Membrane Penetration Research Articles

Macrocyclic Peptides Closed by a Thioether-Bipyridyl Unit That Grants Cell Membrane Permeability.

Membrane permeability is an important factor that determines the virtue of peptides targeting intracellular molecules. By introducing a membrane penetration motif, some peptides exhibit better membrane permeabilities. Previous choices for such motifs have usually been polycationic sequences, but their protease vulnerabilities and modest endosome escapability remain challenging. Here, we report a strategy for macrocyclization of peptides closed by a hydrophobic bipyridyl (BPy) unit, which grants an improvement of their membrane permeability and proteolytic stability compared with the conventional polycationic peptides. We chemically prepared model macrocyclic peptides closed by a thioether-BPy unit and determined their cell membrane permeability, giving 200 nM CP50 (an indicative value of membrane permeability), which is 40-fold better than that of the ordinary thioether macrocycle consisting of the same sequence composition. To discover potent target binders consisting of the BPy unit, we reprogrammed the initiator with chloromethyl-BPy (ClMeBPy) for the peptide library synthesis with a downstream Cys residue(s) and executed RaPID (Random nonstandard Peptide Integrated Discovery) against the bromodomains of BRD4. One of the obtained sequences exhibited a single-digit nanomolar dissociation constant against BRD4 in vitro and showed approximately 2-fold and 10-fold better membrane permeability than positive controls, R9 and Tat peptides, respectively. Moreover, we observed an intracellular activity of the BPy macrocycle tagged with a proteasome target peptide motif (RRRG), resulting in modest but detectable degradation of BRD4. The present demonstration indicates that the combination of the RaPID system with an appropriate hydrophobic unit, such as BPy, would provide a potential approach for devising cell penetrating macrocycles targeting various intracellular proteins.

Structure-Activity Relationships of Cationic Lipidoids against Escherichia coli.

Membrane-active molecules provide a promising strategy to target and kill pathogenic bacteria. Understanding how specific molecular features drive interactions with membrane components and subsequently cause disruption that leads to antimicrobial activity is a crucial step in designing next-generation treatments. Here, we test a library of lipid-like compounds (lipidoids) against Gram-negative bacteria Escherichia coli to garner in-depth structure-activity relationships using antimicrobial assays. Modular lipidoid molecules were synthesized in high-throughput, such that we could analyze 104 compounds with variable combinations of hydrophobic tails and cationic headgroups. Antibacterial activity was strongly correlated to specific structural features, including tail hydrophobicity and headgroup charge density, and also to the overall molecular shape and propensity for self-assembly into curved liquid crystalline phases. Dye permeabilization assays showed that E. coli membranes were permeabilized by lipidoids, confirming their membrane-active nature. The reduced permeabilization, as compared to Gram-positive Bacillus subtilis, alludes to the challenge of permeabilizing the additional outer membrane layer of E. coli. The effect of headgroup solubility in gemini-type lipidoids was also demonstrated, revealing that a headgroup with a more hydrophilic spacer between amine groups had enhanced activity against B. subtilis but not E. coli. This provides insight into features enabling outer membrane penetration and governing selectivity between bacterial species.

An anchor-tether ‘hindered’ HCN1 inhibitor is antihyperalgesic in a rat spared nerve injury neuropathic pain model

BackgroundNeuropathic pain impairs quality of life, is widely prevalent, and incurs significant costs. Current pharmacological therapies have poor/no efficacy and significant adverse effects; safe and effective alternatives are needed. Hyperpolarisation-activated cyclic nucleotide-regulated (HCN) channels are causally implicated in some forms of peripherally mediated neuropathic pain. Whilst 2,6-substituted phenols, such as 2,6-di-tert-butylphenol (26DTB-P), selectively inhibit HCN1 gating and are antihyperalgesic, the development of therapeutically tolerable, HCN-selective antihyperalgesics based on their inverse agonist activity requires that such drugs spare the cardiac isoforms and do not cross the blood–brain barrier. MethodsIn silico molecular dynamics simulation, in vitro electrophysiology, and in vivo rat spared nerve injury methods were used to test whether ‘hindered’ variants of 26DTB-P (wherein a hydrophilic ‘anchor’ is attached in the para-position of 26DTB-P via an acyl chain ‘tether’) had the desired properties. ResultsMolecular dynamics simulation showed that membrane penetration of hindered 26DTB-Ps is controlled by a tethered diol anchor without elimination of head group rotational freedom. In vitro and in vivo analysis showed that BP4L-18:1:1, a variant wherein a diol anchor is attached to 26DTB-P via an 18-carbon tether, is an HCN1 inverse agonist and an orally available antihyperalgesic. With a CNS multiparameter optimisation score of 2.25, a >100-fold lower drug load in the brain vs blood, and an absence of adverse cardiovascular or CNS effects, BP4L-18:1:1 was shown to be poorly CNS penetrant and cardiac sparing. ConclusionsThese findings provide a proof-of-concept demonstration that anchor-tethered drugs are a new chemotype for treatment of disorders involving membrane targets.

Aggregation induced nucleic acids recognition by homodimeric asymmetric monomethyne cyanine fluorochromes in mesenchymal stem cells

In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1′-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1′-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.

Peptide-Based Vectors for Gene Delivery

Gene therapy is the ultimate therapeutic technology for diseases related to gene abnormality. However, the use of DNA alone has serious problems, such as poor stability and difficulty in entering target cells. The development of a safe and efficient gene delivery system is the cornerstone of gene therapy. Of particular interest, multifunctional peptides are rationally designed as non-viral vectors for efficient gene delivery. As components of gene delivery vectors, these peptides play critically important roles in skeleton construction, the implementation of targeting strategies, cell membrane penetration, endosome rupture, and nuclear transport. In recent years, the research of functional peptide-based gene delivery vectors has made important progress in improving transfection efficiency. The latest research progress and future development direction of peptide-based gene delivery vectors are reviewed in this paper.

Theoretical investigation of hydroxylated analogues of valinomycin as potassium transporter

Valinomycin is a potent ionophore known for its ability to transport potassium ions across biological membranes. The study focuses on the hydroxylated analogues of valinomycin (HyVLMs) and compares their energy profiles and capabilities for transporting potassium ions across phospholipid membranes. Using metadynamics, we investigated the energy profiles of wildtype valinomycin (VLM_1) and its three hydroxylated analogues (VLM_2, VLM_3, and VLM_4). We observed that all analogues exhibited energy maxima in the centre of the membrane and preferred positions below the phospholipid heads. Furthermore, the entry barriers for membrane penetration were similar among the analogues, suggesting that the hydroxyl group did not significantly affect their passage through the membrane. Transition state calculations provided insights into the ability of valinomycin analogues to capture potassium ions, with VLM_4 showing the lowest activation energy and VLM_2 displaying the highest. Our findings contribute to understanding the mechanisms of potassium transport by valinomycin analogues and highlight their potential as ionophores. The presence of the hydroxyl group is of particular importance because it paves the way for subsequent chemical modifications and the synthesis of new antiviral agents with reduced intrinsic toxicity.

Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation.

The human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus-host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro. IMPORTANCE Human astroviruses (HAstVs) are an understudied family of viruses that cause mild gastroenteritis but have recent cases associated with a more severe neural pathogenesis. Many important elements of the HAstV life cycle are not well understood, and further elucidating them can help understand the various forms of HAstV pathogenesis. In this study, we utilized an in vitro liposome-based assay to describe and characterize a previously unreported lipid disruption activity. This activity is dependent on the protease cleavage of key sites in HAstV capsid core and can be controlled by site-directed mutagenesis. Our group observed this activity in multiple strains of HAstV and in multiple lipid conditions, indicating this may be a conserved activity across the AstV family. The discovery of this function provides insight into HAstV cellular entry, pathogenesis, and a possible target for future therapeutics.

Design, Synthesis, and Comparison of PLA-PEG-PLA and PEG-PLA-PEG Copolymers for Curcumin Delivery to Cancer Cells.

Curcumin (CUR) has potent anticancer activities, and its bioformulations, including biodegradable polymers, are increasingly able to improve CUR's solubility, stability, and delivery to cancer cells. In this study, copolymers comprising poly (L-lactide)-poly (ethylene glycol)-poly (L-lactide) (PLA-PEG-PLA) and poly (ethylene glycol)-poly (L-lactide)-poly (ethylene glycol) (PEG-PLA-PEG) were designed and synthesized to assess and compare their CUR-delivery capacity and inhibitory potency on MCF-7 breast cancer cells. Molecular dynamics simulations and free energy analysis indicated that PLA-PEG-PLA has a higher propensity to interact with the cell membrane and more negative free energy, suggesting it is the better carrier for cell membrane penetration. To characterize the copolymer synthesis, Fourier transform-infrared (FT-IR) and proton nuclear magnetic resonance (1H-NMR) were employed, copolymer size was measured using dynamic light scattering (DLS), and their surface charge was determined by zeta potential analysis. Characterization indicated that the ring-opening polymerization (ROP) reaction was optimal for synthesizing high-quality polymers. Microspheres comprising the copolymers were then synthesized successfully. Of the two formulations, PLA-PEG-PLA experimentally exhibited better results, with an initial burst release of 17.5%, followed by a slow, constant release of the encapsulated drug up to 80%. PLA-PEG-PLA-CUR showed a significant increase in cell death in MCF-7 cancer cells (IC50 = 23.01 ± 0.85 µM) based on the MTT assay. These data were consistent with gene expression studies of Bax, Bcl2, and hTERT, which showed that PLA-PEG-PLA-CUR induced apoptosis more efficiently in these cells. Through the integration of nano-informatics and in vitro approaches, our study determined that PLA-PEG-PLA-CUR is an optimal system for delivering curcumin to inhibit cancer cells.

Inactivation of fungal spores by performic acid in water: Comparisons with peracetic acid

Performic acid (PFA) has received increasing attention in water disinfection due to its high disinfection efficiency and fewer formation of disinfection by-products. However, the inactivation of fungal spores by PFA has not been investigated. In this study, the results showed that the log-linear regression plus tail model adequately described the inactivation kinetic of fungal spores with PFA. The k values of A. niger and A. flavus with PFA were 0.36 min−1 and 0.07 min−1, respectively. Compared to peracetic acid, PFA was more efficient in inactivating fungal spores and caused more serious damage on cell membrane. Compared to neutral and alkaline conditions, acidic environments demonstrated a greater inactivation efficiency for PFA. The increase of PFA dosage and temperature had a promoting effect on the inactivation efficiency of fungal spores. PFA could kill the fungal spores by damaging cell membrane and penetration of cell membranes. In real water, the inactivation efficiency declined as a result of the existence of background substances such as dissolved organic matter. Moreover, the regrowth potential of fungal spores in R2A medium were severely inhibited after inactivation. This study provides some information for PFA to control fungi pollution and explores the mechanism of PFA inactivation.

Plant Mitochondrial-Targeted Gene Delivery by Peptide/DNA Micelles Quantitatively Surface-Modified with Mitochondrial Targeting and Membrane-Penetrating Peptides.

Plant mitochondria play essential roles in metabolism and respiration. Recently, there has been growing interest in mitochondrial transformation for developing crops with commercially valuable traits, such as resistance to environmental stress and shorter fallow periods. Mitochondrial targeting and cell membrane penetration functions are crucial for improving the gene delivery efficiency of mitochondrial transformation. Here, we developed a peptide-based carrier, referred to as Cytcox/KAibA-Mic, that contains multifunctional peptides for efficient transfection into plant mitochondria. We quantified the mitochondrial targeting and cell membrane-penetrating peptide modification rates to control their functions. The modification rates were easily determined from high-performance liquid chromatography chromatograms. Additionally, the gene carrier size remained constant even when the mitochondrial targeting peptide modification rate was altered. Using this gene carrier, we can quantitatively investigate the relationships between various peptide modifications and transfection efficiency and optimize the gene carrier conditions for mitochondrial transfection.

A conserved tryptophan in the acylated segment of RTX toxins controls their β2 integrin–independent cell penetration

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β2 integrins.

Revealing Low Amplitude Signals of Neuroendocrine Cells through Disordered Silicon Nanowires-Based Microelectrode Array.

Today, the key methodology to study in vitro or in vivo electrical activity in a population of electrogenic cells, under physiological or pathological conditions, is by using microelectrode array (MEA). While significant efforts have been devoted to develop nanostructured MEAs for improving the electrophysiological investigation in neurons and cardiomyocytes, data on the recording of the electrical activity from neuroendocrine cells with MEA technology are scarce owing to their weaker electrical signals. Disordered silicon nanowires (SiNWs) for developing a MEA that, combined with a customized acquisition board, successfully capture the electrical signals generated by the corticotrope AtT-20cells as a function of the extracellular calcium (Ca2+ ) concentration are reported. The recorded signals show a shape that clearly resembles the action potential waveform by suggesting a natural membrane penetration of the SiNWs. Additionally, the generation of synchronous signals observed under high Ca2+ content indicates the occurrence of a collective behavior in the AtT-20cell population. This study extends the usefulness of MEA technology to the investigation of the electrical communication in cells of the pituitary gland, crucial in controlling several essential human functions, and provides new perspectives in recording with MEA the electrical activity of excitable cells.

Novel drug-drug salts of enoxacin with enhanced antibacterial activity: Insights from solubility and lipid-water partition coefficient

Enoxacin (ENX), a third-generation fluoroquinolone, demonstrates broad-spectrum antibacterial activities and is widely applied for the treatment of various bacterial infections. However, it is challenging to improve its antibacterial activity and drug resistance effects based on solubility changes owing to ENX being categorized as a class II drug in the Biopharmaceutics Classification System (BCS). In this experiment, three novel salts formed between ENX and non-steroidal anti-inflammatory drugs (NSAIDs) involving diclofenac (ENX-DFA), flufenamic acid (ENX-FFA) and niflumic acid (ENX-NFA) were firstly synthesized and then characterized. X-ray diffraction, thermodynamic analysis and infrared spectroscopy confirmed the salt formation. Interestingly, all the new salts demonstrated superior antibacterial activities against Escherichia coli (E. coli), Shigella flexneri (Sh. flexneri), Staphylococcus aureus (S. aureus) and Staphylococcus albicans (S. albus) compared with ENX. Further investigation of the pharmaceutical properties of the three salts revealed that they possessed lower aqueous solubility and intrinsic dissolution compared with ENX but exhibited better solubility than the three NSAIDs. Moreover, our results demonstrated that fluctuations in the solubility of ENX salts were pH-dependent. Thus, the solubility changes of ENX salts were assessed using pH-window diagrams. The order of lipid-water partition coefficient (P) was consistent with antibacterial performance (ENX-FFA > ENX-DFA > ENX-NFA > ENX) and was 2.04, 1.95, and 1.69 times greater than that of ENX, indicating that the higher LogP values of salts with strong cell membrane penetration ability improved the efficacy of the drug to inhibit bacterial growth. In summary, this study provides new insights to elucidate antibacterial effects from solubility and LogP values.

Effect of the Lipid Landscape on the Efficacy of Cell-Penetrating Peptides.

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

Graphene Oxide Hosting a pH-Sensitive Prodrug: An In Silico Investigation of Graphene Oxide-Based Nanovehicle toward Cancer Therapy.

Prodrug and drug delivery systems are two effective strategies for improving the selectivity of chemotherapeutics. Herein, via molecular dynamics (MD) simulation and free energy calculation, the effectiveness of the graphene oxide (GO) decorated with the pH-sensitive prodrug (PD) molecules in cancer therapy is investigated. PEI-CA-DOX (prodrug) was loaded onto the GO surface, in which the hydrogen bonding and pi-pi stacking interactions play the main role in the stability of the GO-PD complex. Due to the strong interaction of GO and PD (about -800 kJ/mol), the GO-PD complex remains stable during the membrane penetration process. The obtained results confirm that GO is a suitable surface for hosting the prodrug and passing it through the membrane. Furthermore, the investigation of the release process shows that the PD can be released under acidic conditions. This phenomenon is due to the reduction of the contribution of electrostatic energy in the GO and PD interaction and the entry of water into the drug delivery system. Moreover, it is found that an external electrical field does not have much effect on drug release. Our results provide a deep understanding of the prodrug delivery systems, which helps the combination of nanocarriers and modified chemotherapy drugs in the future.

Is lipophilicity the main factor determining the effective drug penetration through lipid membranes? Combined GIXD and PM-IRRAS studies on the example of anthracyclines

Cell membranes constitute the first barrier for the drugs on their way to the cell. Therefore, their penetration determines the effectivity of the treatment and is defined by the drug-lipid interactions. A systematic, comprehensive study to assess the impact of different aspects influencing the effective penetration of model lipid membranes by the drugs including lipophilicity, charge, ability to form dimers as well as stearic hindrance was performed. Four anthracyclines differing in the structure and with lipophilicity increasing in the following order: doxorubicin (DOx) < pirarubicin (THP) < daunorubicin (DNR) < idarubicin (IDA) were chosen to assess their impact on 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (DMPS) monolayers mimicking cancer cell membranes. The investigation performed at the air–water interface involved the application of multiple surface-sensitive techniques to assess the drug interactions with both polar heads of the phospholipid and the penetration of the acyl chain region. The obtained results allowed us to state that although electrostatic interactions and lipophilicity of the drug are one of the key factors, the ability to form dimers as well as stearic hinderance may strongly prevent from the effective penetration of the phospholipid monolayers even for drugs with higher lipophilicity and therefore have to be also taken into account in the development of new therapeutic agents.

Effects of pH and palmitic acid on the organization and stability of lutein in the phosphatidylcholine membranes – Spectroscopic studies

This paper presents the effect of pH and the presence of palmitic acid (PA) on the organisation and stability of lutein (LUT) in the egg yolk phosphatidylcholine (EYPC) membranes. This is the first known thorough study on the EYPC liposomes formed in different buffers such as acetic at pH 5.0, phosphate at pH 7.4 and borate at pH 9.0. UV–Vis absorption and infra-red (FTIR) spectroscopy were used for molecular studies. Based on analysis of UV–Vis spectra it can be argued that the pH and temperature conditions are crucial for the stability of LUT molecules. After analysing the spectra of samples containing LUT + PA, it can be concluded that the presence of PA in all the applied buffers provides better stability for LUT and perhaps its better solubility. The changes in the absorbance spectrum observed after incubation at 37 °C for 1 h also indicated the beneficial effect of PA presence. Our studies indicate that PA presence in the lipid membranes may have protective effect on LUT, especially at pH 5.0 and that it slows down the degradation process of LUT. Localized pH heterogeneities can induce local dynamical membrane deformations which combined with a certain molecular organization of LUT (such as dimers) can result in lower or higher membrane penetration by LUT molecules.

Determination of non-linear shear strength and volume changes on remolded granitic residual soil due to membrane penetration

Membrane penetration is the term used to describe the displacement of the rubber membrane into the spaces between the cylindrical specimens during triaxial testing in the consolidation stage. Membrane penetration from elevated cell pressure during the consolidation stage might have a negative effect on the interpretation of stress-strain data and specimen volume calculations during the shearing stage. To assist engineers in making knowledgeable judgments, additional study is needed to examine membrane penetration in granitic residual soil. In this study, membrane penetration was investigated using a modified graphical technique. Triaxial tests on grade V granitic residual soil were conducted as part of the study at effective confining pressures of 50 kPa, 100 kPa, 200 kPa, 300 kPa, and 400 kPa. In the tests, real specimens constructed of compacted granitic residual soil with initial diameters of 50mm and 100mm, and dummy specimens made of a thin soil layer containing a solid, rigid cylindrical core, were both employed. Without considering the impact of specimen compression, the behaviour of membrane penetration was only examined in the dummy specimens. The graphical method has to be altered as a result of the fines. The findings demonstrated that the effect of membrane penetration increased with a rise in effective confining pressure and became less relevant at confining pressures of 300 kPa or lower.

Liquefaction Behavior of a Well-Graded Gravelly Soil under Initial Static Shear Stress in Cyclic Triaxial and Simple Shear Conditions

Among other reasons, studies of the liquefaction potential of gravelly soils are limited because of the difficulties involved in preparing uniform specimens, without particle segregation, especially for a well-graded gravelly soil in a dry state for simple shear testing. Errors and difficulties are also involved in compensating for membrane penetration to a gravelly soil specimen in triaxial testing to get reliable data. Thus, innovative approaches for preparing triaxial and simple shear specimens for gravelly soils are introduced and implemented in this study to overcome experimental problems in acquiring accurate test results. In addition to the aim of obtaining reliable testing data on the liquefaction of gravelly soils under initial static shear stress for simulating sloping ground conditions, a study of the effect of various stress paths on the liquefaction resistance of gravelly soils was another goal of this research. In this regard, two sets of cyclic tests using medium-size triaxial and simple shear devices are conducted on a unique soil to compare the liquefaction potential of the tested gravelly soil using these two devices. Results of simple shear tests indicate that as the value of initial static shear stress increases, the cyclic resistance of the tested gravelly soil decreases. However, the results of the triaxial tests show that the variation of cyclic resistance for the tested soil depends on the initial static shear stress level and associated stress reversal conditions. Furthermore, the observed value of the pore water pressure ratio at failure using the strain-based liquefaction criteria for the tested gravelly soil was about 0.85, regardless of the type of testing. In addition, the relationship between the liquefaction resistance of gravelly soils using cyclic triaxial and simple shear devices was obtained.

The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER

Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration mediated by the Sar1 GTPase and further stabilized by assembly of a multilayered complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the efficiency of this process is dramatically reduced. Specifically, secretory cargoes are retained nearly five times longer at ER subdomains when Sar1 is depleted, but they ultimately remain capable of being translocated to the perinuclear region of cells. Taken together, our findings highlight alternative mechanisms by which COPII promotes transport carrier biogenesis.