Rewiring Metabolic Flux in Corynebacterium glutamicum Using a CRISPR/dCpf1-Based Bifunctional Regulation System.

Corynebacterium glutamicum, a microorganism classified as generally recognized as safe for use in the industrial production of food raw materials and additives, has encountered challenges in achieving widespread adoption and popularization as microbial cell factories. These obstacles arise from the intricate nature of manipulating metabolic flux through conventional methods, such as gene knockout and enzyme overexpression. To address this challenge, we developed a CRISPR/dCpf1-based bifunctional regulation system to bidirectionally regulate the expression of multiple genes in C. glutamicum. Specifically, through fusing various transcription factors to the C-terminus of dCpf1, the resulting dCpf1-SoxS exhibited both CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) capabilities in C. glutamicum by altering the binding sites of crRNAs. The bifunctional regulation system was used to fine-tune metabolic flux from shikimic acid (SA) and l-serine biosynthesis, resulting in 27-fold and 10-fold increases in SA and l-serine production, respectively, compared to the original strain. These findings highlight the potential of the CRISPR/dCpf1-based bifunctional regulation system in effectively enhancing the yield of target products in C. glutamicum.

Transcription factor FOXM1 specifies chromatin DNA to extracellular vesicles.

Extracellular vesicle DNAs (evDNAs) hold significant diagnostic value for various diseases and facilitate transcellular transfer of genetic material. Our study identifies transcription factor FOXM1 as a mediator for directing chromatin genes or DNA fragments (termed FOXM1-chDNAs) to extracellular vesicles (EVs). FOXM1 binds to MAP1LC3/LC3 in the nucleus, and FOXM1-chDNAs, such as the DUX4 gene and telomere DNA, are designated by FOXM1 binding and translocated to the cytoplasm before being released to EVs through the secretory autophagy during lysosome inhibition (SALI) process involving LC3. Disrupting FOXM1 expression or the SALI process impairs FOXM1-chDNAs incorporation into EVs. FOXM1-chDNAs can be transmitted to recipient cells via EVs and expressed in recipient cells when they carry functional genes. This finding provides an example of how chromatin DNA fragments are specified to EVs by transcription factor FOXM1, revealing its contribution to the formation of evDNAs from nuclear chromatin. It provides a basis for further exploration of the roles of evDNAs in biological processes, such as horizontal gene transfer.

Metabolic engineering and fermentation optimization strategies for producing organic acids of the tricarboxylic acid cycle by microbial cell factories.

The organic acids of the tricarboxylic acid (TCA) pathway are important platform compounds and are widely used in many areas. The high-productivity strains and high-efficient and low-cost fermentation are required to satisfy a huge market size. The high metabolic flux of the TCA pathway endows microorganisms potential to produce high titers of these organic acids. Coupled with metabolic engineering and fermentation optimization, the titer of the organic acids has been significantly improved in recent years. Herein, we discuss and compare the recent advances in synthetic pathway engineering, cofactor engineering, transporter engineering, and fermentation optimization strategies to maximize the biosynthesis of organic acids. Such engineering strategies were mainly based on the TCA pathway and glyoxylate pathway. Furthermore, organic-acid-secretion enhancement and renewable-substrate-based fermentation are often performed to assist the biosynthesis of organic acids. Further strategies are also discussed to construct high-productivity and acid-resistant strains for industrial large-scale production.

Preparation of core-shell catalyst for the tandem reaction of amino compounds with aldehydes.

Heterogeneous noble metal-based catalysts with stable, precise structures and high catalytic performance are of great research interest for sustainable catalysis. In this article, we designed a novel core-shell catalyst, Pd@UiO-66-NH2@mSiO2, with Pd@UiO-66-NH2 as the core and mesoporous SiO2 (mSiO2) as the shell. Scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) measurement results demonstrated that the obtained catalyst has an excellent core-shell structure. It can significantly prevent the aggregation of Pd nanoparticles (NPs), as well as the leaching of Pd NPs during the reaction process, owing to the protective effect of mSiO2. During the tandem reaction of aniline and benzaldehyde to generate secondary amines, the prepared Pd@UiO-66-NH2@mSiO2 is highly efficient, due to the strong acid sites provided by UiO-66-NH2 and the hydrogenation reduction sites provided by Pd NPs. Meanwhile, the Pd@UiO-66-NH2@mSiO2 with porous structure can also enhance the mass transfer of reactants to improve the reaction efficiency. Additionally, the prepared catalyst was used to catalyze the series reaction of amino compounds and aldehydes, and the results showed that just 5 mg of the catalyst can convert more than 99% of the reactants within 60 minutes in the presence of 1 atm H2 at room temperature. Finally, the selectivity and stability of the as-prepared catalyst were also confirmed.

Metabolic Engineering of Microorganisms to Produce Pyruvate and Derived Compounds.

Pyruvate is a hub of various endogenous metabolic pathways, including glycolysis, TCA cycle, amino acid, and fatty acid biosynthesis. It has also been used as a precursor for pyruvate-derived compounds such as acetoin, 2,3-butanediol (2,3-BD), butanol, butyrate, and L-alanine biosynthesis. Pyruvate and derivatives are widely utilized in food, pharmaceuticals, pesticides, feed additives, and bioenergy industries. However, compounds such as pyruvate, acetoin, and butanol are often chemically synthesized from fossil feedstocks, resulting in declining fossil fuels and increasing environmental pollution. Metabolic engineering is a powerful tool for producing eco-friendly chemicals from renewable biomass resources through microbial fermentation. Here, we review and systematically summarize recent advances in the biosynthesis pathways, regulatory mechanisms, and metabolic engineering strategies for pyruvate and derivatives. Furthermore, the establishment of sustainable industrial synthesis platforms based on alternative substrates and new tools to produce these compounds is elaborated. Finally, we discuss the potential difficulties in the current metabolic engineering of pyruvate and derivatives and promising strategies for constructing efficient producers.

Engineering an AI-based forward-reverse platform for the design of cross-ribosome binding sites of a transcription factor biosensor.

A cross-ribosome binding site (cRBS) adjusts the dynamic range of transcription factor-based biosensors (TFBs) by controlling protein expression and folding. The rational design of a cRBS with desired TFB dynamic range remains an important issue in TFB forward and reverse engineering. Here, we report a novel artificial intelligence (AI)-based forward-reverse engineering platform for TFB dynamic range prediction and de novo cRBS design with selected TFB dynamic ranges. The platform demonstrated superior in processing unbalanced minority-class datasets and was guided by sequence characteristics from trained cRBSs. The platform identified correlations between cRBSs and dynamic ranges to mimic bidirectional design between these factors based on Wasserstein generative adversarial network (GAN) with a gradient penalty (GP) (WGAN-GP) and balancing GAN with GP (BAGAN-GP). For forward and reverse engineering, the predictive accuracy was up to 98% and 82%, respectively. Collectively, we generated an AI-based method for the rational design of TFBs with desired dynamic ranges.

Functional Traits of Male and Female Leaves of Hippophae tibetana on the Eastern Edge of the Tibetan Plateau and Their Altitudinal Variability.

To date, there have been few studies of the functional traits of the dioecious Hippophae tibetana Schlecht leaves, either male or female, in response to ecological factors such as altitude. Elucidating these relationships will establish an important scientific basis for vegetation restoration and reconstruction of the Tibetan Plateau ecosystem. The natural populations of H. tibetana, distributed across three field sites, at 2868 m, 3012 m and 3244 m, in Tianzhu, Gansu, were studied by field survey sampling and laboratory analysis. In particular, the adaptions of leaf functional traits to elevation in these dioecious plants were analyzed. The results show that: (1) there is no "midday depression" of photosynthetic activity in either male or female plants. Over a one-day period, the net photosynthetic rate (Pn) and transpiration rate (Tr) of H. tibetana female plants were higher than those of male plants (p < 0.05). This correlated to the period of vigorous fruit growth in the female plant. The measured Pn and Tr were maximal at the intermediate altitude (3012 m). The light compensation point (LCP) of the leaves of male and female plants were 57.6 and 43.2 µmol·m−2·s−1, respectively, and the light saturation points (LSP) of the leaves were 1857.6 and 1596.8 µmol·m−2·s−1. (2) Altitude had a significant effect on plant and leaf functional traits of male and female H. tibetana (p < 0.05), and no significant difference was noted between plants at the same altitude. The values for leaf area (LA), specific leaf weight (LMA), leaf phosphorus content per unit mass (Pmass) and leaf phosphorus content per unit area (Parea) were also maximal at the intermediate altitude. Leaf nitrogen content per unit area (Narea) and leaf nitrogen content per unit mass (Nmass) increased with altitude. This indicated that the functional traits of male and female plants and leaves of H. tibetana showed a strong "trade-off relationship" with altitude. (3) Pearson correlation analysis showed that there were significant correlations among functional traits of H. tibetana leaves. Redundancy analysis (RDA) showed that soil water content (SWC), altitude (Alt) and soil organic carbon (SOC) had significant effects on the functional traits of H. tibetana leaves (p < 0.05).

High expression of AFAP1-AS1 is associated with poor prognosis of digestive system cancers: A meta-analysis.

BACKGROUND: Actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) is associated with prognosis in many cancers. The aim of this study was to systematically evaluate the potential correlation between AFAP1-AS1 and the prognosis of digestive system cancers (DSC). METHODS: EMBASE, Web of Science, Cochrane Library, PubMed, Wanfang Data (Chinese), and CNKI (Chinese) were comprehensively searched for literature published from the establishment of the database to September 2021.All case-control studies that met the inclusion criteria were retrieved; additionally manual retrieval and literature tracing was performed. After extracting the relevant data, Revman 5.3.5 software was used for meta-analysis. RESULTS: Eighteen studies were included in analyses, high expression of AFAP1-AS1 was significantly correlated with poor prognosis in DSC, including overall survival (HR = 1.93, 95% CI: 1.72-2.17, P < .001) and disease-free survival/progression-free survival (HR = 1.87, 95% CI: 1.56-2.26, P < .001). In addition, the expression of AFAP1-AS1 was significantly correlated with tumor size, tumor stage, and lymph node metastasis. CONCLUSION: High expression of AFAP1-AS1 was associated with poor prognosis in DSC. Therefore, it could be used as a potential marker for evaluating prognosis in DSC.

Extraction of Radix trichosanthis Polysaccharides for Potential Antihyperlipidemic Application.

This study focused on the optimization of ultrasound-assisted compound enzyme extraction for polysaccharides (RTPs) from Radix trichosanthis by orthogonal experiment and response surface methodology, and then its extraction kinetics model and antihyperlipidemic activities were studied. The optimum extraction process was as follows: cellulase-1.0%, papain-1.0%, pectase-0.5%, pH-5, extraction temperature-50°C, and liquid-to-solid ratio-30 mL/g; prediction value of RTPs was 7.54%; the experimental yield of RTPs was 7.22%, while 50 minutes was optimized in Weibull kinetics model. Then high-dose groups of RTP extract could reduce the TC, TG, and LDL-C levels and increase the level of HDL-C in high-fat mice, with the ability to lower the MDA content and enhance SOD level.

Sequential detection of H2S and HOBr with a novel lysosome-targetable fluorescent probe and its application in biological imaging.

Understanding the complex relationship between active small molecules is of great significance in various physiological processes. Herein, we present the design and synthesis of a sequential responsive Lysosome-Naphthalene imide-Azido (lyso-NP-N3) reporter for probing the H2S and HOBr within organelle (lysosome) in living cells. Probe lyso-NP-N3 exhibited high selectivity and sensitivity towards H2S (LOD = 23.5 nM) and HOBr (LOD = 254 nM). Additionally, lyso-NP-N3 possessed an excellent lysosome targeting ability and was utilized to visualize the exogenous/endogenous H2S and HOBr in RAW 264.7, Hela and HepG2 cells. Facilitated by this sequentially activated mechanism, the probe was successfully applied to confirm that the reported scavenger of HOBr, N-acetyl-L-cysteine (NAC) mainly relied on its metabolite H2S to eliminate excess HOBr, thereby playing the role of cell regulation and protection. These results establish the crosstalk between H2S and HOBr in lysosome and provide a promising tool to study metabolite interactions.

One-pot synthesis of a peroxidase-like nanozyme and its application in visual assay for tyrosinase activity.

Both single-atom nanozymes (SAzymes) and protein-template metal nanoparticles have attracted comprehensive attention in several respects owing to their excellent catalytic performance, green facile synthesis process, and robustness. Herein, the peroxidase-like activity of single-atom copper anchored on bovine hemoglobin-template gadolinium nanoparticles (Cu,Gd@BHbFITC NPs) were successfully synthesized and two sensitive turn-on fluorescence strategies for tyrosinase (TYR) activity sensing were proposed for the first time. For strategy Ⅰ, TYR sensing was carried out from 1.00 to 7.80 U/mL with the detection limit (LOD) of 0.20 U/mL based on the fluorescence resonance energy transfer (FRET) between the fluorescein isothiocyanate (FITC) and the in situ generated polydopamine dots (PDA-dots). For strategy Ⅱ, The LOD of TYR was 0.05 U/mL with the linear range of 0.40-19.70 U/mL based on the elimination of inner-filter effect (IEF) between FITC and the reaction product (RC) of phenol and 4-Aminoantipyrine (AAP). The smartphone-assisted sensing platform was applied to construct the on-site detection of TYR with both strategies. The developed probe possessed good selectivity and was successfully utilized to TYR detection in serum samples.

A feasible self-assembled near-infrared fluorescence sensor for acid phosphatase detection and cell imaging.

The single signal amplification strategy is significant for detecting various disease biomarkers but is restricted by its limited accuracy. The multi-signal and multi-mode methods have overcome this deficiency. Acid phosphatase (ACP) is an important intracellular enzyme but one-step cell imaging material-based probes are scarce for ACP. Herein, we designed a one-step self-assembled polymer probe using neutral red (NR), modified-(pyridoxal-5'-phosphate (PLP)) and Eu3+. The polymer exhibited non-emission and excellent stability. Upon the catalytic hydrolysis reaction of ACP, the polymer exhibited two strong fluorescence signals at 373 nm and 613 nm and an appreciable decline of absorbance at 395 nm. The probe has excellent selectivity and higher sensitivity with a limit of detection as low as 0.02 mU mL-1. It possesses favorable biocompatibility and has been successfully used to detect and image intracellular ACP in several living cells.

Transcription-Factor-based Biosensor Engineering for Applications in Synthetic Biology.

Transcription-factor-based biosensors (TFBs) are often used for metabolite detection, adaptive evolution, and metabolic flux control. However, designing TFBs with superior performance for applications in synthetic biology remains challenging. Specifically, natural TFBs often do not meet real-time detection requirements owing to their slow response times and inappropriate dynamic ranges, detection ranges, sensitivity, and selectivity. Furthermore, designing and optimizing complex dynamic regulation networks is time-consuming and labor-intensive. This Review highlights TFB-based applications and recent engineering strategies ranging from traditional trial-and-error approaches to novel computer-model-based rational design approaches. The limitations of the applications and these engineering strategies are additionally reviewed.

[Progress in transcription factor-based metabolite biosensors].

Transcription factor-based biosensors (TFBs) play an essential role in metabolic engineering and synthetic biology. TFBs sense the metabolite concentration signals and convert them into specific signal output. They hold high sensitivity, strong specificity, brief analysis speed, and are widely used in response to target metabolites. Here we reviewe the principles of TFBs, the application examples, and challenges faced in recent years in microbial cells, including detecting target metabolite concentrations, high-throughput screening, adaptive laboratory evolutionary selection, and dynamic control. Simultaneously, to overcome the challenges in the application, we also focus on reviewing the performance tuning strategies of TFBs, mainly including traditional and computer-aided tuning strategies. We also discuss the opportunities and challenges that TFBs may face in practical applications, and propose the future research trend.

Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor.

Currently, predictive translation tuning of regulatory elements to the desired output of transcription factor (TF)-based biosensors remains a challenge. The gene expression of a biosensor system must exhibit appropriate translation intensity, which is controlled by the ribosome-binding site (RBS), to achieve fine-tuning of its dynamic range (i.e. fold change in gene expression between the presence and absence of inducer) by adjusting the translation level of the TF and reporter. However, existing TF-based biosensors generally suffer from unpredictable dynamic range. Here, we elucidated the connections and partial mechanisms between RBS, translation level, protein folding and dynamic range, and presented a design platform that predictably tuned the dynamic range of biosensors based on deep learning of large datasets cross-RBSs (cRBSs). In doing so, a library containing 7053 designed cRBSs was divided into five sub-libraries through fluorescence-activated cell sorting to establish a classification model based on convolutional neural network in deep learning. Finally, the present work exhibited a powerful platform to enable predictable translation tuning of RBS to the dynamic range of biosensors.

Anti-Inflammatory Effects of Berberine Hydrochloride in an LPS-Induced Murine Model of Mastitis.

Berberine hydrochloride is an isoquinoline type alkaloid extracted from Berberidaceae, Rutaceae, and other plants. Previous reports have shown that berberine hydrochloride has anti-inflammatory properties. However, the underlying molecular mechanisms remain unclear. In this study, a lipopolysaccharide- (LPS-) induced murine model of mastitis was established to explore the anti-inflammatory action of berberine hydrochloride. Sixty mice that had been lactating for 5-7 days were randomly divided into six groups, including control, LPS, three berberine hydrochloride treatment groups (5, 10, and 20 mg/kg), and a dexamethasone (DEX) (5 mg/kg) group. Berberine hydrochloride was administered intraperitoneally 1 h before and 12 h after LPS-induced mastitis, and all mice were sacrificed 24 h after LPS induction. The pathological and histopathological changes of the mammary glands were observed. The concentrations and mRNA expressions of TNF-α, IL-1ß, and IL-6 were measured by ELISA and qRT-PCR. The activation of TLR4 and NF-κB signaling pathways was analyzed by Western blot. Results indicated that berberine hydrochloride significantly attenuated neutrophil infiltration and dose-dependently decreased the secretion and mRNA expressions of TNF-α, IL-1ß, and IL-6 within a certain range. Furthermore, berberine hydrochloride suppressed LPS-induced TLR4 and NF-κB p65 activation and the phosphorylation of I-κB. Berberine hydrochloride can provide mice robust protection from LPS-induced mastitis, potentially via the TLR4 and NF-κB pathway.

Andrographolide Inhibits Inflammatory Cytokines Secretion in LPS-Stimulated RAW264.7 Cells through Suppression of NF-κB/MAPK Signaling Pathway.

Andrographolide, the main active component extracted from Andrographis paniculata (Burm.f.) Wall. ex Nees, exerts anti-inflammatory effects; however, the principal molecular mechanisms remain unclear. The objective of this study was to investigate the molecular mechanisms of Andrographolide in modifying lipopolysaccharide- (LPS-) induced signaling pathway in RAW264.7 cells. An in vitro model of inflammation was induced by LPS in mouse RAW264.7 cells in the presence of Andrographolide. The concentration and expression levels of proinflammatory cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The nuclear level of NF-κB was measured by an electrophoretic mobility shift assay (EMSA). The expression levels of NF-κB, p38, ERK, and JNK were determined by western blot. Andrographolide dose-dependently inhibited the release and mRNA expression of TNF-α, IL-6, and IL-1ß in LPS-stimulated RAW264.7 cells. The nuclear level of p65 protein was decreased in Andrographolide treatment group. Western blot analysis showed that Andrographolide suppressed LPS-induced NF-κB activation and the phosphorylation of IkBa, ERK1/2, JNK, and p38. These results suggest that Andrographolide exerts an anti-inflammatory effect by inhibiting the activation of NF-κB/MAPK signaling pathway and the induction of proinflammatory cytokines.

Inhibition of antiviral drug cidofovir on proliferation of human papillomavirus-infected cervical cancer cells.

In order to evaluate the potential application value of cidofovir (CDV) in the prevention of human papillomavirus (HPV) infection and treatment of cervical cancer, the inhibitory effect of CDV on the proliferation of HPV 18-positive HeLa cells in cervical cancer was preliminarily investigated, using cisplatin (DDP) as a positive control. An MTT assay was used to analyze the effects of CDV and DDP on HeLa cell proliferation. In addition, clone formation assay and Giemsa staining were used to examine the extent of HeLa cell apoptosis caused by CDV and DDP. Flow cytometry was also used to detect the shape and size of apoptotic cells following propidium iodide staining, while western blot analysis identified the expression levels of of E6 and p53 proteins in HeLa cells. A cell climbing immunofluorescence technique was used to locate the subcellular position of p53 in HeLa cells. The results demonstrated that CDV and DDP inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Flow cytometry showed that CDV and DDP treatments resulted in cell arrest in the S-phase, and triggered programmed cell death. Furthermore, western blot analysis revealed that CDV and DDP inhibited E6 protein expression and activated p53 expression in HeLa cells. Finally, the immunofluorescence results indicated that CDV and DDP inhibited the nuclear export of p53 by E6 protein, which is required for degradation of endogenous p53 by MDM2 and human papilloma virus E6. In conclusion, CDV and DDP inhibited HeLa cell proliferation in a concentration- and time-dependent manner, reduced the expression of E6 protein, and reinstated p53 protein activity. Thus, CDV regulates cell cycle arrest and apoptosis, and may be a potential cervical cancer therapeutic strategy.

[The research on the construction and characteristics of recombinant engineering bacteria with multi-epitope of Helicobacter pylori].

OBJECTIVE: To construct the multi epitope prokaryotic expression plasmid and appropriate engineering bacteria expressing the multi-epitope fusion protein of urea membrane channel protein (UreI), urease B subunit (UreB) and adhesin (HpaA) of Helicobacter pylori, then study its microbiological characteristics. METHODS: The target sequence contains multi epitope gene sequence of Helicobacter pylori were designed and synthesized, subsequently; it was subcloned into the expression vector pET28a (+), confirmed by restriction enzyme digestion and DNA sequencing. The fusion protein rIBA was expressed in E. coli Rosseta (DE3) and analyzed by Western blot. RESULTS: The plasmid of pET28a(+)/IBA was constructed successfully, confirmed by endonuclease digestion and sequence analyze. The expressed rIBA protein with relative molecular mass about 40 x 10(3) and can be detected by Western blot. CONCLUSION: The prokaryotic engineering bacteria expression multi-epitope of the Helicobacter pylori was constructed successfully. The recombinant protein rIBA expressed by the engineering bacteria can be identified by Sydney strain 1 of Helicobacter pylori (H. pylori SS1) specific antibody IgY, which demonstrated that the rIBA has high correlation with H. pylori SS1.

Nutlin-3 downregulates p53 phosphorylation on serine392 and induces apoptosis in hepatocellular carcinoma cells.

Drug-resistance and imbalance of apoptotic regulation limit chemotherapy clinical application for the human hepatocellular carcinoma (HCC) treatment. The reactivation of p53 is an attractive therapeutic strategy in cancer with disrupted-p53 function. Nutlin-3, a MDM2 antagonist, has antitumor activity in various cancers. The post-translational modifications of p53 are a hot topic, but there are some controversy ideas about the function of phospho-Ser392-p53 protein in cancer cell lines in response to Nutlin-3. Therefore, we investigated the relationship between Nutlin-3 and phospho-Ser392-p53 protein expression levels in SMMC-7721 (wild-type TP53) and HuH-7 cells (mutant TP53). We demonstrated that Nutlin-3 induced apoptosis through down-regulation phospho-Ser392-p53 in two HCC cells. The result suggests that inhibition of p53 phosphorylation on Ser392 presents an alternative for HCC chemotherapy.