Tumor Microenvironment-Responsive Nanocapsule Delivery CRISPR/Cas9 to Reprogram the Immunosuppressive Microenvironment in Hepatoma Carcinoma.

Cancer immunotherapy has demonstrated significant efficacy in various tumors, but its effectiveness in treating Hepatocellular Carcinoma (HCC) remains limited. Therefore, there is an urgent need to identify a new immunotherapy target and develop corresponding intervention strategies. Bioinformatics analysis has revealed that growth differentiation factor 15 (GDF15) is highly expressed in HCC and is closely related to poor prognosis of HCC patients. The previous study revealed that GDF15 can promote immunosuppression in the tumor microenvironment. Therefore, knocking out GDF15 through gene editing could potentially reverse the suppressive tumor immune microenvironment permanently. To deliver the CRISPR/Cas9 system specifically to HCC, nanocapsules (SNC) coated with HCC targeting peptides (SP94) on their surface is utilized. These nanocapsules incorporate disulfide bonds (SNCSS) that release their contents in the tumor microenvironment characterized by high levels of glutathione (GSH). In vivo, the SNCSS target HCC cells, exert a marked inhibitory effect on HCC progression, and promote HCC immunotherapy. Mechanistically, CyTOF analysis showed favorable changes in the immune microenvironment of HCC, immunocytes with killer function increased and immunocytes with inhibitive function decreased. These findings highlight the potential of the CRISPR-Cas9 gene editing system in modulating the immune microenvironment and improving the effectiveness of existing immunotherapy approaches for HCC.

Regulating structural stability and photoelectrical properties of FAPbI3via formamidine cation orientation.

Organic cations play a significant role in the structural stability and photoelectrical properties of organic-inorganic hybrid perovskites. The orientation of organic cations impacts its interaction with inorganic octahedrons [PbI]6-, subsequently modifying the atomic structure and electronic and optical properties of perovskite materials. However, it is still challenging to regulate the stability of perovskites with different orientations. In this work, density functional theory calculations were performed to investigate the effects of the formamidine cation (FA+) located at the angles of 0°, 45°, 90° and 180° (relative to the normal of the crystal plane) along the typical crystal directions ([001], [010], [110] and [111]) on the structural stability and photoelectrical properties of formamidine lead iodide (FAPbI3). The results show that when FA+ is located at 45° along the [111] direction, FAPbI3 achieves the highest stability and excellent photoelectrical properties. The energy evolution curves display that the system with the orientation of [111] has the minimum energy value, signifying stronger stability than the other orientations. Especially, when FA+ is located at 45° along the [111] direction. it exhibits a stronger hydrogen bond between H and I atoms, shorter Pb-I bond length and smaller [PbI]6- octahedral tilt bond angle. The band gap in the [110] direction changes from direct to indirect while FAPbI3 with other FA+ orientations still maintains the direct band structure located at the high symmetric R point. Furthermore, FA+ orientation drives the redshift of FAPbI3 towards the long wavelength region in the [111] crystal direction, which enhances the light absorption coefficient. This work can offer guidance in employing molecular regulation technology for the development of stable perovskite solar cells.

CD63+ cancer-associated fibroblasts confer CDK4/6 inhibitor resistance to breast cancer cells by exosomal miR-20.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) have rapidly received Food and Drug Administration (FDA) approval as a new type of therapy for patients with advanced hormone receptor-positive breast cancer. However, with the widespread application of CDK4/6i, drug resistance has become a new challenge for clinical practice and has greatly limited the treatment effect. Here, the whole microenvironment landscape of ER+ breast cancer tumors was revealed through single-cell RNA sequencing, and a specific subset of cancer-associated fibroblasts (CD63+ CAFs) was identified as highly enriched in CDK4/6i resistant tumor tissues. Then, we found that CD63+ CAFs can distinctly promote resistance to CDK4/6i in breast cancer cells and tumor xenografts. In addition, it was discovered that miR-20 is markedly enriched in the CD63+ CAFs-derived exosomes, which are used to communicate with ER+ breast cancer cells, leading to CDK4/6i resistance. Furthermore, exosomal miR-20 could directly target the RB1 mRNA 3'UTR and negatively regulate RB1 expression to decrease CDK4/6i sensitivity in breast cancer cells. Most importantly, we designed and synthesized cRGD-miR-20 sponge nanoparticles and found that they can enhance the therapeutic effect of CDK4/6i in breast cancer. In summary, our findings reveal that CD63+ CAFs can promote CDK4/6i resistance via exosomal miR-20, which induces the downregulation of RB1 in breast cancer cells, and suggest that CD63+ CAFs may be a novel therapeutic target to enhance CDK4/6i sensitivity.

Biological analysis of the potential pathogenic mechanisms of Infectious COVID-19 and Guillain-Barré syndrome.

Background: Guillain-Barré syndrome (GBS) is a medical condition characterized by the immune system of the body attacking the peripheral nerves, including those in the spinal nerve roots, peripheral nerves, and cranial nerves. It can cause limb weakness, abnormal sensations, and facial nerve paralysis. Some studies have reported clinical cases associated with the severe coronavirus disease 2019 (COVID-19) and GBS, but how COVID-19 affects GBS is unclear. Methods: We utilized bioinformatics techniques to explore the potential genetic connection between COVID-19 and GBS. Differential expression of genes (DEGs) related to COVID-19 and GBS was collected from the Gene Expression Omnibus (GEO) database. By taking the intersection, we obtained shared DEGs for COVID-19 and GBS. Subsequently, we utilized bioinformatics analysis tools to analyze common DEGs, conducting functional enrichment analysis and constructing Protein-protein interaction networks (PPI), Transcription factors (TF) -gene networks, and TF-miRNA networks. Finally, we validated our findings by constructing the Receiver Operating Characteristic (ROC) curves. Results: This study utilizes bioinformatics tools for the first time to investigate the close genetic relationship between COVID-19 and GBS. CAMP, LTF, DEFA1B, SAMD9, GBP1, DDX60, DEFA4, and OAS3 are identified as the most significant interacting genes between COVID-19 and GBS. In addition, the signaling pathway of NOD-like receptors is believed to be essential in the link between COVID-19 and GBS.

Aptamer-based kinetically controlled DNA reactions coupled with metal-organic framework nanoprobes for sensitive detection of SARS-CoV-2 spike protein.

Since the outbreak in 2019, COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become the deadliest infectious disease worldwide for people of all ages, from children to older adults. As a main structural protein of SARS-CoV-2, spike protein is reported to play a key role in the entry of the virus into host cells and is considered as an effective antigenic marker for COVID-19 diagnosis. Herein, we develop a new aptamer-based fluorescence method for SARS-CoV-2 spike protein detection based on using kinetically controlled DNA reactions and metal-organic framework nanoprobes. Specifically, the binding of SARS-CoV-2 spike protein to its aptamer is designed to precisely control the kinetics of a DNA displacement reaction, leading to the release of free signaling probes. By reasonable integration of magnetic enrichment and exonuclease-fuelled recycling, the released probes efficiently disrupt the interaction within metal-organic framework nanoprobes, thereby generating a remarkable fluorescent response. Experimental results show that the method not only exhibits a wide linear range and a low detection limit of 7.8 fg mL-1 for SARS-CoV-2 spike protein detection but also demonstrates desirable specificity and utility in complex samples. Therefore, the method may provide a valuable tool for the detection of SARS-CoV-2 spike protein, and has bright prospects in the rapid diagnosis of COVID-19, which is of great significance for guiding rational treatment during a pandemic of respiratory infectious diseases and reducing the occurrence of severe disease in children.

Identification of genetic mechanisms underlying lipid metabolism-mediated tumor immunity in head and neck squamous cell carcinoma.

OBJECTIVE: To identify the genetic mechanisms underlying lipid metabolism-mediated tumor immunity in head and neck squamous carcinoma (HNSC). MATERIALS AND METHODS: RNA sequencing data and clinical characteristics of HNSC patients were procured from The Cancer Genome Atlas (TCGA) database. Lipid metabolism-related genes were collected from KEGG and MSigDB databases. Immune cells and immune-related genes were obtained from the TISIDB database. The differentially expressed genes (DEGs) in HNSC were identified and weighted correlation network analysis (WGCNA) was performed to identify the significant gene modules. Lasso regression analysis was performed to identify hub genes. The differential gene expression pattern, diagnostic values, relationships with clinical features, prognostic values, relationships with tumor mutation burden (TMB), and signaling pathways involved, were each investigated. RESULTS: One thousand six hundred sixty-eight DEGs were identified as dysregulated between HNSC tumor samples and healthy control head and neck samples. WGCNA analysis and Lasso regression analysis identified 8 hub genes, including 3 immune-related genes (PLA2G2D, TNFAIP8L2 and CYP27A1) and 5 lipid metabolism-related genes (FOXP3, IL21R, ITGAL, TRAF1 and WIPF1). Except CYP27A1, the other hub genes were upregulated in HNSC as compared with healthy control samples, and a low expression of these hub genes indicated a higher risk of death in HNSC. Except PLA2G2D, all other hub genes were significantly and negatively related with TMB in HNSC. The hub genes were implicated in several immune-related signaling pathways including T cell receptor signaling, Th17 cell differentiation, and natural killer (NK) cell mediated cytotoxicity. CONCLUSION: Three immune genes (PLA2G2D, TNFAIP8L2, and CYP27A1) and immune-related pathways (T cell receptor signaling, Th17 cell differentiation, and natural killer (NK) cell mediated cytotoxicity) were predicted to play significant roles in the lipid metabolism-mediated tumor immunity in HNSC.

Exosome-transmitted circCABIN1 promotes temozolomide resistance in glioblastoma via sustaining ErbB downstream signaling.

Although temozolomide (TMZ) provides significant clinical benefit for glioblastoma (GBM), responses are limited by the emergence of acquired resistance. Here, we demonstrate that exosomal circCABIN1 secreted from TMZ-resistant cells was packaged into exosomes and then disseminated TMZ resistance of receipt cells. CircCABIN1 could be cyclized by eukaryotic translation initiation factor 4A3 (EIF4A3) and is highly expressed in GBM tissues and glioma stem cells (GSCs). CircCABIN1 is required for the self-renewal maintenance of GSCs to initiate acquired resistance. Mechanistically, circCABIN1 regulated the expression of olfactomedin-like 3 (OLFML3) by sponging miR-637. Moreover, upregulation of OLFML3 activating the ErbB signaling pathway and ultimately contributing to stemness reprogramming and TMZ resistance. Treatment of GBM orthotopic mice xenografts with engineered exosomes targeting circCABIN1 and OLFML3 provided prominent targetability and had significantly improved antitumor activity of TMZ. In summary, our work proposed a novel mechanism for drug resistance transmission in GBM and provided evidence that engineered exosomes are a promising clinical tool for cancer prevention and therapy.

A Versatile Design-Enabled Analysis of Circulating Extracellular Vesicles in Disease Diagnosis.

Circulating extracellular vesicles (EVs) are considered as potential biomarkers for treatment and diagnosis of many diseases. Most of the existing methods for the EV analysis only have a single function and thus reveal limited information carried by EVs. Herein, a phosphatidylserine-targeting peptide-facilitated design that enables the versatile analysis of circulating EVs for varying requirement is proposed. In the design, DNA probes are inserted into the EV membrane through hydrophobic interactions, and accelerate the removal of protective shielding from DNA-gated metal-organic framework, thereby releasing a large number of methylene blue molecules to amplify the electrochemical signal. Electrochemical results demonstrate equally high sensitivities toward the quantification of EVs derived from different cell sources using an indiscriminative DNA probe. More importantly, the probe can be endowed with extended function for more accurate classification of cell-specific features through the identification of specific EV biomarkers, and demonstrates the potential use in the diagnosis of cardiovascular in a principle-of-proof study for clinical application. Therefore, the method provides a versatile design for the identification of EV features, and may address the needs of clinical diagnosis in the future.

Biosynthesis of L-5-methyltetrahydrofolate by genetically engineered Escherichia coli.

L-5-Methyltetrahydrofolate (L-5-MTHF) is the only biologically active form of folate in the human body. Production of L-5-MTHF by using microbes is an emerging consideration for green synthesis. However, microbes naturally produce only a small amount of L-5-MTHF. Here, Escherichia coli BL21(DE3) was engineered to increase the production of L-5-MTHF by overexpressing the intrinsic genes of dihydrofolate reductase and methylenetetrahydrofolate (methylene-THF) reductase, introducing the genes encoding formate-THF ligase, formyl-THF cyclohydrolase and methylene-THF dehydrogenase from the one-carbon metabolic pathway of Methylobacterium extorquens or Clostridium autoethanogenum and disrupting the gene of methionine synthase involved in the consumption and synthesis inhibition of the target product. Thus, upon its native pathway, an additional pathway for L-5-MTHF synthesis was developed in E. coli, which was further analysed and confirmed by qRT-PCR, enzyme assays and metabolite determination. After optimizing the conditions of induction time, temperature, cell density and concentration of IPTG and supplementing exogenous substances (folic acid, sodium formate and glucose) to the culture, the highest yield of 527.84 µg g-1 of dry cell weight for L-5-MTHF was obtained, which was about 11.8 folds of that of the original strain. This study paves the way for further metabolic engineering to improve the biosynthesis of L-5-MTHF in E. coli.

Recent Advances on the Metal-Organic Frameworks-Based Biosensing Methods for Cancer Biomarkers Detection.

Sensitive and selective detection of cancer biomarkers is crucial for early diagnosis and treatment of cancer, one of the most dangerous diseases in the world. Metal-organic frameworks (MOFs), a class of hybrid porous materials fabricated through the assembly of metal ions/clusters and organic ligands, have attracted increasing attention in the sensing of cancer biomarkers, due to the advantages of adjustable size, high porosity, large surface area and ease of modification. MOFs have been utilized to not only fabricate active sensing interfaces but also arouse a variety of measurable signals. Several representative analytical technologies have been applied in MOF-based biosensing strategies to ensure high detection sensitivity toward cancer biomarkers, such as fluorescence, electrochemistry, electrochemiluminescence, photochemistry and colorimetric methods. In this review, we summarized recent advances on MOFs-based biosensing strategies for the detection of cancer biomarkers in recent three years based on the categories of metal nodes, and aimed to provide valuable references for the development of innovative biosensing platform for the purpose of clinical diagnosis.

Designer Exosomes for Targeted Delivery of a Novel Therapeutic Cargo to Enhance Sorafenib-Mediated Ferroptosis in Hepatocellular Carcinoma.

Sorafenib is one of the few effective first-line drugs approved for the treatment of advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common among individuals with HCC. Recent evidence indicated that the anticancer activity of sorafenib mainly relies on the induction of ferroptosis. Furthermore, in our study, genes that suppress ferroptosis, especially GPX4 and DHODH, were enriched in sorafenib-resistant cells and primary tissues and were associated with poor prognosis of HCC patients who received sorafenib treatment. Therefore, a new ferroptosis inducer comprising a multiplex small interfering RNA (multi-siRNA) capable of simultaneously silencing GPX4 and DHODH was created. Then, exosomes with high multi-siRNA loading and HCC-specific targeting were established by fusing the SP94 peptide and the N-terminal RNA recognition motif (RRM) of U1-A with the exosomal membrane protein Lamp2b. The results from the in vitro and in vivo experiments indicate that this tumor-targeting nano-delivery system (ExoSP94-lamp2b-RRM-multi-siRNA) could enhance sorafenib-induced ferroptosis and overcome sorafenib resistance. Taken together, HCC-targeted exosomes (ExoSP94-Lamp2b-RRM) could specifically deliver multi-siRNA to HCC tissues, enhance sorafenib-induced ferroptosis by silencing GPX4 and DHODH expression and consequently increase HCC sensitivity to sorafenib, which opens a new avenue for clinically overcoming sorafenib resistance from the perspective of ferroptosis.

A Cytoplasmic NAD(P)H-Dependent Polysulfide Reductase with Thiosulfate Reductase Activity from the Hyperthermophilic Bacterium Thermotoga maritima.

Thermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H2 by fermenting carbohydrates. High concentration of H2 inhibits the growth of T. maritima, and S0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx, yeeE, and rnf genes when the strain grew in S0- or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments. IMPORTANCE The reduction of S0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S0, polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S0- or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

The impacts of carbon emissions and voluntary carbon disclosure on firm value.

Investors and other stakeholders are starting to pay attention to firms' carbon emissions and carbon disclosure. This study investigated the effects of voluntary carbon disclosure information and carbon emissions on firm value from listed companies in the Shanghai and Shenzhen 300 (CSI 300) Index. We also apply the Probit model to predict the probability of voluntary carbon disclosure information. The results indicate that the increase in carbon emissions has a negative impact on firm value. The action that companies select to disclose carbon emissions has a positive impact on firm value. The effect of leverage ratio on VCDI is increasing year by year. What is more, the probability of the average size firm carbon disclosure was 30.73% in 2020. Company management needs to pay attention to the risks caused by carbon emissions and ensure the quality of carbon disclosure information, especially the authenticity and reliability of the information.

Toward Deep Adaptive Hinging Hyperplanes.

The adaptive hinging hyperplane (AHH) model is a popular piecewise linear representation with a generalized tree structure and has been successfully applied in dynamic system identification. In this article, we aim to construct the deep AHH (DAHH) model to extend and generalize the networking of AHH model for high-dimensional problems. The network structure of DAHH is determined through a forward growth, in which the activity ratio is introduced to select effective neurons and no connecting weights are involved between the layers. Then, all neurons in the DAHH network can be flexibly connected to the output in a skip-layer format, and only the corresponding weights are the parameters to optimize. With such a network framework, the backpropagation algorithm can be implemented in DAHH to efficiently tackle large-scale problems and the gradient vanishing problem is not encountered in the training of DAHH. In fact, the optimization problem of DAHH can maintain convexity with convex loss in the output layer, which brings natural advantages in optimization. Different from the existing neural networks, DAHH is easier to interpret, where neurons are connected sparsely and analysis of variance (ANOVA) decomposition can be applied, facilitating to revealing the interactions between variables. A theoretical analysis toward universal approximation ability and explicit domain partitions are also derived. Numerical experiments verify the effectiveness of the proposed DAHH.

Pharmacokinetics, distribution, metabolism, and excretion of body-protective compound 157, a potential drug for treating various wounds, in rats and dogs.

Body-protective compound (BPC) 157 demonstrates protective effects against damage to various organs and tissues. For future clinical applications, we had previously established a solid-phase synthesis process for BPC157, verified its biological activity in different wound models, and completed preclinical safety evaluations. This study aimed to investigate the pharmacokinetics, excretion, metabolism, and distribution profiles of BPC157. After a single intravenous (IV) administration, single intramuscular (IM) administrations at three doses in successive increments along with repeated IM administrations, the elimination half-life (t1/2) of prototype BPC157 was less than 30 min, and BPC157 showed linear pharmacokinetic characteristics in rats and beagle dogs at all doses. The mean absolute bioavailability of BPC157 following IM injection was approximately 14%-19% in rats and 45%-51% in beagle dogs. Using [3H]-labeled BPC157 and radioactivity examination, we proved that the main excretory pathways of BPC157 involved urine and bile. [3H]BPC157 was rapidly metabolized into a variety of small peptide fragments in vivo, thus forming single amino acids that entered normal amino acid metabolism and excretion pathways. In conclusion, this study provides the first analysis of the pharmacokinetics of BPC157, which will be helpful for its translation in the clinic.

A Heterodimeric Reduced-Ferredoxin-Dependent Methylenetetrahydrofolate Reductase from Syngas-Fermenting Clostridium ljungdahlii.

The strict anaerobe Clostridium ljungdahlii can ferment CO or H2/CO2 via the Wood-Ljungdahl pathway to acetate, ethanol, and 2,3-butanediol. This ability has attracted considerable interest, since it can be used for syngas fermentation to produce biofuels and biochemicals. However, the key enzyme methylenetetrahydrofolate reductase (MTHFR) in the Wood-Ljungdahl pathway of the strain has not been characterized, and its physiological electron donor is unclear. In this study, we purified the enzyme 46-fold with a benzyl viologen reduction activity of 41.2 U/mg from C. ljungdahlii cells grown on CO. It is composed of two subunits, MetF (31.5 kDa) and MetV (23.5 kDa), and has an apparent molecular mass of 62.2 kDa. The brownish yellow protein contains 0.73 flavin mononucleotide (FMN) and 7.4 Fe, in agreement with the prediction that MetF binds one flavin and MetV binds two [4Fe4S] clusters. It cannot use NAD(P)H as its electron donor or catalyze an electron-bifurcating reaction in combination with ferredoxin as an electron acceptor. The reduced recombinant ferredoxin, flavodoxin, and thioredoxin of C. ljungdahlii can serve as electron donors with specific activities of 91.2, 22.1, and 7.4 U/mg, respectively. The apparent Km values for reduced ferredoxin and flavodoxin were around 1.46 µM and 0.73 µM, respectively. Subunit composition and phylogenetic analysis showed that the enzyme from C. ljungdahlii belongs to MetFV-type MTHFR, which is a heterodimer, and uses reduced ferredoxin as its electron donor. Based on these results, we discuss the energy metabolism of C. ljungdahlii when it grows on CO or H2 plus CO2. IMPORTANCE Syngas, a mixture of CO, CO2, and H2, is the main component of steel mill waste gas and also can be generated by the gasification of biomass and urban domestic waste. Its fermentation to biofuels and biocommodities has attracted attention due to the economic and environmental benefits of this process. Clostridium ljungdahlii is one of the superior acetogens used in the technology. However, the biochemical mechanism of its gas fermentation via the Wood-Ljungdahl pathway is not completely clear. In this study, the key enzyme, methylenetetrahydrofolate reductase (MTHFR), was characterized and found to be a non-electron-bifurcating heterodimer with reduced ferredoxin as its electron donor, representing another example of MetFV-type MTHFR. The findings will form the basis for a deeper understanding of the energy metabolism of syngas fermentation by C. ljungdahlii, which is valuable for developing metabolic engineering strains and efficient syngas fermentation technologies.

GDF15 induces immunosuppression via CD48 on regulatory T cells in hepatocellular carcinoma.

BACKGROUND: A better understanding of the molecular mechanisms that manifest in the immunosuppressive tumor microenvironment (TME) is crucial for developing more efficacious immunotherapies for hepatocellular carcinoma (HCC), which has a poor response to current immunotherapies. Regulatory T (Treg) cells are key mediators of HCC-associated immunosuppression. We investigated the selective mechanism exploited by HCC that lead to Treg cells expansion and to find more efficacious immunotherapies. METHODS: We used matched tumor tissues and blood samples from 150 patients with HCC to identify key factors of Treg cells expansion. We used mass cytometry (CyTOF) and orthotopic cancer mouse models to analyze overall immunological changes after growth differentiation factor 15 (GDF15) gene ablation in HCC. We used flow cytometry, coimmunoprecipitation, RNA sequencing, mass spectrum, chromatin immunoprecipitation and Gdf15-/-, OT-I and GFP transgenic mice to demonstrate the effects of GDF15 on Treg cells and related molecular mechanism. We used hybridoma technology to generate monoclonal antibody to block GDF15 and evaluate its effects on HCC-associated immunosuppression. RESULTS: GDF15 is positively associated with the elevation of Treg cell frequencies in patients wih HCC. Gene ablation of GDF15 in HCC can convert an immunosuppressive TME to an inflammatory state. GDF15 promotes the generation of peripherally derived inducible Treg (iTreg) cells and enhances the suppressive function of natural Treg (nTreg) cells by interacting with a previously unrecognized receptor CD48 on T cells and thus downregulates STUB1, an E3 ligase that mediates forkhead box P3 (FOXP3) protein degradation. GDF15 neutralizing antibody effectively eradicates HCC and augments the antitumor immunity in mouse. CONCLUSIONS: Our results reveal the generation and function enhancement of Treg cells induced by GDF15 is a new mechanism for HCC-related immunosuppression. CD48 is the first discovered receptor of GDF15 in the immune system which provide the possibility to solve the molecular mechanism of the immunomodulatory function of GDF15. The therapeutic GDF15 blockade achieves HCC clearance without obvious adverse events.

An NAD-Specific 6-Hydroxy-3-Succinoyl-Semialdehyde-Pyridine Dehydrogenase from Nicotine-Degrading Agrobacterium tumefaciens Strain S33.

Agrobacterium tumefaciens strain S33 can catabolize nicotine via a hybrid of the pyridine and pyrrolidine pathways. Most of the enzymes involved in this biochemical pathway have been identified and characterized, except for the one catalyzing the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. Based on a previous genomic and transcriptomic analysis, an open reading frame (ORF) annotated to encode aldehyde dehydrogenase (Ald) in the nicotine-degrading cluster was predicted to be responsible for this step. In this study, we heterologously expressed the enzyme and identified its function by biochemical assay and mass spectrum analysis. It was found that Ald catalyzes the NAD-specific dehydrogenation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine. With the nonhydroxylated analog 3-succinoyl-semialdehyde-pyridine (SAP) as a substrate, Ald had a specific activity of 10.05 U/mg at pH 9.0 and apparent Km values of around 58.68 µM and 0.41 mM for SAP and NAD+, respectively. Induction at low temperature and purification and storage in low-salt buffers were helpful to prevent its aggregation and precipitation. Disruption of the ald gene caused a lower growth rate and biomass of strain S33 on nicotine but not on 6-hydroxy-3-succinoylpyridine. Ald has a broad range of substrates, including benzaldehyde, furfural, and acetaldehyde. Recombinant Escherichia coli cells harboring the ald gene can efficiently convert furfural to 2-furoic acid at a specific rate of 0.032 mmol min-1 g dry cells-1, extending the application of Ald in the catalysis of bio-based furan compounds. These findings provide new insights into the biochemical mechanism of the nicotine-degrading hybrid pathway and the possible application of Ald in industrial biocatalysis. IMPORTANCE Nicotine is one of the major toxic N-heterocyclic aromatic alkaloids produced in tobacco plants. Manufacturing tobacco and smoking may lead to some environmental and public health problems. Microorganisms can degrade nicotine by various biochemical pathways, but the biochemical mechanism for nicotine degradation has not been fully elucidated. In this study, we identified an aldehyde dehydrogenase responsible for the oxidation of 6-hydroxy-3-succinoyl-semialdehyde-pyridine to 6-hydroxy-3-succinoylpyridine; this was the only uncharacterized enzyme in the hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33. Similar to the known aldehyde dehydrogenase, the NAD-specific homodimeric enzyme presents a broad substrate range with high activity in alkaline and low-salt-containing buffers. It can catalyze not only the aldehyde from nicotine degradation but also those of benzaldehyde, furfural, and acetaldehyde. It was found that recombinant Escherichia coli cells harboring the ald gene could efficiently convert furfural to valuable 2-furoic acid, demonstrating its potential application for enzymatic catalysis.

Renewable fatty acid ester production in Clostridium.

Bioproduction of renewable chemicals is considered as an urgent solution for fossil energy crisis. However, despite tremendous efforts, it is still challenging to generate microbial strains that can produce target biochemical to high levels. Here, we report an example of biosynthesis of high-value and easy-recoverable derivatives built upon natural microbial pathways, leading to improvement in bioproduction efficiency. By leveraging pathways in solventogenic clostridia for co-producing acyl-CoAs, acids and alcohols as precursors, through rational screening for host strains and enzymes, systematic metabolic engineering-including elimination of putative prophages, we develop strains that can produce 20.3 g/L butyl acetate and 1.6 g/L butyl butyrate. Techno-economic analysis results suggest the economic competitiveness of our developed bioprocess. Our principles of selecting the most appropriate host for specific bioproduction and engineering microbial chassis to produce high-value and easy-separable end products may be applicable to other bioprocesses.