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International Journal of Molecular... Jun 2024Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms...
Pollen from common ragweed is an important allergen source worldwide and especially in western and southern Romania. More than 100 million patients suffer from symptoms of respiratory allergy (e.g., rhinitis, asthma) to ragweed pollen. Among the eleven characterized allergens, Amb a 6 is a non-specific lipid transfer protein (nsLTP). nsLTPs are structurally stable proteins in pollen and food from different unrelated plants capable of inducing severe reactions. The goal of this study was to produce Amb a 6 as a recombinant and structurally folded protein (rAmb a 6) and to characterize its physicochemical and immunological features. rAmb a 6 was expressed in cells as a secreted protein and characterized by mass spectrometry and circular dichroism (CD) spectroscopy regarding molecular mass and fold, respectively. The IgE-binding frequency towards the purified protein was evaluated using sera from 150 clinically well-characterized ragweed-allergic patients. The allergenic activities of rAmb a 6 and the nsLTP from the weed (Par j 2) were evaluated in basophil activation assays. rAmb a 6-specific IgE reactivity was associated with clinical features. Pure rAmb a 6 was obtained by insect cell expression. Its deduced molecular weight corresponded to that determined by mass spectrometry (i.e., 10,963 Da). rAmb a 6 formed oligomers as determined by SDS-PAGE under non-reducing conditions. According to multiple sequence comparisons, Amb a 6 was a distinct nsLTP with less than 40% sequence identity to currently known plant nsLTP allergens, except for nsLTP from (i.e., 52%). rAmb a 6 is an important ragweed allergen recognized by 30% of ragweed pollen allergic patients. For certain patients, rAmb a 6-specific IgE levels were higher than those specific for the major ragweed allergen Amb a 1 and analysis also showed a higher allergenic activity in the basophil activation test. rAmb a 6-positive patients suffered mainly from respiratory symptoms. The assumption that Amb a 6 is a source-specific ragweed allergen is supported by the finding that none of the patients showing rAmb a 6-induced basophil activation reacted with Par j 2 and only one rAmb a 6-sensitized patient had a history of plant food allergy. Immunization of rabbits with rAmb a 6 induced IgG antibodies which strongly inhibited IgE binding to rAmb a 6. Our results demonstrate that Amb a 6 is an important source-specific ragweed pollen allergen that should be considered for diagnosis and allergen-specific immunotherapy of ragweed pollen allergy.
Topics: Humans; Allergens; Immunoglobulin E; Antigens, Plant; Animals; Carrier Proteins; Plant Proteins; Female; Rhinitis, Allergic, Seasonal; Male; Adult; Ambrosia; Spodoptera; Recombinant Proteins; Amino Acid Sequence; Sf9 Cells; Middle Aged; Plant Extracts
PubMed: 38928218
DOI: 10.3390/ijms25126513 -
International Journal of Molecular... Jun 2024Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the...
Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.
Topics: Formaldehyde; Animals; Mice; Brain; Tissue Fixation; Sequence Analysis, RNA; Alzheimer Disease; Polymers; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA
PubMed: 38928210
DOI: 10.3390/ijms25126504 -
International Journal of Molecular... Jun 2024Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the...
Complex gut microbiota increases chickens' resistance to enteric pathogens. However, the principles of this phenomenon are not understood in detail. One of the possibilities for how to decipher the role of gut microbiota in chickens' resistance to enteric pathogens is to systematically characterise the gene expression of individual gut microbiota members colonising the chicken caecum. To reach this aim, newly hatched chicks were inoculated with bacterial species whose whole genomic sequence was known. Total protein purified from the chicken caecum was analysed by mass spectrometry, and the obtained spectra were searched against strain-specific protein databases generated from known genomic sequences. , sp. and did not utilise carbohydrates when colonising the chicken caecum. On the other hand, , , , , , , , and fermented carbohydrates. was the only motile bacterium, and expressed the type VI secretion system. Classification of in vivo expression is key for understanding the role of individual species in complex microbial populations colonising the intestinal tract. Knowledge of the expression of motility, the type VI secretion system, and preference for carbohydrate or amino acid fermentation is important for the selection of bacteria for defined competitive exclusion products.
Topics: Animals; Chickens; Gastrointestinal Microbiome; Amino Acids; Type VI Secretion Systems; Carbohydrate Metabolism; Cecum; Bacteria
PubMed: 38928209
DOI: 10.3390/ijms25126505 -
International Journal of Molecular... Jun 2024Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation,...
Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 μM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.
Topics: Protein Multimerization; Humans; Cytoskeletal Proteins; Nerve Tissue Proteins; Animals
PubMed: 38928159
DOI: 10.3390/ijms25126454 -
International Journal of Molecular... Jun 2024is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of would improve its biological control effect. Chemosensory...
is the dominant parasitic natural enemy of aphids. Elucidating the molecular mechanism of host recognition of would improve its biological control effect. Chemosensory proteins (CSPs) play a crucial role in insect olfactory systems and are mainly involved in host localization. In this study, a total of nine CSPs of with complete open reading frames were identified based on antennal transcriptome data. Phylogenetic analysis revealed that AgifCSPs were mainly clustered into three subgroups (AgifCSP1/2/7/8, AgifCSP3/9, and AgifCSP4/5/6). showed high expression in the antennae of both sexes. Moreover, was found to be specifically expressed in the antennae. In addition, fluorescent binding assays revealed that AifCSP5 had greater affinities for 7 of 32 volatile odor molecules from various sources. Molecular docking and site-directed mutagenesis results revealed that the residue at which AgifCSP5 binds to these seven plant volatiles is Tyr75. Behavior tests further confirmed that -2-nonenal, one of the seven active volatiles in the ligand binding test, significantly attracted female adults at a relatively low concentration of 10 mg/mL. In conclusion, AgifCSP5 may be involved in locating aphid-infested crops from long distances by detecting and binding -2-nonenal. These findings provide a theoretical foundation for further understanding the olfactory recognition mechanisms and indirect aphid localization behavior of from long distances by first identifying the host plant of aphids.
Topics: Animals; Aphids; Insect Proteins; Phylogeny; Female; Male; Host-Parasite Interactions; Arthropod Antennae; Molecular Docking Simulation; Amino Acid Sequence; Receptors, Odorant; Wasps
PubMed: 38928098
DOI: 10.3390/ijms25126392 -
International Journal of Molecular... Jun 2024The downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk...
The downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.
Topics: src Homology Domains; Drosophila Proteins; Molecular Dynamics Simulation; Protein Binding; Animals; Humans; Receptor Protein-Tyrosine Kinases; GRB2 Adaptor Protein; Molecular Docking Simulation; Binding Sites; Amino Acid Sequence; Magnetic Resonance Spectroscopy
PubMed: 38928093
DOI: 10.3390/ijms25126386 -
International Journal of Molecular... Jun 2024Lung adenocarcinoma (LUAD) is the most widespread cancer in the world, and its development is associated with complex biological mechanisms that are poorly understood....
Lung adenocarcinoma (LUAD) is the most widespread cancer in the world, and its development is associated with complex biological mechanisms that are poorly understood. Here, we revealed a marked upregulation in the mRNA level of C1orf131 in LUAD samples compared to non-tumor tissue samples in The Cancer Genome Atlas (TCGA). Depletion of C1orf131 suppressed cell proliferation and growth, whereas it stimulated apoptosis in LUAD cells. Mechanistic investigations revealed that C1orf131 knockdown induced cell cycle dysregulation via the AKT and p53/p21 signalling pathways. Additionally, C1orf131 knockdown blocked cell migration through the modulation of epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. Notably, we identified the C1orf131 protein nucleolar localization sequence, which included amino acid residues 137-142 (KKRKLT) and 240-245 (KKKRKG). Collectively, C1orf131 has potential as a novel therapeutic marker for patients in the future, as it plays a vital role in the progression of lung adenocarcinoma.
Topics: Humans; Adenocarcinoma of Lung; Proto-Oncogene Proteins c-akt; Signal Transduction; Lung Neoplasms; Cell Proliferation; Gene Expression Regulation, Neoplastic; Cell Movement; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Apoptosis; Disease Progression; Tumor Suppressor Protein p53; Nuclear Proteins; A549 Cells
PubMed: 38928092
DOI: 10.3390/ijms25126381 -
International Journal of Molecular... Jun 2024The secreted proteins of human body fluid have the potential to be used as biomarkers for diseases. These biomarkers can be used for early diagnosis and risk prediction...
The secreted proteins of human body fluid have the potential to be used as biomarkers for diseases. These biomarkers can be used for early diagnosis and risk prediction of diseases, so the study of secreted proteins of human body fluid has great application value. In recent years, the deep-learning-based transformer language model has transferred from the field of natural language processing (NLP) to the field of proteomics, leading to the development of protein language models (PLMs) for protein sequence representation. Here, we propose a deep learning framework called ESM Predict Secreted Proteins (ESMSec) to predict three types of proteins secreted in human body fluid. The ESMSec is based on the ESM2 model and attention architecture. Specifically, the protein sequence data are firstly put into the ESM2 model to extract the feature information from the last hidden layer, and all the input proteins are encoded into a fixed 1000 × 480 matrix. Secondly, multi-head attention with a fully connected neural network is employed as the classifier to perform binary classification according to whether they are secreted into each body fluid. Our experiment utilized three human body fluids that are important and ubiquitous markers. Experimental results show that ESMSec achieved average accuracy of 0.8486, 0.8358, and 0.8325 on the testing datasets for plasma, cerebrospinal fluid (CSF), and seminal fluid, which on average outperform the state-of-the-art (SOTA) methods. The outstanding performance results of ESMSec demonstrate that the ESM can improve the prediction performance of the model and has great potential to screen the secretion information of human body fluid proteins.
Topics: Humans; Body Fluids; Biomarkers; Deep Learning; Natural Language Processing; Proteomics; Proteins; Neural Networks, Computer; Computational Biology
PubMed: 38928078
DOI: 10.3390/ijms25126371 -
Bioengineering (Basel, Switzerland) Jun 2024The significant growth of the global protein drug market, including fusion proteins, emphasizes the crucial role of optimizing amino acid sequences to enhance the...
The significant growth of the global protein drug market, including fusion proteins, emphasizes the crucial role of optimizing amino acid sequences to enhance the productivity and bioefficacy. Among these fusion proteins, RBP-IIIA-IB, comprising retinol-binding protein in conjunction with the albumin domains, IIIA and IB, has displayed efficacy in alleviating liver fibrosis by inhibiting the activation of hepatic stellate cells (HSCs). This study aimed to address the issue of the low productivity in RBP-IIIA-IB. To induce structural changes, the linking sequence, EVDD, between domain IIIA and IB in RBP-IIIA-IB was modified to DGPG, AAAA, and GGPA. Among these, RBP-IIIA-AAAA-IB demonstrated an increase in yield (>4-fold) and a heightened inhibition of HSC activation. Furthermore, we identified amino acid residues that could form disulfide bonds when substituted with cysteine. Through the mutation of N453S-V480S in RBP-IIIA-AAAA-IB, the productivity further increased by over 9-fold, accompanied by an increase in anti-fibrotic activity. Overall, there was a more than 30-fold increase in the fusion protein's yield. These findings demonstrate the effectiveness of modifying linker sequences and introducing extra disulfide bonds to improve both the production yield and biological efficacy of fusion proteins.
PubMed: 38927853
DOI: 10.3390/bioengineering11060617 -
Genes Jun 2024(Poaceae Bromeae) is a forage grass with high adaptability and ecological and economic value. Here, we sequenced , , , and chloroplast genomes and compared them with... (Comparative Study)
Comparative Study
(Poaceae Bromeae) is a forage grass with high adaptability and ecological and economic value. Here, we sequenced , , , and chloroplast genomes and compared them with four previously described species. The genome sizes of species ranged from 136,934 bp () to 137,189 bp (, ), with a typical quadripartite structure. The studied species had 129 genes, consisting of 83 protein-coding, 38 tRNA-coding, and 8 rRNA-coding genes. The highest GC content was found in the inverted repeat (IR) region (43.85-44.15%), followed by the large single-copy (LSC) region (36.25-36.65%) and the small single-copy (SSC) region (32.21-32.46%). There were 33 high-frequency codons, with those ending in A/U accounting for 90.91%. A total of 350 simple sequence repeats (SSRs) were identified, with single-nucleotide repeats being the most common (61.43%). A total of 228 forward and 141 palindromic repeats were identified. No reverse or complementary repeats were detected. The sequence identities of all sequences were very similar, especially with respect to the protein-coding and inverted repeat regions. Seven highly variable regions were detected, which could be used for molecular marker development. The constructed phylogenetic tree indicates that is a monophyletic taxon closely related to Triticum. This comparative analysis of the chloroplast genome of provides a scientific basis for species identification and phylogenetic studies.
Topics: Genome, Chloroplast; Phylogeny; Microsatellite Repeats; Bromus; Base Composition
PubMed: 38927750
DOI: 10.3390/genes15060815