A metagenomics study reveals the gut microbiome as a sex-specific modulator of healthy aging in Hainan centenarians.

BACKGROUND: Sex differences in health status and life expectancy are widely accepted to exist. The mechanisms underlying it are still poorly understood. In this study, we aimed to clarify the influences and contributions of sex on the gut microbiome in healthy centenarians and to explore the different roles played by the gut microbiome in healthy aging between the sexes. RESULTS: Taking covariates of different dimensions into account (social demographics, anthropometry, the activities of daily living, dietary structure, mental state, blood tests, lifestyle and disease history), our data showed that sex was one of the most significant covariates affecting the gut microbiome of healthy centenarians at both the species and Kyoto Encyclopedia of Genes and Genomes Orthology (KO) levels. The beta diversity between the sexes were significantly different (Adonis test: p = 0.011, R2 = 0.031), and the male centenarians had a greater alpha diversity than the females (Simpson and Shannon test: P<0.05). At the species level, we identified 31 species enriched in males and 7 species enriched in females. The composition and function patterns of the microbiome varied between the sexes. Further functional analysis showed that males' gut microbiome exhibited greater resistance to oxidative stress compared to females. In contrast to men, the species associated with healthy aging dominated among healthy female centenarians, while the species associated with unhealthy aging were relatively rare. CONCLUSIONS: The present study reveals that the gut microbiome structure and resistance to oxidative stress in healthy centenarians differ between the sexes and provides new insights into the possible sex-specific role of the gut microbiome in healthy aging.

Hereditary hemorrhagic telangiectasia involving portal venous system: A case report and review of the literature.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder with an incidence of approximately 1 in 5000 in the general population. It is characterized by vasodilation, which affects specific organs, such as the skin, mucous membranes, brain, lungs, gastrointestinal tract, liver, and others. However, HHT rarely involves the portal venous system to cause serious clinical complications. CASE SUMMARY: A 68-year-old woman was admitted to the emergency department due to four consecutive days of abdominal pain and bloody stool and was subsequently diagnosed with HHT. Computed tomography angiography confirmed the presence of an arteriovenous fistula (AVFs). Considering this specific manifestation, whole exome sequencing was performed. After a comprehensive evaluation, a selective superior mesenteric artery embolization was prioritized to avoid intestinal ischemia. The postoperative symptoms of the patient were quickly relieved. Unfortunately, two months post-procedure the patient died from intestinal necrosis and abdominal infection related to remaining AVFs. CONCLUSION: For patients with diffuse superior mesenteric AVFs, selective mesenteric arterial embolization may lead to positive short-term outcomes.

Regulation of serotonin production by specific microbes from piglet gut.

BACKGROUND: Serotonin is an important signaling molecule that regulates secretory and sensory functions in the gut. Gut microbiota has been demonstrated to affect serotonin synthesis in rodent models. However, how gut microbes regulate intestinal serotonin production in piglets remains vague. To investigate the relationship between microbiota and serotonin specifically in the colon, microbial composition and serotonin concentration were analyzed in ileum-cannulated piglets subjected to antibiotic infusion from the ileum when comparing with saline infusion. Microbes that correlated positively with serotonin production were isolated from piglet colon and were further used to investigate the regulation mechanisms on serotonin production in IPEC-J2 and a putative enterochromaffin cell line RIN-14B cells. RESULTS: Antibiotic infusion increased quantities of Lactobacillus amylovorus (LA) that positively correlated with increased serotonin concentrations in the colon, while no effects observed for Limosilactobacillus reuteri (LR). To understand how microbes regulate serotonin, representative strains of LA, LR, and Streptococcus alactolyticus (SA, enriched in feces from prior observation) were selected for cell culture studies. Compared to the control group, LA, LR and SA supernatants significantly up-regulated tryptophan hydroxylase 1 (TPH1) expression and promoted serotonin production in IPEC-J2 cells, while in RIN-14B cells only LA exerted similar action. To investigate potential mechanisms mediated by microbe-derived molecules, microbial metabolites including lactate, acetate, glutamine, and γ-aminobutyric acid were selected for cell treatment based on computational and metabolite profiling in bacterial supernatant. Among these metabolites, acetate upregulated the expression of free fatty acid receptor 3 and TPH1 while downregulated indoleamine 2,3-dioxygenase 1. Similar effects were also recapitulated when treating the cells with AR420626, an agonist targeting free fatty acid receptor 3. CONCLUSIONS: Overall, these results suggest that Lactobacillus amylovorus showed a positive correlation with serotonin production in the pig gut and exhibited a remarkable ability to regulate serotonin production in cell cultures. These findings provide evidence that microbial metabolites mediate the dialogue between microbes and host, which reveals a potential approach using microbial manipulation to regulate intestinal serotonin biosynthesis.

Lactobacillus oris improves non-alcoholic fatty liver in mice and inhibits endogenous cholesterol biosynthesis.

We previously confirmed that a strain of Lactobacillus oris isolated from the fecal samples of healthy Hainan centenarian having potent lipid-lowering ability in HepG2 cells; and this study was to investigate the effect of the stain on non-alcoholic fatty liver in mice in vivio. The Lactobacillus oris strain isolated from Hainan centenarian fecal samples were frozen stored in our laboratory. Thirty ob/ob mice (10 in each group) were orally gavaged with Lactobacillus oris (Lactobacillus, 5 × 109 cfu), mixed probiotics (Mixed, 5 × 109 cfu, a mixture with known lipid-lowering ability), or culture medium (Control) respectively. Lactobacillus oris isolated from fecal samples of Hainan centenarians showed significantly in vivo lipid lowering ability compared with the controls, and the ability was comparable with mixed probiotics strains in mice The possible mechanisms of lipid-lowering of probiotics and Lactobacillus oris may be associated with HMGR inhibition to suppress the synthesis of endogenous cholesterol; bile acids reabsorption, and intestinal FXR-FGF15 signaling pathways promoting the cholesterol conversion into bile acids secretion.

Lipid Nanoparticles Deliver the Therapeutic VEGFA mRNA In Vitro and In Vivo and Transform Extracellular Vesicles for Their Functional Extensions.

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells' response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs.

Interaction Kinetics of Individual mRNA-Containing Lipid Nanoparticles with an Endosomal Membrane Mimic: Dependence on pH, Protein Corona Formation, and Lipoprotein Depletion.

Lipid nanoparticles (LNPs) have emerged as potent carriers for mRNA delivery, but several challenges remain before this approach can offer broad clinical translation of mRNA therapeutics. To improve their efficacy, a better understanding is required regarding how LNPs are trapped and processed at the anionic endosomal membrane prior to mRNA release. We used surface-sensitive fluorescence microscopy with single LNP resolution to investigate the pH dependency of the binding kinetics of ionizable lipid-containing LNPs to a supported endosomal model membrane. A sharp increase of LNP binding was observed when the pH was lowered from 6 to 5, accompanied by stepwise large-scale LNP disintegration. For LNPs preincubated in serum, protein corona formation shifted the onset of LNP binding and subsequent disintegration to lower pH, an effect that was less pronounced for lipoprotein-depleted serum. The LNP binding to the endosomal membrane mimic was observed to eventually become severely limited by suppression of the driving force for the formation of multivalent bonds during LNP attachment or, more specifically, by charge neutralization of anionic lipids in the model membrane due to their association with cationic lipids from earlier attached LNPs upon their disintegration. Cell uptake experiments demonstrated marginal differences in LNP uptake in untreated and lipoprotein-depleted serum, whereas lipoprotein-depleted serum increased mRNA-controlled protein (eGFP) production substantially. This complies with model membrane data and suggests that protein corona formation on the surface of the LNPs influences the nature of the interaction between LNPs and endosomal membranes.

Amino acid utilization allows intestinal dominance of Lactobacillus amylovorus.

The mammalian intestine harbors heterogeneous distribution of microbes among which specific taxa (e.g. Lactobacillus) dominate across mammals. Deterministic factors such as nutrient availability and utilization may affect microbial distributions. Due to physiological complexity, mechanisms linking nutrient utilization and the dominance of key taxa remain unclear. Lactobacillus amylovorus is a predominant species in the small intestine of pigs. Employing a pig model, we found that the small intestine was dominated by Lactobacillus and particularly L. amylovorus, and enriched with peptide-bound amino acids (PBAAs), all of which were further boosted after a peptide-rich diet. To investigate the bacterial growth dominance mechanism, a representative strain L. amylovorus S1 was isolated from the small intestine and anaerobically cultured in media with free amino acids or peptides as sole nitrogen sources. L. amylovorus S1 grew preferentially with peptide-rich rather than amino acid-rich substrates, as reflected by enhanced growth and PBAA utilization, and peptide transporter upregulations. Utilization of free amino acids (e.g. methionine, valine, lysine) and expressions of transporters and metabolic enzymes were enhanced simultaneously in peptide-rich substrate. Additionally, lactate was elevated in peptide-rich substrates while acetate in amino acid-rich substrates, indicating distinct metabolic patterns depending on substrate forms. These results suggest that an increased capability of utilizing PBAAs contributes to the dominance of L. amylovorus, indicating amino acid utilization as a deterministic factor affecting intestinal microbial distribution. These findings may provide new insights into the microbe-gut nutrition interplay and guidelines for dietary manipulations toward gut health especially small intestine health.

Deep insights into the gut microbial community of extreme longevity in south Chinese centenarians by ultra-deep metagenomics and large-scale culturomics.

The gut microbes play important roles in human longevity and the gut microbiota profile of centenarians shows some unique features from young adults. Nowadays, most microbial studies on longevity are commonly based on metagenomic sequencing which may lose information about the functional microbes with extremely low abundance. Here, we combined in-depth metagenomic sequencing and large-scale culturomics to reveal the unique gut microbial structure of a Chinese longevity population, and to explore the possible relationship between intestinal microbes and longevity. Twenty-five healthy Hainan natives were enrolled in the study, including 12 centenarians and 13 senior neighbors. An average of 51.1 Gb raw sequencing data were obtained from individual fecal sample. We assembled 1778 non-redundant metagenomic assembled genomes (MAGs), 33.46% of which cannot be classified into known species. Comparison with the ordinary people in Hainan province, the longevous cohort displayed significantly decreased abundance of butyrate-producing bacteria and largely increased proportion of Escherichia coli, Desulfovibrio piger and Methanobrevibacter smithii. These species showed a constant change with aging. We also isolated 8,030 strains from these samples by large-scale culturomics, most of which belonged to 203 known species as identified by MALDI-TOF. Surprisingly, only 42.17% of the isolated species were also detected by metagenomics, indicating obvious complementarity between these two approaches. Combination of two complement methods, in-depth metagenomic sequencing and culturomics, provides deeper insights into the longevity-related gut microbiota. The uniquely enriched gut microbes in Hainan extreme decades population may help to promote health and longevity.

Transcriptome profiling of mRNAs in muscle tissue of Pinan cattle and Nanyang cattle.

Mammalian muscle development is regulated by complex gene networks at the molecular level. The revelation of gene regulatory mechanisms is an important basis for the study of muscle development and molecular breeding. To analyze the excellent meat performance of Pinan cattle at the molecular level, we performed high-throughput RNA sequencing to analyze the key regulatory genes that determine the muscle quality traits in Pinan cattle (n = 3) and Nanyang cattle (n = 3). We identified 57 differentially expressed genes in muscle tissue of Pinan cattle compared to that of Nanyang cattle, including 32 upregulated and 25 downregulated genes. GO enrichment analysis showed that these genes were significantly enriched in 'molecular function', including voltage-gated ion channel activity, calcium channel activity and calcium ion binding, and KEGG pathway analysis results revealed that adrenergic signaling in cardio myocytes, cell adhesion molecules and inositol phosphate metabolism pathway were significantly enriched. We identified the reliability of RNA-Seq data through RT-qPCR. Meanwhile, we found that GSTA3, PLCB1 and ISYNA1 genes are highly expressed in muscle tissue of Pinan cattle, and these genes play important roles in PI3K/Akt, MEK1/2-ERK and p53-ISYNA1 signaling pathway. In summary, our results suggested that these differentially expressed genes may play important roles in muscle development in Pinan cattle. However, the functions and mechanism of these significantly differential expressed genes should be investigated in future studies.

Circadian Gene PER2 Silencing Downregulates PPARG and SREBF1 and Suppresses Lipid Synthesis in Bovine Mammary Epithelial Cells.

PER2, a circadian clock gene, is associated with mammary gland development and lipid synthesis in rodents, partly via regulating peroxisome proliferator-activated receptor gamma (PPARG). Whether such a type of molecular link existed in bovines was unclear. We hypothesized that PER2 was associated with lipid metabolism and regulated cell cycles and apoptosis in bovine mammary epithelial cells (BMECs). To test this hypothesis, BMECs isolated from three mid-lactation (average 110 d postpartum) cows were used. The transient transfection of small interfering RNA (siRNA) was used to inhibit PER2 transcription in primary BMECs. The silencing of PER2 led to lower concentrations of cellular lipid droplets and triacylglycerol along with the downregulation of lipogenic-related genes such as ACACA, FASN, LPIN1, and SCD, suggesting an overall inhibition of lipogenesis and desaturation. The downregulation of PPARG and SREBF1 in response to PER2 silencing underscored the importance of circadian clock signaling and the transcriptional regulation of lipogenesis. Although the proliferation of BMECs was not influenced by PER2 silencing, the number of cells in the G2/GM phase was upregulated. PER2 silencing did not affect cell apoptosis. Overall, the data provided evidence that PER2 participated in the coordination of mammary lipid metabolism and was potentially a component of the control of lipid droplets and TAG synthesis in ruminant mammary cells. The present data suggested that such an effect could occur through direct effects on transcriptional regulators.

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

Metagenomics Study Reveals Changes in Gut Microbiota in Centenarians: A Cohort Study of Hainan Centenarians.

Several studies based on 16SrDNA analysis have revealed certain unique characteristics of gut microbiome in centenarians. We established a prospective cohort of fecal microbiota and conducted the first metagenomics-based study among centenarians. The objective was to explore the dynamic changes of gut microbiota in healthy centenarians and centenarians approaching end of life and to unravel the characteristics of aging-associated microbiome. Seventy-five healthy centenarians residing in three regions of Hainan participated in follow-up surveys and collection of fecal samples at intervals of 3 months. Data pertaining to dietary status, health status scores, cause of disease and death, and fecal specimens were collected for 15 months. Twenty participants died within 20 months during the follow-up period. The median survival time was 8-9 months (range, 1-17) and the mortality rate was 14.7% per year. The health status scores before death were significantly lower than those at 3 months before the end of the follow-up period [median score: 3 (range, 1-5), P < 0.05]. At this time, the participants mainly exhibited symptoms of anorexia and reduced dietary intake and physical activity. Metagenomics sequencing and analysis were carried out to characterize the gut microbiota changes in the centenarians during their transition from healthy status to death. Anosim analysis showed a significant change in gut microbiota from 7 months prior to death (R = 0.10, P = 0.02). All participants were grouped with 7 months before death as cut-off; no significant difference in α diversity was found between the two groups (P = 0.45). Semi-supervised monitoring and log rank sum analysis revealed significant changes in the abundance of ten bacterial species before death; of these, eight species were significantly reduced (Akkermansia muciniphila, Alistipes finegoldii, Alistipes shahii, Bacteroides faecis, Bacteroides intestinalis, Butyrivibrio crossotus, Bacteroides stercoris, and Prevotella stercorea) while two were significantly increased before death (Bifidobacterium longum and Ruminococcus bromii). Compared to centenarians in northern Italy, Hainan centenarians exhibited unique characteristics of gut microbiome. The abundance of ten bacterial species showed significant changes starting from 7 months before death. We speculate that these changes might occur before the clinical symptoms of deterioration in health status.

Asymmetric cationic liposomes designed for heat-activated association with cells.

Improved anticancer drugs and drug carriers are needed in combination therapies, such as hyperthermia-assisted chemotherapy. Liposomal drug carriers with advanced functions are attractive candidates for targeted accumulation and drug release in response to heat stimulus. We report on the design of liposomes with a heat-activated surface function. Our design is based on asymmetric lipid membranes with a defined gel to liquid-crystalline phase-transition temperature around 41°C. Asymmetry between the inner and the outer membrane leaflets was generated through selective PEGylation of cationic lipids in the outer membrane leaflet. In a physiological buffer, the PEGylated asymmetric liposomes had a neutral zeta potential and did not bind to planar anionic model membranes. In contrast, following upon heat-activation, binding of liposomes to the model membranes occurred. Release of a hydrophilic dye encapsulated in the asymmetric liposomes occurred at 40°C. Enhanced uptake of the asymmetric liposomes by hypopharyngeal carcinoma cells (FaDu cells) was observed when hyperthermia was applied compared to experiments performed at 37°C. These results show the potential of asymmetric liposomes for localized delivery of drugs into cells in response to (external) temperature stimulus.

Temperature and electric-field induced phase transitions, and full tensor properties of [011] C -poled domain-engineered tetragonal 0.63Pb(Mg1/3Nb2/3)-0.37PbTiO3 single crystals.

The phase-transition sequence of 0.67Pb(Mg1/3Nb2/3)-0.37PbTiO3 (PMN-0.37PT) single crystals driven by the electric (E) field and temperature is comprehensively studied. Based on the strain-E field loop, polarization-E field loop, and the evolution of domain configurations, the E field along the [011] C induced phase transitions have been confirmed to be as follows: tetragonal (T) → monoclinic (MC ) → single domain orthorhombic (O) phase. As the E field decreases, the induced O phase cannot be maintained and transformed to the MC phase, then to the coexistence state of MC and T phases. In addition, the complete sets of dielectric, piezoelectric, and elastic constants for the [011] C -poled domain-engineered PMN-0.37PT single crystal were measured at room temperature, which show high longitudinal dielectric, piezoelectric, and electromechanical properties ([Formula: see text], d33 = 1052 pC/N, and k33 = 0.766). Our results revealed that the MC phase plays an important role in the high electromechanical properties of this domain-engineered single crystal. The temperature dependence of the domain configuration revealed that the volume fraction of the MC phase decreases with temperature accompanied by the reduction of [Formula: see text], d31, and k31 due to the substantially smaller intrinsic properties of the T phase.

Effects of Al(3+) on Phosphocholine and Phosphoglycerol Containing Solid Supported Lipid Bilayers.

Aluminum has attracted great attention recently as it has been suggested by several studies to be associated with increased risks for Alzheimer's and Parkinson's disease. The toxicity of the trivalent ion is assumed to derive from structural changes induced in lipid bilayers upon binding, though the mechanism of this process is still not well understood. In the present study we elucidate the effect of Al(3+) on supported lipid bilayers (SLBs) using fluorescence microscopy, the quartz crystal microbalance with dissipation (QCM-D) technique, dual-polarization interferometry (DPI), and molecular dynamics (MD) simulations. Results from these techniques show that binding of Al(3+) to SLBs containing negatively charged and neutral phospholipids induces irreversible changes such as domain formation. The measured variations in SLB thickness, birefringence, and density indicate a phase transition from a disordered to a densely packed ordered phase.

Heat-activated liposome targeting to streptavidin-coated surfaces.

There is a great need of improved anticancer drugs and corresponding drug carriers. In particular, liposomal drug carriers with heat-activated release and targeting functions are being developed for combined hyperthermia and chemotherapy treatments of tumors. The aim of this study is to demonstrate the heat-activation of liposome targeting to biotinylated surfaces, in model experiments where streptavidin is used as a pretargeting protein. The design of the heat-activated liposomes is based on liposomes assembled in an asymmetric structure and with a defined phase transition temperature. Asymmetry between the inside and the outside of the liposome membrane was generated through the enzymatic action of phospholipase D, where lipid head groups in the outer membrane leaflet, i.e. exposed to the enzyme, were hydrolyzed. The enzymatically treated and purified liposomes did not bind to streptavidin-modified surfaces. When activation heat was applied, starting from 22°C, binding of the liposomes occurred once the temperature approached 33±0.5°C. Moreover, it was observed that the asymmetric structure remained stable for at least 2 weeks. These results show the potential of asymmetric liposomes for the targeted binding to cell membranes in response to (external) temperature stimulus. By using pretargeting proteins, this approach can be further developed for personalized medicine, where tumor-specific antibodies can be selected for the conjugation of pretargeting agents.

Recent advances on the circadian gene PER2 and metabolic rhythm of lactation of mammary gland.

Due to regulation by circadian rhythm, the lactation of the mammary gland has rhythmicity. As one of prominent members of period protein family which regulates biological rhythms, PER2 plays an important role in developing the milk duct and maintaining the polarity and the morphology of the mammary epithelium; at the same time, it is also closely related with the metabolism of milk protein and milk fat. This paper summarized recent researches on PER2 gene and related researches on mammary gland development and metabolism to provide some information for the studies of the theory and technology on physiological functions of the mammary gland and milk quality control.

Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size.

DPPC liposomes ranging from 90 nm to 160 nm in diameter were prepared and used for studies of the formation of supported lipid membranes on silica (SiO2) at temperatures below and above the gel to liquid-crystalline phase transition temperature (Tm = 41 °C), and by applying temperature gradients through Tm. The main method was the quartz crystal microbalance with dissipation (QCM-D) technique. It was found that liposomes smaller than 100 nm spontaneously rupture on the silica surface when deposited at a temperature above Tm and at a critical surface coverage, following a well-established pathway. In contrast, DPPC liposomes larger than 160 nm do not rupture on the surface when adsorbed at 22 °C or at 50 °C. However, when liposomes of this size are first adsorbed at 22 °C and at a high enough surface coverage, after which they are subject to a constant temperature gradient up to 50 °C, they rupture and fuse to a bilayer, a process that is initiated around Tm. The results are discussed and interpreted considering a combination of effects derived from liposome-surface and liposome-liposome interactions, different softness/stiffness and shape of liposomes below and above Tm, the dynamics and thermal activation of the bilayers occurring around Tm and (for liposomes containing 33% of NaCl) osmotic pressure. These findings are valuable both for preparation of supported lipid bilayer cell membrane mimics and for designing temperature-responsive material coatings.

Phase transition-controlled flip-flop in asymmetric lipid membranes.

Lipid membrane asymmetry is of fundamental importance for biological systems and also provides an attractive means for molecular control over biomaterial surface properties (including drug carriers). In particular, temperature-dependent changes of surface properties can be achieved by taking advantage of distinct phase transitions in lipid membrane coatings where lipids exchange (flip-flop) between leaflets. In this study, temperature is used to control flip-flop of lipids in asymmetric lipid membranes on planar solid supports, where the two leaflets of the lipid membrane are in different phase states. More specifically, the lower leaflet is prepared from a supported lipid membrane composed of a high Tm lipid mixture of phosphocholine (PC), phosphatidylserine (PS), and a bioactive lipid on TiO2, followed by selective removal of the top leaflet by detergent. Next, at a lower temperature, where the remaining leaflet is in the gel state, a top leaflet of a different lipid composition and in the fluid phase is formed. Phase transition-induced changes in membrane surface properties following upon temperature-activation of the prepared asymmetric membrane are demonstrated by the detection of biotinylated lipids, which were initially located (thus "hidden") in the lower-gel phase leaflet, at the surface of the top leaflet. These processes were monitored in real-time by the quartz crystal microbalance with dissipation (QCM-D) and the dual polarization interferometry (DPI) techniques, allowing modeling of the mass and the anisotropic property of the lipid structures in different phase states.