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Parasites & Vectors Oct 2022Recombinant antigens rK39 (based on kinesin sequence) and rK28 (comprising kinesin and HASPB sequences) are a mainstay of serological diagnosis for visceral...
BACKGROUND
Recombinant antigens rK39 (based on kinesin sequence) and rK28 (comprising kinesin and HASPB sequences) are a mainstay of serological diagnosis for visceral leishmaniasis (VL). However, their key epitopes and the significance of their structural conformation are not clearly defined, particularly in relation to reported cross-reactivity with sera from patients with malaria, schistosomiasis, and tuberculosis.
METHODS
To assess the effect of conformation on antigenicity with Sudanese VL sera, antigens rK39 and rK28 were heat-denatured at 95 °C for 10 min and then assayed by enzyme-linked immunosorbent assay (ELISA). Amino acid sequences of rK39 and rK28 were submitted to NCBI BLASTp to assess homology with Plasmodium, Schistosoma, and Mycobacterium.
RESULTS
Heat denaturation significantly diminished the antigenicity of rK39 compared to non-denatured antigen (P = 0.001), but not for rK28 (P = 0.275). In BLASTp searches, HASPB sequences from rK28 had similarities with sequences from Plasmodium, encompassing software-predicted B-cell epitopes.
CONCLUSIONS
The antigenicity of rK39 appears to be dependent on structural conformation, whereas that of rK28 depends on linear sequence. HASPB sequence homology with Plasmodium may be responsible for the reported cross-reactivity of rK28 with malaria sera. Further work is warranted to refine the specificity of these antigens.
Topics: Humans; Leishmaniasis, Visceral; Antigens, Protozoan; Kinesins; Epitopes, B-Lymphocyte; Protozoan Proteins; Sensitivity and Specificity
PubMed: 36273150
DOI: 10.1186/s13071-022-05495-1 -
Analytical Chemistry Aug 2019Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research...
Electrospray ionization mass spectrometry (ESI-MS) is a ubiquitously used analytical method applied across multiple departments in biopharma, ranging from early research discovery to process development. Accurate, efficient, and consistent protein MS spectral deconvolution across multiple instrument and detector platforms (time-of-flight, Orbitrap, Fourier-transform ion cyclotron resonance) is essential. When proteins are ionized during the ESI process, a distribution of consecutive multiply charged ions are observed on the / scale, either positive [M + H] or negative [M - H] depending on the ionization polarity. The manual calculation of the neutral molecular weight (MW) of single proteins measured by ESI-MS is simple; however, algorithmic deconvolution is required for more complex protein mixtures to derive accurate MWs. Multiple deconvolution algorithms have evolved over the past two decades, all of which have their advantages and disadvantages, in terms of speed, user-input parameters (or ideally lack thereof), and whether they perform optimally on proteins analyzed under denatured or native-MS and solution conditions. Herein, we describe the utility of a parsimonious deconvolution algorithm (explaining the observed spectra with a minimum number of masses) to process a wide range of highly diverse biopharma relevant and research grade proteins and complexes (PEG-GCSF; an IgG1; IgG1- and IgG2-biotin covalent conjugates; the membrane protein complex AqpZ; a highly polydisperse empty MSP1D1 nanodisc and the tetradecameric chaperone protein complex GroEL) analyzed under native-MS, denaturing LC-MS, and positive and negative modes of ionization, using multiple instruments and therefore multiple data formats. The implementation of a comb filter and peak sharpening option is also demonstrated to be highly effective for deconvolution of highly polydisperse and enhanced separation of a low level lysine glycation post-translational modification (+162.1 Da), partially processed heavy chain lysine residues (+128.1 Da), and loss of -acetylglucosamine (GlcNAc; -203.1 Da).
Topics: Algorithms; Antibodies, Monoclonal; Biopharmaceutics; Glycosylation; Immunoconjugates; Lysine; Membrane Proteins; Molecular Weight; Proteins; Spectrometry, Mass, Electrospray Ionization
PubMed: 31194911
DOI: 10.1021/acs.analchem.9b00062 -
Frontiers in Microbiology 2020This review describes the researches performed in the last years to assess the impact of pesticide sub-lethal doses on soil microorganisms and non-target organisms in... (Review)
Review
This review describes the researches performed in the last years to assess the impact of pesticide sub-lethal doses on soil microorganisms and non-target organisms in agricultural soil ecosystems. The overview was developed through the careful description and a critical analysis of three methodologies based on culture-independent approaches involving DNA extraction and sequencing (denaturing gradient gel electrophoresis, DGGE; next-generation sequencing, NGS) to characterize the microbial population and DNA damage assessment (comet assay) to determine the effect on soil invertebrates. The examination of the related published articles showed a continuous improvement of the possibility to detect the detrimental effect of the pesticides on soil microorganisms and non-target organisms at sub-lethal doses, i.e., doses which have no lethal effect on the organisms. Considering the overall critical discussion on microbial soil monitoring in the function of pesticide treatments, we can confirm the usefulness of PCR-DGGE as a screening technique to assess the genetic diversity of microbial communities. Nowadays, DGGE remains a preliminary technique to highlight rapidly the main differences in microbial community composition, which is able to give further information if coupled with culture-dependent microbiological approaches, while thorough assessments must be gained by high-throughput techniques such as NGS. The comet assay represents an elective technique for assessing genotoxicity in environmental biomonitoring, being mature after decades of implementation and widely used worldwide for its direct, simple, and affordable implementation. Nonetheless, in order to promote the consistency and reliability of results, regulatory bodies should provide guidelines on the optimal use of this tool, strongly indicating the most reliable indicators of DNA damage. This review may help the European Regulation Authority in deriving new ecotoxicological endpoints to be included in the Registration Procedure of new pesticides.
PubMed: 33013727
DOI: 10.3389/fmicb.2020.01892 -
Ciencia & Saude Coletiva Sep 2022The purpose of this study was to evaluate, through a scope review, studies that address the perceptions and attitudes of dentists regarding the care of women in... (Review)
Review
The purpose of this study was to evaluate, through a scope review, studies that address the perceptions and attitudes of dentists regarding the care of women in situations of violence. Using the descriptors women violence, dentist attendance or dentist care, 473 articles were identified, of which 13 were included at the end of the selection process. Although the need for training was predominant, it was not sufficient. There is a weakness in understanding violence as a health problem, in understanding the role of the professional in solving this problem, and the factors that can contribute to its growth or its control. The results revealed that the dentist had greater difficulty than other professionals in coping with the issue and required extensive training. The recognition of these cases of abuse by the dentist requires the incorporation of educational measures that cause cultural changes, deconstruction of gender norms and the denaturalization of this social phenomenon.
Topics: Attitude of Health Personnel; Dentists; Emergency Medical Services; Female; Humans; Violence
PubMed: 36000658
DOI: 10.1590/1413-81232022279.22532021 -
Molecules (Basel, Switzerland) Jan 2022The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual...
The chaperone DNAJB6b delays amyloid formation by suppressing the nucleation of amyloid fibrils and increases the solubility of amyloid-prone proteins. These dual effects on kinetics and equilibrium are related to the unusually high chemical potential of DNAJB6b in solution. As a consequence, the chaperone alone forms highly polydisperse oligomers, whereas in a mixture with an amyloid-forming protein or peptide it may form co-aggregates to gain a reduced chemical potential, thus enabling the amyloid peptide to increase its chemical potential leading to enhanced solubility of the peptide. Understanding such action at the level of molecular driving forces and detailed structures requires access to highly pure and sequence homogeneous DNAJB6b with no sequence extension. We therefore outline here an expression and purification protocol of the protein "as is" with no tags leading to very high levels of pure protein based on its physicochemical properties, including size and charge. The versatility of the protocol is demonstrated through the expression of an isotope labelled protein and seven variants, and the purification of three of these. The activity of the protein is bench-marked using aggregation assays. Two of the variants are used to produce a palette of fluorescent DNAJB6b labelled at an engineered N- or C-terminal cysteine.
Topics: Ammonium Sulfate; Amyloidogenic Proteins; Chemical Precipitation; Chromatography, Gel; Escherichia coli; Fluorescent Dyes; HSP40 Heat-Shock Proteins; Humans; Hydrogen-Ion Concentration; Molecular Chaperones; Nerve Tissue Proteins; Protein Denaturation; Protein Engineering; Recombinant Proteins; Rhodamines; Solubility; Sulfonic Acids
PubMed: 35056736
DOI: 10.3390/molecules27020418 -
Viruses Jun 2023There is little doubt that final victories over pandemics, such as COVID-19, are attributed to herd immunity, either through post-disease convalescence or active... (Review)
Review
There is little doubt that final victories over pandemics, such as COVID-19, are attributed to herd immunity, either through post-disease convalescence or active immunization of a high percentage of the world's population with vaccines, which demonstrate protection from infection and transmission and are available in large quantities at reasonable prices. However, it is assumable that humans with immune defects or immune suppression, e.g., as a consequence of allograft transplantation, cannot be immunized actively nor produce sufficient immune responses to prevent SARS-CoV-2 infections. These subjects desperately need other strategies, such as sophisticated protection measures and passive immunization. Hypertonic salt solutions attack vulnerable core areas of viruses; i.e., salt denatures surface proteins and thus prohibits virus penetration of somatic cells. It has to be ensured that somatic proteins are not affected by denaturation regarding this unspecific virus protection. Impregnating filtering facepieces with hypertonic salt solutions is a straightforward way to inactivate viruses and other potential pathogens. As a result of the contact of salt crystals on the filtering facepiece, these pathogens become denatured and inactivated almost quantitatively. Such a strategy could be easily applied to fight against the COVID-19 pandemic and other ones that may occur in the future. Another possible tool to fight the COVID-19 pandemic is passive immunization with antibodies against SARS-CoV-2, preferably from human origin. Such antibodies can be harvested from human patients' sera who have successfully survived their SARS-CoV-2 infection. The disadvantage of a rapid decrease in the immunoglobulin titer after the infection ends can be overcome by immortalizing antibody-producing B cells via fusion with, e.g., mouse myeloma cells. The resulting monoclonal antibodies are then of human origin and available in, at least theoretically, unlimited amounts. Finally, dry blood spots are a valuable tool for surveilling a population's immunity. The add-on strategies were selected as examples for immediate, medium and long-term assistance and therefore did not raise any claim to completeness.
Topics: Animals; Mice; Humans; COVID-19; SARS-CoV-2; Pandemics; Vaccination; Antibodies, Viral; Antibodies, Neutralizing
PubMed: 37376671
DOI: 10.3390/v15061372 -
Archives of Biochemistry and Biophysics Mar 2022PADI4 (protein-arginine deiminase, also known as protein l-arginine iminohydrolase) is one of the human isoforms of a family of Ca-dependent proteins catalyzing the...
PADI4 (protein-arginine deiminase, also known as protein l-arginine iminohydrolase) is one of the human isoforms of a family of Ca-dependent proteins catalyzing the conversion of arginine to citrulline. Although the consequences of this process, known as citrullination, are not fully understood, all PADIs have been suggested to play essential roles in development and cell differentiation. They have been found in a wide range of cells and tissues and, among them, PADI4 is present in macrophages, monocytes, granulocytes and cancer cells. In this work, we focused on the biophysical features of PADI4 and, more importantly, how its expression was altered in cancer cells. Firstly, we described the different expression patterns of PADI4 in various cancer cell lines and its colocalization with the tumor-related protein p53. Secondly, we carried out a biophysical characterization of PADI4, by using a combination of biophysical techniques and in silico molecular dynamics simulations. Our biochemical results suggest the presence of several forms of PADI4 with different subcellular localizations, depending on the cancer cell line. Furthermore, PADI4 could have a major role in tumorigenesis by regulating p53 expression in certain cancer cell lines. On the other hand, the native structure of PADI4 was strongly pH-dependent both in the absence or presence of Ca, and showed two pH-titrations at basic and acidic pH values. Thus, there was a narrow pH range (from 6.5 to 8.0) where the protein was dimeric and had a native structure, supporting its role in histones citrullination. Thermal denaturations were always two-state, but guanidinium-induced ones showed that PADI4 unfolded through at least one intermediate. Our simulation results suggest that the thermal melting of PADI4 structure was rather homogenous throughout its sequence. The overall results are discussed in terms of the functional role of PADI4 in the development of cancer.
Topics: Arginine; Biomarkers, Tumor; Carcinogenesis; Catalysis; Cell Differentiation; Cell Line, Tumor; Citrulline; Gene Expression Regulation; Humans; Molecular Dynamics Simulation; Protein Binding; Protein Processing, Post-Translational; Protein-Arginine Deiminase Type 4; Protein-Arginine Deiminases; Signal Transduction; Tumor Suppressor Protein p53
PubMed: 35081374
DOI: 10.1016/j.abb.2022.109125 -
Chembiochem : a European Journal of... May 2022Here we demonstrate a switchable DNA electron-transfer catalyst, enabled by selective destabilization of secondary structure by the denaturant, perchlorate. The system...
Here we demonstrate a switchable DNA electron-transfer catalyst, enabled by selective destabilization of secondary structure by the denaturant, perchlorate. The system is comprised of two strands, one of which can be selectively switched between a G-quadruplex and duplex or single-stranded conformations. In the G-quadruplex state, it binds hemin, enabling peroxidase activity. This switching ability arises from our finding that perchlorate, a chaotropic Hofmeister ion, selectively destabilizes duplex over G-quadruplex DNA. By varying perchlorate concentration, we show that the DNA structure can be switched between states that do and do not catalyze electron-transfer catalysis. State switching can be achieved in three ways: thermally, by dilution, or by concentration.
Topics: DNA; G-Quadruplexes; Hemin; Perchlorates; Peroxidases
PubMed: 35245408
DOI: 10.1002/cbic.202200090 -
Scientific Reports Nov 2019Protein electrochemistry represents a powerful technique for investigating the function and structure of proteins. Currently available biochemical assays provide limited... (Comparative Study)
Comparative Study
Protein electrochemistry represents a powerful technique for investigating the function and structure of proteins. Currently available biochemical assays provide limited information related to the conformational state of proteins and high costs. This work provides novel insights into the electrochemical investigation of the metalloprotein p53 and its redox products using label-free direct electrochemistry and label-based antibody-specific approaches. First, the redox activities of different p53 redox products were qualitatively investigated on carbon-based electrodes. Then, focusing on the open p53 isoform (denatured p53), a quantitative analysis was performed, comparing the performances of different bulk and nanostructured materials (carbon and platinum). Overall, four different p53 products could be successfully discriminated, from wild type to denatured. Label-free analysis suggested a single electron exchange with electron transfer rate constants on the order of 1 s. Label-based analysis showed decreasing affinity of pAb240 towards denatured, oxidized and nitrated p53. Furthermore, platinum nanostructured electrodes showed the highest enhancement of the limit of detection in the quantitative analysis (100 ng/ml). Overall, the obtained results represent a first step towards the implementation of highly requested complex integrated devices for clinical practices, with the aim to go beyond simple protein quantification.
Topics: Antibodies; Carbon; Electrochemical Techniques; Electrodes; Gold; Humans; Limit of Detection; Models, Molecular; Nanostructures; Platinum; Protein Conformation; Protein Denaturation; Tumor Suppressor Protein p53
PubMed: 31758050
DOI: 10.1038/s41598-019-53994-6 -
ELife Dec 2019Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both...
Previous studies demonstrated importance of C-mannosylation for efficient protein secretion. To study its impact on protein folding and stability, we analyzed both C-mannosylated and non-C-mannosylated thrombospondin type 1 repeats (TSRs) of netrin receptor UNC-5. In absence of C-mannosylation, UNC-5 TSRs could only be obtained at low temperature and a significant proportion displayed incorrect intermolecular disulfide bridging, which was hardly observed when C-mannosylated. Glycosylated TSRs exhibited higher resistance to thermal and reductive denaturation processes, and the presence of C-mannoses promoted the oxidative folding of a reduced and denatured TSR in vitro. Molecular dynamics simulations supported the experimental studies and showed that C-mannoses can be involved in intramolecular hydrogen bonding and limit the flexibility of the TSR tryptophan-arginine ladder. We propose that in the endoplasmic reticulum folding process, C-mannoses orient the underlying tryptophan residues and facilitate the formation of the tryptophan-arginine ladder, thereby influencing the positioning of cysteines and disulfide bridging.
Topics: Animals; Arginine; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cysteine; Disulfides; Drosophila melanogaster; Endoplasmic Reticulum; Glycosylation; Hydrogen Bonding; Mannose; Membrane Proteins; Molecular Dynamics Simulation; Protein Conformation; Protein Folding; Receptors, Cell Surface; Thrombospondins; Tryptophan
PubMed: 31868591
DOI: 10.7554/eLife.52978