ABSTRACT
The E26 transformation-specific or E-twenty-six (ETS) genes encode a superfamily of transcription factors involved in diverse biological processes. Here, we report the identification and characterization of a previously unidentified member of the ETS transcription factors, Spi2, that is found exclusively in the ray-finned fish kingdom. We show that the expression of spi2 is restricted to hemogenic endothelial cells (HECs) and to hematopoietic stem and progenitor cells (HSPCs) in zebrafish. Using bacteria artificial chromosome transgenesis, we generate a spi2 reporter line, TgBAC(spi2:P2a-GFP), which manifests the GFP pattern recapitulating the endogenous spi2 expression. Genetic ablation of spi2 has little effect on HEC formation and the endothelial-to-hematopoietic transition, but results in compromised proliferation of HSPCs in the caudal hematopoietic tissue (CHT) during early development and in severe myeloid lineage defect in adulthood. Epistatic analysis shows that spi2 acts downstream of runx1 in regulating HSPC development in the CHT. Our study identifies Spi2 as an essential regulator for definitive hematopoietic cell development and creates a TgBAC(spi2:P2a-GFP) reporter line for tracking HECs, HSPCs, myeloid cells and thrombocytes from early development to adulthood.
Subject(s)
Hemangioblasts , Zebrafish , Animals , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Hemangioblasts/metabolism , Hematopoiesis/genetics , Proto-Oncogene Proteins c-ets/genetics , Proto-Oncogene Proteins c-ets/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Type I and type II IFNs are important immune modulators in both innate and adaptive immunity. They transmit signaling by activating JAK-STAT pathways. Sirtuin 1 (SIRT1), a class III NAD+-dependent deacetylase, has multiple functions in a variety of physiological processes. Here, we characterized the novel functions of SIRT1 in the regulation of type I and type II IFN-induced signaling. Overexpression of SIRT1 inhibited type I and type II IFN-induced interferon-stimulated response element activation. In contrast, knockout of SIRT1 promoted type I and type II IFN-induced expression of ISGs and inhibited viral replication. Treatment with SIRT1 inhibitor EX527 had similar positive effects. SIRT1 physically associated with STAT1 or STAT3, and this interaction was enhanced by IFN stimulation or viral infection. By deacetylating STAT1 at K673 and STAT3 at K679/K685/K707/K709, SIRT1 downregulated the phosphorylation of STAT1 (Y701) and STAT3 (Y705). Sirt1+/- primary peritoneal macrophages and Sirt1+/- mice exhibited enhanced IFN-induced signaling and antiviral activity. Thus, SIRT1 is a novel negative regulator of type I and type II IFN-induced signaling through its deacetylase activity.IMPORTANCESIRT1 has been reported in the precise regulation of antiviral (RNA and DNA) immunity. However, its functions in type I and type II IFN-induced signaling are still unclear. In this study, we deciphered the important functions of SIRT1 in both type I and type II IFN-induced JAK-STAT signaling and explored the potential acting mechanisms. It is helpful for understanding the regulatory roles of SIRT1 at different levels of IFN signaling. It also consolidates the notion that SIRT1 is an important target for intervention in viral infection, inflammatory diseases, or even interferon-related therapies.
Subject(s)
Interferon Type I , Sirtuin 1 , Virus Diseases , Animals , Mice , Immunity, Innate , Interferon Type I/metabolism , Interferon-gamma , Phosphorylation , Signal Transduction , Sirtuin 1/genetics , Sirtuin 1/metabolism , STAT1 Transcription Factor/metabolism , Virus Diseases/immunologyABSTRACT
The fundamental understanding of sluggish hydrogen evolution reaction (HER) kinetics on a platinum (Pt) surface in alkaline media is a topic of considerable debate. Herein, we combine cyclic voltammetry (CV) and electrical transport spectroscopy (ETS) approaches to probe the Pt surface at different pH values and develop molecular-level insights into the pH-dependent HER kinetics in alkaline media. The change in HER Tafel slope from â¼110 mV/decade in pH 7-10 to â¼53 mV/decade in pH 11-13 suggests considerably enhanced kinetics at higher pH. The ETS studies reveal a similar pH-dependent switch in the ETS conductance signal at around pH 10, suggesting a notable change of surface adsorbates. Fixed-potential calculations and chemical bonding analysis suggest that this switch is attributed to a change in interfacial water orientation, shifting from primarily an O-down configuration below pH 10 to a H-down configuration above pH 10. This reorientation weakens the O-H bond in the interfacial water molecules and modifies the reaction pathway, leading to considerably accelerated HER kinetics at higher pH. Our integrated studies provide an unprecedented molecular-level understanding of the nontrivial pH-dependent HER kinetics in alkaline media.
ABSTRACT
A new method of harmonic beam coaxial combination (HBCC) from two intra-cavity frequency doubling branches was demonstrated. Firstly, two identical nanosecond (ns) 532â nm green lasers with high power and good beam quality were created. Each green laser was constructed of an intra-cavity frequency doubling branch based on a laser diode (LD) end-pumped acousto-optical (AO) Q-switched 1064â nm Nd:YVO4 laser in a LiB3O5 (LBO) nonlinear crystal. Each branch generated about 45â W green output at a 50â kHz pulse repetition rate (PRR) with diffraction limited beam quality. The first green beam was injected into the LBO crystal in the second branch, and the pulses from the two branches did not exist simultaneously. Then, the HBCC was performed. Consequently, an 83â W combined green output power at 532â nm was obtained with a combination efficiency of 92.2%. The PRR of the HBCC pulse was doubled to be 100â kHz, with a pulse width of about 22â ns, corresponding to a single pulse energy of 0.83â mJ and a peak power of 37.73â kW. The combined beam quality factor was measured to be M x2 = 1.80 in the x direction and M y2 = 1.71 in the y direction, respectively. Moreover, many more beams could also be combined with this method for further scaling the green power.
ABSTRACT
In solid state systems, group representation theory is powerful in characterizing the behavior of quasiparticles, notably the energy degeneracy. While conventional group theory is effective in answering yes-or-no questions related to symmetry breaking, its application to determining the magnitude of energy splitting resulting from symmetry lowering is limited. Here, we propose a theory on quasisymmetry and near degeneracy, thereby expanding the applicability of group theory to address questions regarding large-or-small energy splitting. Defined within the degenerate subspace of an unperturbed Hamiltonian, quasisymmetries form an enlarged symmetry group eliminating the first-order splitting. This framework ensures that the magnitude of splitting arises as a second-order effect of symmetry-lowering perturbations, such as external fields and spin-orbit coupling. We systematically tabulate the quasisymmetry groups within 32 crystallographic point groups and find all the possible unitary quasisymmetry group structures regarding double degeneracy. Applying our theory to the realistic material AgLa, we predict a "quasi-Dirac semimetal" phase characterized by two tiny-gap band anticrossings.
ABSTRACT
The mesenchymal epithelial transition factor (c-Met) is frequently overexpressed in numerous cancers and has served as a validated anticancer target. Inter- and intra-tumor heterogeneity of c-Met, however, challenges the use of anti-MET therapies, highlighting an urgent need to develop an alternative tool for visualizing whole-body c-Met expression quantitatively and noninvasively. Here we firstly reported an 18F labeled, small-molecule quinine compound-based PET probe, 1-(4-(5-amino-7-(trifluoromethyl) quinolin-3-yl) piperazin-1-yl)-2-(fluoro-[18F]) propan-1-one, herein referred as [18F]-AZC. METHODS: [18F]-AZC was synthesized via a one-step substitution reaction and characterized by radiochemistry methods. [18F]-AZC specificity and affinity toward c-Met were assessed by cell uptake assay, with or without cold compound [19F]-AZC or commercial c-Met inhibitor blocking. MicroPET/CT imaging and biodistribution studies were conducted in subcutaneous murine xenografts of glioma. Additionally, [18F]-AZC was then further evaluated in orthotopic glioma xenografts, by microPET/CT imaging accompanied with MRI and autoradiography for co-registration of the tumor. Immunofluorescence staining was also carried out to qualitatively evaluate the c-Met expression in tumor tissue, co-localizes with H&E staining. RESULTS: This probe shows easy radiosynthesis, high stability in vitro and in vivo, high targeting affinity, and favorable lipophilicity and brain transport coefficient. [18F]-AZC demonstrates excellent tumor imaging properties in vivo and can delineate c-Met positive glioma specifically at 1 h after intravenous injection of the probe. Moreover, favorable correlation was observed between the [18F]-AZC accumulation and the amount of c-Met expression in tumor. CONCLUSION: This novel imaging probe could be applied as a valuable tool for management of anti-c-Met therapies in patients in the future.
Subject(s)
Glioma , Positron-Emission Tomography , Humans , Mice , Animals , Tissue Distribution , Positron-Emission Tomography/methods , Glioma/pathology , Biological Transport , Cell Line, Tumor , Fluorine RadioisotopesABSTRACT
OBJECTIVE: This Bayesian network meta-analysis was performed to analyze the associations between clinicopathological characteristics and BRAF mutations in ameloblastoma (AM) patients and to evaluate the diagnostic accuracy. MATERIALS AND METHODS: Four electronic databases were searched from 2010 to 2024. The search terms used were specific to BRAF and AM. Observational studies or randomized controlled trials were considered eligible. The incidence of BRAF mutation and corresponding clinicopathological features in AM patients were subjected to Bayesian network analyses and diagnostic accuracy evaluation. RESULTS: A total of 937 AM patients from 20 studies were included. The pooled prevalence of BRAF mutations in AM patients was 72%. According to the Bayesian network analysis, BRAF mutations are more likely to occur in younger (odds ratio [OR], 2.3; credible interval [CrI]: 1.2-4.5), mandible site (OR, 3.6; 95% CrI: 2.7-5.2), and unicystic (OR, 1.6; 95% CrI: 1.1-2.4) AM patients. Similarly, higher diagnostic accuracy was found in the younger, mandible, and unicystic AM groups. CONCLUSIONS: The incidence, risk, and diagnostic accuracy of BRAF mutation in AM were greater in younger patients, those with mandible involvement, and those with unicystic AM than in patients with other clinicopathological features. In addition, there was a strong concordance in the diagnostic accuracy between molecular tests and immunohistochemical analysis.
Subject(s)
Ameloblastoma , Bayes Theorem , Mutation , Proto-Oncogene Proteins B-raf , Ameloblastoma/genetics , Ameloblastoma/pathology , Humans , Proto-Oncogene Proteins B-raf/genetics , Jaw Neoplasms/genetics , Jaw Neoplasms/pathology , Network Meta-Analysis , Male , Female , Adult , Middle AgedABSTRACT
Bruton's tyrosine kinase (BTK) has emerged as a therapeutic target for B-cell malignancies, which is substantiated by the efficacy of various irreversible or reversible BTK inhibitors. However, on-target BTK mutations facilitating evasion from BTK inhibition lead to resistance that limits the therapeutic efficacy of BTK inhibitors. In this study we employed structure-based drug design strategies based on established BTK inhibitors and yielded a series of BTK targeting compounds. Among them, compound S-016 bearing a unique tricyclic structure exhibited potent BTK kinase inhibitory activity with an IC50 value of 0.5 nM, comparable to a commercially available BTK inhibitor ibrutinib (IC50 = 0.4 nM). S-016, as a novel irreversible BTK inhibitor, displayed superior kinase selectivity compared to ibrutinib and significant therapeutic effects against B-cell lymphoma both in vitro and in vivo. Furthermore, we generated BTK inhibitor-resistant lymphoma cells harboring BTK C481F or A428D to explore strategies for overcoming resistance. Co-culture of these DLBCL cells with M0 macrophages led to the polarization of M0 macrophages toward the M2 phenotype, a process known to support tumor progression. Intriguingly, we demonstrated that SYHA1813, a compound targeting both VEGFR and CSF1R, effectively reshaped the tumor microenvironment (TME) and significantly overcame the acquired resistance to BTK inhibitors in both BTK-mutated and wild-type BTK DLBCL models by inhibiting angiogenesis and modulating macrophage polarization. Overall, this study not only promotes the development of new BTK inhibitors but also offers innovative treatment strategies for B-cell lymphomas, including those with BTK mutations.
ABSTRACT
OBJECTIVE: In this study, our aim was to develop and validate the effectiveness of diverse radiomic models for distinguishing between gnathic fibrous dysplasia (FD) and ossifying fibroma (OF) before surgery. MATERIALS AND METHODS: We enrolled 220 patients with confirmed FD or OF. We extracted radiomic features from nonenhanced CT images. Following dimensionality reduction and feature selection, we constructed radiomic models using logistic regression, support vector machine, random forest, light gradient boosting machine, and eXtreme gradient boosting. We then identified the best radiomic model using receiver operating characteristic (ROC) curve analysis. After combining radiomics features with clinical features, we developed a comprehensive model. ROC curve and decision curve analysis (DCA) demonstrated the models' robustness and clinical value. RESULTS: We extracted 1834 radiomic features from CT images, reduced them to eight valuable features, and achieved high predictive efficiency, with area under curves (AUC) exceeding 0.95 for all the models. Ultimately, our combined model, which integrates radiomic and clinical data, displayed superior discriminatory ability (AUC: training cohort 0.970; test cohort 0.967). DCA highlighted its optimal clinical efficacy. CONCLUSION: Our combined model effectively differentiates between FD and OF, offering a noninvasive and efficient approach to clinical decision-making.
ABSTRACT
Chemoresistance is the primary reason for poor prognosis in patients with pancreatic cancer (PC). Recent studies have indicated that ferroptosis may improve chemoresistance, but the underlying mechanisms remain unclear. In this study, significant upregulation of heat shock protein 90α (Hsp90α) expression is detected in the peripheral blood and tissue samples of patients with chemoresistant PC. Further studies reveal that Hsp90α promotes the proliferation, migration, and invasion of a chemoresistant pancreatic cell line (Panc-1-gem) by suppressing ferroptosis. Hsp90α competitively binds to Kelch-like ECH-associated protein 1 (Keap1), liberating nuclear factor erythroid 2-related factor 2 (Nrf2) from Keap1 sequestration. Nrf2 subsequently translocates into the nucleus and activates the glutathione peroxidase 4 (GPX4) pathway, thereby suppressing ferroptosis. This process further worsens the chemoresistance of PC cells. This study provides valuable insight into potential molecular targets to overcome chemoresistance in PC. It sheds light on the intricate mechanisms linking Hsp90α and ferroptosis to chemoresistance in PC and provides a theoretical foundation for the development of novel therapeutic strategies.
ABSTRACT
Cadmium (Cd) pollution in soil poses a global concern due to its serious impacts on human health and ecological security. In plants, tremendous efforts have been made to identify some key genes and pathways in Cd stress responses. However, studies on the roles of epigenetic factors in response to Cd stress were still limited. In the study, we first gain insight into the gene expression dynamics for maize seedlings under 0â¯h, 12â¯h, and 72â¯hâ¯Cd stress. As a result, six distinct groups of genes were identified by hierarchical clustering and principal component analysis. The key pathways associated with 12â¯hâ¯Cd stress were protein modifications including protein ubiquitination, signal transduction by protein phosphorylation, and histone modification. Whereas, under 72â¯h stress, main pathways were involved in biological processes including phenylalanine metabolism, response to oxygen-containing compounds and metal ions. Then to be noted, one of the most highly expressed genes at 12â¯h under Cd treatment is annotated as histone demethylases (ZmJMJ20). The evolutionary tree analysis and domain analysis showed that ZmJMJ20 belonged to the JmjC-only subfamily of the Jumonji-C (JmjC) family, and ZmJMJ20 was conserved in rice and Arabidopsis. After 72â¯h of Cd treatment, the zmjmj20 mutant created by EMS treatment manifested less severe chlorosis/leaf yellowing symptoms compared with wild-type plants, and there was no significant difference in Fv/Fm and φPSII value before and after Cd treatment. Moreover, the expression levels of several photosynthesis-related down-regulated genes in EMS mutant plants were dramatically increased compared with those in wild-type plants at 12â¯h under Cd treatment. Our results suggested that ZmJMJ20 plays an important role in the Cd tolerance response pathway and will facilitate the development of cultivars with improved Cd stress tolerance.
Subject(s)
Cadmium , Gene Expression Profiling , Gene Expression Regulation, Plant , Soil Pollutants , Stress, Physiological , Zea mays , Zea mays/genetics , Zea mays/drug effects , Cadmium/toxicity , Soil Pollutants/toxicity , Stress, Physiological/drug effects , Gene Expression Regulation, Plant/drug effects , Histone Demethylases/genetics , Histone Demethylases/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Seedlings/drug effects , Seedlings/geneticsABSTRACT
Periodic assembly of heterogeneous nanoparticles provides a strategy for integrating distinct nanocatalyst blocks where their synergic effects can be explored for diverse applications. To achieve the synergistic enhancement, an intimate clean interface is preferred which however is usually plagued by the bulky surfactant molecules used in the synthesis and assembly process. Herein, we showed the creation of one-dimensional Pt-Au nanowires (NWs) with periodic alternating Pt and Au nanoblocks, by assembling Pt-Au Janus nanoparticles with the assistance of peptide T7 (Ac-TLTTLTN-CONH2). It is demonstrated that the Pt-Au NWs showed much-improved performance in the methanol oxidation reaction (MOR), exhibiting 5.3 times higher specific activity and 2.5 times higher mass activity than the current state-of-the-art commercial Pt/C catalyst. In addition, the periodic heterostructure also improves the stability of Pt-Au NWs in the MOR, where the Pt-Au NWs retained 93.9% of their initial mass activity much higher than commercial Pt/C (30.6%).
ABSTRACT
BACKGROUND: Amino acids (AAs) are the building blocks of proteins, but they also serve as biological compounds in biochemical processes, and d-AA isomers are increasingly being recognized as important signaling molecules. As the main organic substrate used by cells in the intestinal tract, the role of the chiral specificity of glutamine is still largely ignored. RESULTS: In a previous study, we found that d-glutamine affected the quorum sensing of Lactiplantibacillus plantarum A3, promoted the release of signaling molecule AI-2 and up-regulated the expression of the LuxS gene. The results showed that when d-glutamine and L. plantarum A3 were simultaneously applied to a mouse model, the diversity and abundance of intestinal flora in both male and female mice were increased. Interestingly, the simultaneous effect of d-glutamine and L. plantarum A3 on the bacterial diversity and abundance of male mice was significantly higher than that of female mice. In addition, the combination of d-glutamine and L. plantarum A3 can improve the host microecology by enhancing the population of Firmicutes such as Lactobacillus and Lachnospiraceae, reducing the population of Fusobacterium and Bacteroides and affecting metabolic pathways such as AA metabolism and transporter transport. CONCLUSION: d-Glutamine, as a signaling molecule, can better stimulate the endogenous d-glutamine synthesis in mice and be utilized by L. plantarum A3. Furthermore, sex differences in the changes of intestinal microflora are also found in this research. This research sheds some light on the adoption of d-AAs combined with lactic acid bacteria in intestinal tract health treatment. © 2024 Society of Chemical Industry.
Subject(s)
Gastrointestinal Microbiome , Glutamine , Probiotics , Quorum Sensing , Animals , Mice , Gastrointestinal Microbiome/drug effects , Female , Male , Quorum Sensing/drug effects , Probiotics/administration & dosage , Probiotics/pharmacology , Glutamine/metabolism , Glutamine/pharmacology , Lactobacillus plantarum/metabolism , Lactobacillus plantarum/chemistry , Bacteria/genetics , Bacteria/classification , Bacteria/metabolism , Bacteria/drug effects , Bacteria/isolation & purificationABSTRACT
Long interspersed elements-1(LINE-1) is the only autonomous transposon in human genomeï¼and its retrotransposition results in change of cellular genome structure and function, leading occurrence of various severe diseases. As a central key intermediated component during life cycle of LINE-1 retrotransposition, the host modification of LINE-1 mRNA affects the LINE-1 transposition directly. N6-adenosine methylation(m6A), the most abundant epigenetic modification on eukaryotic RNA, is dynamically reversible. m6A modification is also found on LINE-1 mRNA, and it participants regulation of the whole LINE-1 replication cycle, with affecting LINE-1 retrotransposition as well as its adjacent genes expression, followed by influencing genomic stability, cellular self-renewal, and differentiation potential, which plays important roles in human development and diseases. In this review, we summarize the research progress in LINE-1 m6A modification, including its modification positions, patterns and related mechanisms, hoping to provide a new sight on the mechanism research and treatment of related diseases.
Subject(s)
Adenosine/analogs & derivatives , Genome, Human , RNA , Humans , Methylation , RNA/metabolism , RNA, Messenger/geneticsABSTRACT
Although monkeypox (mpox) has been endemic in Western and Central Africa for 50 years, it has not received sufficient prophylactic and therapeutical attention to avoid evolving into an epidemic. From January 2022 to January 2023, more than 84,000 of mpox cases were reported from 110 countries worldwide. Case numbers appear to be rising every day, making mpox an increasing global public health threat for the foreseeable future. In this perspective, we review the known biology and epidemiology of mpox virus, together with the latest therapeutic options available for mpox treatment. Further, small molecule inhibitors against mpox virus and the future directions in this field are discussed as well.
Subject(s)
Mpox (monkeypox) , Humans , Public Health , BiologyABSTRACT
Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real-time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain- and loss-of-function methods. The association of CircSLC8A1 and miR-144-3p, along with miR-144-3p and RUNX1, was predicted using bioinformatics. RNA pull-down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL-mouse model was also investigated using adeno-associated virus. CircSLC8A1 was found to be downregulated in PPOL patients' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR-144-3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR-144-3p and participated in PPOL through the miR-144-3p/RUNX1 pathway in hBMSCs. Micro-CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR-144-3p/RUNX1 pathway may provide a potential target for prevention of PPOL.
Subject(s)
Mesenchymal Stem Cells , MicroRNAs , Osteolysis , Animals , Mice , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Circular/genetics , RNA, Circular/metabolism , Osteogenesis/genetics , Core Binding Factor Alpha 2 Subunit/genetics , Core Binding Factor Alpha 2 Subunit/metabolism , Osteolysis/genetics , Osteolysis/metabolism , Cell Differentiation/genetics , Mesenchymal Stem Cells/metabolism , Osteocalcin/metabolism , Cells, CulturedABSTRACT
The heterodimerization of WT Cu, Zn superoxide dismutase-1 (SOD1), and mutant SOD1 might be a critical step in the pathogenesis of SOD1-linked amyotrophic lateral sclerosis (ALS). Rates and free energies of heterodimerization (ΔGHet) between WT and ALS-mutant SOD1 in mismatched metalation states-where one subunit is metalated and the other is not-have been difficult to obtain. Consequently, the hypothesis that under-metalated SOD1 might trigger misfolding of metalated SOD1 by "stealing" metal ions remains untested. This study used capillary zone electrophoresis and mass spectrometry to track heterodimerization and metal transfer between WT SOD1, ALS-variant SOD1 (E100K, E100G, D90A), and triply deamidated SOD1 (modeled with N26D/N131D/N139D substitutions). We determined that rates of subunit exchange between apo dimers and metalated dimers-expressed as time to reach 30% heterodimer-ranged from t30% = 67.75 ± 9.08 to 338.53 ± 26.95 min; free energies of heterodimerization ranged from ΔGHet = -1.21 ± 0.31 to -3.06 ± 0.12 kJ/mol. Rates and ΔGHet values of partially metalated heterodimers were more similar to those of fully metalated heterodimers than apo heterodimers, and largely independent of which subunit (mutant or WT) was metal-replete or metal-free. Mass spectrometry and capillary electrophoresis demonstrated that mutant or WT 4Zn-SOD1 could transfer up to two equivalents of Zn2+ to mutant or WT apo-SOD1 (at rates faster than the rate of heterodimerization). This result suggests that zinc-replete SOD1 can function as a chaperone to deliver Zn2+ to apo-SOD1, and that WT apo-SOD1 might increase the toxicity of mutant SOD1 by stealing its Zn2+.
Subject(s)
Amyotrophic Lateral Sclerosis , Humans , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/chemistry , Amyotrophic Lateral Sclerosis/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase/chemistry , Metals , Zinc/chemistry , MutationABSTRACT
In Bacillus subtilis, sporulation is a sequential and highly regulated process. Phosphorylation events by histidine kinases are key points in the phosphorelay that initiates sporulation, but serine/threonine protein kinases also play important auxiliary roles in this regulation. PrkA has been proposed to be a serine protein kinase expressed during the initiation of sporulation and involved in this differentiation process. Additionally, the role of PrkA in sporulation has been previously proposed to be mediated via the transition phase regulator ScoC, which in turn regulates the transcriptional factor σK and its regulon. However, the kinase activity of PrkA has not been clearly demonstrated, and neither its autophosphorylation nor phosphorylated substrates have been unambiguously established in B. subtilis. We demonstrated here that PrkA regulation of ScoC is likely indirect. Following bioinformatic homology searches, we revealed sequence similarities of PrkA with the ATPases associated with diverse cellular activities ATP-dependent Lon protease family. Here, we showed that PrkA is indeed able to hydrolyze α-casein, an exogenous substrate of Lon proteases, in an ATP-dependent manner. We also showed that this ATP-dependent protease activity is essential for PrkA function in sporulation since mutation in the Walker A motif leads to a sporulation defect. Furthermore, we found that PrkA protease activity is tightly regulated by phosphorylation events involving one of the Ser/Thr protein kinases of B. subtilis, PrkC. Taken together, our results clarify the key role of PrkA in the complex process of B. subtilis sporulation.
Subject(s)
ATP-Dependent Proteases , Bacillus subtilis , Bacterial Proteins , Spores, Bacterial , ATP-Dependent Proteases/genetics , ATP-Dependent Proteases/metabolism , Bacillus subtilis/genetics , Bacillus subtilis/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Protein Serine-Threonine Kinases/genetics , Spores, Bacterial/genetics , Spores, Bacterial/physiologyABSTRACT
Data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used in proteomic analyses. A well-performed LC-MS/MS workflow, which involves multiple procedures and interdependent metrics, is a prerequisite for deep proteome profiling. Researchers have previously evaluated LC-MS/MS performance mainly based on the number of identified peptides and proteins. However, this is not a comprehensive approach. This motivates us to develop MSRefine, which aims to evaluate and optimize the performance of the LC-MS/MS workflow for data-dependent acquisition (DDA) proteomics. It extracts 47 kinds of metrics, scores the metrics, and reports visual results, assisting users in evaluating the workflow, locating problems, and providing optimizing strategies. In this study, we compared and analyzed multiple pairs of datasets spanning different samples, methods, and instruments and demonstrated that the comprehensive visual metrics and scores in MSRefine enable us to evaluate the performance of the various experiments and provide optimal strategies for the identification of more peptides and proteins.
Subject(s)
Proteome , Tandem Mass Spectrometry , Chromatography, Liquid/methods , Proteome/analysis , Tandem Mass Spectrometry/methods , Workflow , Proteomics/methods , Peptides/chemistryABSTRACT
CRISPR-based directed evolution is an effective breeding biotechnology to improve agronomic traits in plants. However, its gene diversification is still limited using individual single guide RNA. We described here a multiplexed orthogonal base editor (MoBE), and a randomly multiplexed sgRNAs assembly strategy to maximize gene diversification. MoBE could induce efficiently orthogonal ABE (<36.6%), CBE (<36.0%), and A&CBE (<37.6%) on different targets, while the sgRNA assembling strategy randomized base editing events on various targets. With respective 130 and 84 targets from each strand of the 34th exon of rice acetyl-coenzyme A carboxylase (OsACC), we observed the target-scaffold combination types up to 27 294 in randomly dual and randomly triple sgRNA libraries. We further performed directed evolution of OsACC using MoBE and randomly dual sgRNA libraries in rice, and obtained single or linked mutations of stronger herbicide resistance. These strategies are useful for in situ directed evolution of functional genes and may accelerate trait improvement in rice.