Ocr-mediated suppression of BrxX unveils a phage counter-defense mechanism.

The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial defense mechanisms, such as the BREX system, which utilizes the methyltransferase BrxX to protect against phage infection. This study presents the first in vitro characterization of BrxX from Escherichia coli, revealing its substrate-specific recognition and catalytic activity. We demonstrate that BrxX exhibits nonspecific DNA binding but selectively methylates adenine within specific motifs. Kinetic analysis indicates a potential regulation of BrxX by the concentration of its co-substrate, S-adenosylmethionine, and suggests a role for other BREX components in modulating BrxX activity. Furthermore, we elucidate the molecular mechanism by which the T7 phage protein Ocr (Overcoming classical restriction) inhibits BrxX. Despite low sequence homology between BrxX from different bacterial species, Ocr effectively suppresses BrxX's enzymatic activity through high-affinity binding. Cryo-electron microscopy and biophysical analyses reveal that Ocr, a DNA mimic, forms a stable complex with BrxX, highlighting a conserved interaction interface across diverse BrxX variants. Our findings provide insights into the strategic counteraction by phages against bacterial defense systems and offer a foundational understanding of the complex interplay between phages and their bacterial hosts, with implications for the development of phage therapy to combat antibiotic resistance.

Competitive Inhibition of Okanin against Plasmodium falciparum Tyrosyl-tRNA Synthetase.

Malaria is a severe disease that presents a significant threat to human health. As resistance to current drugs continues to increase, there is an urgent need for new antimalarial medications. Aminoacyl-tRNA synthetases (aaRSs) represent promising targets for drug development. In this study, we identified Plasmodium falciparum tyrosyl-tRNA synthetase (PfTyrRS) as a potential target for antimalarial drug development through a comparative analysis of the amino acid sequences and three-dimensional structures of human and plasmodium TyrRS, with particular emphasis on differences in key amino acids at the aminoacylation site. A total of 2141 bioactive compounds were screened using a high-throughput thermal shift assay (TSA). Okanin, known as an inhibitor of LPS-induced TLR4 expression, exhibited potent inhibitory activity against PfTyrRS, while showing limited inhibition of human TyrRS. Furthermore, bio-layer interferometry (BLI) confirmed the high affinity of okanin for PfTyrRS. Molecular dynamics (MD) simulations highlighted the stable conformation of okanin within PfTyrRS and its sustained binding to the enzyme. A molecular docking analysis revealed that okanin binds to both the tyrosine and partial ATP binding sites of the enzyme, preventing substrate binding. In addition, the compound inhibited the production of Plasmodium falciparum in the blood stage and had little cytotoxicity. Thus, okanin is a promising lead compound for the treatment of malaria caused by P. falciparum.

Integrated transcriptome and metabolome analysis unveils the mechanism of color-transition in Edgeworthia chrysantha tepals.

BACKGROUND: Edgeworthia chrysantha, a deciduous shrub endemic to China, is known for its high ornamental value, extensive cultivation history, and wide-ranging applications. However, theoretical research on this plant is severely lacking. While its flowering process displays striking color transitions from green (S1) to yellow (S2) and then to white (S3), the scientific exploration of this phenomenon is limited, and the underlying regulatory mechanisms are yet to be elucidated. RESULTS: Correlation analysis between phenotypic measurements and pigment content revealed that carotenoids and chlorophyll are the key pigments responsible for the color changes. Metabolomic analysis of carotenoids demonstrated that lutein and ß-carotene were present at higher levels in S1, while S2 exhibited increased diversity and quantity of carotenoids compared to other stages. Notably, antheraxanthin, zeaxanthin, lycopene, and α-cryptoxanthin showed significant increases. In S3, apart from the colorless phytoene, other carotenoid metabolites were significantly reduced to extremely low levels. Transcriptomic data indicated that PSY, Z-ISO, crtZ, ZEP, PDS and ZDS are key genes involved in carotenoid biosynthesis and accumulation, while NCED plays a crucial role in carotenoid degradation. SGR was identified as a key gene contributing to the progressive decline in chlorophyll content. Additionally, three transcription factors potentially regulating carotenoid metabolism were also identified. CONCLUSIONS: This study represents the first systematic investigation, spanning from phenotypic to molecular levels, of the color-changing phenomenon in E. chrysantha. The study elucidates the crucial pigments, metabolites, genes, and transcription factors responsible for flower color changes during the flowering process, thereby providing preliminary understanding of the intrinsic regulatory mechanisms. These findings establish a theoretical foundation for the genetic improvement of flower color in E. chrysantha.

Structure-Based Virtual Screening, ADMET Properties Prediction and Molecular Dynamics Studies Reveal Potential Inhibitors of Mycoplasma pneumoniae HPrK/P.

Mycoplasma pneumoniae pneumonia (MPP) is a frequent cause of community-acquired pneumonia (CAP) in children. The incidence of childhood pneumonia caused by M. pneumoniae infection has been rapidly increasing worldwide. M. pneumoniae is naturally resistant to beta-lactam antibiotics due to its lack of a cell wall. Macrolides and related antibiotics are considered the optimal drugs for treating M. pneumoniae infection. However, clinical resistance to macrolides has become a global concern in recent years. Therefore, it is imperative to urgently identify new targets and develop new anti-M. pneumoniae drugs to treat MMP. Previous studies have shown that deficiencies in HPrK/P kinase or phosphorylase activity can seriously affect carbon metabolism, growth, morphology, and other cellular functions of M. pneumoniae. To identify potential drug development targets against M. pneumoniae, this study analyzed the sequence homology and 3D structure alignment of M. pneumoniae HPrK/P. Through sequence and structure analysis, we found that HPrK/P lacks homologous proteins in the human, while its functional motifs are highly conserved in bacteria. This renders it a promising candidate for drug development. Structure-based virtual screening was then used to discover potential inhibitors among 2614 FDA-approved drugs and 948 bioactive small molecules for M. pneumoniae HPrK/P. Finally, we identified three candidate drugs (Folic acid, Protokylol and Gluconolactone) as potential HPrK/P inhibitors through molecular docking, molecular dynamics (MDs) simulations, and ADMET predictions. These drugs offer new strategies for the treatment of MPP.

Histone demethylase KDM5A regulates the functions of human periodontal ligament stem cells during periodontitis via the miR-495-3p/HOXC8 axis.

Introduction: Periodontitis is the sixth most common human disease and epigenetic regulation is identified to affect the functions of stem cells. This research aims to analyze the role of histone demethylase Lysine-specific demethylase 5A (KDM5A) in human periodontal ligament stem cells (hPDLSCs) with periodontitis. Methods: hPDLSCs were treated with porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) and subjected to osteogenic induction. The expression of KDM5A was detected by RT-qPCR and Western blot. Then, KDM5A expression patterns in hPDLSCs were measured and then silenced using shRNA to explore its role in osteogenic differentiation (OD), proliferation, and migration of hPDLSCs. ChIP assay was used to analyze the relationship between KDM5A and miR-495-3p, Western blot was used to detect H3K4me3 and RT-qPCR was used to detect miR-495-3p expression. CPI-455 (specific KDM5 inhibitor) was adopted to confirm the role of H3K4me3, and dual-Luciferase assay indicted the relationship between miR-495-3p and homeobox C8 (HOXC8). A functional rescue experiment was designed to analyze the role of miR-495-3p in hPDLSCs with periodontitis. Results: KDM5A was highly expressed in LPS-treated hPDLSCs. Downregulation of KDM5A promoted OD, proliferation, and migration of hPDLSCs. Mechanically, KDM5A inhibited miR-495-3p expression by demethylation of H3K4me3 to enhance HOXC8 transcription. Downregulation of miR-495-3p could weaken the effect of sh-KDM5A to promote OD, proliferation, and migration of hPDLSCs. Conclusions: KDM5A could bind to the miR-495-3p promoter and inhibit miR-495-3p expression by demethylation of H3K4me3 to enhance HOXC8 transcription, thereby increasing the HOXC8 and limiting OD, proliferation, and migration of hPDLSCs with periodontitis.

Use of ixekizumab in an HIV-positive patient with psoriatic arthritis.

Psoriasis is a chronic immune-mediated disease of the skin. The incidence of psoriasis among people living with HIV (PLHIV) is higher than that in the general population. The mechanism is complex, the manifestations are varied, and the treatment is difficult. Biotherapy has greatly alleviated psoriasis, but clinical trials often exclude PLHIV, and evidence is limited to case reports. Here, we report a man living with psoriatic arthritis who had poor response to traditional treatments. After receiving the anti-interleukin (IL)-17 monoclonal antibody (ixekizumab), the arthritis symptoms were significantly relieved, while CD4+ T cell count increased and the viral load of HIV-1 remained undetectable in combination with antiretroviral therapy (ART). In conclusion, anti-IL-17 monoclonal antibody is a promising and safe treatment for psoriatic arthritis in HIV-positive patients.

Rapid, sensitive and cost-effective determination of immune checkpoint inhibitor activity using a magnetic bead-based binding assay.

Immune checkpoint Inhibitors (ICIs) are effective immunno-therapeutic agents for cancer. Rapid and sensitive determination of the blocking activity of ICIs is important for ICIs development and immunological research. Among various immune checkpoint (IC) binding assays, cell-based binding assays are widely regarded, and the functional ELISA is a convenient alternative. However, these methodologies are limited by time-consuming preparation of cell lines stably expressing IC molecules, or long turnaround time with high cost. In this study, two magnetic bead based binding assays were developed to evaluate activity of ICIs, which was determined by a soluble ligand/bead immobilized receptor based binding assay (sL/bR binding assay) that assessed efficacy to block binding of one soluble IC ligand on its cognate receptor immobilized beads, or by a soluble receptor/bead immobilized ligand based binding assay (sR/bL binding assay) that assessed efficacy to block binding of soluble IC receptor on its cognate ligand immobilized beads. Half maximal inhibitory concentration (IC50) values of ICIs were calculated to determine ICIs activity. The sL/bR binding assay accurately determined the activity of two TIGIT blocking antibodies, since the relative blocking activity of two TIGIT antibodies determined by the sL/bR binding assay established in this study and that by the cell based binding assay were almost identical. In contrast, the sR/bL binding assay showed significantly improved sensitivity to determine activity of two PD-1 blocking antibodies than the sL/bR binding assay that was tested in this study and previous reports. Moreover, both amount of the used recombinant protein of ICI receptor/ligand and turnaround time of the two binding assays were more than 10 times less than those of the functional ELISA. These data indicate that the two magnetic bead based binding assays are sensitive, rapid and cost-effective methods to determine blocking activity of ICIs.

Characterization of the liver tissue interstitial fluid (TIF) proteome indicates potential for application in liver disease biomarker discovery.

Tissue interstitial fluid (TIF) forms the interface between circulating body fluids and intracellular fluid. Pathological alterations of liver cells could be reflected in TIF, making it a promising source of liver disease biomarkers. Mouse liver TIF was extracted, separated by SDS-PAGE, analyzed by linear ion trap mass spectrometer, and 1450 proteins were identified. These proteins may be secreted, shed from membrane vesicles, or represent cellular breakdown products. They show different profiling patterns, quantities, and possibly modification/cleavage of intracellular proteins. The high solubility and even distribution of liver TIF supports its suitability for proteome analysis. Comparison of mouse liver TIF data with liver tissue and plasma proteome data identified major proteins that might be released from liver to plasma and serve as blood biomarkers of liver origin. This result was partially supported by comparison of human liver TIF data with human liver and plasma proteome data. Paired TIFs from tumor and nontumor liver tissues of a hepatocellular carcinoma patient were analyzed and the profile of subtracted differential proteins supports the potential for biomarker discovery in TIF. This study is the first analysis of the liver TIF proteome and provides a foundation for further application of TIF in liver disease biomarker discovery.

Quantitative proteomic survey of endoplasmic reticulum in mouse liver.

To gain a better understanding of the critical function of the endoplasmic reticulum (ER) in liver, we carried out a proteomic survey of mouse liver ER. The ER proteome was profiled with a new three-dimensional, gel-based strategy. From 6152 and 6935 MS spectra, 903 and 1042 proteins were identified with at least two peptides matches at 95% confidence in the rough (r) and smooth (s) ER, respectively. Comparison of the rER and sER proteomes showed that calcium-binding proteins are significantly enriched in the sER suggesting that the ion-binding function of the ER is compartmentalized. Comparison of the rat and mouse ER proteomes showed that 662 proteins were common to both, comprising 53.5% and 49.3% of those proteomes, respectively. We proposed that these proteins were stably expressed proteins that were essential for the maintenance of ER function. GO annotation with a hypergeometric model proved this hypothesis. Unexpectedly, 210 unknown proteins and some proteins previously reported to occur in the cytosol were highly enriched in the ER. This study provides a reference map for the ER proteome of liver. Identification of new ER proteins will enhance our current understanding of the ER and also suggest new functions for this organelle.

Embryonic stem cells derived from somatic cloned and fertilized blastocysts are post-transcriptionally indistinguishable: a MicroRNA and protein profile comparison.

Therapeutic cloning, whereby somatic cell nuclear transfer is used to generate customized embryonic stem cells (NT-ES) from differentiated somatic cells of specific individuals, has been successfully performed in mice and non-human primates. Safety concerns have prevented this technology from being potentially applied to humans, as severely abnormal phenotypes have been observed in cloned animals. Although it has been demonstrated that the transcriptional profiles and developmental potentials of ES cells derived from cloned blastocysts are identical to those of ES cells derived from fertilized blastocysts (F-ES), a systematic analysis of the post-transcriptional profiles of NT-ES cell lines has not yet been performed. To investigate whether NT-ES cells are comparable to F-ES cells post-transcriptionally, we compared the microRNA and protein profiles of five NT- and matching F-ES cell lines by microRNA microarray, 2-D DIGE and bioinformatic analyses. Stem-loop real-time PCR and MS/MS assays were further performed to verify the expression of specific microRNAs and characterize differentially expressed proteins. Our results demonstrate that the ES cell lines derived from cloned and fertilized mouse blastocysts have highly similar microRNA and protein expression profiles, consistent with their similar developmental potentials and transcriptional profiles.

Differential proteome analysis of bone marrow mesenchymal stem cells from adolescent idiopathic scoliosis patients.

Adolescent idiopathic scoliosis (AIS) is a complex three-dimensional deformity of the spine. The cause and pathogenesis of scoliosis and the accompanying generalized osteopenia remain unclear despite decades of extensive research. In this study, we utilized two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS) to analyze the differential proteome of bone marrow mesenchymal stem cells (BM-MSCs) from AIS patients. In total, 41 significantly altered protein spots were detected, of which 34 spots were identified by MALDI-TOF/TOF analysis and found to represent 25 distinct gene products. Among these proteins, five related to bone growth and development, including pyruvate kinase M2, annexin A2, heat shock 27 kDa protein, γ-actin, and ß-actin, were found to be dysregulated and therefore selected for further validation by Western blot analysis. At the protein level, our results supported the previous hypothesis that decreased osteogenic differentiation ability of MSCs is one of the mechanisms leading to osteopenia in AIS. In summary, we analyzed the differential BM-MSCs proteome of AIS patients for the first time, which may help to elucidate the underlying molecular mechanisms of bone loss in AIS and also increase understanding of the etiology and pathogenesis of AIS.

Proteomic alteration of PK-15 cells after infection by classical swine fever virus.

Viral infections usually result in alterations in the host cell proteome which determine the fate of the infected cells and the progress of pathogenesis. To uncover cellular protein responses in classical swine fever virus-infected PK-15 cells, a proteomic analysis was conducted using 2D PAGE followed by MALDI-TOF-MS/MS identification. Altered expression of 35 protein spots in infected cells at 48 h p.i. were identified in 2D gels, with 21 of these being characterized by MALDI-TOF-MS/MS, including 16 upregulated proteins and 5 down-regulated proteins. Western-blot analysis confirmed the up-regulation of annexin 2 and down-regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The altered proteins could be sorted into 7 groups according to cellular function: cytoskeleton, energy metabolism, replication/transcription and translation processes, protein processing, antioxidative stress proteins, heat shock proteins and signal transduction. The altered expression of these proteins provides a response profile of PK-15 host cells to CSFV infection. Further study of these altered proteins may facilitate understanding the mechanisms of CSFV infection and pathogenesis.

Liver proteome analysis of adaptive response in rat immediately after partial hepatectomy.

The extraordinary ability of the liver to regenerate after resection continues to be an important fascination to mammalian liver researchers. However, at present, there are still several central questions regarding the process of liver regeneration that are not clear. In our study, we try to clarify how the liver is able to maintain its functions as well as to initiate liver regeneration after a significant loss of two-thirds. Here differentially expressed proteins in rat livers at 1 h after partial hepatectomy (PHx) and sham operation were analyzed using 2-DE combined with MALDI-TOF/TOF MS. After the analysis, 24 significantly changed spots (ratio> or =2, p<0.05) were identified. Those proteins are involved in important liver functions such as metabolism, detoxification, and inflammation. Based on the changes in the protein levels found in our data, we identified two aspects of remnant liver immediately after PHx, which focused on the hepatic adaptation and the inflammatory response associated with the initiation of liver regeneration after PHx. For the first time, the differential expression of pyruvate dehydrogenase complex (PDHX), paraoxonase 1 (PON1), thyroid hormone receptor beta, GAP43 (where GAP stands for growth-associated protein), and interleukin-2 (IL2), after PHx, were validated by Western blot.

Sample preparation project for the subcellular proteome of mouse liver.

Organelle proteome has become one of the most important fields of proteomics, and the subcellular fractionation with high purity and yield has always been a challenge for cell biologists and also for the Human Liver Proteome Project (HLPP). The liver of a C57BL/6J mouse was chosen as the model to find the optimum method for subcellular preparation. The method we selected could obtain the multiple fractions including plasma membrane, mitochondria, nucleus, ER, and cytosol from a single homogenate. With the same procedure, it is for the first time that the preparation method of frozen homogenized livers was compared with that of the fresh livers and frozen livers. We systematically evaluated the purity, efficiency, and integrity by protein yield, immunoblotting, and transmission electron microscopy. Taken together, the method of multiple fractions from a single tissue is effective enough for subcellular fractionation of mouse liver. We give a selective sample preparation method for frozen homogenized livers, for rare clinical samples, which cannot easily be used for subcellular separation immediately. But the frozen livers are not recommended for organelles isolation. This result is especially useful for sample preparation of human liver for subcellular fractionation of HLPP.

A novel missense mutation of the EDA gene in a Mongolian family with congenital hypodontia.

X-linked hypohidrotic ectodermal dysplasia (HED) is a rare disease characterized by the hypoplasia or absence of eccrine glands, dry skin, scant hair, and dental abnormalities. Here, we report a Mongolian family with congenital absence of teeth inherited in an X-linked fashion. The affected members of the family did not show other HED characteristics, except hypodontia. We successfully mapped the affected locus to chromosome Xq12-q13.1, and then found a novel missense mutation, c.193C>G, in the ectodysplasin A (EDA) gene in all affected males and carrier females. The mutation causes arginine to be replaced by glycine in codon 65 (R65G) in the juxtamembrane region of EDA. In addition, 33% (3/9) of female carriers have a skewed X-chromosome inactivation pattern. Our result strongly suggests that the c.193C>G mutation is the disease-causing mutation in this family.