Ribosomal Proteins Research Articles

Deciphering early responsive signature genes in rice blast disease: an integrated temporal transcriptomic study.

Rice blast disease, caused by Magnaporthe oryzae, reigns as the top-most cereal killer, jeopardizing global food security. This necessitates the timely scouting of pathogen stress-responsive genes during the early infection stages. Thus, we integrated time-series microarray (GSE95394) and RNA-Seq (GSE131641) datasets to decipher rice transcriptome responses at 12- and 24-h post-infection (Hpi). Our analysis revealed 1580 differentially expressed genes (DEGs) overlapped between datasets. We constructed a protein-protein interaction (PPI) network for these DEGs and identified significant subnetworks using the MCODE plugin. Further analysis with CytoHubba highlighted eight plausible hub genes for pathogenesis: RPL8 (upregulated) and RPL27, OsPRPL3, RPL21, RPL9, RPS5, OsRPS9, and RPL17 (downregulated). We validated the expression levels of these hub genes in response to infection, finding that RPL8 exhibited significantly higher expression compared with other downregulated genes. Remarkably, RPL8 formed a distinct cluster in the co-expression network, whereas other hub genes were interconnected, with RPL9 playing a central role, indicating its pivotal role in coordinating gene expression during infection. Gene Ontology highlighted the enrichment of hub genes in the ribosome and protein translation processes. Prior studies suggested that plant immune defence activation diminishes the energy pool by suppressing ribosomes. Intriguingly, our study aligns with this phenomenon, as the identified ribosomal proteins (RPs) were suppressed, while RPL8 expression was activated. We anticipate that these RPs could be targeted to develop new stress-resistant rice varieties, beyond their housekeeping role. Overall, integrating transcriptomic data revealed more common DEGs, enhancing the reliability of our analysis and providing deeper insights into rice blast disease mechanisms.

Functional connexion of bacterioferritin in antibiotic production and morphological differentiation in Streptomyces coelicolor

BackgroundSeveral two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor.ResultsThe ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869.ConclusionThis bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

Transient Adaptation of Toxoplasma gondii to Exposure by Thiosemicarbazone Drugs That Target Ribosomal Proteins Is Associated with the Upregulated Expression of Tachyzoite Transmembrane Proteins and Transporters.

Thiosemicarbazones and their metal complexes have been studied for their biological activities against bacteria, cancer cells and protozoa. Short-term in vitro treatment with one gold (III) complex (C3) and its salicyl-thiosemicarbazone ligand (C4) selectively inhibited proliferation of T. gondii. Transmission Electron Microscopy (TEM) detected transient structural alterations in the parasitophorous vacuole membrane and the tachyzoite cytoplasm, but the mitochondrial membrane potential appeared unaffected by these compounds. Proteins potentially interacting with C3 and C4 were identified using differential affinity chromatography coupled with mass spectrometry (DAC-MS). Moreover, long-term in vitro treatment was performed to investigate parasitostatic or parasiticidal activity of the compounds. DAC-MS identified 50 ribosomal proteins binding both compounds, and continuous drug treatments for up to 6 days caused the loss of efficacy. Parasite tolerance to both compounds was, however, rapidly lost in their absence and regained shortly after re-exposure. Proteome analyses of six T. gondii ME49 clones adapted to C3 and C4 compared to the non-adapted wildtype revealed overexpression of ribosomal proteins, of two transmembrane proteins involved in exocytosis and of an alpha/beta hydrolase fold domain-containing protein. Results suggest that C3 and C4 may interfere with protein biosynthesis and that adaptation may be associated with the upregulated expression of tachyzoite transmembrane proteins and transporters, suggesting that the in vitro drug tolerance in T. gondii might be due to reversible, non-drug specific stress-responses mediated by phenotypic plasticity.

Translation in Bacillus subtilis is spatially and temporally coordinated during sporulation

The transcriptional control of sporulation in Bacillus subtilis is reasonably well understood, but its translational control is underexplored. Here, we use RNA-seq, ribosome profiling and fluorescence microscopy to study the translational dynamics of B. subtilis sporulation. We identify two events of translation silencing and describe spatiotemporal changes in subcellular localization of ribosomes during sporulation. We investigate the potential regulatory role of ribosomes during sporulation using a strain lacking zinc-independent paralogs of three zinc-dependent ribosomal proteins (L31, L33 and S14). The mutant strain exhibits delayed sporulation, reduced germination efficiency, dysregulated translation of metabolic and sporulation-related genes, and disruptions in translation silencing, particularly in late sporulation.

Unravelling transcriptional responses of the willow to Fusarium kuroshium infection

Fusarium kuroshium and the ambrosia beetle Euwallacea kuroshio attack several tree species, including the arroyo willow (Salix lasiolepis). Despite the importance of this pest, there is no molecular information about this plant-fungus interaction. Using an in vitro pathosystem of S. lasiolepis leaves – F. kurosihum and the RNA-seq approach, this study analyzed for the first time the transcriptome of both organisms at 4-, 8-, and 12-days post-inoculation. De novo assembly was done to generate the willow transcriptome, and a total of 361,2151 unique transcripts (UniGenes) were found. 4,583 UniGenes responded to the infection, and the posterior analysis showed that it contains the molecular elements to develop both pattern- and effector-triggered immunity responses (PTI and ETI, respectively). Significant expression changes of genes involved in an oxidative burst, Ca2+ signaling, and mutually antagonistic interaction between salicylic acid- and jasmonic acid/ethylene-dependent signaling suggest that the willow recognized the fungus and triggered downstream responses. Additionally, chromatin remodelers and non-coding RNA play a significant role in plant defense. Concerning F. kuroshium, 178 induced genes that show homology with genes involved in the pathogenicity in other ascomycetes fungi plus transport proteins, ribosomal proteins, and uncharacterized proteins with specific domains were identified. Collectively these data provide a basis for future research on the molecular response of this transcendental tree during biotic stress, specifically during Fusarium dieback disease.

Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response

Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.

Mutations in nuclear genes encoding mitochondrial ribosome proteins restore pollen fertility in S male-sterile maize.

The interaction of plant mitochondrial and nuclear genetic systems is exemplified by mitochondria-encoded cytoplasmic male sterility (CMS) under the control of nuclear restorer-of-fertility genes. The S type of CMS in maize is characterized by a pollen collapse phenotype and a unique paradigm for fertility restoration in which numerous nuclear restorer-of-fertility lethal mutations rescue pollen function but condition homozygous-lethal seed phenotypes. Two nonallelic restorer mutations recovered from Mutator transposon-active lines were investigated to determine the mechanisms of pollen fertility restoration and seed lethality. Mu Illumina sequencing of transposon-flanking regions identified insertion alleles of nuclear genes encoding mitochondrial ribosomal proteins RPL6 and RPL14 as candidate restorer-of-fertility lethal mutations. Both candidates were associated with lowered abundance of mitochondria-encoded proteins in developing maize pollen, and the rpl14 mutant candidate was confirmed by independent insertion alleles. While the restored pollen functioned despite reduced accumulation of mitochondrial respiratory proteins, normal-cytoplasm plants heterozygous for the mutant alleles showed a significant pollen transmission bias in favor of the nonmutant Rpl6 and Rpl14 alleles. CMS-S fertility restoration affords a unique forward genetic approach to investigate the mitochondrial requirements for, and contributions to, pollen and seed development.

Insights into macrolide resistance in Arcobacter butzleri: potential resistance mechanisms and impact on bacterial fitness and virulence.

Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri, as well as the effects on the fitness of this species. Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR, causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains.

Diametral influence of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) on the growth of Campylobacter jejuni with consequences on the bacterial transcriptome

BackgroundDeoxynivalenol (DON) is a type B trichothecene mycotoxin that is commonly found in cereals and grains worldwide. The presence of this fungal secondary-metabolite raises public-health concerns at both the agriculture and food industry level. Recently, we have shown that DON has a negative impact on gut integrity, a feature also noticed for Campylobacter (C.) jejuni. We further demonstrated that DON increased the load of C. jejuni in the gut and inner organs. In contrast, feeding the less toxic DON metabolite deepoxy-deoxynivalenol (DOM-1) to broilers reduced the Campylobacter load in vivo. Consequently, it can be hypothesized that DON and DOM-1 have a direct effect on the growth profile of C. jejuni. The aim of the present study was to further resolve the nature of this interaction in vitro by co-incubation and RNA-sequencing.ResultsThe co-incubation of C. jejuni with DON resulted in significantly higher bacterial growth rates from 30 h of incubation onwards. On the contrary, the co-incubation of C. jejuni with DOM-1 reduced the CFU counts, indicating that this DON metabolite might contribute to reduce the burden of C. jejuni in birds, altogether confirming in vivo data. Furthermore, the transcriptomic profile of C. jejuni following incubation with either DON or DOM-1 differed. Co-incubation of C. jejuni with DON significantly increased the expression of multiple genes which are critical for Campylobacter growth, particularly members of the Flagella gene family, frr (ribosome-recycling factor), PBP2 futA-like (Fe3+ periplasmic binding family) and PotA (ATP-binding subunit). Flagella are responsible for motility, biofilm formation and host colonization, which may explain the high Campylobacter load in the gut of DON-fed broiler chickens. On the contrary, DOM-1 downregulated the Flagella gene family and upregulated ribosomal proteins.ConclusionThe results highlight the adaptive mechanisms involved in the transcriptional response of C. jejuni to DON and its metabolite DOM-1, based on the following effects: (a) ribosomal proteins; (b) flagellar proteins; (c) engagement of different metabolic pathways. The results provide insight into the response of an important intestinal microbial pathogen against DON and lead to a better understanding of the luminal or environmental acclimation mechanisms in chickens.

Loss of ADAR1 protein induces changes in small RNA landscape in hepatocytes.

In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis.

Protein Fold Usages in Ribosomes: Another Glance to the Past.

The analysis of protein fold usage, similar to codon usage, offers profound insights into the evolution of biological systems and the origins of modern proteomes. While previous studies have examined fold distribution in modern genomes, our study focuses on the comparative distribution and usage of protein folds in ribosomes across bacteria, archaea, and eukaryotes. We identify the prevalence of certain 'super-ribosome folds,' such as the OB fold in bacteria and the SH3 domain in archaea and eukaryotes. The observed protein fold distribution in the ribosomes announces the future power-law distribution where only a few folds are highly prevalent, and most are rare. Additionally, we highlight the presence of three copies of proto-Rossmann folds in ribosomes across all kingdoms, showing its ancient and fundamental role in ribosomal structure and function. Our study also explores early mechanisms of molecular convergence, where different protein folds bind equivalent ribosomal RNA structures in ribosomes across different kingdoms. This comparative analysis enhances our understanding of ribosomal evolution, particularly the distinct evolutionary paths of the large and small subunits, and underscores the complex interplay between RNA and protein components in the transition from the RNA world to modern cellular life. Transcending the concept of folds also makes it possible to group a large number of ribosomal proteins into five categories of urfolds or metafolds, which could attest to their ancestral character and common origins. This work also demonstrates that the gradual acquisition of extensions by simple but ordered folds constitutes an inexorable evolutionary mechanism. This observation supports the idea that simple but structured ribosomal proteins preceded the development of their disordered extensions.

Ribosome Biogenesis and Cancer: Insights into NOB1 and PNO1 Mechanisms.

Post-transcriptional modifications (PTMs) are pivotal in the regulation of gene expression, and pseudouridylation is emerging as a critical player. This modification, facilitated by enzymes such as NOB1 (PNO1), is integral to ribosome biogenesis. PNO1, in collaboration with the NIN1/RPN12 binding protein 1 homolog (NOB1), is vital for the maturation of ribosomes, transitioning 20S pre-rRNA into functional 18S rRNA. Recent studies have highlighted PNO1's potential involvement in cancer progression; however, its underlying mechanisms remain unclear. Relentless growth characterizing cancer underscores the burgeoning significance of epitranscriptomic modifications, including pseudouridylation, in oncogenesis. Given PNO1's emerging role, it is imperative to delineate its contribution to cancer development to identify novel therapeutic interventions. This review summarizes the current literature regarding the role of PNO1 in cancer progression and its molecular underpinnings in oncogenesis. Overexpression of PNO1 was associated with unfavorable prognosis and increased tumor malignancy. At the molecular level, PNO1 facilitates cancer progression by modulating mRNA stability, alternative splicing, and translation efficiency. Its role in pseudouridylation of oncogenic and tumor-suppressor transcripts further underscores its significance in cancer biology. Although disruption of ribosome biogenesis is known to precipitate oncogenesis, the precise mechanisms by which these alterations contribute to cancer remain unclear. This review elucidates the intricate process of ribosomal small subunit maturation, highlighting the roles of crucial ribosomal proteins (RPs) and RNA-binding proteins (RBPs) as well as the positioning and function of NOB1 and PNO1 within the 40S subunit. The involvement of these components in the maturation of the small subunit (SSU) and their significance in the context of cancer therapeutics has been thoroughly explored. PNO1's burgeoning significance in oncology makes it a potential target for cancer therapies. Strategies aimed at modulating PNO1-mediated pseudouridylation may provide new avenues for cancer treatment. However, further research is essential to unravel the complete spectrum of PNO1 mechanisms in cancer and harness this knowledge for the development of targeted and more efficacious anticancer therapies.

Multiomics-Based Biocargo Components Analysis in Enterococcus faecium Membrane Vesicles.

Enterococcus spp. have been shown to have gastrointestinal tract protective functions; our recent results suggest that membrane vesicles (MVs) play an important role in the gastric protection of Enterococcus faecium (E. faecium). The specific function is determined by molecular compositions of MVs. To resolve biocargo components in E. faecium MVs (EfmMVs), MVs were isolated from E. faecium culture. Transcriptomics, label-free quantitative proteomics, and untargeted metabolomics were performed to obtain information about the complexity of ribonucleic acids (RNAs), proteins, and metabolites biocargo they carry, respectively. RNA-sequencing identified a total of 2122 transcripts. The top 20 transcripts accounted for 27.63% of total counts, which, including enzymes, participate in glycolysis, ribosomal proteins, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, molecules associated with protein post-translational processing and transport, and peptidoglycan lyases. Label-free quantitative proteomics analysis identified a total of 711 proteins. The top 20 proteins accounted for 48.02% of all identified proteins, which including ribosomal proteins, enzymes participate in glycolysis, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, peptidoglycan lyases, and autolysin. Untargeted metabolomics analysis identified a total of 519 metabolites. The top 20 metabolites accounted for 79.55% of all identified metabolites, which included amino acids, substrates, or products in the metabolism of amino acids, natural organic acids, products in the metabolism of organic acids, ketone compounds, and two other compounds. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the identified biocargo components enriched in metabolism, genetic, and environmental information processing. Overall, we hope that the current exploration of multiple "-omics" analyses of this EfmMVs will provide useful information and further groundwork for future studies on E. faecium application.

Putting It All Together: The Roles of Ribosomal Proteins in Nucleolar Stages of 60S Ribosomal Assembly in the Yeast Saccharomyces cerevisiae.

Here we review the functions of ribosomal proteins (RPs) in the nucleolar stages of large ribosomal subunit assembly in the yeast Saccharomyces cerevisiae. We summarize the effects of depleting RPs on pre-rRNA processing and turnover, on the assembly of other RPs, and on the entry and exit of assembly factors (AFs). These results are interpreted in light of recent near-atomic-resolution cryo-EM structures of multiple assembly intermediates. Results are discussed with respect to each neighborhood of RPs and rRNA. We identify several key mechanisms related to RP behavior. Neighborhoods of RPs can assemble in one or more than one step. Entry of RPs can be triggered by molecular switches, in which an AF is replaced by an RP binding to the same site. To drive assembly forward, rRNA structure can be stabilized by RPs, including clamping rRNA structures or forming bridges between rRNA domains.

Fragment Screening to Identify Inhibitors Targeting Ribosome Binding of Shiga Toxin 2.

Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.

A Combined Transcriptomic and Proteomic Analysis of Monkeypox Virus A23 Protein on HEK293T Cells.

Monkeypox virus (MPXV) is a cross-kingdom pathogen infecting both humans and wildlife, which poses a significant health risk to the public. Although MPXV attracts broad attention, there is a lack of adequate studies to elucidate pathogenic mechanisms associated with viral infections. In this study, a high-throughput RNA sequencing (RNA-seq) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach was used to explore the transcriptional and metabolic responses of MPXV A23 protein to HEK293T cells. The protein-protein interactions and signaling pathways were conducted by GO and KEGG analyses. The localization of A23 protein in HEK293T cells was detected by immunofluorescence. A total of 648 differentially expressed genes (DEGs) were identified in cells by RNA-Seq, including 314 upregulated genes and 334 downregulated genes. Additionally, liquid chromatography-tandem mass spectrometry (LC-MS/MS) detected 115 cellular proteins that interact with the A23 proteins. Transcriptomic sequencing analysis revealed that transfection of MPXV A23 protein modulated genes primarily associated with cellular apoptosis and DNA damage repair. Proteomic analysis indicated that this protein primarily interacted with host ribosomal proteins and histones. Following the identification of the nuclear localization sequence RKKR within the A23 protein, a truncated mutant A23ΔRKKR was constructed to investigate the subcellular localization of A23 protein. The wild-type A23 protein exhibits a significantly higher nuclear-to-cytoplasmic ratio, exceeding 1.5, in contrast to the mutant A23ΔRKKR, which has a ratio of approximately 1. Immunofluorescence assays showed that the A23 protein was mainly localized in the nucleus. The integration of transcriptomics and proteomics analysis provides a comprehensive understanding of the interaction between MPXV A23 protein and the host. Our findings highlight the potential role of this enzyme in suppressing host antiviral immune responses and modulating host gene expression.

Machine Learning Assisted MALDI Mass Spectrometry for Rapid Antimicrobial Resistance Prediction in Clinicals.

Antimicrobial susceptibility testing (AST) plays a critical role in assessing the resistance of individual microbial isolates and determining appropriate antimicrobial therapeutics in a timely manner. However, conventional AST normally takes up to 72 h for obtaining the results. In healthcare facilities, the global distribution of vancomycin-resistant Enterococcus fecium (VRE) infections underscores the importance of rapidly determining VRE isolates. Here, we developed an integrated antimicrobial resistance (AMR) screening strategy by combining matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) with machine learning to rapidly predict VRE from clinical samples. Over 400 VRE and vancomycin-susceptible E. faecium (VSE) isolates were analyzed using MALDI-MS at different culture times, and a comprehensive dataset comprising 2388 mass spectra was generated. Algorithms including the support vector machine (SVM), SVM with L1-norm, logistic regression, and multilayer perceptron (MLP) were utilized to train the classification model. Validation on a panel of clinical samples (external patients) resulted in a prediction accuracy of 78.07%, 80.26%, 78.95%, and 80.54% for each algorithm, respectively, all with an AUROC above 0.80. Furthermore, a total of 33 mass regions were recognized as influential features and elucidated, contributing to the differences between VRE and VSE through the Shapley value and accuracy, while tandem mass spectrometry was employed to identify the specific peaks among them. Certain ribosomal proteins, such as A0A133N352 and R2Q455, were tentatively identified. Overall, the integration of machine learning with MALDI-MS has enabled the rapid determination of bacterial antibiotic resistance, greatly expediting the usage of appropriate antibiotics.

Transcriptomic analyses provide molecular insight into the cold stress response of cold-tolerant alfalfa

BackgroundDaye No.3 is a novel cultivar of alfalfa (Medicago sativa L.) that is well suited for cultivation in high-altitude regions such as the Qinghai‒Tibet Plateau owing to its high yield and notable cold resistance. However, the limited availability of transcriptomic information has hindered our investigation into the potential mechanisms of cold tolerance in this cultivar. Consequently, we conducted de novo transcriptome assembly to overcome this limitation. Subsequently, we compared the patterns of gene expression in Daye No. 3 during cold acclimatization and exposure to cold stress at various time points.ResultsA total of 15 alfalfa samples were included in the transcriptome assembly, resulting in 141.97 Gb of clean bases. A total of 441 DEGs were induced by cold acclimation, while 4525, 5016, and 8056 DEGs were identified at 12 h, 24 h, and 36 h after prolonged cold stress at 4 °C, respectively. The consistency between the RT‒qPCR and transcriptome data confirmed the accuracy and reliability of the transcriptomic data. KEGG enrichment analysis revealed that many genes related to photosynthesis were enriched under cold stress. STEM analysis demonstrated that genes involved in nitrogen metabolism and the TCA cycle were consistently upregulated under cold stress, while genes associated with photosynthesis, particularly antenna protein genes, were downregulated. PPI network analysis revealed that ubiquitination-related ribosomal proteins act as hub genes in response to cold stress. Additionally, the plant hormone signaling pathway was activated under cold stress, suggesting its vital role in the cold stress response of alfalfa.ConclusionsUbiquitination-related ribosomal proteins induced by cold acclimation play a crucial role in early cold signal transduction. As hub genes, these ubiquitination-related ribosomal proteins regulate a multitude of downstream genes in response to cold stress. The upregulation of genes related to nitrogen metabolism and the TCA cycle and the activation of the plant hormone signaling pathway contribute to the enhanced cold tolerance of alfalfa.

Metabolic disruption impairs ribosomal protein levels, resulting in enhanced aminoglycoside tolerance.

Aminoglycoside antibiotics target ribosomes and are effective against a wide range of bacteria. Here, we demonstrated that knockout strains related to energy metabolism in Escherichia coli showed increased tolerance to aminoglycosides during the mid-exponential growth phase. Contrary to expectations, these mutations did not reduce the proton motive force or aminoglycoside uptake, as there were no significant changes in metabolic indicators or intracellular gentamicin levels between wild-type and mutant strains. Our comprehensive proteomics analysis unveiled a noteworthy upregulation of proteins linked to the tricarboxylic acid (TCA) cycle in the mutant strains during the mid-exponential growth phase, suggesting that these strains compensate for the perturbation in their energy metabolism by increasing TCA cycle activity to maintain their membrane potential and ATP levels. Furthermore, our pathway enrichment analysis shed light on local network clusters displaying downregulation across all mutant strains, which were associated with both large and small ribosomal binding proteins, ribosome biogenesis, translation factor activity, and the biosynthesis of ribonucleoside monophosphates. These findings offer a plausible explanation for the observed tolerance of aminoglycosides in the mutant strains. Altogether, this research provides valuable insights into the mechanisms of aminoglycoside tolerance, paving the way for novel strategies to combat such cells.

Complement C3d enables protective immunity capable of distinguishing spontaneously transformed from non-transformed cells.

We show that intracellular soluble fragment 3d of complement (C3d) induces regression of spontaneous multiple myeloma in mice reducing tumor burden by 10 fold, after 8 weeks. C3d enables cell-mediated immunity to target multiple myeloma clones sparing non-transformed polyclonal B cells and plasma cells with lower mutation loads. We show that C3d increases the expression of ribosomal subunits associated with the translation of defective ribosomal products (DRiPs). C3d also decreases expression of protein arginine methyl transferase (PRMT) 5 which in turn relieves E2f1 repression increasing the expression of Lnc RNAs and derived peptides that evoke anti-tumor cellular immunity. The approach increases MHC-I expression by tumor cells and generates a CMI response that overcomes tumor immune-evasion strategies. Tumors are immunogenic in part because of somatic mutations that originate novel peptides that once presented on MHC engage cell-mediated immunity (CMI). However, in spite of the higher mutation load most tumors evade immunity. We discovered that a component of the complement system (C3d) overcomes tumor immune evasion by augmenting expression of ribosomal proteins and lncRNAs linked to the presentation of novel peptides by tumor cells. C3d induced CMI targets cancer cells sparing non transformed cells uncovering a novel function for complement in immune surveillance.