Multiplex detection of bacterial pathogens by PCR/SERS assay.

Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making.

Exploring the Potential of Bioactive Peptides: From Natural Sources to Therapeutics.

Bioactive peptides, specific protein fragments with positive health effects, are gaining traction in drug development for advantages like enhanced penetration, low toxicity, and rapid clearance. This comprehensive review navigates the intricate landscape of peptide science, covering discovery to functional characterization. Beginning with a peptidomic exploration of natural sources, the review emphasizes the search for novel peptides. Extraction approaches, including enzymatic hydrolysis, microbial fermentation, and specialized methods for disulfide-linked peptides, are extensively covered. Mass spectrometric analysis techniques for data acquisition and identification, such as liquid chromatography, capillary electrophoresis, untargeted peptide analysis, and bioinformatics, are thoroughly outlined. The exploration of peptide bioactivity incorporates various methodologies, from in vitro assays to in silico techniques, including advanced approaches like phage display and cell-based assays. The review also discusses the structure-activity relationship in the context of antimicrobial peptides (AMPs), ACE-inhibitory peptides (ACEs), and antioxidative peptides (AOPs). Concluding with key findings and future research directions, this interdisciplinary review serves as a comprehensive reference, offering a holistic understanding of peptides and their potential therapeutic applications.

Adapted method for rapid detection and quantification of pathogen Campylobacter jejuni from environmental water samples.

Building on a previously developed workflow for rapid and sensitive pathogen detection by qPCR, this work has established a sample treatment strategy that produces consistent quantification efficiencies (QEs) for Campylobacter jejuni against a complex and highly variable sample matrix from a suburban river. The individual treatments most effective at minimizing the inhibitory effects of the sample matrix were pH buffering with HEPES (50 mM, pH 5.7) and addition of the surfactant Tween 20 (2% v/v). Unexpectedly, sample acidification (pH 4-5) resulting from the use of aged Tween 20 that had undergone partial hydrolysis, appeared to play a key role in enhancing QE. This effect could be replicated by direct pH adjustment with dilute hydrochloric acid and may be linked to the solubilization and removal of inhibitory particles at an acidic pH. While the effectiveness of each individual treatment method varied, a combined treatment of either HEPES buffer + Tween 20, or direct pH adjustment + Tween 20, consistently produced QEs of 60%-70% and up to 100%, respectively, over a sampling period of one year. The consistency and scalability of this workflow make it a suitable alternative to culture-based ISO methods for detecting Campylobacter spp.

Bioremediation of Industrial Pollutants by Insects Expressing a Fungal Laccase.

Inadequate management of household and industrial wastes poses major challenges to human and environmental health. Advances in synthetic biology may help address these challenges by engineering biological systems to perform new functions such as biomanufacturing of high-value compounds from low-value waste streams and bioremediation of industrial pollutants. The current emphasis on microbial systems for biomanufacturing, which often requires highly preprocessed inputs and sophisticated infrastructure, is not feasible for many waste streams. Furthermore, concerns about transgene biocontainment have limited the release of engineered microbes or plants for bioremediation. Engineering of animals may provide opportunities for utilizing various waste streams that are not suitable for microbial biomanufacturing while effective transgene biocontainment options should enable in situ bioremediation. Here, we engineer the model insect Drosophila melanogaster to express a functional laccase from the fungus Trametes trogii. Laccase-expressing flies reduced concentrations of the endocrine disruptor bisphenol A by more than 50% when present in their growth media. A lyophilized powder prepared from engineered adult flies retained substantial enzymatic activity, degrading more than 90% of bisphenol A and the textile dye indigo carmine in aqueous solutions. Our results demonstrate that transgenic animals may be used to bioremediate environmental contaminants in vivo and serve as novel production platforms for industrial enzymes. These results support further development of insects, and possibly other animals, as bioproduction platforms and their potential use in bioremediation.

Universal Enzyme-Based Field Workflow for Rapid and Sensitive Quantification of Water Pathogens.

A universal filtration and enzyme-based workflow has been established to allow for the rapid and sensitive quantification of leading pathogens Cryptosporidium parvum, Giardia gamblia, Campylobacter jejuni, and Escherichia coli from tap water samples with volumes up to 100 mL, and the potential to scale up to larger volumes. qPCR limits of quantification as low as four oocysts for Cryptosporidium, twelve cysts for Giardia, two cells for C. jejuni, and nineteen cells for E. coli per reaction were achieved. A polycarbonate filter-based sampling method coupled with the prepGEM enzyme-based DNA extraction system created a single-step transfer workflow that required as little as 20 min of incubation time and a 100 µL reaction mix. The quantification via qPCR was performed directly on the prepGEM extract, bypassing time-consuming, labour-intensive conventional culture-based methods. The tap water samples were shown to contain insoluble particles that inhibited detection by reducing the quantification efficiency of a representative pathogen (C. jejuni) to 30-60%. This sample inhibition was effectively removed by an on-filter treatment of 20% (v/v) phosphoric acid wash. Overall, the established workflow was able to achieve quantification efficiencies of 92% and higher for all four leading water pathogens, forming the basis of a rapid, portable, and low-cost solution to water monitoring.

Bioengineering a Light-Responsive Encapsulin Nanoreactor: A Potential Tool for In Vitro Photodynamic Therapy.

Encapsulins, a prokaryotic class of self-assembling protein nanocompartments, are being re-engineered to serve as "nanoreactors" for the augmentation or creation of key biochemical reactions. However, approaches that allow encapsulin nanoreactors to be functionally activated with spatial and temporal precision are lacking. We report the construction of a light-responsive encapsulin nanoreactor for "on demand" production of reactive oxygen species (ROS). Herein, encapsulins were loaded with the fluorescent flavoprotein mini-singlet oxygen generator (miniSOG), a biological photosensitizer that is activated by blue light to generate ROS, primarily singlet oxygen (1O2). We established that the nanocompartments stably encased miniSOG and in response to blue light were able to mediate the photoconversion of molecular oxygen into ROS. Using an in vitro model of lung cancer, we showed that ROS generated by the nanoreactor triggered photosensitized oxidation reactions which exerted a toxic effect on tumor cells, suggesting utility in photodynamic therapy. This encapsulin nanoreactor thus represents a platform for the light-controlled initiation and/or modulation of ROS-driven processes in biomedicine and biotechnology.

Smartphone technology facilitates point-of-care nucleic acid diagnosis: a beginner's guide.

The reliable detection of nucleic acids at low concentrations in clinical samples like blood, urine and saliva, and in food can be achieved by nucleic acid amplification methods. Several portable and hand-held devices have been developed to translate these laboratory-based methods to point-of-care (POC) settings. POC diagnostic devices could potentially play an important role in environmental monitoring, health, and food safety. Use of a smartphone for nucleic acid testing has shown promising progress in endpoint as well as real-time analysis of various disease conditions. The emergence of smartphone-based POC devices together with paper-based sensors, microfluidic chips and digital droplet assays are used currently in many situations to provide quantitative detection of nucleic acid targets. State-of-the-art portable devices are commercially available and rapidly emerging smartphone-based POC devices that allow the performance of laboratory-quality colorimetric, fluorescent and electrochemical detection are described in this review. We present a comprehensive review of smartphone-based POC sensing applications, specifically on microbial diagnostics, assess their performance and propose recommendations for the future.

Molecular tools and applications of Euglena gracilis: From biorefineries to bioremediation.

Euglena gracilis is a promising source of commercially important metabolites such as vitamins, wax esters, paramylon, and amino acids. However, the molecular tools available to create improved Euglena strains are limited compared to other microorganisms that are currently exploited in the biotechnology industry. The complex poly-endosymbiotic nature of the Euglena genome is a major bottleneck for obtaining a complete genome sequence and thus represents a notable shortcoming in gaining molecular information of this organism. Therefore, the studies and applications have been more focused on using the wild-type strain or its variants and optimizing the nutrient composition and cultivation conditions to enhance the production of biomass and valuable metabolites. In addition to producing metabolites, the E. gracilis biorefinery concept also provides means for the production of biofuels and biogas as well as residual biomass for the remediation of industrial and municipal wastewater. Using Euglena for bioremediation of environments contaminated with heavy metals is of special interest due to the strong ability of the organism to accumulate and sequester these compounds. The published draft genome and transcriptome will serve as a basis for further molecular studies of Euglena and provide a guide for the engineering of metabolic pathways of relevance for the already established as well as novel applications.

Alternative carbohydrate pathways - enzymes, functions and engineering.

Metabolic engineering is crucial in the development of production strains for platform chemicals, pharmaceuticals and biomaterials from renewable resources. The central carbon metabolism (CCM) of heterotrophs plays an essential role in the conversion of biomass to the cellular building blocks required for growth. Yet, engineering the CCM ultimately aims toward a maximization of flux toward products of interest. The most abundant dissimilative carbohydrate pathways amongst prokaryotes (and eukaryotes) are the Embden-Meyerhof-Parnas (EMP) and the Entner-Doudoroff (ED) pathways, which build the basics for heterotrophic metabolic chassis strains. Although the EMP is regarded as the textbook example of a carbohydrate pathway owing to its central role in production strains like Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis, it is either modified, complemented or even replaced by alternative carbohydrate pathways in different organisms. The ED pathway also plays key roles in biotechnological relevant bacteria, like Zymomonas mobilis and Pseudomonas putida, and its importance was recently discovered in photoautotrophs and marine microorganisms. In contrast to the EMP, the ED pathway and its variations are not evolutionary optimized for high ATP production and it differs in key principles such as protein cost, energetics and thermodynamics, which can be exploited in the construction of unique metabolic designs. Single ED pathway enzymes and complete ED pathway modules have been used to rewire carbon metabolisms in production strains and for the construction of cell-free enzymatic pathways. This review focuses on the differences of the ED and EMP pathways including their variations and discusses the use of alternative pathway strategies for in vivo and cell-free metabolic engineering.

The Effect of Oligomerization on A Solid-Binding Peptide Binding to Silica-Based Materials.

The bifunctional linker-protein G (LPG) fusion protein comprises a peptide (linker) sequence and a truncated form of Streptococcus strain G148 protein G (protein G). The linker represents a multimeric solid-binding peptide (SBP) comprising 4 × 21-amino acid sequence repeats that display high binding affinity towards silica-based materials. In this study, several truncated derivatives were investigated to determine the effect of the SBP oligomerization on the silica binding function of LPG (for the sake of clarity, LPG will be referred from here on as 4 × LPG). Various biophysical characterization techniques were used to quantify and compare the truncated derivatives against 4 × LPG and protein G without linker (PG). The derivative containing two sequence repeats (2 × LPG) showed minimal binding to silica, while the truncated derivative with only a single sequence (1 × LPG) displayed no binding. The derivative containing three sequence repeats (3 × LPG) was able to bind to silica with a binding affinity of KD = 53.23 ± 4.5 nM, which is 1.5 times lower than that obtained for 4 × LPG under similar experimental conditions. Circular dichroism (CD) spectroscopy and fluorescence spectroscopy studies indicated that the SBP degree of oligomerization has only a small effect on the secondary structure (the linker unravels the beginning of the protein G sequence) and chemical stability of the parent protein G. However, based on quartz crystal microbalance with dissipation monitoring (QCM-D), oligomerization is an important parameter for a strong and stable binding to silica. The replacement of three sequence repeats by a (GGGGS)12 glycine-rich spacer indicated that the overall length rather than the SBP oligomerization mediated the effective binding to silica.

Rapid and specific duplex detection of methicillin-resistant Staphylococcus aureus genes by surface-enhanced Raman spectroscopy.

Methicillin-resistant Staphylococcus aureus (MRSA) is considered to be one of the important hospital-acquired pathogens. MRSA is also commonly associated with hospital-acquired infections and mortality. Quantitative and precise detection of MRSA is essential for rapid diagnosis and subsequent effective disease management strategies. We herein developed a highly specific method for rapid MRSA detection that combines surface-enhanced Raman spectroscopy (SERS) nanotags and polymerase chain reaction (PCR). SERS provided the sensitivity and spectral multiplexing capability while PCR provided the specificity required for the assay. The method was tested by the simultaneous detection of two MRSA specific genes (mecA and femA) amplified from genomic DNA isolated from clinical specimens. Magnetic isolation and rapid duplex detection were performed to obtain a detectable signal down to 104 input copies within 80 min. This demonstrated the potential of the SERS-PCR based approach for the accurate identification of MRSA at an early-diagnosis stage. This study also provides an alternative approach to the existing methods for detecting clinical targets without compromising sensitivity and selectivity, and with minimal handling steps. We thus believe that this approach will find a broad application in clinical applications.

Probing the Role of the Chloroplasts in Heavy Metal Tolerance and Accumulation in Euglena gracilis.

The E. gracilis Zm-strain lacking chloroplasts, characterized in this study, was compared with the earlier assessed wild type Z-strain to explore the role of chloroplasts in heavy metal accumulation and tolerance. Comparison of the minimum inhibitory concentration (MIC) values indicated that both strains tolerated similar concentrations of mercury (Hg) and lead (Pb), but cadmium (Cd) tolerance of the Z-strain was twice that of the Zm-strain. The ability of the Zm-strain to accumulate Hg was higher compared to the Z-strain, indicating the existence of a Hg transportation and accumulation mechanism not depending on the presence of chloroplasts. Transmission electron microscopy (TEM) showed maximum accumulation of Hg in the cytosol of the Zm-strain and highest accumulation of Cd in the chloroplasts of the Z-strain indicating a difference in the ability of the two strains to deposit heavy metals in the cell. The highly abundant heavy metal transporter MTP2 in the Z-strain may have a role in Cd transportation to the chloroplasts. A multidrug resistance-associated protein highly increased in abundance in the Zm-strain could be a potential Hg transporter to either cytosol or mitochondria. Overall, the chloroplasts appear to have major role in the tolerance and accumulation of Cd in E. gracilis.

A novel framework for the cell-free enzymatic production of glucaric acid.

Glucaric acid (GlucA) is a valuable glucose-derived chemical with promising applications as a biodegradable and biocompatible chemical in the manufacturing of plastics, detergents and drugs. Recently, there has been a significant focus on producing GlucA microbially (in vivo) from renewable materials such as glucose, sucrose and myo-inositol. However, these in vivo GlucA production processes generally lack efficiency due to toxicity problems, metabolite competition and suboptimal enzyme ratios. Synthetic biology and accompanying cell-free biocatalysis have been proposed as a viable approach to overcome many of these limitations. However, cell-free biocatalysis faces its own limitations for industrial applications due to high enzyme costs and cofactor consumption. We have constructed a cell-free GlucA pathway and demonstrated a novel framework to overcome limitations of cell-free biocatalysis by i) the combination of both thermostable and mesophilic enzymes, ii) incorporation of a cofactor regeneration system and iii) immobilisation and recycling of the pathway enzymes. The cell-free production of GlucA was achieved from glucose-1-phosphate with a titre of 14.1 ±â€¯0.9 mM (3.0 ±â€¯0.2 g l-1) and a molar yield of 35.2 ±â€¯2.3% using non-immobilised enzymes, and a titre of 8.1 ±â€¯0.2 mM (1.70 ±â€¯0.04 g l-1) and a molar yield of 20.2 ±â€¯0.5% using immobilised enzymes with a total reaction time of 10 h. The resulting productivities (0.30 ±â€¯0.02 g/h/l for free enzymes and 0.170 ±â€¯0.004 g/h/l for immobilised enzymes) are the highest productivities so far reported for glucaric acid production using a synthetic enzyme pathway.

Cell-Free Enzymatic Conversion of Spent Coffee Grounds Into the Platform Chemical Lactic Acid.

The coffee industry produces over 10 billion kg beans per year and generates high amounts of different waste products. Spent coffee grounds (SCG) are an industrially underutilized waste resource, which is rich in the polysaccharide galactomannan, a polysaccharide consisting of a mannose backbone with galactose side groups. Here, we present a cell-free reaction cascade for the conversion of mannose, the most abundant sugar in SCG, into L-lactic acid. The enzymatic conversion is based on a so far unknown oxidative mannose metabolism from Thermoplasma acidophilum and uses a previously characterized mannonate dehydratase to convert mannose into lactic acid via 4 enzymatic reactions. In comparison to known in vivo metabolisms the bioconversion is free of phosphorylated intermediates and cofactors. Assessment of enzymes, adjustment of enzyme loadings, substrate and cofactor concentrations, and buffer ionic strength allowed the identification of crucial reaction parameters and bottlenecks. Moreover, reactions with isotope labeled mannose enabled the monitoring of pathway intermediates and revealed a reverse flux in the conversion process. Finally, 4.4 ± 0.1 mM lactic acid was produced from 14.57 ± 0.7 mM SCG-derived mannose. While the conversion efficiency of the process can be further improved by enzyme engineering, the reaction demonstrates the first multi-enzyme cascade for the bioconversion of SCG.

Functionalized Upconversion Nanoparticles for Targeted Labelling of Bladder Cancer Cells.

Bladder cancer is the ninth most common cancer worldwide. Due to a high risk of recurrence and progression of bladder cancer, every patient needs long-term surveillance, which includes regular cystoscopy, sometimes followed by a biopsy of suspicious lesions or resections of recurring tumours. This study addresses the development of novel biohybrid nanocomplexes representing upconversion nanoparticles (UCNP) coupled to antibodies for photoluminescent (PL) detection of bladder cancer cells. Carrying specific antibodies, these nanoconjugates selectively bind to urothelial carcinoma cells and make them visible by emitting visible PL upon excitation with deeply penetrating near-infrared light. UCNP were coated with a silica layer and linked to anti-Glypican-1 antibody MIL38 via silica-specific solid-binding peptide. Conjugates have been shown to specifically attach to urothelial carcinoma cells with high expression of Glypican-1. This result highlights the potential of produced conjugates and conjugation technology for further studies of their application in the tumour detection and fluorescence-guided resection.

Elucidating the Binding Mechanism of a Novel Silica-Binding Peptide.

Linker-protein G (LPG) is a bifunctional fusion protein composed of a solid-binding peptide (SBP, referred as the "linker") with high affinity to silica-based compounds and a Streptococcus protein G (PG), which binds antibodies. The binding mechanisms of LPG to silica-based materials was studied using different biophysical techniques and compared to that of PG without the linker. LPG displayed high binding affinity to a silica surface (KD = 34.77 ± 11.8 nM), with a vertical orientation, in comparison to parent PG, which exhibited no measurable binding affinity. Incorporation of the linker in the fusion protein, LPG, had no effect on the antibody-binding function of PG, which retained its secondary structure and displayed no alteration of its chemical stability. The LPG system provided a milder, easier, and faster affinity-driven immobilization of antibodies to inorganic surfaces when compared to traditional chemical coupling techniques.

Developing Protein-Based Nanoparticles as Versatile Delivery Systems for Cancer Therapy and Imaging.

In recent years, it has become apparent that cancer nanomedicine's reliance on synthetic nanoparticles as drug delivery systems has resulted in limited clinical outcomes. This is mostly due to a poor understanding of their "bio-nano" interactions. Protein-based nanoparticles (PNPs) are rapidly emerging as versatile vehicles for the delivery of therapeutic and diagnostic agents, offering a potential alternative to synthetic nanoparticles. PNPs are abundant in nature, genetically and chemically modifiable, monodisperse, biocompatible, and biodegradable. To harness their full clinical potential, it is important for PNPs to be accurately designed and engineered. In this review, we outline the recent advancements and applications of PNPs in cancer nanomedicine. We also discuss the future directions for PNP research and what challenges must be overcome to ensure their translation into the clinic.

Bioproducts From Euglena gracilis: Synthesis and Applications.

In recent years, the versatile phototrophic protist Euglena gracilis has emerged as an interesting candidate for application-driven research and commercialisation, as it is an excellent source of dietary protein, pro(vitamins), lipids, and the ß-1,3-glucan paramylon only found in euglenoids. From these, paramylon is already marketed as an immunostimulatory agent in nutraceuticals. Bioproducts from E. gracilis can be produced under various cultivation conditions discussed in this review, and their yields are relatively high when compared with those achieved in microalgal systems. Future challenges include achieving the economy of large-scale cultivation. Recent insights into the complex metabolism of E. gracilis have highlighted unique metabolic pathways, which could provide new leads for product enhancement by genetic modification of the organism. Also, development of molecular tools for strain improvement are emerging rapidly, making E. gracilis a noteworthy challenger for microalgae such as Chlorella spp. and their products currently on the market.

Linker-protein G mediated functionalization of polystyrene-encapsulated upconversion nanoparticles for rapid gene assay using convective PCR.

The authors report on a simplified approach to encapsulate upconversion nanoparticles (UCNPs) in polystyrene spheres by mini-emulsion polymerisation. The resulting particles (PS-UCNP) are hydrophilic, stable and suitable for biomolecular recognition and biosensing applications. Also, a strategy was developed for bioconjugation of antibodies onto the surface of the PS-UCNPs by using the bifunctional fusion protein linker-protein G (LPG). LPG mediates the functionalisation of PS-UCNPs with antibodies against digoxigenin allowing for specific labelling of convective PCR (cPCR) amplicons. Lambda DNA was amplified using cPCR on a heat block for 30 min using the digoxigenin labelled forward and biotin labelled reverse primers. The antibody functionalised PS-UCNPs bind to the digoxigenin end of the cPCR amplicons. Finally, the streptavidin labelled magnetic beads were used to selectively capture the PS-UCNP-labelled cPCR amplicons and the upconversion signal was detected at 537 nm under 980 nm excitation. This sandwich approach enables direct recognition of the target lambda DNA with a detection limit of 103 copies µL-1. The upconversion signal decreased proportionally to the concentration of the lambda DNA with a linear response between 107 and 103 copies of DNA. Graphical abstract Schematic representation of polystyrene-encapsulated upconversion nanoparticles (PS-UCNPs) prepared by mini-emulsion polymerisation. The PS-UCNPs were functionalised with anti-digoxigenin antibody using the fusion protein linker-protein G (LPG). Detection of digoxigenin-labelled amplicons is achieved (a) by using the antibody-functionalised LPG@PS-UCNP labels; (b) magnetic separation, and (c) 980 nm laser light for detection of the green upconversion luminescence peaking at 537 nm.

Mixed-mode liquid chromatography for the rapid analysis of biocatalytic glucaric acid reaction pathways.

Glucaric acid (GlucA) has been identified as one of the top 10 potential bio-based chemicals for replacement of oil-based chemicals. Several synthetic enzyme pathways have been engineered in bacteria and yeast to produce GlucA from glucose and myo-inositol. However, the yields and titres achieved with these systems remain too low for the requirements of a bio-based GlucA industry. A major limitation for the optimisation of GlucA production via synthetic enzymatic pathways are the laborious analytical procedures required to detect the final product (GlucA) and pathway intermediates. We have developed a novel method for the simple and simultaneous analysis of GlucA and pathway intermediates to address this limitation using mixed mode (MM) HILIC and weak anion exchange chromatography (WAX), referred to as MM HILIC/WAX, coupled with RID. Isocratic mobile phase conditions and the sample solvent were optimised for the separation of GlucA, glucose-1-phosphate (G1P), glucose-6-phosphate (G6P), inositol-1-phosphate (I1P), myo-inositol and glucuronic acid (GA). The method showed good repeatability, precision and excellent accuracy with detection and quantitation limits (LOD and LOQ) of 1.5-2 and 577 mM, respectively. The method developed was used for monitoring the enzymatic synthesis of the final step in the GlucA pathway, and showed that GlucA was produced from GA with near 100% conversion and a titre of 9.2 g L-1.