Beclin 2 functions in autophagy, degradation of G protein-coupled receptors, and metabolism.

The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism.

Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.

Author Correction: Disruption of the beclin 1-BCL2 autophagy regulatory complex promotes longevity in mice.

In this Letter, the graphs in Fig. 2a and c were inadvertently the same owing to a copy and paste error from the original graphs in Prism. The Source Data files containing the raw data were correct. Fig. 2c has been corrected online.

Autophagy plays a positive role in induction of epidermal proliferation.

Autophagy is a multistage catabolic process that mediates stress responses. However, the role of autophagy in epidermal proliferation, particularly under conditions when the epidermis becomes "activated" (hyperproliferative), remains unclear. We have shown that inhibition of Beclin 1, a key activator in the initiation phase of autophagy, attenuates imiquimod (IMQ)-induced epidermal hyperplasia in adult mice as well as naturally occurring hyperproliferation in neonatal mouse epidermis. Inhibition of Beclin 1 did not change the levels of several key inflammatory molecules or the numbers of immune cells in lesional skins. This indicates that autophagy does not affect inflammatory regulators in IMQ-treated mouse skin. Bioinformatic analysis combined with gene expression quantitative assays, revealed that a deficiency in autophagy decreases the expression of PDZ Binding Kinase (PBK), a regulator of the cell cycle, in mouse epidermis and human epidermal keratinocytes (HEKs). Interestingly, the decrease in PBK results in inhibition of proliferation in HEKs and such reduced proliferation can be rescued by activation of p38, the downstream signaling of PBK. Collectively, autophagy plays a positive role in epidermal proliferation, which is in part via regulating PBK expression.

Free fatty acid receptor 3 differentially contributes to ß-cell compensation under high-fat diet and streptozotocin stress.

The free fatty acid receptor 3 (FFA3) is a nutrient sensor of gut microbiota-generated nutrients, the short-chain fatty acids. Previously, we have shown that FFA3 is expressed in ß-cells and inhibits islet insulin secretion ex vivo. Here, we determined the physiological relevance of the above observation by challenging wild-type (WT) and FFA3 knockout (KO) male mice with 1) hyperglycemia and monitoring insulin response via highly sensitive hyperglycemic clamps, 2) dietary high fat (HF), and 3) chemical-induced diabetes. As expected, FFA3 KO mice exhibited significantly higher insulin secretion and glucose infusion rate in hyperglycemic clamps. Predictably, under metabolic stress induced by HF-diet feeding, FFA3 KO mice exhibited less glucose intolerance compared with the WT mice. Moreover, similar islet architecture and ß-cell area in HF diet-fed FFA3 KO and WT mice was observed. Upon challenge with streptozotocin (STZ), FFA3 KO mice initially exhibited a tendency for an accelerated incidence of diabetes compared with the WT mice. However, this difference was not maintained. Similar glycemia and ß-cell mass loss was observed in both genotypes 10 days post-STZ challenge. Higher resistance to STZ-induced diabetes in WT mice could be due to higher basal islet autophagy. However, this difference was not protective because in response to STZ, similar autophagy induction was observed in both WT and FFA3 KO islets. These data demonstrate that FFA3 plays a role in modulating insulin secretion and ß-cell response to stressors. The ß-cell FFA3 and autophagy link warrant further research.

A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease.

Impairment of the autophagy pathway has been observed during the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder characterized by abnormal deposition of extracellular and intracellular amyloid ß (Aß) peptides. Yet the role of autophagy in Aß production and AD progression is complex. To study whether increased basal autophagy plays a beneficial role in Aß clearance and cognitive improvement, we developed a novel genetic model to hyperactivate autophagy in vivo. We found that knock-in of a point mutation F121A in the essential autophagy gene Beclin 1/Becn1 in mice significantly reduces the interaction of BECN1 with its inhibitor BCL2, and thus leads to constitutively active autophagy even under non-autophagy-inducing conditions in multiple tissues, including brain. Becn1F121A-mediated autophagy hyperactivation significantly decreases amyloid accumulation, prevents cognitive decline, and restores survival in AD mouse models. Using an immunoisolation method, we found biochemically that Aß oligomers are autophagic substrates and sequestered inside autophagosomes in the brain of autophagy-hyperactive AD mice. In addition to genetic activation of autophagy by Becn1 gain-of-function, we also found that ML246, a small-molecule autophagy inducer, as well as voluntary exercise, a physiological autophagy inducer, exert similar Becn1-dependent protective effects on Aß removal and memory in AD mice. Taken together, these results demonstrate that genetically disrupting BECN1-BCL2 binding hyperactivates autophagy in vivo, which sequestrates amyloid oligomers and prevents AD progression. The study establishes new approaches to activate autophagy in the brain, and reveals the important function of Becn1-mediated autophagy hyperactivation in the prevention of AD.

Low-complexity recombined SLM scheme for PAPR reduction in IM/DD optical OFDM systems.

High peak-to-average power ratio (PAPR) causes nonlinear impairments in intensity modulation direct detection (IM/DD) optical orthogonal frequency division multiplexing (O-OFDM) systems. Selective mapping (SLM) is a well-known effective PAPR reduction technique, but it suffers from high computational complexity due to the bank of inverse fast Fourier transforms (IFFTs) required to generate the set of candidate signals. In this paper, we propose a recombined SLM scheme that can generate up to 2U2 symbol candidates with U IFFTs. The candidate sequences are first partitioned and then recombined to generate new candidate signals, where the addition operation replaces the IFFT block and reduces the computational complexity significantly. Simulations and a real-time end-to-end IM/DD O-OFDM transmission system with line rate 10.5 Gb/s are set up to verify the performance of the proposed scheme. It is demonstrated that compared with conventional SLM, the proposed scheme achieves similar PAPR reduction performance with considerably lower computational complexity and no bit error rate degradation.

Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis.

Exercise has beneficial effects on human health, including protection against metabolic disorders such as diabetes. However, the cellular mechanisms underlying these effects are incompletely understood. The lysosomal degradation pathway, autophagy, is an intracellular recycling system that functions during basal conditions in organelle and protein quality control. During stress, increased levels of autophagy permit cells to adapt to changing nutritional and energy demands through protein catabolism. Moreover, in animal models, autophagy protects against diseases such as cancer, neurodegenerative disorders, infections, inflammatory diseases, ageing and insulin resistance. Here we show that acute exercise induces autophagy in skeletal and cardiac muscle of fed mice. To investigate the role of exercise-mediated autophagy in vivo, we generated mutant mice that show normal levels of basal autophagy but are deficient in stimulus (exercise- or starvation)-induced autophagy. These mice (termed BCL2 AAA mice) contain knock-in mutations in BCL2 phosphorylation sites (Thr69Ala, Ser70Ala and Ser84Ala) that prevent stimulus-induced disruption of the BCL2-beclin-1 complex and autophagy activation. BCL2 AAA mice show decreased endurance and altered glucose metabolism during acute exercise, as well as impaired chronic exercise-mediated protection against high-fat-diet-induced glucose intolerance. Thus, exercise induces autophagy, BCL2 is a crucial regulator of exercise- (and starvation)-induced autophagy in vivo, and autophagy induction may contribute to the beneficial metabolic effects of exercise.

DNA methylation mechanism of intracellular zinc deficiency-induced injury in primary hippocampal neurons in the rat brain.

OBJECTIVE: To explore Zn2+ deficiency-induced neuronal injury in relation to DNA methylation, providing valuable data and basic information for clarifying the mechanism of Zn2+ deficiency-induced neuronal injury. METHODS: Cultured hippocampal neurons were exposed to the cell membrane-permeant Zn2+ chelator N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (2 µM), and to TPEN (2 µM) plus ZnSO4 (5 µM) for 24 hours. We analyzed intracellular Zn2+ levels, neuronal viability, and protein/mRNA levels for DNA (cytosine-5) methyltransferase 1 (DNMT1), DNA (cytosine-5-) methyltransferase 3 alpha (DNMT3a), methyl CpG binding protein 2 (MeCP2), Brain-derived neurotrophic factor (BDNF), and growth arrest and DNA-damage-inducible, beta (GADD45b) in the treated neurons. RESULTS: We found that exposure of hippocampal neurons to TPEN (2 µM) for 24 hours significantly reduced intracellular Zn2+ concentration and neuronal viability. Furthermore, DNMT3a, DNMT1, BDNF, and GADD45b protein levels in TPEN-treated neurons were significantly downregulated, whereas MeCP2 levels were, as expected, upregulated. In addition, DNMT3a and DNMT1 mRNA levels in TPEN-treated neurons were downregulated, while MeCP2, GADD45b, and BDNF mRNA were largely upregulated. Addition of ZnSO4 (5 µM) almost completely reversed the TPEN-induced alterations. CONCLUSION: Our data suggest that free Zn2+ deficiency-induced hippocampal neuronal injury correlates with free Zn2+ deficiency-induced changes in methylation-related protein gene expression including DNMT3a/DNMT1/MeCP2 and GADD45b, as well as BDNF gene expression.

Regulation mechanisms and signaling pathways of autophagy.

Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases. We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis. We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.

TPEN, a Specific Zn2+ Chelator, Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis, Glutamate Signaling, and Voltage-Gated K+ and Na+ Channels.

Hypoxia-ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia-ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic-ischemic conditions. The effects of N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn2+-chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-D-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K+ and Na+ channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I K as well as decreased GluR2 protein expression and Na+ channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K+ and Na+ channels in neurons. Hence, Zn2+ chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.

The cognitive impairment induced by zinc deficiency in rats aged 0∼2 months related to BDNF DNA methylation changes in the hippocampus.

OBJECTIVE: This study was carried out to understand the effects of zinc deficiency in rats aged 0∼2 months on learning and memory, and the brain-derived neurotrophic factor (BDNF) gene methylation status in the hippocampus. METHODS: The lactating mother rats were randomly divided into three groups (n = 12): zinc-adequate group (ZA: zinc 30 mg/kg diet), zinc-deprived group (ZD: zinc 1 mg/kg diet), and a pair-fed group (PF: zinc 30 mg/kg diet), in which the rats were pair-fed to those in the ZD group. After weaning (on day 23), offspring were fed the same diets as their mothers. After 37 days, the zinc concentrations in the plasma and hippocampus were measured, and the behavioral function of the offspring rats was measured using the passive avoidance performance test. We then assessed the DNA methylation patterns of the exon IX of BDNF by methylation-specific quantitative real-time PCR and the mRNA expression of BDNF in the hippocampus by RT-PCR. RESULTS: Compared with the ZA and PF groups, rats in the ZD group had shorter latency period, lower zinc concentrations in the plasma and hippocampus (P < 0.05). Interestingly, the DNA methylation of the BDNF exon IX was significantly increased in the ZD group, compared with the ZA and PF groups, whereas the expression of the BDNF mRNA was decreased. In addition, the DNMT1 mRNA expression was significantly upregulated and DNMT3A was downregulated in the ZD group, but not in the ZA and PF groups. CONCLUSION: The learning and memory damage in offspring may be a result of the epigenetic changes of the BDNF genes in response to the zinc-deficient diet during 0∼2 month period. Furthermore, this work supports the speculative notion that altered DNA methylation of BDNF in the hippocampus is one of the main causes of cognitive impairment by zinc deficiency.

A comparative study of aroma-active compounds between dark and milk chocolate: relationship to sensory perception.

BACKGROUND: The most important aroma-active compounds of two types of chocolate and cocoa liquor used for their production were analysed by gas chromatography-olfactometry-mass spectrometry (GC-O-MS) and aroma extract dilution analysis (AEDA). Furthermore, the relationship between odorants and sensory perception of chocolate was measured by quantitative analysis, sensory evaluation and correlation analysis. In addition, some chemicals were added to the original dark or milk chocolate to validate their roles in the aroma property of chocolate. RESULTS: A total of 32 major aroma-active compounds were identified in the chocolate with the flavour dilution factors of 27-729 by AEDA, including seven aldehydes, six pyrazines, three pyrroles, four carboxylic acids, four lactones, two alcohols, two ketones, one ester, one pyrone, one furan and one sulfur-containing compound. Further quantitative analysis showed that dark chocolate had higher contents of pyrazine, pyrrole, carboxylic acids, alcohols and Strecker aldehydes, whereas the concentration of lactones, esters, long chain aldehydes and ketones were higher in the milk type. CONCLUSION: Differences in volatile composition and descriptive flavour attributes between the dark and milk chocolate were observed. The relationship between aroma-active compounds and sensory perception in the chocolate was verified.

Exercise-activated hepatic autophagy via the FN1-α5ß1 integrin pathway drives metabolic benefits of exercise.

How exercise elicits systemic metabolic benefits in both muscles and non-contractile tissues is unclear. Autophagy is a stress-induced lysosomal degradation pathway that mediates protein and organelle turnover and metabolic adaptation. Exercise activates autophagy in not only contracting muscles but also non-contractile tissues including the liver. However, the role and mechanism of exercise-activated autophagy in non-contractile tissues remain mysterious. Here, we show that hepatic autophagy activation is essential for exercise-induced metabolic benefits. Plasma or serum from exercised mice is sufficient to activate autophagy in cells. By proteomic studies, we identify fibronectin (FN1), which was previously considered as an extracellular matrix protein, as an exercise-induced, muscle-secreted, autophagy-inducing circulating factor. Muscle-secreted FN1 mediates exercise-induced hepatic autophagy and systemic insulin sensitization via the hepatic receptor α5ß1 integrin and the downstream IKKα/ß-JNK1-BECN1 pathway. Thus, we demonstrate that hepatic autophagy activation drives exercise-induced metabolic benefits against diabetes via muscle-secreted soluble FN1 and hepatic α5ß1 integrin signaling.

Outcomes of single- vs two-stage primary joint arthroplasty for septic arthritis: a systematic review and meta-analysis.

Purpose: Septic arthritis (SA) is an intra-articular infection caused by purulent bacteria and the only effective method is surgical intervention. Two-stage arthroplasty is considered the gold standard treatment for SA, but recent studies have found that single-stage arthroplasty can achieve the same efficacy as two-stage arthroplasty. This study aimed to compare the efficacy of single- vs two-stage arthroplasty in the treatment of (acute or quiescent) SA. Methods: The review process was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched the PubMed, EMBASE, Medline, and Cochrane Library databases to identify all literature on the treatment of SA using single- and two-stage arthroplasty from the date of database inception to November 10, 2022. Data on reinfection rates were expressed as odds ratios and 95% CIs. Results: Seven retrospective studies with a total of 413 patients were included. Pooled analysis showed no difference in the reinfection rate between single- and two-stage arthroplasty. Subgroup analysis found no difference between the single- and two-stage arthroplasty groups in the incidence of purulent infection of the hip and knee. Cumulative meta-analysis showed gradual stabilization of outcomes. Conclusions: Based on our meta-analysis of available retrospective studies, we found no significant difference in reinfection rates between single- and two-stage arthroplasty for SA. Further prospective cohort studies are needed to confirm our results, although our meta-analysis provides important insights into the current literature on this topic.

Integrated single-cell and spatial transcriptomics reveals heterogeneity of fibroblast and pivotal genes in psoriasis.

Psoriasis, which is one of the most common skin diseases, involves an array of complex immune constituents including T cells, dendritic cells and monocytes. Particularly, the cytokine IL17A, primarily generated by TH17 cells, assumes a crucial function in the etiology of psoriasis. In this study, a comprehensive investigation utilizing bulk RNA analysis, single-cell RNA sequencing, and spatial transcriptomics was employed to elucidate the underlying mechanisms of psoriasis. Our study revealed that there is an overlap between the genes that are differentially expressed in psoriasis patients receiving three anti-IL17A monoclonal antibody drugs and the genes that are differentially expressed in lesion versus non-lesion samples in these patients. Further analysis using single-cell and spatial data from psoriasis samples confirmed the expression of hub genes that had low expressions in psoriasis tissue but were up-regulated after anti-IL17A treatments. These genes were found to be associated with the treatment effects of brodalumab and methotrexate, but not adalimumab, etanercept, and ustekinumab. Additionally, these genes were predominantly expressed in fibroblasts. In our study, fibroblasts were categorized into five clusters. Notably, hub genes exhibited predominant expression in cluster 3 fibroblasts, which were primarily engaged in the regulation of the extracellular matrix and were predominantly located in the reticular dermis. Subsequent analysis unveiled that cluster 3 fibroblasts also established communication with epithelial cells and monocytes via the ANGPTL-SDC4 ligand-receptor configuration, and their regulation was governed by the transcription factor TWIST1. Conversely, cluster 4 fibroblasts, responsible for vascular endothelial regulation, were predominantly distributed in the papillary dermis. Cluster 4 predominantly engaged in interactions with endothelial cells via MDK signals and was governed by the distinctive transcription factor, ERG. By means of an integrated analysis encompassing bulk transcriptomics, single-cell RNA sequencing, and spatial transcriptomics, we have discerned genes and clusters of fibroblasts that potentially contribute to the pathogenesis of psoriasis.

Effects of Europium Complex on Thermal and Photoluminescence Properties of Polyurethane-Europium Materials.

A europium complex with double bonds was synthesized with crotonic acid as the ligand and a europium ion as the center ion. Then, the obtained europium complex was added to synthesized poly(urethane-acrylate) macromonomers to prepare the bonded polyurethane-europium materials by the polymerization of the double bonds in the complex and the poly(urethane-acrylate) macromonomers. The prepared polyurethane-europium materials had high transparency, good thermal stability and good fluorescence. The storage moduli of polyurethane-europium materials are obviously higher than those of pure polyurethane. Polyurethane-europium materials exhibit bright red light with good monochromaticity. The light transmittance of the material decreases slightly with increases in the europium complex content, but the luminescence intensity gradually increases. In particular, polyurethane-europium materials possess a long luminescence lifetime, which has potential applications for optical display instruments.