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.

A Widespread Radical-Mediated Glycolysis Pathway.

Glycyl radical enzymes (GREs) catalyze mechanistically diverse radical-mediated reactions, playing important roles in the metabolism of anaerobic bacteria. The model bacterium Escherichia coli MG1655 contains two GREs of unknown function, YbiW and PflD, which are widespread among human intestinal bacteria. Here, we report that YbiW and PflD catalyze ring-opening C-O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C-O cleavage. This "anhydroglycolysis" pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch. Discovery of the anhydroglycolysis pathway expands the known catalytic repertoire of GREs, clarifies the hitherto unknown physiological functions of the well-studied enzymes FsaA, FsaB, and GldA, and demonstrates how enzyme discovery efforts can cast light on prevalent yet overlooked metabolites in the microbiome.

Label-Free Imaging of Cell Apoptosis by a Light-Addressable Electrochemical Sensor.

Label-free electrochemical visualization of cancer cell apoptosis is essential for cancer therapies. In this work, we proposed a noninvasive imaging method using a light-addressable electrochemical sensor (LAES) for label-free imaging of drug-induced tumor cell apoptosis. The dynamic AC photocurrent changes on MCF-7 human breast adenocarcinoma cells after inducing by tamoxifen were imaged. And the reasons for photocurrent changes on the cells were explored by monitoring the changes in the ζ potentials of cells and Faradic impedance. The results demonstrated that the AC photocurrent on apoptotic MCF-7 cells increased, and the apoptosis degree of each cell was heterogeneous. Moreover, the AC photocurrent increase was attributed to the increased cell membrane permeability and the increased gap between the cell basal surface and the substrate caused by cell apoptosis. This study provides a brand new approach for label-free visualizing cell apoptosis heterogeneity, which has great potential in apoptosis-associated drug screening or drug efficacy evaluation.

Blocking Rice Shoot Gravitropism by Altering One Amino Acid in LAZY1.

Tiller angle is an important trait that determines plant architecture and yield in cereal crops. Tiller angle is partially controlled during gravistimulation by the dynamic re-allocation of LAZY1 (LA1) protein between the nucleus and plasma membrane, but the underlying mechanism remains unclear. In this study, we identified and characterized a new allele of LA1 based on analysis of a rice (Oryza sativa L.) spreading-tiller mutant la1G74V, which harbors a non-synonymous mutation in the predicted transmembrane (TM) domain-encoding region of this gene. The mutation causes complete loss of shoot gravitropism, leading to prostrate growth of plants. Our results showed that LA1 localizes not only to the nucleus and plasma membrane but also to the endoplasmic reticulum. Removal of the TM domain in LA1 showed spreading-tiller phenotype of plants similar to la1G74V but did not affect the plasma membrane localization; thus, making it distinct from its ortholog ZmLA1 in Zea mays. Therefore, we propose that the TM domain is indispensable for the biological function of LA1, but this domain does not determine the localization of the protein to the plasma membrane. Our study provides new insights into the LA1-mediated regulation of shoot gravitropism.

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.

Comprehensive analysis of presurgical factors predicting psychiatric disorders in patients with refractory temporal lobe epilepsy and mesial temporal sclerosis underwent cortico-amygdalohippocampectomy.

BACKGROUND: This study aimed to evaluate the predictive value of presurgical factors for psychiatric disorders (PD) in refractory temporal lobe epilepsy and mesial temporal sclerosis (TLE-MTS) patients underwent cortico-amygdalohippocampectomy (CAH). METHODS: A total of 98 refractory TLE-MTS patients underwent CAH were consecutively enrolled in this cohort study. Several presurgical factors were recorded, such as married status, employment status, highest education, disease duration, family history of epilepsy, and disorganized VEEG background activity. RESULTS: There were 17 (17.3%) refractory TLE-MTS patients occurring PD after CAH, including 8 (8.2%) mood disorders, 7 (7.1%) anxiety disorders, 8 (8.2%) psychoses, and 1 (1.0%) interictal dysphoric disorder. Employed status correlated with low PD occurrence, while disease duration and asymmetric VEEG background activity positively correlated with PD occurrence. Multivariate logistic analysis revealed employed status (P = 0.009) could independently predict lower PD occurrence, while highest education (P = 0.027), disease duration (P = 0.028), seizure frequencies (P = 0.015), and asymmetric VEEG background activity (P = 0.034) could independently predict higher PD occurrence. Receiver operating characteristic curve showed combination of these five factors (area under curve (AUC) = 0.871, 95%CI: 0.783-0.960) disclosed a great predictive value of PD occurrence. The sensitivity and specificity were 70.6% and 92.6% at the best cutoff point. In addition, the percentage of PD was increased with higher Engel classification (P = 0.003). CONCLUSION: Employed status, highest education, disease duration, seizure frequencies, and asymmetric VEEG background activity correlate with PD occurrence independently in epileptic patients.

Invariant natural killer T cells promote immunogenic maturation of lung dendritic cells in mouse models of asthma.

Our previous study showed that invariant natural killer T (iNKT) cells might act as an adjuvant to promote Th2 inflammatory responses in an OVA-induced mouse model of allergic asthma, but the mechanism remains unknown. To clarify the underlying mechanism through which iNKT cells promote Th2 inflammatory responses, we investigated the modulatory influence of iNKT cells on phenotypic and functional maturation of lung dendritic cells (LDCs) using iNKT cell-knockout mice, specific iNKT cell activation, coculture experiments, and adoptive transfer of iNKT cells in mouse models of asthma. Our data showed that iNKT cell deficiency could downregulate surface maturation markers and proinflammatory cytokine secretion of LDCs from a mouse model of asthma. However, elevated activation of iNKT cells by α-galactosylceramide and adoptive transfer of iNKT cells could upregulate surface maturation markers and proinflammatory cytokine secretion of LDCs from mouse models of asthma. Meanwhile, iNKT cells significantly influenced the function of LDCs, markedly enhancing Th2 responses in vivo and in vitro. In addition, iNKT cell can induce LDCs expression of CD206 and RELM-α, reflecting alternative activation of LDCs in a mouse model of asthma. α-Galactosylceramide treatment significantly enhanced expression of CD40L of lung iNKT cells from a mouse model of asthma, and the coculture experiment of LDCs with iNKT cells showed that the blockade of CD40L strongly suppressed surface maturation markers and proinflammatory cytokine production by LDCs. Our data suggest that iNKT cells can promote immunogenic maturation of LDCs to enhance Th2 responses in mouse models of asthma.

One-step construction of multiplexed enzymatic biosensors using light-addressable electrochemistry on a single silicon photoelectrode.

The multiplexed detection of metabolites in parallel within a single biosensor plate is sufficiently valuable but also challenging. Herein, we combine the inherent light addressability of silicon with the high selectivity of enzymes, for the construction of multiplexed photoelectrochemical enzymatic biosensors. To conduct a stable electrochemistry and reagentless biosensing on silicon, a new strategy involving the immobilization of both redox mediators and enzymes using an amide bond-based hydrogel membrane was proposed. The membrane characterization results demonstrated a covalent coupling of ferrocene mediator to hydrogel, in which the mediator acted as not only a signal generator but also a renewable sacrifice agent. By adding corresponding enzymes on different spots of hydrogel membrane modified silicon and recording local photocurrents with a moveable light pointer, this biosensor setup was used successfully to detect multiple metabolites, such as lactate, glucose, and sarcosine, with good analytical performances. The limits of detection of glucose, sarcosine and lactate were found to be 179 µM, 16 µM, and 780 µM with the linear ranges of 0.5-2.5 mM, 0.3-1.5 mM, and 1.0-3.0 mM, respectively. We believe this proof-of-concept study provides a simple and rapid one-step immobilization approach for the fabrication of reagentless enzymatic assays with silicon-based light-addressable electrochemistry.

Carbonized polydopamine layer-protected silicon substrates for light-addressable electrochemical sensing and imaging.

The application of silicon (Si) substrate as photoelectrode in light-addressable electrochemistry (LAE) is severely limited due to its ease of surface oxidation. The resulted silicon oxide (SiOx) layer is electronically insulating and blocks charge transfer between the electrode and electrolyte. Keeping the Si from being oxidized is a key challenge for its practical use as a semiconductor electrode. In this work, we find that by developing a thin layer of polydopamine film on the surface of Si substrate, followed by carbonization at 550 °C, the natural oxidation of Si substrate can be successfully forestalled. When applied as an electrode, it is further found that the carbonized polydopamine (cPDA) layer can also prevent anodic oxidation of Si. The cPDA layer-modified Si substrate exhibits good photoelectrochemical performance and great stability, with no obvious signal decrease under ambient environment over 32 h. Our work here provides a new modification strategy for anti-oxidation of Si substrate and it is promising in the application of light-addressable electrochemical sensing and imaging.

Potential Roles of the Gut Microbiota in Pancreatic Carcinogenesis and Therapeutics.

The intestinal microenvironment is composed of normal gut microbiota and the environment in which it lives. The largest microecosystem in the human body is the gut microbiota, which is closely related to various diseases of the human body. Pancreatic cancer (PC) is a common malignancy of the digestive system worldwide, and it has a 5-year survival rate of only 5%. Early diagnosis of pancreatic cancer is difficult, so most patients have missed their best opportunity for surgery at the time of diagnosis. However, the etiology is not entirely clear, but there are certain associations between PC and diet, lifestyle, obesity, diabetes and chronic pancreatitis. Many studies have shown that the translocation of the gut microbiota, microbiota dysbiosis, imbalance of the oral microbiota, the interference of normal metabolism function and toxic metabolite products are closely associated with the incidence of PC and influence its prognosis. Therefore, understanding the correlation between the gut microbiota and PC could aid the diagnosis and treatment of PC. Here, we review the correlation between the gut microbiota and PC and the research progresses for the gut microbiota in the diagnosis and treatment of PC.