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Analytical Methods : Advancing Methods... May 2024This work describes an analytical procedure, single particle-inductively coupled plasma-time-of-flight-mass spectrometry (SP-ICP-TOF-MS), that was developed to determine...
This work describes an analytical procedure, single particle-inductively coupled plasma-time-of-flight-mass spectrometry (SP-ICP-TOF-MS), that was developed to determine the platinum binding efficiency of protein-coated magnetic microparticles. SP-ICP-TOF-MS is advantageous due to its ability to quasi-simultaneously detect all nuclides (Li-Pu), allowing for both platinum and iron (composition of magnetic microparticles) to be measured concurrently. This method subsequently allows for the differentiation between bound and unbound platinum. The 1 μm magnetic microparticles were fully characterized for their iron concentration, particle concentration, and trace element composition by bulk digestion-ICP-MS and SP-ICP-TOF-MS. The results of both approaches agreed with the certificate values. Using the single particle methodology the platinum loading was quantified to be to 0.18 ± 0.02 fg per particle and 0.32 ± 0.02 fg per particle, for the streptavidin-coated and azurin-coated microparticles, respectively. Both streptavidin-coated and the azurin-coated microparticles had a particle-platinum association of >65%. Platinum bound samples were also analyzed bulk digestion-based ICP-MS. The bulk ICP-MS results overestimated platinum loading due to free platinum in the samples. This highlights the importance of single particle analysis for a closer inspection of platinum binding performance. The SP-ICP-TOF-MS approach offers advantages over typical bulk digestion methods by eliminating laborious sample preparation, enabling differentiation between bound/unbound platinum in a solution, and quantification of platinum on a particle-by-particle basis. The procedure presented here enables quantification of metal content per particle, which could be broadly implemented for other single particle applications.
Topics: Platinum; Mass Spectrometry; Microspheres; Iron; Streptavidin; Particle Size; Magnetite Nanoparticles
PubMed: 38639200
DOI: 10.1039/d4ay00268g -
Clinical Laboratory May 2024The goal was to improve the clinical cognition of Ph-positive mixed phenotype acute leukemia and avoid misdiagnosis or delayed diagnosis.
BACKGROUND
The goal was to improve the clinical cognition of Ph-positive mixed phenotype acute leukemia and avoid misdiagnosis or delayed diagnosis.
METHODS
The clinical manifestations and laboratory results (bone marrow cell morphology, multiparameter flow cytometry, and cytogenetics) of a case of Ph-positive mixed phenotype acute leukemia were analyzed, and related literature was reviewed.
RESULTS
Blood routine: WBC 386.35 x 109/L, HGB 117.00 g/L, PLT 31 x 109/L; 80% of the original cells can be seen by artificial classification. Morphological examination of bone marrow cells showed that the proliferation of nucleated cells was obviously active, and the original cells accounted for 76%. The size of the original cells was somewhat uniform, most of the cells had less mass, were stained light grayish blue, the cytoplasm particles were not obvious, the nuclei were mostly round or quasi-round, some of them showed distortion and nuclear notch, and the chromatin was coarse. Some of the cells were rich in mass, small azurin granules were seen, the nuclei were regular, most of them were round, the chromatin was fine, the myeloperoxidase and esterase staining were negative, the eosinophils accounted for 2.5%, and the basophils accounted for 0.5%. Flow cytometry immunotyping: Two groups of abnormal cells were seen in the bone marrow. 1. A group included 12.32% of nuclear cells and showed abnormal myeloid primitive cell phenotype. Main expression: CD117, CD34, CD38, HLA-DR, CD33, CD64, CD123, weak expression: CD13, CD19. 2. The other group included 45.61% of the nuclear cells and had a B-lymphoblastic phenotype. Main expression: CD34, CD38, HLA-DR, CD123, CD19, CD10, CD9, cCD79a, TDT, weak expression of CD13, CD22. Mixed phenotype acute leukemia (M/B) immunophenotype was considered. Chromosome: 46,XY,t(9; 22)(q34;q11.2) [20]. BCR-ABL (P210) fusion gene was positive.
CONCLUSIONS
Mixed phenotype acute leukemia (MPAL) is a rare type of malignant hematologic disease. Its diagnosis is based on the comprehensive evaluation of bone marrow cell morphology, immunophenotype, molecular and cytogenetic features.
Topics: Humans; Phenotype; Flow Cytometry; Male; Immunophenotyping; Bone Marrow Cells; Philadelphia Chromosome; Leukemia, Biphenotypic, Acute; Leukemia; Adult; Female; Middle Aged
PubMed: 38747916
DOI: 10.7754/Clin.Lab.2023.231220 -
Infection, Genetics and Evolution :... Aug 2023Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it...
Reverse vaccinology approaches to introduce promising immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae: Thinking outside the box in current prevention and treatment.
Gonorrhea is an urgent antimicrobial resistance threat and its therapeutic options are continuously getting restricted. Moreover, no vaccine has been approved against it so far. Hence, the present study aimed to introduce novel immunogenic and drug targets against antibiotic-resistant Neisseria gonorrhoeae strains. In the first step, the core proteins of 79 complete genomes of N. gonorrhoeae were retrieved. Next, the surface-exposed proteins were evaluated from different aspects such as antigenicity, allergenicity, conservancy, and B-cell and T-cell epitopes to introduce promising immunogenic candidates. Then, the interactions with human Toll-like receptors (TLR-1, 2, and 4), and immunoreactivity to elicit humoral and cellular immune responses were simulated. On the other hand, to identify novel broad-spectrum drug targets, the cytoplasmic and essential proteins were detected. Then, the N. gonorrhoeae metabolome-specific proteins were compared to the drug targets of the DrugBank, and novel drug targets were retrieved. Finally, the protein data bank (PDB) file availability and prevalence among the ESKAPE group and common sexually transmitted infection (STI) agents were assessed. Our analyses resulted in the recognition of ten novel and putative immunogenic targets including murein transglycosylase A, PBP1A, Opa, NlpD, Azurin, MtrE, RmpM, LptD, NspA, and TamA. Moreover, four potential and broad-spectrum drug targets were identified including UMP kinase, GlyQ, HU family DNA-binding protein, and IF-1. Some of the shortlisted immunogenic and drug targets have confirmed roles in adhesion, immune evasion, and antibiotic resistance that can induce bactericidal antibodies. Other immunogenic and drug targets might be associated with the virulence of N. gonorrhoeae as well. Thus, further experimental studies and site-directed mutations are recommended to investigate the role of potential vaccine and drug targets in the pathogenesis of N. gonorrhoeae. It seems that the efforts for proposing novel vaccines and drug targets appear to be paving the way for a prevention-treatment strategy against this bacterium. Additionally, a combination of bactericidal monoclonal antibodies and antibiotics is a promising approach to curing N. gonorrhoeae.
Topics: Humans; Neisseria gonorrhoeae; Anti-Bacterial Agents; Vaccinology; Gonorrhea; Membrane Proteins
PubMed: 37225067
DOI: 10.1016/j.meegid.2023.105449 -
Physical Chemistry Chemical Physics :... Oct 2023Polarizability is a fundamental property of all molecular systems describing the deformation of the molecular electronic density in response to an applied electric...
Comment on "Applicability of perturbed matrix method for charge transfer studies at bio/metallic interfaces: a case of azurin" by O. Kontkanen, D. Biriukov and Z. Futera, , 2023, , 12479.
Polarizability is a fundamental property of all molecular systems describing the deformation of the molecular electronic density in response to an applied electric field. The question of whether polarizability of the active site needs to be included in theories of enzymatic activity remains open. Hybrid quantum mechanical/molecular mechanical calculations are hampered by difficulties faced by many quantum-chemistry algorithms to provide sufficiently accurate estimates of the anisotropic second-rank tensor of molecular polarizability. In this Comment, we provide general theoretical arguments for the values of polarizability of the quantum region or a molecule which have to be reproduced by electronic structure calculations.
PubMed: 37782532
DOI: 10.1039/d3cp03178k -
Cell Reports. Medicine May 2024Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate...
Bacteria-based therapies are powerful strategies for cancer therapy, yet their clinical application is limited by a lack of tunable genetic switches to safely regulate the local expression and release of therapeutic cargoes. Rapid advances in remote-control technologies have enabled precise control of biological processes in time and space. We developed therapeutically active engineered bacteria mediated by a sono-activatable integrated gene circuit based on the thermosensitive transcriptional repressor TlpA. Through promoter engineering and ribosome binding site screening, we achieved ultrasound (US)-induced protein expression and secretion in engineered bacteria with minimal noise and high induction efficiency. Specifically, delivered either intratumorally or intravenously, engineered bacteria colonizing tumors suppressed tumor growth through US-irradiation-induced release of the apoptotic protein azurin and an immune checkpoint inhibitor, a nanobody targeting programmed death-ligand 1, in different tumor mouse models. Beyond developing safe and high-performance designer bacteria for tumor therapy, our study illustrates a sonogenetics-controlled therapeutic platform that can be harnessed for bacteria-based precision medicine.
Topics: Animals; Mice; Humans; Neoplasms; Disease Models, Animal; Cell Line, Tumor; Female; B7-H1 Antigen; Immune Checkpoint Inhibitors; Escherichia coli
PubMed: 38608697
DOI: 10.1016/j.xcrm.2024.101513 -
The Journal of Physical Chemistry. B Feb 2024Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such...
Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a β-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the β-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the S-S bonded and S-S nonbonded () forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for azurin compared to that of the form (420 K). As observed in the case of azurin, the unfolding process of the form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual β-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.
Topics: Azurin; Copper; Metalloproteins; Disulfides; Temperature; Ions; Protein Folding
PubMed: 38236012
DOI: 10.1021/acs.jpcb.3c07089 -
Molecular Pharmaceutics Dec 2023Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a...
Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein hisEGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the binding study, where the analogue of ephrinA, hisEGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The hisEGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.
Topics: Male; Humans; Receptors, Eph Family; Azurin; Receptor, EphA6; Precision Medicine; Prostatic Neoplasms; Ephrins
PubMed: 37906960
DOI: 10.1021/acs.molpharmaceut.3c00387 -
Biomacromolecules Jan 2024A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is...
A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is designed, cloned, expressed, and purified. A simple method of inverse transition cycle (ITC) is employed to purify the fusion protein azurin-ELP diblock copolymer (d-bc). The molecular weight of the azurin-ELP fusion protein is ∼32 kDa. Further, its self-assembly properties are investigated. Interestingly, the engineered azurin-ELP d-bc in response to increasing temperature shows a dual-step phase separation into biofunctional nanostructures. Around the physiological temperature, azurin-ELP d-bc forms stable coacervates, which is dependent on the concentration and time of incubation. These coacervates are formed below the lower critical solubility temperature (LCST) of the ELP block at physiological temperature. Above LCST, i.e., 50-55°C, micelles of size ranging from 25 to 30 nm are formed. The cytotoxicity of azurin-ELP d-bc depends on the size of the coacervates formed and their cellular uptake at physiological temperature. Further, MTT assay of azurin-ELP d-bc in the cross-linked micelles prepared ex situ shows > six times higher killing of LNCaP cells than the unimeric form of azurin-ELP at 5 μM concentration. The flow cytometric results of these micelles at 20 μM concentration show ∼97% LNCaP cells in the apoptotic phase. Thus, azurin-ELP cross-linked micelles have enhanced potential for anticancer therapy due to their higher avidity.
Topics: Humans; Male; Elastin-Like Polypeptides; Micelles; Azurin; Peptides; Elastin; Prostatic Neoplasms
PubMed: 38047916
DOI: 10.1021/acs.biomac.3c01125 -
Proteins Mar 2024
PubMed: 37881118
DOI: 10.1002/prot.26624 -
Journal of Inorganic Biochemistry May 2024Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to...
Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.
Topics: Copper; Histidine; Binding Sites; Methionine; Anti-Infective Agents; Ions; Azurin
PubMed: 38364337
DOI: 10.1016/j.jinorgbio.2024.112503