The influence of "small private online course + flipped classroom" teaching on physical education students' learning motivation from the perspective of self-determination theory.

Objective: The study aimed to enhance the learning motivation of college physical education students and improve their learning outcomes. Based on the perspective of the self-determination theory, this study explores the influence of "Small Private Online Course (SPOC) + flipped classroom" teaching on the learning motivation of students majoring in physical education and profoundly analyzes the influencing factors and promotion paths of learning motivation using this model. Materials and methods: A total of four classes (64 students) of physical education majors in a university were selected and randomly divided into an experimental group (34 students) and a control group (30 students). The experimental group received "SPOC + flipped classroom" teaching, the control group received traditional teaching. Before and after the 16-week intervention, learning motivation, teacher support perception, basic psychological need satisfaction, and academic emotions of the 64 students were measured, and the data were analyzed by repeated-measures analysis of variance and partial least square regression. Results: (1) The instructional intervention reduced non-regulation, external regulation, and introjected regulation, while increased identified regulation, intrinsic regulation, and self-determination levels in the students. The levels of non-regulation, external regulation, identified regulation, and self-determination were also significantly different from those of the control group. (2) After the intervention, the scores of support for autonomy, support for competence, support for relatedness, and need for relatedness in the experimental group were significantly higher than those in the control group. (3) Support for autonomy, support for competence, support for relatedness, need for competence and need for relatedness positively predicted the self-determination level, and intrinsic regulation and identified regulation negatively predicted non-regulation, external regulation, and introjected regulation. Conclusion: "SPOC + flipped classroom" teaching has a positive impact on students' learning motivation of basketball skills and promotes students' motivation autonomy. The improvement of support for autonomy, support for competence, support for relatedness, need for competence, and need for relatedness may be related to the improvement of learning motivation of college students majoring in Physical Education (PE). "SPOC + flipped classroom" teaching enables students to obtain more demand satisfaction by giving them more demand support, while demand support and demand satisfaction can promote the internalization of learning motivation so that students can maintain high autonomy motivation.

The innate immunity protein IFITM3 modulates γ-secretase in Alzheimer's disease.

Innate immunity is associated with Alzheimer's disease1, but the influence of immune activation on the production of amyloid-ß is unknown2,3. Here we identify interferon-induced transmembrane protein 3 (IFITM3) as a γ-secretase modulatory protein, and establish a mechanism by which inflammation affects the generation of amyloid-ß. Inflammatory cytokines induce the expression of IFITM3 in neurons and astrocytes, which binds to γ-secretase and upregulates its activity, thereby increasing the production of amyloid-ß. The expression of IFITM3 is increased with ageing and in mouse models that express familial Alzheimer's disease genes. Furthermore, knockout of IFITM3 reduces γ-secretase activity and the formation of amyloid plaques in a transgenic mouse model (5xFAD) of early amyloid deposition. IFITM3 protein is upregulated in tissue samples from a subset of patients with late-onset Alzheimer's disease that exhibit higher γ-secretase activity. The amount of IFITM3 in the γ-secretase complex has a strong and positive correlation with γ-secretase activity in samples from patients with late-onset Alzheimer's disease. These findings reveal a mechanism in which γ-secretase is modulated by neuroinflammation via IFITM3 and the risk of Alzheimer's disease is thereby increased.

Lasso peptides: structure, function, biosynthesis, and engineering.

Lasso peptides are a class of ribosomally-synthesized and posttranslationally-modified natural products with diverse bioactivities. This review describes the structure and function of all known lasso peptides (as of mid-2012) and covers our current knowledge about the biosynthesis of those molecules. The isolation and characterization of lasso peptides are also covered as are bioinformatics strategies for the discovery of new lasso peptides from genomic sequence data. Several studies on the engineering of new or improved function into lasso peptides are highlighted, and unanswered questions in the field are also described.

The role of a conserved threonine residue in the leader peptide of lasso peptide precursors.

The conserved threonine (Thr) residue in the penultimate position of the leader peptide of lasso peptides microcin J25 and capistruin can be effectively replaced by several amino acids close in size and shape to Thr. These findings suggest a model for lasso peptide biosynthesis in which the Thr sidechain is a recognition element for the lasso peptide maturation machinery.

Construction of a single polypeptide that matures and exports the lasso peptide microcin J25.

Roped in: The lasso peptide microcin J25 (MccJ25) is matured by two enzymes and is exported by a putative ABC transporter. We probed the function of the maturation enzymes using mutagenesis. We demonstrate that fusions of the enzymes with intervening linkers can produce MccJ25. Even a 151 kDa tripartite fusion between the ABC transporter and the two enzymes is capable of producing and exporting MccJ25.

Sequence diversity in the lasso peptide framework: discovery of functional microcin J25 variants with multiple amino acid substitutions.

Microcin J25 (MccJ25) is a ribosomally synthesized antimicrobial peptide that has an unusual threaded lasso structure in which the C-terminal "tail" of the peptide is fed through a macrocyclic "ring" formed by the N-terminal residues. Production of MccJ25 in Escherichia coli is dependent upon a four-gene cluster encoding the structural gene mcjA, two maturation enzymes mcjB and mcjC, and an immunity factor, mcjD, in the form of an MccJ25 export pump. Here we have developed a system for orthogonal control of the expression of mcjA and mcjD, thus permitting independent control of MccJ25 production and export/immunity in E. coli. We used this system to screen saturation mutagenesis libraries targeted to either the ring or tail portions of MccJ25 and discovered nearly 100 new MccJ25 variants that retain antimicrobial function. While multiple amino acid substitutions in the tail portion of the peptide are well-tolerated, mutagenesis of the ring portion of the peptide is detrimental to the antimicrobial function of MccJ25. We demonstrated that the decreased function of the ring variants is due to the inability of these variants to be transported to the cytoplasm of susceptible strains. Additionally, we found several MccJ25 variants from the tail library with improved efficacy toward the MccJ25-sensitive strains E. coli and Salmonella enterica serovar Newport with the best variants exhibiting a nearly 5-fold increase in potency. The results described here provide further evidence that diverse amino acid sequences can be tolerated by the rigid lasso peptide fold.

Computational design of the lasso peptide antibiotic microcin J25.

Microcin J25 (MccJ25) is a 21 amino acid (aa) ribosomally synthesized antimicrobial peptide with an unusual structure in which the eight N-terminal residues form a covalently cyclized macrolactam ring through which the remaining 13 aa tail is fed. An open question is the extent of sequence space that can occupy such an extraordinary, highly constrained peptide fold. To begin answering this question, here we have undertaken a computational redesign of the MccJ25 peptide using a two-stage sequence selection procedure based on both energy minimization and fold specificity. Eight of the most highly ranked sequences from the design algorithm, each of which contained two or three amino acid substitutions, were expressed in Escherichia coli and tested for production and antimicrobial activity. Six of the eight variants were successfully produced by E.coli at production levels comparable with that of the wild-type peptide. Of these six variants, three retain detectable antimicrobial activity, although this activity is reduced relative to wild-type MccJ25. The results here build upon previous findings that even rigid, constrained structures like the lasso architecture are amenable to redesign. Furthermore, this work provides evidence that a large amount of amino acid variation is tolerated by the lasso peptide fold.

Much of the microcin J25 leader peptide is dispensable.

The antimicrobial peptide microcin J25 (MccJ25) is matured by two enzymes, McjB and McjC, from a 58 amino acid (aa) preprotein, McjA, into its final 21 aa lasso topology. Herein we have investigated the role of the leader peptide of McjA and found that only the eight C-terminal amino acids of this leader peptide are required for maturation of MccJ25. There is a high content of lysine residues in the McjA leader peptide, but herein we also demonstrate that these charged amino acids do not play a major role in the maturation of MccJ25. Alanine scanning mutagenesis studies revealed that the Thr-35 residue in the leader peptide is critical for correct processing of McjA into mature MccJ25. In the absence of detailed structural and biochemical data about McjB and McjC, these studies allow us to propose a putative role for the leader peptide as a simple motif for docking of the McjA preprotein in the maturation enzymes.

Engineered gene clusters for the production of the antimicrobial peptide microcin J25.

Microcin J25 (MccJ25) is an antimicrobial peptide produced by isolates of Escherichia coli with activity against closely related species. Production and export of mature MccJ25 in E. coli requires four genes that are organized on a plasmid-borne cluster in natural producer strains. In these strains, MccJ25 production does not commence until the cells reach stationary phase, and, according to previous literature, the highest titers of MccJ25 are obtained from cells grown in nutrient-poor medium. We sought to design an engineered MccJ25 gene cluster that alleviated the growth phase and media limitations of the natural cluster. In contrast to previous reports, we observe here that production of MccJ25 from its natural cluster is efficient in rich media, such as Luria-Bertani (LB). The engineered gene cluster functions in several E. coli strains and produces titers of MccJ25 that are moderately increased (1.5- to 2-fold) relative to the natural cluster. RT-PCR experiments and translational GFP fusion experiments confirm that the engineered cluster produces MccJ25 throughout exponential phase. Furthermore, we provide evidence that control of the natural MccJ25 gene cluster is at the transcriptional level. The observations herein provide design parameters for large-scale production of MccJ25 for biotechnological applications.