Characterization Of A Highly Active Polyhydroxyalkanoate Synthase

Polyhydroxyalkanoate (PHA) synthase from a locally isolated Chromobacterium sp. USM2 (PhaCCs) exhibited superior polymerizing ability and broad in vivo substrate specificity with preferences for short chain length (SCL) [3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV)] and medium chain length (MCL) [3-hydroxyhexanoate (3HHx)] monomers. For further characterization of the synthase, a Strep2-tagged PhaCCs for expression in and purification from Escherichia coli, was constructed in this study. In vitro enzymatic assay revealed an activity of 253 ± 13 U/mg for polymerization of 3-hydroxybutyryl-coenzyme A (3HB-CoA), which was approximately fivefold higher than that of model PHAproducing strain Cupriavidus necator (39 ± 5 U/mg)

A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants

Molecular characterisation of phaCAB from Comamonas sp. EB172 for functional expression in Escherichia coli JM109

In this study, PHA biosynthesis operon of Comamonas sp. EB172, an acid-tolerant strain, consisting of three genes encoding acetyl-CoA acetyltransferase (phaACo gene, 1182 bp), acetoacetyl-CoA reductase (phaBCo gene, 738 bp) and PHA synthase, class I (phaCCo gene, 1694 bp) were identified. Sequence analysis of the phaACo, phaBCo and phaCCo genes revealed that they shared more than 85%, 89% and 69% identity, respectively, with orthologues from Delftia acidovorans SPH-1 and Acidovorax ebreus TPSY. The PHA biosynthesis genes (phaCCo and phaABCo) were successfully cloned in a heterologous host, Escherichia coli JM109. E. coli JM109 transformants harbouring pGEM′-phaCCoABRe and pGEM′-phaCReABCo were shown to be functionally active synthesising 33 wt.% and 17 wt.% of poly(3-hydroxybutyrate) [P(3HB)]. E. coli JM109 transformant harbouring the three genes from the acid-tolerant Comamonas sp. EB172 (phaCABCo) under the control of native promoter from Cupriavidus necator, in vivo polymerised P(3HB) when fed with glucose and volatile mixed organic acids (acetic acid:propionic acid:n-butyric acid) in ration of 3:1:1, respectively. The E. coli JM109 transformant harbouring phaCABCo could accumulate P(3HB) at 2 g/L of propionic acid. P(3HB) contents of 40.9% and 43.6% were achieved by using 1% of glucose and mixed organic acids, respectively

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Exploring Various Techniques for the Chemical and Biological Synthesis of Polymeric Nanoparticles

Nanoparticles (NPs) have remarkable properties for delivering therapeutic drugs to the body’s targeted cells. NPs have shown to be significantly more efficient as drug delivery carriers than micron-sized particles, which are quickly eliminated by the immune system. Biopolymer-based polymeric nanoparticles (PNPs) are colloidal systems composed of either natural or synthetic polymers and can be synthesized by the direct polymerization of monomers (e.g., emulsion polymerization, surfactant-free emulsion polymerization, mini-emulsion polymerization, micro-emulsion polymerization, and microbial polymerization) or by the dispersion of preformed polymers (e.g., nanoprecipitation, emulsification solvent evaporation, emulsification solvent diffusion, and salting-out). The desired characteristics of NPs and their target applications are determining factors in the choice of method used for their production. This review article aims to shed light on the different methods employed for the production of PNPs and to discuss the effect of experimental parameters on the physicochemical properties of PNPs. Thus, this review highlights specific properties of PNPs that can be tailored to be employed as drug carriers, especially in hospitals for point-of-care diagnostics for targeted therapies

Self-Assembled Peptide-Based System for Mitochondrial-Targeted Gene Delivery: Functional and Structural Insights

Human mitochondrial dysfunction can lead to severe and often deadly diseases, for which there are no known cures. Although the targeted delivery of therapeutic gene to mitochondria is a promising approach to alleviate these disorders, gene carrier systems for the selective delivery of functional DNA into the mitochondria of living mammalian cells are currently unavailable. Here we rationally developed dual-domain peptides containing DNA-condensing/cell-penetrating/endosome-disruptive and mitochondria-targeting sequences. Secondary structures of the dual-domain peptides were analyzed, and variations in the physicochemical properties (stability, size, and ζ potential) of peptide/DNA complexes were studied as a function of peptide-to-DNA ratio and serum addition. An optimized formulation, identified through qualitative and quantitative studies, fulfills the fundamental prerequisites for mitochondria-specific DNA delivery, successfully transfecting a high proportion (82 ± 2%) of mitochondria in a human cell line with concomitant biocompatibility. Nuclear magnetic resonance studies confirmed the effectiveness of our bipartite peptide design with segregated functions: a helical domain necessary for mitochondrial import and an unstructured region for interaction with DNA involving lysine residues. Further analyses revealed that the lysine-specific interaction assisted the self-organization of the peptide and the DNA cargo, leading to a structural arrangement within the formed complex that is crucial for its biological efficiency. Thus the reported gene vector represents a new and reliable tool to uncover the complexity of mitochondrial transfection

Influence of Hydroxyl Groups on the Cell Viability of Polyhydroxyalkanoate (PHA) Scaffolds for Tissue Engineering

Polyhydroxyalkanoates (PHAs) are biopolyesters that have been studied as tissue engineering materials because of their biodegradability, biocompatibility, and low cytotoxicity. In this study, poly­(3-hydroxybutyrate-<i>co</i>-3-hydroxyvalerate-<i>co</i>-2,3-dihydroxybutyrate) [PHBVDB] containing hydroxyl groups was produced by recombinant <i>Ralstonia eutropha</i>. <i>R. eutropha</i> were constructed to express the propionate-coenzymeA transferase (<i>pct</i>) gene from <i>Megasphaera elsdenii</i>, and glycolate was used as the carbon source. Disruption of <i>phaA</i> encoding β-ketothiolase in the <i>phaCAB</i> operon increased 2,3-dihydroxybutyrate (2,3-DHBA) compositions to 3 mol %. The PHBVDB film showed a lower water contact angle compared with other PHA films, indicating increased hydrophilicity due to the hydroxyl groups. The mechanical properties of the PHBVDB scaffold met the requirements for a soft tissue matrix. The effect of hydroxyl groups on cytotoxicity was evaluated with human mesenchymal stem cells. Results of cell proliferation and live/dead assays showed that the PHBVDB scaffold did not exhibit significant cytotoxicity toward the cells. These results indicate that PBVDB containing hydroxyl groups could be applied as a hydrophilicity-controlled scaffold for soft tissue engineering

Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide–DNA Complexes

Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals

Enzyme-Mimic Peptide Assembly To Achieve Amidolytic Activity

Amyloid fibers are classified as a new generation of tunable bionanomaterials that exhibit new functions related to their distinctive characteristics, such as their universality, tunability, and stiffness. Here, we introduce the catalytic residues of serine protease into a peptide catalyst (PC) via an enzyme-mimic approach. The rational design of a repeating pattern of polar and nonpolar amino acids favors the conversion of the peptides into amyloid-like fibrils via self-assembly. Distinct fibrous morphologies have been observed at different pH values and temperatures, which indicates that different fibril packing schemes can be designed; hence, fibrillar peptides can be used to generate efficient artificial catalysts for amidolytic activities at mild pH values. The results of atomic force microscopy, Raman spectroscopy, and wide-angle X-ray scattering analyses are used to discuss and compare the fibril structure of a fibrillar PC with its amidolytic activity. The pH of the fibrillation reaction crucially affects the p<i>K</i>_a of the side chains of the catalytic triads and is important for stable fibril formation. Temperature is another important parameter that controls the self-assembly of peptides into highly stacked and laminated morphologies. The morphology and stability of fibrils are crucial and represent important factors for demonstrating the capability of the peptides to exert amidolytic activity. The observed amidolytic activity of PC4, one of the PCs, was validated using an inhibition assay, which revealed that PC4 can perform enzyme-like amidolytic catalysis. These results provide insights into the potential use of designed peptides in the generation of efficient artificial enzymes