A Supramolecular Host for Phosphatidylglycerol (PG) Lipids with Antibacterial Activity

Lipids fulfill a variety of important physiological functions, such as energy storage, providing a hydrophobic barrier, and signal transduction. Despite this plethora of biological roles, lipids are rarely considered a...

Rapid, Simple and Inexpensive Fabrication of Paper-Based Analytical Devices by Parafilm® Hot Pressing

Paper-based analytical devices have been substantially developed in recent decades. Many fabrication techniques for paper-based analytical devices have been demonstrated and reported. Herein, we report a relatively rapid, simple, and inexpensive method for fabricating paper-based analytical devices using parafilm hot pressing. We studied and optimized the effect of the key fabrication parameters, namely pressure, temperature, and pressing time. We discerned the optimal conditions, including a pressure of 3.8 MPa, temperature of 80 °C, and 3 min of pressing time, with the smallest hydrophobic barrier size (821 µm) being governed by laminate mask and parafilm dispersal from pressure and heat. Physical and biochemical properties were evaluated to substantiate the paper functionality for analytical devices. The wicking speed in the fabricated paper strips was slightly lower than that of non-processed paper, resulting from a reduced paper pore size after hot pressing. A colorimetric immunological assay was performed to demonstrate the protein binding capacity of the paper-based device after exposure to pressure and heat from the fabrication. Moreover, mixing in a two-dimensional paper-based device and flowing in a three-dimensional counterpart were thoroughly investigated, demonstrating that the paper devices from this fabrication process are potentially applicable as analytical devices for biomolecule detection. Fast, easy, and inexpensive parafilm hot press fabrication presents an opportunity for researchers to develop paper-based analytical devices in resource-limited environments.

The Good and the Bad of Cell Membrane Electroporation

Electroporation is used to increase the permeability of the cell membrane through high-voltage electric pulses. Nowadays, it is widely used in different areas, such as medicine, biotechnology, and the food industry. Electroporation induces the formation of hydrophilic pores in the lipid bilayer of cell membranes, to allow the entry or exit of molecules that cannot otherwise cross this hydrophobic barrier. In this article, we critically review the basic principles of electroporation, along with the advantages and drawbacks of this method. We discuss the effects of electroporation on the key components of biological membranes, as well as the main applications of this procedure in medicine, such as electrochemotherapy, gene electrotransfer, and tissue ablation. Finally, we define the most relevant challenges of this romising area of research.

Rapid, Simple and Inexpensive Fabrication of Paper-based Analytical Devices by Parafilm® Hot Pressing

Paper-based analytical devices have been substantially developed in recent decades. Many fabrication techniques for paper-based analytical devices have been demonstrated and reported. Herein we report a relatively rapid, simple, and inexpensive method for fabricating paper-based analytical devices using parafilm hot pressing. We studied and optimized the effect of the key fabrication parameters, namely pressure, temperature, and pressing time. We discerned the optimal conditions, including pressure of 3.8 MPa (3 tons), temperature of 80oC, and 3 minutes of pressing time, with the smallest hydrophobic barrier size (821 µm) being governed by laminate mask and parafilm dispersal from pressure and heat. Physical and biochemical properties were evaluated to substantiate the paper functionality for analytical devices. Wicking speed in the fabricated paper strips was slightly slower than that of non-processed paper, resulting from reducing paper pore size. A colorimetric immunological assay was performed to demonstrate the protein binding capacity of the paper-based device after exposure to pressure and heat from the fabrication. Moreover, mixing in two-dimensional paper-based device and flowing in a three-dimensional counterpart were thoroughly investigated, demonstrating that the paper device from this fabrication process is potentially applicable as analytical devices for biomolecule detection. Fast, easy, and inexpensive parafilm hot press fabrication presents an opportunity for researchers to develop paper-based analytical devices in resource-limited environments.

Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis

As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.

Fluid Control with Hydrophobic Pillars in Paper-Based Microfluidics

Paper-based microfluidics has been widely used in chemical and medical analysis applications. In the conventional paper-based microfluidic approach, fluid is propagating inside the porous structure, and the flow direction of the fluid propagation is usually controlled with the pre-defined hydrophobic barrier (e.g. wax). However, the fluid propagation velocity inside the paper-based microfluidic devices largely depends on the material properties of paper and fluid, the relative control method is rarely reported. In this study, a fluid propagation velocity control method is proposed for paper-based microfluidics: hydrophobic pillar arrays with different configurations were deposited in the microchannels in paper-based microfluidics for flow speed control, result indicates the deposited hydrophobic pillar arrays can effectively slow down the fluid propagation at different levels and can be used to passively control the fluid propagation inside microchannels for paper-based microfluidics. For the demonstration of the proposed fluid control methods, a paper-based microfluidic device for nitrite test in water was also fabricated. The proposed fluid control method for paper-based microfluidics may have significant importance for applications that involve sequenced reactions and more actuate fluid manipulation.

Equisetin Restores Colistin Sensitivity against Multi-Drug Resistant Gram-Negative Bacteria

The overuse of antibiotics and the scarcity of new drugs have led to a serious antimicrobial resistance crisis, especially for multi-drug resistant (MDR) Gram-negative bacteria. In the present study, we investigated the antimicrobial activity of a marine antibiotic equisetin in combination with colistin against Gram-negative bacteria and explored the mechanisms of synergistic activity. We tested the synergistic effect of equisetin in combination with colistin on 23 clinical mcr-1 positive isolates and found that 4 µg/mL equisetin combined with 1 µg/mL colistin showed 100% inhibition. Consistently, equisetin restored the sensitivity of 10 species of mcr-1 positive Gram-negative bacteria to colistin. The combination of equisetin and colistin quickly killed 99.9% bacteria in one hour in time-kill assays. We found that colistin promoted intracellular accumulation of equisetin in colistin-resistant E. coli based on LC-MS/MS analysis. Interestingly, equisetin boosted ROS accumulation in E. coli in the presence of colistin. Moreover, we found that equisetin and colistin lost the synergistic effect in two LPS-deficient A. baumannii strains. These findings suggest that colistin destroys the hydrophobic barrier of Gram-negative bacteria, facilitating equisetin to enter the cell and exert its antibacterial effect. Lastly, equisetin restored the activity of colistin in a G. mellonella larvae infection model. Collectively, these results reveal that equisetin can potentiate colistin activity against MDR Gram-negative bacteria including colistin-resistant strains, providing an alternative approach to address Gram-negative pathogens associated with infections in clinics.

Exploration of synergistic action of cell wall-degrading enzymes against Mycobacterium tuberculosis

Background The major global health threat tuberculosis is caused by Mycobacterium tuberculosis (Mtb). Mtb has a complex cell envelope – a partially covalently linked composite of polysaccharides, peptidoglycan and lipids, including a mycolic acid layer – which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating gram-positive and -negative infections with cell wall degrading enzymes, we investigated such approach for Mtb. Objectives (i) Development of an Mtb microtiter growth inhibition assay that allows undisturbed cell envelope formation, to overcome the invalidation of results by typical clumped Mtb-growth in surfactant-free assays. (ii) Exploring anti-Mtb potency of cell wall layer-degrading enzymes. (iii) Investigation of the concerted action of several such enzymes. Methods We inserted a bacterial luciferase-operon in an auxotrophic Mtb strain to develop a microtiter assay that allows proper evaluation of cell wall degrading anti-Mtb enzymes. We assessed growth-inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase (LysB), fungal α-amylase and human and chicken egg white lysozymes) and combinations thereof, in presence or absence of biopharmaceutically acceptable surfactant. Results Our biosafety level-2 assay identified both LysB and lysozymes as potent Mtb-inhibitors, but only in presence of surfactant. Moreover, most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase and polysorbate 80. Conclusions Synergistically acting cell wall degrading enzymes are potently inhibiting Mtb – which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

Design of Chitosan and Alginate Emulsion-Based Formulations for the Production of Monolayer Crosslinked Edible Films and Coatings

This study aimed to develop edible monolayer emulsion-based barriers with polysaccharides as film-forming components (chitosan and sodium alginate), soy lecithin as a surfactant and olive oil as a hydrophobic barrier. Monolayer barriers in the form of films were prepared by casting filmogenic emulsions composed of 2% w/v chitosan (dissolved in lactic acid 1% v/v) or 1% w/v sodium alginate, with different lipid contents (25, 50 and 100% w/w biopolymer basis) and different surfactant concentrations (5, 10 and 25% w/w, lipid basis). Glycerol was used as a plasticizer (25 % w/w, biopolymer basis). After the emulsion drying process, the obtained stand-alone films were sprayed with a crosslinking solution, achieving an optimized crosslinker content of 3.2 mgCa2+/cm2 alginate film and 4 mg tripolyphosphate/cm2 chitosan film. The effect of oil and lecithin contents, as well the presence of crosslinking agents, on the film’s water vapour permeability (WVP), water vapour sorption capacity, mechanical properties and colour parameters, was evaluated. The results have shown that the lowest WVP values were obtained with formulations containing 25% lipid and 25% surfactant for chitosan films, and 100% lipid and 25% surfactant for alginate films. The application of the crosslinking agents decreased even further the WVP, especially for chitosan films (by 30%). Crosslinking also increased films’ resistance to deformation under tensile tests. Overall, the films developed present a good potential as polysaccharide-based barriers with increased resistance to water, which envisages the use of the designed formulations to produce either edible/biodegradable films or edible coatings.