Lipid and protein interactions at air-water interface: correlating structural organization with rheological properties

Biological compounds such as lipids and proteins are building blocks of all living species and therefore, understanding how interactions between these molecules contribute to proper biological functioning is of great importance. Among the various perspectives of understanding lipid and protein interactions, this thesis is focused on the interaction of these molecules at the air-water interface. Lipid and proteins at the liquid interfaces are both abundant and influential. A few of the biological and physiologically relevant interfaces include cell membrane, tear film, and lung surfactant. While biological interactions of lipids and proteins regulate cell process, enable the lung function and keep us alive, some of these interactions could also be unfavorable in industrial applications. For example, interaction of proteins at the interfaces has been a source of concern in pharmaceutical industry due to the protein aggregation and particle formation initiated or increased in presence of the interfaces. This thesis has been focused in both biologically and industrially relevant studies of lipids and proteins at the air-water interface to learn how to better measure and predict these interactions. An active microrheology, imaging techniques, and surface energy measurements have been used to visualize and characterize the chemical-mechanical behavior of the lipids and proteins at the interface. The results showed interesting points on the influence of lipid chemistry, including headgroup charge, size, and saturation of the tail, on its interfacial rheological properties. The effect of subphase properties such as pH had a great influence on lipid packing. Additionally the interaction of lipids at the interfaces is greatly influenced by presence of even small ratios of protein or nanoparticles; the lipid-protein mixtures were significantly more viscose than lipid mixtures. Moreover our studies using a model IgG1 mAb had shown the role of air-water interface and its renewal due to mechanical stress on protein particle formation. The interfacial properties of the protein films were influenced greatly by the liquid buffer properties such as pH. Future studies on correlating the chemical-mechanical properties of lipids and proteins and their macroscale behavior at interface could shed light on important physiologically and industrially relevant questions

Monitoring phases and phase transitions in phosphatidylethanolamine monolayers using active interfacial microrheology

This is the published version. Copyright 2015 Royal Society of ChemistryActive interfacial microrheology is a sensitive tool to detect phase transitions and headgroup order in phospholipid monolayers. The re-orientation of a magnetic nickel nanorod is used to explore changes in the surface rheology of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), which differ by two CH2 groups in their alkyl chains. Phosphatidylethanolamines such as DLPE and DMPE are a major component of cell membranes in bacteria and in the nervous system. At room temperature, DLPE has a liquid expanded (LE) phase for surface pressure, Π < ∼38 mN m−1; DMPE has an LE phase for Π < ∼7 mN m−1. In their respective LE phases, DLPE and DMPE show no measurable change in surface viscosity with Π, consistent with a surface viscosity <10−9 N s m−1, the resolution of our technique. However, there is a measurable, discontinuous change in the surface viscosity at the LE to liquid condensed (LC) transition for both DLPE and DMPE. This discontinuous change is correlated with a significant increase in the surface compressibility modulus (or isothermal two-dimensional bulk modulus). In the LC phase of DMPE there is an exponential increase in surface viscosity with Π consistent with a two-dimensional free area model. The second-order LC to solid (S) transition in DMPE is marked by an abrupt onset of surface elasticity; there is no measurable elasticity in the LC phase. A measurable surface elasticity in the S phase suggests a change in the molecular ordering or interactions of the DMPE headgroups that is not reflected in isotherms or in grazing incidence X-ray diffraction. This onset of measurable elasticity is also seen in DLPE, even though no indication of a LC–S transition is visible in the isotherms

Ethanol production from sugarcane bagasse by means of on-site produced and commercial enzymes; a comparative study

In this study ethanol was produced without using expensive commercial enzymes from sugarcane bagasse. Alkali pretreatment was used to prepare biomass before enzymatic hydrolysis. The comparison between NaOH, KOH and Ca(OH)_2 revealed that NaOH has been more effective on bagasse structure. The required enzymes for biomass hydrolysis were produced by bagasse solid state fermentation using three fungi: Trichoderma longibrachiatum, T. reesei and Aspergillus niger. Results indicated enzyme solution produced by A. niger has functioned better than the other two in cellulose conversion during sole hydrolysis. Ethanol was produced by simultaneous saccharification and fermentation (SSF) with on-site prepared crude enzyme solutions and yeast Saccharomyces cerevisiae. Here, T. longibrachiatum had the best performance in ethanol production. To evaluate this procedure, SSF of pretreated bagasse applying Celluclast 1.5L by Novozymes was also investigated. The yield of ethanol production by commercial enzyme and T. longibrac hiatum enzyme solution were 81% and 52.5% respectively

Effect of Lipid Headgroup Charge and pH on the Stability and Membrane Insertion Potential of Calcium Condensed Gene Complexes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la504970n.Noncovalently condensed complexes of genetic material, cell penetrating peptides (CPPs), and calcium chloride present a nonviral route to improve transfection efficiency of nucleic acids (e.g., pDNA and siRNA). However, the exact mechanisms of membrane insertion and delivery of macromolecule complexes to intracellular locations as well as their stability in the intracellular environment are not understood. We show that calcium condensed gene complexes containing different hydrophilic (i.e., dTAT, K9, R9, and RH9) and amphiphilic (i.e., RA9, RL9, and RW9) CPPs formed stable cationic complexes of hydrodynamic radii 100 nm at neutral pH. However, increasing the acidity caused the complexes to become neutral or anionic and increase in size. Using zwitterionic and anionic phospholipid monolayers as models that mimic the membrane composition of the outer leaflet of cell membranes and intracellular vesicles and pHs that mimic the intracellular environment, we study the membrane insertion potential of these seven gene complexes (CPP/pDNA/Ca2+ complexes) into model membranes. At neutral pH, all gene complexes demonstrated the highest insertion potential into anionic phospholipid membranes, with complexes containing amphiphilic peptides showing the maximum insertion. However, at acidic pH, the gene complexes demonstrated maximum monolayer insertion into zwitterionic lipids, irrespective of the chemical composition of the CPP in the complexes. Our results suggest

Viscoelastic Properties of ECM-Rich Embryonic Microenvironments

This work is licensed under a Creative Commons Attribution 4.0 International License.The material properties of tissues and their mechanical state is an important factor in development, disease, regenerative medicine and tissue engineering. Here we describe a microrheological measurement technique utilizing aggregates of microinjected ferromagnetic nickel particles to probe the viscoelastic properties of embryonic tissues. Quail embryos were cultured in a plastic incubator chamber located at the center of two pairs of crossed electromagnets. We found a pronounced viscoelastic behavior within the ECM-rich region separating the mesoderm and endoderm in Hamburger Hamilton stage 10 quail embryos, consistent with a Zener (standard generalized solid) model. The viscoelastic response is about 45% of the total response, with a characteristic relaxation time of 1.3 s.NIH (R01GM102801)American Heart Association (19IPLOI34760594)Hungarian Scholarship Board's Eotvos Scholarshi

pH-Induced Changes in the Surface Viscosity of Unsaturated Phospholipids Monitored Using Active Interfacial Microrheology

Lipid membranes, a major component of cells, are subjected to significant changes in pH depending on their location in the cell: the outer leaflet of the cell membrane is exposed to a pH of 7.4 whereas lipid membranes that make up late endosomes and lysosomes are exposed to a pH of as low as 4.4. The purpose of this study is to evaluate how changes in the environmental pH within cells alter the fluidity of phospholipid membranes. Specifically, we studied pH-induced alterations in the surface arrangement of monounsaturated lipids with zwitterionic headgroups (phos­pho­ethanol­amine (PE) and phos­pho­choline (PC)) that are abundant in plasma membranes as well as anionic lipids (phos­pha­tidyl­serine (PS) and phos­pha­tidyl­glycerol (PG)) that are abundant in inner membranes using a combination of techniques including surface tension vs area measurements, interfacial microrheology, and fluorescence/atomic force microscopy. Using an active interfacial microrheology technique, we find that phospholipids with zwitterionic headgroups show a significant increase in their surface viscosity at acidic pH. This increase in surface viscosity is also found to depend on the size of the lipid headgroup, with a smaller headgroup showing a greater increase in viscosity. The observed pH-induced increase in viscosity is also accompanied by an increase in the cohesion pressure between zwitterionic molecules at acidic pH and a decrease in the average molecular area of the lipids, as measured by fitting the surface pressure isotherms to well-established equations of state. Because fluorescent images show no change in the phase of the lipids, we attribute this change in surface viscosity to the pH-induced reorientation of the P^––N⁺ dipoles that form part of the polar lipid headgroup, resulting in increased lipid–lipid interactions. Anionic PG headgroups do not demonstrate this pH-induced change in viscosity, suggesting that the presence of a net negative charge on the headgroup causes electrostatic repulsion between the headgroups. Our results also show that active interfacial microrheology is a sensitive technique for detecting minute changes in the lipid headgroup orientation induced by changes in the local membrane environment, even in unsaturated phospholipids where the surface viscosity is close to the experimental detection limit

Effect of Lipid Headgroup Charge and pH on the Stability and Membrane Insertion Potential of Calcium Condensed Gene Complexes

Noncovalently condensed complexes of genetic material, cell penetrating peptides (CPPs), and calcium chloride present a nonviral route to improve transfection efficiency of nucleic acids (e.g., pDNA and siRNA). However, the exact mechanisms of membrane insertion and delivery of macromolecule complexes to intracellular locations as well as their stability in the intracellular environment are not understood. We show that calcium condensed gene complexes containing different hydrophilic (i.e., dTAT, K9, R9, and RH9) and amphiphilic (i.e., RA9, RL9, and RW9) CPPs formed stable cationic complexes of hydrodynamic radii 100 nm at neutral pH. However, increasing the acidity caused the complexes to become neutral or anionic and increase in size. Using zwitterionic and anionic phospholipid monolayers as models that mimic the membrane composition of the outer leaflet of cell membranes and intracellular vesicles and pHs that mimic the intracellular environment, we study the membrane insertion potential of these seven gene complexes (CPP/pDNA/Ca²⁺ complexes) into model membranes. At neutral pH, all gene complexes demonstrated the highest insertion potential into anionic phospholipid membranes, with complexes containing amphiphilic peptides showing the maximum insertion. However, at acidic pH, the gene complexes demonstrated maximum monolayer insertion into zwitterionic lipids, irrespective of the chemical composition of the CPP in the complexes. Our results suggest that in the neutral environment the complexes are unable to penetrate the zwitterionic lipid membranes but can penetrate through the anionic lipid membranes. However, the acidic pH mimicking the local environment in the late endosomes leads to a significant increase in adsorption of the complexes to zwitterionic lipid headgroups and decreases for anionic headgroups. These membrane–gene complex interactions may be responsible for the ability of the complexes to efficiently enter the intracellular environment through endocytosis and escape from the endosomes to effectively deliver their genetic payload

The Effects of Testosterone on Cognitive Function Impairments and Brain Edema Induced by Methamphetamine in Gonadectomized Rats

Background and purpose: Methamphetamine is one of the stimulant drugs that leads to the occurrence of cognitive and behavioral dysfunctions. Among the disorders that occur after methamphetamine abuse are learning and memory disorders as well as social interactions and anxiety. On the other hand, the function of sex steroids in the development of brain regions involved in reproduction has been the subject of extensive research over the years. In this context, testosterone plays an important role in neuronal and glial organization in prenatal and postnatal periods. Recent studies showed the effect of sex hormones on the nervous system and highlight the brain as a main target tissue for androgens. On the other hand, studies have shown that testosterone has neuroprotective effects against cognitive and behavioral disorders. Clinical manifestations of testosterone-related brain changes include cognitive impairments such as reduced memory efficiency related to the frontal cortex, as well as reduced long-term memory related to the hippocampus and other temporal regions of the midbrain. Androgens, especially testosterone, play an important role in memory function whereby the decrease and absence of testosterone leads to cognitive impairment and also neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Therefore, in this study, the role of testosterone hormone in social memory, novel object recognition memory, as well as anxiety-like behavior and brain edema induced by a neurotoxic regimen of methamphetamine were investigated. Materials and methods: This study was conducted on 48 gonadectomized rats in 6 groups including control groups, methamphetamine (6mg/kg), solvent (sesame oil), methamphetamine + testosterone (0.25mg /kg), methamphetamine + testosterone (0.5 mg/kg) and methamphetamine + testosterone (1 mg/kg) were performed. At first, mice were anesthetized and gonadectomy surgery was performed. After 2 weeks of recovery, the evaluation of social memory was done using the three-chamber social interaction test, and anxiety-like behavior index using the open field test. Finally, the animals were killed and the brain tissue was evaluated for brain edema. Results: The findings of the study showed that social memory was significantly impaired in rats that received methamphetamine compared to the control group (P<0.001). On the other hand, results showed that there was a significant increase in anxiety-like behaviors as well as brain edema in rats that received methamphetamine (P<0.001). Statistical analysis also showed that the administration of testosterone hormone was able to improve cognitive functions and reduce anxiety-like behaviors and brain edema caused by methamphetamine (P<0.05). Conclusion: Overall, the present study suggests that the male sex hormone testosterone can be effective in improving behavioral disorders and anxiety, as well as brain edema caused by the administration of methamphetamine. Further research is needed to investigate the cellular and molecular mechanisms of the effects of this hormone