, the generation of PDA becomes slower, while the sensitivity improves. By adding PCs at the molar ratio of lipid:DA = 1:1, the sensitivity of PDA can be varied: the PCs with lower phase transition temperatures (Tm) made PDA insensitive, while the PCs with higher Tm improved the sensitivity as compared to pure poly(PCDA). It is concluded that the photopolymerization of DAs with a lower membrane fluidity induces highly sensitive PDA, while the photopolymerization of DAs with a higher membrane fluidity induces insensitive PDA with robustness toward stimuli. [ABSTRACT FROM AUTHOR]

Arsenic (As) in groundwater and soils for soybean (Glycine max L.) irrigation and cultivation constitutes a major agronomic problem since the metalloid can be incorporated into plant tissues. Phospholipids (PL), and their constituent fatty acids (FA) are main components of cell membranes, so they are one of the biomolecules that firstly sense external conditions. Since the role of PL and FA in the response of soybean to As is unknown, our objective was to analyze possible changes in these molecules profiles in roots exposed to arsenate (AsV) and arsenite (AsIII). For that, PL and FA from soybean plants treated during 1 and 8 days with As-enriched solutions were analyzed by HPLC–MS/MS and GC–MS. A decrease in the relative amount of unsaturated FA together with an increase in saturated FA was found in roots exposed to both chemical forms of As compared to control condition. As a result, less unsaturated FA/saturated FA ratio and double bond index (DBI) occurred under exposure to As. Changes in PL composition were more evident after 8 d, including a decrease in the total content of phosphatidylethanolamine, with the consequent rise in the phosphatidylcholine/phosphatidylethanolamine ratio, and an increase in the total content of phosphatidic acid. This remodeling of PL and FA in soybean roots could be associated with a reduction in the fluidity and permeability of cell membranes, which would be a key aspect in the adaptive response of root tissue to As-stress. [ABSTRACT FROM AUTHOR]

Abstract Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation. [ABSTRACT FROM AUTHOR]

Voltage‐clamp fluorometry (VCF) enables the study of voltage‐sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage‐gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine‐conjugated flavonoid (4‐oxo‐2‐phenyl‐4H‐chromene‐7‐yl)‐phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)‐carboxytetramethylrhodamine‐(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane‐bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein. [ABSTRACT FROM AUTHOR]

Metal pollutants are a growing concern due to increased use in mining and other industrial processes. Moreover, the use of metals in daily life is becoming increasingly prevalent. Metals such as manganese (Mn), cobalt (Co), and nickel (Ni) are toxic in high amounts whereas lead (Pb) and cadmium (Cd) are acutely toxic at low µM concentrations. These metals are associated with system dysfunction in humans including cancer, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, and other cellular process'. One known but lesser studied target of these metals are lipids that are key membrane building blocks or serve signalling functions. It was shown that Mn, Co, Ni, Pb, and Cd cause rigidification of liposomes and increase the phase transition in membranes composed of both saturated or partly unsaturated phosphatidic acid (PA) and phosphatidylserine (PS). The selected metals showed differential effects that were more pronounced on saturated lipids. In addition, more rigidity was induced in the biologically relevant liquid-crystalline phase. Moreover, metal affinity, induced rigidification and liposome size increases also varied with the headgroup architecture, whereby the carboxyl group of PS appeared to play an important role. Thus, it can be inferred that Mn, Co, Ni, Cd, and Pb may have preferred binding coordination with the lipid headgroup, degree of acyl chain unsaturation, and membrane phase. [ABSTRACT FROM AUTHOR]

The growing issue of salinity is a significant threat to global agriculture, affecting diverse regions worldwide. Nitric oxide (NO) serves as an essential signal molecule in regulating photosynthetic performance under physiological and stress conditions. The present study reveals the protective effects of different concentrations (0–300 µM) of sodium nitroprusside (SNP, a donor of NO) on the functions of the main complexes within the photosynthetic apparatus of maize (Zea mays L. Kerala) under salt stress (150 mM NaCl). The data showed that SNP alleviates salt-induced oxidative stress and prevents changes in the fluidity of thylakoid membranes (Laurdan GP) and energy redistribution between the two photosystems (77K chlorophyll fluorescence ratio F735/F685). Chlorophyll fluorescence measurements demonstrated that the foliar spray with SNP under salt stress prevents the decline of photosystem II (PSII) open reaction centers (qP) and improves their efficiency (Φexc), thereby influencing QA− reoxidation. The data also revealed that SNP protects the rate constants for two pathways of QA− reoxidation (k1 and k2) from the changes caused by NaCl treatment alone. Additionally, there is a predominance of QA− interaction with plastoquinone in comparison to the recombination of electrons in QA QB− with the oxygen-evolving complex (OEC). The analysis of flash oxygen evolution showed that SNP treatment prevents a salt-induced 10% increase in PSII centers in the S0 state, i.e., protects the initial S0–S1 state distribution, and the modification of the Mn cluster in the OEC. Moreover, this study demonstrates that SNP-induced defense occurs on both the donor and acceptor sides of the PSII, leading to the protection of overall photosystems performance (PIABS) and efficient electron transfer from the PSII donor side to the reduction of PSI end electron acceptors (PItotal). This study clearly shows that the optimal protection under salt stress occurs at approximately 50–63 nmoles NO/g FW in leaves, corresponding to foliar spray with 50–150 µM SNP. [ABSTRACT FROM AUTHOR]

Cyanobacteria inhabit areas with a broad range of light, temperature and nutrient conditions. The robustness of cyanobacterial cells, which can survive under different conditions, may depend on the resilience of photosynthetic activity. Cyanothece sp. PCC 8801 (Cyanothece), a freshwater cyanobacterium isolated from a Taiwanese rice field, had a higher repair activity of photodamaged photosystem II (PSII) under intense light than Synechocystis sp. PCC 6803 (Synechocystis), another freshwater cyanobacterium. Cyanothece contains myristic acid (14:0) as the major fatty acid at the sn -2 position of the glycerolipids. To investigate the role of 14:0 in the repair of photodamaged PSII, we used a Synechocystis transformant expressing a T-1274 encoding a lysophosphatidic acid acyltransferase (LPAAT) from Cyanothece. The wild-type and transformant cells contained 0.2 and 20.1 mol% of 14:0 in glycerolipids, respectively. The higher content of 14:0 in the transformants increased the fluidity of the thylakoid membrane. In the transformants, PSII repair was accelerated due to an enhancement in the de novo synthesis of D1 protein, and the production of singlet oxygen (1O2), which inhibited protein synthesis, was suppressed. The high content of 14:0 increased transfer of light energy received by phycobilisomes to PSI and CP47 in PSII and the content of carotenoids. These results indicated that an increase in 14:0 reduced 1O2 formation and enhanced PSII repair. The higher content of 14:0 in the glycerolipids may be required as a survival strategy for Cyanothece inhabiting a rice field under direct sunlight. [ABSTRACT FROM AUTHOR]

Mycobacterial plasma membrane, together with the peptidoglycan-arabinogalactan cell wall and waxy outer membrane, creates a robust permeability barrier against xenobiotics. The fact that several antituberculosis drugs target plasma membrane-embedded enzymes underscores the importance of the plasma membrane in bacterial physiology and pathogenesis. Nevertheless, its accurate phospholipid composition remains undefined, with conflicting reports on the abundance of phosphatidylinositol mannosides (PIMs), physiologically important glycolipids evolutionarily conserved among mycobacteria and related bacteria. Some studies indicate cardiolipin, phosphatidylethanolamine, and phosphatidylinositol as dominant structural phospholipids. Conversely, some suggest PIMs dominate the plasma membrane. A striking example of the latter is the use of reverse micelle extraction, showing diacyl phosphatidylinositol dimannoside (Ac2PIM2) as the most abundant phospholipid in a model organism, Mycobacterium smegmatis. Our recent work reveals a rapid response mechanism to membrane-fluidizing stress in mycobacterial plasma membrane: monoacyl phosphatidylinositol dimannoside and hexamannoside (AcPIM2 and AcPIM6) are converted to diacyl forms (Ac2PIM2 and Ac2PIM6). Given the dynamic nature of PIMs, we aimed to resolve the conflicting data in the literature. We show that unstressed M. smegmatis lacks an Ac2PIM2-dominated plasma membrane. Ac2PIM2 accumulation is induced by experimental conditions involving sodium docusate, a component of the reverse micellar solution. Using chemically synthesized PIMs as standards, we accurately quantified phospholipid ratio in M. smegmatis through liquid chromatography-mass spectrometry, revealing that mycobacterial plasma membrane is dominated by cardiolipin, phosphatidylethanolamine, and phosphatidylinositol. PIMs are quantitatively minor but responsive to environmental stresses in M. smegmatis. Our study paves the way for accurate modeling of mycobacterial plasma membrane.

ABSTRACT Porphyromonas gingivalis has been associated with progression of periodontitis, characterized by inflammation and destruction of periodontal tissues. Here, we report that matcha, a product of Camellia sinensis, hampers the adherence and survival of P. gingivalis through multiple tactics. Matcha extract (ME) inhibited the growth not only of P. gingivalis but also of Prevotella nigrescens and Fusobacterium nucleatum, while it did not inhibit growth of nine species of oral streptococci and Aggregatibacter actinomycetemcomitans. ME-mediated P. gingivalis growth inhibition was characterized by both morphological and physiological changes at the bacterial envelope, which were accompanied by nano-particle formation and decreased membrane fluidity/permeability without loss of membrane integrity. ME also triggered autoaggregation of P. gingivalis in a major fimbriae (FimA)-dependent manner. In addition, adherence of P. gingivalis was dramatically inhibited by ME, irrespective of fimbriae. Furthermore, a structure-activity relationship study tested a series of catechins isolated from ME and identified the pyrogallol-type B-ring of catechins as essential for P. gingivalis growth inhibition. In a clinical study to assess the microbiological and therapeutic effects of matcha mouthwash in patients with periodontitis, the P. gingivalis number in saliva was significantly reduced by matcha mouthwash compared to the pre-intervention level. A tendency toward improvement in probing pocket depth was observed in the matcha group, although the difference was not statistically significant. Taken together, we present a proof of concept, based on the multimodal inhibitory effect of matcha against P. gingivalis, and that matcha may have clinical applicability for prevention and treatment of periodontitis.IMPORTANCEPeriodontitis, a multifactorial inflammatory disease of the oral cavity, results in alveolar bone destruction, and is a major cause of tooth loss of humans. In addition, emerging evidence has demonstrated associations between periodontitis and a wide range of other chronic inflammation-driven disorders, including diabetes mellitus, preterm birth, cardiovascular disease, aspiration pneumonia, rheumatoid arthritis, cognitive disorder, and cancer. In the present study, we report that matcha, a product of Camellia sinensis, hampers Porphyromonas gingivalis, a major periodontal pathobiont, in not only a series of in vitro experiments but also a pilot intervention clinical trial of patients with periodontitis, in which matcha mouthwash statistically significantly reduced the P. gingivalis number in saliva, as compared to the pre-intervention level. Taken together, we suggest that matcha may have clinical applicability for prevention and treatment of periodontitis.

Supported cell membrane coatings meet many requirements set to bioactive nanocarriers and materials, provided sidedness and fluidity of the natural membrane are maintained upon coating. However, the properties of a support-surface responsible for maintaining correct sidedness and fluidity are unknown. Here, we briefly review the properties of natural membranes and membrane-isolation methods, with focus on the asymmetric distribution of functional groups in natural membranes (sidedness) and the ability of molecules to float across a membrane to form functional domains (fluidity). This review concludes that hydrophilic sugar-residues of glycoproteins in the outer-leaflet of cell membranes direct the more hydrophobic inner-leaflet towards a support-surface to create a correctly-sided membrane coating, regardless of electrostatic double-layer interactions. On positively-charged support-surfaces however, strong, electrostatic double-layer attraction of negatively-charged membranes can impede homogeneous coating. In correctly-sided membrane coatings, fluidity is maintained regardless of whether the surface carries a positive or negative charge. However, membranes are frozen on positively-charged, highly-curved, small nanoparticles and localized nanoscopic structures on a support-surface. This leaves an unsupported membrane coating in between nanostructures on planar support-surfaces that is in dual-sided contact with its aqueous environment, yielding enhanced fluidity in membrane coatings on nanostructured, planar support-surfaces as compared with smooth ones.

Abstract Background This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). Results An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P

BACKGROUND: Yeast is often used to build cell factories to produce various chemicals or nutrient substances, which means the yeast has to encounter stressful environments. Previous research reported that unsaturated fatty acids were closely related to yeast stress resistance. Engineering unsaturated fatty acids may be a viable strategy for enhancing the stress resistance of cells. RESULTS: In this study, two desaturase genes, OLE1 and FAD2 from Z. rouxii, were overexpressed in S. cerevisiae to determine how unsaturated fatty acids affect cellular stress tolerance of cells. After cloning and plasmid recombination, the recombinant S. cerevisiae cells were constructed. Analysis of membrane fatty acid contents revealed that the recombinant S. cerevisiae with overexpression of OLE1 and FAD2 genes contained higher levels of fatty acids C16:1 (2.77 times), C18:1 (1.51 times) and C18:2 (4.15 times) than the wild‐type S. cerevisiae pY15TEF1. In addition, recombinant S. cerevisiae cells were more resistant to multiple stresses, and exhibited improved membrane functionality, including membrane fluidity and integrity. CONCLUSION: These findings demonstrated that strengthening the expression of desaturases was beneficial to stress tolerance. Overall, this study may provide a suitable means to build a cell factory of industrial yeast cells with high tolerance during biological manufacturing. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Aims: Improving the composition of circulating fatty acids (FA) leads to a reduction in cardiovascular diseases (CVD) in high‐risk individuals. The membrane fluidity of red blood cells (RBC), which reflects circulating FA status, may be a valid biomarker of cardiovascular (CV) risk in type 2 diabetes (T2D). Methods: Red blood cell membrane fluidity, quantified as general polarization (GP), was assessed in 234 subjects with T2D, 86 with prior major CVD. Based on GP distribution, a cut‐off of.445 was used to divide the study cohort into two groups: the first with higher GP, called GEL, and the second, defined as lower GP (LGP). Lipidomic analysis was performed to evaluate FA composition of RBC membranes. Results: Although with comparable CV risk factors, the LGP group had a greater percentage of patients with major CVD than the GEL group (40% vs 24%, respectively, p <.05). Moreover, in a logistic regression analysis, a lower GP value was independently associated with the presence of macrovascular complications. Lipidomic analysis showed a clear shift of LGP membranes towards a pro‐inflammatory condition due to higher content of arachidonic acid and increased omega 6/omega 3 index. Conclusions: Increased membrane fluidity is associated with a higher CV risk in subjects with T2D. If confirmed in prospective studies, membrane fluidity could be a new biomarker for residual CV risk assessment in T2D. [ABSTRACT FROM AUTHOR]

Pathogenic Rhodococcus equi release the virulence‐associated protein A (VapA) within macrophage phagosomes. VapA permeabilizes phagosome and lysosome membranes and reduces acidification of both compartments. Using biophysical techniques, we found that VapA interacts with model membranes in four steps: (i) binding, change of mechanical properties, (ii) formation of specific membrane domains, (iii) permeabilization within the domains, and (iv) pH‐specific transformation of domains. Biosensor data revealed that VapA binds to membranes in one step at pH 6.5 and in two steps at pH 4.5 and decreases membrane fluidity. The integration of VapA into lipid monolayers was only significant at lateral pressures <20 mN m−1 indicating preferential incorporation into membrane regions with reduced integrity. Atomic force microscopy of lipid mono‐ and bilayers showed that VapA increased the surface heterogeneity of liquid disordered domains. Furthermore, VapA led to the formation of a new microstructured domain type and, at pH 4.5, to the formation of 5 nm high domains. VapA binding, its integration and lipid domain formation depended on lipid composition, pH, protein concentration and lateral membrane pressure. VapA‐mediated permeabilization is clearly distinct from that caused by classical microbial pore formers and is a key contribution to the multiplication of Rhodococcus equi in phagosomes. [ABSTRACT FROM AUTHOR]

Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H2O2) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila’ stress/virulence response. [ABSTRACT FROM AUTHOR]

Tomatidine (TO) is a natural narrow-spectrum antibiotic acting on the Staphylococcus aureus small colony variant (SCV) with a minimal inhibitory concentration (MIC) of 0.06 µg/mL while it shows no activity against prototypical strains (MIC > 128 µg/mL). To expand the spectrum of activity of TO, the 3β-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, TM-02 (C3-β) and TM-03 (C3-α). These molecules are equally potent against prototypical S. aureus and E. coli strains (MIC 8 and 32 µg/mL, respectively), whereas TM-02 is more potent against SCV (MIC 0.5 µg/mL) and hyperpermeable E. coli strains (MIC 1 µg/mL). The differences in their modes of action were investigated. We used membrane vesicles to confirm the inhibition of the bacterial ATP synthase, the documented target of TO, and measured effects on bacterial cell membranes. Both molecules inhibited E. coli ATP synthase, with Ki values of 1.1 µM and 3.5 µM for TM-02 and TM-03, respectively, and the bactericidal effect of TM-02 was linked to ATP synthase inhibition. Furthermore, TM-02 had no major effect on the membrane fluidity and gradually reduced membrane potential. In contrast, TM-03 caused structural damages to membranes and completely disrupted the membrane potential (>90%). We were successful in broadening the spectrum of activity of TO. C3-β-diastereoisomers may have more specific antibacterial action than C3-α. [ABSTRACT FROM AUTHOR]

The lanthanide metal gadolinium has been used in the healthcare industry as a paramagnetic contrast agent for years. Gadolinium deposition in brain tissue and kidneys has been reported following gadolinium-based contrast agent administration to patients undergoing MRI. This study demonstrates the detrimental effects of gadolinium exposure at the level of the cell membrane. Biophysical analysis using fluorescence spectroscopy and dynamic light scattering illustrates differential interactions of gadolinium ions with key classes of brain membrane lipids, including phosphatidylcholines and sphingomyelins, as well as brain polar extracts and biomimetic brain model membranes. Electrostatic attraction to negatively charged lipids like phosphatidylserine facilitates metal complexation but zwitterionic phosphatidylcholine and sphingomyelin interaction was also significant, leading to membrane rigidification and increases in liposome size. Effects were stronger for fully saturated over monounsaturated acyl chains. The metal targets key lipid classes of brain membranes and these biophysical changes could be very detrimental in biological membranes, suggesting that the potential negative impact of gadolinium contrast agents will require more scientific attention. [ABSTRACT FROM AUTHOR]

Background: This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). Results: An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05). Conclusions: In conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters. [ABSTRACT FROM AUTHOR]

ABSTRACT Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The Bacillus subtilis histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the des system: activation of the Pdes promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of des, desK, and desR deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the des system as reporter for antibiotic-induced changes in membrane thickness, we did observe that des expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the des system contributes to fluidity adaptation under these conditions.IMPORTANCEThe B. subtilis des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied in vitro. Our study shows that our understanding of its function in vivo and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the des system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the des system contributes to adaptation of overall membrane fluidity.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1−/− mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1−/− mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1−/− mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1−/− mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

Investigation of new materials for biomedical applications has represented a relevant subject in the latest decade, enhancing versatile properties of lipids. It has been documented that the capabilities of lipid-based systems improve when they combine with polymers, proteins, and sugars. In this field, understanding the driving forces behind such hybrid systems is mandatory for biomedical applications. From this perspective, it is crucial to investigate the biophysical properties of this kind of material. Here, we investigate the biophysical properties of hybrid membranes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol, and octyl-β-D-glucopyranoside (OGP). Lipid/sugar materials could have potential properties to use as nanovesicles for drug delivery. We encapsulate ibuprofen in lipid/sugar vesicles and evaluate their thermodynamics, hydrodynamics, and morphological properties by differential scanning calorimetry, dynamic light scattering, and scanning electron microscopy. We found that OGP combined with cholesterol modifies thermodynamic parameters of membranes such as phase transition temperature, enthalpy change, and cooperativity. Lipid vesicles containing OGP at 6.0 mM loaded with ibuprofen demonstrated good stability after 3 months of storage. Furthermore, electronic microscopy revealed the presence of well-defined liposomes. We conclude that cholesterol and OGP can act synergistically in polar–nonpolar spaces of the DPPC bilayer, where the hydrophobic nature of ibuprofen leads to incorporation into this hybrid core, which implies changes in the fluidity and compactness of the membrane occurring at temperatures of biological relevance. This investigation provides crucial knowledge regarding the biophysical properties of thermo-responsive biohybrid vesicles potentially to use in nanomedicine, which could be of practical reference for designing innovative drug delivery systems. [ABSTRACT FROM AUTHOR]

Lipid biosensors are molecular tools used both in vivo and in vitro applications, capable of selectively detecting specific types of lipids in biological membranes. However, despite their extensive use, there is a lack of systematic characterization of their binding properties in various membrane conditions. The purpose of this study was to investigate the impact of membrane properties, such as fluidity and membrane charge, on the sensitivity of two lipid biosensors, LactC2 and P4M, to their target lipids, phosphatidylserine (PS) or phosphatidylinositol 4-phosphate (PI4P), respectively. Dual-color fluorescence cross-correlation spectroscopy, employed in this study, provided a useful technique to investigate interactions of these recombinant fluorescent biosensors with liposomes of varying compositions. The results of the study demonstrate that the binding of the LactC2 biosensor to low levels of PS in the membrane is highly supported by the presence of anionic lipids or membrane fluidity. However, at high PS levels, the presence of anionic lipids does not further enhance binding of LactC2. In contrast, neither membrane charge, nor membrane fluidity significantly affect the binding affinity of P4M to PI4P. These findings provide valuable insights into the role of membrane properties on the binding properties of lipid biosensors. • Fluorescence cross-correlation reveals binding properties of lipid biosensors. • Lipid biosensors binding responses might be affected by bulk membrane properties. • LactC2 sensitivity to PS is influenced by membrane charge and fluidity. • P4M is not susceptible to various membrane conditions when recognizing PI4P. [ABSTRACT FROM AUTHOR]

Nanomedicines engineered to deliver molecules with therapeutic potentials, overcoming drawbacks such as poor solubility, toxicity or a short half-life, are targeted towards their cellular destination either passively or through various elements of cell membranes. The differences in the physicochemical properties of the cell membrane between tumor and nontumor cells have been reported, but they are not systematically used for drug delivery purposes. Thus, in this study, a new approach based on a match between the liposome compositions, i.e., membrane fluidity, to selectively interact with the targeted cell membrane was used. Lipid-based carriers of two different fluidities were designed and used to deliver 4(RS)-4-F4t-Neuroprostane (F4t-NeuroP), a potential antitumor molecule derived from docosahexaenoic acid (DHA). Based on its hydrophobic character, F4t-NeuroP was added to the lipid mixture prior to liposome formation, a protocol that yielded over 80% encapsulation efficiency in both rigid and fluid liposomes. The presence of the active molecule did not modify the liposome size but increased the liposome negative charge and the liposome membrane fluidity, which suggested that the active molecule was accommodated in the lipid membrane. F4t-NeuroP integration in liposomes with a fluid character allowed for the selective targeting of the metastatic prostate cell line PC-3 vs. fibroblast controls. A significant decrease in viability (40%) was observed for the PC-3 cancer line in the presence of F4t-NeuroP fluid liposomes, whereas rigid F4t-NeuroP liposomes did not alter the PC-3 cell viability. These findings demonstrate that liposomes encapsulating F4t-NeuroP or other related molecules may be an interesting model of drug carriers based on membrane fluidity. [ABSTRACT FROM AUTHOR]

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins. [ABSTRACT FROM AUTHOR]

Viruses interact with receptors on the cell surface to initiate and coordinate infection. The distribution of receptors on host cells can be a key determinant of viral tropism and host infection. Unravelling the complex nature of virus-receptor interactions is, therefore, of fundamental importance to understanding viral pathogenesis. Noroviruses are non-enveloped, icosahedral, positive-sense RNA viruses of global importance to human health, with no approved vaccine or antiviral agent available. Here, we use murine norovirus as a model to study the molecular mechanisms of virus-receptor interactions. We show that variation at a single amino acid residue in the major viral capsid protein, VP1 301, has a key impact on the interaction between virus and receptor. This variation did not affect virion replication or virus growth kinetics, but a specific amino acid was rapidly selected through evolution experiments and significantly improved cellular attachment when infecting cells in suspension. However, modulating plasma membrane mobility counteracted this phenotype, suggesting a role for membrane fluidity in norovirus cellular attachment. When the infectivity of a panel of recombinant viruses with single amino acid substitutions at this residue was compared in vivo, there were differences in the tissue distribution of viruses in a murine host, suggesting a role for VP1 301 in dissemination in vivo. Overall, these results highlight how capsid evolution can influence infectivity and dissemination in the host. [ABSTRACT FROM AUTHOR]

The lipidome of cells and organelles is more complex than originally anticipated. It has become clear that biomembranes are active materials bearing a tremendous regulatory potential. Cells control the collective biophysical properties of their organelle membranes in order to maintain the organization and functionality of the membrane proteome. The concept of homeoviscous adaptation provided an intuitive interpretation for the changes of membrane compositions with temperature. However, the mechanistic relevance of membrane viscosity for many cellular processes including those regulating the lipid fatty acyl chain composition has been recently challenged. The crucial role of membrane compressibility and thickness in organizing the membrane proteome, however, has gained fresh emphasis. The transverse membrane compressibility of the endoplasmic reticulum (ER) membrane modulates crucial aspects of transmembrane protein biology including their bilayer insertion and extraction, their conformational dynamics and activity, as well as their sorting along the secretory pathway and inheritance from mother to daughter cells. The unfolded protein response (UPR) can sense aberrant ER membrane stiffening and surfaces as a prime candidate for balancing membrane lipid and protein production at the ER as a mechanism of biophysical membrane homeostasis during cellular stress. Biomembranes are complex materials composed of lipids and proteins that compartmentalize biochemistry. They are actively remodeled in response to physical and metabolic cues, as well as during cell differentiation and stress. The concept of homeoviscous adaptation has become a textbook example of membrane responsiveness. Here, we discuss limitations and common misconceptions revolving around it. By highlighting key moments in the life cycle of a transmembrane protein, we illustrate that membrane thickness and a finely regulated membrane compressibility are crucial to facilitate proper membrane protein insertion, function, sorting, and inheritance. We propose that the unfolded protein response (UPR) provides a mechanism for endoplasmic reticulum (ER) membrane homeostasis by sensing aberrant transverse membrane stiffening and triggering adaptive responses that re-establish membrane compressibility. [ABSTRACT FROM AUTHOR]

High levels of carbon dioxide are known to inhibit the growth of microorganisms. A total of twenty strains of filamentous fungi and yeasts were isolated from habitats with enriched carbon dioxide concentration. Most strains were derived from modified atmosphere packed (MAP) food products or mofettes and were cultivated under an atmosphere of 20% CO2 and 80% O2. The influence of CO2 on fungal cell membrane fatty acid profiles was examined in this study. Major changes were the increase in linolenic acid (C18:3 cis 9, 12, 15) and, additionally in most strains, linoleic acid (C18:2 cis 9, 12) with a maximum of 24.8%, at the expense of oleic (C18:1 cis 9), palmitic (C16:0), palmitoleic (C16:1 cis 9) and stearic acid (C18:0). The degree of fatty acid unsaturation increased for all of the strains in the study, which consequently led to lower melting temperatures of the cell membranes after incubation with elevated levels of CO2, indicating fluidization of the membrane and a potential membrane malfunction. Growth was reduced in 18 out of 20 strains in laboratory experiments and a change in pigmentation was observed in several strains. Two of the isolated strains, strain WT5 and strain WR1, were found to represent a hitherto undescribed yeast for which the new genus and species Stenotrophomyces fumitolerans (MB# 849906) is proposed. [ABSTRACT FROM AUTHOR]

In every bacterial cell, the plasma membrane plays a key role in viability as it forms a selective barrier between the inside of the cell and its environment. This barrier function depends on the physical state of the lipid bilayer and the proteins embedded or associated with the bilayer. Over the past decade or so, it has become apparent that many membrane‐organizing proteins and principles, which were described in eukaryote systems, are ubiquitous and play important roles in bacterial cells. In this minireview, we focus on the enigmatic roles of bacterial flotillins in membrane compartmentalization and bacterial dynamins and ESCRT‐like systems in membrane repair and remodeling. [ABSTRACT FROM AUTHOR]

The growing issue of salinity is a significant threat to global agriculture, affecting diverse regions worldwide. Nitric oxide (NO) serves as an essential signal molecule in regulating photosynthetic performance under physiological and stress conditions. The present study reveals the protective effects of different concentrations (0–300 µM) of sodium nitroprusside (SNP, a donor of NO) on the functions of the main complexes within the photosynthetic apparatus of maize (Zea mays L. Kerala) under salt stress (150 mM NaCl). The data showed that SNP alleviates salt-induced oxidative stress and prevents changes in the fluidity of thylakoid membranes (Laurdan GP) and energy redistribution between the two photosystems (77K chlorophyll fluorescence ratio F735/F685). Chlorophyll fluorescence measurements demonstrated that the foliar spray with SNP under salt stress prevents the decline of photosystem II (PSII) open reaction centers (qP) and improves their efficiency (Φexc), thereby influencing QA− reoxidation. The data also revealed that SNP protects the rate constants for two pathways of QA− reoxidation (k1 and k2) from the changes caused by NaCl treatment alone. Additionally, there is a predominance of QA− interaction with plastoquinone in comparison to the recombination of electrons in QA QB− with the oxygen-evolving complex (OEC). The analysis of flash oxygen evolution showed that SNP treatment prevents a salt-induced 10% increase in PSII centers in the S0 state, i.e., protects the initial S0–S1 state distribution, and the modification of the Mn cluster in the OEC. Moreover, this study demonstrates that SNP-induced defense occurs on both the donor and acceptor sides of the PSII, leading to the protection of overall photosystems performance (PIABS) and efficient electron transfer from the PSII donor side to the reduction of PSI end electron acceptors (PItotal). This study clearly shows that the optimal protection under salt stress occurs at approximately 50–63 nmoles NO/g FW in leaves, corresponding to foliar spray with 50–150 µM SNP.

Abstract Background Membrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors. Methods Since changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John’s wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 μM] PBMCs with Ze 117 [10–50 µg/ml] were analyzed by mass spectrometry. Results Cortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 μM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments. Conclusion The increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract.

The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid–lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM. [ABSTRACT FROM AUTHOR]

α-Synuclein has a critical role in Parkinson's disease, but the mechanism of how extracellular α-synuclein aggregates lead to astrocytic degeneration remains unknown. Our recent study in astrocytes highlighted that α-synuclein aggregates undergo lower endocytosis than the monomeric-form, even while displaying a higher impact on glutathione-machinery and glutamate-metabolism under sublethal conditions. As optimal intracellular calcium levels are essential for these functions, we aimed to study the effect of extracellular α-synuclein aggregates on ER calcium entry. We assessed the association of extracellular aggregated-α-synuclein (WT and A30P/A53T double-mutant) with the astrocytic membrane (lipid rafts) and studied its effects on membrane fluidity, ER stress, and ER calcium refilling in three systems—purified rat primary midbrain astrocyte culture, human iPSC-derived astrocytes, and U87 cells. The corresponding timeline effect on mitochondrial membrane potential was also evaluated. Post-24 h exposure to extracellular WT and mutant α-synuclein aggregates, fluorescence-based studies showed a significant increase in astrocyte membrane rigidity over control, with membrane association being significantly higher for the double mutant aggregates. α-Synuclein aggregates also showed preferentially higher association with lipid rafts of astrocytic membrane. A simultaneous increase in ER stress markers (phosphorylated PERK and CHOP) with significantly higher SOCE was also observed in aggregate-treated astrocytes, with higher levels for double mutant variant. These observations correlate with increased expression of SOCE markers, especially Orai3, on plasma membrane. Alterations in mitochondrial membrane potential were only noted post-48 h of exposure to α-synuclein aggregates. We therefore suggest that in astrocytes, α-synuclein-aggregates preferentially associate with lipid rafts of membrane, altering membrane fluidity and consequently inducing ER stress mediated by interaction with membrane SOCE proteins, resulting in higher Ca2+ entry. A distinct cascade of events of sequential impairment of ER followed by mitochondrial alteration is observed. The study provides novel evidence elucidating relationships between extracellular α-synuclein aggregates and organellar stress in astrocytes and indicates the therapeutic potential in targeting the association of α-synuclein aggregates with astrocytic membrane. [ABSTRACT FROM AUTHOR]

An acidophilic heterotrophic gram-negative Acidiphilium symbioticum strain H8 is an important bacterium possessing a high capacity for heavy metal binding. As these bacteria inhabit acidic mine regions they are subjected to occasional temperature stress; thus their membrane profile is very important. Cell morphology determined by scanning and transmission electron microscopy revealed the surface and internal adaptation of the organelles. Phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidic acid are the major phospholipids affected by growth temperature. The membrane fluidity in response to temperature stress was investigated by measuring fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe. Significant changes in membrane fluidity revealed the transition temperature midpoints as 11 and 38°C. To maintain the optimum fluidity increasing the percentage of cis-vaccenic acid at suboptimal and palmitic acid at the upper cardinal temperature support the evidence of membrane remodeling in different growth temperatures. The protein profile from SDS-PAGE provided information about different polypeptide bands which may be responsible for the different metal binding abilities of the strain. [ABSTRACT FROM AUTHOR]

The action of UVA radiation (both that derived from solar radiation and that used in the treatment of skin diseases) modifies the function and composition of keratinocyte membranes. Therefore, this study aimed to assess the effects of phytocannabinoids (CBD and CBG), used singly and in combination, on the contents of phospholipids, ceramides, lipid rafts and sialic acid in keratinocyte membranes exposed to UVA radiation, together with their structure and functionality. The phytocannabinoids, especially in combination (CBD+CBG), partially prevented increased levels of phosphatidylinositols and sialic acid from occurring and sphingomyelinase activity after the UVA exposure of keratinocytes. This was accompanied by a reduction in the formation of lipid rafts and malondialdehyde, which correlated with the parameters responsible for the integrity and functionality of the keratinocyte membrane (membrane fluidity and permeability and the activity of transmembrane transporters), compared to UVA-irradiated cells. This suggests that the simultaneous use of two phytocannabinoids may have a protective effect on healthy cells, without significantly reducing the therapeutic effect of UV radiation used to treat skin diseases such as psoriasis. [ABSTRACT FROM AUTHOR]

Lipid microdomains, ordered membrane phases containing cholesterol and glycosphingolipids, play an essential role in cancer cell adhesion and ultimately metastasis. Notably, elevated levels of cholesterol-rich lipid microdomains are found in cancer cells relative to their normal counterparts. Therefore, alterations of lipid microdomains through cholesterol modulation could be used as a strategy to prevent cancer metastasis. In this study, methyl-beta-cyclodextrin (MbCD), sphingomyelinase (SMase), and simvastatin (Simva) were used to investigate the effects of cholesterol on the adhesive behaviors of four non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A549) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule that initiates circulating tumor cell recruitment at metastatic sites. Under hemodynamic flow conditions, the number of adherent NSCLC cells on E-selectin significantly decreased by MbCD and Simva treatments, whereas SMase treatment did not show a significant effect. Significant increases in rolling velocities were detected only for H1299 and H23 cells after MbCD treatment. In contrast, cholesterol depletion did not affect SCLC cell attachment and rolling velocities. Moreover, cholesterol depletion by MbCD and Simva induced CD44 shedding and resulted in an enhanced membrane fluidity in the NSCLC cells, whereas it did not affect the membrane fluidity of the SCLC cells which lacked detectable expression of CD44. Our finding suggests that cholesterol regulates the E-selectin-mediated adhesion of NSCLC cells by redistributing the CD44 glycoprotein and thus modulating the membrane fluidity. NEW & NOTEWORTHY This study investigates the effects of cholesterol on the adhesive behaviors of lung cancer cells in recruitment at metastatic sites. Using cholesterol-modulating compounds, we found that reducing cholesterol decreases the adhesion of non-small cell lung cancer (NSCLC) cells while having no significant effect on small cell lung cancer (SCLC) cells. The study suggests that cholesterol regulates NSCLC cell metastasis by redistributing the adhesion proteins on the cells and modulating cells' membrane fluidity. [ABSTRACT FROM AUTHOR]

For industrial processes, a fast, precise, and reliable method of determining the physiological state of yeast cells, especially viability, is essential. However, an increasing number of processes use magnetic nanoparticles (MNPs) for yeast cell manipulation, but their impact on yeast cell viability and the assay itself is unclear. This study tested the viability of Saccharomyces pastorianus ssp. carlsbergensis and Pichia pastoris by comparing traditional colourimetric, high-throughput, and growth assays with membrane fluidity. Results showed that methylene blue staining is only reliable for S. pastorianus cells with good viability, being erroneous in low viability (R2 = 0.945; σ ^ = 5.78%). In comparison, the fluorescence microscopy–based assay of S. pastorianus demonstrated a coefficient of determination of R2 = 0.991 at α = 0 ( σ ^ = 2.50%) and flow cytometric viability determination using carboxyfluorescein diacetate (CFDA), enabling high-throughput analysis of representative cell numbers; R2 = 0.972 ( α = 0 ; σ ^ = 3.89%). Membrane fluidity resulted in a non-linear relationship with the viability of the yeast cells ( α ≠ 0 ). We also determined similar results using P. pastoris yeast. In addition, we demonstrated that MNPs affected methylene blue staining by overestimating viability. The random forest model has been shown to be a precise method for classifying nanoparticles and yeast cells and viability differentiation in flow cytometry by using CFDA. Moreover, CFDA and membrane fluidity revealed precise results for both yeasts, also in the presence of nanoparticles, enabling fast and reliable determination of viability in many experiments using MNPs for yeast cell manipulation or separation. [ABSTRACT FROM AUTHOR]

Every cell biological textbook teaches us that the main role of the plasma membrane is to separate cells from their neighborhood to allow for a controlled composition of the intracellular space. The mostly hydrophobic nature of the cell membrane presents an impenetrable barrier for most hydrophilic molecules larger than 1 kDa. On the other hand, cell-penetrating peptides (CPPs) are capable of traversing this barrier without compromising membrane integrity, and they can do so on their own or coupled to cargos. Coupling biologically and medically relevant cargos to CPPs holds great promise of delivering membrane-impermeable drugs into cells. If the cargo is able to interact with certain cell types, uptake of the CPP–drug complex can be tailored to be cell-type-specific. Besides outlining the major membrane penetration pathways of CPPs, this review is aimed at deciphering how properties of the membrane influence the uptake mechanisms of CPPs. By summarizing an extensive body of experimental evidence, we argue that a more ordered, less flexible membrane structure, often present in the very diseases planned to be treated with CPPs, decreases their cellular uptake. These correlations are not only relevant for understanding the cellular biology of CPPs, but also for rationally improving their value in translational or clinical applications. [ABSTRACT FROM AUTHOR]

The purpose of this study was to provide new ideas for the antibacterial mechanism of monolauroyl-galactosylglycerol (MLGG) from the perspective of cell membranes. The changes in cell membrane properties of Bacillus cereus (B. cereus) CMCC 66,301 exposed to different concentrations (1 × MIC (minimum inhibitory concentration), 2 × MIC, 1 × MBC (minimum bacterial concentration)) of MLGG were evaluated. It was found that the lag phase of B. cereus cells was prolonged at low concentration MLGG (1 × MIC and 2 × MIC), while about 2 log CFU/mL reduction in B. cereus populations were observed when exposed to high concentration MLGG (1 × MBC). MLGG treated B. cereus displayed obvious membrane depolarization, while membrane permeability had no change using PI (propidium iodide) staining. Significant increase in the membrane fluidity in response to MLGG exposure occurred, which was consistent with the modification of membrane fatty acids compositions, where the relative content of straight-chain fatty acids (SCFAs) and unsaturated fatty acids (UFAs) increased, while branched-chain fatty acids (BCFAs) decreased significantly. The decreased transition Tm value and cell surface hydrophobicity was also observed. Additionally, effect of MLGG on bacterial membrane compositions were explored at the submolecular level by infrared spectroscopy. Resistance tests of B. cereus to MLGG had demonstrated the advantages of MLGG as a bacteriostatic agent. Collectively, these studies indicate that modifying the fatty acid composition and properties of cellular membranes through MLGG exposure is crucial for inhibiting bacteria growth, providing new insights into the antimicrobial mechanisms of MLGG. Key points: • Monolauroyl-galactosylglycerol inserted into B. cereus lipid bilayer membrane • Monolauroyl-galactosylglycerol treatment caused B. cereus membrane depolarization • Monolauroyl-galactosylglycerol resulted in B. cereus membrane fatty acids alteration [ABSTRACT FROM AUTHOR]

Lipophilic index (LI) has been introduced to assess the overall fatty acid lipophilicity and as a simple estimate of membrane fluidity. However, little is known on effect of diet on LI. We tested if Camelina sativa oil (CSO) high in ALA, fatty fish (FF) or lean fish (LF) affect LI as compared to control diet and, secondarily, if the LI is associated with HDL lipids and functionality and LDL lipidome. We used data from two randomized clinical trials. The AlfaFish intervention lasted 12 weeks and 79 subjects with impaired glucose tolerance were randomized to FF, LF, CSO or control group. In the Fish trial, 33 subjects with myocardial infarction or unstable ischemic heart attack were randomized to FF, LF or control group for 8 weeks. LI was calculated from erythrocyte membrane fatty acids in AlfaFish and from serum phospholipids in Fish trial. HDL lipids were measured using high-throughput proton nuclear magnetic resonance spectroscopy. There was a significant decrease in LI in the FF group in the AlfaFish (fold change 0.98 ± 0.03) and in the Fish trial (0.95 ± 0.04) and the decrease differed from that of control group in both trials and from CSO group in the AlfaFish study. There were no significant changes in LI in LF or CSO groups. The mean diameter of HDL particles and concentration of large HDL particles were inversely associated with LI. FF consumption decreased LI indicating better membrane fluidity in subjects with impaired glucose tolerance or coronary heart disease. • Low lipophilic index is considered as marker of better membrane fluidity. • Fatty fish consumption decreased lipophilic index. • Lower lipophilic index was associated with larger mean HDL size. [ABSTRACT FROM AUTHOR]

We evaluated the in vivo effects of melatonin treatment on oxidative damage in the liver in an experimental model of ischemia–reperfusion. A total of 37 male Sprague-Dawley rats were randomly divided into four groups: control, ischemia, ischemia + reperfusion, and ischemia + reperfusion + melatonin. Hepatic ischemia was maintained for 20 min, and the clamp was removed to initiate vascular reperfusion for 30 min. Melatonin (50 mg/kg body weight) was intraperitoneally administered. Fluidity was measured by polarization changes in 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluene sulfonate). After 20 min of ischemia, no significant changes were observed in cell and mitochondrial membrane fluidity levels, lipid peroxidation, and protein carbonylation. However, after 30 min of reperfusion, membrane fluidity decreased compared to controls. Increases in lipid and protein oxidation were also seen in hepatic homogenates of animals exposed to reperfusion. Melatonin injected 30 min before ischemia and reperfusion fully prevented membrane rigidity and both lipid and protein oxidation. Livers from ischemia–reperfusion showed histopathological alterations and positive labeling with antibodies to oxidized lipids and proteins. Melatonin reduced the severity of these morphological changes and protected against in vivo ischemia–reperfusion-induced toxicity in the liver. Therefore, melatonin might be a candidate for co-treatment for patients with hepatic vascular occlusion followed by reperfusion. [ABSTRACT FROM AUTHOR]

The goal of this study is to obtain and characterize the complex of quercetin with glycyrrhizic acid, which is known to serve as a drug delivery system. Quercetin is a flavonoid with a wide range of biological activities, including an antimicrobial effect. However, quercetin instability and low bioavailability that limits its use in medical practice makes it necessary to look for new nanoformulations of it. The formation of the GAQ complex (2:1) was confirmed by using UV and FT-IR spectroscopies. It was found that the GAQ exhibited antimicrobial and antihemolytical activities against S. aureus bacteria and its main virulent factor—α-hemolysin. The IC50 value for the antihemolytical effect of GAQ was 1.923 ± 0.255 µg/mL. Using a fluorescence method, we also showed that the GAQ bound tightly to the toxin that appears to underlie its antihemolytic activity. In addition, another mechanism of the antihemolytic activity of the GAQ against α-hemolysin was shown, namely, its ability to increase the rigidity of the outer layer of the erythrocyte membrane and thus inhibit the incorporation of α-hemolysin into the target cells, increasing their resistance to the toxin. Both of these effects of GAQ were observed at concentrations below the MIC value for S. aureus growth, indicating the potential of the complex as an antivirulence agent. [ABSTRACT FROM AUTHOR]

The fluidity and polar environment of ~100 nm hybrid vesicles combining dipalmitoylphosphatidylcholine (DPPC) and poly(1,2-butadiene)-block-polyethylene oxide (PBd-PEO, average molecular weight 950 g/mol) were studied upon vesicle heating using the fluorescence spectroscopy techniques of DPH anisotropy and laurdan generalized polarization (GP). These techniques indicated PBd-PEO membranes are less ordered than solid DPPC, but slightly more ordered than fluid DPPC or dioleoylphosphatidylcholine (DOPC) membranes. We find the DPH anisotropy values are less than expected from additivity of the components' anisotropies in the fluid phase mixture of DPPC and PBd-PEO, inferring that DPPC strongly fluidizes the PBd-PEO. We use transitions in DPH anisotropy and laurdan GP to create a temperature/composition phase diagram for DPPC/PBd-PEO which we find displays a significantly broader solid/fluid phase coexistence region than DPPC/DOPC, showing that DPPC partitions less readily into fluid PBd-PEO than into fluid DOPC. The existence of a broad solid/fluid phase coexistence region in DPPC/PBd-PEO vesicles is verified by Förster resonance energy transfer results and the visualization of phase separation in giant unilamellar vesicles containing up to 95% PBd-PEO and a single phase in 100% PBd-PEO vesicles at room temperature. These results add to the limited knowledge of phase behavior and phase diagrams of hybrid vesicles, and should be useful in understanding and tailoring membrane surface architecture toward biomedical applications such as drug delivery or membrane protein reconstitution.

BackgroundThe deep ocean is characterized by low temperatures, high hydrostatic pressures, and low concentrations of organic matter. While these conditions likely select for distinct genomic characteristics within prokaryotes, the attributes facilitating adaptation to the deep ocean are relatively unexplored. In this study, we compared the genomes of seven strains within the genus Colwellia, including some of the most piezophilic microbes known, to identify genomic features that enable life in the deep sea.ResultsSignificant differences were found to exist between piezophilic and non-piezophilic strains of Colwellia. Piezophilic Colwellia have a more basic and hydrophobic proteome. The piezophilic abyssal and hadal isolates have more genes involved in replication/recombination/repair, cell wall/membrane biogenesis, and cell motility. The characteristics of respiration, pilus generation, and membrane fluidity adjustment vary between the strains, with operons for a nuo dehydrogenase and a tad pilus only present in the piezophiles. In contrast, the piezosensitive members are unique in having the capacity for dissimilatory nitrite and TMAO reduction. A number of genes exist only within deep-sea adapted species, such as those encoding d-alanine-d-alanine ligase for peptidoglycan formation, alanine dehydrogenase for NADH/NAD+ homeostasis, and a SAM methyltransferase for tRNA modification. Many of these piezophile-specific genes are in variable regions of the genome near genomic islands, transposases, and toxin-antitoxin systems.ConclusionsWe identified a number of adaptations that may facilitate deep-sea radiation in members of the genus Colwellia, as well as in other piezophilic bacteria. An enrichment in more basic and hydrophobic amino acids could help piezophiles stabilize and limit water intrusion into proteins as a result of high pressure. Variations in genes associated with the membrane, including those involved in unsaturated fatty acid production and respiration, indicate that membrane-based adaptations are critical for coping with high pressure. The presence of many piezophile-specific genes near genomic islands highlights that adaptation to the deep ocean may be facilitated by horizontal gene transfer through transposases or other mobile elements. Some of these genes are amenable to further study in genetically tractable piezophilic and piezotolerant deep-sea microorganisms.