Construction of an artificial phosphoketolase pathway that efficiently catabolizes multiple carbon sources to acetyl-CoA.

The canonical glycolysis pathway is responsible for converting glucose into 2 molecules of acetyl-coenzyme A (acetyl-CoA) through a cascade of 11 biochemical reactions. Here, we have designed and constructed an artificial phosphoketolase (APK) pathway, which consists of only 3 types of biochemical reactions. The core enzyme in this pathway is phosphoketolase, while phosphatase and isomerase act as auxiliary enzymes. The APK pathway has the potential to achieve a 100% carbon yield to acetyl-CoA from any monosaccharide by integrating a one-carbon condensation reaction. We tested the APK pathway in vitro, demonstrating that it could efficiently catabolize typical C1-C6 carbohydrates to acetyl-CoA with yields ranging from 83% to 95%. Furthermore, we engineered Escherichia coli stain capable of growth utilizing APK pathway when glycerol act as a carbon source. This novel catabolic pathway holds promising route for future biomanufacturing and offering a stoichiometric production platform using multiple carbon sources.

Application of starch-based nanoparticles and cyclodextrin for prebiotics delivery and controlled glucose release in the human gut: a review.

Starches are a major constituent of staple foods and are the main source of energy in the human diet (55-70%). In the gastrointestinal tract, starches are hydrolyzed into glucose by α-amylase and α-glucosidase, which leads to a postprandial glucose elevation. High levels of blood glucose levels over sustained periods may promote type 2 diabetes mellitus (T2DM) and obesity. Increasing consumption of starchy foods with a lower glycemic index may therefore contribute to improved health. In this paper, the preparation and properties of several starch-based nanoparticles (SNPs) and cyclodextrins (CDs) derivatives are reviewed. In particular, we focus on the various mechanisms responsible for the ability of these edible nanomaterials to modulate glucose release and the gut microbiome in the gastrointestinal tract. The probiotic functions are achieved through encapsulation and protection of prebiotics or bioactive components in foods or the human gut. This review therefore provides valuable information that could be used to design functional foods for improving human health and wellbeing.

Effects of body parameters on renal cortical stiffness measured by shear-wave elastography in patients with kidney transplantation. / 人体参数对剪切波弹性成像定量移植肾皮质硬度的影响.

OBJECTIVES: The results of elastic imaging in evaluating the function and histopathological changes of allogeneic renal transplantation are contradictory, one of the important reasons may be that there are differences in human parameters related to kidney transplantation among individuals. The purpose of this study is to explore the related human body parameters on shear-wave elastography (SWE) effects on quantitative stiffness of graft cortex. METHODS: From March 2021 to November 2021, a total of 63 patients with allogeneic kidney transplantation in the Department of Ultrasonography, Third Xiangya Hospital, Central South University, were selected to collect the parameters of two-dimensional, color Doppler and SWE. The subjects were divided into a <20% group and a 20%-30% group according to the variation of cortical hardness measurement. Mann Whitney U test was used to compare the differences in relevant human parameters, and Spearman rank correlation was used to analyze the correlation between relevant human parameters and cortical hardness of transplanted kidney. RESULTS: There was no significant difference between the 2 groups in age, sex, postoperative time, resistance index of interlobar artery, SCr, blood uric acid, ratio of fat layer to muscle layer, and BMI (all P>0.05). Compared with the <20% group, the patients in the 20%-30% group had smaller cortical hardness of the transplanted kidney, greater total distance between the transplanted kidney and the skin surface, and thicker fat layer or muscle layer in front of the transplanted kidney (all P<0.05). The age of patients, the total distance from the transplanted kidney to the skin surface, the thickness of fat layer and muscle layer, the ratio of fat layer to muscle layer, BMI, and the variation of cortical hardness were significantly negatively correlated with the cortical hardness of the transplanted kidney (all P<0.05). CONCLUSIONS: Human parameters relevant to kidney transplantation affect the accuracy of SWE in measuring the cortical hardness of the transplanted kidney. It is very important to obtain the highly stabile elastic measurement value and interpret the elastic measurement results according to different levels of human body related parameters in combination with individual conditions.

Biocatalytic C-C Bond Formation for One Carbon Resource Utilization.

The carbon-carbon bond formation has always been one of the most important reactions in C1 resource utilization. Compared to traditional organic synthesis methods, biocatalytic C-C bond formation offers a green and potent alternative for C1 transformation. In recent years, with the development of synthetic biology, more and more carboxylases and C-C ligases have been mined and designed for the C1 transformation in vitro and C1 assimilation in vivo. This article presents an overview of C-C bond formation in biocatalytic C1 resource utilization is first provided. Sets of newly mined and designed carboxylases and ligases capable of catalyzing C-C bond formation for the transformation of CO2, formaldehyde, CO, and formate are then reviewed, and their catalytic mechanisms are discussed. Finally, the current advances and the future perspectives for the development of catalysts for C1 resource utilization are provided.

Systematic design and in vitro validation of novel one-carbon assimilation pathways.

The utilization of one-carbon (C1) assimilation pathways to produce chemicals and fuels from low-cost C1 compounds could greatly reduce the substrate-related production costs, and would also alleviate the pressure of the resource supply for bio-manufacturing. However, the natural C1 assimilation pathways normally involve ATP consumption or the loss of carbon resources as CO2, resulting in low product yields, making the design of novel pathways highly pertinent. Here we present several new ATP-independent and carbon-conserving C1 assimilation cycles with 100% theoretical carbon yield, which were discovered by computational analysis of metabolic reaction set with 6578 natural reactions from MetaCyc database and 73 computationally predicted aldolase reactions from ATLAS database. Then, kinetic evaluation of these cycles was conducted and the cycles without kinetic traps were chosen for further experimental verification. Finally, we used the two engineered enzymes Gals and TalBF178Y for the artificial reactions to construct a novel C1 assimilation pathway in vitro and optimized the pathway to achieve 88% carbon yield. These results demonstrate the usefulness of computational design in finding novel metabolic pathways for the efficient utilization of C1 compounds and shedding light on other promising pathways.

Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture.

Identification and characterization of a potential strain for the production of polyhydroxyalkanoate from glycerol.

While poly (3-hydroxybutyrate) (PHB) holds promise as a bioplastic, its commercial utilization has been hampered by the high cost of raw materials. However, glycerol emerges as a viable feedstock for PHB production, offering a sustainable production approach and substantial cost reduction potential. Glycerol stands out as a promising feedstock for PHB production, offering a pathway toward sustainable manufacturing and considerable cost savings. The identification and characterization of strains capable of converting glycerol into PHB represent a pivotal strategy in advancing PHB production research. In this study, we isolated a strain, Ralstonia sp. RRA (RRA). The strain exhibits remarkable proficiency in synthesizing PHB from glycerol. With glycerol as the carbon source, RRA achieved a specific growth rate of 0.19 h-1, attaining a PHB content of approximately 50% within 30 h. Through third-generation genome and transcriptome sequencing, we elucidated the genome composition and identified a total of eight genes (glpR, glpD, glpS, glpT, glpP, glpQ, glpV, and glpK) involved in the glycerol metabolism pathway. Leveraging these findings, the strain RRA demonstrates significant promise in producing PHB from low-cost renewable carbon sources.

Cytochrome P450 Enzyme Design by Constraining the Catalytic Pocket in a Diffusion Model.

Although cytochrome P450 enzymes are the most versatile biocatalysts in nature, there is insufficient comprehension of the molecular mechanism underlying their functional innovation process. Here, by combining ancestral sequence reconstruction, reverse mutation assay, and progressive forward accumulation, we identified 5 founder residues in the catalytic pocket of flavone 6-hydroxylase (F6H) and proposed a "3-point fixation" model to elucidate the functional innovation mechanisms of P450s in nature. According to this design principle of catalytic pocket, we further developed a de novo diffusion model (P450Diffusion) to generate artificial P450s. Ultimately, among the 17 non-natural P450s we generated, 10 designs exhibited significant F6H activity and 6 exhibited a 1.3- to 3.5-fold increase in catalytic capacity compared to the natural CYP706X1. This work not only explores the design principle of catalytic pockets of P450s, but also provides an insight into the artificial design of P450 enzymes with desired functions.

CircSLC39A8 attenuates paclitaxel resistance in ovarian cancer by regulating the miR­185­5p/BMF axis.

Chemoresistance to paclitaxel (PTX) is one of the main reasons for treatment failure and poor prognosis in patients with advanced ovarian cancer. Therefore, it is imperative to explore the mechanisms related to chemotherapy resistance in ovarian cancer to find potential therapeutic targets. Circular RNAs (circRNAs) play important roles in cancer development and progression. However, their biological functions and clinical significance in ovarian cancer have not been fully elucidated. Therefore, in this study, we aimed to investigate the function and underlying mechanism of hsa_circ_0002782 (circSLC39A8), identified by circRNA sequencing, in regulating PTX resistance. The effects of circSLC39A8 on PTX resistance was assessed by cell viability, colony formation, flow cytometry assays and an in vivo subcutaneous xenografted tumor mouse model. RNA immunoprecipitation and dual-luciferase reporter assays were performed to verify the interaction between circSLC39A8 and the miR-185-5p/BMF signal axis. We found that circSLC39A8 was downregulated in PTX-resistant ovarian cancer cells and tissues, and its low expression was associated with poor prognosis. Biologically, circSLC39A8 knockdown promoted PTX resistance in vitro and in vivo, while circSLC39A8 overexpression showed the opposite effect. Mechanistically, circSLC39A8, acting as an endogenous sponge for miR-185-5p, could relieve the inhibition of miR-185-5p on the expression of its downstream target, BMF; thus enhancing the sensitivity of ovarian cancer to PTX. Our findings demonstrate that circSLC39A8 can promote PTX sensitivity by regulating the miR-185-5p/BMF axis. This may be a valuable prognostic biomarker and a promising therapeutic target for patients with ovarian cancer.

Enantioselective and solvent-controlled diastereoselective Mannich reaction of isatin imines with hydroxyacetone: synthesis of 3-substituted 3-aminooxindoles.

The diastereoselectively switchable enantioselective Mannich reaction of isatin imines with hydroxyacetone is reported. The chiral primary amino acid catalyzed this Mannich reaction to afford both anti- and syn-Mannich adducts in high yields, good diastereoselectivities, and enantioselectivities. The reason for the solvent control of the diastereoselectivity phenomenon was investigated.

Curcumin enhances the anti-cancer efficacy of paclitaxel in ovarian cancer by regulating the miR-9-5p/BRCA1 axis.

Background: Patients with late-stage ovarian cancer still have a very poor prognosis due to chemotherapy resistance. Curcumin has been shown to synergistically enhance the therapeutic effects of multiple chemotherapeutic agents, but the potential involvement of curcumin in ovarian cancer is largely unknown. This study aimed to investigate whether curcumin has synergistic anti-cancer effects with paclitaxel in ovarian cancer and its underlying mechanism. Methods: Ovarian cancer cell lines (SKOV3 and A2780) were treated with curcumin, alone or combined with paclitaxel. Cell viability, colony formation, EdU incorporation assays, and flow cytometry were used to assess cell proliferation, apoptosis, and cell cycle progression. The cytotoxic synergistic effect of curcumin and paclitaxel was detected by Calcusyn software. RNA immunoprecipitation assay was used to verify the interaction between miR-9-5p and BRCA1. qRT-PCR and Western blot were performed to detect gene and protein expression. Results: We found that curcumin and paclitaxel synergistically inhibited proliferation and promoted apoptosis in ovarian cancer cells. Furthermore, curcumin and paclitaxel combination resulted in decreased miR-9-5p expression and increased BRCA1 expression. Functionally, miR-9-5p overexpression counteracted the synergistic effect of curcumin and paclitaxel on cell proliferation and apoptosis by targeting BRCA1. Meanwhile, in vivo experiments revealed that curcumin and paclitaxel combination dramatically suppressed the growth of transplanted tumors, while miR-9-5p mimics eliminated the growth inhibition of xenografts induced by the combined treatment. Conclusion: Curcumin enhanced the anti-cancer efficacy of paclitaxel in ovarian cancer by regulating the miR-9-5p/BRCA1 axis. These findings provide strong evidence for clinical investigation of curcumin and paclitaxel combination as a novel strategy for ovarian cancer patients, and identify miR-9-5p and BRCA1 as key targets for regulating sensitivity to this therapy.

Safety and efficacy of wrist-ankle acupuncture in treating catheter-related bladder discomfort after transurethral resection of the prostate: a double-blind randomized clinical trial.

Background: Benign prostatic hyperplasia (BPH) is an age-related condition and its prevalence has increased as China's population ages. Transurethral resection of the prostate (TURP) remains the gold standard for treating moderate to severe BPH. Routine placement of a urinary catheter after TURP is often associated with catheter-related bladder discomfort (CRBD). The development of CRBD is related to an increased synthesis of prostaglandin (PG), and wrist-ankle acupuncture (WAA) can inhibit the expression of PG at the site of inflammation, thus alleviating CRBD symptoms. Here we evaluated the efficacy of WAA in alleviating CRBD in patients undergoing TURP. Methods: A total of 46 patients who underwent elective TURP in Hebei Provincial Hospital of Traditional Chinese Medicine from June 2022 to July 2022 were randomly divided into two groups according to the complete randomization method. The WAA group (n=23) and the control group (n=23). The WAA group received WAA, and the needles were retained for 24 h. The control group was treated with sham needles that did not penetrate the skin, and the needles were also retained for 24 h. At T1 (0 h after entering the ward), T2 (0.5 h after entering the ward), T3 (6 h after entering the ward), and T4 (24 h after entering the ward), CRBD severity score, visual analogue scale (VAS) and vital signs monitor were used for assessment. Accidents were recorded in the case report form. Graded data using Wlicoxon signed rank sum test, repeated measures using repeated measures analysis of variance. Results: A total of 46 patients participated in this study, and 44 patients completed the experiment. At T2, T3, and T4, the severity of CRBD in the WAA group was significantly lower than that in the control group (all P<0.05), and the VAS pain score was significantly lower in the WAA group than in the control group (all P<0.05). In contrast, the vital signs, including mean arterial pressure (MAP), heart rate (HR), and blood oxygen saturation, showed no statistical significance (all P>0.05). No accident occurred in both groups. Conclusions: WAA can effectively relieve CRBD symptoms after TURP. WAA deserves further research and assessment for clinical practice. Trial Registration: Chinese Clinical Trial Registry identifier: ChiCTR2200061525..

Construction of functional soybean peptide-cyclodextrin carboxylate nanoparticles and their interaction with porcine pancreatic α-amylase.

In this study, soybean peptide-succinic acid-modified cyclodextrin (SPT-SACD) nanoparticles (NPs) were successfully fabricated by combining SPT and SACD using an antisolvent precipitation approach. The effects of the average molecular weight of SPT and the SPT/SACD mass ratio on the structure and properties of the SACD-SPT NPs were investigated. Under optimal conditions, the SPT/SACD mass ratio was 2:1, and the SPT average molecular weight was 300 Da. SPT-SACD NPs were prepared under these conditions were spherical and had good uniformity. The particle sizes by DLS of SPT1 (300 Da) /SACD and SPT2 (500 Da) /SACD were in the range of 250-400 nm. The interaction between α-amylase and SPT-SACD NPs was investigated using ultraviolet visible (UV-Vis) absorption, fluorescence, and circular dichroism (CD) spectroscopy. The results of the fluorescence spectra and CD spectroscopy suggested that the presence of SPT-SACD NPs changed the microenvironment of the aromatic amino acid residues, which leads to the change of enzyme protein structure. The SPT-SACD NPs statically quenched the intrinsic fluorescence of the α-amylase by forming a complex with the enzyme. Moreover, the SPT-SACD NPs significantly improved the inhibitory effect of EGCG on α-amylase. The semi-inhibitory concentration (IC50) decreased from 0.324 to 0.248 mg/mL. This study provides an improved understanding of the interaction mechanism between polypeptide-cyclodextrin complexes and digestive enzymes, which may facilitate the design of functional foods.

[Artificial bioconversion of carbon dioxide].

Utilization of carbon dioxide (CO2) is a huge challenge for global sustainable development. Biological carbon fixation occurs in nature, but the low energy efficiency and slow speed hamper its commercialization. Physical-chemical carbon fixation is efficient, but relies on high energy consumption and often generates unwanted by-products. Combining the advantages of biological, physical and chemical technologies for efficient utilization of CO2 remains to be an urgent scientific and technological challenge to be addressed. Here, based on the development of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences in the past decade, we summarize the important progress in the design and construction of functional parts, pathways and systems for artificial bioconversion of carbon dioxide, including the breakthrough on the artificial synthesis of starch from CO2. Moreover, we prospect how to further develop the technologies for artificial bioconversion of carbon dioxide. These progress and perspectives provide new insight for achieving the goal of "carbon peaking and carbon neutrality".

PLAA suppresses ovarian cancer metastasis via METTL3-mediated m6A modification of TRPC3 mRNA.

Wide metastasis contributes to a high death rate in ovarian cancer, and understanding of the molecular mechanism helps to find effective targets for metastatic ovarian cancer therapy. It has been found that phospholipase A2-activating protein (PLAA) is inactivated in some cancers, but its role in cancer metastasis remains unknown. Here, we found that PLAA was significantly downregulated in ovarian cancer highly metastatic cell lines and patients, and the low expression of PLAA was associated with poorer prognosis and high-risk clinicopathological features of patients. PLAA inhibited the migration and invasion of ovarian cancer cells and metastasis of transplanted tumor in the orthotopic xenograft mouse model. Meanwhile, PLAA inhibited metastasis of ovarian cancer by inhibiting transient receptor potential channel canonical 3 (TRPC3)-mediated the intracellular Ca2+ level. Mechanistically, PLAA inhibited methyltransferase-like 3 (METTL3) expression through the ubiquitin-mediated degradation, and METTL3 stabilized TRPC3 mRNA expression via N6-methyladenosine (m6A) modification. Our study verified the function and mechanism of the PLAA-METTL3-TRPC3 axis involved in ovarian cancer metastasis, with a view to providing a potential therapeutic approach for ovarian cancer.

Directed Evolution of Propionyl-CoA Carboxylase for Succinate Biosynthesis.

Due to low carboxylase activity, CO2 biotransformation is challenging to achieve using natural CO2 fixation pathways. Liu et al. have improved the activity of propionyl-CoA carboxylase (PCC) 94-fold, enabling the efficient synthesis of succinate from acetyl-CoA and paving the way for CO2 assimilation via the 3-hydroxypropionate (3-HP) bicycle or 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle.

Neuroregeneration and functional recovery after stroke: advancing neural stem cell therapy toward clinical application.

Stroke is a main cause of death and disability worldwide. The ability of the brain to self-repair in the acute and chronic phases after stroke is minimal; however, promising stem cell-based interventions are emerging that may give substantial and possibly complete recovery of brain function after stroke. Many animal models and clinical trials have demonstrated that neural stem cells (NSCs) in the central nervous system can orchestrate neurological repair through nerve regeneration, neuron polarization, axon pruning, neurite outgrowth, repair of myelin, and remodeling of the microenvironment and brain networks. Compared with other types of stem cells, NSCs have unique advantages in cell replacement, paracrine action, inflammatory regulation and neuroprotection. Our review summarizes NSC origins, characteristics, therapeutic mechanisms and repair processes, then highlights current research findings and clinical evidence for NSC therapy. These results may be helpful to inform the direction of future stroke research and to guide clinical decision-making.

Chromosome-level genome of Himalayan yew provides insights into the origin and evolution of the paclitaxel biosynthetic pathway.

Taxus, commonly known as yew, is a well-known gymnosperm with great ornamental and medicinal value. In this study, by assembling a chromosome-level genome of the Himalayan yew (Taxus wallichiana) with 10.9 Gb in 12 chromosomes, we revealed that tandem duplication acts as the driving force of gene family evolution in the yew genome, resulting in the main genes for paclitaxel biosynthesis, i.e. those encoding the taxadiene synthase, P450s, and transferases, being clustered on the same chromosome. The tandem duplication may also provide genetic resources for the nature to sculpt the core structure of taxoids at different positions and subsequently establish the complex pathway of paclitaxel by neofunctionalization. Furthermore, we confirmed that there are two genes in the cluster encoding isoenzymes of a known enzyme in the paclitaxel biosynthetic pathway. The reference genome of the Himalayan yew will serve as a platform for decoding the complete biosynthetic pathway of paclitaxel and understanding the chemodiversity of taxoids in gymnosperms.

Combining protein and metabolic engineering to construct efficient microbial cell factories.

Microbial cell factories offer great opportunities for the feasible biosynthesis of various value-added products from renewable resources. In recent years, protein engineering has served as a powerful approach for generating enzymes or proteins with desirable properties. As an effective toolbox, many efforts have been employed into protein engineering for the improvement of biological networks by enhancing, regulating, expanding, and innovating metabolic pathways. In combination with traditional metabolic engineering strategies, protein engineering has demonstrated tremendous promise in facilitating the production of many bio-based products. In this review, we present recent trends and strategies available for protein engineering, and highlight the successful interplay between protein and metabolic engineering in boosting the production of target compounds using diverse microbial cell factories. The perspectives for protein design in metabolic engineering are also briefly discussed.

[Biocatalysis of formaldehyde to L-xylose].

It is of great significance to use biosynthesis to transform the inorganic substance formaldehyde into organic sugars. Most important in this process was to find a suitable catalyst combination to achieve the dimerization of formaldehyde. In a recent report, an engineered glycolaldehyde synthase was reported to catalyze this reaction. It could be combined with engineered D-fructose-6-phosphate aldolase, a "one-pot enzyme" method, to synthesize L-xylose using formaldehyde and the conversion rate could reach up to 64%. This process also provides a reference for the synthesis of other sugars. With the increasing consumption of non-renewable resources, it was of great significance to convert formaldehyde into sugar by biosynthesis.