Root-knot nematodes secrete effectors into plant cells to facilitate parasitism. A candidate effector, MiMSP8, of Meloidogyne incognita has been shown to localise within the nucleus when transiently expressed in Nicotiana benthamiana leaf cells, but its role is still unknown. We demonstrate that the MiMSP8 protein is expressed in the dorsal gland of M. incognita juveniles and it can also be detected within giant cells induced by nematodes. Silencing of MiMSP8 impairs nematode parasitism while overexpression of MiMSP8 increases susceptibility to nematode infection. MiMSP8 interacts with SlU2AF35, the small subunit of the U2 snRNP auxiliary factor (U2AF) in tomato. This interaction competes with SlU2AF65 for binding to SlU2AF35, thereby disrupting the formation of heterodimers of the key splicing factor U2AF. Overexpression of MiMSP8 in tomato hairy roots leads to genome-wide alternative splicing changes involved in multiple biological processes. MiMSP8 interferes with the binding between SlU2AF35 and pre-mRNAs of a subset of genes. Silencing SlU2AF35 results in abnormal gene splicing in plants and increases their sensitivity to nematode parasitism. Collectively, our findings reveal that M. incognita deploys a nuclear localised effector to target a key component of the spliceosome and disrupt the splicing of plant pre-mRNA to promote parasitism.

Mitochondrial RNA polymerase (POLRMT) plays a pivotal role in various mitochondrial functions. The upregulation of POLRMT in prostate cancer (PCa) has been well-documented, underscoring its potential as a therapeutic target. In this study, we described the rational design, optimization, and comprehensive biological evaluation of a novel series of POLRMT inhibitors. The most potent compound YH-0623 demonstrated a robust antiproliferative effect on 22Rv1 cell line, correlating with a remarkable reduction in the expression of mitochondrial-related genes. Particularly, YH-0623 significantly inhibited cell growth, colony formation, and the expression of proteins associated with OXPHOS. Furthermore, YH-0623 exhibited a superior pharmacokinetic profile, with an oral bioavailability of 88.9%, indicating favorable absorption upon oral administration. Notably, YH-0623 demonstrated promising therapeutic efficacy, with substantial tumor growth inhibition observed in a PCa xenograft mice model. The potent, selective, and orally available POLRMT inhibitors represent a new class of compounds as potential therapeutics against PCa.

The root-knot nematode Meloidogyne incognita is an obligate biotrophic pathogen that causes extensive losses to agriculture worldwide. Effectors secreted by the parasite play an essential role during nematode infection through suppressing plant innate immunity. Here, we identify and characterize a M. incognita effector designated as MiV86, which is secreted into plant cells and positively regulates nematode parasitism. We show that MiV86 interacts with RING finger protein 217 (NbRNF217), an RBR-type E3 ubiquitin ligase of Nicotiana benthamiana, which negatively regulates plant immunity in an enzymatic activity-dependent manner. Moreover, we demonstrate that NbRNF217 targets and ubiquitinates the helper nucleotide-binding leucine-rich repeat receptor protein NRC4, resulting in its relocation and degradation through the 26S proteasome and endosomal/vacuolar pathways. NbRNF217 regulates its homeostasis through self-catalyzed ubiquitination or external ubiquitination modifications, and we show that MiV86 inhibits the ubiquitination of NbRNF217 in planta without affecting its activity, thereby promoting the degradation of NRC4, which also contributes to the resistance of N. benthamiana against M. incognita. Our findings reveal a mechanism by which a nematode effector hijacks an E3 ubiquitin ligase to attenuate NRC4-mediated plant immunity, facilitating nematode parasitism.

Acute myeloid leukemia (AML) is recognized as a complex disease involving hematopoietic stem cell disorders. Despite advanced in therapy, the prognosis for AML patients remains poor. Previous studies by our group and others have demonstrated that krüppel-like factor 4 (KLF4), a transcription factor, inhibited AML through microRNA networks or p57 signaling pathway. In our previous studies, we found that overexpression of the YAMANAK factor (OSKM) in AML cells induced apoptosis, with KLF4 playing a significant role. However, the mechanisms through which KLF4 regulates gene transcription and histone modifications in AML remain elusive and require further investigation. In this study, we demonstrated that the overexpression of KLF4 inhibited AML proliferation, induced cell apoptosis, and prolonged the survival of AML mice. Co-inmmunoprecipitation (Co-IP) and mass spectrometry analysis verified that KLF4 interacted with the MLL3 CAMPASS complex, including MLL3/4, WDR5, RBBP5 and others. To investigate the mechanisms, we generated different truncated forms of KLF4 and showed that the zinc finger (ZnF) and TRD domains of KLF4 were crucial for the elimination of AML cells, as evidenced by cell viability experiments. In addition, we conducted RNA-seq, ATAC-seq and ChIP-seq in KLF4 overexpressing THP-1 cells. The results indicated that KLF4 regulated the transcription of NRBP2. Interestingly, downregulation of MLL3 led to a decrease in the luciferase activity of NRBP2. Furthermore, the increased cell apoptosis and impaired cell proliferation induced by overexpression of KLF4 could be rescued by knocking down NRBP2. Consequently, KLF4 promoted AML pathogenesis by interacting with the MLL3 CAMPASS complex and facilitating the transcription of NRBP2. These findings indicate that the KLF4-MLL3 CAMPASS-NRBP2 axis plays a crucial role in AML cells and suggests a potential therapeutic target for the disease.

Molecule-based electronic devices, using the intrinsic electronic structure of molecules as device units and constructing electronic devices at the molecular scale, serve as an ideal experimental platform for studying molecular charge transfer mechanisms. They also provide a novel strategy for achieving new functional electronic devices at the micro-nano scale. The realization of a micro-nano electrode gap and a reliable electrode-molecule connection are key factors in developing highly reproducible molecular devices. Carbon materials have been widely applied in the construction of molecular devices due to their remarkable chemical stability and abundant surface chemistry. This review summarizes the research status of using carbon as electrodes in molecular device construction, showcasing the prominent advantages of carbon materials, such as high stability, low cost, and scalability, as well as their applications and research progress in large-area molecular devices and single-molecule devices. The review presents a wealth of achievements in the construction of functional molecular devices, such as molecular switches and rectifiers, using carbon electrodes, as well as the study of the structure-performance relationship in molecular-electron transport. Lastly, this work analyzes the challenges currently faced in carbon-based molecular device research and provides prospects for the chemical connection of carbon electrode-molecular interface and functionalization of carbon-based molecular devices, as well as the integration of future molecular devices

Histone modifications are typically recognized by chromatin-binding protein modules (referred to as 'readers') to mediate fundamental processes such as transcription. Lysine β-hydroxybutyrylation (Kbhb) is a new type of histone mark that couples metabolism to gene expression. However, the readers that prefer histone Kbhb remain elusive. This knowledge gap should be filled in order to reveal the molecular mechanism of this epigenetic regulation. Herein, we developed a chemical proteomic approach, relying upon multivalent photoaffinity probes to capture binders of the mark, and identified ENL as a novel target of H3K9bhb. Biochemical studies and CUT&Tag analysis further suggested that ENL favorably binds to H3K9bhb, and co-localizes with it on promoter regions to modulate gene expression. Notably, disrupting the interaction between H3K9bhb and ENL via structure-based mutation led to the suppressed expression of genes such MYC that drive cell proliferation. Together, our work offered a chemoproteomics approach and identified ENL as a novel histone β-hydroxybutyrylation effector that regulates gene transcription, providing new insight into the regulation mechanism and function of histone Kbhb.

Transcription factors collaborate with chromatin's epigenetic states to regulate cell fate decisions. While recent in vitro work using cryo-EM (Sinha et al., Nature, 2023) suggested that histone modifications could regulate the activity of pioneer transcription factors, it was not clear whether these findings applied in vivo, or if chromatin states could influence transcription factor binding to affect cell fate. Our previous work (Liu et al., Leukemia,2014; Wang et al., Nat Commun,2019) showed that reprogramming factors (Oct4, Sox2, Klf4, and cMyc, collectively known as OSKM) selectively eliminated leukemia cells in vivo, with minimal impact on normal Hematopoietic Stem and Progenitor Cells (HSPCs). This suggested that cell fates induced by OSKM could be influenced by pre-existing chromatin states. Building on this hypothesis, we profiled the pre-existing chromatin states of leukemia cells and HSPCs using MNase-ChIP-seq, ATAC-seq, and WGBS-seq. In our Tet-on-induced models, Klf4 activation resulted in the targeting of distinct gene sets and exhibited similar dynamic changes in chromatin accessibility, despite these regions sharing the same active chromatin state. Using machine learning algorithms, we identified H3K18ac –a feature of the active chromatin states preferred by Klf4– as a dominant factor influencing Klf4's genomic binding. We then altered H3K18ac through an enzymatically deficient SIRT7, which is an H3K18ac deacetylase, and examined where Klf4 binds. We observed an increase in Klf4 binding at sites where H3K18ac levels were elevated. This result suggested that Klf4 binding was influenced by changes in H3K18ac. We exposed human CD34+ cells, several leukemia cell lines, and samples from leukemia patients to ATPO-253. This small molecule, currently in phase 1 clinical trials, has been found to enhance the levels of the KLF4 protein. This treatment led to an increase in KLF4 protein levels and we observed activation of apoptosis genes in the tested leukemia cell lines that possessed high pre-existing H3K18ac levels. In addition, we observed a decline across all tested leukemia samples, while the population of CD34+ cells essentially remained stable. In conclusion, our work demonstrated that pre-existing chromatin states, such as H3K18ac, regulate Klf4's genomic binding, thereby influencing distinct cell fates in leukemia cells and HSPCs. This opens potential avenues for clinical applications in targeted leukemia cell clearance using KLF4 or ATPO-253.

Genetic diversity is an important factor affecting the efficiency of adaptive laboratory evolution (ALE). The recent development of precise tools and strategies for genomic engineering has greatly accelerated mutant library construction for ALE. Here, a global regulator library based on the CRISPR-enabled trackable genome engineering (CREATE) technology was first used to accelerate adaptive evolution for improved furfural tolerance in Escherichia coli, and the furfural tolerance was increased approximately 2-fold in the genetically diversified CREATE-based strains. The evolved strain tolerated up to 4.7 g/L furfural and also showed marked cross-tolerance to NaCl, isobutanol, butanol, ethanol, and high temperature. Whole-genome sequencing and mutant reconstruction analysis revealed for the first time that rpoBP153L mutation leads to greatly increased furfural tolerance. The expression of genes coding central carbon and energy metabolism was significantly altered according to transcriptome analysis. In particular, it was confirmed for the first time that the knockout of sRNA sgrS and the overexpression of sRNA arrS significantly increased furfural tolerance. This study provides evidence that combined ALE and the CREATE technology can not only obtain highly efficient strains with favorable mutation combinations but also accelerate ALE by providing much greater genomic and functional diversity.

As a new interdisciplinary field, synthetic biology has led to valuable innovations in the fields of medicine, chemistry, agriculture, energy and environment. In this paper, we systematically review the development status of global synthetic biology in the past six years, and make an in-depth benchmarking analysis of the field in China. With the aid of Scopus and SciVal, we analyze the scholarly output of synthetic biology in the world and individual countries, including publication distribution, popular journals and eminent institutions. Furthermore, the research focus and concepts, citation impact and collaborations are also examined using numerical index methods such as the field-weighted citation impact (FWCI) and relative activity index (RAI), showing the differences between data more intuitively. This study aims to offer a comprehensive understanding of the research status of synthetic biology in China and the world, offering a benchmarked overview of the results as a reference to guide the development of this field in the future.

To explore the suitability of Corynebacterium glutamicum as a chassis for diacetyl production from glucose, diacetyl metabolic pathway and the respiratory chain were linked to achieve redox balance. The carbon flux was redirected from pyruvate to diacetyl by overexpressing the α-acetolactate synthase, in combination with disruption the biosynthetic pathways of lactate, acetoin, 2,3-butanediol and acetate in C. glutamicum ATCC 13032. These modifications resulted in a sharp increase of the NADH/NAD+ ratio from 0.53 to 1.10, and produced 0.58 g/L diacetyl under aerobic conditions, representing a 58-fold increase over the wild type. Although the modification of the by-product pathways is an effective strategy, these disruption led to intracellular cofactor imbalance. NADH re-oxidization was further successfully solved by overexpressing of cytochrome bd oxidase. We constructed an efficient respiration-dependent cell factory by modification of the respiratory chain, improving diacetyl titer to 1.29 g/L in CGC11, decreased NADH/NAD+ ratio to 0.45, increased the ATP concentration from 8.51 to 10.64 μM/gDCW. To our best knowledge, this is the first report of diacetyl synthesis in C. glutamicum. Intracellular cofactor imbalance can be reduced by modification of the respiratory chain for production of diacetyl as well as other bio-based products with cofactor imbalance in C. glutamicum.

It is important to expedite our understanding of antibiotic resistance to address the increasing numbers of fatalities and environmental pollution due to the emergence of antibiotic resistance and multidrug-resistant strains. Here, we combined the CRISPR-enabled trackable genome engineering (CREATE) technology and transcriptomic analysis to investigate antibiotic tolerance in Escherichia coli We developed rationally designed site saturation mutagenesis libraries targeting 23 global regulators to identify fitness-conferring mutations in response to diverse antibiotic stresses. We identified seven novel mutations that confer resistance to the ribosome-targeting antibiotics doxycycline, thiamphenicol, and gentamicin in E. coli To the best of our knowledge, these mutations that we identified have not been reported previously during treatment with the indicated antibiotics. Transcriptome sequencing-based transcriptome analysis was further employed to evaluate the genome-wide changes in gene expression in E. coli for SoxR G121P and cAMP receptor protein (CRP) V140W reconstructions, and improved fitness in response to doxycycline and gentamicin was seen. In the case of doxycycline, we speculated that SoxR G121P significantly increased the expression of genes involved in carbohydrate metabolism and energy metabolism to promote cell growth for improved adaptation. In the CRP V140W mutant with improved gentamicin tolerance, the expression of several amino acid biosynthesis genes and fatty acid degradation genes was significantly changed, and these changes probably altered the cellular energy state to improve adaptation. These findings have important significance for understanding such nonspecific mechanisms of antibiotic resistance and developing new antibacterial drugs.IMPORTANCE The growing threat of antimicrobial resistance poses a serious threat to public health care and motivates efforts to understand the means by which resistance acquisition occurs and how this can be combatted. To address these challenges, we expedited the identification of novel mutations that enable complex phenotypic changes that result in improved tolerance to antibiotics by integrating CREATE and transcriptomic analysis of global regulators. The results give us a better understanding of the mechanisms of resistance to tetracycline antibiotics and aminoglycoside antibiotics and also indicate that the method may be used for quickly identifying resistance-related mutations.

Histone posttranslational modifications (HPTMs) play important roles in eukaryotic transcriptional regulation. Recently, it has been suggested that combinatorial modification codes that comprise two or more HPTMs can recruit readers of HPTMs, performing complex regulation of gene expression. However, the characterization of the multiplex interactions remains challenging, especially for the molecular network of histone PTMs, readers and binding complexes. Here, we developed an integrated method that combines a peptide library, affinity enrichment, mass spectrometry (MS) and bioinformatics analysis for the identification of the interaction between HPTMs and their binding proteins. Five tandem-domain-reader proteins (BPTF, CBP, TAF1, TRIM24 and TRIM33) were designed and prepared as the enriched probes, and a group of histone peptides with multiple PTMs were synthesized as the target peptide library. First, the domain probes were used to pull down the PTM peptides from the library, and then the resulting product was characterized by MS. The binding interactions between PTM peptides and domains were further validated and measured by isothermal titration calorimetry analysis (ITC). Meanwhile, the binding proteins were enriched by domain probes and identified by HPLC-MS/MS. The interaction network of histone PTMs-readers-binding complexes was finally analyzed via informatics tools. Our results showed that the integrated approach combining MS analysis with ITC assay enables us to understand the interaction between the combinatorial HPTMs and reading domains. The identified network of "HPTMs-reader proteins-binding complexes" provided potential clues to reveal HPTM functions and their regulatory mechanisms.

乙酸是微生物发酵生产常见的副产物,也可作为碳源存在于木质纤维素水解液等非粮原料发酵培养基中.培养基中含有高浓度的乙酸/乙酸盐时会抑制细胞生长、降低生物量,影响目标产品的产量和产率.研究乙酸耐受性机制,改进菌株的乙酸耐受性,构建具有高乙酸耐受性工程菌株,对于以乙酸为碳源或利用含乙酸的原料进行高附加值产品发酵生产具有重要意义.本文综述了通过代谢工程、实验室适应性进化、全局转录机器工程和基于CRISPR可追踪基因组工程等方法构建大肠杆菌乙酸耐受性菌株的研究进展,进一步从乙酸同化代谢、氨基酸依赖型代谢、离子转运系统调节和细胞膜成分修饰等4个方面阐述了大肠杆菌乙酸耐受性菌株的耐受性应答机制,总结了大肠杆菌乙酸耐受菌株的生产应用,展望了提高大肠杆菌乙酸耐受方法和大肠杆菌乙酸耐受机制的研究方向.

Lysine 2-hydroxyisobutyrylation (Khib) has recently been shown to be an evolutionarily conserved histone mark. Here, we report that CobB serves as a lysine de-2-hydroxyisobutyrylation enzyme that regulates glycolysis and cell growth in prokaryotes. We identified the specific binding of CobB to Khib using a novel self-assembled multivalent photocrosslinking peptide probe and demonstrated that CobB can catalyze lysine de-2-hydroxyisobutyrylation both in vivo and in vitro. R58 of CobB is a critical site for its de-2-hydroxyisobutyrylase activity. Using a quantitative proteomics approach, we identified 99 endogenous substrates that are targeted by CobB for de-2-hydroxyisobutyrylation. We further demonstrated that CobB can regulate the catalytic activities of enolase (ENO) by removing K343hib and K326ac of ENO simultaneously, which account for changes of bacterial growth. In brief, our study dissects a Khib-mediated molecular mechanism that is catalyzed by CobB for the regulation of the activity of metabolic enzymes as well as the cell growth of bacteria.

Histone post-translational modifications (HPTMs) provide signaling platforms to recruit proteins or protein complexes (e.g., transcription factors, the so-called "readers" of the histone code), changing DNA accessibility in the regulation of gene expression. Thus, it is an essential task to identify HPTM readers for understanding of epigenetic regulation. Herein we designed and prepared a novel HPTM probe based on self-assembled multivalent photo-cross-linking technique for selective enrichment and identification of HPTM readers. By use of trimethylation of histone H3 lysine 4, we showcased that the functionalized HPTM probe was able to capture its reader with high enrichment efficiency and remarkable specificity even in a complex environment. Notably, this approach was readily applicable for exploring crosstalk among multiple HPTMs. Combining the probes with a mass spectrometry-based proteomic approach, our approach reached a fairly high coverage of known H3K4me3 readers. We further demonstrated that the HPTM probes can enrich a new type of HPTM readers and uncovered several novel putative binders of crotonylation of histone H3 lysine 9, expanding the repertoire of readers for this epigenetic mark. More broadly, our work provides a general strategy for rapid and robust interrogating HPTM readers and will be of great importance to elucidate epigenetic mechanism in regulating gene activity.

High levels of uric acid (UA) could lead to serious renal disease. However, the underlying mechanisms are not very clear yet. This study used human renal mesangial cells (H RMC) as a model and found that cell proliferation of HRMC was significantly suppressed with increasing concentrations of UA in culture medium. Furthermore, a mass spectrometry-based proteomics approach was used to measure changes of proteins with and without treatment of UA. Among 1,977 proteins quantified, 314 were abnormally expressed in the stimulation of UA. Bioinformatics revealed that differential proteins were enriched in the pathways of endoplasmic reticulum (ER) stress and apoptosis. Several key proteins were further verified by Western blotting assay. Flow cytometry experiments also confirmed the effects of UA on apoptosis of HRMC. Finally, this study discussed the mechanisms underlying apoptosis pathways promoted by UA in HRMC. This work provides an overview of protein expression changes in HRMC cells treated by high levels of UA and potentially contributes to further study of cell apoptosis in renal disease patients.