Small molecules reprogram reactive astrocytes into neuronal cells in the injured adult spinal cord.

INTRODUCTION: Ectopic expression of transcription factor-mediated in vivo neuronal reprogramming provides promising strategy to compensate for neuronal loss, while its further clinical application may be hindered by delivery and safety concerns. As a novel and attractive alternative, small molecules may offer a non-viral and non-integrative chemical approach for reprogramming cell fates. Recent definitive evidences have shown that small molecules can convert non-neuronal cells into neurons in vitro. However, whether small molecules alone can induce neuronal reprogramming in vivo remains largely unknown. OBJECTIVES: To identify chemical compounds that can induce in vivo neuronal reprogramming in the adult spinal cord. METHODS: Immunocytochemistry, immunohistochemistry, qRT-PCR and fate-mapping are performed to analyze the role of small molecules in reprogramming astrocytes into neuronal cells in vitro and in vivo. RESULTS: By screening, we identify a chemical cocktail with only two chemical compounds that can directly and rapidly reprogram cultured astrocytes into neuronal cells. Importantly, this chemical cocktail can also successfully trigger neuronal reprogramming in the injured adult spinal cord without introducing exogenous genetic factors. These chemically induced cells showed typical neuronal morphologies and neuron-specific marker expression and could become mature and survive for more than 12 months. Lineage tracing indicated that the chemical compound-converted neuronal cells mainly originated from post-injury spinal reactive astrocytes. CONCLUSION: Our proof-of-principle study demonstrates that in vivo glia-to-neuron conversion can be manipulated in a chemical compound-based manner. Albeit our current chemical cocktail has a lowreprogramming efficiency, it will bring in vivo cell fate reprogramming closer to clinical application in brain and spinal cord repair. Future studies should focus on further refining our chemical cocktail and reprogramming approach to boost the reprogramming efficiency.

Complete Depolymerization of PET Wastes by an Evolved PET Hydrolase from Directed Evolution.

PETase displays great potential in PET depolymerization. Directed evolution has been limited to engineer PETase due to the lack of high-throughput screening assay. In this study, a novel fluorescence-based high-throughput screening assay employing a newly designed substrate, bis (2-hydroxyethyl) 2-hydroxyterephthalate (termed BHET-OH), was developed for PET hydrolases. The best variant DepoPETase produced 1407-fold more products towards amorphous PET film at 50 °C and showed a 23.3 °C higher Tm value than the PETase WT. DepoPETase enabled complete depolymerization of seven untreated PET wastes and 19.1 g PET waste (0.4 % Wenzyme /WPET ) in liter-scale reactor, suggesting that it is a potential candidate for industrial PET depolymerization processes. The molecular dynamic simulations revealed that the distal substitutions stabilized the loops around the active sites and transmitted the stabilization effect to the active sites through enhancing inter-loop interactions network.

Systems metabolic engineering of Escherichia coli for hyper-production of 5­aminolevulinic acid.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) is a promising biostimulant, feed nutrient, and photodynamic drug with wide applications in modern agriculture and therapy. Although microbial production of 5-ALA has been improved realized by using metabolic engineering strategies during the past few years, there is still a gap between the present production level and the requirement of industrialization. RESULTS: In this study, pathway, protein, and cellular engineering strategies were systematically employed to construct an industrially competitive 5-ALA producing Escherichia coli. Pathways involved in precursor supply and product degradation were regulated by gene overexpression and synthetic sRNA-based repression to channel metabolic flux to 5-ALA biosynthesis. 5-ALA synthase was rationally engineered to release the inhibition of heme and improve the catalytic activity. 5-ALA transport and antioxidant defense systems were targeted to enhance cellular tolerance to intra- and extra-cellular 5-ALA. The final engineered strain produced 30.7 g/L of 5-ALA in bioreactors with a productivity of 1.02 g/L/h and a yield of 0.532 mol/mol glucose, represent a new record of 5-ALA bioproduction. CONCLUSIONS: An industrially competitive 5-ALA producing E. coli strain was constructed with the metabolic engineering strategies at multiple layers (protein, pathway, and cellular engineering), and the strategies here can be useful for developing industrial-strength strains for biomanufacturing.

Preference of Virtual Reality Games in Psychological Pressure and Depression Treatment: Discrete Choice Experiment.

BACKGROUND: Virtual reality (VR) can be used to build many different scenes aimed at reducing study-related stress. However, only few academic experiments on university students for preference testing have been performed. OBJECTIVE: This study aims to assess the preference of VR games for stress and depression treatment using a discrete choice experiment (DCE). METHODS: A total of 5 different attributes were selected based on the depression therapy parameters and attributes related to VR: (1) treatment modality; (2) therapy duration; (3) perceived remission rate; (4) probability of adverse events; and the (5) monthly cost of adding treatment to a discrete choice experiment. By comparing different attributes and levels, we could draw some conclusions about the depression therapy testing preference for university students; 1 university student was responsible for VR scene development and 1 for participant recruitment. RESULTS: The utility value of different attributes for "0% Probability of adverse events" was higher than others (99.22), and the utility value of VR treatment as the most popular treatment method compared with counseling and medicine treatment was 80.95. Three parameter aspects (different treatments for depression) were statistically significant (P<.001), including "0%" and "50%" of "Probability of adverse events" and "¥500" (a currency exchange rate of ¥1 [Chinese yuan]=US $0.15 is applicable) of "The monthly cost of treatment." Most individuals preferred 12 months as the therapy duration, and the odds ratio of "12 months" was 1.095 (95% CI 0.945-1.270) when compared with the reference level (6 months). Meanwhile, the cheapest price (¥500) of depression therapy was the optimum choice for most students. CONCLUSIONS: People placed great preference on VR technology psychological intervention methods, which indicates that VR may have a potential market in the treatment of psychological problems. However, adverse events and treatment costs need to be considered. This study can be used to guide policies that are relevant to the development of the application of VR technology in the field of psychological pressure and depression treatment.

Healthcare preferences of the general Chinese population in the hierarchical medical system: A discrete choice experiment.

Background: Chinese health insurance system faces resource distribution challenges. A patient-centric approach allows decision-makers to be keenly aware of optimized medical resource allocation. Objective: This study aims to use the discrete choice model to determine the main factors affecting the healthcare preferences of the general Chinese population and their weights in the three scenarios (chronic non-communicable diseases, acute infectious diseases, and major diseases). Methods: This study firstly identified the key factors affecting people's healthcare preferences through literature review and qualitative interviews, and then designed the DCE questionnaire. An online questionnaire produced by Lighthouse Studio (version 9.9.1) software was distributed to voluntary respondents recruited from mainland China's entire population from January 2021 to June 2021. Participants were required to answer a total of 21 questions of three scenarios in the questionnaire. The multinomial logit model and latent class model were used to analyze the collected data. Results: A total of 4,156 participants from mainland China were included in this study. The multinomial logit and latent class model analyses showed that medical insurance reimbursement is the most important attribute in all three disease scenarios. In the scenario of "non-communicable diseases," the attributes that participants valued were, from the most to the least, medical insurance reimbursement (45.0%), hospital-level (21.6%), distance (14.4%), cost (9.7%), waiting time (8.3%), and care provider (1.0%). As for willingness to pay (WTP), participants were willing to pay 204.5 yuan, or 1,743.8 yuan, to change from private hospitals or community hospitals to tertiary hospitals, respectively. Conclusions: This study explores the healthcare preferences of Chinese residents from a new perspective, which can provide theoretical reference for the refinement of many disease medical reimbursement policies, such as developing different reimbursement ratios for various common diseases and realizing rational configuration of medical resources.

Lifestyles and health-related quality of life in Chinese people: a national family study.

BACKGROUND: There were few studies that investigated health-related quality of life (HRQoL) of the general population in China, and many of them reported limitations in sampling. OBJECTIVE: To investigate the relationship between lifestyles and HRQoL in the Chinese population in both individual and family levels. METHOD: Online questionnaires were distributed across China to collect demographic information and participants' HRQoL using EuroQoL 5 Dimension scales. The EuroQoL Group's 5 Dimension scale (EQ-5D) index and EuroQoL Group's visual analog scale (EQ VAS) score were calculated to evaluate the HRQoL. RESULTS: A total of 1305 valid questionnaires were included. Higher HRQoL was found in people with intend to lower oil intake, intend to lower salt intake, intend to lower sugar intake, balanced diet, moderate sports every week, a sport hobby and joining a fitness organization (all p<.05). HRQoL was higher among male (female as reference), healthy weight (unhealthy weight as reference) (both p<.05). Negative correlation was found between HRQoL and clinical medical history and drinking history. Small families (1-2 persons, 83.19 ± 20.14) had poorer HRQoL (EQ VAS score) than big families (≥3 persons, 85.00 ± 17.96, p <.05). CONCLUSION: In China, people with healthy dietary habits, regular sports habits, healthy weight and male groups tended to have better HRQoL. Clinical medical history and drinking history were negatively related to HRQoL. Small families tend to have poorer HRQoL than big families. The finding implicated influence of the number of family members on people's perception of health and provided scientific evidence for the current policies to encourage birth in China. For a better HRQoL, we suggest people live in big families and take measures to lower salt/sugar/oil intake and exercise regularly in daily life.

Topoisomerase IIA in adult NSCs regulates SVZ neurogenesis by transcriptional activation of Usp37.

Topoisomerase IIA (TOP2a) has traditionally been known as an important nuclear enzyme that resolves entanglements and relieves torsional stress of DNA double strands. However, its function in genomic transcriptional regulation remains largely unknown, especially during adult neurogenesis. Here, we show that TOP2a is preferentially expressed in neurogenic niches in the brain of adult mice, such as the subventricular zone (SVZ). Conditional knockout of Top2a in adult neural stem cells (NSCs) of the SVZ significantly inhibits their self-renewal and proliferation, and ultimately reduces neurogenesis. To gain insight into the molecular mechanisms by which TOP2a regulates adult NSCs, we perform RNA-sequencing (RNA-Seq) plus chromatin immunoprecipitation sequencing (ChIP-Seq) and identify ubiquitin-specific protease 37 (Usp37) as a direct TOP2a target gene. Importantly, overexpression of Usp37 is sufficient to rescue the impaired self-renewal ability of adult NSCs caused by Top2a knockdown. Taken together, this proof-of-principle study illustrates a TOP2a/Usp37-mediated novel molecular mechanism in adult neurogenesis, which will significantly expand our understanding of the function of topoisomerase in the adult brain.

NOTCH1 signaling regulates the latent neurogenic program in adult reactive astrocytes after spinal cord injury.

Background: Direct reprogramming of astrocytes into neurons opens up a new avenue for neuroregenerative medicine. However, the poor understanding of the molecular mechanisms underpinning the latent neurogenic program in astrocytes has largely restricted this strategy towards safe and effective clinical therapies. Methods: Immunocytochemistry, immunohistochemistry, western blotting, qRT-PCR, gene knockdown and fate-mapping are performed to analyze the role of NOTCH1 signaling in regulation of the latent neurogenic program in reactive astrocytes after spinal cord injury. Results: Western blotting analysis highlights that NOTCH1 is a key signaling mediating Ascl1- and Neurog2-driven astrocyte-to-neuron conversion. Inhibition of NOTCH1 signaling in cultured astrocytes by shRNA or DAPT (a NOTCH1 inhibitor) is sufficient to reprogram them into neurons by upregulating the expression of pro-neural transcription factors, including NeuroD1, NeuroD2, Pax6, Lmx1a and Lhx6. In the spinal cord of adult mouse, the expression of Notch1 is detected in resident astrocytes, which was significantly increased after spinal cord injury (SCI). Genetical knockdown of NOTCH1 signaling alone successfully triggers endogenous reactive astrocytes reprogramming into neurons in the injured adult spinal cord. Importantly, pharmacologically blocking NOTCH1 signaling with small molecule DAPT alone can also induce in situ astrocyte-to-neuron conversion after SCI. Conclusions: We identify NOTCH1 as a key common signaling pathway in reactive astrocyte that provides a barrier for cell fate conversion. This proof-of-principle study will significantly expand our molecular understanding of astroglial-lineage reprogramming and overcoming the NOTCH1 gatekeeper with small molecules may provide a transgene-free approach for in vivo chemical neuronal reprogramming with potential clinical application in neuroregeneration.

Chemoenzymatic conversion of glycerol to lactic acid and glycolic acid.

Catalytic valorization of raw glycerol derived from biodiesel into high-value chemicals has attracted great attention. Here, we report chemoenzymatic cascade reactions that convert glycerol to lactic acid and glycolic acid. In the enzymatic step, a coenzyme recycling system was developed to convert glycerol into 1,3-dihydroxyacetone (DHA) with a yield of 92.3% in potassium phosphate buffer (300 mM, pH 7.1) containing 100 mM glycerol, 2 mM NAD+, 242 U/mL glycerol dehydrogenase-GldA and NADH oxidase-SpNoxK184R at 30 °C. Subsequently, NaOH or NaClO2 catalyzes the formation of lactic acid and glycolic acid from DHA. The high yield of lactic acid (72.3%) and glycolic acid (78.2%) verify the benefit of the chemoenzymatic approaches.

Directed Evolution and Rational Design of Mechanosensitive Channel MscCG2 for Improved Glutamate Excretion Efficiency.

Mechanosensitive amino acid exporters have drawn increasing attention due to their important roles in extracellular accumulation of the target amino acids. Protein engineering is a powerful approach to tailor the properties of amino acid exporters and illustrate structure-function relationships. Here we report the first protein engineering effort on the mechanosensitive glutamate exporter MscCG2 from Corynebacterium glutamicum for improved excretion efficiency of glutamate and understanding of the structure-function relationship. MscCG2 was engineered through directed evolution and computer-assisted design with a coupled assay in microtiter plate format. Improved MscCG2 variants were identified with up to 2.5-fold increase in the level of glutamate excretion in the early stage of fermentation and 1.5-fold in the late stage of fermentation under experimental conditions. Furthermore, the identified variants exhibited enhanced efflux of 4-fluoroglutamate (4-FG), an analog of glutamate. Structure analysis employing homology modeling and molecular dynamics (MD) simulation reveal that identified amino acid substitutions enlarge the size of the seven portals on the equator of MscCG2 and expand the narrowest rim of its inner channel, respectively. This study demonstrates the great potential of protein engineering in improving the secretion efficiency of exporters for enhanced bioproduction.

Engineered LPMO Significantly Boosting Cellulase-Catalyzed Depolymerization of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the enzymatic depolymerization of cellulose through oxidative cleavage of the glycosidic bond in the highly recalcitrant crystalline cellulose region. Improving the activity of LPMOs is of considerable importance for second-generation biorefinery. In this study, we identified a beneficial amino acid substitution (N526S) located in the cellulose binding module (CBM) of HcLPMO10 (LPMO of Hahella chejuensis) using directed evolution. The improved variant HcLPMO10 M1 (N526S) exhibits 2.1-fold higher activity for the H2O2 production, 2.7-fold higher oxidation activity, and 1.9-fold higher binding capacity toward cellulose compared with those of the wild type (WT). Furthermore, M1 shows 2.1-fold higher activity for degradation of crystalline cellulose in synergy with cellulase, compared to the WT. Structural analysis through molecular modeling and molecular dynamics (MD) simulation revealed that the substitution N526S located in the CBM likely stabilizes the cellulose binding surface and enhances the binding capacity of HcLPMO10 to cellulose, thereby enhancing enzyme activity. These findings demonstrate the important role of the CBM in the catalytic function of LPMO.

Direct neuronal reprogramming of olfactory ensheathing cells for CNS repair.

Direct conversion of readily available non-neural cells from patients into induced neurons holds great promise for neurological disease modeling and cell-based therapy. Olfactory ensheathing cells (OECs) is a unique population of glia in olfactory nervous system. Based on the regeneration-promoting properties and the relative clinical accessibility, OECs are attracting increasing attention from neuroscientists as potential therapeutic agents for use in neural repair. Here, we report that OECs can be directly, rapidly and efficiently reprogrammed into neuronal cells by the single transcription factor Neurogenin 2 (NGN2). These induced cells exhibit typical neuronal morphologies, express multiple neuron-specific markers, produce action potentials, and form functional synapses. Genome-wide RNA-sequencing analysis shows that the transcriptome profile of OECs is effectively reprogrammed towards that of neuronal lineage. Importantly, these OEC-derived induced neurons survive and mature after transplantation into adult mouse spinal cords. Taken together, our study provides a direct and efficient strategy to quickly obtain neuronal cells from adult OECs, suggestive of promising potential for personalized disease modeling and cell replacement-mediated therapeutic approaches to neurological disorders.

Inhibition of Glioma Development by ASCL1-Mediated Direct Neuronal Reprogramming.

Direct conversion of non-neural cells into induced neurons holds great promise for brain repair. As the most common malignant tumor in the central nervous system, glioma is currently incurable due to its exponential growth and invasive behavior. Given that neurons are irreversible postmitotic cells, reprogramming glioma cells into terminally differentiated neuron-like cells represents a potential approach to inhibit brain tumor development. We here show that human glioma cells can be directly, rapidly and efficiently reprogrammed into terminally differentiated neuron-like cells by the single transcription factor ASCL1 (Achaete-scute complex-like 1, also known as MASH1). These induced cells exhibit typical neuron-like morphology and express multiple neuron-specific markers. Importantly, ASCL1-mediated neuronal reprogramming drives human glioma cells to exit the cell cycle and results in dramatic inhibition of proliferation, both in vitro and in vivo. Taken together, this proof-of-principle study demonstrates a potential strategy for impeding brain tumor development by ASCL1-induced direct neuronal reprogramming.

Conditional ablation of reactive astrocytes to dissect their roles in spinal cord injury and repair.

Astrocytes become reactive in response to spinal cord injury (SCI) and ultimately form a histologically apparent glial scar at the lesion site. It is controversial whether astrocytic scar is detrimental or beneficial to the axonal regeneration and SCI repair. Therefore, much effort has focused on understanding the functions of reactive astrocytes. Here, we used a lentivirus-mediated herpes simplex thymidine kinase/ganciclovir (HSVtk/GCV) system to selectively kill scar-forming reactive proliferating astrocytes. The suicide gene expression was regulated by human glial fibrillary acidic protein (hGFAP) promoter, which is active primarily in astrocytes. Conditional ablation of reactive astrocytes in a mouse SCI model with crush injury impeded glial scar formation and resulted in widespread infiltration of inflammatory cells, increased neuronal loss, and severe tissue degeneration, which ultimately led to the failure of spontaneous functional recovery. These results suggest that reactive proliferating astrocytes play key roles in the healing process after SCI, shedding light on the potential benefit for the repair after central nervous system (CNS) injury.

Region-Restrict Astrocytes Exhibit Heterogeneous Susceptibility to Neuronal Reprogramming.

The adult CNS has poor ability to replace degenerated neurons following injury or disease. Recently, direct reprogramming of astrocytes into induced neurons has been proposed as an innovative strategy toward CNS repair. As a cell population that shows high diversity on physiological properties and functions depending on their spatiotemporal distribution, however, whether the astrocyte heterogeneity affect neuronal reprogramming is not clear. Here, we show that astrocytes derived from cortex, cerebellum, and spinal cord exhibit biological heterogeneity and possess distinct susceptibility to transcription factor-induced neuronal reprogramming. The heterogeneous expression level of NOTCH1 signaling in the different CNS regions-derived astrocytes is shown to be responsible for the neuronal reprogramming diversity. Taken together, our findings demonstrate that region-restricted astrocytes reveal different intrinsic limitation of the response to neuronal reprogramming.

Enhancing thermostability and removing hemin inhibition of Rhodopseudomonas palustris 5-aminolevulinic acid synthase by computer-aided rational design.

OBJECTIVE: To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy. RESULTS: Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 µM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively. CONCLUSION: H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.

Identification of characteristic genes distinguishing neural stem cells from astrocytes.

BACKGROUND: Neural stem cells (NSCs) have unique biological characteristics such as continuous proliferation and multipotential differentiation, providing a possible method for restoration of central nervous system (CNS) function after injury or disease. NSCs and astrocytes share many similar biological properties including cell morphology and molecular expression and can trans-differentiate into each other under certain conditions. However, characteristic genes specifically expressed by NSCs have not been well described. METHODS: To provide insights into the characteristic expression of NSCs, bioinformatics analysis of two microarrays of mouse NSCs and astrocytes was performed. Compared to astrocytes, the differentially expressed genes (DEGs) in NSCs were identified and annotated by GO, KEGG and GSEA analysis, respectively. Then key genes were screened by protein-protein interaction (PPI) network and modules analysis, and were verified using multiple high-throughput sequencing resources. Finally, the expression difference between the two cell types was confirmed by Real-time Quantitative PCR (qPCR), western blotting and immunochemical analysis. RESULTS: In the present study, 282 and 250 NSC-enriched genes from two microarrays were identified and annotated respectively, and the 77 overlapping DEGs were then selected. From the PPI network 24 key genes in three modules were screened out. Importantly, sequencing data of tissues showed that these 24 key genes tended to be highly expressed in NSCs compared with astrocytes. Furthermore, qPCR and western blot analysis of cultured NSCs and astrocytes showed two genes (KIF2C and TOP2A) were not only differentially expressed in RNA level but also at the protein level. Importantly, the NSC-specific genes KIF2C and TOP2A were validated by immunohistochemistry in vivo. CONCLUSION: In present study, we identified 2 hub genes (KIF2C and TOP2A) that might serve as potential biomarkers for distinguishing NSCs from astrocytes, contributing to our comprehensive understanding of the biological properties and functions of NSCs.

Enhancement of the thermal and alkaline pH stability of Escherichia coli lysine decarboxylase for efficient cadaverine production.

OBJECTIVE: To enhance the thermal and alkaline pH stability of the lysine decarboxylase from Escherichia coli (CadA) by engineering the decameric interface and explore its potential for industrial applications. RESULTS: The mutant T88S was designed for improved structural stability by computational analysis. The optimal pH and temperature of T88S were 7.0 and 55 °C (5.5 and 50 °C for wild-type). T88S showed higher thermostability with a 2.9-fold increase in the half-life at 70 °C (from 11 to 32 min) and increased melting temperature (from 76 to 78 °C). Additionally, the specific activity and pH stability (residual activity after 10 h incubation) of T88S at pH 8.0 were increased to 164 U/mg and 78% (58 U/mg and 57% for wild-type). The productivity of cadaverine with T88S (284 g L-lysine L-1 and 5 g DCW L-1) was 40 g L-1 h-1, in contrast to 28 g L-1 h-1 with wild-type. CONCLUSION: The mutant T88S showed high thermostability, pH stability, and activity at alkaline pH, indicating that this mutant is a promising biocatalyst for industrial production of cadaverine.

Establishment and characterization of primary astrocyte culture from adult mouse brain.

As a major class of glial cells, astrocytes have been indicated to play multi-roles in physiological and pathological brain. Astrocyte cultures derived from postnatal mouse brains have been extensively used to characterize their biological properties. However, the inability to culture adult mouse primary astrocytes has long stymied studies of function in adult brain. Here, we developed a protocol to successfully establish highly enriched astrocyte cultures from the brains of adult mouse. Cortical tissues were collected to prepare cell suspension by enzymatic digestion and mechanical dissociation, and then plated onto vessels pre-coated with gelatin and matrigel and cultured in DMEM medium containing 20% fetal bovine serum (FBS). Forskolin (FSK) and glial-derived neurotrophic factor (GDNF) were use to promote astrocyte proliferation and survival respectively. These adult astrocyte cultures were identified by immunocytochemical, immunobloting and PCR analysis. Furthermore, biological and functional analysis indicated that they possess the biochemical and physiological properties of astrocytes, suggestive of a useful cell model for astroglial studies in adult brain.