ABSTRACT
Bacterial adaptation is largely shaped by horizontal gene transfer, xenogeneic silencing mediated by lineage-specific DNA bridgers (H-NS, Lsr2, MvaT and Rok), and various anti-silencing mechanisms. No xenogeneic silencing DNA bridger is known for α-proteobacteria, from which mitochondria evolved. By investigating α-proteobacterium Sinorhizobium fredii, a facultative legume microsymbiont, here we report the conserved zinc-finger bearing MucR as a novel xenogeneic silencing DNA bridger. Self-association mediated by its N-terminal domain (NTD) is required for DNA-MucR-DNA bridging complex formation, maximizing MucR stability, transcriptional silencing, and efficient symbiosis in legume nodules. Essential roles of NTD, CTD (C-terminal DNA-binding domain), or full-length MucR in symbiosis can be replaced by non-homologous NTD, CTD, or full-length protein of H-NS from γ-proteobacterium Escherichia coli, while NTD rather than CTD of Lsr2 from Gram-positive Mycobacterium tuberculosis can replace the corresponding domain of MucR in symbiosis. Chromatin immunoprecipitation sequencing reveals similar recruitment profiles of H-NS, MucR and various functional chimeric xenogeneic silencers across the multipartite genome of S. fredii, i.e. preferring AT-rich genomic islands and symbiosis plasmid with key symbiosis genes as shared targets. Collectively, the convergently evolved DNA bridger MucR predisposed α-proteobacteria to integrate AT-rich foreign DNA including symbiosis genes, horizontal transfer of which is strongly selected in nature.
Subject(s)
Alphaproteobacteria , Gene Expression Regulation, Bacterial , Alphaproteobacteria/genetics , Bacterial Proteins/metabolism , DNA , Escherichia coli/genetics , Escherichia coli/metabolism , SymbiosisABSTRACT
BACKGROUND: The behaviors and ontogeny of Aedes aegypti are closely related to the spread of diseases caused by dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV) viruses. During the life cycle, Ae. aegypti undergoes drastic morphological, metabolic, and functional changes triggered by gene regulation and other molecular mechanisms. Some essential regulatory factors that regulate insect ontogeny have been revealed in other species, but their roles are still poorly investigated in the mosquito. RESULTS: Our study identified 6 gene modules and their intramodular hub genes that were highly associated with the ontogeny of Ae. aegypti in the constructed network. Those modules were found to be enriched in functional roles related to cuticle development, ATP generation, digestion, immunity, pupation control, lectins, and spermatogenesis. Additionally, digestion-related pathways were activated in the larvae and adult females but suppressed in the pupae. The integrated proteinâprotein network also identified cilium-related genes. In addition, we verified that the 6 intramodular hub genes encoding proteins such as EcKinase regulating larval molt were only expressed in the larval stage. Quantitative RTâPCR of the intramodular hub genes gave similar results as the RNA-Seq expression profile, and most hub genes were ontogeny-specifically expressed. CONCLUSIONS: The constructed gene coexpression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. Ultimately, these findings will be key in identifying potential molecular targets for disease control.
Subject(s)
Aedes , Dengue , Yellow Fever , Zika Virus Infection , Zika Virus , Male , Animals , Female , Yellow Fever/genetics , Zika Virus/genetics , Gene Regulatory Networks , Mosquito Vectors , Proteins/genetics , LarvaABSTRACT
We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.
Subject(s)
Click Chemistry/methods , Proteome/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , Animals , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , HeLa Cells , High-Throughput Nucleotide Sequencing/methods , Humans , Mass Spectrometry/methods , Mice , Protein Interaction Maps , RNA/genetics , RNA-Binding Proteins/genetics , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
Prokaryotes benefit from having accessory genes, but it is unclear how accessory genes can be linked with the core regulatory network when developing adaptations to new niches. Here we determined hierarchical core/accessory subsets in the multipartite pangenome (composed of genes from the chromosome, chromid and plasmids) of the soybean microsymbiont Sinorhizobium fredii by comparing twelve Sinorhizobium genomes. Transcriptomes of two S. fredii strains at mid-log and stationary growth phases and in symbiotic conditions were obtained. The average level of gene expression, variation of expression between different conditions, and gene connectivity within the co-expression network were positively correlated with the gene conservation level from strain-specific accessory genes to genus core. Condition-dependent transcriptomes exhibited adaptive transcriptional changes in pangenome subsets shared by the two strains, while strain-dependent transcriptomes were enriched with accessory genes on the chromid. Proportionally more chromid genes than plasmid genes were co-expressed with chromosomal genes, while plasmid genes had a higher within-replicon connectivity in expression than chromid ones. However, key nitrogen fixation genes on the symbiosis plasmid were characterized by high connectivity in both within- and between-replicon analyses. Among those genes with host-specific upregulation patterns, chromosomal znu and mdt operons, encoding a conserved high-affinity zinc transporter and an accessory multi-drug efflux system, respectively, were experimentally demonstrated to be involved in host-specific symbiotic adaptation. These findings highlight the importance of integrative regulation of hierarchical core/accessory components in the multipartite genome of bacteria during niche adaptation and in shaping the prokaryotic pangenome in the long run.
Subject(s)
Adaptation, Biological/genetics , Gene Expression Regulation, Bacterial , Plasmids/genetics , Sinorhizobium fredii/genetics , Symbiosis/genetics , Bacterial Proteins/genetics , Genes, Bacterial/genetics , Genome, Bacterial , Nitrogen Fixation/genetics , Replicon/genetics , Glycine max/microbiology , TranscriptomeABSTRACT
The intestinal epithelium has a high rate of cell turn over and is an excellent system to study stem cell-mediated tissue homeostasis. The Misshapen subfamily of the Ste20 kinases in mammals consists of misshapen like kinase 1 (MINK1), mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), and TRAF2 and NCK interacting kinase (TNIK). Recent reports suggest that this subfamily has a novel function equal to the Hippo/MST subfamily as upstream kinases for Warts/Large tumor suppressor kinase (LATS) to suppress tissue growth. To study the in vivo functions of Mink1, Map4k4, and Tnik, we generated a compound knockout of these three genes in the mouse intestinal epithelium. The intestinal epithelia of the mutant animals were phenotypically normal up to approximately 12 months. The older animals then exhibited mildly increased proliferation throughout the lower GI tract. We also observed that the normally spatially organized Paneth cells in the crypt base became dispersed. The expression of one of the YAP pathway target genes Sox9 was increased while other target genes including CTGF did not show a significant change. Therefore, the Misshapen and Hippo subfamilies may have highly redundant functions to regulate growth in the intestinal epithelium, as illustrated in recent tissue culture models.
Subject(s)
Aging , Cell Proliferation/physiology , Intestinal Mucosa/metabolism , Stem Cells/metabolism , Animals , Mice, Transgenic , Phosphorylation/physiologyABSTRACT
Receiving nodulation and nitrogen fixation genes does not guarantee rhizobia an effective symbiosis with legumes. Here, variations in gene content were determined for three Sinorhizobium species showing contrasting symbiotic efficiency on soybeans. A nitrate-reduction gene cluster absent in S. sojae was found to be essential for symbiotic adaptations of S. fredii and S. sp. III. In S. fredii, the deletion mutation of the nap (nitrate reductase), instead of nir (nitrite reductase) and nor (nitric oxide reductase), led to defects in nitrogen-fixation (Fix- ). By contrast, none of these core nitrate-reduction genes were required for the symbiosis of S. sp. III. However, within the same gene cluster, the deletion of hemN1 (encoding oxygen-independent coproporphyrinogen III oxidase) in both S. fredii and S. sp. III led to the formation of nitrogen-fixing (Fix+ ) but ineffective (Eff- ) nodules. These Fix+ /Eff- nodules were characterized by significantly lower enzyme activity of glutamine synthetase indicating rhizobial modulation of nitrogen-assimilation by plants. A distant homologue of HemN1 from S. sojae can complement this defect in S. fredii and S. sp. III, but exhibited a more pleotropic role in symbiosis establishment. These findings highlighted the lineage-dependent optimization of symbiotic functions in different rhizobial species associated with the same host.
Subject(s)
Glycine max/microbiology , Nitrite Reductases/genetics , Nitrogen Fixation/genetics , Sinorhizobium fredii/genetics , Sinorhizobium fredii/metabolism , Symbiosis/genetics , Coproporphyrinogen Oxidase/genetics , Glutamate-Ammonia Ligase/metabolism , Multigene Family/genetics , Oxidation-Reduction , Oxidoreductases/genetics , Rhizobium/geneticsABSTRACT
To achieve effective symbiosis with legume, rhizobia should fine-tune their background regulation network in addition to activating key genes involved in nodulation (nod) and nitrogen fixation (nif). Here, we report that an ancestral zinc finger regulator, MucR1, other than its paralog, MucR2, carrying a frameshift mutation, is essential for supporting nitrogen fixation of Sinorhizobium fredii CCBAU45436 within soybean nodules. In contrast to the chromosomal mucR1, mucR2 is located on symbiosis plasmid, indicating its horizontal transfer potential. A MucR2 homolog lacking the frameshift mutation, such as the one from S. fredii NGR234, can complement phenotypic defects of the mucR1 mutant of CCBAU45436. RNA-seq analysis revealed that the MucR1 regulon of CCBAU45436 within nodules exhibits significant difference compared with that of free-living cells. MucR1 is required for active expression of transporters for phosphate, zinc, and elements essential for nitrogenase activity (iron, molybdenum, and sulfur) in nodules but is dispensable for transcription of key genes (nif/fix) involved in nitrogen fixation. Further reverse genetics suggests that S. fredii uses high-affinity transporters to meet the demand for zinc and phosphate within nodules. These findings, together with the horizontal transfer potential of the mucR homolog, imply an intriguing evolutionary role of this ancestral regulator in supporting nitrogen fixation.
Subject(s)
Bacterial Proteins/metabolism , Glycine max/microbiology , Nitrogen Fixation/physiology , Root Nodules, Plant/microbiology , Sinorhizobium fredii/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Carrier Proteins , Frameshift Mutation , Gene Expression Regulation, Bacterial/physiology , Ion Transport , Phylogeny , RNA, Bacterial/genetics , Real-Time Polymerase Chain Reaction , Transcription, GeneticABSTRACT
RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvß3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvß3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.
Subject(s)
Neoplasms/therapy , Peptides, Cyclic/administration & dosage , RNA Interference , RNA, Small Interfering/administration & dosage , Animals , Cell Line, Tumor , Cells, Cultured , Female , Gene Knockdown Techniques , HeLa Cells , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice, Inbred BALB C , Mice, Nude , Neoplasms/blood supply , Neovascularization, Physiologic , Peptides, Cyclic/analysis , Peptides, Cyclic/chemistry , RNA, Small Interfering/chemistry , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Vascular Endothelial Growth Factor Receptor-2/genetics , Zebrafish/embryologyABSTRACT
NF-κB/p65 is retained in the cytoplasm until it is activated in response to stress. Nuclear import of p65 is regulated by importin α in a nuclear localization signal (NLS)-dependent manner. However, the role of importin ß family members in the nuclear translocation of p65 is largely unclear. In this study, using high-content siRNA screening, we identified three of 17 importin ß family members that are involved in the nuclear import of p65. Our data showed that knockdown of KPNB1, XPO7 and IPO8 reduced the amount of nuclear p65 following tumor necrosis factor-α (TNF-α) stimulation, resulting in lower NF-κB activity. KPNB1 was the major importin ß receptor for p65 import, and this import was dependent on the NLS of p65. However, NLS-mutated p65 still entered the nucleus and bound to XPO7 and IPO8. Interestingly, among the six members of the importin α family, KPNA2 was most important for p65 import. Taken together, our results show that the import of p65 mainly relies on the canonical KPNA2/KPNB1 pathway; however, p65 is also imported by an alternative pathway that is independent of its NLS. Redundant importin receptors are likely to maintain the important function of p65 according to need.
Subject(s)
Cell Nucleus/metabolism , Karyopherins/metabolism , Transcription Factor RelA/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus , HEK293 Cells , HeLa Cells , Humans , Karyopherins/genetics , Nuclear Localization Signals , Protein Binding , Transcription Factor RelA/chemistry , Transcription Factor RelA/genetics , alpha Karyopherins/genetics , alpha Karyopherins/metabolism , beta Karyopherins/genetics , ran GTP-Binding ProteinABSTRACT
Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.
ABSTRACT
Electrodeposited chromium plating continues to be widely used in a number of specialized areas, such as weapons, transport, aerospace, etc. However, the formation of texture, hydrogen content and residual stress can degrade the serviceability and lead to material failure. The effect of post heat treatment processes on the relationship of texture, hydrogen content, residual stress and corrosion resistance of hexavalent [Cr(VI)] chromium coatings deposited on Cr-Ni-Mo-V steel substrates was investigated. Macrotexture was measured by XRD. Microtexture, dislocation density and grain size were studied by EBSD. With the increase of the heat treatment temperature, it was found that the fiber texture strength of the (222) plane tended to increase and subsequently decrease. Below 600 °C, the increase in the (222) plane texture carried a decrease in the hydrogen content, residual stress, microhardness and an increase in the corrosion resistance. In addition, crack density and texture strength were less affected by the heat treatment time. Notably, relatively fewer crack densities of 219/cm2, a lower corrosion current density of 1.798 × 10-6 A/dm2 and a higher microhardness of 865 HV were found under the preferred heat treatment temperature and time of 380 °C and 4 h, respectively. The hydrogen content and residual stress were 7.63 ppm and 61 MPa, with 86% and 75% reduction rates compared to the as-plated state, respectively. In conclusion, in our future judgement of the influence of heat treatment on coating properties, we can screen or determine to a certain extent whether the heat treatment process is reasonable or not by measuring only the macrotexture.
ABSTRACT
The COVID-19 pandemic caused by SARS-CoV-2 has had a persistent and significant impact on global public health for 4 years. Recently, there has been a resurgence of seasonal influenza transmission worldwide. The co-circulation of SARS-CoV-2 and seasonal influenza viruses results in a dual burden on communities. Additionally, the pandemic potential of zoonotic influenza viruses, such as avian Influenza A/H5N1 and A/H7N9, remains a concern. Therefore, a combined vaccine against all these respiratory diseases is in urgent need. mRNA vaccines, with their superior efficacy, speed in development, flexibility, and cost-effectiveness, offer a promising solution for such infectious diseases and potential future pandemics. In this study, we present FLUCOV-10, a novel 10-valent mRNA vaccine created from our proven platform. This vaccine encodes hemagglutinin (HA) proteins from four seasonal influenza viruses and two avian influenza viruses with pandemic potential, as well as spike proteins from four SARS-CoV-2 variants. A two-dose immunization with the FLUCOV-10 elicited robust immune responses in mice, producing IgG antibodies, neutralizing antibodies, and antigen-specific cellular immune responses against all the vaccine-matched viruses of influenza and SARS-CoV-2. Remarkably, the FLUCOV-10 immunization provided complete protection in mouse models against both homologous and heterologous strains of influenza and SARS-CoV-2. These results highlight the potential of FLUCOV-10 as an effective vaccine candidate for the prevention of influenza and COVID-19.IMPORTANCEAmidst the ongoing and emerging respiratory viral threats, particularly the concurrent and sequential spread of SARS-CoV-2 and influenza, our research introduces FLUCOV-10. This novel mRNA-based combination vaccine, designed to counteract both influenza and COVID-19, by incorporating genes for surface glycoproteins from various influenza viruses and SARS-CoV-2 variants. This combination vaccine was highly effective in preclinical trials, generating strong immune responses and ensuring protection against both matching and heterologous strains of influenza viruses and SARS-CoV-2. FLUCOV-10 represents a significant step forward in our ability to address respiratory viral threats, showcasing potential as a singular, adaptable vaccine solution for global health challenges.
ABSTRACT
Detection of RNA synthesis in cells to measure the rate of total transcription is an important experimental technique. To screen the best nucleoside analogue for labeling RNA synthesis, a series of alkyne-modified nucleoside analogues, including 5-ethynylcytidine (EC) and 8-ethynyladenosine (EA), were successfully synthesized by the Sonogashira coupling reaction. The synthesis of RNA or DNA was assayed based on the biosynthetic incorporation of these analogues into newly transcribed RNA or replicating DNA. Analogue-labeled cellular RNA or DNA was detected quickly and with high sensitivity via "click" chemistry with fluorescent azides, followed by fluorescence microscopic imaging. The results showed that EC was efficiently incorporated into RNA, but not into DNA, in seven cell lines, as also previously shown for 5-ethynyluridine (EU). Moreover, EC was able to assay transcription rates of various tissues in animals and the rate of metabolism of EC was much faster than that of EU.
Subject(s)
Cytidine/analogs & derivatives , Microscopy, Fluorescence , RNA/analysis , Alkynes/chemistry , Animals , Azides/chemistry , Cell Line , Click Chemistry , Cytidine/chemical synthesis , Cytidine/chemistry , Fluorescent Dyes/chemistry , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Intestine, Large/metabolism , Kidney/metabolism , Liver/metabolism , Mice , RNA/metabolismABSTRACT
microRNAs play an important roles in cell growth, differentiation, proliferation and apoptosis. They can function either as tumor suppressors or oncogenes. We found that the overexpression of miR-192 inhibited cell proliferation in A549, H460 and 95D cells, and inhibited tumorigenesis in a nude mouse model. Both caspase-7 and the PARP protein were activated by the overexpression of miR-192, thus suggesting that miR-192 induces cell apoptosis through the caspase pathway. Further studies showed that retinoblastoma 1 (RB1) is a direct target of miR-192. Over-expression of miR-192 decreased RB1 mRNA and protein levels and repressed RB1-3'-UTR reporter activity. Knockdown of RB1 using siRNA resulted in a similar cell morphology as that observed for overexpression of miR-192. Additionally, RB1-siRNA treatment inhibited cell proliferation and induced cell apoptosis in lung cancer cells. Analysis of miRNA expression in clinical samples showed that miR-192 is significantly downregulated in lung cancer tissues compared to adjacent non-cancerous lung tissues. In conclusion, our results demonstrate that miR-192 is a tumor suppressor that can target the RB1 gene to inhibit cell proliferation and induce cell apoptosis in lung cancer cells. Furthermore, miR-192 was expressed at low levels in lung cancer samples, indicating that it might be a promising therapeutic target for lung cancer treatment.
Subject(s)
Apoptosis , Lung Neoplasms/genetics , MicroRNAs/metabolism , Retinoblastoma Protein/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, Nude , RNA Interference , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolismABSTRACT
Recent advancements in the production, modification, and cellular delivery of RNA molecules facilitated the expansion of RNA-based therapeutics. The increasing understanding of RNA biology initiated a corresponding growth in RNA therapeutics. In this review, the general concepts of five classes of RNA-based therapeutics, including RNA interference-based therapies, antisense oligonucleotides, small activating RNA therapies, circular RNA therapies, and messenger RNA-based therapeutics, will be discussed. Moreover, we also provide an overview of RNA-based therapeutics that have already received regulatory approval or are currently being evaluated in clinical trials, along with challenges faced by these technologies. RNA-based drugs demonstrated positive clinical trial results and have the ability to address previously "undruggable" targets, which delivers great promise as a disruptive therapeutic technology to fulfill its full clinical potentiality.
Subject(s)
Oligonucleotides, Antisense , RNA, Small Interfering/genetics , RNA, Small Interfering/therapeutic use , RNA Interference , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/therapeutic use , RNA, Messenger/geneticsABSTRACT
The distribution and abundance of transposable elements across the tree of life have significantly shaped the evolution of cellular organisms, but the underlying mechanisms shaping these ecological patterns remain elusive. Here we establish a "common garden" approach to study causal ecological interactions between a xenogeneic conditional lethal sacB gene and the community of transposable insertion sequences (ISs) in a multipartite prokaryote genome. Xenogeneic sacB of low, medium, or high GC content was individually inserted into three replicons of a model bacterium Sinorhizobium fredii, and exhibited replicon- and GC-dependent variation in genetic stability. This variation was largely attributable to multidimensional niche differentiation for IS community members. The transposition efficiency of major active ISs depended on the nucleoid-associated xenogeneic silencer MucR. Experimentally eliminating insertion activity of specific ISs by deleting MucR strongly demonstrated a dominant role of niche differentiation among ISs. This intracellular common garden approach in the experimental evolution context allows not only for evaluating genetic stability of natural and synthetic xenogeneic genes of different sequence signatures in host cells but also for tracking and testing causal relationships in unifying ecological principles in genome ecology.
Subject(s)
DNA Transposable Elements , Genome, Bacterial , Bacteria/genetics , Prokaryotic Cells , RepliconABSTRACT
Migration from rhizosphere to rhizoplane is a key selecting process in root microbiome assembly, but not fully understood. Rhizobiales members are overrepresented in the core root microbiome of terrestrial plants, and here we report a genome-wide transposon-sequencing of rhizoplane fitness genes of beneficial Sinorhizobium fredii on wild soybean, cultivated soybean, rice, and maize. There were few genes involved in broad-host-range rhizoplane colonization. The fadL mutant lacking a fatty acid transporter exhibited high colonization rates, while mutations in exoFQP (encoding membrane proteins directing exopolysaccharide polymerization and secretion), but not those in exo genes essential for exopolysaccharide biosynthesis, led to severely impaired colonization rates. This variation was not explainable by their rhizosphere and rhizoplane survivability, and associated biofilm and exopolysaccharide production, but consistent with their migration ability toward rhizoplane, and associated surface motility and the mixture of quorum-sensing AHLs (N-acylated-L-homoserine lactones). Genetics and physiology evidences suggested that FadL mediated long-chain AHL uptake while ExoF mediated the secretion of short-chain AHLs which negatively affected long-chain AHL biosynthesis. The fadL and exoF mutants had elevated and depleted extracellular long-chain AHLs, respectively. A synthetic mixture of long-chain AHLs mimicking that of the fadL mutant can improve rhizobial surface motility. When this AHL mixture was spotted into rhizosphere, the migration toward roots and rhizoplane colonization of S. fredii were enhanced in a diffusible way. This work adds novel parts managing extracellular AHLs, which modulate bacterial migration toward rhizoplane. The FadL-ExoFQP system is conserved in Alphaproteobacteria and may shape the "home life" of diverse keystone rhizobacteria.
Subject(s)
Rhizobium , Bacteria/genetics , Quorum Sensing , Biofilms , Fatty Acids , Acyl-Butyrolactones/metabolismABSTRACT
Introduction: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant has rapidly spread around the globe. With a substantial number of mutations in its Spike protein, the SARS-CoV-2 Omicron variant is prone to immune evasion and led to the reduced efficacy of approved vaccines. Thus, emerging variants have brought new challenges to the prevention of COVID-19 and updated vaccines are urgently needed to provide better protection against the Omicron variant or other highly mutated variants. Materials and methods: Here, we developed a novel bivalent mRNA vaccine, RBMRNA-405, comprising a 1:1 mix of mRNAs encoding both Delta-derived and Omicron-derived Spike proteins. We evaluated the immunogenicity of RBMRNA-405 in BALB/c mice and compared the antibody response and prophylactic efficacy induced by monovalent Delta or Omicron-specific vaccine with the bivalent RBMRNA-405 vaccine in the SARSCoV-2 variant challenge. Results: Results showed that the RBMRNA-405 vaccine could generate broader neutralizing antibody responses against both Wuhan-Hu-1 and other SARS-CoV-2 variants, including Delta, Omicron, Alpha, Beta, and Gamma. RBMRNA-405 efficiently blocked infectious viral replication and lung injury in both Omicron- and Delta-challenged K18-ACE2 mice. Conclusion: Our data suggest that RBMRNA-405 is a promising bivalent SARS-CoV-2 vaccine with broad-spectrum efficacy for further clinical development.
Subject(s)
COVID-19 Vaccines , COVID-19 , Animals , Humans , Mice , SARS-CoV-2 , COVID-19/prevention & control , Mice, Inbred BALB C , RNA, Messenger , Vaccines, Combined , mRNA VaccinesABSTRACT
MicroRNAs (miRNAs) are emerging critical regulators of cell function that frequently reside in clusters throughout the genome. They influence a myriad of cell functions, including the generation of induced pluripotent stem cells, also termed reprogramming. Here, we have successfully delivered entire miRNA clusters into reprogramming fibroblasts using retroviral vectors. This strategy avoids caveats associated with transient transfection of chemically synthesized miRNA mimics. Overexpression of 2 miRNA clusters, 106a-363 and in particular 302-367, allowed potent increases in induced pluripotent stem cell generation efficiency in mouse fibroblasts using 3 exogenous factors (Sox2, Klf4, and Oct4). Pathway analysis highlighted potential relevant effectors, including mesenchymal-to-epithelial transition, cell cycle, and epigenetic regulators. Further study showed that miRNA cluster 302-367 targeted TGFß receptor 2, promoted increased E-cadherin expression, and accelerated mesenchymal-to-epithelial changes necessary for colony formation. Our work thus provides an interesting alternative for improving reprogramming using miRNAs and adds new evidence for the emerging relationship between pluripotency and the epithelial phenotype.
Subject(s)
MicroRNAs/metabolism , Transforming Growth Factor beta/metabolism , Animals , Cell Adhesion , Epithelial Cells/cytology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Factor 4 , Male , Mesoderm/cytology , Mice , MicroRNAs/genetics , Phenotype , Stem Cells/cytologyABSTRACT
Nuclear translocation of Smad proteins is a critical step in signal transduction of transforming growth factor beta (TGF-beta) and bone morphogenetic proteins (BMPs). Using nuclear accumulation of the Drosophila Smad Mothers against Decapentaplegic (Mad) as the readout, we carried out a whole-genome RNAi screening in Drosophila cells. The screen identified moleskin (msk) as important for the nuclear import of phosphorylated Mad. Genetic evidence in the developing eye imaginal discs also demonstrates the critical functions of msk in regulating phospho-Mad. Moreover, knockdown of importin 7 and 8 (Imp7 and 8), the mammalian orthologues of Msk, markedly impaired nuclear accumulation of Smad1 in response to BMP2 and of Smad2/3 in response to TGF-beta. Biochemical studies further suggest that Smads are novel nuclear import substrates of Imp7 and 8. We have thus identified new evolutionarily conserved proteins that are important in the signal transduction of TGF-beta and BMP into the nucleus.