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Annals of Laboratory Medicine Jun 2024Droplet digital (dd)PCR is a new-generation PCR technique with high precision and sensitivity; however, the positive and negative droplets are not always effectively...
BACKGROUND
Droplet digital (dd)PCR is a new-generation PCR technique with high precision and sensitivity; however, the positive and negative droplets are not always effectively separated because of the "rain" phenomenon. We aimed to develop a practical optimization and evaluation process for the ddPCR assay and to apply it to the detection of V600E in fine-needle aspiration (FNA) specimens of thyroid nodules, as an example.
METHODS
We optimized seven ddPCR parameters that can affect "rain." Analytical and clinical performance were analyzed based on histological diagnosis after thyroidectomy using a consecutive prospective series of 242 FNA specimens.
RESULTS
The annealing time and temperature, number of PCR cycles, and primer and probe concentrations were found to be more important considerations for assay optimization than the denaturation time and ramp rate. The limit of blank and 95% limit of detection were 0% and 0.027%, respectively. The sensitivity of ddPCR for histological papillary thyroid carcinoma (PTC) was 82.4% (95% confidence interval [CI], 73.6%-89.2%). The pooled sensitivity of V600E in FNA specimens for histological PTC was 78.6% (95% CI, 75.9%-81.2%, I=60.6%).
CONCLUSIONS
We present a practical approach for optimizing ddPCR parameters that affect the separation of positive and negative droplets to reduce rain. Our approach to optimizing ddPCR parameters can be expanded to general ddPCR assays for specific mutations in clinical laboratories. The highly sensitive ddPCR can compensate for uncertainty in cytological diagnosis by detecting low levels of V600E.
PubMed: 38872331
DOI: 10.3343/alm.2023.0405 -
Food Chemistry Jun 2024This study aimed to explore how pulsed electric field (PEF) treatment affects the structural, physicochemical, and emulsification properties of porcine-derived...
Mechanisms underlying the changes in the structural, physicochemical, and emulsification properties of porcine myofibrillar proteins induced by prolonged pulsed electric field treatment.
This study aimed to explore how pulsed electric field (PEF) treatment affects the structural, physicochemical, and emulsification properties of porcine-derived myofibrillar proteins (MPs). Increasing PEF treatment induced partial polarization and protein unfolding, resulting in notable denaturation that affected both the secondary and tertiary structures. PEF treatment also improved the solubility and emulsification ability of MPs by reducing their pH and surface hydrophobicity. Confocal laser scanning microscopy confirmed the effective adsorption of MPs and PEF-treated MPs at the oil/water interface, resulting in well-fabricated Pickering emulsions. A weak particle network increased the apparent viscosity in short-term PEF-treated Pickering emulsions. Conversely, in emulsions with long-term PEF-treated MP, rheological variables decreased, and dispersion stability increased. These results endorse the potential application of PEF-treated porcine-derived MPs as efficient Pickering stabilizers, offering valuable insights into the creative use of PEF for enhancing high-quality meat products, meeting the increasing demand for clean-label choices.
PubMed: 38870818
DOI: 10.1016/j.foodchem.2024.140024 -
Lab on a Chip Jun 2024Physical properties of blood plasma, such as viscosity, serve as crucial indicators of disease. The inherent capillary effect of paper microchannels, coupled with...
Physical properties of blood plasma, such as viscosity, serve as crucial indicators of disease. The inherent capillary effect of paper microchannels, coupled with minimal sample requirement, stimulated the advancement of paper-based viscometers. This study presents a precise, non-contact optoelectronic system using a microfluidic platform for the measurement of blood plasma viscosity. Microchannels were defined onto the filter paper using an available and inexpensive wax crayon, without the need for conventional wax printing equipment. The time required for the 5 μL sample to pass a specific distance was measured using two pairs of infrared sensors. Subsequently, this data was sent to the microcontroller, which automatically calculated the viscosity. Throughout the measurements, sample temperature was maintained at a constant 37 °C through an integrated heater with automated control. The microfluidic platform successfully processed real samples, yielding viscosity measurements in under three minutes. Evaluation with fetal bovine serum, spiked with varying protein concentrations in both native and denatured states, demonstrated a precision exceeding 96% compared to conventional Ostwald viscometer readings. For human subjects exhibiting pathologies affecting serum and plasma viscosity compared to physiological norms, strong correlations were observed between resultant values and clinical diagnoses. The proposed device aims to replace expensive and complex optical equipment, offering a safer alternative for measuring plasma viscosity. Unlike similar devices, it eliminates the risk of component deformation due to chemical contact or unsafe irradiation.
Topics: Blood Viscosity; Humans; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Cattle; Animals; Point-of-Care Systems; Equipment Design
PubMed: 38869225
DOI: 10.1039/d4lc00211c -
Biometals : An International Journal on... Jun 2024Schiff bases of existing antimicrobial drugs are an area, which is still to be comprehensively explored to improve drug efficiency against consistently resisting...
Schiff bases of existing antimicrobial drugs are an area, which is still to be comprehensively explored to improve drug efficiency against consistently resisting bacterial species. In this study, we have targeted a new and eco-friendly method of condensation reaction that allows the "green synthesis" as well as improved biological efficacy. The transition metal complexes of cefpodoxime with well-enhanced biological activities were synthesized. The condensation reaction product of cefpodoxime and vanillin was further reacted with suitable metal salts of [Mn (II), Cu (II), Fe (II), Zn (II), and Ni (II)] with 1:2 molar ratio (metal: ligand). The characterization of all the products were carried out by using UV-Visible, elemental analyzer, FTIR, H-NMR, ICP-OES, and LC-MS. Electronic data obtained by UV-Visible proved the octahedral geometry of metal complexes. The biological activities Schiff base ligand and its transition metal complexes were tested by using in-vitro anti-bacterial analysis against various Gram-negative, as well as Gram-positive bacterial strains. Proteinase and protein denaturation inhibition assays were utilized to evaluate the products in-vitro anti-inflammatory activities. The in vitro antioxidant activity of the ligand and its complexes was evaluated by utilizing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) in-vitro method. The final results proved metal complexes to be more effective against bacterial microorganisms as compared to respective parent drug as well as their free ligands. Patch Dock, a molecular docking tool, was used to dock complexes 1a-5e with the crystal structure of GlcN-6-P synthase (ID: 1MOQ). According to the docking results, complex 2b exhibited a highest score (8,882; ACE = -580.43 kcal/mol) that is well correlated with a high inhibition as compared to other complexes which corresponds to the antibacterial screening outcomes.
PubMed: 38864936
DOI: 10.1007/s10534-024-00601-5 -
International Journal of Biological... Jun 2024Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high...
Given the severe protein denaturation and self-aggregation during the high-temperature desolubilization, denatured soy meal (DSM) is limited by its low reactivity, high viscosity, and poor water solubility. Preparing low-cost and high-performance adhesives with DSM as the key feedstock is still challenging. Herein, this study reveals a double-enzyme co-activation method targeting DSM with the glycosidic bonds in protein-carbohydrate complexes and partial amide bonds in protein, increasing the protein dispersion index from 10.2 % to 75.1 % improves the reactivity of DSM. The green crosslinker transglutaminase (TGase) constructs a robust adhesive isopeptide bond network with high water-resistant bonding strength comparable to chemical crosslinkers. The adhesive has demonstrated high dry/wet shear strength (2.56 and 0.93 MPa) for plywood. After molecular recombination by enzyme strategy, the adhesive had the proper viscosity, high reactivity, and strong water resistance. This research showcases a novel perspective on developing a DSM-based adhesive and blazes new avenues for changes in protein structural function and adhesive performance.
PubMed: 38862054
DOI: 10.1016/j.ijbiomac.2024.133054 -
Chemistry & Biodiversity Jun 2024Any pathogenic attack, infection, or disease can initiate inflammation. It results in significant adverse consequences like inflammatory bowel disease, rheumatoid...
A Structure-Based Design Strategy with Pyrazole-Pyridine Derivatives Targeting TNFα as Anti-inflammatory Agents: E-pharmacophore, Dynamic Simulation, Synthesis and In Vitro Evaluation.
Any pathogenic attack, infection, or disease can initiate inflammation. It results in significant adverse consequences like inflammatory bowel disease, rheumatoid arthritis, etc. TNFα is one of the major pro-inflammatory cytokines for the progression of inflammation-the present study designed a series of hybrid compounds consisting of the pyrazole-pyridine moiety. Virtual screening was performed utilizing the e-pharmacophore hypothesis with the co-ligand of TNFα, screening, docking, and ADMET study. Induced fit docking, DFT analysis, and molecular dynamic simulation showed that the four best molecules - Dh1- Dh4 - showed crucial interaction with Tyrosine, higher dock scores, and better stability than Diclofenac. Following the synthesis of hit molecules, an in vitro albumin denaturation IC50 of Dh1 was found to be 118.01μM. Further in-depth in vitro and in vivo analyses of these pyrazole-pyridine small compounds may serve as potential space for creating new anti-inflammatory leads.
PubMed: 38861376
DOI: 10.1002/cbdv.202400778 -
Soft Matter Jun 2024Biogenic CaCO formation is regulated by crystallization proteins during crystal growth. Interactions of proteins with nascent mineral surfaces trigger proteins to be...
Biogenic CaCO formation is regulated by crystallization proteins during crystal growth. Interactions of proteins with nascent mineral surfaces trigger proteins to be incorporated into the crystal lattice. As a result of incorporation, these intracrystalline proteins are protected in the lattice, an example of which is ancient eggshell proteins that have persisted in CaCO for thousands of years even under harsh environmental conditions. OC17 is an eggshell protein known to interact with CaCO during eggshell formation during which OC17 becomes incorporated into the lattice. Understanding protein incorporation into CaCO could offer insights into protein stability inside crystals. Here, we study the protection of OC17 in the CaCO lattice. Using thermogravimetric analysis we show that the effect of temperature on intracrystalline proteins of eggshells is negligible below 250 °C. Next, we show that lattice incorporation protects the OC17 structure despite a heat-treatment step that is shown to denature the protein. Because incorporated proteins need to be released from crystals, we verify metal chelation as a safe crystal dissolution method to avoid protein denaturation during reconstitution. Finally, we optimize the recombinant expression of OC17 which could allow engineering OC17 for engineered intracrystalline entrapment studies.
Topics: Calcium Carbonate; Egg Proteins; Crystallization; Animals; Temperature
PubMed: 38860646
DOI: 10.1039/d4sm00371c -
Journal of the Science of Food and... Jun 2024Morden advanced analytical tools offer valuable information into the understanding of molecular mechanism of traditional food processing. Chopping temperature is...
BACKGROUND
Morden advanced analytical tools offer valuable information into the understanding of molecular mechanism of traditional food processing. Chopping temperature is well-known to affect the surimi gel quality of silver carp, but the detailed molecular mechanism is not very clear. In this study, a gel-based proteomics was performed on the extracted surimi proteins under different chopping temperatures (0, 5, 10, and 25 °C) along with other physicochemical characterization of surimi proteins and gels.
RESULTS
With increased chopping temperature, protein extractability (in 3% sodium chloride) generally decreased, while the extracted protein generally exhibited larger surface hydrophobicity, reduced intrinsic fluorescence intensity, lower sulfhydryl content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile of extracted protein showed a clear difference at 25 °C when compared with the other three temperatures, and more protein fragmentation occurred. Proteomic analysis of selected bands indicated that major myofibrillar proteins react differently with chopping temperatures, especially at 25 °C. The selected bands contained a variety of other proteins or their fragments, including adenosine triphosphate (ATP) synthase, glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate isomerase, heat shock protein, parvalbumin, collagen, and so forth. For the surimi gel, water-holding capacity and gel strength generally decreased with increased chopping temperature.
CONCLUSION
Our results suggested that chopping at 0-10 °C is acceptable for the production of silver carp surimi in terms of gel strength and water-holding capacity. However, a chopping temperature near 0 °C led to less protein oxidation and denaturation. The inferior gel quality at 25 °C is linked to a decreased concentration of extracted protein and degradation of major myofibrillar protein, the latter is likely crosslinked with sarcoplasmic proteins. © 2024 Society of Chemical Industry.
PubMed: 38860545
DOI: 10.1002/jsfa.13654 -
Communications Biology Jun 2024In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering...
In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface. Our results demonstrate that the IAAG grid effectively enriches PA-tagged target proteins and overcomes preferred orientation issues. Furthermore, we demonstrate the utility of our IAAG strategy for on-grid purification of low-abundance target complexes from cell lysates, enabling atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification, feasible for high-resolution cryo-EM. This approach holds potential for broader applicability in both cryo-EM and cryo-electron tomography (cryo-ET).
Topics: Cryoelectron Microscopy; Antibodies, Immobilized; Graphite; Humans
PubMed: 38858498
DOI: 10.1038/s42003-024-06406-z -
Protein Expression and Purification Jun 2024Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate,...
Chlamydia trachomatis (CT) is the bacterial pathogen responsible for causing the most common sexually transmitted disease in the United States. This obligate, intracellular Gram-negative bacterium has a type III secretion system (T3SS) to invade host cells. CopN is an important effector, plug protein that mediates early interactions between the host and Chlamydia. CopN is chaperoned by a heterodimer, T3SS chaperone complex containing Scc4 and Scc1. Scc4 is a unique, bifunctional protein that, in addition to its T3SS chaperone activity, acts as an RNA polymerase (RNAP) binding protein. We hypothesized that the two functions occur at different points in CT's developmental cycle with Scc4 acting alone in the early-to-mid stages and the Scc4:Scc1 complex chaperoning CopN in the mid-to-late stages. To study the Scc4:Scc1 complex by NMR, we previously explored various methods of associating Scc4 and Scc1 in vitro to produce the complex with chain-selective isotopic labeling. Though co-expressed Scc4 and Scc1 form a stable complex, the in vitro association studies suggest that partial protein denaturation and/or components in E. coli lysate are necessary to form the stable complex. In this study Scc4 and Scc1 were sequentially expressed in E. coli under the control of different promoters, allowing separate isotopic labeling of each chain and complex formation in vivo. Sequential expression resulted in no or unstable complex formation depending on the culture medium used. These results, taken together with previous in vitro association studies, suggest that Scc4 and Scc1 assemble co-translationally to form the stable Scc4:Scc1 complex in E. coli.
PubMed: 38857716
DOI: 10.1016/j.pep.2024.106532