Search Results (27)

S-adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the polyamine biosynthesis pathway and plays a key role in the synthesis of the polyamines spermidine and spermine, polycationic alkylamines that are present in millimolar levels in mammalian cells. Polyamines are metabolic molecules that are involved in many fundamental processes, including regulation of protein and nucleic acid synthesis, stabilization of chromatin, differentiation, apoptosis, protection from oxidation, and regulation of ion channels. Multiple oncogenic pathways lead to dysregulation of polyamines, making polyamines a potential biomarker for cancer and polyamine biosynthesis a target for therapeutic intervention. This study uses multi-temperature crystallography to probe the structure and dynamics of AdoMetDC by collecting diffraction data at 100 K, 273 K, and 293 K. Differential loop behavior is observed across the collected datasets, with dramatic residue rearrangements. In the loop containing residues 20–28, the ambient temperature datasets show a large motion relative to the cryo structure. In a second loop containing residues 164–174, previous cryo structures do not report ordered positions. This loop is ordered in our 100 K structure, while assuming different conformations in the 273 K and 293 K data. These results further illustrate the usefulness of ambient data collection for understanding the structure and dynamics of proteins, especially in loop regions which are less restrained than protein cores. Full article

Microbial detection in milk is crucial for food safety and quality, as beneficial and harmful microorganisms can affect consumer health and dairy product integrity. Identifying and quantifying these microorganisms helps prevent contamination and spoilage. The study employs advanced molecular techniques to detect and quantify the genomic DNA for the target hydrolytic enzyme coding genes lipA and aprX based on the multi-align sequence conserved region, specific primer pair, and hydrolysis probes designed using the singleplex qPCR and multiplex qPCR. Cultured isolates and artificially contaminated sterilized ultra-high-temperature (UHT) milk were analyzed for their specificity, cross-reactivity, and sensitivity. The finding indicated that strains with lipA and aprX genes were amplified while the other strains were not amplified. This indicated that the designed primer pairs/probes were very specific to the target gene of interest. The specificity of each design primer pair was checked using SYBR Green qPCR using 16 different isolate strains from the milk sample. The quantification specificity of each strain target gene was deemed to be with a mean Ct value for positive pseudomonas strain > 16.98 ± 1.76 (p < 0.0001), non-pseudomonas positive strain ≥ 27.47 ± 1.25 (p < 0.0001), no Ct for the negative control and molecular grade water. The sensitivity limit of detection (LOD) analyzed based on culture broth and milk sample was >10⁵ and >10⁴ in PCR amplification while it was >10⁴ and >10³ in real-time qPCR, respectively. At the same time, the correlation regression coefficient of the standard curve based on the pure culture cell DNA as the DNA concentration serially diluted (20 ng/µL to 0.0002 ng/µL) was obtained in multiplex without interference and cross-reactivity, yielding R² ≥ 0.9908 slope (−3.2591) and intercepting with a value of 37, where the efficiency reached the level of 95–102% sensitivity reached up to 0.0002 ng/µL concentration of DNA, and sensitivity of microbial load was up to 1.2 × 10² CFU/mL. Therefore, multiplex TaqMan qPCR simultaneous amplification was considered the best method developed for the detection of the lipA and aprX genes in a single tube. This will result in developing future simultaneous (three- to four-gene) detection of spoilage psychrotrophic bacteria in raw milk. Full article

Vibrio parahaemolyticus is a foodborne pathogen commonly associated with the consumption of contaminated seafood, particularly oysters. While PCR and real-time PCR are widely used to detect its pathogenicity through tdh and trh gene detection, these methods may not be practical in resource-limited settings such as field environments. To address this limitation, a rapid, sensitive, and specific duplex detection method was developed using the multienzyme isothermal rapid amplification (MIRA) assay in combination with lateral flow dipstick (LFD) technology. The assay utilized specific primer sets and probes to simultaneously amplify tdh and trh fragments tagged with 3′-FAM and 5′-Digoxigenin or Biotin during MIRA amplification, enabling the detection via respective antibody capture on the LFD strip. This duplex MIRA-LFD assay demonstrated a detection limit of 100 fg of DNA, 300 CFU/reaction for bacterial culture, and 3000 CFU/reaction for seeded oyster samples at 40 °C within 20 min. Notably, the assay exhibited no cross-reactivity with nine other Vibrio species or 18 foodborne pathogens, confirming its high specificity. Due to its simplicity, rapid turnaround time, and high sensitivity, this duplex MIRA-LFD assay offers a valuable tool for the surveillance of V. parahaemolyticus pathogenicity, aiding in public health protection and supporting the local seafood industry. Full article

Insulin-like growth factor-1 (IGF-1) is a regulatory factor closely associated with diabetes, obesity, and breast cancer, and it also acts as one of the most abundant growth factors in bovine colostrum. Current methods generally have the problem of low sensitivity, a time-consuming nature, and low stability, which makes it difficult to crack down on the false advertising of IGF-1 content in dairy products. In this work, an ultrasensitive fluorescent enzyme-linked immunosorbent assay (ELISA) is proposed, where the antibody and the target are combined in the form of a “sandwich” to ensure the accuracy and specificity of the assay. IGF-1 is quantified based on an effective hydrogen peroxide (H₂O₂) probe with 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) as the fluorogenic substrate. The proposed fluorescent sandwich ELISA has a low limit of detection (LOD) of 77.29 pg/mL, fast experimental process within 1 h, and stable signal of 1 h. Furthermore, multi-step pretreatment methods for bovine colostrum powders are established to remove interfering substances, including fat, casein, and binding proteins, achieving the accurate and specific detection of IGF-1. IGF-1 recovery studies on treated bovine colostrum powders exhibit good recovery rates ranging from 91.71% to 102.32%, which proves the feasibility of detecting IGF-1 in real bovine colostrum. Full article

Understanding brain function requires advanced neural probes to monitor electrical and chemical signaling across multiple timescales and brain regions. Microelectrode arrays (MEAs) are widely used to record neurophysiological activity across various depths and brain regions, providing single-unit resolution for extended periods. Recent advancements in flexible MEAs, built on micrometer-thick polymer substrates, have improved integration with brain tissue by mimicking the brain’s soft nature, reducing mechanical trauma and inflammation. These flexible, subcellular-scale MEAs can record stable neural signals for months, making them ideal for long-term studies. In addition to electrical recording, MEAs have been functionalized for electrochemical neurotransmitter detection. Electroactive neurotransmitters, such as dopamine, serotonin, and adenosine, can be directly measured via electrochemical methods, particularly on carbon-based surfaces. For non-electroactive neurotransmitters like acetylcholine, glutamate, and γ-aminobutyric acid, alternative strategies, such as enzyme immobilization and aptamer-based recognition, are employed to generate electrochemical signals. This review highlights recent developments in flexible MEA fabrication and functionalization to achieve both electrochemical and electrophysiological recordings, minimizing sensor fowling and brain damage when implanted long-term. It covers multi-time scale neurotransmitter detection, development of conducting polymer and nanomaterial composite coatings to enhance sensitivity, incorporation of enzyme and aptamer-based recognition methods, and the integration of carbon electrodes on flexible MEAs. Finally, it summarizes strategies to acquire electrochemical and electrophysiological measurements from the same device. Full article

Cellular senescence is a recently emerged research topic in modern biology. Often described as a double-edged sword, it encompasses numerous essential biological processes, including beneficial effects such as wound healing and embryonic development, as well as detrimental contributions to chronic inflammation and tumor development. Consequently, there is an increasing need to unravel the intricate networks of senescence and develop reliable detection methods to distinguish it from related phenomena. To address these challenges, a variety of detection methods have been developed. In particular, small-molecule fluorescent probes offer distinct advantages such as suitability for real-time live cell monitoring and in vivo imaging, superior tunable properties, and versatile applications. In this review, we explored recent advancements in the development of small-molecule fluorescent probes toward monitoring cellular senescence by targeting various senescence-related biological phenomena. These phenomena include the upregulation of senescence-associated enzymes, perturbation of the subcellular environment, and increased endogenous ROS levels. Moreover, multi-senescence biomarker-targeting approaches are also discussed to improve their sensitivities and specificities for the detection of cellular senescence. With recent advances in senescence probe development, current challenges in this field are also discussed to facilitate further progress. Full article

Organic fluorescent probes have attracted attention for bioimaging due to their advantages, including high sensitivity, biocompatibility, and multi-functionality. However, some limitations related to low signal-to-background ratio and false positive and negative signals make them difficult for in situ target detection. Recently, organelle targeting self-assembled fluorescent probes have been studied to meet this demand. Most of the dye molecules suffer from a quenching effect, but, specifically, some dyes like Pyrene, Near-Infrared (NIR), Nitrobenzoxadiazole (NBD), Fluorescein isothiocyanate (FITC), Naphthalenediimides (NDI), and Aggregation induced emission (AIE) show unique characteristics when they undergo self-assembly or aggregation. Therefore, in this review, we classified the molecules according to the dye type and provided an overview of the organelle-targeting strategy with an emphasis on the construction of fluorescent nanostructures within complex cellular environments. Results demonstrated that fluorescent probes effectively target and localized inside the organelles (mitochondria, lysosome, and golgi body) and undergo self-assembly to form various nanostructures that possess bio-functionality with long retention time, organelles membrane disruption/ROS generation/enzyme activity suppression ability, and enhanced photodynamic properties for anticancer treatment. Furthermore, we systematically discussed the challenges that remain to be resolved for the high performance of these probes and mentioned some of the future directions for the design of molecules. Full article

Their unique physicochemical properties and multi-enzymatic activity make CeO₂ nanoparticles (CeO₂ NPs) the most promising active component of the next generation of theranostic drugs. When doped with gadolinium ions, CeO₂ NPs constitute a new type of contrast agent for magnetic resonance imaging, possessing improved biocatalytic properties and a high level of biocompatibility. The present study is focused on an in-depth analysis of the enzyme-like properties of gadolinium-doped CeO₂ NPs (CeO₂:Gd NPs) and their antioxidant activity against superoxide anion radicals, hydrogen peroxide, and alkylperoxyl radicals. Using an anion-exchange method, CeO₂:Gd NPs (~5 nm) with various Gd-doping levels (10 mol.% or 20 mol.%) were synthesized. The radical-scavenging properties and biomimetic activities (namely SOD- and peroxidase-like activities) of CeO₂:Gd NPs were assessed using a chemiluminescent method with selective chemical probes: luminol, lucigenin, and L-012 (a highly sensitive luminol analogue). In particular, gadolinium doping has been shown to enhance the radical-scavenging properties of CeO₂ NPs. Unexpectedly, both bare CeO₂ NPs and CeO₂:Gd NPs did not exhibit SOD-like activity, acting as pro-oxidants and contributing to the generation of reactive oxygen species. Gadolinium doping caused an increase in the pro-oxidant properties of nanoscale CeO₂. At the same time, CeO₂:Gd NPs did not significantly inhibit the intrinsic activity of the natural enzyme superoxide dismutase, and CeO₂:Gd NPs conjugated with SOD demonstrated SOD-like activity. In contrast to SOD-like properties, peroxidase-like activity was observed for both bare CeO₂ NPs and CeO₂:Gd NPs. This type of enzyme-like activity was found to be pH-dependent. In a neutral medium (pH = 7.4), nanoscale CeO₂ acted as a prooxidant enzyme (peroxidase), while in an alkaline medium (pH = 8.6), it lost its catalytic properties; thus, it cannot be regarded as a nanozyme. Both gadolinium doping and conjugation with a natural enzyme were shown to modulate the interaction of CeO₂ NPs with the key components of redox homeostasis. Full article

Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are time-consuming and require sophisticated equipment. In this study, an assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) was developed for the efficient and rapid detection of DHBV. The primary reaction condition of the MIRA assay for DHBV detection was 10 min at 38 °C without a temperature cycler. Combined with the LFD assay, the complete procedure of the newly developed MIRA assay for DHBV detection required only 15 min, which is about one-fourth of the reaction time for routine polymerase chain reaction assay. And electrophoresis and gel imaging equipment were not required for detection and to read the results. Furthermore, the detection limit of MIRA was 45.6 copies per reaction, which is approximately 10 times lower than that of a routine polymerase chain reaction assay. The primer set and probe had much simpler designs than loop-mediated isothermal amplification, and they were only specific to DHBV, with no cross-reactivity with duck hepatitis A virus subtype 1 and duck hepatitis A virus subtype 3, goose parvovirus, duck enteritis virus, duck circovirus, or Riemerella anatipestifer. In this study, we offer a simple, fast, and accurate assay method to identify DHBV in clinical serum samples of ducks and geese, which would be suitable for widespread application in field clinics. Full article

Colostrum, the first milk produced by mammals, is rich in various bioactive components that provide numerous health benefits to newborns, such as growth factors, hormones, immunoglobulins, cytokines, and enzymes. Topical application of bovine or equine colostrum has been found to improve regeneration, accelerate cutaneous wound healing, and have moisturizing, protective, and anti-aging properties. The aim of this study was to examine the effect of a cosmetic preparation containing sheep colostrum on skin with signs of aging in mature women. Fifty-two women, aged 40–70, were randomized into two groups to receive either colostrum or placebo cream. The participants applied the cream for eight weeks. Skin hydration, TEWL, sebum, erythema, and tone were measured using a standardized Courage + Khazaka electronic GmbH Multi Probe Adapter; skin elasticity was measured with a cutometer, and images were taken by FotoMedicus. The treatment increased skin moisture, reduced TEWL, and improved skin firmness. These findings were confirmed by the subjective survey. The participants reported, inter alia, improved skin softness and less redness and hypersensitivity. Sheep colostrum cream was more effective at improving skin conditions than placebo cream. Colostrum creams can improve certain aspects of skin quality, especially the hydrolipid barrier, and overall rejuvenation. Full article

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an N^ε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression. Full article

Contamination of deoxynivalenol (DON) in grains has attracted widespread concern. It is urgently needed to develop a highly sensitive and robust assay for DON high-throughput screening. Antibody against DON was assembled on the surface of immunomagnetic beads orientationally by the aid of Protein G. AuNPs were obtained under the scaffolding of poly(amidoamine) dendrimer (PAMAM). DON-horseradish peroxidase (HRP) was combined on the periphery of AuNPs/PAMAM by a covalent link to develop DON-HRP/AuNPs/PAMAM. Magnetic immunoassay based on DON-HRP/AuNPs/PAMAM was optimized and that based on DON-HRP/AuNPs and DON-HRP was adopted as comparison. The limits of detection (LODs) were 0.447 ng/mL, 0.127 ng/mL and 0.035 ng/mL for magnetic immunoassays based on DON-HRP, DON-HRP/Au and DON-HRP/Au/PAMAM, respectively. Magnetic immunoassay based on DON-HRP/AuNPs/PAMAM displayed higher specificity towards DON and was utilized to analyze grain samples. The recovery for the spiked DON in grain samples was 90.8–116.2% and the method presented a good correlation with UPLC/MS. It was found that the concentration of DON was in the range of ND-3.76 ng/mL. This method allows the integration of dendrimer–inorganic NPs with signal amplification properties for applications in food safety analysis. Full article

Developing models able to predict interactions between drugs and enzymes is a primary goal in computational biology since these models may be used for predicting both new active drugs and the interactions between known drugs on untested targets. With the compilation of a large dataset of drug–enzyme pairs (62,524), we recognized a unique opportunity to attempt to build a novel multi-target machine learning (MTML) quantitative structure-activity relationship (QSAR) model for probing interactions among different drugs and enzyme targets. To this end, this paper presents an MTML-QSAR model based on using the features of topological drugs together with the artificial neural network (ANN) multi-layer perceptron (MLP). Validation of the final best model found was carried out by internal cross-validation statistics and other relevant diagnostic statistical parameters. The overall accuracy of the derived model was found to be higher than 96%. Finally, to maximize the diffusion of this model, a public and accessible tool has been developed to allow users to perform their own predictions. The developed web-based tool is public accessible and can be downloaded as free open-source software. Full article

Polymers are well-recognized carriers useful for delivering therapeutic drug and imaging probes to the target specified in the defined pathophysiological site. The functional drug molecules and imaging agents were chemically attached or physically loaded in the carrier polymer matrix via cleavable spacers. Using appropriate targeting moieties, these polymeric carriers (PCs) loaded with functional molecules were designed to realize target-specific delivery at the cellular level. The biodistribution of these carriers can be tracked using imaging agents with suitable imaging techniques. The drug molecules can be released by cleaving the spacers either by endogenous stimuli (e.g., pH, redox species, glucose level and enzymes) at the targeted physiological site or exogenous stimuli (e.g., light, electrical pulses, ultrasound and magnetism). Recently, two-photon absorption (2PA)-mediated drug delivery and imaging has gained significant attention because TPA from near-infrared light (700–950 nm, NIR) renders light energy similar to the one-photon absorption from ultraviolet (UV) light. NIR has been considered biologically safe unlike UV, which is harmful to soft tissues, cells and blood vessels. In addition to the heat and reactive oxygen species generating capability of 2PA molecules, 2PA-functionalized PCs were also found to be useful for treating diseases such as cancer by photothermal and photodynamic therapies. Herein, insights attained towards the design, synthesis and biomedical applications of 2PA-activated PCs are reviewed. In particular, specific focus is provided to the imaging and drug delivery applications with a special emphasis on multi-responsive platforms. Full article

The outer mitochondrial membrane (OMM) is involved in multiple cellular functions such as apoptosis, inflammation and signaling via its membrane-associated and -embedded proteins. Despite the central role of the OMM in these vital phenomena, the structure and dynamics of the membrane have regularly been investigated in silico using simple two-component models. Accordingly, the aim was to generate the realistic multi-component model of the OMM and inspect its properties using atomistic molecular dynamics (MD) simulations. All major lipid components, phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), were included in the probed OMM models. Because increased levels of anionic PS lipids have potential effects on schizophrenia and, more specifically, on monoamine oxidase B enzyme activity, the effect of varying the PS concentration was explored. The MD simulations indicate that the complex membrane lipid composition (MLC) behavior is notably different from the two-component PC-PE model. The MLC changes caused relatively minor effects on the membrane structural properties such as membrane thickness or area per lipid; however, notable effects could be seen with the dynamical parameters at the water-membrane interface. Increase of PS levels appears to slow down lateral diffusion of all lipids and, in general, the presence of anionic lipids reduced hydration and slowed down the PE headgroup rotation. In addition, sodium ions could neutralize the membrane surface, when PI was the main anionic component; however, a similar effect was not seen for high PS levels. Based on these results, it is advisable for future studies on the OMM and its protein or ligand partners, especially when wanting to replicate the correct properties on the water-membrane interface, to use models that are sufficiently complex, containing anionic lipid types, PI in particular. Full article