Enhancing plastic biodegradation process: strategies and opportunities.

Plastic biodegradation has emerged as a sustainable approach and green alternative in handling the ever-increasing accumulation of plastic wastes in the environment. The complete biodegradation of polyethylene terephthalate is one of the most recent breakthroughs in the field of plastic biodegradation. Despite the success, the effective and complete biodegradation of a wide variety of plastics is still far from the practical implementation, and an on-going effort has been mainly devoted to the exploration of novel microorganisms and enzymes for plastic biodegradation. However, alternative strategies which enhance the existing biodegradation process should not be neglected in the continuous advancement of this field. Thus, this review highlights various strategies which have shown to improve the biodegradation of plastics, which include the pretreatment of plastics using UV irradiation, thermal, or chemical treatments to increase the susceptibility of plastics toward microbial action. Alternative pretreatment strategies are also suggested and compared with the existing techniques. Besides, the effects of additives such as pro-oxidants, natural polymers, and surfactants on plastic biodegradation are discussed. In addition, considerations governing the biodegradation performance, such as the formulation of biodegradation medium, cell-free biocatalysis, and physico-chemical properties of plastics, are addressed. Lastly, the challenges and future prospects for the advancement of plastic biodegradation are also highlighted.

In silico screening and heterologous expression of soluble dimethyl sulfide monooxygenases of microbial origin in Escherichia coli.

Sequence-based screening has been widely applied in the discovery of novel microbial enzymes. However, majority of the sequences in the genomic databases were annotated using computational approaches and lacks experimental characterization. Hence, the success in obtaining the functional biocatalysts with improved characteristics requires an efficient screening method that considers a wide array of factors. Recombinant expression of microbial enzymes is often hampered by the undesirable formation of inclusion body. Here, we present a systematic in silico screening method to identify the proteins expressible in soluble form and with the desired biological properties. The screening approach was adopted in the recombinant expression of dimethyl sulfide (DMS) monooxygenase in Escherichia coli. DMS monooxygenase, a two-component enzyme consisting of DmoA and DmoB subunits, was used as a model protein. The success rate of producing soluble and active DmoA is 71% (5 out of 7 genes). Interestingly, the soluble recombinant DmoA enzymes exhibited the NADH:FMN oxidoreductase activity in the absence of DmoB (second subunit), and the cofactor FMN, suggesting that DmoA is also an oxidoreductase. DmoA originated from Janthinobacterium sp. AD80 showed the maximum NADH oxidation activity (maximum reaction rate: 6.6 µM/min; specific activity: 133 µM/min/mg). This novel finding may allow DmoA to be used as an oxidoreductase biocatalyst for various industrial applications. The in silico gene screening methodology established from this study can increase the success rate of producing soluble and functional enzymes while avoiding the laborious trial and error involved in the screening of a large pool of genes available. KEY POINTS: • A systematic gene screening method was demonstrated. • DmoA is also an oxidoreductase capable of oxidizing NADH and reducing FMN. • DmoA oxidizes NADH in the absence of external FMN.

Pickering emulsion-templated ionotropic gelation of tocotrienol microcapsules: effects of alginate and chitosan concentrations and gelation process parameters.

BACKGROUND: Throughout the past decade, Pickering emulsion has been increasingly utilized for the encapsulation of bioactive compounds due to its high stability and biocompatibility. In the present work, palm tocotrienols were initially encapsulated in a calcium carbonate Pickering emulsion, which was then subjected to alginate gelation and subsequent chitosan coating. The effects of wall material (alginate and chitosan) concentrations, gelation pH and time, and chitosan coating time on the encapsulation efficiency of palm tocotrienols were explored. RESULTS: Our findings revealed that uncoated alginate microcapsules ruptured upon drying and exhibited low encapsulation efficiency (13.81 ± 2.76%). However, the addition of chitosan successfully provided a more complex and rigid external wall structure to enhance the stability of the microcapsules. By prolonging the crosslinking time from 5 to 30 min and increasing the chitosan concentration from 0.1% to 0.5%, the oil encapsulation efficiency was increased by 28%. Under the right gelation pH (pH 4), the extension of gelation time from 1 to 12 h resulted in an increase in alginate-Ca2+ crosslinkings, thus strengthening the microcapsules. CONCLUSION: With the optimum formulation and process parameters, a high encapsulation efficiency (81.49 ± 1.75%) with an elevated oil loading efficiency (63.58 ± 2.96%) were achieved. The final product is biocompatible and can potentially be used for the delivery of palm tocotrienols. © 2021 Society of Chemical Industry.

Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches.

Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.

Size-selective purification of hepatitis B virus-like particle in flow-through chromatography: Types of ion exchange adsorbent and grafted polymer architecture.

Hepatitis B virus-like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow-through chromatography mode. The virus-like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus-like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch-chain length) of the grafted polymer. The branch-chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 µm) had an approximately twofold increase in the flow-through recovery, as compared to the smaller adsorbent (30 µm). Generally, polymer-grafted adsorbents improved the exclusion of the virus-like particles. Overall, the middle branch-chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow-through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer-grafted adsorbent showed that a better exclusion of virus-like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus-like particles, which preserved the particles' structure.

Towards more accurate prediction of protein folding rates: a review of the existing Web-based bioinformatics approaches.

The understanding of protein-folding mechanisms is often considered to be an important goal that will enable structural biologists to discover the mysterious relationship between the sequence, structure and function of proteins. The ability to predict protein-folding rates without the need for actual experimental work will assist the research work of structural biologists in many ways. Many bioinformatics tools have emerged in the past decade, and each has showcased different features. In this article, we review and compare eight web-based prediction tools that are currently available and that predominantly predict the protein-folding rate. The prediction performance, usability and utility, together with the prediction tool development and validation methodologies for these tools, are critically reviewed. This article is presented in a comprehensible manner to assist readers in the process of selecting the most appropriate bioinformatics tools to meet their needs.

Bioinformatics approaches for improved recombinant protein production in Escherichia coli: protein solubility prediction.

The solubility of recombinant protein expressed in Escherichia coli often represents the production yield. However, up-to-date, instances of successful production of soluble recombinant proteins in E. coli expression system with high yield remain scarce. This is mainly due to the difficulties in improving the overall production capacity, as most of the well-established strategies usually involve a series of trial and error steps with unguaranteed success. One way to concurrently improve the production yield and minimize the production cost would be incorporating the potency of bioinformatics tools to conduct in silico studies, which forecasts the outcome before actual experimental work. In this article, we review and compare seven prediction tools available, which predict the solubility of protein expressed in E. coli, using the following criteria: prediction performance, usability, utility, prediction tool development and validation methodologies. This comprehensive review will be a valuable resource for researchers with limited prior experience in bioinformatics tools. As such, this will facilitate their choice of appropriate tools for studies related to enhancement of intracellular recombinant protein production in E. coli.

Controlled assembly of superparamagnetic iron oxide nanoparticle into nanoliposome for Pickering emulsion preparation.

There has been a surge in effort in the development of various solid nanoparticles as Pickering emulsion stabilizers in the past decades. Regardless, the exploration of stabilizers that simultaneously stabilize and deliver bioactive has been limited. For this, liposomes with amphiphilic nature have been introduced as Pickering emulsion stabilizers but these nano-sized vesicles lack targeting specificity. Therefore in this study, superparamagnetic iron oxide nanoparticles (SPION) encapsulated within liposomes (MLP) were used as Pickering emulsion stabilizers to prepare pH and magnetic-responsive Pickering emulsions. A stable MLP-stabilized Pickering emulsion formulation was established by varying the MLP pH, concentration, and oil loading during the emulsification process. The primary stabilization mechanism of the emulsion under pH variation was identified to be largely associated with the MLP phosphate group deprotonation. When subjected to sequential pH adjustment to imitate the gastrointestinal digestion pH environment, a recovery in Pickering emulsion integrity was observed as the pH changes from acidic to alkaline. By incorporating SPION, the Pickering emulsion can be guided to the targeted site under the influence of a magnetic field without compromising emulsion stability. Overall, the results demonstrated the potential of MLP-stabilized Pickering emulsion as a dual pH- and magnetic-responsive drug delivery carrier with the ability to co-encapsulate hydrophobic and hydrophilic bioactive.

Native agarose gel electrophoresis and electroelution: A fast and cost-effective method to separate the small and large hepatitis B capsids.

Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self-assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost-effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE-EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE-EE method are monodisperse with polydispersity values of ∼15% and ∼13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE-EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ∼84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.

Dual (pH and thermal) stimuli-responsive Pickering emulsion stabilized by chitosan-carrageenan composite microgels.

Formulation of water-in-oil (W/O) Pickering emulsion (PE) for food applications has been largely restricted by the limited choices of food-grade Pickering emulsifiers. In this study, composite microgels made of chitosan and carrageenan were explored as a dual (pH and thermal) stimuli-responsive Pickering emulsifier for the stabilization of W/O PE. The chitosan-carrageenan (CS-CRG) composite microgels not only exhibited pH- and thermo-responsiveness, but also displayed enhanced lipophilicity as compared to the discrete polymers. The stability of the CS-CRG-stabilized W/O PE system (CS-CRG PE) was governed by CS:CRG mass ratio and oil fractions used. The CS-CRG PE remained stable at acidic pH and at temperatures below 40 °C. The instability of CS-CRG composite microgels at alkaline pH and at temperatures above 40 °C rendered the demulsification of CS-CRG PE. This stimuli-responsive W/O PE could unlock new opportunities for the development of stimuli-responsive W/O PE using food-grade materials.

Hydroxychloroquine: Key therapeutic advances and emerging nanotechnological landscape for cancer mitigation.

Hydroxychloroquine (HCQ) is a unique class of medications that has been widely utilized for the treatment of cancer. HCQ plays a dichotomous role by inhibiting autophagy induced by the tumor microenvironment (TME). Preclinical studies support the use of HCQ for anti-cancer therapy, especially in combination with conventional anti-cancer treatments since they sensitize tumor cells to drugs, potentiating the therapeutic activity. However, clinical evidence has suggested poor outcomes for HCQ due to various obstacles, including non-specific distribution, low aqueous solubility and low bioavailability at target sites, transport across tissue barriers, and retinal toxicity. These issues are addressable via the integration of HCQ with nanotechnology to produce HCQ-conjugated nanomedicines. This review aims to discuss the pharmacodynamic, pharmacokinetic and antitumor properties of HCQ. Furthermore, the antitumor performance of the nanoformulated HCQ is also reviewed thoroughly, aiming to serve as a guide for the HCQ-based enhanced treatment of cancers. The nanoencapsulation or nanoconjugation of HCQ with nanoassemblies appears to be a promising method for reducing the toxicity and improving the antitumor efficacy of HCQ.

Nanoglue: an alternative way to display cell-internalizing peptide at the spikes of hepatitis B virus core nanoparticles for cell-targeting delivery.

Cell-internalizing peptides (CIPs) can be used to mediate specific delivery of nanoparticles across cellular membrane. The objective of this study was to develop a display technique using hepatitis B virus (HBV) capsid-binding peptide as a "nanoglue" to present CIPs on HBV nanoparticles for cell-targeting delivery. A CIP was selected from a phage display library and cross-linked specifically at the tips of the spikes of the HBV capsid nanoparticle via the "nanoglue" by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Fluorescent oligonucleotides packaged in the nanoparticles and the fluorescein molecules conjugated on the nanoparticles were delivered to cells by using this display technique. This study demonstrated a proof of principle for cell-targeting delivery via "nanoglue" bioconjugation.

Statistical optimization of green fluorescent protein production from Escherichia coli BL21(DE3).

An optimized cultivation condition is needed to maximize the functional green fluorescent protein (GFP) production. Six process variables (agitation rate, temperature, initial medium pH, concentration of inducer, time of induction, and inoculum density) were screened using the fractional factorial design. Three variables (agitation rate, temperature, and time of induction) exerted significant effects on functional GFP production in E. coli shake flask cultivation and were optimized subsequently using the Box-Behnken design. An agitation rate of 206 rpm at 31°C and induction of the protein expression when the cell density (OD(600nm)) reaches 1.04 could enhance the yield of functional GFP production from 0.025 g/L to 0.241 g/L, which is about ninefold higher than the unoptimized conditions. Unoptimized cultivation conditions resulted in protein aggregation and hence reduced the quantity of functional GFP. The model and regression equation based on the shake flask cultivation could be applied to a 2-L bioreactor for maximum functional GFP production.

Stimuli-responsive nanoassemblies for targeted delivery against tumor and its microenvironment.

Despite the emergence of various cancer treatments, such as surgery, chemotherapy, radiotherapy, and immunotherapy, their use remains restricted owing to their limited tumor elimination efficacy and side effects. The use of nanoassemblies as delivery systems in nanomedicine for tumor diagnosis and therapy is flourishing. These nanoassemblies can be designed to have various shapes, sizes, and surface charges to meet the requirements of different applications. It is crucial for nanoassemblies to have enhanced delivery of payloads while inducing minimal to no toxicity to healthy tissues. In this review, stimuli-responsive nanoassemblies capable of combating the tumor microenvironment (TME) are discussed. First, various TME characteristics, such as hypoxia, oxidoreduction, adenosine triphosphate (ATP) elevation, and acidic TME, are described. Subsequently, the unique characteristics of the vascular and stromal TME are differentiated, and multiple barriers that have to be overcome are discussed. Furthermore, strategies to overcome these barriers for successful drug delivery to the targeted site are reviewed and summarized. In conclusion, the possible challenges and prospects of using these nanoassemblies for tumor-targeted delivery are discussed. This review aims at inspiring researchers to develop stimuli-responsive nanoassemblies for tumor-targeted delivery for clinical applications.

Sonoproduction of nanobiomaterials - A critical review.

Ultrasound (US) demonstrates remarkable potential in synthesising nanomaterials, particularly nanobiomaterials targeted towards biomedical applications. This review briefly introduces existing top-down and bottom-up approaches for nanomaterials synthesis and their corresponding synthesis mechanisms, followed by the expounding of US-driven nanomaterials synthesis. Subsequently, the pros and cons of sono-nanotechnology and its advances in the synthesis of nanobiomaterials are drawn based on recent works. US-synthesised nanobiomaterials have improved properties and performance over conventional synthesis methods and most essentially eliminate the need for harsh and expensive chemicals. The sonoproduction of different classes and types of nanobiomaterials such as metal and superparamagnetic nanoparticles (NPs), lipid- and carbohydrate-based NPs, protein microspheres, microgels and other nanocomposites are broadly categorised based on the physical and/or chemical effects induced by US. This review ends on a good note and recognises US-driven synthesis as a pragmatic solution to satisfy the growing demand for nanobiomaterials, nonetheless some technical challenges are highlighted.

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli.

Optimization of the fermentation process for recombinant protein production (RPP) is often resource-intensive. Machine learning (ML) approaches are helpful in minimizing the experimentations and find vast applications in RPP. However, these ML-based tools primarily focus on features with respect to amino-acid-sequence, ruling out the influence of fermentation process conditions. The present study combines the features derived from fermentation process conditions with that from amino acid-sequence to construct an ML-based model that predicts the maximal protein yields and the corresponding fermentation conditions for the expression of target recombinant protein in the Escherichia coli periplasm. Two sets of XGBoost classifiers were employed in the first stage to classify the expression levels of the target protein as high (>50 mg/L), medium (between 0.5 and 50 mg/L), or low (<0.5 mg/L). The second-stage framework consisted of three regression models involving support vector machines and random forest to predict the expression yields corresponding to each expression-level-class. Independent tests showed that the predictor achieved an overall average accuracy of 75% and a Pearson coefficient correlation of 0.91 for the correctly classified instances. Therefore, our model offers a reliable substitution of numerous trial-and-error experiments to identify the optimal fermentation conditions and yield for RPP. It is also implemented as an open-access webserver, PERISCOPE-Opt (http://periscope-opt.erc.monash.edu).

Quartz crystal microbalance-based biosensing of hepatitis B antigen using a molecularly imprinted polydopamine film.

Quartz crystal microbalance (QCM)-based biosensors are highly attractive as rapid diagnostic devices for detecting infectious diseases. However, the fabrication of QCM-based biosensors often involves tedious processes due to the poor stability of the biological recognition elements. In this work, the simple self-polymerisation of dopamine was used to functionalise the QCM crystal surface with a molecularly imprinted polydopamine (MIPDA) sensing film for detecting the hepatitis B core antigen (HBcAg), a serological biomarker of hepatitis B. Recognition cavities that complemented the size and shape of HBcAg were observed on the QCM crystal surface after functionalisation with the MIPDA film. The MIPDA-QCM biosensor showed a selective affinity for HBcAg, recording frequency responses up to 7.8 folds larger towards HBcAg compared to human serum albumin at the same analyte concentrations. The biosensor response was enhanced by using the optimal concentrations of 10 mg mL-1 of dopamine and 1 mg mL-1 of template for MIPDA film formation, resulting in a low detection limit (0.88 µg mL-1) that enables the detection of clinically relevant titres of HBcAg. The detection process could be completed within 10 min after sample loading without additional steps for signal amplification, highlighting the practical advantages of the MIPDA-QCM biosensor for point-of-care detection of hepatitis B.

Stimuli-controllable iron oxide nanoparticle assemblies: Design, manipulation and bio-applications.

Despite its wide establishment over the years, iron oxide nanoparticle (IONP) still draws extensive interest in the biomedical fields due to its biocompatibility, biodegradability, magnetivity and surface tunable properties. IONP has been used for the MRI, magnetic targeting, drug delivery and hyperthermia of various diseases. However, their poor stability, low diagnostic sensitivity and low disease-specificity have resulted in unsatisfying diagnostic and therapeutic outputs. The surface functionalization of IONP with biocompatible and colloidally stable components appears to be promising to improve its circulation and colloidal stability. Importantly, through surface functionalization with designated functional components, IONP-based assemblies with multiple stimuli-responsivity could be formed to achieve an accurate and efficient delivery of IONP to disease sites for an improved disease diagnosis and therapy. In this work, we first described the design of biocompatible and stable IONP assemblies. Further, their stimuli-driven manipulation strategies are reviewed. Next, the utilization of IONP assemblies for disease diagnosis, therapy and imaging-guided therapy are discussed. Then, the potential toxicity of IONPs and their clinical usages are described. Finally, the intrinsic challenges and future outlooks of IONP assemblies are commented. This review provides recent insights into IONP assemblies, which could inspire researchers on the future development of multi-responsive and disease-targetable nanoassemblies for biomedical utilization.

Spray-dried alginate-coated Pickering emulsion stabilized by chitosan for improved oxidative stability and in vitro release profile.

The commercial application of liquid-state Pickering emulsions in food systems remains a major challenge. In this study, we developed a spray-dried Pickering emulsion powder using chitosan as a Pickering emulsifier and alginate as a coating material. The functionality of the powder was evaluated in terms of its oxidative stability, pH-responsiveness, mucoadhesivity, and lipid digestibility. The Pickering emulsion powder was oxidatively more stable than the conventional emulsion powder stabilized by gum Arabic. The powder exhibited pH-responsiveness, whereby it remained intact in acidic pH, but dissolved to release the emulsion in 'Pickering form' at near-neutral pH. The Pickering emulsion powder was also mucoadhesive and could be digested by lipase in a controlled manner. These findings suggested that the multi-functional Pickering emulsion powder could be a potential delivery system for applications in the food industry.

Synthesis of bio-inspired cellulose nanocrystals-soy protein isolate nanoconjugate for stabilization of oil-in-water Pickering emulsions.

The development of hybrid polysaccharide-protein complexes as Pickering emulsion stabilizers has attracted increasing research interest in recent years. This work presents an eco-friendly surface modification strategy to functionalize hydrophilic cellulose nanocrystals (CNC) using hydrophobic soy protein isolate (SPI) via mussel adhesive-inspired poly (l-dopa) (PLD) to develop improved nanoconjugates as stabilizers for oil-in-water Pickering emulsion. The physicochemical properties of the CNC-PLD-SPI nanoconjugate were evaluated by solid-state 13C NMR, FT-IR, TGA, XRD, contact angle analysis, and TEM. The modified CNC (conjugation content of 38.22 ± 1.21%) had lowered crystallinity index, higher thermal stability, and more hydrophobic than unmodified CNC, with an average particle size of 309.9 ± 8.0 nm. Use of amphiphilic CNC-PLD-SPI nanoconjugate with greater conformational flexibility as Pickering stabilizer produced oil-in-water emulsions with greater physical stability.