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Journal of Biochemistry Apr 2024The ribosome, the protein synthesizing machinery composed of dozens of proteins and several ribosomal RNAs (rRNAs), is essential for life. In vitro reconstitution of the...
The ribosome, the protein synthesizing machinery composed of dozens of proteins and several ribosomal RNAs (rRNAs), is essential for life. In vitro reconstitution of the ribosome holds significance for understanding biosynthesis, applications in biotechnology and potential contributions to synthetic biology. There is a long history of in vitro reconstitution of bacterial ribosomes, originating in the 1970s when the 30S ribosome of Escherichia coli was reconstituted from the protein and rRNA components prepared from native ribosome. Since then, the reconstitution using in vitro transcribed rRNAs has been established, and more recently, the reconstitution using recombinant ribosomal proteins has also become possible. A recent report by Aoyama et al. (J. Biochem. 2022; 171:227-237), the reconstitution of the 50S ribosome using 33 recombinant ribosomal proteins, is a new leap toward complete reconstitution of the holo ribosome complex from recombinant proteins and in vitro transcribed rRNAs. This commentary also discusses future challenges.
Topics: Escherichia coli; Escherichia coli Proteins; Recombinant Proteins; Ribosomal Proteins; Ribosomes; RNA, Ribosomal
PubMed: 38236695
DOI: 10.1093/jb/mvad121 -
Sensors (Basel, Switzerland) May 2024Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface...
Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface functionalization of biosensors is a crucial aspect that influences their overall performance, resulting in the accurate, sensitive, and specific detection of target molecules. Here, the surface of a microring resonator (MRR)-based biosensor was functionalized for the detection of protein biomarkers. Among the several existing functionalization methods, a strategy based on aptamers and mercaptosilanes was selected as the most highly performing approach. All steps of the functionalization protocol were carefully characterized and optimized to obtain a suitable protocol to be transferred to the final biosensor. The functionalization protocol comprised a preliminary plasma treatment aimed at cleaning and activating the surface for the subsequent silanization step. Different plasma treatments as well as different silanes were tested in order to covalently bind aptamers specific to different biomarker targets, i.e., C-reactive protein, SARS-CoV-2 spike protein, and thrombin. Argon plasma and 1% / mercaptosilane were found as the most suitable for obtaining a homogeneous layer apt to aptamer conjugation. The aptamer concentration and time for immobilization were optimized, resulting in 1 µM and 3 h, respectively. A final passivation step based on mercaptohexanol was also implemented. The functionalization protocol was then evaluated for the detection of thrombin with a photonic biosensor based on microring resonators. The preliminary results identified the successful recognition of the correct target as well as some limitations of the developed protocol in real measurement conditions.
Topics: Biosensing Techniques; Aptamers, Nucleotide; Silanes; Humans; Thrombin; C-Reactive Protein; Spike Glycoprotein, Coronavirus; SARS-CoV-2; Biomarkers; Surface Properties; COVID-19
PubMed: 38793970
DOI: 10.3390/s24103107 -
Acta Tropica Jun 2024Cervids are highly exposed to ticks, however, their role in the life cycle of these rickettsiae has not been fully elucidated. Given the expanding distribution and...
Cervids are highly exposed to ticks, however, their role in the life cycle of these rickettsiae has not been fully elucidated. Given the expanding distribution and growing population of deer species in Portugal, coupled with their direct and indirect interactions with humans during hunting, it becomes crucial to explore their role as sentinels and potential reservoirs of Rickettsia. The present investigation aimed to detect and evaluate exposure to Rickettsia in free-living deer from Portugal. Blood samples (n = 77) were collected from hunted game animals (red deer and fallow deer) from different areas throughout Portugal (Idanha-a-Nova, Monte Fidalgo, Montalvão and Arraiolos) and sera were tested by immunofluorescence assay, to detect antibodies. Additionally, blood DNA samples were screened for SFGR by nested-polymerase chain reaction targeting a fragment of the outer membrane protein B (ompB) gene, as well as for Anaplasma and Ehrlichia spp. targeting the 16S rRNA gene. Thirty-five per cent (25 deer and two fallow deer) tested positive (sera with a titer ≥1:64) for IgG antibodies against Rickettsia conorii. No rickettsial DNA was detected by PCR for the ompB gene, and all DNA samples tested negative for Anaplasma and Ehrlichia. As far as we know, this study is the first screening of cervid species in Portugal for Rickettsia antibodies. The findings suggest that these animals serve as useful sentinel indicators for the circulation of rickettsiae, offering a complementary perspective to studies focused on ticks. The increasing numbers of hunted deer in Portugal and the potential zoonotic features of Rickettsia spp. highlight the importance of continued surveillance directed at tick-borne diseases, especially those involving wild animals.
Topics: Animals; Portugal; Deer; Antibodies, Bacterial; Rickettsia; Rickettsia Infections; Sentinel Species; DNA, Bacterial; Immunoglobulin G; Polymerase Chain Reaction; RNA, Ribosomal, 16S; Anaplasma; Ehrlichia; Rickettsia conorii; Bacterial Outer Membrane Proteins; Male
PubMed: 38565332
DOI: 10.1016/j.actatropica.2024.107202 -
Nutrients Apr 2024As gluten may trigger gastrointestinal disorders (GIDs), its presence or absence in the diet can change the diversity and proportion of gut microbiota. The effects of... (Randomized Controlled Trial)
Randomized Controlled Trial
As gluten may trigger gastrointestinal disorders (GIDs), its presence or absence in the diet can change the diversity and proportion of gut microbiota. The effects of gluten after six weeks of a double-blind, placebo-controlled intervention with a gluten-free diet (GFD) were studied in participants with GIDs suffering from migraines and atopic dermatitis (n = 46). Clinical biomarkers, digestive symptoms, stool, the Migraine Disability Assessment questionnaire, and zonulin levels were analyzed. Next-generation sequencing was used to amplify the 16S rRNA gene of bacteria and the internal transcribed spacer (ITS) regions of fungi. The GFD increased Chao1 fungal diversity after the intervention, while the fungal composition showed no changes. Bacterial diversity and composition remained stable, but a positive association between bacterial and fungal Chao1 diversity and a negative association between Dothideomycetes and were observed. GIDs decreased in both groups and migraines improved in the placebo group. Our findings may aid the development of GID treatment strategies.
Topics: Humans; Migraine Disorders; Gastrointestinal Microbiome; Female; Male; Gastrointestinal Diseases; Adult; Double-Blind Method; Glutens; Middle Aged; Diet, Gluten-Free; Dermatitis, Atopic; Feces; Bacteria; Fungi; RNA, Ribosomal, 16S; Protein Precursors; Haptoglobins
PubMed: 38674918
DOI: 10.3390/nu16081228 -
Journal of Colloid and Interface Science Aug 2024Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms...
Protein body (PB) formation in wheat seeds is a critical process influencing seed content and nutritional quality. In this study, we investigate the potential mechanisms governing PB formation through an in vitro approach, focusing on γ-gliadin, a key wheat storage protein. We used a microfluidic technique to encapsulate γ-gliadin within giant unilamellar vesicles (GUVs) and tune the physicochemical conditions in a controlled and rapid way. We examined the influence of pH and protein concentration on LLPS and protein-membrane interactions using various microscopy and spectroscopy techniques. We showed that γ-gliadin encapsulated in GUVs can undergo a pH-triggered liquid-liquid phase separation (LLPS) by two distinct mechanisms depending on the γ-gliadin concentration. At low protein concentrations, γ-gliadins phase separate by a nucleation and growth-like process, while, at higher protein concentration and pH above 6.0, γ-gliadin formed a bi-continuous phase suggesting a spinodal decomposition-like mechanism. Fluorescence and microscopy data suggested that γ-gliadin dense phase exhibited affinity for the GUV membrane, forming a layer at the interface and affecting the reversibility of the phase separation.
Topics: Gliadin; Triticum; Hydrogen-Ion Concentration; Unilamellar Liposomes; Water; Membrane Lipids; Phase Separation
PubMed: 38678881
DOI: 10.1016/j.jcis.2024.04.136 -
Biological Chemistry May 2024Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action...
Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells.
Topics: Humans; Antigens, Differentiation; Protein Conformation; Recombinant Proteins; Antiviral Agents
PubMed: 38379409
DOI: 10.1515/hsz-2023-0327 -
Food Chemistry Dec 2023In this study, extrusion was used to induce Maillard reaction between soy protein isolate (SPI) and oat β-glucan (OG) and effect of extrusion temperature (70, 90, 110...
In this study, extrusion was used to induce Maillard reaction between soy protein isolate (SPI) and oat β-glucan (OG) and effect of extrusion temperature (70, 90, 110 and 130 °C) on the structure and emulsifying properties of extruded SPI-OG was investigated. SDS-PAGE and fluorescence spectroscopy provided evidence for the formation of SPI-OG conjugates during extrusion. The results showed that 90 °C and 110 °C extruded SPI-OG had the highest level of degree of glycosylation (were 14.34% and 13.70%, respectively, p > 0.05). Structural analysis found that α-helix content of extruded SPI-OG decreased by 8.93-13.14% compared to mixture of SPI and OG. Meanwhile, extruded SPI-OG had lower protein solubility (29.83-34.38%) and surface hydrophobicity (1549-2027), larger average particle size (2363-4807 nm) and higher emulsion stability (74.33-90.15%). Therefore, these findings may provide a theoretical basis for the development of novel food emulsion stabilizers.
Topics: Soybean Proteins; Emulsions; Temperature; Avena; Hydrophobic and Hydrophilic Interactions; Surface Properties; Particle Size
PubMed: 37478603
DOI: 10.1016/j.foodchem.2023.136787 -
International Journal of Biological... May 2024In recent years, Flammulina velutipes (F. velutipes) has attracted consequential attention in various research fields due to its rich composition of proteins, vitamins,... (Review)
Review
Research progress and future development potential of Flammulina velutipes polysaccharides in the preparation process, structure analysis, biology, and pharmacology: A review.
In recent years, Flammulina velutipes (F. velutipes) has attracted consequential attention in various research fields due to its rich composition of proteins, vitamins, amino acids, polysaccharides, and polyphenols. F. velutipes polysaccharides (FVPs) are considered as key bioactive components of F. velutipes, demonstrating multiple physiological activities, including immunomodulatory, anti-inflammatory, and antibacterial properties. Moreover, they offer health benefits such as antioxidant and anti-aging properties, which have exceptionally valuable clinical applications. Polysaccharides derived from different sources exhibit a wide range of biomedical functions and distinct biological activities. The varied biological functions of polysaccharides, coupled with their extensive application in functional foods and clinical applications, have prompted a heightened focus on polysaccharide research. Additionally, the extraction, deproteinization, and purification of FVPs are fundamental to investigate the structure and biological activities of polysaccharides. Therefore, this review provides a comprehensive and systematic overview of the extraction, deproteinization, purification, characterization, and structural elucidation of FVPs. Furthermore, the biological activities and mechanisms of FVPs have been further explored through in vivo and in vitro experiments. This review aims to provide a theoretical foundation and guide future research and development of FVPs.
Topics: Flammulina; Humans; Antioxidants; Animals; Anti-Inflammatory Agents; Polysaccharides; Fungal Polysaccharides; Immunologic Factors; Anti-Bacterial Agents
PubMed: 38599436
DOI: 10.1016/j.ijbiomac.2024.131467 -
Plant Foods For Human Nutrition... Jun 2024This study focused on studying the bioaccesible phenolic compounds (PCs) from yellow pea flour (F) and protein isolate (I). Total phenolic contents (TPC), PCs...
This study focused on studying the bioaccesible phenolic compounds (PCs) from yellow pea flour (F) and protein isolate (I). Total phenolic contents (TPC), PCs composition and antioxidant activities were analysed in ethanol 60% extracts obtained by applying ultrasound assisted extraction (UAE, 15 min/40% amplitude). The preparation of I under alkaline conditions and the elimination of some soluble components at lower pH produced a change of PCs profile and antioxidant activity. After simulated gastrointestinal digestion (SGID) of both ingredients to obtain the digests FD and ID, notable changes in the PCs concentration and profiles could be demonstrated. FD presented a higher ORAC activity than ID (IC = 0.022 and 0.039 mg GAE/g dm, respectively), but lower ABTS activity (IC = 0.8 and 0.3 mg GAE/g dm, respectively). After treatment with cholestyramine of extracts from FD and ID in order to eliminate bile salts and obtain the bioaccesible fractions FD and ID, ROS scavenging in HO-induced Caco2-TC7 cells was evaluated, registering a greater activity for ID respect to FD (IC = 0.042 and 0.017 mg GAE/mL, respectively). These activities could be attributed to the major bioaccesible PCs: OH-tyrosol, polydatin, trans-resveratrol, rutin, (-)-epicatechin and (-)-gallocatechin gallate for FD; syringic (the most concentrated) and ellagic acids, trans-resveratrol, and (-)-gallocatechin gallate for ID, but probably other compounds such as peptides or amino acids can also contribute.
Topics: Antioxidants; Pisum sativum; Phenols; Flour; Humans; Caco-2 Cells; Plant Extracts; Plant Proteins; Pea Proteins; Digestion
PubMed: 38602652
DOI: 10.1007/s11130-024-01172-z -
Journal of Virological Methods Oct 2023Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and...
Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 10 and 1.0 × 10 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.
Topics: Capripoxvirus; Recombinases; Nucleic Acid Amplification Techniques; Viral Proteins; Poxviridae Infections; Animals; Cattle; Sheep; Goats; Sensitivity and Specificity
PubMed: 37517457
DOI: 10.1016/j.jviromet.2023.114788