Recent advances in liposome development for studying protein-lipid interactions.

Protein-lipid interactions are crucial for various cellular biological processes like intracellular signaling, membrane transport, and cytoskeletal dynamics. Therefore, studying these interactions is essential to understand and unravel their specific functions. Nevertheless, the interacting proteins of many lipids are poorly understood and still require systematic study. Liposomes are the most well-known and familiar biomimetic systems used to study protein-lipid interactions. Although liposomes have been widely used for studying protein-lipid interactions in classical methods such as the co-flotation assay (CFA), co-sedimentation assay (CSA), and flow cytometric assay (FCA), an overview of their current applications and developments in high-throughput methods is not yet available. Here, we summarize the liposome development in low and high-throughput methods to study protein-lipid interactions. Besides, a constructive comment for each platform is presented to stimulate the advancement of these technologies in the future.

Label-Free Bimetallic In Situ-Grown 3D Nickel-Foam-Supported NH2-MIL-88B(Fe2Co)-MOF-based Impedimetric Immunosensor for the Detection of Cardiac Troponin I.

In this study, a simple and competent metal-organic framework (MOF)-based nickel foam (NF)-supported three-dimensional (3D) immunosensor (Ab-NH2-MIL-88B(Fe2Co)-MOF/NF) was constructed and utilized for the specific recognition of the biomarker cardiac troponin (I) (cTnI). In the present work, biosensor fabrication was progressed through the modification of the NF substrate with the MOF material (NH2-MIL-88B(Fe2Co)-MOF) to enable an amine-functionalized electrode. This amine-functionalized NF electrodes (NH2-MIL-88B(Fe2Co)-MOF/NF) were then biointerfaced with anti-cTnI antibodies, which ended up as Ab-NH2-MIL-88B(Fe2Co)-MOF/NF electrodes. Analytical executions of the constructed bioelectrode were investigated for the quantitative analysis of cTnI in both buffered and serum solutions. Then, the electrochemical studies were carried out using the electrochemical impedance spectroscopy (EIS) method by monitoring changes concerning the charge transfer resistance (Rct) characteristics. The limit of detection (LOD) of the Ab-NH2-MIL-88B(Fe2Co)-MOF/NF immunosensor was achieved to be 13 fg/mL with great specificity. This kind of immunosensor imparts a new platform for the construction and application of MOF-hybrid 3D electrode materials with enhanced electrochemical behavior in cTnI sensing for the first time.

Analyzing polymeric matrix for fabrication of a biodegradable microneedle array to enhance transdermal delivery.

Traditional drug delivery systems, using invasive, transdermal, and oral routes, are limited by various factors, such as the digestive system environment, skin protection, and sensory nerve stimulation. To improve the drug delivery system, we fabricated a polysaccharide-based, dissolvable microneedle-based array, which combines the advantages of both invasive and transdermal delivery systems, and promises to be an innovative solution for minimally invasive drug delivery. In this study, we designed a reusable aluminum mold that greatly improved the efficiency and convenience of microneedle fabrication. Physical characterization of the polysaccharides, individual or mixed at different ratios, was performed to identify a suitable molecule to fabricate the dissolvable microneedle. We used a vacuum deposition-based micro-molding method at low temperature to fabricate the model. Using a series of checkpoints from material into product, a systematic feedback mechanism was built into the "all-in-one" fabrication step, which helped to improve production yields. The physical properties of the fabricated microneedle were assessed. The cytotoxicity analysis and animal testing of the microneedle demonstrated the safety and compatibility of the microneedle, and the successful penetration and effective release of a model protein.

Molecular and Functional Characterization of FLOWERING LOCUS T Homologs in Allium cepa.

Onion bulbing is an important agricultural trait affecting economic value and is regulated by flowering-related genes. FLOWERING LOCUS T (FT)-like gene function is crucial for the initiation of flowering in various plant species and also in asexual reproduction in tuber plants. By employing various computational analysis using RNA-Seq data, we identified eight FT-like genes (AcFT) encoding PEBP (phosphatidylethanolamine-binding protein) domains in Allium cepa. Sequence and phylogenetic analyses of FT-like proteins revealed six proteins that were identical to previously reported AcFT1-6 proteins, as well as one (AcFT7) with a highly conserved region shared with AcFT6 and another (comp106231) with low similarity to MFT protein, but containing a PEBP domain. Homology modelling of AcFT7 proteins showed similar structures and conservation of amino acids crucial for function in AtFT (Arabidopsis) and Hd3a (rice), with variation in the C-terminal region. Further, we analyzed AcFT expression patterns in different transitional stages, as well as under SD (short-day), LD (long-day), and drought treatment in two contrasting genotypic lines EM (early maturation, 36101) and LM (late maturation, 36122). The FT transcript levels were greatly affected by various environmental factors such as photoperiod, temperature and drought. Our results suggest that AcFT7 is a member of the FT-like genes in Allium cepa and may be involved in regulation of onion bulbing, similar to other FT genes. In addition, AcFT4 and AcFT7 could be involved in establishing the difference in timing of bulb maturity between the two contrasting onion lines.

Exchange of active site residues alters substrate specificity in extremely thermostable ß-glycosidase from Thermococcus kodakarensis KOD1.

ß-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with ß-glucosidase, ß-mannosidase, ß-fucosidase and ß-galactosidase activities. Sequence alignment with other ß-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic ß-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its ß-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for ß-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures.

Synthesis of zinc oxide nanoparticles on graphene-carbon nanotube hybrid for glucose biosensor applications.

Synthesis of zinc oxide nanoparticles incorporated graphene-carbon nanotubes hybrid (GR-CNT-ZnO) through a simple, one-pot method is demonstrated. The as-synthesized GR-CNT-ZnO composite is applied to fabricate an enzyme based glucose biosensor. The GOx immobilized on GR-CNT-ZnO composite exhibits well-defined redox peaks with a peak potential separation (ΔEp) of about 26 mV with enhanced peak currents, indicating a fast electron transfer at the modified electrode surface. The cyclic voltammetry measurements revealed that the modified film has high electrocatalytic ability towards glucose detection in the presence of oxygen. The proposed sensor has a wide linear detection range from 10 µM to 6.5 mM of glucose with a limit of detection (LOD) of 4.5 (±0.08) µM. In addition, the sensor possessed appreciable repeatability, reproducibility and remarkable stability for the sensitive determination of glucose. The practicality of this sensor has been demonstrated in human serum samples, with results being in good agreement with those determined using a standard photometric method.

An intermolecular disulfide bond is required for thermostability and thermoactivity of ß-glycosidase from Thermococcus kodakarensis KOD1.

Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. ß-glycosidase, Tkßgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkßgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the kcat/Km of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkßgly enzyme.