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The outer membrane (OM) is an essential component of the Gram-negative bacterial cell envelope. Restricted diffusion of integral OM proteins and lipopolysaccharide (LPS) that constitute the outer leaflet of the OM support a model in which the OM is in a semi-crystalline state. The low fluidity of the OM has been suggested to be an important property of this membrane that even contributes to cell rigidity. The LPS characteristics strongly determine the properties of the OM and the LPS layer fluidity has been measured using different techniques that require specific conditions or are technically challenging. Here, we characterize the Escherichia coli LPS fluidity by evaluating the lateral diffusion of the styryl dye FM4-64FX in fluorescence recovery after photobleaching experiments. This technique allowed us to determine the effect of different conditions and genetic backgrounds on the LPS fluidity. Our results show that a fraction of the LPS can slowly diffuse and that the fluidity of the LPS layer adapts by modifying the diffusion of the LPS and the fraction of mobile LPS molecules.

Voltage-gated proton channels (HV 1) have been found in many mammalian cells and play a crucial role in the immune system, male fertility, and cancer progression. Glycosaminoglycans play a significant role in various aspects of cell physiology, including the modulation of membrane receptors and ion channel function. We present here evidence that mechanosensitivity of the dimeric HV 1 channel transduce changes on cell membrane fluidity related to the defective biosynthesis of chondroitin sulphate and heparan sulphate in Chinese Hamster Ovary (CHO-745) cells into a leftward shift in the activation voltage dependence. This effect was accompanied by an increase in the H+ current, and an acceleration of the activation kinetics, under symmetrical or asymmetrical pH gradient (ΔpH) conditions. Similar gating alterations were evoked by two naturally occurring HV 1 N-terminal truncated isoforms expressed in wild-type CHO-K1 and CHO-745 cells. On three different monomeric HV 1 constructs, no alterations in the biophysical parameters were observed. Moreover, we have showed that HV 1 gating can be modulated by manipulating CHO-K1 cell membrane fluidity. Our results suggest that the defective biosynthesis of chondroitin sulphate and heparan sulphate on CHO-745 cell increases membrane fluidity and allosterically modulates the coupling between voltage- and ΔpH-sensing through the dimeric HV 1 channel.

In bacteria, exposure to changes in environmental conditions can alter membrane fluidity, thereby affecting its essential functions in cell physiology. To adapt to these changes, bacteria maintain appropriate fluidity by varying the composition of the fatty acids of membrane phospholipids, a phenomenon known as homeophasic adaptation. In Pseudomonas aeruginosa, this response is achieved mainly by two mechanisms of fatty acid desaturation: the FabA-FabB and DesA-DesB systems. This study analyzed the effect of ultraviolet-A (UVA) radiation-the major fraction of solar UV radiation reaching the Earth's surface-on the homeophasic process. The prototypical strain PAO1 was grown under sublethal UVA doses or in the dark, and the profiles of membrane fatty acids were compared at early logarithmic, logarithmic and stationary growth phases. In the logarithmic growth phase, it was observed that growth under sublethal UVA doses induced the expression of the desaturase-encoding genes desA and desB and increased the proportion of unsaturated fatty acids; in addition, membrane fluidity could also increase, as suggested by the indices used as indicators of this parameter. The opposite effect was observed in the stationary growth phase. These results demonstrate the relevant role of UVA on the homeophasic response at transcriptional level.

Ex-vivo interaction of NaYF4 :Yb,Er nanophosphors with isolated mitochondria has been investigated. The nanophosphors were synthesised using hydrothermal method. The synthesised NaYF4 :Yb,Er nanophosphors were characterised for physicochemical properties. The NaYF4 :Yb,Er nanophosphors showed successful upconversion with excitation wavelength lying near-infrared region. The effect of synthesised NaYF4 :Yb,Er nanophosphors on mitochondria isolated from chicken heart tissue has been examined through ROS generation capacity, membrane fluidity and complex II activity. The exposer of NaYF4 :Yb,Er nanophosphors to isolated mitochondria inhibits ROS generation activity as compared to control. The mitochondria membrane fluidity of lipid bilayer and complex-II activity of mitochondria was observed to be unaltered after Interaction with NaYF4 :Yb,Er nanoparticles. The results confirm that synthesised NaYF4 :Yb,Er nanoparticles can be used as a safe contrast agent. This article is protected by copyright. All rights reserved.

Background Chilling injury (CI) is a physiological disorder that results in a limitation for cold storage (CS) of many fruits and vegetables. The low temperature-induced changes in the properties and composition of cell membranes are involved in the response to chilling temperature and in the mechanism of CI and tolerance. Results We compared the changes in the lipid composition by gas chromatography-mass spectrometry before, immediately after CS, as well as during a 3-day subsequent period, of tomato fruits with different chilling-sensitivity: Micro-Tom (tolerant) and Minitomato (susceptible). The changes in linolenic acid content, double bond index and digalactosyldiacylglycerol/monogalactosyldiacylglycerol ratio (DGDG/MGDG) showed membrane fluidity adjustment, depending on the temperature. By a database search, we identified 18 membrane-bound fatty acid desaturase (FAD) genes and five DGDG synthases (DGD) genes that phylogenetically clustered into four and two subfamilies, respectively. The FAD and DGD genes were differentially expressed in response to CS, as determined by quantitative reverse transcriptase-polymerase chain reaction analysis. Conclusion The data strongly suggest that reversion of CS-induced changes during the recovery period is important for the proper function of the membrane and tolerance to postharvest CI in tomato fruit. © 2021 Society of Chemical Industry.

Since the phasing out and eventual ban on the production of organohalogen flame retardants, the use of organophosphate flame retardants (OPFRs) has increased rapidly. This has led to the detection of OPFRs in various environments including the Arctic. Two of the most prevalent OPFRs found in the Arctic are tris(2-chloroisopropyl) phosphate (TCPP), and 2-ethylhexyl diphenyl phosphate (EHDPP). The impacts of exposure to OPFRs on Arctic organisms is poorly understood. The objective of the present study was to determine the effects of exposure to TCPP, EHDPP, and a mixture of OPFRs on gene expression patterns in Atlantic cod, Gadus morhua. Precision-cut liver slices from Atlantic cod in vitro were exposed to either TCPP or EHDPP alone or in a mixture and sampled at 2 different time points to quantify gene expression patterns using RNA sequencing. We exposed the liver slices to 2 concentrations of TCPP and EHDPP, one of which was chosen based on the levels found in the Arctic environment. The RNA sequencing results demonstrated differential expression of hundreds of genes in response to exposure. The genes representing cholesterol biosynthesis and lipid metabolism pathway were significantly enriched in all the treatment groups. Almost all the cholesterol biosynthesis genes were significantly down-regulated in response to OPFR exposure. The effects on these pathways could involve various physiological processes including reproduction, growth, and behavior as well as adaptation to changing temperatures. Membrane fluidity is an important adaptive mechanism among aquatic organisms. Altered cholesterol homeostasis could have long-term consequences by altering the adaptive potential of aquatic organisms to changing water temperatures, particularly those living in polar environments. These results suggest that OPFRs could have unique effects on the organisms living in the Arctic compared with other environments. Further studies are needed to understand the long-term impacts of exposure to environmentally realistic concentrations using laboratory and field-based studies. Environ Toxicol Chem 2021;40:1639-1648. © 2021 SETAC.

Hypothermic storage of boar semen may allow antibiotic-free semen preservation but is limited due to chilling sensitivity of boar spermatozoa. Progress in this area requires sensitive tools to detect chilling injury. Therefore, multiparameter flow cytometry panels were evaluated to ascertain whether they are useful tools for identifying sublethal damage of sperm function at a single cell level, thus considering the high intrinsic sperm heterogeneity in a sample. The first fluorochrome panel consisted of Hoechst 33342 to identify DNA-containing events, Yo-Pro 1 to detect viability, merocyanine 540 to describe membrane fluidity, and PNA-Alexa Fluor™ 647 to identify acrosomic integrity. The second fluorochrome panel consisted of SiR700-DNA to identify DNA-containing events, JC-1 to characterize the mitochondrial transmembrane potential (MMP), and Calbryte 630 to assess the intracellular calcium level. Extended boar semen was stored either at 17°C (control) or 5°C (chilled). It is shown that chilling increased membrane fluidity in the viable (Yo-Pro 1 negative) sperm population at 24 h (p < 0.05). At 144 h, the viable, acrosomic intact sperm population with low membrane fluidity was similar for both storage temperatures. Moreover, chilling reduced the main sperm population with high MMP, medium fluorescence for JC-1 monomer and low intracellular calcium level (p < 0.05). However, after in vitro sperm capacitation, this population did not differ between the two storage temperatures. Exemplary computational data visualization in t-distributed stochastic neighbor embedding (t-SNE) maps and moving radar plots revealed similar subpopulations as identified by three-dimensional stacked bar charts. In conclusion, sperm surviving an initial chilling injury withstand long-term storage and respond in a similar manner to capacitation conditions as sperm stored conventionally at 17°C. Multicolor flow cytometry is a valuable tool for detecting chilling-induced alterations of cell function in sperm subpopulations.

Transmembrane anion transport is an important biological process in maintaining cellular functions. Thus, synthetic anion transporters are widely developed for their biological applications. Imidazolinium was introduced as anion recognition site to a multiblock amphiphilic structure that consists of octa(ethylene glycol) and aromatic units. Ion transport assay using halide-sensitive lucigenin and pH-sensitive 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) revealed that imidazolinium-based multiblock amphiphile (IMA) transports anions and showed high selectivity for nitrate, which plays crucial roles in many biological events. Temperature-dependent ion transport assay using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) indicated that IMA works as a mobile carrier. 1 H NMR titration experiments indicated that the C2 proton of the imidazolinium ring recognizes anions via a (C-H)+ ⋅⋅⋅X- hydrogen bond. Furthermore, all-atom molecular dynamics simulations revealed a dynamic feature of IMA within the membranes during ion transportation.

Myriocin damaged Fon cell membrane. The damage to the cell membrane of Fon was caused by the altered or disrupted expression of some membrane‐related genes and proteins at the mRNA and protein levels; these genes and proteins mainly including those related to sphingolipid metabolism, glycerophospholipid metabolism, steroid biosynthesis, ABC transporters, and protein processing in the endoplasmic reticulum., Summary Myriocin, which is produced by Bacillus amyloliquefaciens LZN01, can inhibit the growth of Fusarium oxysporum f. sp. niveum (Fon). In the present study, the antifungal mechanism of myriocin against Fon was investigated with a focus on the effects of myriocin on the cell membrane. Myriocin decreased the membrane fluidity and destroyed the membrane integrity of Fon. Significant microscopic morphological changes, including conidial shrinkage, the appearance of larger vacuoles and inhomogeneity of electron density, were observed in myriocin‐treated cells. A membrane‐targeted mechanism of action was also supported by transcriptomic and proteomic analyses; a total of 560 common differentially expressed genes (DEGs) and 285 common differentially expressed proteins (DEPs) were identified. The DEGs were further verified by using RT‐qPCR. The combined analysis between the transcriptome and proteome revealed that the expression of some membrane‐related genes and proteins, mainly those related to sphingolipid metabolism, glycerophospholipid metabolism, steroid biosynthesis, ABC transporters and protein processing in the endoplasmic reticulum, was disordered. Myriocin affected the serine palmitoyl transferase (SPT) activity as evidenced through molecular docking. Our results indicate that myriocin has significant antifungal activity owing to its ability to induce membrane damage in Fon.

To unravel the underlying principles of membrane adaptation in small systems like bacterial cells, robust approaches to characterize membrane fluidity are needed. Currently available relevant methods require advanced instrumentation and are not suitable for high‐throughput settings needed to elucidate the biochemical pathways involved in adaptation. We developed a fast, robust, and financially accessible quantitative method to measure the microviscosity of lipid membranes in bulk suspension using a commercially available plate reader. Our approach, which is suitable for high‐throughput screening, is based on the simultaneous measurements of absorbance and fluorescence emission of a viscosity‐sensitive fluorescent dye, 9‐(2,2‐dicyanovinyl)julolidine (DCVJ), incorporated into a lipid membrane. We validated our method using artificial membranes with various lipid compositions over a range of temperatures and observed values that were in good agreement with previously published results. Using our approach, we were able to detect a lipid phase transition in the ruminant pathogen Mycoplasma mycoides., Membrane adaptation: We developed a robust and financially accessible quantitative method to measure the microviscosity of lipid membranes that is suitable for high‐throughput screening. The approach is based on the simultaneous measurements of absorbance and fluorescence emission of a fluorescent dye and allows monitoring of the temperature dependence of viscosity in bacterial membranes.

Mitochondrial respiration (R) is central to plant physiology and responds dynamically to daily short-term temperature changes. In the longer-term, changes in energy demand and membrane fluidity can decrease leaf R at a common temperature and increase the temperature at which leaf R peaks (Tmax). However, leaf R functionality is more susceptible to short-term heatwaves. Catalysis increases with rising leaf temperature, driving faster metabolism and leaf R demand, despite declines in photosynthesis restricting assimilate supply and growth. Proteins denature as temperatures increase further, adding to maintenance costs. Excessive heat also inactivates respiratory enzymes, with a concomitant limitation on the capacity of the R system. These competing push-and-pull factors are responsible for the diminishing acceleration in leaf R rate as temperature rises. Under extreme heat, membranes become overly fluid and enzymes such as the cytochrome c oxidase are impaired. Such changes can lead to over-reduction of the energy system culminating in reactive oxygen species production. This ultimately leads to the total breakdown of leaf R, setting the limit of leaf survival. Understanding the heat stress responses of leaf R is imperative given the continued rise in frequency and intensity of heatwaves and the importance of R for plant fitness and survival.

Sphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO linein whichserine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In this study, LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, that is, 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 hours. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of growth rates analogous to those of control LY-B cells. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led only to a sixfold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Smaller, though significant, changes were also detected in glycerophospholipids under SL-restriction conditions., This work was supported in part by grants from the Spanish Ministry of Economy (grant FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grants No. IT1264-19 and IT1270-19), as well as Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government, and by the Swiss National Science Foundation (310030-184949).

Amyloid β-protein (Aβ) molecules tend to aggregate and subsequently form low MW (LMW) oligomers, high MW (HMW) aggregates such as protofibrils, and ultimately fibrils. These Aβ species can generally form amyloid plaques implicated in the neurodegeneration of Alzheimer disease (AD), but therapies designed to reduce plaque load have not demonstrated clinical efficacy. Recent evidence implicates amyloid oligomers in AD neuropathology, but the precise mechanisms are uncertain. We examined the mechanisms of neuronal dysfunction from HMW-Aβ1-42 exposure by measuring membrane integrity, reactive oxygen species (ROS) generation, membrane lipid peroxidation, membrane fluidity, intracellular calcium regulation, passive membrane electrophysiological properties, and long-term potentiation (LTP). HMW-Aβ1-42 disturbed membrane integrity by inducing ROS generation and lipid peroxidation, resulting in decreased membrane fluidity, intracellular calcium dysregulation, depolarization, and impaired LTP. The damaging effects of HMW-Aβ1-42 were significantly greater than those of LMW-Aβ1-42. Therapeutic reduction of HMW-Aβ1-42 may prevent AD progression by ameliorating direct neuronal membrane damage.-Yasumoto, T., Takamura, Y., Tsuji, M., Watanabe-Nakayama, T., Imamura, K., Inoue, H., Nakamura, S., Inoue, T., Kimura, A., Yano, S., Nishijo, H., Kiuchi, Y., Teplow, D. B., Ono, K. High molecular weight amyloid β1-42 oligomers induce neurotoxicity via plasma membrane damage.

Effects of various growth temperatures on cell membrane fatty acid composition of Salmonella Typhimurium (S. Typhimurium) and the resistance to Pulsed Electric Fields (PEF) treatments, as well as PEF combining with mild‐thermal (35, 45, 55 °C) treatments were investigated. Results indicated that the PEF resistance of S. Typhimurium at stationary phase was varied markedly at different growth temperatures. S. Typhimurium grown at 45 °C exhibited greater PEF resistance than cells grown at a lower temperature. Alteration of membrane fatty acid composition decreased in the unsaturated to saturated fatty acids ratio (UFA/SFA) and increased in cyclopropane fatty acids (CFA) proportion as growth temperature increased. It was found that the PEF resistance of S. Typhimurium at stationary phase was in a membrane fluidity dependent manner. Thermal combined PEF treatment improved the PEF lethality which indicated that the PEF resistance of S. Typhimurium was greatly affected by the fluidity of cytomembrane.

The importance of diadinoxanthin (Ddx) de-epoxidation in the short-term modulation of the temperature effect on photosynthetic membranes of the diatom Phaeodactylum tricornutum was demonstrated by electron paramagnetic resonance (EPR), Laurdan fluorescence spectroscopy, and high-performance liquid chromatography. The 5-SASL spin probe employed for the EPR measurements and Laurdan provided information about the membrane area close to the polar head groups of the membrane lipids, whereas with the 16-SASL spin probe, the hydrophobic core, where the fatty acid residues are located, was probed. The obtained results indicate that Ddx de-epoxidation induces a two component mechanism in the short-term regulation of the membrane fluidity of diatom thylakoids during changing temperatures. One component has been termed the "dynamic effect" and the second the "stable effect" of Ddx de-epoxidation. The "dynamic effect" includes changes of the membrane during the time course of de-epoxidation whereas the "stable effect" is based on the rigidifying properties of Dtx. The combination of both effects results in a temporary increase of the rigidity of both peripheral and internal parts of the membrane whereas the persistent increase of the rigidity of the hydrophobic core of the membrane is solely based on the "stable effect."

Objectives Silicate bioactive glass (BG) has been widely demonstrated to stimulate both of the hard and soft tissue regeneration, in which ion products released from BG play important roles. However, the mechanism by which ion products act on cells on cells is unclear. Materials and methods Human umbilical vein endothelial cells and human bone marrow stromal cells were used in this study. Fluorescence recovery after photobleaching and generalized polarization was used to characterize changes in cell membrane fluidity. Migration, differentiation and apoptosis experiments were carried out. RNA and protein chip were detected. The signal cascade is simulated to evaluate the effect of increased cell membrane fluidity on signal transduction. Results We have demonstrated that ion products released from BG could effectively enhance cell membrane fluidity in a direct and physical way, and Si ions may play a major role. Bioactivities of BG ion products on cells, such as migration and differentiation, were regulated by membrane fluidity. Furthermore, we have proved that BG ion products could promote apoptosis of injured cells based on our conclusion that BG ion products increased membrane fluidity. Conclusions This study proved that BG ion products could develop its bioactivity on cells by directly enhancing cell membrane fluidity and subsequently affected cell behaviours, which may provide an explanation for the general bioactivities of silicate material., Ion products released from 45S5 Bioglass can significantly enhance cell membrane fluidity, showing a faster fluorescence recovery rate in the bleached area. As the fluidity of the cell membrane increases, the signal transduction and cell recognition processes on the cell membrane are accelerated, leading to increased cell migration and differentiation, even membrane‐related apoptosis.

Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.

Placement of dental implants initiates inflammatory foreign body response, in which macrophages play a central role and affect the subsequent tissue healing process such as bone formation. The purpose of this study was to fabricate phosphatidylserine (PS)-containing supported lipid bilayers (SLBs) on a titanium surface to regulate the polarization of macrophages, a critical factor that affects following tissue healing and regeneration. The fluorescent recovery after photobleaching images showed that the percentage of PS had a critical influence on the fluidity, and 20% PS had the highest fluidity. Furthermore, more expanded and elongated cells were observed in the SLB-coated groups. transforming growth factor-β1 and vascular endothelial growth factor, the key cytokine markers of M2 macrophage polarization, were increased in the SLB-coated groups, especially in the 20% PS group. Consistently, cells cultured on the SLB-coated titanium exhibited the distribution of CD206+ , which is a M2 macrophage specific maker. The results of this study demonstrated M2 polarization of macrophages on PS-SLB-coated titanium discs, which suggests the application of PS-SLB as an immune-regulating coating material to improve tissue reactions to dental implants. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2625-2633, 2018.

Na+ , K+ -ATPase (NKA) activity, which establishes the sodium and potassium gradient across the cell membrane and is instrumental in the propagation of the nerve impulses, is altered in a number of neurological and neuropsychiatric disorders, including autism spectrum disorders (ASD). In the present work, we examined a wide range of biochemical and cellular parameters in the attempt to understand the reason(s) for the severe decrease in NKA activity in erythrocytes of ASD children that we reported previously. NKA activity in leukocytes was found to be decreased independently from alteration in plasma membrane fluidity. The different subunits were evaluated for gene expression in leukocytes and for protein expression in erythrocytes: small differences in gene expression between ASD and typically developing children were not apparently paralleled by differences in protein expression. Moreover, no gross difference in erythrocyte plasma membrane oxidative modifications was detectable, although oxidative stress in blood samples from ASD children was confirmed by increased expression of NRF2 mRNA. Interestingly, gene expression of some NKA subunits correlated with clinical features. Excess inhibitory metals or ouabain-like activities, which might account for NKA activity decrease, were ruled out. Plasma membrane cholesterol, but not phosphatidylcholine and phosphatidlserine, was slighty decreased in erythrocytes from ASD children. Although no compelling results were obtained, our data suggest that alteration in the erytrocyte lipid moiety or subtle oxidative modifications in NKA structure are likely candidates for the observed decrease in NKA activity. These findings are discussed in the light of the relevance of NKA in ASD. Autism Res 2018, 11: 1388-1403. © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The activity of the cell membrane enzyme NKA, which is instrumental in the propagation of the nerve impulses, is severely decreased in erythrocytes from ASD children and in other brain disorders, yet no explanation has been provided for this observation. We strived to find a biological/biochemical cause of such alteration, but most queries went unsolved because of the complexity of NKA regulation. As NKA activity is altered in many brain disorders, we stress the relevance of studies aimed at understanding its regulation in ASD.

The volatile anesthetic isoflurane (ISO) has previously been shown to increase the fluidity of artificial lipid membranes, but very few studies have used biological cell membranes. Therefore, to investigate whether ISO affects the mobility of membrane proteins, fluorescence-labeled transferrin receptor (TfR) and glycosylphosphatidylinositol (GPI)-anchored protein were expressed in human embryonic kidney 293T cells and neural cells and lateral diffusion was examined using fluorescence recovery after photobleaching. Lateral diffusion of the TfR increased with ISO treatment. On the other hand, there was no effect on GPI-anchored protein. We also used GC/MS to confirm that there was no change in the concentration of ISO due to vaporization during measurement. These results suggest that ISO affects the mobility of transmembrane protein molecules in living cells.

The marine cyanobacteria of the genus Synechococcus are important primary producers, displaying a wide latitudinal distribution that is underpinned by diversification into temperature ecotypes. The physiological basis underlying these ecotypes is poorly known. In many organisms, regulation of membrane fluidity is crucial for acclimating to variations in temperature. Here, we reveal the detailed composition of the membrane lipidome of the model strain Synechococcus sp. WH7803 and its response to temperature variation. Unlike freshwater strains, membranes are almost devoid of C18, mainly containing C14 and C16 chains with no more than two unsaturations. In response to cold, we observed a rarely observed process of acyl chain shortening that likely induces membrane thinning, along with specific desaturation activities. Both of these mechanisms likely regulate membrane fluidity, facilitating the maintenance of efficient photosynthetic activity. A comprehensive examination of 53 Synechococcus genomes revealed clade-specific gene sets regulating membrane lipids. In particular, the genes encoding desaturase enzymes, which is a key to the temperature stress response, appeared to be temperature ecotype-specific, with some of them originating from lateral transfers. Our study suggests that regulation of membrane fluidity has been among the important adaptation processes for the colonization of different thermal niches by marine Synechococcus.

Omega-3 (ω-3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are known to have important roles in human health and disease. Besides being utilized as fuel, ω-3 FAs have specific functions based on their structural characteristics. These functions include serving as ligands for several receptors, precursors of lipid mediators, and components of membrane glycerophospholipids (GPLs). Since ω-3 FAs (especially DHA) are highly flexible, the levels of DHA in GPLs may affect membrane biophysical properties such as fluidity, flexibility, and thickness. Here, we summarize some of the cellular mechanisms for incorporating DHA into membrane GPLs and propose biological effects and functions of DHA-containing membranes of several cell and tissue types. This article is protected by copyright. All rights reserved.

The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier-systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) towards mechanical stress, typically prevailing in stirred-tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23 W kg−1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6 % release of the previously encapsulated model dye calcein was recognized at 25°C within 48 h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two-phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case. This article is protected by copyright. All rights reserved

Exposure of cells and nanoparticles to near-infrared nanosecond pulsed laser light can lead to efficient intracellular delivery of molecules while maintaining high cell viability by a photoacoustic phenomenon known as transient nanoparticle energy transduction (TNET). Here, we examined the influence of cytoskeletal mechanics and plasma membrane fluidity on intracellular uptake of molecules and loss of cell viability due to TNET. We found that destabilization of actin filaments using latrunculin A led to greater uptake of molecules and less viability loss caused by TNET. Stabilization of actin filaments using jasplakinolide had no significant effect on uptake or viability loss caused by TNET. To study the role of plasma membrane fluidity, we increased fluidity by depletion of membrane cholesterol using methyl-β-cyclodextrin and decreased fluidity by enrichment of the membrane with cholesterol using water-soluble cholesterol. Neither of these membrane fluidity changes significantly altered cellular uptake or viability loss caused by TNET. We conclude that weakening mechanical integrity of the cytoskeleton can increase intracellular uptake and decrease loss of cell viability, while plasma membrane fluidity does not appear to play a significant role in uptake or viability loss caused by TNET. The positive effects of cytoskeletal weakening may be due to an enhanced ability of the cell to recover from the effects of TNET and maintain viability. Biotechnol. Bioeng. 2017;114: 2390-2399. © 2017 Wiley Periodicals, Inc.

Nitrated fatty acids (NO2 -FAs) act as anti-inflammatory signal mediators, albeit the molecular mechanisms behind NO2 -FAs' influence on diverse metabolic and signaling pathways in inflamed tissues are essentially elusive. Here, we combine fluorescence measurements with surface-specific sum frequency generation vibrational spectroscopy and coarse-grained computer simulations to demonstrate that NO2 -FAs alter lipid organization by accumulation at the membrane-water interface. As the function of membrane proteins strongly depends on both, protein structure as well as membrane properties, we consecutively follow the structural dynamics of an integral membrane protein in presence of NO2 -FAs. Based on our results, we suggest a molecular mechanism of the NO2 -FA in vivo activity: Driven by the NO2 -FA-induced lipid layer reorganization, the structure and function of membrane-associated (signaling) proteins is indirectly affected.

The intention of this study was to investigate the role of polyunsaturated fatty acids (PUFA) in the cold adaptation of Rhodosporidium kratochvilovae YM25235 by knockout of the Δ12/Δ15-fatty acid desaturase gene (RKD12) to inactivate Δ12/Δ15-fatty acid desaturase. Polymerase chain reaction (PCR) amplification was used to detect the genomic structure of RKD12 gene in YM25235. The RKD12 gene was knocked out by DNA homologous recombination to inhibit the biosynthesis of PUFA. Then, the contents of linoleic acid (LNA) and α-linolenic acid (ALA) after gene knockout were investigated using a gas chromatography-mass spectrometer, followed by determination of the growth rate and membrane fluidity of YM25235 at low temperature. After PCR amplification, a 1611 bp genomic fragment was amplified from YM25235. When the RKD12 gene was knocked out, the contents of LNA and ALA in YM25235 significantly decreased. The growth rate and membrane fluidity of YM25235 decreased significantly at low temperature. Inhibition of PUFA biosynthesis by RKD12 gene knockout influenced cold adaptation of YM25235 by decreasing the PUFA content in cell membranes and reducing the growth rate and membrane fluidity of YM25235 at low temperature.

The behavior of a single Aβ40 molecule within a dipalmitoylphosphatidylcholine (DPPC) bilayer was studied by all-atom molecular dynamics simulations. The effect of membrane structure was investigated on Aβ40 behavior, secondary structure, and insertion depth. Simulations were performed at three temperatures (323, 310, and 300 K) to probe three different bilayer fluidities. Results show that at all above temperatures, the peptide contains two short helices, coil, bend, and turn structures. At 300 K, the peptide contains a region with β structure in C-terminal region. Our results also show that Aβ decreases the bilayer thickness and the order of lipids in its vicinity which leads to water insertion into the bilayer and concomitant increase in the local fluidity. The peptide remains embedded in the bilayer at all temperatures, and become inserted into the bilayer up to several residues at 323 and 310 K. At 310 and 300 K, the dominant interaction energy between Aβ and bilayer changes from electrostatic to van der Waals. It can be proposed that at higher temperatures (e.g., 323 K), Lys28 and the C-terminal region of the peptide play the role of two anchors that keep Aβ inside the top leaflet. This study demonstrates that Aβ molecule can perturb the integrity of cellular membranes. Proteins 2017; 85:1298-1310. © 2017 Wiley Periodicals, Inc.

Ganoderma lucidum has become a potential model system for evaluating how environmental factors regulate the secondary metabolism of basidiomycetes. Heat stress (HS) is one of the most important environmental factors. It was previously reported that HS could induce the biosynthesis of ganoderic acids (GA). In this study, we found that HS increased GA biosynthesis and also significantly increased cell membrane fluidity. Furthermore, our results showed that addition of the membrane rigidifier dimethylsulfoxide (DMSO) could revert the increased GA biosynthesis elicited by HS. These results indicate that an increase in membrane fluidity is associated with HS-induced GA biosynthesis. Further evidence showed that the GA content was decreased in D9des-silenced strains and could be reverted to WT levels by addition of the membrane fluidizer benzyl alcohol (BA). In contrast, GA content was increased in D9des-overexpression strains and could be reverted to WT levels by the addition of DMSO. Furthermore, both membrane fluidity and GA biosynthesis induced by HS could be reverted by DMSO in WT and D9des-silenced strains. To the best of our knowledge, this is the first report demonstrating that membrane fluidity is involved in the regulation of heat stress induced secondary metabolism in filamentous fungi.

Contents The objective was to assess the effect of cooling to different subzero temperatures around ice formation (−5°C) on dog sperm cryosurvival and plasma membrane fluidity. Semen was centrifuged, and sperm were resuspended in a Tris-egg yolk medium (3% glycerol). Diluted sperm were cooled from 22 to 5°C, and then, a Tris-egg yolk medium containing 7% glycerol was added (final concentration of 5% glycerol and 200 × 106 cells/ml). Sperm were packaged in 0.5-ml plastic straws, and equilibration was done 16 hr at 5°C before freezing. I. Straws (n = 47) at 5°C were exposed to nitrogen vapours to determine the freezing point. II. Other straws (from different ejaculates) processed as mentioned, were further cooled to −3, −5 or −7°C and immediately rewarmed in a water bath at 37°C. Motility, plasma membrane functionality and acrosome integrity were assessed. III. Other straws (from different ejaculates) processed as mentioned were further cooled to −3 or −5°C, frozen over nitrogen vapours and stored in liquid nitrogen for one month. Straws were thawed in a water bath at 38°C for 30 s. Motility, plasma membrane functionality, plasma membrane integrity, acrosome integrity, capacitation status and plasma membrane fluidity were assessed. Ice nucleation temperature was −14.3 ± 2.05°C (mean ± SD); cooling to +5, −3, −5 and −7°C, without freezing, produces no differences on sperm quality between target temperatures; cooling to +5, −3, and −5°C produced no differences on sperm survival and plasma membrane fluidity after freeze–thawing. In conclusion, cooling of dog spermatozoa to different subzero temperatures did not improve sperm cryosurvival and had no effect on plasma membrane fluidity after thawing.

New Findings What is the central question of this study? What is the influence of a single bout of exercise on the properties of erythrocyte fractions at different ages? What is the main finding and its importance? A single bout of exercise in untrained men induced oxidative stress in erythrocytes and had an influence on antioxidant defense in these cells. Old erythrocytes were more sensitive to oxidative damage than young and middle-aged cells. Higher levels of glutathione in old erythrocyte fractions did not protect them against oxidative stress. It seems that exercise may promote the removal of old erythrocytes from the circulation. The objective of this study was to establish the role of exercise-induced oxidative stress in the erythrocyte fractions [young (YF), middle-aged (MAF) and old (OF)] of young untrained men after acute exercise. Blood samples were collected before exercise, immediately after and 1 h after exercise. The maximal power generated was 292 ± 27 W, and exercise duration was 8.73 ± 0.9 min. Different optical properties and oxidative stress parameters were found in each erythrocyte fraction. Total thiols in YF and MAF after exercise and after 1 h rest were similar to values before exercise; however, in OF {32.7 ± 9.8 nmol [mg haemoglobin (Hb)]−1} the concentration was lower in comparison to YF [55.5 ± 3.2 nmol (mg Hb)−1] and MAF [56.8 ± 7.7 nmol (mg Hb)−1] and increased 1 h later (P

Several reports described different modes of cell death triggered by antimicrobial peptides (AMPs) due to direct effects on membrane disruption, and more recently by apoptosis and necrosis-like patterns. Cytotoxic curves of four β-hairpin AMPs (gomesin, protegrin, tachyplesin, and polyphemusin) were obtained from several human leukemic lineages and normal monocytes and Two cell lines were then selected based on their cytotoxic sensitivity. One was sensitive to AMPs (K562) and the other resistant (KG-1) and their effect compared between these lineages. Thus, these lineages were chosen to further investigate biological features related with their cytotoxicities to AMPs. Stimulation with AMPs produced cell death, with activation of caspase-3, in K562 lineage. Increase on the fluidity of plasmatic membrane by reducing cholesterol potentiated cytotoxicity of AMPs in both lineages. Quantification of internal and external gomesin binding to the cellular membrane of both K562 and KG-1 cells showed that more peptide is accumulated inside of K562 cells. Additionally, evaluation of multi-drug resistant pumps activity showed that KG-1 has more activity than K562 lineage. A comparison of intrinsic gene patterns showed great differences between K562 and KG-1, but stimulation with gomesin promoted few changes in gene expression patterns. Differences in internalization process through the plasma membrane, multidrug resistance pumps activity, and gene expression pattern are important features to AMPs regulated cell death. J. Cell. Biochem. 118: 1764-1773, 2017. © 2016 Wiley Periodicals, Inc.

Summary Psychrobacter sp. PAMC 21119, isolated from Antarctic permafrost soil, grows and proliferates at subzero temperatures. However, its major mechanism of cold adaptation regulation remains poorly understood. We investigated the transcriptomic and proteomic responses of this species to cold temperatures by comparing profiles at −5°C and 20°C to understand how extreme microorganisms survive under subzero conditions. We found a total of 2,906 transcripts and 584 differentially expressed genes (≥ twofold, P

The easy-to-use in vivo model, zebrafish larva, is being increasingly used to screen chemical-induced hepatotoxicity, with a good predictivity for various mechanisms of liver injury. However, nothing is known about its applicability in exploring the mechanism called membrane remodeling, depicted as changes in membrane fluidity or lipid raft properties. The aim of this study was, therefore, to substantiate the zebrafish larva as a suitable in vivo model in this context. Ethanol was chosen as a prototype toxicant because it is largely described, both in hepatocyte cultures and in rodents, as capable of inducing a membrane remodeling leading to hepatocyte death and liver injury. The zebrafish larva model was demonstrated to be fully relevant as membrane remodeling was maintained even after a 1-week exposure without any adaptation as usually reported in rodents and hepatocyte cultures. It was also proven to exhibit a high sensitivity as it discriminated various levels of cytotoxicity depending on the extent of changes in membrane remodeling. In this context, its sensitivity appeared higher than that of WIF-B9 hepatic cells, which is suited for analyzing this kind of hepatotoxicity. Finally, the protection afforded by a membrane stabilizer, ursodeoxycholic acid (UDCA), or by a lipid raft disrupter, pravastatin, definitely validated zebrafish larva as a reliable model to quickly assess membrane remodeling involvement in chemical-induced hepatotoxicity. In conclusion, this model, compatible with a high throughput screening, might be adapted to seek hepatotoxicants via membrane remodeling, and also drugs targeting membrane features to propose new preventive or therapeutic strategies in chemical-induced liver diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Summary The effect of culture temperature on the conformation of membrane lipid of Salmonella Typhimurium as well as their pulsed electric field (PEF) resistance was investigated. Results revealed that S. Typhimurium cells cultured at relatively lower temperature (10 and 25 °C) were more easily inactivated by PEF treatment than those cultured at relatively higher temperature (37 and 45 °C). Using a Weibull model, it was determined that the PEF treatment time to inactivate 90% S. Typhimurium cells cultured at 45 °C was almost fourfold compared to those cultured at 10 °C. Results of micro-Raman spectroscopy indicated that as the culture temperature increased, the order degree of C–C and lateral packing order of membrane lipid chain also increased, resulting in a drop in the membrane fluidity. These results are important in considering the use of heat and PEF to inactivate microbe contaminants in food.

Scope Alzheimer's disease is a neurodegenerative condition marked by the formation and aggregation of amyloid-β (Aβ) peptides. There exists, to this day, no cure or effective prevention for the disease; however, there is evidence that a healthy diet and certain food products can slow down first occurrence and progression. To investigate if food ingredients can interact with peptide aggregates, synthetic membranes that contained aggregates consisting of cross-β sheets of the membrane active fragment A β 25 -- 35 are prepared. Methods and results The impact of resveratrol, found in grapes, caffeine, the main active ingredient in coffee, β-carotene, found in orange fruits and vegetables, and epigallocatechin gallate (EGCG), a component of green tea, on the size and volume fraction of Aβ aggregates is studied using optical and fluorescence microscopy, X-ray diffraction, UV-vis spectroscopy, and molecular dynamics simulations. All compounds are membrane active and spontaneously partitioned in the synthetic brain membranes. While resveratrol and caffeine lead to membrane thickening and reduced membrane fluidity, β-carotene and EGCG preserve or increase fluidity. Conclusion Resveratrol and caffeine do not reduce the volume fraction of peptide aggregates while β-carotene significantly reduces plaque size. Interestingly, EGCG dissolves peptide aggregates and significantly decreases the corresponding cross-β and β-sheet signals.

Sphingolipidoses arise from inherited loss of function of key enzymes regulating the sphingolipid (SL) metabolism and the accumulation of large quantities of these lipids in affected cells. Most frequently, toxicity is manifested in the nervous system, where survival and function of neurons and glial cells are most affected. Although detailed information is available on neuroglial alterations during terminal stages of the disease, the initial pathogenic mechanisms triggering neuropathology are largely unclear. Because they are key components of biological membranes, changes in the local concentration of SLs are likely to impact the organization of membrane domains and functions. This Commentary proposes that SL toxicity involves initial defects in the integrity of lipid domains, membrane fluidity, and membrane bending, leading to membrane deformation and deregulation of cell signaling and function. Understanding how SLs alter membrane architecture may provide breakthroughs for more efficient treatment of sphingolipidoses. © 2016 Wiley Periodicals, Inc.

Crustaceans found in metal-contaminated regions are able to survive, and the authors investigated the physiological mechanisms involved by comparing populations from contaminated and noncontaminated areas. The objective of the present study was to measure the cellular transport of a nonessential metal (cadmium [Cd]) in gills and hepatopancreas of Ucides cordatus, together with cell membrane fluidity, metallothionein levels, and lipid peroxidation. The 2 populations compared were from a polluted and a nonpolluted mangrove area of Sao Paulo State, Brazil. The authors found, for the first time, larger Cd transport in gills and hepatopancreatic cells from crabs living in polluted mangrove areas. The cells also had lower plasma membrane fluidity, increased lipid peroxidation and less metallothionein compared to those from nonpolluted regions. The authors also found larger amounts of Cd in intracellular organelles of gills, but not in the hepatopancreas, from crabs in polluted regions. Therefore, in polluted areas, these animals showed higher Cd transport and lower plasma membrane fluidity and storage of Cd intracellularly in gill cells, whereas hepatopancreatic cells used metallothionein as their main line of defense. The findings suggest that crabs from polluted areas can accumulate Cd more easily than crabs from nonpolluted areas, probably because of an impairment of the regulatory mechanisms of cell membrane transport. Environ Toxicol Chem 2017;36:361-371. © 2016 SETAC.

One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 μm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity.

Membrane fluidity change has long been suggested as the primary mechanism by which, plants adapt to cold stress, but the underlying molecular mechanisms are not completely established. In this study, we found that a knockout of acyl-lipid/CoA desaturase 1 gene (ADS1; EC 1.14.99) enhances freezing tolerance after cold acclimation (CA). Fatty acid composition analysis demonstrated that 18:1 content in ads1 mutant plants was 20% lower than in wild-type (WT) grown at 23°C. Lipidomics revealed that 34C-species of monogalactosyl diacylglycerol (MGDG) content in ads1 mutants were 3.3-14.9% lower than in WT. Lipid positional analysis identified 10% lower 18:1 fatty acid content at the sn-2 position of MGDG. The cytosolic calcium content in ads1 mutant plants was also approximately two-times higher than that of WT in response to cold shock. Each of these biochemical differences between WT and ads1 mutant disappeared after CA. Subcellular localization of C- and N-terminal enhanced-fluorescence-fusion proteins indicated that ADS1 localized exclusively to chloroplasts. These observations suggest that ADS1-mediated alteration of chloroplast membrane fluidity is required to prime a CA response, and is the upstream event of cytosolic calcium signaling.

In order to reveal the growth mechanism of gold nanorods (GNRs) in a binary surfactant system, we synthesized various GNRs by changing the concentration of the surfactants, AgNO3 , and HBr in the growth solution. We found that the benzyldimethylhexadecylammoniumchloride surfactant had weak interaction with the gold ions, but it could reduce the membrane fluidity. In addition, we could dramatically decrease the cetyltrimethylammonium bromide concentration required for GNR growth by adding an HBr solution. Notably, Ag+ ions were necessary to break the symmetry of the seed crystals for GNR growth, but increasing the concentration of Ag+ and Br− ions caused a decrease in the template size.

The effect of chitosan on growth of Trichoderma spp., a cosmopolitan genus widely exploited for their biocontrol properties was evaluated. Based on genotypic (ITS of 18S rDNA) characters, four isolates of Trichoderma were identified as T. pseudokoningii FLM16, T. citrinoviride FLM17, T. harzianum EZG47, and T. koningiopsis VSL185. Chitosan reduces radial growth of Trichoderma isolates in concentration-wise manner. T. koningiopsis VSL185 was the most chitosan tolerant isolate in all culture media amended with chitosan (0.5-2.0 mg ml(-1) ). Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) were determined showing that T. koningiopsis VSL185 displays higher chitosan tolerance with MIC value >2000 μg ml(-1) while for other Trichoderma isolates MIC values were around 10 μg ml(-1) . Finally, free fatty acid composition reveals that T. koningiopsis VSL185, chitosan tolerant isolate, displays lower linolenic acid (C18:3) content than chitosan sensitive Trichoderma isolates. Our findings suggest that low membrane fluidity is associated with chitosan tolerance in Trichoderma spp.

Mechanical stretching and topographical cues are both effective mechanical stimulations for regulating cell morphology, orientation, and behaviors. The competition of these two mechanical stimulations remains largely underexplored. Previous studies have suggested that a small cyclic mechanical strain is not able to reorient cells that have been pre-aligned by relatively large linear microstructures, but can reorient those pre-aligned by small linear micro/nanostructures if the characteristic dimension of these structures is below a certain threshold. Likewise, for micro/nanostructures with a given characteristic dimension, the strain must exceed a certain magnitude to overrule the topographic cues. There are however no in-depth investigations of such "thresholds" due to the lack of close examination of dynamic cell orientation during and shortly after the mechanical loading. In this study, the time-dependent combinatory effects of active and passive mechanical stimulations on cell orientation are investigated by developing a micromechanical stimulator. The results show that the cells pre-aligned by linear micro/nanostructures can be altered by cyclic in-plane strain, regardless of the structure size. During the loading, the micro/nanostructures can resist the reorientation effects by cyclic in-plane strain while the resistive capability (measured by the mean orientation angle change and the reorientation speed) increases with the increasing characteristic dimension. The micro/nanostructures also can recover the cell orientation after the cessation of cyclic in-plane strain, while the recovering capability increases with the characteristic dimension. The previously observed thresholds are largely dependent on the observation time points. In order to accurately evaluate the combinatory effects of the two mechanical stimulations, observations during the active loading with a short time interval or endpoint observations shortly after the loading are preferred. This study provides a microengineering solution to investigate the time-dependent combinatory effects of the active and passive mechanical stimulations and is expected to enhance our understanding of cell responses to complex mechanical environments. Biotechnol. Bioeng. 2016;113: 2191-2201. © 2016 Wiley Periodicals, Inc.

Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol-rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl-β-cyclodextrin (β-MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike β-MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101-109, 2017. © 2016 Wiley Periodicals, Inc.