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Frontiers in Microbiology 2024species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and...
INTRODUCTION
species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of , a model organism that infects mice, in the context of its copper sensing and tolerance.
METHODS AND RESULTS
The query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in , significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of exposed to various CuSO concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO concentration probed (100 μM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies - homology-based annotation, SEC-ICP-MS/IMAC/LC-MS/MS, and RNA-seq - yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods.
DISCUSSION
The insights contribute to the broader knowledge of copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of pathogenicity and copper stress survival should consider the entire spectrum of identified genes.
PubMed: 38812685
DOI: 10.3389/fmicb.2024.1383737 -
Frontiers in Bioscience (Landmark... May 2024Human immunodeficiency virus (HIV) infection is associated with pronounced oxidative stress, leading to the development of various virus-associated pathologies. A wealth...
BACKGROUND
Human immunodeficiency virus (HIV) infection is associated with pronounced oxidative stress, leading to the development of various virus-associated pathologies. A wealth of evidence suggests that, along with canonical enzymes of reactive oxygen species regulation, human blood contains antibodies with peroxidase, superoxide dismutase, and catalase activities. Here we show that the catalase activity of IgGs and their κκ-IgG, λλ-IgG, and κλ-IgG subfractions of HIV-infected individuals is significantly different compared to the healthy donors.
METHODS
Protein G-Sepharose sorbent was used to resolve IgG from blood of healthy donors and HIV-infected patients by affinity chromatography. Subfractions of κκ-IgG, λλ-IgG, and κλ-IgG were separated from IgGs samples of each group by affinity chromatography on sorbents containing immobilized antibodies to κ or λ light human chains. The IgG catalase activity level was measured spectrophotometrically by evaluating the decrease in optical density (A240) due to hydrogen peroxide decomposition.
RESULTS
The relative catalase activity of antibodies from HIV-infected patients ( = (1.41 ± 0.92) × 103 min-1, 95% CI: [1.01-1.81]) was statistically significant, 1.6 times higher ( = 0.014) compared to apparently healthy donors ((0.86 ± 0.49) × 103, 95% CI: [0.69-1.03]). The activity level of κκ-IgG HIV-infected patients ((0.44 ± 0.04) × 103 min-1) was 1.4 times higher than that of λλ-IgGs ((0.31 ± 0.025) × 103 min-1); the opposite was observed for κκ-IgGs from apparently healthy donors, which activity ((0.17 ± 0.015) × 103 min-1) was 3.1 times lower compared to λλ-IgGs ((0.53 ± 0.045) × 103 min-1).
CONCLUSIONS
Thus, the data obtained may indicate that IgG with increased catalase activity may prevent harmful processes arising from oxidative stress in HIV-infected patients, acting as an additional natural molecular mechanism of regulation of hydrogen peroxide level.
Topics: Humans; Catalase; Immunoglobulin G; HIV Infections; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Adult; Case-Control Studies; Male; Female; Chromatography, Affinity; Oxidative Stress; Middle Aged
PubMed: 38812328
DOI: 10.31083/j.fbl2905191 -
Microorganisms Apr 2024Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, morphologically resembles ,...
Malaria is one of the most prevalent diseases worldwide with high incidence and mortality. Among the five species that can infect humans, morphologically resembles , resulting in misidentification and confusion in diagnosis, and is responsible for malarial disease relapse due to the formation of hypnozoites. receives relatively less attention compared to other major parasites, such as and , primarily due to its lower pathogenicity, mortality rates, and prevalence rates. To efficiently produce lactate dehydrogenase (LDH), a major target for diagnosing malaria, this study used three strains, BL21(DE3), BL21(DE3)pLysS, and Rosetta(DE3), commonly used for recombinant protein production. These strains were characterized to select the optimal strain for LDH (PoLDH) production. Gene cloning for recombinant PoLDH production and transformation of the three strains for protein expression were performed. The optimal PoLDH overexpression and washing buffer conditions in nickel-based affinity chromatography were established to ensure high-purity PoLDH. The yields of PoLDH expressed by the three strains were as follows: BL21(DE3), 7.6 mg/L; BL21(DE3)pLysS, 7.4 mg/L; and Rosetta(DE3), 9.5 mg/L. These findings are expected to be highly useful for PoLDH-specific diagnosis and development of antimalarial therapeutics.
PubMed: 38792706
DOI: 10.3390/microorganisms12050876 -
Molecules (Basel, Switzerland) May 2024This study investigates the chemical composition of the essential oil obtained from the leaves of (Mart.) R.E.Fr (Annonaceae), examining its effectiveness in combating...
This study investigates the chemical composition of the essential oil obtained from the leaves of (Mart.) R.E.Fr (Annonaceae), examining its effectiveness in combating both the larvae and adult forms of mosquitoes. Additionally, for a deeper understanding of the insecticidal activity, toxicity properties and molecular docking calculations were conducted using the main compounds of this essential oil. GC/MS analysis revealed the presence of 26 constituents, representing 95.2% of the essential oil, with the major components identified as the sesquiterpenes -selinene, -selinene, and -elemene. Larvicidal assays demonstrated potent activity of this essential oil with significant LC values of 40.8 and 39.4 μg/mL at 24 and 48 h, respectively. Adulticidal assessments highlighted strong efficacy with LC of 12.5 µg/mL. Molecular docking analysis identified optimal interaction activities of -selinene and -selinene with key proteins. The in silico studies comparing synthetic insecticides with the major sesquiterpenes of the essential oil revealed that -selinene exhibited a significantly higher binding affinity compared to the other two sesquiterpenes. Also, ADMET studies of the three main sesquiterpenes indicated acceptable drug-like properties. In these findings, safety evaluations showed low toxicity and skin sensitization for the main sesquiterpenes, contrasting with commercial synthetic insecticides. Therefore, in silico analyses suggest promising interactions with proteins, indicating its potential as an effective alternative to conventional insecticides These results show the larvicidal and adulticidal potential of the essential oil from against , supported by its predominant constituents, -selinene, -selinene and -elemene.
Topics: Animals; Aedes; Oils, Volatile; Insecticides; Molecular Docking Simulation; Larva; Plant Leaves; Sesquiterpenes; Gas Chromatography-Mass Spectrometry
PubMed: 38792102
DOI: 10.3390/molecules29102240 -
International Journal of Molecular... May 2024Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the...
Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable -terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.
Topics: Humans; Antigens, Plant; Immunoglobulin E; Animals; Allergens; Ambrosia; Recombinant Proteins; Female; Adult; Plant Proteins; Rhinitis, Allergic, Seasonal; Male; Middle Aged; Plant Extracts
PubMed: 38791214
DOI: 10.3390/ijms25105175 -
Toxins May 2024Scorpion envenomation poses a global public health issue, with an estimated 1,500,000 cases worldwide annually resulting in 2600 deaths. North Africa, particularly... (Review)
Review
Scorpion envenomation poses a global public health issue, with an estimated 1,500,000 cases worldwide annually resulting in 2600 deaths. North Africa, particularly Morocco, experiences severe envenomations, mainly attributed to and in Morocco, and and in Algeria and Tunisia, with case numbers often underestimated. Current treatment relies mainly on symptomatic approaches, except in Morocco, where management is limited to symptomatic treatment due to controversies regarding specific treatment. In Morocco, between 30,000 and 50,000 scorpion envenomation cases are reported annually, leading to hundreds of deaths, mainly among children. Controversies among clinicians persist regarding the appropriate course of action, often limiting treatments to symptomatic measures. The absence of a specific antivenom for the venoms of the most lethal scorpions further exacerbates the situation. This study aims to address this gap by developing a monovalent antivenom against the endemic and most dangerous scorpion, . The antivenom was produced by immunizing albino rabbits with a mixture of venom collected from high-risk areas in Morocco. Immunizations were performed by subcutaneous injections at multiple sites near the lymphatic system, following an immunization schedule. Production control of neutralizing antibody titers was conducted through immunodiffusion. Once a sufficient antibody titer was achieved, blood collection was performed, and the recovered plasma underwent affinity chromatography. The efficacy of purified IgG was evaluated by determining the ED in mice, complemented by histological and immunohistochemical studies on its ability to neutralize venom-induced tissue alterations and the neutralization of toxins bound to receptors in the studied organs. The monovalent antivenom demonstrated specificity against venom and effective cross-protection against the venom of the scorpions and , highly implicated in lethal envenomations in the Maghreb. This study shows that the developed monovalent antivenom exhibits notable efficacy against local scorpions and a surprising ability to neutralize the most lethal envenomations in North Africa. These results pave the way for a new, more specific, and promising therapeutic approach to countering severe scorpion envenomations, especially in Morocco, where specific treatment is lacking.
Topics: Antivenins; Animals; Morocco; Scorpion Stings; Scorpion Venoms; Scorpions; Humans; Africa, Northern
PubMed: 38787066
DOI: 10.3390/toxins16050214 -
Nanomaterials (Basel, Switzerland) May 2024The affinity between carbon nanotubes (CNTs) and organic compounds is of substantial importance since it strongly relates to the dispersibility of CNTs in those...
The affinity between carbon nanotubes (CNTs) and organic compounds is of substantial importance since it strongly relates to the dispersibility of CNTs in those compounds. Several affinity evaluation methods have been developed so far, and the concept of the Hansen solubility parameter is a representative method widely used in the field of nanocarbon materials. Here, we demonstrate that CNT-loaded silica columns can effectively assess the affinity of organic compounds for CNT surface by exploiting the chromatographic retention time as a criterion. Obtained trends of the affinity of organic compounds for CNT were compared to those based on Hansen solubility parameter distance values. Most organic compounds showed similar trends, but one exceptional compound was observed. Simple CNT dispersion tests were conducted with these organic compounds to demonstrate the advantage of the chromatographic assessment. Further, we conducted comparison experiments using a pyrene-functionalized column and other CNT-loaded columns to elucidate the characteristics of each CNT column. The chromatographic approaches using CNT columns would be beneficial for realizing CNT suspensions with improved CNT dispersibility.
PubMed: 38786781
DOI: 10.3390/nano14100824 -
Gels (Basel, Switzerland) Apr 2024Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan...
Copper-chelated chitosan microgels were investigated as an immobilized metal affinity chromatography (IMAC) phase for peptide separation. The copper-crosslinked chitosan beads were shown to strongly interact with a range of amino acids, in a wide range of pH and saline conditions. The beads exhibited an affinity that seemed to depend on the isoelectric point of the amino acid, with the extent of uptake increasing with decreasing isoelectric point. This selective interaction with anionic amino acids resulted in a significant relative enrichment of the supernatant solution in cationic amino acids. The beads were then studied as a novel fractionation system for complex milk hydrolysates. The copper chitosan beads selectively removed larger peptides from the hydrolysate aqueous solution, yielding a solution relatively enriched in medium and smaller peptides, which was characterized both quantitatively and qualitatively by size exclusion chromatography (SEC). Liquid chromatography-mass spectrometry (LCMS) work provided comprehensive data on a peptide sequence level and showed that a depletion of the anionic peptides by the beads resulted in a relative enrichment of the cationic peptides in the supernatant solution. It could be concluded that after fractionation a dramatic relative enrichment in respect to small- and medium-sized cationic peptides in the solution, characteristics that have been linked to bioactivities, such as anti-microbial and cell-penetrating properties. The results demonstrate the use of the chitosan copper gel bead system in lab scale fractionation of complex hydrolysate mixtures, with the potential to enhance milk hydrolysate bioactivity.
PubMed: 38786205
DOI: 10.3390/gels10050289 -
Biomolecules Apr 2024Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due...
Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from NRC-1 was cloned and successfully expressed in . The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using -nitrophenyl valerate as substrate (K = 78 µM, k = 0.67 s). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, -hexane, -heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.
Topics: Cloning, Molecular; Carboxylesterase; Recombinant Proteins; Substrate Specificity; Halobacterium salinarum; Enzymes, Immobilized; Hydrogen-Ion Concentration; Kinetics; Enzyme Stability; Archaeal Proteins; Temperature
PubMed: 38785941
DOI: 10.3390/biom14050534 -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the...
The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.
Topics: tau Proteins; Animals; Antibodies, Monoclonal; Humans; Mice; Alzheimer Disease; Enzyme-Linked Immunosorbent Assay; Recombinant Proteins; Hybridomas; Mice, Inbred BALB C; Antibody Specificity; Protein Domains; Epitopes
PubMed: 38783817
DOI: 10.13345/j.cjb.230655