Negatively-charged Membranes Research Articles

Elucidating the mechanisms underlying the difference between chloride and nitrate rejection in nanofiltration

Despite the strong similarity between chloride (Cl-) and nitrate (NO3-) anions in terms of their hydrated radius and charge, Cl- is rejected more favorably than NO3- by nanofiltration (NF) membranes. The main goal of this study is to provide a better understanding of the removal mechanisms favoring the higher rejection of Cl- over NO3- in NF. A series of experiments with polyamide (NF270) and cellulose acetate (CK) NF membranes at different pH values, followed by calculation of the activation energies for Cl- and NO3- passage through the membranes, showed that the higher Cl- than NO3- rejection is attributed to both size-exclusion and Donnan (charge)-exclusion mechanisms. At a neutral membrane charge, a size-exclusion mechanism dominates the rejection of both anions. In this case, we observe higher rejection of Cl- over NO3- due to the lower hydration energy of NO3-, which corresponds to higher degree of dehydration and thus higher rate of passage through the NF membrane pores. At a negative membrane charge, the smaller volume of Cl- compared to NO3-, corresponding to higher ionic charge density, results in a stronger electrostatic repulsion of Cl- by the negatively charged membrane and therefore higher Cl- rejection than NO3-. The coupling of size- and Donnan-exclusion mechanisms with the NF270 membrane results in a maximum Cl- to NO3- rejection ratio at near the isoelectric pH where the membrane is slightly negatively charged. At a positive membrane charge, the sodium (Na+) counter ions dictate salt rejection independently of the anion type, resulting in almost similar rejections of Cl- and NO3-. Based on the insight gained from these experiments, a layer-by-layer (LbL) polyelectrolyte modification was applied to the NF270 membrane to control its surface charge. This modification showed that shifting the isoelectric point of the NF270 membrane from its original value (pH 4–5) to higher values (pH 6–9) increased the Cl- to NO3- rejection ratio at near neutral pH conditions, thus providing further support for our proposed mechanism underlying the difference between Cl- and NO3- rejection by NF membranes.

Probing the disparate effects of arginine and lysine residues on antimicrobial peptide/bilayer association

Antimicrobial peptides (AMPs) are key components of the innate immune response and represent promising templates for the development of broad-spectrum alternatives to conventional antibiotics. Most AMPs are short, cationic peptides that interact more strongly with negatively charged prokaryotic membranes than net neutral eukaryotic ones. Both AMPs and synthetic analogues with arginine-like side chains are more active against bacteria than those with lysine-like amine groups, though the atomistic mechanism for this increase in potency remains unclear. To examine this, we conducted comparative molecular dynamics simulations of a model negatively-charged membrane system interacting with two mutants of the AMP KR-12: one with lysine residues mutated to arginines (R-KR12) and one with arginine residues mutated to lysine (K-KR12). Simulations show that both partition analogously to the bilayer and display similar preferences for hydrogen bonding with the anionic POPGs. However, R-KR12 binds stronger to the bilayer than K-KR12 and forms significantly more hydrogen bonds, leading to considerably longer interaction times. Additional simulations with methylated R-KR12 and charge-modified K-KR12 mutants show that the extensive interaction seen in the R-KR12 system is partly due to arginine's strong atomic charge distribution, rather than being purely an effect of the greater number of hydrogen bond donors. Finally, free energy simulations reveal that both peptides are disordered in solution but form an amphipathic α-helix when inserted into the bilayer headgroup region. Overall, these results highlight the role of charge and hydrogen bond strength in peptide bilayer insertion, and offer potential insights for designing more potent analogues in the future.

Phospholipid-Biomimetic Fluorescent Mitochondrial Probe with Ultrahigh Selectivity Enables In Situ and High-Fidelity Tissue Imaging.

In situ and directly imaging mitochondria in tissues instead of isolated cells can offer more native and accurate information. Particularly, in the clinical diagnose of mitochondrial diseases such as mitochondrial myopathy, it is a routine examination item to directly observe mitochondrial morphology and number in muscle tissues from patients. However, it is still a challenging task because the selectivity of available probes is inadequate for exclusively tissue imaging. Inspired by the chemical structure of amphiphilic phospholipids in mitochondrial inner membrane, we synthesized a phospholipid-biomimetic amphiphilic fluorescent probe (Mito-MOI) by modifying a C18-alkyl chain to the lipophilic side of carbazole-indolenine cation. Thus, the phospholipid-like Mito-MOI locates at mitochondrial inner membrane through electrostatic interaction between its cation and inner membrane negative charge. Simultaneously, the C18-alkyl chain, as the second targeting group, is deeply embedded into the hydrophobic region of inner membrane through hydrophobic interaction. Therefore, the dual targeting groups (cation and C18-alkyl chain) actually endow Mito-MOI with ultrahigh selectivity. As expected, high-resolution microscopic photos showed that Mito-MOI indeed stained mitochondrial inner membrane. Moreover, in situ and high-fidelity tissue imaging has been achieved, and particularly, four kinds of mitochondria and their crystal-like structure in muscle tissues were visualized clearly. Finally, the dynamic process of mitochondrial fission in living cells has been shown. The strategy employing dual targeting groups should have reference value for designing fluorescent probes with ultrahigh selectivity to various intracellular membranous components.

Charge inversion and external salt effect in semi-permeable membrane electrostatics

Understanding the electrostatics of a charged semi-permeable plasma membrane is central to quantifying the membrane potential jump and the membrane osmotic pressure that play key roles in a myriad of biological processes ranging from control of intracellular calcium and apoptosis in cancer cells to regulation of cell-antibody or cell-vesicle interactions. In this study, we demonstrate a most remarkable charge inversion (CI) like electrostatics in the cytosol (cell) side of a negatively-charged plasma membrane for the case when the membrane is permeable to a cation and impermeable to an anion. This CI like electrostatics implies a switching of the sign of electrostatic potential from negative to positive from the membrane-cytosol interface to locations deep within the cytosol. Classical electric double layer (EDL) studies have reported such electrostatics manifesting switching of the sign of the electrostatic potential primarily during the occurrence of the CI phenomenon in presence multivalent counterions or ions with finite sizes – here, on the contrary, we witness such an effect on account of the combined influence of the specific sign of the charge and the specific permeability behavior of the cell membrane. We further demonstrate the role of an external permeable salt (both cations and anions of the salt permeate through the membrane) in the membrane electrostatics – presence of the salt invariably lowers the effect of semipermeability by either completely eradicating the CI like electrostatics phenomenon or by substantially reducing the membrane potential jump. Finally, we study the disjoining pressure triggered by the membrane-semipermeability-induced asymmetry in ionic distribution and show the distinct influences of the CI like electrostatics and external salt effects on the disjoining pressure.

Electrostatic Interactions between Tau Peptides and Model Biological Membranes: A Folding-Upon-Binding Mechanism

Found in neuronal cells of the central nervous system, tau proteins primarily function in cytoskeletal stability by associating with microtubules. During Alzheimer's disease and other tauopathies, misfolded tau proteins form neurofibrillary tangles (NFTs), thus impairing neuronal function and causing various neurodegenerative effects. The microtubule binding domain of tau consists of four repeat segments (R1-R4), and aggregation of these segments has previously been observed within NFTs. Previous research has also found misfolded tau components associated with the Golgi membrane, but not the plasma membrane. Here, we have used circular dicroism (CD) and isothermal titration calorimetry (ITC) to examine interactions between repeat segments R1-R4 and lipid bilayers of varying charge (DOPC and DOPC/DOPG 80:20). Our data indicate that significant structural changes are induced by charged membrane mimics and an entropy-driven binding process occurs during this interaction. Indeed, binding of tau occurs concomitantly with a classical hydrophobic effect. Thus, we propose a folding-upon-binding event occurring between negatively-charged membranes and the more promiscuous tau repeat segments. Results from calcein leakage assays and membrane fusion FRET measurements will also be presented to further probe the mechanism of bilayer disruption. These results will help explain a possible mechanism of tau misfolding during Alzheimer's disease, and are potentially beneficial to design strategies that prevent or reverse the aggregation process.

Natural Killer Cells Transfer Antimicrobial and Antitumoral Histone H2AZ to Kill Multiple Myeloma Cells Contributing to Transmissible Cytotoxicity

Natural Killer (NK) cells are antitumoral, antiviral and antimicrobial cells. The first antitumor mechanism described for NK cells was the "missing self" recognition which happens between the inhibitory "Killer Immunoglobulin-like Receptors" (KIRs) on NK cells and the HLA-I present in all nucleated cells. Thus, down-regulation or absence of HLA-I in tumor cells leads to "missing self" recognition activating NK cells. As a consequence, NK cells deliver Granzyme-B activating apoptotic cell death. However, NK cells can mediate Granzyme-B and Caspase-independent cell death against tumor cells expressing HLA-I, such as multiple myeloma (MM) cells. The cytotoxic mediators mediating non-apoptotic cell death remain unknown. Mechanisms mediating NK cell antimicrobial activity have been less studied, being recognized the role of Granulysin mediating Caspase independent cell death. Cord blood (CB) derived NK cells (CB-NK) is a clinically applicable strategy for the generation of highly functional NK cells which can be used to treat MM and potentially other haematological malignancies. We have demonstrated that CB-NK cytotoxicity against MM is Granzyme B and Caspase-independent. This CB-NK cytotoxicity is transmissible among MM cells, meaning that the initial MM recipient cells (termed primary MM cells or 1ºMM cells)-which formed direct contact with CB-NK are able to transfer lipid-protein vesicles to neighbouring unexposed MM cells (termed secondary MM cells or 2ºMM cells) causing a transmissible cytotoxicity to 2ºMM cells, which have not been in direct contact with CB-NK. We aimed to determine the key cytotoxic mediators transferred from CB-NK to 1ºMM and afterwards to 2ºMM cells that mediate secondary cytotoxicity. To do that, we performed TRANS-SILAC proteomics to determine proteome trafficking between CB-NK and MM cells. TRANS-SILAC proteomics demonstrated increased cell-cell communication between MM cells after CB-NK exposure, with secondary communication between MM cells to transfer the previously received cytotoxic CB-NK material. This secondary transfer represents a dilution of the CB-NK cytotoxic material. At the same time, MM cells transferred their own proteome to neighboring MM cells. Histones were among the CB-NK transferred proteins to 1ºMM, and 2ºMM cells. We selected the Histone H2AZ variant 1 (H2AZ) for validation. Time lapse in vitro confocal microscopy with CB-NK over-expressing H2AZ confirmed dynamic transfer of H2AZ from CB-NK to MM cells. Transfer occurred through vesicles and large intercellular structures. Extracellular DNA staining confirmed that these structures with H2AZ were accompanied by DNA. At the extracellular level, Histones exhibit significant antimicrobial activity, being the main antimicrobial effectors of neutrophils. The multiple positive charges and hydrophobic residues present in Histones allow them to bind to and invade negatively-charged cell membranes which are present in both microbial pathogens and tumor cells and interact with the DNA. We determined the cytotoxic role of H2AZ. H2AZ over-expression caused Caspase independent cell death in four different MM cell lines (p<0.0001). No differences were found on K562 cells. H2AZ over-expression increased DNA damage in MM cell lines. Furthermore, MM cell lines over-expressing H2AZ transferred to neighboring MM cells both H2AZ and the DNA damage. H2AZ transfer and DNA damage transfer also translated into a transmissible cytotoxicity between MM cells, as MM cells over-expressing H2AZ transferred cytotoxicity to neighboring MM cells who received this H2AZ. H2AZ involvement in CB-NK cytotoxicity against MM cells was demonstrated as knock-down and over-expression of H2AZ in CB-NK reduced and increased, respectively, CB-NK cytotoxicity against MM cell lines (p<0.05). For K562, no impact was observed confirming again a different CB-NK cytotoxicity mechanism for MM cell lines. Knock-down and over-expression of H2AZ in CB-NK reduced and increased, respectively, CB-NK antimicrobial activity against Candida albicans and Escherichia coli, demonstrating the antimicrobial role of H2AZ. Last, the antitumoral and antimicrobial properties of the recombinant protein H2AZ against MM cell lines and against bacteria were also demonstrated. Taken together, we demonstrate for first time the antitumoral and antimicrobial role of CB-NK Histones, revealing new therapeutic targets DisclosuresNo relevant conflicts of interest to declare.

Manipulation and Immobilization of a Single Fluorescence Nanosensor for Selective Injection into Cells.

Manipulation and injection of single nanosensors with high cell viability is an emerging field in cell analysis. We propose a new method using fluorescence nanosensors with a glass nanoprobe and optical control of the zeta potential. The nanosensor is fabricated by encapsulating a fluorescence polystyrene nanobead into a lipid layer with 1,3,3-trimethylindolino-6′-nitrobenzopyrylospiran (SP), which is a photochromic material. The nanobead contains iron oxide nanoparticles and a temperature-sensitive fluorescent dye, Rhodamine B. The zeta potential of the nanosensor switches between negative and positive by photo-isomerization of SP with ultraviolet irradiation. The positively-charged nanosensor easily adheres to a negatively-charged glass nanoprobe, is transported to a target cell, and then adheres to the negatively-charged cell membrane. The nanosensor is then injected into the cytoplasm by heating with a near-infrared (NIR) laser. As a demonstration, a single 750 nm nanosensor was picked-up using a glass nanoprobe with optical control of the zeta potential. Then, the nanosensor was transported and immobilized onto a target cell membrane. Finally, it was injected into the cytoplasm using a NIR laser. The success rates of pick-up and cell immobilization of the nanosensor were 75% and 64%, respectively. Cell injection and cell survival rates were 80% and 100%, respectively.

1,2,3-Triazolyl ester of Ketorolac: A “Click Chemistry”-based highly potent PAK1-blocking cancer-killer

An old anti-inflammatory/analgesic drug called Toradol is a racemic form of Ketorolac (50% R-form and 50% S-form) that blocks the oncogenic RAC-PAK1-COX-2 (cyclooxygenase-2) signaling, through the direct inhibition of RAC by the R-form and of COX-2 by the S-form, eventually down-regulating the production of prostaglandins. However, due to its COOH moiety which is clearly repulsive to negatively-charged phospholipid-based plasma membrane, its cell-permeability is rather poor (the IC50 against the growth of human cancer cells such as A549 is around 13 μM). In an attempt to boost its anti-cancer activity, hopefully by increasing its cell-permeability through abolishing the negative charge, yet keeping its water-solubility, here we synthesized a 1,2,3-triazolyl ester of Toradol through “Click Chemistry”. The resultant water-soluble “azo” derivative called “15K” was found to be over 500 times more potent than Toradol with the IC50 around 24 nM against the PAK1-dependent growth of A549 cancer cells, inactivating PAK1 in cell culture with the apparent IC50 around 65 nM, and inhibiting COX-2 in vitro with the IC50 around 6 nM. Furthermore, the Click Chemistry boosts the anti-cancer activity of Ketorolac by 5000 times against the PAK1-independent growth of B16F10 melanoma cells. Using a multi-drug-resistant (MDR) cancer cell line (EMT6), we found that the esterization of Ketorolac boosts its cell-permeability by at least 10 folds. Thus, the Click Chemistry dramatically boosts the anti-cancer activity of Ketorolac, at least in three ways: increasing its cell-permeability, the anti-PAK1 activity of R-form and anti-COX-2 activity of S-form. The resultant “15K” is so far among the most potent PAK1-blockers, and therefore would be potentially useful for the therapy of many different PAK1-dependent diseases/disorders such as cancers.

Significance of surface functionalization of Gold Nanorods for reduced effect on IgG stability and minimization of cytotoxicity

Gold nanorods (AuNRs) used for biomedical applications could be encountered by biomolecules in the bloodstream, of which IgG is the most abundant antibody. With a view to mitigate their side effect on encountered proteins, the effect of Au concentration (5–40μM) and functionalization (CTAB-positive;PSS-negative; PEG-neutral) of AuNRs was investigated on the stability of a model protein, IgG (1μM). Electron microscopic images and particle size analyses indicated least aggregation behavior for PEG-AuNRs, which can be correlated to their neutral charge (from zeta potential analyses) or stearic hindrance of PEG chains. Variations in tryptophan domain were probed by UV–visible absorption and fluorescence quenching studies. Synchronous fluorescence study helped to provide information regarding variations in the hydrophobic region of IgG. The denaturation studies also indicated the stability of AuNR–IgG complex formation. These studies showed that positively charged IgG (pI: 7.8±1.0) was mostly affected by negatively charged PSS-AuNRs and least affected by PEG-AuNRs. This was verified by secondary structural investigations performed using CD and FTIR spectroscopy. For cytotoxicity studies on human lymphocytes, CTAB-AuNRs are known to show higher toxicity compared to PSS-AuNRs and PEG-AuNRs (least). Though PSS-functionalized AuNRs were shown to affect cells to a lesser degree based on the negative charge of cell membrane, they could hamper with positively charged biomolecules in the bloodstream before they reach the target, which must also be considered for choosing the right AuNR functionalization. Thus, this work indicates the effect of different AuNR functionalization on protein and cellular toxicity and stresses the necessity to use neutral particles to mitigate their side effect for theranostic applications.

The control of N-nitrosodimethylamine, Halonitromethane, and Trihalomethane precursors by Nanofiltration

Nanofiltration (NF) is a promising technology for removing precursors of disinfection byproducts (DBPs) from source waters prior to oxidant addition in water treatment. The aims of this study were to investigate (i) the removal efficiencies of N-nitrosodimethylamine (NDMA), halonitromethane (HNM), and trihalomethane (THM) precursors by NF membranes from different source water types (i.e. surface water, wastewater impacted surface water, and municipal and industrial wastewater treatment effluents), (ii) the impact of membrane type, and (iii) the effects of background water components (i.e., pH, ionic strength, and Ca2+) on the removal of selected DBP precursors from different source waters. The results showed the overall precursor removal efficiencies were 57–83%, 48–87%, and 72–97% for NDMA, HNM, and THM precursors, respectively. The removal of NDMA precursors decreased with the increases in average molecular weight cut off and negative surface charge of NF membranes tested, while the removal of THM precursors was slightly affected. pH changes increased the removal of NDMA precursors, but pH did not affect the removal of THM and HNM precursors in municipal WWTP effluent. On the other hand, pH changes had little or no effect on DBP removal from industrial WWTP effluent. In addition, regardless of the membrane type or background water type/characteristics, ionic strength did not show any impact on DBP precursor removals. Lastly, an increase in Ca2+ concentration enhanced the removal of NDMA precursors while a slight decrease and no effect was observed for THM and HNM precursors, respectively, in municipal WWTP effluent.

Phase Structure Transition and Properties of Salt-Free Phosphoric Acid/Non-ionic Surfactants in Water.

Precise control of phase structure transition for the synthesis of multi-dimensional soft materials is a fascinating target in amphiphilic molecule self-assembly. Here, we demonstrate a spontaneous formation of a closely packed lamellar phase consisting of uni- and multi-lamellar vesicles through the incorporation of a small amount of an extractant, di(2-ethylhexyl)phosphoric acid (DEHPA), into the highly swollen, planar lamellar phase of a non-ionic tetraethylene glycol monododecyl ether (C12EO4) surfactant in water. It is figured out that the introduction of negative membrane charges results in the electrostatic repulsion among the lamellae, which suppresses the Helfrich undulation and induces a phase structure transition from planar lamellae to closely packed vesicles. Our results provide important insight into amphiphilic molecule self-assembly, where additives and pH can satisfy the opportunities for the precise tuning of the lamellar structures, which makes a way for the development of lamellar soft materials.

Nanoparticles Suitable for BCAA Isolation Can Serve for Use in Magnetic Lipoplex-Based Delivery System for L, I, V, or R-rich Antimicrobial Peptides.

This paper investigates the synthesis of paramagnetic nanoparticles, which are able to bind branched chain amino acids (BCAAs)—leucine, valine, and isoleucine and, thus, serve as a tool for their isolation. Further, by this, we present an approach for encapsulation of nanoparticles into a liposome cavity resulting in a delivery system. Analyses of valine and leucine in entire complex show that 31.3% and 32.6% recoveries are reached for those amino acids. Evaluation of results shows that the success rate of delivery in Escherichia coli (E. coli) is higher in the case of BCAAs on nanoparticles entrapped in liposomes (28.7% and 34.7% for valine and leucine, respectively) when compared to nanoparticles with no liposomal envelope (18.3% and 13.7% for valine and leucine, respectively). The nanoparticles with no liposomal envelope exhibit the negative zeta potential (−9.1 ± 0.3 mV); however, their encapsulation results in a shift into positive values (range of 28.9 ± 0.4 to 33.1 ± 0.5 mV). Thus, electrostatic interactions with negatively-charged cell membranes (approx. −50 mV in the case of E. coli) leads to a better uptake of cargo. Our delivery system was finally tested with the leucine-rich antimicrobial peptide (FALALKALKKALKKLKKALKKAL) and it is shown that hemocompatibility (7.5%) and antimicrobial activity of the entire complex against E. coli, Staphylococcus aureus (S. aureus), and methicilin-resistant S. aureus (MRSA) is comparable or better than conventional penicillin antibiotics.

Optimization of microelectrophoresis to select highly negatively charged sperm.

The sperm membrane undergoes extensive surface remodeling as it matures in the epididymis. During this process, the sperm is encapsulated in an extensive glycocalyx layer, which provides the membrane with its characteristic negative electrostatic charge. In this study, we develop a method of microelectrophoresis and standardize the protocol to isolate sperm with high negative membrane charge. Under an electric field, the percentage of positively charged sperm (PCS), negatively charged sperm (NCS), and neutrally charged sperm was determined for each ejaculate prior to and following density gradient centrifugation (DGC), and evaluated for sperm DNA damage, and histone retention. Subsequently, PCS, NCS, and neutrally charged sperm were selected using an ICSI needle and directly analyzed for DNA damage. When raw semen was analyzed using microelectrophoresis, 94% were NCS. In contrast, DGC completely or partially stripped the negative membrane charge from sperm resulting PCS and neutrally charged sperm, while the charged sperm populations are increased with an increase in electrophoretic current. Following DGC, high sperm DNA damage and abnormal histone retention were inversely correlated with percentage NCS and directly correlated with percentage PCS. NCS exhibited significantly lower DNA damage when compared with control (P < 0.05) and PCS (P < 0.05). When the charged sperm population was corrected for neutrally charged sperm, sperm DNA damage was strongly associated with NCS at a lower electrophoretic current. The results suggest that selection of NCS at lower current may be an important biomarker to select healthy sperm for assisted reproductive treatment.

Selective Membrane Disruption Mechanism of an Antibacterial γ-Aapeptide Defined by EPR Spectroscopy

γ-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. How γ-AApeptides interact with bacterial membranes and alter lipid assembly and properties are unclear, but such information is essential in order to understand their antimicrobial activities. Using electron paramagnetic resonance (EPR) techniques, we characterized the membrane interaction and destabilizing activities of a lipo-cyclic-γ-AApeptide. The analyses revealed that the γ-AApeptide binding increases the membrane permeability of POPC/POPG liposomes, which mimics negatively-charged bacterial membranes. Moreover, the γ-AApeptide interacts strongly with POPC/POPG liposomes, thereby inhibiting membrane fluidity and reducing solvent accessibility around the lipid head group region. Furthermore, binding of the γ-AApeptide induces significant lipid-lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon the γ-AApeptide binding for liposomes mimicking mammalian cell membranes, consisting of neutral lipids and cholesterol. Our findings suggest that the γ-AApeptide interact and disrupt bacterial membranes through a “carpet-like” mechanism. The results illustrated that the intrinsic features of γ-AApeptides are important for their ability to selectively disrupt bacterial membranes, the implications of which extend to developing new antibacterial biomaterials.

Using Molecular Dynamics Simulations to Characterize the Role Played by Basic Residues in Interactions of HDAPs and Bacterial Lipid Membranes

Antimicrobial peptides are small, cationic peptides that induce bacterial cell death via cell lysis or interactions with intracellular components. Currently, with growing bacterial resistance to existing antimicrobial drugs there is an urgent need to study the potentcy of antimicrobial peptides and find ways to improve their activity. Most AMPs include positively charged amino acids that allow binding to negatively-charged bacterial membranes. Previous studies have shown that an increased composition of arginine versus lysine enhances the activity of two histone-derived antimicrobial peptides (HDAPs), buforin II (BF2) and DesHDAP1. Variants of these peptides with all basic residues modified to arginine had increased antibacterial activity and membrane translocation but unchanged membrane permeabilization. In this study, we use molecular dynamics simulations to study how the arginine-modified peptides interact with model bacterial lipid membranes (1:3 POPG:POPE) differently than wild-type and lysine-modified peptides. Simulations were performed with one and four peptides for wild type BF2 (BF2wt), arginine-modified BF2 (BF2R), and lysine-modified BF2 (BF2K). The results of 100ns simulations of each system show that BF2R has increased interactions with the lipid membrane and structural stability compared to the other peptides. The analysis of hydrogen bonding per lipids supports that anionic POPG lipids interact more with the peptides than zwitterionic POPE lipids, further emphasizing the role of the basic residues. Analogous simulations considering the lipid interactions of DesHDAP1 and its arginine and lysine mutants are ongoing. Our findings elucidate mechanistic details of peptide-lipid interactions that will become helpful in the development of AMPs with improved activity.

Comparison of flat-sheet and spiral-wound negatively-charged wide-pore ultrafiltration membranes for whey protein concentration

This work compared laboratory-scale flat-sheet and pilot plant-scale spiral-wound wide-pore, negatively-charged ultrafiltration membranes for concentration of whey proteins. By placing a negative charge on the surface of ultrafiltration membranes, a wider pore size could be used to concentrate whey proteins because negatively-charged proteins were rejected by electrostatic repulsion and not simply sized-based sieving. Negatively-charged 100 kDa regenerated cellulose membranes had an 85% higher flux than unmodified 10 kDa membranes, and equivalent protein retention. The pilot plant-scale spiral-wound membranes had 70-fold more area, and a different membrane geometry than the laboratory-scale flat-sheet membranes, yet both membranes were successful in retaining >98% of the whey protein.

Novel negatively-charged membrane adsorbers made using combination of photopolymerization and immersion precipitation

A novel method combining a traditional immersion precipitation process for making membranes with photopolymerization and crosslinking of functional monomers included in the casting solution was used to prepare asymmetric polyethersulfone membranes with submicron particles incorporating glycidyl methacrylate copolymer. In order to introduce sulfonic groups epoxide rings of glycidyl methacrylate were opened using two methods. The first method was functionalization with sodium sulfite, and the second method was functionalization with sulfuric acid and then grafting with 2-acrylamido-2-methylpropane sulfonic acid. Obtained membranes were characterized using infrared spectroscopy, conductometric titration and water permeability measurements. Scanning electron microscopy and atomic force microscopy were used to investigate the surface morphology and topology of membrane. Dynamic adsorption of Rhodamine B as a model dye was used to demonstrate suitability of these novel membranes for membrane adsorption since the adsorption capacity for dye cations was much better for both functionalized membrane with sodium sulfite and grafted membrane with 2-acrylamido-2-methylpropane sulfonic acid compared to the nonfunctionalized membrane.

Membrane Charge Directs the Outcome of F-BAR Domain Lipid Binding and Autoregulation.

F-BAR domain proteins regulate and sense membrane curvature by interacting with negatively charged phospholipids and assembling into higher-order scaffolds. However, regulatory mechanisms controlling these interactions are poorly understood. Here, we show that Drosophila Nervous Wreck (Nwk) is autoregulated by a C-terminal SH3 domain module that interacts directly with its F-BAR domain. Surprisingly, this autoregulation does not mediate a simple "on-off" switch for membrane remodeling. Instead, the isolated Nwk F-BAR domain efficiently assembles into higher-order structures and deforms membranes only within a limited range of negative membrane charge, and autoregulation elevates this range. Thus, autoregulation could either reduce membrane binding or promote higher-order assembly, depending on local cellular membrane composition. Our findings uncover an unexpected mechanism by which lipid composition directs membrane remodeling.

PolySTAT-modified chitosan gauzes for improved hemostasis in external hemorrhage

Positively-charged chitosan gauzes stop bleeding from wounds by electrostatically interacting with negatively-charged cell membranes of erythrocytes to cause erythrocyte agglutination and by sealing wounds through tissue adhesion. In the following work, nonwoven chitosan gauze was impregnated with PolySTAT, a synthetic polymer that enhances coagulation by cross-linking fibrin, to generate PolySTAT/chitosan gauzes with improved hemostatic efficacy. When comparing nonwoven chitosan and PolySTAT/chitosan to a commercially-available chitosan-containing gauze (Celox® Rapid), no appreciable differences were observed in fiber size, morphology, and pore size. However, PolySTAT/chitosan demonstrated more rapid blood absorption compared to Celox® Rapid. In a rat model of femoral artery injury, PolySTAT/chitosan gauzes reduced blood loss and improved survival rate compared to non-hemostatic controls and Celox® Rapid. While Celox® Rapid had stronger adherence to tissues compared to PolySTAT/chitosan gauzes, blood loss was greater due to hematoma formation under the Celox® dressing. Animals treated with PolySTAT/chitosan gauzes required less saline infusion to restore and maintain blood pressure above the target blood pressure (60mmHg) while other treatment groups required more saline due to continued bleeding from the wound. These results suggest that PolySTAT/chitosan gauzes are able to improve blood clotting and withstand increasing arterial pressure with the addition of a fibrin cross-linking hemostatic mechanism. Statement of significanceBlood loss remains one of the leading causes of death after traumatic injury in civilian populations and on the battlefield. Advanced biomaterials that interact with blood components and/or accelerate the clotting process to form a hemostatic plug are necessary to staunch bleeding after injury. Chitosan-based gauzes, which stop bleeding by causing red blood cell aggregation, are currently used on the battlefield and have shown variable performance under high pressure arterial blood flow in animal studies, suggesting that red blood cell aggregates require further mechanical stabilization for more reliable performance. In this work, we investigate the binding and cross-linking of fibrin, a major component in blood clots, on chitosan gauze fiber surfaces to structurally reinforce red blood cell aggregates.

Mammalian Antimicrobial Peptides: Promising Therapeutic Targets Against Infection and Chronic Inflammation.

Antimicrobial peptides (AMPs) are integral components of the host innate immune system and functional throughout the plant and animal kingdoms. AMPs are short cationic molecules and lethal against a wide range of bacteria, viruses, fungi, yeast and protozoa due to their membranolytic effects on the negatively-charged microbial membranes. In addition, they exert multiple immunomodulatory roles like chemotaxis, modulation of cytokine and chemokine expression, leukocyte activation etc. Since AMPs suffer loss of microbicidal properties under serum and tissue environments, their capacity to modulate the immune system may predominates under the physiological conditions. Discovery of new antibiotics is lagging far behind the rapidly spreading drug resistance among the microorganisms. Both natural and synthetic AMPs have shown promise as 'next generation antibiotics' due to their unique mode of action, which minimises the chance of development of microbial resistance. In addition, they have therapeutic potential against non-infectious diseases like chronic inflammation and cancer. Many of the synthetic AMPs are currently undergoing clinical trials for the treatment of debilitating diseases, such as catheter-related infections, diabetic foot ulcers, chemotherapy-associated infections etc. Some of them have already entered the market as topical preparations. In this review, we synopsise the current literature of natural and synthetic AMPs in different infectious and inflammatory diseases of human microfloral habitats, especially the gastrointestinal, respiratory and genitourinary tracts and the skin. We also discuss the classification of AMPs, their mode action and antimicrobial spectrum, including the pathogen evasion mechanisms. In short, we tried to present the locus standi of AMPs in relation to human diseases and highlight the most promising synthetic peptides emerging from the clinical trials. Finally, we focused on the limitations and hurdles in terms of cost of production, bioavailability, pharmacokinetic stability and toxicity faced by commercial development and clinical use of the AMPs and strategies to overcome these hurdles.