Directed Evolution of Escherichia coli Nissle 1917 to Utilize Allulose as Sole Carbon Source.

Sugar substitutes are popular due to their akin taste and low calories. However, excessive use of aspartame and erythritol can have varying effects. While D-allulose is presently deemed a secure alternative to sugar, its excessive consumption is not devoid of cellular stress implications. In this study, the evolution of Escherichia coli Nissle 1917 (EcN) is directed to utilize allulose as sole carbon source through a combination of adaptive laboratory evolution (ALE) and fluorescence-activated droplet sorting (FADS) techniques. Employing whole genome sequencing (WGS) and clustered regularly interspaced short palindromic repeats interference (CRISPRi) in conjunction with compensatory expression displayed those genetic mutations in sugar and amino acid metabolic pathways, including glnP, glpF, gmpA, nagE, pgmB, ybaN, etc., increased allulose assimilation. Enzyme-substrate dynamics simulations and deep learning predict enhanced substrate specificity and catalytic efficiency in nagE A247E and pgmB G12R mutants. The findings evince that these mutations hold considerable promise in enhancing allulose uptake and facilitating its conversion into glycolysis, thus signifying the emergence of a novel metabolic pathway for allulose utilization. These revelations bear immense potential for the sustainable utilization of D-allulose in promoting health and well-being.

Low-Dose LPS Modulates Microglia/Macrophages Phenotypic Transformation to Amplify Rehabilitation Effects in Chronic Spinal Cord Injured (CSCI) Mice.

Spinal cord injury (SCI) results in stalled motor function recovery under the chronic phase. One of the reasons due to the presence of ongoing inflammation. Therefore, regulating the status of immune cells may help reopen the window for neural repair, which represents a potential therapeutic target. In this study, we aimed to investigate whether this could be achieved in mice with cervical 5 crush CSCI (4 W) by utilizing a concentration of 0.5 mg/kg of lipopolysaccharide (LPS) to stimulate microglia/macrophages. Additionally, the mice underwent rehabilitation training for another 6 weeks. Our results showed that systemic injection of LPS enhanced the effects of forelimb rehabilitation training, as evaluated through single pellet grasping (SPG). Electrophysiological studies revealed the restoration of cortical drive to the injured side's forelimb muscles in the training combined with LPS group. Tract tracing studies demonstrated the reconstruction of cortical innervation to the cervical spinal cord. Furthermore, the levels of pro-inflammatory phenotype markers, such as inducible nitric oxide synthase (INOS) and CD68, decreased, while the expression of anti-inflammatory phenotype markers, including arginase 1 (ARG-1) and CD206, increased. Importantly, this phenotypic switch in microglia/macrophages was accompanied by an increase in phagocytic activity markers as indicated by BODIPY + IBA1 + staining. Collectively, our data suggests that low-dose LPS improves the effects of rehabilitation training by regulating the phenotypic transformation of microglia/macrophages in CSCI. This study provides a fresh perspective and intervention direction for the clinical treatment of chronic spinal cord injuries.

Myelin repair of spinal cord injury in adult mice induced by treadmill training upregulated peroxisome proliferator-activated receptor gamma coactivator 1 alpha.

Growing evidence has proven the efficacy of physical exercise in remyelination and motor function performance after spinal cord injury (SCI). However, the molecular mechanisms of treadmill training on myelin repair and functional recovery after SCI have not yet been fully studied. Here, we explored the effect of treadmill training on upregulating peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α)-mediated myelin repair and functional recovery in a mouse model of thoracic T10 contusion injury. A 4-week treadmill training scheme was conducted on mice with SCI. The expression levels of oligodendrogenesis-related protein and PGC1α were detected by immunofluorescence, RNA fluorescence in situ hybridization and western blotting. Transmission electron microscopy (TEM) was used to observe myelin structure. The Basso Mouse Scale (BMS) and CatWalk automated gait analysis system were used for motor function recovery evaluation. Motor evoked potentials (MEPs) were also identified. In addition, adeno-associated virus (AAV)-mediated PGC1α knockdown in OLs was used to further unravel the role of PGC1α in exercise-induced remyelination. We found that treadmill training boosts oligodendrocyte precursor cells (OPCs) proliferation, potentiates oligodendrocytes (OLs) maturation, and increases myelin-related protein and myelin sheath thickness, thus impelling myelin repair and hindlimb functional performance as well as the speed and amplitude of nerve conduction after SCI. Additionally, downregulating PGC1α through AAV attenuated these positive effects of treadmill training. Collectively, our results suggest that treadmill training enhances remyelination and functional recovery by upregulating PGC1α, which should provide a step forward in the understanding of the effects of physical exercise on myelin repair.

IGF-1 Combined with OPN Promotes Neuronal Axon Growth in Vitro Through the IGF-1R/Akt/mTOR Signaling Pathway in Lipid Rafts.

This study aims to investigate the effect of insulin-like growth factor 1 (IGF-1) combined with osteopontin (OPN) on the protein expression levels and growth of neuronal axons and its possible mechanism. In this study, IGF-1 combined with OPN promoted neuronal axon growth through the IGF-1R/Akt/mTOR signaling pathway in lipid rafts, and the effect was better than that of either agent alone. This effect was suppressed when given the mTOR inhibitor rapamycin or the lipid raft cholesterol extraction agent methyl-ß-cyclodextrin (M-ß-CD). Rapamycin could inhibit the expression of phosphorylated ribosomal S6 protein (p-S6) and phosphorylated protein kinase B (p-Akt) and limit axon growth. In addition to the above effects, M-ß-CD significantly downregulated the expression of phosphorylated insulin-like growth factor 1 receptor (p-IR). To further investigate the changes in lipid rafts when stimulated by different recombinant proteins, membrane lipid rafts were isolated to observe the changes by western blot. The expression levels of insulin-like growth factor 1 receptor (IR) and P-IR in the IGF-1 combined with OPN group were the highest. When M-ß-CD was administered to the lipid rafts of neurons, the enrichment of IR by IGF-1 combined with OPN was weakened, and the p-IR was decreased. Our study found that IGF-1 combined with OPN could promote axon growth by activating the IGF-1R/Akt/mTOR signaling pathway in neuronal lipid rafts.

Multimodal rehabilitation promotes axonal sprouting and functional recovery in a murine model of spinal cord injury (SCI).

Spinal cord injury (SCI) is a devastating neurological disorder affecting millions of people worldwide, resulting in severe and permanent disabilities that significantly impact the individual's life. Rehabilitation is a commonly accepted and effective clinical treatment modality for neurological disabilities. A single form of rehabilitation training is, however, limited. Indeed, recent studies have reported that a combination of various training strategies may be more promising in promoting functional recovery. However, few studies have focused on combining different forms of rehabilitative training. Here, we investigated the effect of combining treadmill training and single pellet grasping in a well-established model of murine SCI to assess whether combining rehabilitation approaches improve outcomes. In brief, one week following crush SCI, mice were subjected to the treadmill and single pellet grasping training (SPG) for a period of six weeks. Biotinylated dextran amine (BDA) was used to anterogradely trace corticospinal tract axons to assess functionally relevant axonal sprouting. Our results revealed that the combined training upregulated p-S6 expression, facilitated axonal sprouting, increased the formation of functional synaptic connections, and promoted functional recovery of the upper limb. Our study provides experimental evidence for the benefit of combining multiple modalities of rehabilitative strategies.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 µg ml-1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

MYB gene family in the diatom Phaeodactylum tricornutum revealing their potential functions in the adaption to nitrogen deficiency and diurnal cycle.

The MYB transcription factor (TF) family is one of the largest and most important TF families, regulating the growth and response of microalgae to stress. However, the gene structure and characteristics of Phaeodactylum tricornutum MYB TFs, and their functions under nitrogen deficiency, have not been explored yet. To identify all P. tricornutum MYB (PtMYB) genes, the MYB gene family was analyzed at the genome-wide level in this study. A total ofm26 PtMYB genes were identified from the genome of P. tricornutum. These PtMYB genes were divided into 5 subfamilies: 5R-MYB, 4R-MYB, R2R3-MYB, R1R2R3-MYB, and MYB-related proteins. Phylogenetical motif and gene structure analyses of MYB genes indicated that the number and proportion of MYB TFs were species-specific, and MYB genes exhibited a lot of duplication events from microalgae to higher plants. Furthermore, the differentially expressed patterns of all 26 PtMYB TFs implied that PtMYB genes might have functional specificity under nitrogen deficiency. Homology analysis of MYB genes revealed that PtMYB3, PtMYB15, and PtMYB21 might play important roles in the regulation of the diurnal cycle and response to nitrogen stress in P. tricornutum. PtMYB3, PtMYB15, and PtMYB21 genes might be used as potential candidate genes for further studying the regulatory mechanisms of P. tricornutum under nitrogen deficiency. This work provides an important foundation for the future research of the potential functions of PtMYB genes and its diurnal regulatory mechanisms under nitrogen deficiency.