Time-resolved FRET and NMR analyses reveal selective binding of peptides containing the LC3-interacting region to ATG8 family proteins.

Selective autophagy sequesters cytoplasmic cargo for lysosomal degradation via the binding of autophagy receptors to Atg8 (autophagy-related 8) family proteins on the autophagic membrane. The sole yeast Atg8 gene has six mAtg8 (mammalian Atg8) homologs, including the MAP1LC3 (microtubule-associated protein-1 light chain 3) family and the GABA receptor-associated proteins. Selective autophagy receptors interact with two conserved hydrophobic pockets (termed the W-site and L-site) of mATG8 proteins through a linear motif called the LC3-interacting region (LIR) with the general composition (W/F/Y)XX(I/L/V). To address a lack in our knowledge regarding LIR peptide specificity toward each mATG8 homolog, here we used competitive time-resolved FRET to sensitively and quantitatively characterize the interactions between LIRs and mAtg8. We report that 14 representative LIR-containing peptides display differential binding affinities toward the mAtg8 proteins and identified the LIR domain peptide of TP53INP1 as exhibiting high affinity for all six mATG8 proteins. Using peptide truncation studies, we found that both N- and C-terminal acidic residues, as well as the C-terminal Cys residue of the TP53INP1 LIR peptide, are required for its high-affinity binding to LC3A and LC3B, whereas binding to the GABARAP subfamily proteins was facilitated by residues either N-terminal or C-terminal to the core motif. Finally, we used NMR chemical shift perturbation analysis to gain molecular insights into these findings. Collectively, our results may aid in the development of molecules that selectively disrupt specific mATG8-LIR interactions to dissect the biological roles of the six mATG8 homologs for potential therapeutic applications.

Paediatric nurses' general self-efficacy, perceived organizational support and perceived professional benefits from Class A tertiary hospitals in Jilin province of China: the mediating effect of nursing practice environment.

BACKGROUND: General self-efficacy is considered one of the most influential parameters affecting the quality of clinical practice and nurses' perceived professional benefits (NPPB). Perceived organizational support (POS) is regarded as being central in understanding job-related attitudes, and it is important to enhance POS for nurses to maintain their current employment. NPPB can further reduce nurses' job burnout and turn-over intention. Many studies have explored the relationships among general self-efficacy, POS, nursing practice environment (NPE) and NPPB. However, a moderating effect of NPE has not been fully explored in nurses, especially among paediatric nurses. METHODS: A descriptive cross-sectional study was conducted from July to October 2018 with 300 paediatric nurses from 3 Class A tertiary hospitals in Jilin Province. The respondents completed the General Self-Efficacy Scale, Perceived Organizational Support Scale, Practice Environment Scale and Nurses' Perceived Professional Benefits Scale. The data were analyzed using path analysis and SPSS (version 23.0, IBM). RESULTS: General self-efficacy and POS were significantly positively associated with NPPB, which showed that the model had a good fit to the data. NPE was found to play a partial mediating role between POS and NPPB and also had a complete mediating role between general self-efficacy and NPPB. CONCLUSIONS: The results suggest that general self-efficacy indirectly influences NPPB, and POS directly and indirectly influences NPPB by NPE. Effective measures should be taken to improve nurses' practice environment in hospitals to raise nurses' enthusiasm and confidence in their work.

Quantification of size effect on protein rotational mobility in cells by 19F NMR spectroscopy.

Protein diffusion in living cells might differ significantly from that measured in vitro. Little is known about the effect of globular protein size on rotational diffusion in cells because each protein has distinct surface properties, which result in different interactions with cellular components. To overcome this problem, the B1 domain of protein G (GB1) and several concatemers of the protein were labeled with 5-fluorotryptophan and studied by 19F NMR in Escherichia coli cells, Xenopus laevis oocytes, and in aqueous solutions crowded with glycerol, or Ficoll70™ and lysozyme. Relaxation data show that the size dependence of protein rotation in cells is due to weak interactions of the target protein with cellular components, but the effect of these interactions decreases as protein size increases. The results provide valuable information for interpreting protein diffusion data acquired in living cells. Graphical abstract Size matters. The protein rotational mobility in living cells was assessed by 19F NMR. The size dependence effect may arise from weak interactions between protein and cytoplasmic components.

Labeling strategy and signal broadening mechanism of Protein NMR spectroscopy in Xenopus laevis oocytes.

We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in-cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high-resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. (19) F labeling is more promising than (15) N, (13) C, and (2) H enrichment. We also used (19) F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells.

Direct observation of Ca(2+) -induced calmodulin conformational transitions in intact Xenopus laevis oocytes by (19) F†NMR spectroscopy.

The Ca(2+) -mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by (19) F NMR spectroscopy of the transition of the Ca(2+) -free and -bound forms in Xenopus laevis oocytes at different Ca(2+) levels. We find that the Ca(2+) -bound CaM population increased greatly upon binding the target protein myosin light-chain kinase (MLCK) at the same Ca(2+) level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long-range structural constraints in cells. Our study shows that (19) F NMR spectroscopy can be used to obtain long-range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants.

Strategies for protein NMR in Escherichia coli.

In-cell NMR spectroscopy provides insight into protein conformation, dynamics, and function at atomic resolution in living cells. Systematic evaluation of isotopic-labeling strategies is necessary to observe the target protein in the sea of other molecules in the cell. Here, we investigate the detectability, sensitivity, and resolution of in-cell NMR spectra of the globular proteins GB1, ubiquitin, calmodulin, and bcl-xl-cutloop, resulting from uniform (15)N enrichment (with and without deuteration), selective (15)N-Leu enrichment, (13)C-methyl enrichment of isoleucine, leucine, valine, and alanine, fractional (13)C enrichment, and (19)F labeling. Most of the target proteins can be observed by (19)F labeling and (13)C enrichment with direct detection because selectively labeling suppresses background signals and because deuteration improves in-cell spectra. Our results demonstrate that the detectability of proteins is determined by weak interactions with intercellular components and that choosing appropriate labeling strategies is critical for the success of in-cell protein NMR studies.

Prevalence of hypertension among type 2 diabetes mellitus patients in China: a systematic review and meta-analysis.

This study aimed to estimate the prevalence of hypertension in Chinese patients with type 2 diabetes mellitus (T2DM) using the results of a regional study. The studies were reviewed using PubMed, EMBASE, Web of Science, the Cochrane Library, China National Knowledge Infrastructure, China Biology Medicine, Wan Fang Data and Chinese Science Citation Database. After screening articles and data extraction, Stata V.16 was used for statistical analysis. This study was registered in the Prospective Register Systematic Reviews (CRD42020170649). A total of 2126 articles were identified, and nine papers were finally included. Random-effects modelling showed that the pooled prevalence of hypertension among Chinese patients with T2DM was 54% (95% CI 47 to 61%). Subgroup analysis showed that the prevalence in men (59.8%; 95% CI 49.0 to 70.7%) was higher than that in women (40.2%; 95% CI 29.3 to 59.2%). The prevalence in North China was higher than that in East China. The prevalence in institution-based settings (56%; 95% CI 48 to 64%) was higher than that in community-based settings (51%; 95% CI 34 to 69%). Appropriate preventive measures should be undertaken, such as health education, to control and reduce the risk of hypertension in diabetic patients and reduce the burden of cardiovascular disease.

What's in an E3: role of highly curved membranes in facilitating LC3-phosphatidylethanolamine conjugation during autophagy.

During autophagosome formation, ATG3, an E2-like enzyme, catalyzes the transfer of LC3-family proteins (including Atg8 in yeast and LC3- and GABARAP-subfamily members in more complex eukaryotes) from the covalent conjugated ATG3-LC3 intermediate to PE lipids in targeted membranes. A recent study has shown that the catalytically important regions of human ATG3 (hereafter referred to as ATG3), including residues 262 to 277 and 291 to 300, in cooperation with its N-terminal curvature-sensing amphipathic helix (NAH), directly interact with the membrane. These membrane interactions are functionally necessary for in vitro conjugation and in vivo cellular assays. They provide a molecular mechanism for how the membrane curvature-sensitive interaction of the NAH of ATG3 is closely coupled to its conjugase activity. Together, the data are consistent with a model in which the highly curved phagophore rims facilitate the recruitment of the ATG3-LC3 complex and promote the conjugation of LC3 to PE lipids. Mechanistically, the highly curved membranes of the phagophore rims act in much the same manner as classical E3 enzymes in the sumo/ubiquitin system, bringing substrates into proximity and rearranging the catalytic center of ATG3. Future studies will investigate how this multifaceted membrane interaction of ATG3 works with the putative E3 complex, ATG12-ATG5-ATG16L1, to promote LC3-PE conjugation.

Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure.

VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.

(19) F NMR spectroscopy as a probe of cytoplasmic viscosity and weak protein interactions in living cells.

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of (19) F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy.

Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy.

Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims.

[Effects of moxibustion at Yongquan (KI 1) on cognition function and lower limb motor function in patients with post-stroke cognitive impairment of kidney essence deficiency].

OBJECTIVE: To observe the effects of moxibustion at Yongquan（KI 1） on the cognitive function and lower limb motor function in patients with post-stroke cognitive impairment of kidney essence deficiency. METHODS: Eighty-four patients with post-stroke cognitive impairment of kidney essence deficiency were randomly divided into an observation group（42 cases，1 case dropped off）and a control group（42 cases，1 case dropped off）.The control group was treated with medication，electroacupuncture，rehabilitation training and repetitive transcranial magnetic stimulation（rTMS）；on the basis of the treatment as the control group，moxibustion at bilateral Yongquan（KI 1）was adopted in the observation group.Both groups were treated once a day，5 days a week with 2-day interval，4 weeks were required. The Montreal cognitive assessment (MoCA) score, mini-mental state examination (MMSE) score, Fugl-Meyer assessment-lower extremity (FMA-LE) score, Berg balance scale (BBS) score, functional independence measure (FIM) score, modified fall efficacy scale (MFES) score and scale for the differentiation of syndromes of vascular dementia (SDSVD) score before and after treatment were observed in the two groups. RESULTS: After treatment，the MoCA, MMSE, FMA-LE, BBS, FIM and MFES scores were higher than those before treatment in both groups (P<0.05), and the scores in the observation group were higher than those in the control group (P<0.05). After treatment，the SDSVD scores were lower than those before treatment in both groups (P< 0.05), and the SDSVD score in the observation group was lower than that in the control group (P< 0.05). CONCLUSION: Moxibustion at Yongquan（KI 1） can improve the cognitive function and motor and balance function of lower limbs in patients with post-stroke cognitive impairment of kidney essence deficiency，reduce the risk of fall and improve the quality of life.

Social support as a mediator of the relationship between forgiveness and post-traumatic growth in hemodialysis patients: A structural equation modeling approach.

Background: Post-traumatic growth (PTG) refers to the positive psychological changes experienced with individuals after struggling with highly challenging life circumstances. Forgiveness can facilitate positive outcomes such as reduced distress, anxiety, and depression. Many studies have tested the relationships among forgiveness, social support, and PTG; however, a mechanism of social support has not been completely explored in hemodialysis patients. Objective: To test the relationship between forgiveness and post-traumatic growth and verify the mediating factor of social support on the relationship between forgiveness and PTG in hemodialysis patients. Materials and methods: In a descriptive cross-sectional study using convenience sampling from March to May 2021, 497 hemodialysis patients from nine hospitals filled out the Perceived Social Support Scale (PSSS), Heartland Forgiveness Scale (HFS), Post-traumatic Growth Inventory (PTGI), and general information. Data were analyzed using SPSS, and structural equation modeling was used to explore the relationships among forgiveness, social support, and PTG. Results: Forgiveness was significantly positively associated with PTG (P < 0.01). The proposed model provided a good fit to the data. Social support was found to play a partial mediating role between forgiveness and PTG (a*b = 0.122, BCa 95% CI: 0.078∼0.181). Conclusion: The results imply that forgiveness significantly directly and indirectly is related to PTG. Forgiveness in hemodialysis patients should be detected and effectively managed to ameliorate positive effects on PTG. It is necessary for nurses to consider implementing forgiveness interventions with an emphasis on building social support strategies to help hemodialysis patients enhance their PTG.

Developing a new individual earthquake resilience questionnaire: A reliability and validity test.

Earthquakes pose serious threats to the world. Good individual resilience can cope with disaster well, but there were few appropriate assessment tools. The purpose of this study was to develop a new individual earthquake resilience questionnaire and test its reliability and validity. First, we built the framework of the individual earthquake resilience questionnaire based on expert interviews. Then, we established the initial version of questionnaire and used the Delphi method and item selection to modify it by qualitative and quantitative methods. Finally, we built the final version of questionnaire (contained 4 dimensions and 17 items) and tested the reliability and validity. The Cronbach's α values of the four dimensions were between 0.79 and 0.91, the split-half reliabilities were between 0.85 and 0.93, and the test-retest reliabilities were between 0.72 and 0.80. The item content validity indexes were between 0.87-1, and the average questionnaire content validity index was 0.94. The correlation coefficients between each item and dimension with the total questionnaire ranged from 0.79-0.90 and 0.66-0.79, respectively. We used exploratory factor analysis to identify four common factors with a cumulative variance contribution rate of 74.97%. The questionnaire is a valid and reliable tool to measure individual resilience in the context of earthquake disasters.

NMR resonance assignments of human Atg3 in aqueous solution and bicelles.

Human Atg3 (hAtg3) is an E2-like enzyme that catalyzes the conjugation of LC3 family proteins to phosphatidylethanolamine (PE) lipids in the autophagosomal membrane during autophagy. The reaction product, LC3-PE, acts as a marker for autophagic cargo and is required for the effective construction of functional autophagosomes. However, the structural and molecular basis of this conjugation reaction remains elusive, at least in part, because of the absence of lipid bilayers in structural studies conducted to date. Here, we report a sequential resonance assignment for an hAtg3 construct both in aqueous solution and in bicelles. hAtg3 has 314 residues, and our construct lacks the unstructured region from residues 90 to 190. Our results demonstrate a structural rearrangement of hAtg3 N-terminus when it interacts with membranes.

An N-terminal conserved region in human Atg3 couples membrane curvature sensitivity to conjugase activity during autophagy.

During autophagy the enzyme Atg3 catalyzes the covalent conjugation of LC3 to the amino group of phosphatidylethanolamine (PE) lipids, which is one of the key steps in autophagosome formation. Here, we have demonstrated that an N-terminal conserved region of human Atg3 (hAtg3) communicates information from the N-terminal membrane curvature-sensitive amphipathic helix (AH), which presumably targets the enzyme to the tip of phagophore, to the C-terminally located catalytic core for LC3-PE conjugation. Mutations in the putative communication region greatly reduce or abolish the ability of hAtg3 to catalyze this conjugation in vitro and in vivo, and alter the membrane-bound conformation of the wild-type protein, as reported by NMR. Collectively, our results demonstrate that the N-terminal conserved region of hAtg3 works in concert with its geometry-selective AH to promote LC3-PE conjugation only on the target membrane, and substantiate the concept that highly curved membranes drive spatial regulation of the autophagosome biogenesis during autophagy.

Mediating effects of general self-efficacy on the relationship between the source of meaning in life and prosocial behaviours in vocational college nursing students: A cross-sectional study.

OBJECTIVES: To examine the mediating effect of general self-efficacy on the relationship between the source of meaning in life (SML) and prosocial behaviours in vocational college nursing students. METHODS: Between March and June 2019, a cross-sectional descriptive study was conducted, and 799 nursing students from three vocational colleges completed the Source of Meaning in Life Scale, General Self-Efficacy Scale and Prosocial Behaviours Scale. Data were analyzed using structural equation modelling and statistical analysis by SPSS (version 23.0, IBM). RESULTS: The average SML, general self-efficacy and prosocial behaviours scores of the 799 nursing students were 6.43±0.83, 2.48±0.59 and 3.69±0.62, respectively. Correlation analysis showed that SML, general self-efficacy and prosocial behaviours were positively correlated (P<0.01). General self-efficacy partially mediated the relationship between SML and prosocial behaviours (P<0.01); this mediating effect contributed 22.97% of the total effect and explained 17.6% of the variance in the dependent variable. CONCLUSIONS: Educators should focus on cultivating nursing students' cognition and experience of meaning in life and their efficacy in life, study and work, which can improve students' "people-oriented" service and prosocial behaviour and the quality of nursing services.

Phosphorylation dependent α-synuclein degradation monitored by in-cell NMR.

Here we report the dephosphorylation and proteolysis of phosphorylated α-synuclein, a Parkinson's disease-related protein, in living cells in a time resolved manner using in-cell NMR.

Positively Charged Tags Impede Protein Mobility in Cells as Quantified by 19F NMR.

Proteins are often tagged for visualization or delivery in the "sea" of other macromolecules in cells but how tags affect protein mobility remains poorly understood. Here, we employ in-cell 19F NMR to quantify the mobility of proteins with charged tags in Escherichia coli cells and Xenopus laevis oocytes. We find that the transient charge-charge interactions between the tag and cellular components affect protein mobility. More specifically, positively charged tags impede protein mobility.

Moderating Effects of Forgiveness on Relationship Between Empathy and Health-Related Quality of Life in Hemodialysis Patients: A Structural Equation Modeling Approach.

CONTEXT: Health-related quality of life (QOL) is a recommended clinical tool to assess hemodialysis patients and a primary end point to observe the effectiveness of overall disease management. Empathy is associated with positive outcomes such as pain relief and reduced anxiety and distress. Numerous studies have tested the relationships among empathy, forgiveness, and QOL; however, a mechanism of forgiveness has not been fully explored in hemodialysis patients. OBJECTIVES: To test the relationship among empathy and health-related QOL and confirm the moderating effects of forgiveness on relationship between empathy and health-related QOL among hemodialysis patients. METHODS: In a descriptive cross-sectional study conducted from September to December 2017, 457 hemodialysis patients from five hospitals filled out the Heartland Forgiveness Scale, Interpersonal Reactivity Index-C, Kidney Disease Questionnaire, and general information. The data were analyzed using SPSS, and structural equation modeling was used to address the relationships among empathy, forgiveness, and health-related QOL. RESULTS: Empathy was significantly positively associated with health-related QOL. The proposed model had a good fit to the data. Forgiveness was found to play a partial mediating role between empathy and health-related QOL. CONCLUSION: The results imply that empathy significantly directly and indirectly influences health-related QOL. Empathy among hemodialysis patients should be monitored and effectively managed to improve positive effects on their health-related QOL. Nurses should consider implementing empathy interventions with an emphasis on building forgiveness strategies to help hemodialysis patients improve their health-related QOL.