Construction and application of artificial lipoproteins using adiposomes.

Lipoproteins are complex particles comprised of a neutral lipid core wrapped with a phospholipid monolayer membrane and apolipoproteins on the membrane, which is closely associated with metabolic diseases. To facilitate the elucidation of its formation and dynamics, as well as its applications, we developed an in vitro system in which adiposomes, consisting of a hydrophobic core encircled by a monolayer-phospholipid membrane, were engineered into artificial lipoproteins (ALPs) by recruiting one or more kinds of apolipoproteins, for example, apolipoprotein (Apo) A-I, ApoE, ApoA-IV, and ApoB. In vitro and in vivo studies demonstrated the stability and biological activity of ALPs derived from adiposomes, which resembles native lipoproteins. Of note, adiposomes bearing ApoE were internalized via clathrin-mediated endocytosis following LDLR binding and were delivered to lysosomes. On the other hand, adiposomes bearing ApoA-IV mimicked the existing form of endogenous ApoA-IV and exhibited significant improvement in glucose tolerance in mice. In addition, the construction process was simple, precise, reproducible, as well as easy to adjust for mass production. With this experimental system, different apolipoproteins can be recruited to build ALPs for some biological goals and potential applications in biomedicine.

Measurement of ATGL activity using adiposomes.

Adipose triacylglycerol lipase (ATGL) is a dynamic lipid droplet-associated protein involved in cellular lipolysis, which is conserved from bacteria to humans. Recent methods that measure the enzymatic activity of ATGL in vitro are established using lipid emulsions. However, the lipid emulsion platforms contain various membranous structures which reduce the accuracy of enzymatic activity determination. Therefore, a new platform and corresponding method are required for accurate measurement of ATGL enzymatic activity that represents cellular lipid and energy homeostasis. Adiposomes are artificial lipid nanostructures mimicking lipid droplets. Employing adiposome as a platform, we have developed an assay to measure the enzymatic activity of ATGL in vitro. Here, a detailed protocol is described to explain how to measure the activity of ATGL using adiposomes. This method successfully proves the concept of lipid droplet-mimetic lipase activity determining platform and provides a tool to identify the active sites of lipases.

One amino acid drives the lipid droplet targeting sequence of a new noncoding RNA-encoded protein to mitochondrion.

Over the past decade, the majority of the mammalian genome considered to be noncoding has been revealed to be able to produce proteins. Many RNA molecules, mis-annotated as noncoding, actually are predicted to code for proteins. Some of those proteins have been identified and verified to play critical roles in multiple biological processes. The lipid droplet (LD) is a unique cellular organelle bound with a phospholipid monolayer membrane, and is closely associated with cellular lipid metabolism and metabolic disorders. However, it is still unclear how a protein targets to LDs. Here we identified a new protein on LDs, LDANP2, which is encoded by noncoding RNA, through a proteomics-based strategy. The key sequence for its localization on LDs, Truncation 3, is predicted to form an amphipathic helix. Surprisingly, the deletion of the first amino acid in Truncation 3 resulted in mitochondrial localization. How the types of amino acids would determine the LD or mitochondrial localizations of the protein was studied. The findings introduce a useful strategy to mine for new proteins and would provide clues to the understanding of how a protein would find its right organelle, with phospholipid monolayer or bilayer membrane.

Comparison of Different Isolation Methods for Plasma-Derived Extracellular Vesicles in Patients with Hyperlipidemia.

Extracellular vesicles are commonly found in human body fluids and can reflect current physiological conditions of human body and act as biomarkers of disease. The quality of isolated extracellular vesicles facilitates the early diagnosis of various diseases accompanied by hyperlipidemia. Nonetheless, there are no reports on which special methods are suitable for isolating extracellular vesicles from the plasma of patients with hyperlipidemia. Thus, this study compared three different research-based extracellular vesicle isolation approaches, namely ultracentrifugation (UC), polyethylene glycol (PEG) precipitation, and size exclusion chromatography (SEC), and determined which of them was the most effective method. We selected blood samples from 12 patients with clinically diagnosed hyperlipidemia and isolated plasma-derived extracellular vesicles using three methods. The morphology of the isolated extracellular vesicles was observed using transmission electron microscopy, while the concentration was detected by asymmetric flow field-flow fractionation and multi-angle light scattering. Marker proteins were identified by Western blotting, and protein composition was evaluated by silver staining. Both determined the contaminations in the extracellular vesicle samples. The results showed that the three methods can be successfully used for the isolation of extracellular vesicles. The extracellular vesicles isolated by UC were larger in size, and the yield was much lower. Although the yield of extracellular vesicles isolated by PEG precipitation was greatly improved, the contamination was increased. Of the three methods, only the SEC-isolated extracellular vesicles were characterized by high yield and low contamination. Therefore, our data suggested that the SEC was a more ideal method for isolating extracellular vesicles from the plasma of patients with hyperlipidemia.

Protocol for using artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins.

Here, we present a protocol to construct artificial lipid droplets to study the binding affinity of lipid droplet-associated proteins. We provide procedures to construct adiposomes and prepare recombinant lipid droplet-associated proteins. Then we describe approaches to measure the number density of perilipin 2 on natural lipid droplets, construct artificial lipid droplets, and determine the binding affinity of perilipin 2 on artificial lipid droplets. This protocol can be adapted to determine the binding properties of various lipid droplet-associated proteins. For complete details on the use and execution of this protocol, please refer to Ma et al. (2021).

Validating an artificial organelle: Studies of lipid droplet-specific proteins on adiposome platform.

New strategies are urgently needed to characterize the functions of the lipid droplet (LD). Here, adiposome, an artificial LD mimetic platform, was validated by comparative in vitro bioassays. Scatchard analysis found that the binding of perilipin 2 (PLIN2) to the adiposome surface was saturable. Phosphatidylinositol (PtdIns) was found to inhibit PLIN2 binding while it did not impede perilipin 3 (PLIN3). Structural analysis combined with mutagenesis revealed that the 73rd glutamic acid of PLIN2 is significant for the effect of PtdIns on the PLIN2 binding. Furthermore, adiposome was also found to be an ideal platform for in situ enzymatic activity measurement of adipose triglyceride lipase (ATGL). The significant serine mutants of ATGL were found to cause the loss of lipase activity. Our study demonstrates the adiposome as a powerful, manipulatable model system that mimics the function of LD for binding and enzymatic activity studies of LD proteins in vitro.

Synthesis of sandwich-structured silver@polydopamine@silver shells with enhanced antibacterial activities.

The unique antibacterial characteristics of Ag nanomaterials offer a wide potential range of applications, but achieving rapid and durable antibacterial efficacy is challenging. This is because the speed and durability of the antibacterial function make conflicting demands on the structural design: the former requires the direct exposure of Ag to the surrounding environment, whereas the durability requires Ag to be protected from the environment. To overcome this incompatibility, we synthesize sandwich-structured polydopamine shells decorated both internally and externally with Ag nanoparticles, which exhibit prompt and lasting bioactivity in applications. These shells are biocompatible and can be used in vivo to counter bacterial infection caused by methicillin-resistant Staphylococcus aureus superbugs and to inhibit biofilm formation. This work represents a new paradigm for the design of composite materials with enhanced antibacterial properties.

Formation of Alkanethiol Supported Hybrid Membranes Revisited.

A phospholipid monolayer supported on an alkanethiol self-assembled monolayer (SAM) constitutes a supported hybrid membrane, a model of biological membranes optimized for electronic access through the underlying metal support surface. It is believed that phospholipids, when deposited from aqueous liposome suspension, spontaneously cover the alkanethiol-modified surface, owing to the reduction of surface free energy of the hydrophobic alkane surface exposed to the solution. However, the formation of the hybrid layer has to overcome significant energy barriers in rupturing the vesicle and "unzipping" the membrane leaflets; hence drivers of the spontaneous hybrid membrane formation are unclear. In this work, the authors studied the efficiency of the liposome deposition method to form hybrid membranes on octanethiol and hexadecanethiol SAMs in aqueous environment. Using quartz crystal microbalance to monitor the deposition process it was found that the hybrid membrane did not form spontaneously; the deposit was dominated by hemi-fused liposomes that can only be removed by applying osmotic stress. However, osmotic stress yielded a reproducible layer characterized by ≈-5Hz frequency change that is also confirmed by fluorescence microscopy imaging, irrespective of lipid concentration and the chain length of the SAMs. The frequency change is ≈20% of the frequency change expected for a tightly bound bilayer membrane, or 40% of a single leaflet, suggesting that the lipid layer is in a different conformation compared to a bilayer membrane: the acyl chains are most likely parallel to the SAM surface, likely due to strong hydrophobic interaction. Comparing these results to the literature it appears that the initial formation of hybrid membranes is inhibited by the ionic environment, while osmotic stress leads to the observed unique layer conformation.

Dual-Functional Polyethylene Glycol-b-polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities.

In recent years, microbial colonization on the surface of biomedical implants/devices has become a severe threat to human health. Herein, surface-immobilized guanidine derivative block copolymers create an antimicrobial and antifouling dual-functional coating. We report the preparation of an antimicrobial and antifouling block copolymer by the conjugation of polyhexanide (PHMB) with either allyl glycidyl ether or allyloxy polyethylene glycol (APEG; MW 1200 and 2400). The allyl glycidyl ether modified PHMB (A-PHMB) and allyloxy polyethylene glycol1200/2400 modified PHMB (APEG1200/2400-PHMB) copolymers were grafted onto a silicone rubber surface as a bottlebrush-like coating, respectively, using a plasma-UV-assisted surface-initiated polymerization. Both A-PHMB and APEG1200/2400-PHMB coatings exhibited excellent broad-spectrum antimicrobial properties against Gram-negative/positive bacteria and fungi. The APEG2400-PHMB coating displayed an improved antibiofilm as well as antifouling properties and a long reusable cycle, compared with two other coatings, due to its abundant PEG blocks among those copolymers. Also, the APEG2400-PHMB-coated silicone coupons were biocompatible toward mammalian cells, as revealed by in vitro hemocompatibile and cytotoxic assays. An in vivo study showed a significant decline of Escherichia coli colonies with a 5-log reduction, indicating the APEG2400-PHMB coating surface worked effectively in the rodent subcutaneous infection model. This PHMB-based block copolymer coating is believed to be an effective strategy to prevent biomaterial-associated infections.

Autoclaving-Derived Surface Coating with In Vitro and In Vivo Antimicrobial and Antibiofilm Efficacies.

Biomedical device-associated infections which engender severe threat to public health require feasible solutions. In this study, block copolymers consisting of antimicrobial, antifouling, and surface-tethering segments in one molecule are synthesized and grafted on polymeric substrates by a facile plasma/autoclave-assisted method. Hetero-bifunctional polyethylene glycol (PEG) with allyl and tosyl groups (APEG-OTs) is first prepared. PEGs with different molecular weights (1200 and 2400 Da) are employed. Polyhexamethylene guanidine (PHMG) which has excellent broad-spectrum antimicrobial activity and thermal/chemical stability, is conjugated with APEG-OTs to generate the block copolymer (APEG-PHMG). Allyl terminated PHMG (A-PHMG) without PEG segments is also synthesized by reacting PHMG with allyl glycidyl ether. The synthesized copolymers are thermal initiated by autoclaving and grafted on plasma pretreated silicone surface, forming permanently bonded bottlebrush-like coatings. Both A-PHMG and APEG1200/2400 -PHMG coatings exhibit potent antimicrobial activity against gram-positive/negative bacteria and fungus, whereas APEG1200/2400 -PHMG coatings show superior antifouling activity and long-term reusability to A-PHMG coating. APEG2400 -PHMG coating demonstrates the most effective in vitro antibiofilm and protein/platelet-resistant properties, as well as excellent hemo/biocompatibility. Furthermore, APEG2400 -PHMG greatly reduces the bacteria number with 5-log reduction in a rodent subcutaneous infection model. This rationally designed dual-functional antimicrobial and antifouling coating has great potential in combating biomedical devices/implant-associated infections.

White-rot fungal pretreatment of wheat straw with Phanerochaete chrysosporium for biohydrogen production: simultaneous saccharification and fermentation.

This paper demonstrates biohydrogen production was enhanced by white-rot fungal pretreatment of wheat straw (WS) through simultaneous saccharification and fermentation (SSF). Wheat straw was pretreated by Phanerochaete chrysosporium at 30 °C under solid state fermentation for 12 days, and lignin was removed about 28.5 ± 1.3 %. Microscopic structure observation combined thermal gravity and differential thermal gravity analysis further showed that the lignocellulose structure obviously disrupted after fungal pretreatment. Subsequently, the pretreated WS and crude cellulases prepared from Trichoderma atroviride were applied in SSF for hydrogen production using Clostridium perfringens. The maximum hydrogen yield was obtained to be 78.5 ± 3.4 ml g(-1)-pretreated WS, which was about 1.8-fold than the unpretreated group. Furthermore, the modified Gompertz model was applied study the progress of cumulative H(2) production. This work developed a novel bio-approach to improve fermentative H(2) yield from lignocellulosic biomass.

Comparison of various pretreatment methods for biohydrogen production from cornstalk.

To establish high-efficiency pretreatments of cornstalk (CS) for hydrogen fermentative production, various pretreatment strategies have been investigated and contrasted in this work. Five pretreatment methods, including acid-soaking pretreatment, base-soaking pretreatment, high-temperature-assisted acid pretreatment, high-temperature-assisted base pretreatment and ultrasonic-assisted acid pretreatment (UAP), were performed on CS. The results showed that UAP significantly promoted the hydrogen production by CS compared with other pretreatments. The optimum UAP process, pretreating substrate with ultrasonication in 2.0% sulfuric acid solution for 1.5 h at the liquid-solid ratio of 20:1, obtained the maximum specific hydrogen accumulation of 142.59 mL g(-1)-CS and an average hydrogen production rate of 17.03 mL g(-1)-CS h(-1). Furthermore, the scanning electron microscope analysis of CS samples supports the hydrogen production results as well. The present work demonstrates that UAP is an efficient and practical CS pretreatment for hydrogen production from agricultural waste straws.