Toxicity evaluation of zinc oxide nanoparticles green synthesized using papaya extract in zebrafish.

In green synthesis of zinc oxide nanoparticles (ZnO NPs), the use of papaya extract as a capping and reducing agent shows promise for potential applications of these particles in biomedicine. However, toxicity evaluation is necessary to ensure the safety of humans and the environment. The zebrafish model is used to assess toxicity with embryo developmental observation as it is a rapid, simple method for screening of toxicity. The objective of the present study was to assess the toxicological characteristics of ZnO NPs produced from papaya extract using a zebrafish model. The preparation of plant extracts from papaya using two solvents (water and methanol) and characterization of bioactive compounds in the extracts were reported. ZnO NPs were synthesized from both plant extracts and characterized with scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. Toxicity evaluation was conducted on zebrafish embryos for 96 h. ZnO NPs synthesized from aqueous and methanol extracts had mean crystallite diameters of 13 and 12 nm, respectively. Mortality, hatching rate and malformation of zebrafish embryos were assessed at different concentrations of ZnO NPs. Both NPs showed high mortality rates at high concentrations, with 100 (aqueous) and 20 mg/l (methanol extract) being lethal for all embryos. Concentrations <10 mg/l for both synthesized ZnO NPs had similar results to the negative control, indicating a safe dosage for embryos. The hatching rate and malformation were also affected, with higher concentrations of NPs causing a delayed hatching rate and malformation in pericardial and yolk sac edema. Whole embryo mRNA expression of immune-associated genes, including IL-1 and -10 and TNF-α, was upregulated following lethal concentration 50 (LC50) ZnO NP exposure. ZnO NPs synthesized from papaya extract (both in aqueous and methanol environments) had a dose- and time-dependent embryonic toxicity effect. Hence, the present study demonstrated initial toxicity screening of ZnO NPs synthesized from plant extract.

Synthesis of zinc oxide nanoparticles using methanol propolis extract (Pro-ZnO NPs) as antidiabetic and antioxidant.

In recent times, the overall health of individuals has been declining due to unhealthy lifestyles, leading to various diseases, including diabetes. To address this issue, antidiabetic and antioxidant agents are required to back-up human well-being. Zinc oxide (ZnO) is one such substance known for its antidiabetic and antioxidant effects. To enhance its capability and effectiveness, propolis was utilized to synthesize zinc oxide nanoparticles (Pro-ZnO NPs). The objective of this study was to synthesize Pro-ZnO NPs and assess their performance by conducting inhibition assays against α-amylase and α-glucosidase enzymes, as well as a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. The results showed that Pro-ZnO NPs were formed in a hexagonal wurtzite structure, with particle sizes ranging from 30 to 50 nm and an absorption band observed at 341 nm. The stability, chemical properties, and crystallography of Pro-ZnO NPs were also thoroughly examined using appropriate methods. The Pro-ZnO NPs demonstrated significant inhibitory effects against α-amylase and α-glucosidase enzymes, with inhibition rates reaching 69.52% and 73.78%, respectively, whereas the antioxidant activity was as high as 70.76%. Consequently, with their high inhibition rates, the Pro-ZnO NPs demonstrate the potential to be employed as a natural agent for combating diabetes and promoting antioxidant effects.

Structural Characterization of Polycrystalline Titania Nanoparticles on C. striata Biosilica for Photocatalytic POME Degradation.

The biosilica shell of marine diatoms has emerged as a unique matrix for photocatalysis, owing to its sophisticated architecture with hierarchical nanopores and large surface area. Although the deposition of titania nanoparticles on diatom biosilica has been demonstrated previously, their photocatalytic activity has been tested only for degradation of pure compounds, such as dyes, nitrogen oxide, and aldehydes. The efficiency of such photocatalysts for degradation of mixtures, for instance, industrial wastewaters, is yet to be investigated. Furthermore, reports on the lattice structures and orientation of nanotitania crystals on biosilica are considerably limited, especially for the underexplored tropical marine diatoms. Here, we report an extensive characterization of titania-loaded biosilica from the tropical Cyclotella striata diatom, starting from freshly grown cell cultures to photodegradation of wastewaters, namely, the palm oil mill effluent (POME). As Indonesia is the largest palm oil producer in the world, photocatalytic technology could serve as a sustainable alternative for local treatment of POME. In this study, we achieved a 54% loading of titania on C. striata TBI strain biosilica, as corroborated by XRF analyses, which was considerably high compared to previous studies. Through visualization using HR-TEM, supported by SAED and XRD analyses, nanocrystal TiO2 appeared to be trapped in an anatase phase with polycrystalline characteristics and distinct crystallographic orientations. Importantly, the presence of C. striata biosilica lowered the band gap of titania from 3.41 eV to around 3.2 eV upon deposition, enabling photodegradation of POME using a broad-range xenon lamp as the light source, mimicking the sunlight. Kinetic analyses revealed that POME degradation using the photocatalysts followed quasi-first-order kinetics, in which the highest titania content resulted in the highest photocatalytic activity (i.e., up to 47% decrease in chemical oxygen demand) and exhibited good photostability throughout the reaction cycles. Unraveling the structure and photoactivity of titania-biosilica catalysts allows transforming marine diatoms into functional materials for wastewater photodegradation.

Optimization of haloacid dehalogenase production by recombinant E. coli BL21 (DE3)/pET-hakp1 containing haloacid dehalogenase gene from Klebsiella pneumoniae ITB1 using Response Surface Methodology (RSM).

Organohalogens, including monochloroacetic acid (MCA), are abundantly synthesized compounds for various industrial purposes. MCA is widely used as a raw material or as an intermediate compound for the production of pesticides, herbicides, fungicides, plastics, surfactants, shampoos, liquid soaps, and emulsion agents. Nonetheless, widespread and large-scale utilization of organohalogens might negatively impact life quality as these compounds are toxic to organisms and persistently present in the environment. An effort to decrease the effect of MCA pollutant is by performing bioremediation, taking advantage of microorganisms that produce haloacid dehalogenases, a class of enzymes that catalyze the breakage of carbon halogen bonds. In this sense, we have isolated Klebsiella pneumoniae ITB1 that could degrade MCA. The haloacid dehalogenase gene from this bacterium has been successfully cloned into pGEM-T vector and subcloned into pET-30a(+) expression vector to yield pET-hakp1 recombinant clone in Escherichia coli BL21 (DE) host cell. This research aimed to find an optimum condition for producing haloacid dehalogenase from this recombinant clone using Response Surface Methodology (RSM). Among the independent variables studied were the concentration of inducer, incubation temperature after the induction, and incubation period after the induction. We obtained the crude extract of the enzyme as cells' lysate after sonicating the bacterial cells. Haloacid dehalogenase activity against MCA substrate was determined by measuring the amount of chloride ions released into the medium of the enzymatic reaction using the colorimetry method, according to Bergmann and Sanik. The result indicated that the optimum condition for haloacid dehalogenase production by E. coli BL21 (DE3)/pET-hakp1 was observed when using 1.8 mM IPTG (isopropyl-ß-D-1-thiogalactopyranoside) as the inducer, followed by 4 h incubation with shaking at 37 °C, which was predicted to result in a maximum of 0.48 mM chloride ions from 0.50 mM of MCA substrate. This report provides an insight into applying RSM for optimization of enzyme production from E. coli recombinant clones.

Molecularly Imprinted Affinity Membrane: A Review.

A molecularly imprinted affinity membrane (MIAM) can perform separation with high selectivity due to its unique molecular recognition introduced from the molecular-printing technique. In this way, a MIAM is able to separate a specific or targeted molecule from a mixture. In addition, it is possible to achieve high selectivity while maintaining membrane permeability. Various methods have been developed to produce a MIAM with high selectivity and productivity, with their respective advantages and disadvantages. In this paper, the MIAM is reviewed comprehensively, from the fundamentals of the affinity membrane to its applications. First, the development of a MIAM and various preparation methods are presented. Then, applications of MIAMs in sensor, metal ion separation, and organic compound separation are discussed. The last part of the review discusses the outlook of MIAMs for future development.

Recent Developments of Liquid Chromatography Stationary Phases for Compound Separation: From Proteins to Small Organic Compounds.

Compound separation plays a key role in producing and analyzing chemical compounds. Various methods are offered to obtain high-quality separation results. Liquid chromatography is one of the most common tools used in compound separation across length scales, from larger biomacromolecules to smaller organic compounds. Liquid chromatography also allows ease of modification, the ability to combine compatible mobile and stationary phases, the ability to conduct qualitative and quantitative analyses, and the ability to concentrate samples. Notably, the main feature of a liquid chromatography setup is the stationary phase. The stationary phase directly interacts with the samples via various basic mode of interactions based on affinity, size, and electrostatic interactions. Different interactions between compounds and the stationary phase will eventually result in compound separation. Recent years have witnessed the development of stationary phases to increase binding selectivity, tunability, and reusability. To demonstrate the use of liquid chromatography across length scales of target molecules, this review discusses the recent development of stationary phases for separating macromolecule proteins and small organic compounds, such as small chiral molecules and polycyclic aromatic hydrocarbons (PAHs).

Photoprogramming Allostery in Human Serum Albumin.

Developing strategies to interfere with allosteric interactions in proteins not only promises to deepen our understanding of vital cellular processes but also allows their regulation using external triggers. Light is particularly attractive as a trigger being spatiotemporally selective and compatible with the physiological environment. Here, we engineered a hybrid protein in which irradiation with light opens a new allosteric communication route that is not inherent to the natural system. We select human serum albumin, a promiscuous protein responsible for transporting a variety of ligands in plasma, and show that by covalently incorporating a synthetic photoswitch to subdomain IA we achieve optical control of the ligand binding in subdomain IB. Molecular dynamics simulations confirm the allosteric nature of the interactions between IA and IB in the engineered protein. Specifically, upon illumination, photoconversion of the switch is found to correlate with a less-coordinated motion of the two subdomains and an increased flexibility of the binding pocket in subdomain IB, whose fluctuations are cooperatively enhanced by the presence of ligands, ultimately facilitating their release. Our combined experimental and computational work demonstrates how harnessing artificial molecular switches enables photoprogramming the allosteric regulation of binding activities in such a prominent protein.

CCMV-Based Enzymatic Nanoreactors.

Protein-based nanoreactors are generated by encapsulating an enzyme inside the capsid of the cowpea chlorotic mottle virus (CCMV). Here, three different noncovalent methods are described to efficiently incorporate enzymes inside the capsid of these viral protein cages. The methods are based on pH, leucine zippers, and electrostatic interactions respectively, as a driving force for encapsulation. The methods are exclusively described for the enzymes horseradish peroxidase, glucose oxidase, and Pseudozyma antarctica lipase B, but they are also applicable for other enzymes.

Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.

Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems.

Assembling Enzymatic Cascade Pathways inside Virus-Based Nanocages Using Dual-Tasking Nucleic Acid Tags.

The packaging of proteins into discrete compartments is an essential feature for cellular efficiency. Inspired by Nature, we harness virus-like assemblies as artificial nanocompartments for enzyme-catalyzed cascade reactions. Using the negative charges of nucleic acid tags, we develop a versatile strategy to promote an efficient noncovalent co-encapsulation of enzymes within a single protein cage of cowpea chlorotic mottle virus (CCMV) at neutral pH. The encapsulation results in stable 21-22 nm sized CCMV-like particles, which is characteristic of an icosahedral T = 1 symmetry. Cryo-EM reconstruction was used to demonstrate the structure of T = 1 assemblies templated by biological soft materials as well as the extra-swelling capacity of these T = 1 capsids. Furthermore, the specific sequence of the DNA tag is capable of operating as a secondary biocatalyst as well as bridging two enzymes for co-encapsulation in a single capsid while maintaining their enzymatic activity. Using CCMV-like particles to mimic nanocompartments can provide valuable insight on the role of biological compartments in enhancing metabolic efficiency.

Self-Assembly of Proteins: Towards Supramolecular Materials.

The study of protein self-assembly has attracted great interest over the decades, due to the important role that proteins play in life. In contrast to the major achievements that have been made in the fields of DNA origami, RNA, and synthetic peptides, methods for the design of self-assembling proteins have progressed more slowly. This Concept article provides a brief overview of studies on native protein and artificial scaffold assemblies and highlights advances in designing self-assembling proteins. The discussions are focused on design strategies for self-assembling proteins, including protein fusion, chemical conjugation, supramolecular, and computational-aided de novo design.

Labelling Bacterial Nanocages with Photo-switchable Fluorophores.

The robustness and biocompatibility of bacterial nanocages holds promise for bio-nanotechnologies. The propensity of these nano-carriers to penetrate cells has been demonstrated, which calls for the development of tracking strategies, both in vitro and in vivo. Here, we label bacterial nanocages with photo-switchable fluorophores, to facilitate their imaging by super-resolution microscopy. We demonstrate the functionalization of the encapsulin from Brevibacterium linens with a spiropyran, which is not fluorescent, by covalent attachment to the amine residues at the outer encapsulin shell. Upon alternating irradiation with ultraviolet and visible light, the spiropyran switches forth and back to its fluorescent merocyanine photo-isomer and thus the fluorescence can be switched on and off, reversibly. We also show that the bacterial compartments preserve their structural integrity upon covalent modification and over at least five irradiation cycles.

Self-assembled cage-like protein structures.

Proteins and protein-based assemblies represent the most structurally and functionally diverse molecules found in nature. Protein cages, viruses and bacterial microcompartments are highly organized structures that are composed primarily of protein building blocks and play important roles in molecular ion storage, nucleic acid packaging and catalysis. The outer and inner surface of protein cages can be modified, either chemically or genetically, and the internal cavity can be used to template, store and arrange molecular cargo within a defined space. Owing to their structural, morphological, chemical and thermal diversity, protein cages have been investigated extensively for applications in nanotechnology, nanomedicine and materials science. Here we provide a concise overview of the most common icosahedral viral and nonviral assemblies, their role in nature, and why they are highly attractive scaffolds for the encapsulation of functional materials.